FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the invention: METHOD FOR MESENCHYMAL STEM CELL ISOLATION AND
OSTEOBLAST DIFFERENTIATION
2. Applicant(s)
NAME NATIONALITY ADDRESS
REGROW BIOSCIENCES Indian 2-ABC, ACME Plaza, Andheri-
PRIVATE LIMITED Kurla Road, Andheri (E),
Mumbai-400 059, Maharashtra,
India
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.

FIELD OF INVENTION
[001] The present disclosure relates to cell therapy, preferably cell therapy in humans, particularly isolated progenitor cells from clotted bone marrow, a method for deriving such cells, and methods to differentiate such cells in culture.
BACKGROUND OF INVENTION
[002] In stem cell research, the regenerative properties of human mesenchymal stem cells (MSCs) from bone marrow for the cells' potential therapeutic applications is an important topic of research. MSCs are a promising source of adult stem cells for regenerative medicine, however many senescent cells are found in the heterogeneous ensemble of progenitors and lineage-committed cells that are associated with loss of proliferation potential and differentiation potential. Regenerative properties are highly variable among MSC subsets. Consequently, identification and isolation of progenitor subsets in heterogeneous MSC cultures are essential to the development of highly efficacious stem cell therapies. In other words, the elimination of senescent cells from heterogeneous MSC cultures may improve the treatment outcome of autologous MSC therapies by increasing both cells yield and enhancing the integrity of regenerated tissue.
[003] The bone marrow stroma was originally thought to function mainly as a structural framework for the hematopoietic component of the marrow. Subsequently, it has become well established that the stroma consists of a heterogeneous population of cells, a subset of which exerts both positive and negative regulatory effects on the proliferation and differentiation of hematopoietic stem cells (HSC) in the marrow through a combination of physical and chemical signals. The stroma also contains other non-hematopoietic cells termed mesenchymal stem cells (MSC), which are capable of both self-renewal and differentiation into osteoblasts, adipocytes, myoblasts and chondroblasts. The number of HSCs in bone marrow is about 10-100 times greater than that of MSCs. MSCs also give rise to a variety of mature cell types via a step-wise maturation process similar to haematopoiesis, termed mesengenesis. Functions that have been

attributed to MSCs include, for example, the daily control of inflammation, immune response, haematopoiesis and organ integrity.
[004] Despite the features ascribed to MSC populations by in-vitro differentiation capabilities of theirs, the mechanisms governing their proliferation and multi-lineage differentiation capacity have been poorly understood.
[005] A current barrier to realizing the therapeutic potential of MSCs is the inability to identify different MSC populations in a heterogeneous culture. The heterogeneous cultures which include cells with lower proliferation and multipotent potential results in substantial variation and decreases the effectiveness of stem cell therapies with MSCs.
[006] Ex-vivo preparations of bone marrow aspirates can show a great diversity of cell types. Even when such preparations are enriched for MSCs (e.g. by adherence), there is a remarkable diversity and heterogeneity. Methods exist in the art to enrich and expand such MSC's in culture, nonetheless, heterogeneity is observed at biochemical, genetic, and phenotypic levels.
[007] Even so-called “pure MSC” preparations demonstrate heterogeneity with variation of therapeutic effect, potency, differentiation capacity, mitotic activity, and so forth. For example, MSCs are known to undergo phenotypic rearrangements during ex vivo manipulations, losing expression of some markers while acquiring new ones (see Augello et al. “The Regulation of Differentiation in Mesenchymal Stem Cells” HUMAN GENE THERAPY 21:1226-1238 (October 2010)). Depending on culture conditions, various MSC subsets are preferentially expanded in culture, differing, for example, in expression of surface markers and other proteins, differentiation capacity, proliferation, and morphology (see Baksh et al. “Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy,” J. Cell. Mol. Med. Vol 8, No 3, 2004 pp. 301-316; Bobis et al. “Mesenchymal stem cells: characteristics and clinical applications,” FOLIA HISTOCHEMICA ET CYTOBIOLOGICA. Vol. 44, No. 4, 2006; pp. 215-230). [008] The art is replete with reports of poorly characterized preparations based on a mixed variety of cellular phenotypes derived from a mixed variety of manufacturing techniques. While there is a tendency among some to combine

teachings from such reports, such combinations can lead to false conclusions when the reports represent different MSC populations. To advance the understanding of MSC biology and therapy, it will be important to fully characterize the phenotype of MSCs in a preparation and to recognize heterogeneity where it exists. [009] JP5148873B2 discloses the isolation of a homogeneous population of cells from umbilical cord tissue containing no blood, cell renewal, expansion and differentiation in culture, where one of the process steps involves the use of dispase, hyaluronidase, and collagenase for isolation of cells.
[0010] US20070265558 discloses a process for extraction and separation of bone marrow cells that enables the increase of their therapeutic potency, where the process involves the steps of (a) centrifuging the bone marrow; (b) collecting the buffy coat layer formed after step (a); (c) centrifuging the buffy coat obtained at step (b); and collecting the buffy coat layer formed after step (c).
[0011] US8337827B2 discloses a process for obtaining osteoprogenitors, osteoblasts or osteoblast phenotype cells from human bone marrow stem cells in-vitro or ex-vivo, comprising contacting the bone marrow stem cells with human plasma or serum and a growth factor or a biologically active variant or derivative thereof.
[0012] Schlaefli et al. (“An enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples” Journal of visualized experiments: JoVE, 98 52694. 12 Apr. 2015, doi:10.3791/52694), discloses that upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system, leading to a loss of MSCs for expansion culture and direct stem cell therapy. It further discloses in the tested 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Said document further discloses a protocol for enzymatic digestion using Urokinase of those clots to avoid labor-intense and costly bone marrow resampling while maintaining the quality of cells.
[0013] Muraglia et al. (“Culture Medium Supplements Derived from Human
Platelet and Plasma: Cell Commitment and Proliferation Support” Frontiers in
bioengineering and biotechnology vol. 5 66. 20 Nov. 2017,

doi:10.3389/fbioe.2017.00066), discloses additives which are (i) a heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. When used with MSCs, these additives maintained their differentiation potential and did not show alterations in their karyotype.
[0014] Ruggiu et al. (“The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration,” Biomaterials, Vol. 34, No. 37, 2013; pp. 9318-9330), assesses the regenerative potential of platelet lysates for bone repair and discloses that platelet lysate treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential.
[0015] Thus, what is needed in the art is the ability to manufacture uniform preparations of MSCs in numbers sufficient for one or more therapeutically-effective dose, having a reproducible therapeutic action, and having a phenotype that is stable during ex-vivo expansion and following cryogenic preservation. Also required is a method of expanding and differentiating MSCs in culture under conditions that recapitulate bone remodelling in-vivo so as to reach transplantation-ready osteoblasts, for example, to treat patients with bone defects and injuries so as to repopulate, rejuvenate and fix such bone tissues.
SUMMARY OF THE INVENTION
[0016] In an aspect of the present invention, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature of at least 35℃ for a time of at least 20 minutes to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium

to obtain a suspension; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer to obtain a filtrate; (h) centrifuging the filtrate to obtain a cell pellet; and (i) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate.
[0017] In an aspect of the present invention, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (h) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (i) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate.
[0018] In another aspect of the present invention, there is provided a method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (i) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a process comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a

temperature in a range of 35°C to 39°C for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (h) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (i) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, to obtain culture flask-adhered mesenchymal stem cells; (ii) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (iii) supplementing the nutrient medium of step (i) with differentiation factors and growth factors to obtain a differentiation medium; (iv) complementing the differentiation nutrient medium of step (iii) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (v) sub-culturing the population of pre-osteoblast cells of step (iv) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (vi) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells. [0019] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0020] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may

be better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[0021] Figure 1 illustrates a representative clotted bone marrow biopsy, in
accordance with an embodiment of the present disclosure.
[0022] Figure 2 illustrates a representative pipetting bone marrow biopsy, in
accordance with an embodiment of the present disclosure.
[0023] Figure 3 illustrates a representative sample that was filtered with 100µM
cell strainer, in accordance with an embodiment of the present disclosure.
[0024] Figure 4 illustrates bone marrow mesenchymal cells (BM-MSCs) before
staining at 100X magnification, in accordance with an embodiment of the present
disclosure.
[0025] Figure 5 illustrates Crystal violet staining showing stained BM-MSC
colonies in Petri plates, in accordance with an embodiment of the present
disclosure.
[0026] Figure 6 illustrates in part (i) and (ii) the BM-MSC-derived CFU-F colonies
from clotted bone marrow sample at 100X magnification after Crystal violet
staining, in accordance with an embodiment of the present disclosure.
[0027] Figure 7 illustrates demonstrates representative FACS data that shows
immunophenotypic results using flow cytometry for bone marrow derived MSCs at
14 ± 3 days of culture for CD90, CD105 positive expression and CD34 negative
expression, in accordance with an embodiment of the present disclosure. P1 depicts
forward scattering v/s side scattering data, P2 with FITC depicts results for CD90,
P3 with PE depicts results for CD105, and P4 with APC depicts results for CD34.
[0028] Figure 8 illustrates representative FACS data that shows
immunophenotypic results using flow cytometry for bone marrow derived MSCs at 14 ± 3 days of culture for CD73 positive expression and HLA-DR negative expression, in accordance with an embodiment of the present disclosure. P1 depicts forward scattering v/s side scattering data, P2 with FITC does not gate for any marker, P3 with PE depicts results for CD73, and P4 with APC depicts results for HLA-DR.

[0029] Figure 9 illustrates in parts (i) the RT-PCR results for down-regulation of Oct-4, Nanog, Sox-2, icam, leptin and Ephrin genes in MSCs, (ii) the RT-PCR results for down-regulation of Oct-4, Nanog, Sox-2, icam, leptin and Ephrin genes in pre-osteoblasts, and (iii) the RT-PCR results for down-regulation of Oct-4, Nanog, Sox-2, icam, leptin and Ephrin genes in mature osteoblasts, in accordance with an embodiment of the present disclosure.
[0030] Figure 10 illustrates in parts (i) the RT-PCR results for up-regulation of ALP, Collagen 1, runx-2, Osterix, MEPE and EphrinB genes in MSCs, (ii) the RT-PCR results for up-regulation of ALP, Collagen 1, runx-2, Osterix, MEPE and EphrinB genes in pre-osteoblasts, and (iii) the RT-PCR results for up-regulation of ALP, Collagen 1, runx-2, Osterix, MEPE and EphrinB genes in mature osteoblasts, in accordance with an embodiment of the present disclosure.
[0031] Figure 11 illustrates a representative 6-months cryopreservation data having cell count and ALP expression, in accordance with an embodiment of the present disclosure.
[0032] Figure 12 illustrates a representative 6-months cryopreservation data having cell viability, in accordance with an embodiment of the present disclosure. [0033] Figure 13 illustrates a representative 12-months cryopreservation data having cell count, in accordance with an embodiment of the present disclosure. [0034] Figure 14 illustrates a representative 12-months cryopreservation data having cell viability, in accordance with an embodiment of the present disclosure. [0035] Figure 15 illustrates a representative 24-months cryopreservation data having cell count and ALP expression, in accordance with an embodiment of the present disclosure.
[0036] Figure 16 illustrates a representative 24-months cryopreservation data having cell viability, in accordance with an embodiment of the present disclosure. [0037] Figure 17 illustrates a representative image of stained calcified nodules, i.e., final product of cultured osteoblast cells in the culture using Alizarin red mineralization stain, which were taken under 100X of inverted microscope, in accordance with an embodiment of the present disclosure.

[0038] Figure 18 illustrates a representative image of stained calcified nodules, i.e.,
final product of cultured osteoblast cells in the culture using Alizarin red
mineralization stain, which were taken under 40X of inverted microscope, in
accordance with an embodiment of the present disclosure.
[0039] Figure 19 illustrates a representative karyotyping analysis of final product
of cultured osteoblasts cells, in accordance with an embodiment of the present
disclosure.
[0040] Figure 20 illustrates a graphical presentation of a representative karyotyping
analysis final product of cultured osteoblasts cells, in accordance with an
embodiment of the present disclosure.
[0041] Figure 21 illustrates a representative FACS data and graphs of
differentiated and cultured Osteoblasts cells culture showed positive expression for
bone Alkaline phosphatase (BALP) (>85%), in accordance with an embodiment of
the present disclosure. A total five samples were differentiated and cultured for
28±3 days of culture of osteoblasts. P1 depicts forward scattering v/s side
scattering data, P3 with PE depicts results for BALP.
[0042] Figure 22 illustrates a representative sample 1 showing Alizarin Red S
staining in Osteoblasts cultured at 28 days ± 3 days, in accordance with an
embodiment of the present disclosure.
[0043] Figure 23 illustrates a representative sample 2 showing Alizarin Red S
staining in Osteoblasts cultured at 28 days ± 3 days, in accordance with an
embodiment of the present disclosure.
[0044] Figure 24 illustrates a representative sample 3 showing Alizarin Red S
staining in Osteoblasts cultured at 28 days ± 3 days, in accordance with an
embodiment of the present disclosure.
[0045] Figure 25 illustrates a representative sample 4 showing Alizarin Red S
staining in Osteoblasts cultured at 28 days ± 3 days, in accordance with an
embodiment of the present disclosure.
[0046] Figure 26 illustrates a representative sample 5 showing Alizarin Red S
staining in Osteoblasts cultured at 28 days ± 3 days, in accordance with an
embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0047] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and
modifications. The disclosure also includes all such steps, features, compositions,
and compounds referred to or indicated in this specification, individually or
collectively, and any and all combinations of any or more of such steps or features.
[0048] Sequences 1 to 24 as disclosed herein (Table 6) has been provided as a
computer readable text file.
Definitions
[0049] For convenience, before further description of the present disclosure,
certain terms employed in the specification, and examples are delineated here.
These definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the
meanings recognized and known to those of skill in the art, however, for
convenience and completeness, particular terms and their meanings are set forth
below.
[0050] The articles “a”, “an” and “the” are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0051] The terms “comprise” and “comprising” are used in the inclusive, open
sense, meaning that additional elements may be included. It is not intended to be
construed as “consists of only”.
[0052] Throughout this specification, unless the context requires otherwise the
word “comprise”, and variations such as “comprises” and “comprising”, will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or
steps.
[0053] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.

[0054] The term “clotted bone marrow” as used herein means harvested bone marrow from clotting due to coagulation.
[0055] The term “buffer” as used herein means solution to maintain stable ph in the solution as they can neutralize small quantities of additional acid or base. [0056] The term “at least” as used herein means not less than the following amount.
[0057] The term “lysate” as used herein includes cellular lysates, platelet lysates, plasma and combinations thereof that are procured following cellular lysis. Cellular lysis may be brought on by freeze/thaw cycles, osmotic changes, other physical and chemical means known in the field.
[0058] The term “platelet-rich plasma” (PRP) as used herein means the less light plasma portion of the blood that comprises platelets in a concentration above one million per microliter.
[0059] The term “platelet lysate” as used herein means cell lysates produced from regular platelet transfusion units by lysis.
[0060] The term “platelet” as used herein refers to cells which are small a-nucleated structures of hematopoietic origin which contribute to homeostasis and wound healing by secreting growth factors and cytokines. They are produced by the fragmentation of megakaryocytes and released into the bloodstream, where they circulate for 7–10 days before being replaced.
[0061] The term “umbilical cord blood” (UCB) as used herein means the blood that remains in the placenta and in the attached umbilical cord after childbirth. Cord blood is collected because it contains stem cells, which can be used to treat hematopoietic and genetic disorders. Generally, a lot of this rich biological resource is discarded. The preferred source is human.
[0062] The process for umbilical cord blood (UCB) collection entails: (a) confirming the identity of a subject, (b) cleaning the segment of the umbilical cord with 10% povidone Iodine and 70% alcohol (Ethyl Alcohol /Isopropyl Alcohol) thrice alternately, swabbing away from the collection area, before collection, (c) after spirit evaporates, removing outer gloves to prevent contamination, (d) holding cord blood collection bag with sterile inner gloves, (e) inserting one end of a needle

in the umbilical cord vein near the cord clamp and the other end into a blood
collection bag, (f) gently and properly mixing the cord blood flowing into the
collection bag with an anticoagulant, (g) once umbilical cord appears empty and
whitish and all blood has been removed, stopping the blood link through the
needle, checking for leakage, if any, and cleaning the blood collection bag with the
collected umbilical cord blood (UCB) with sterile gauze. The preferred source is
human.
[0063] The term “maternal blood” (MB) as used herein means the blood collected
from a mother pre- and post- delivery. The maternal blood (MB) collection may
take place at a time immediately before/after cord blood collection, at the time of
admission for delivery (after initiation of labour) or before transfusion/infusion of
any intravenous fluid (colloids/crystalloids/blood products). The preferred source is
human.
[0064] The term “umbilical cord blood and maternal blood” (UCB+MB) as used
herein means any combination of an umbilical cord blood and maternal blood. Said
combination may come from autologous and/or allogenic sources. The preferred
source is human.
[0065] The term “a combination of umbilical cord blood derived platelets and
maternal blood derived platelets” as used herein refers to mixing or combination of
platelets derived from processing umbilical cord blood and maternal blood,
whether allogenous or autogenous by origin, and mixed in a ratio in a range of 10:1
to 30:1, preferably, 10:1 to 26:1.
[0066] The MSCs may be from an autologous or allogenous source, where the
source is human for another subject human.
[0067] The term “transplantation-ready” as used herein refers to mature osteoblast
cells that need not undergo any further expansion and are ready to be transplanted
into a subject in need. Further, the cell number for such transplantation-ready
osteoblasts are determined based on the size and characteristics of the site of
transplant.
[0068] Abbreviations as used herein include, α-MEM – α Minimum Essential
Medium, DMEM – Dulbecco’s Modified Eagle Medium, IMDM - Iscove's

Modified Dulbecco's Medium, EMEM - Eagle’s Minimum Essential Medium, FGF - Fibroblast Growth Factor, TGF - Transforming Growth Factor, IGF -Insulin-like growth factor 1, VEGF - Vascular endothelial growth factor, PDGF -Platelet-derived growth factor, BMP-2 – Bone Morphogenic Protein-2, CD90 -Cluster of Differentiation 90, CD73 - Cluster of Differentiation 73, CD105 -Cluster of Differentiation 105, CD34 - Cluster of Differentiation 34, HLA-DR -Human Leukocyte Antigen – DR isotype, umbilical cord blood (UCB), maternal blood (MB), platelet-rich plasma (PRP), platelet lysate (PL), mesenchymal stem cells (MSCs), OCT-4 - octamer-binding transcription factor 4, Sox-2 – Sex determining region Y box 2, ALP – Alkaline phosphatase, bone alkaline phosphatase
[0069] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. [0070] To address the problems encountered while isolating and culturing mammalian or human (primary) mesenchymal stem cells and their differentiation to osteoblastic cells in the presence optimized media composition results in enhanced expansion and the formation of aggregates or clusters of mesenchymal stem cells or of osteoblastic cells evenly distributed in the culture medium ultimately for therapeutic applications. The present disclosure provides a method that is applicable for both allogenous and autogenous sources of osteoprogenitors. [0071] In particular, the present disclosure is based on the insight that culturing human or mammalian mesenchymal stem cells or osteoblastic cells in the presence of an optimized media composition derivative over a restricted period of time, such as over two weeks or less, in particular over a time of one week or less, allows to obtain aggregates or clusters of osteoblastic cells having a unique cell count. [0072] More particularly the present disclosure is believed to be more advantageous over the conventional technique of producing pure osteoblastic

culture, i.e., delay in the transformation of osteoblastic cell aggregates to osteocyte aggregates, that is, the mineralization of the osteoblastic cell aggregates in vitro and in vivo in the presence of optimized culture media of appropriate kind. [0073] The present disclosure relates to: a method of preparing an osteoblast from an MSCs population of a mammal, the method including, enzymatic and mechanical chopping of the subject sample introducing enzyme combination with sets of different media composition for differentiation or expansion or proliferation of osteoblast or osteoprogenitors thereof mimicking in vivo bone remodeling system through the expression of genetic markers independently into the cell, the bone-related gene including at least one kind selected from the group consisting of OCT-4, Nanog, Sox2, ALP, Collagen-1, RunX2, Ephrin B4, Osterix, MEPE, ICAM-1, Leptin receptor, Ephrin B2 and an osteoblast prepared by the method. [0074] Further, it is observed that in presence of the optimized media derivative of the invention enhances cell proliferation even more; concomitantly the expression of osteoblastic cell markers indicative of differentiation towards mature osteoblasts in the respective cell culture medium. The present invention allows to obtain such cell aggregates faster and in substantially greater amounts than by known culturing processes.
[0075] The physical nature of the aggregates or clusters of the invention makes them suitable for injection into sites of bone defect in a living bone, for drug delivery, and for implant integration.
[0076] A method for producing an osteoblast or osteoprogenitor composition for treating varied bone diseases and bone fractures.
[0077] The method comprises: isolating osteoprogenitors, the like MSCs from a clotted bone marrow and differentiating them in to osteoblasts followed by culturing/proliferating the osteoblasts in a plurality of media composition comprising of but not limiting to Dulbecco's Modified Eagle's Medium (DMEM) or Minimum Essential Medium, Alpha Modification (α-MEM) or F-12 or RPMI to prepare an osteoblast suspension.
[0078] Then, mixing the resulting osteoblast suspension with a coagulation factor and a mix of enzymes to prepare an osteoblast therapeutic agent.

[0079] In an embodiment of the present disclosure; the human iliac crest and sternum are used as the major source of bone marrow with a considerable amount of bone marrow and therefore serves as main site of aspiration. However, the quality of the aspirate decreases with increasing volume of bone marrow withdrawn. The first 10-20 ml of bone marrow aspirate generally contain good quantity and quality of MSCs and further withdrawal of larger volumes leads to dilution of the aspirate with peripheral blood from the highly vascularized bone. Due to presence of megakaryocytes and platelets, bone marrow aspirates are prone to clotting, unless anticoagulants are used. Sometime with anticoagulants, clots may occur, and to improper mixing of bone marrow with anticoagulant is one of the causes
[0080] In an embodiment of the present disclosure, a blood clot, that is, a clump of blood changes from a liquid to a gel-like or semisolid state. Bone marrow aspirates (BMA) is prone to clot. BMA clot formation is usually considered a complication hampering the procedures on MSC expansion. Bone marrow is one of the major sources of MSCs, which are interesting for autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all the hematopoietic system. Bone marrow sample contains megakaryocytes and platelets, bone marrow aspirates are prone to clotting, unless anticoagulants are used. But even with anticoagulants, clots may occur due to delay in transferring bone marrow to anticoagulant solution, bone marrow needle not properly heparinized, improper mixing of bone marrow with anticoagulants or less concentrations of anticoagulants used, etc. Therefore, the resultant clots containing MSCs are lost for the expansion culture and thereby its therapeutic purposes.
[0081] In another embodiment of the present disclosure, it envisages use of enzymatic cleavage of the clotted blood sample. Urokinase mixed with enzymes including and not limiting to collagenase enzymes used for clotted bone marrow sample to isolate multipotent MSCs. This mix of enzymes not only dissolve the coagulated clots but could also allow the MSCs to migrate from the potentially existed undissolved small clots, resulting in the “liberation” of MSCs from the dense fibrin arrangement. Further the differentiation properties of MSCs isolated

from clots remain unaffected, if they are properly isolated and cultured. Therefore, enzymatic treatment proves a best method to release MSCs in the coagulated blood clots. Enzymatic digestion is an optimized technique of these clots to avoid labor-intense and costly bone marrow resampling. Further, bone marrow harvesting is a tedious procedure which involves specialized hospital set up and patient needs to go anesthetic conditions twice if harvested bone marrow doesn’t serve its purpose. Again, if the sample is clotted or knotted, then the processing of the sample becomes difficult as it takes 1-2 days to reach the processing facility depending on time and in such cases sometimes re-biopsy of the patient is not possible, which may result in an intervention for the treatment of the patient. Under such cases, enzyme urokinase mixed with enzymes including and not limiting to enzyme collagenase clears away the bone marrow clots completely. Consequently, clot treated bone marrow aspirates yield similar numbers of MSCs as like unclotted bone marrow samples. Also, after enzymatic treatment the cells can undergo metabolic activity and the ability to differentiate into osteogenic lineages. Our method recovers MSCs from clotted blood of bone marrow samples without affecting the quality of the cells and further well differentiated into osteoblasts. This obsoletes resampling, considerably reduces sampling costs and enables the use of isolated and/or differentiated cells from clotted samples for therapeutic purpose. [0082] In bone marrow, MSCs represent only a small proportion of the total cell pool and need to be expanded in culture if required for therapeutic applications. The quality of such a culture largely depends on the initial cell pool, i.e., diversity and a high starting number. Low numbers of MSCs from withdrawals may be partly explained by donor variability. On the other hand, MSCs from low quality samples require longer time in culture and extended passaging to reach the desired number of cells. In either case, extended passaging is a source of cell senescence and can lead to the loss of differentiation potential in vivo as well as in vitro. Therefore, optimized method that can maximize cell yield and prevent from unfavorable effects must be developed.
[0083] An embodiment of the present disclosure envisages bone cells to communicate with each other using various cell surface molecules, including and

more particularly expressing the in vivo bone remodeling system of bone union. Several ephrins and Ephs are expressed in osteoclasts and osteoblasts and regulate bone mineral metabolism through bidirectional signaling into not only receptor-expressing cells but also into ligand-expressing cells. Therefore, the interaction between ephrinB2-expressing osteoclasts and EphB4-expressing osteoblasts facilitates the transition from bone resorption to bone formation during bone remodeling.
[0084] Expression of the three transcription factors, OCT-4, SOX-2 and NANOG, is essential for the major properties of stem cells, self-renewal and pluripotency. OCT-4 belongs to the family of Pou-domain transcriptional factors, and it is found in developing embryos, developing endoderm as well as developing neurectoderm. SOX-2 is a member of the SRY-related HMG-box (SOX) transcription factor family with a diverse role in stem cell potency and maintenance. SOX-2 closely co-regulate alongside core pluripotency factors OCT-4 and NANOG. OCT-4 together with SOX-2 up-regulates the expression of NANOG. Upon differentiation expression, the pluripotency transcriptional factors were downregulated. Thus, OCT-4 and SOX-2 expression has been confirmed in bone marrow. The expression of OCT-4, NANOG and SOX-2 in cultured bone marrow-derived mesenchymal stem cells and differentiated osteoblasts of human origin were evaluated. [0085] BLCs express cell surface markers characteristic of mesenchymal stem/progenitors that are largely absent in osteoblasts including ICAM-1 and Leptin Receptor. ICAM‐1 positive osteoblast plays a role in osteoclastogenesis. Immunolocalization of this intercellular adhesion molecule were performed in bone sections. Bone lining cells proved to negate its presence in the in vitro culturing of osteoblasts through RT-PCR results.
[0086] Osteoprogenitor cells partially differentiate to pre-osteoblasts, which are characterized by their expression of alkaline phosphatase (ALP), an early marker of osteoblast differentiation. The early expression of the transcription factors Runx2 and OSX as well as osteoblast differentiation markers including collagen 1 and OC should be increased via detection by real-time PCR. Furthermore, ALP assays and mineralization staining such as von Kossa and alizarin red staining should be used.

[0087] Accordingly, the osteoblast composition of the present disclosure has
capability to achieve the bone reformation or regeneration for performing bone
union without the clinical graft rejection.
[0088] It is possible to uniformly distribute the osteoblast composition or
suspension into the affected area of the bone fracture or bone defect via a unique
delivery system.
[0089] Therefore, the osteoblast or osteoprogenitor composition is effectively and
rapidly cure the bone defects via a constant dosage supplement into the affected
area.
[0090] Following are the steps incurred in the present disclosure:
• Collection
• Transportation
• Isolation
• Initial characterization by Flow cytometry & MSC CFU assay
• Differentiation (diff media and diff factor)
• Expansion and comparation with FBS
• Characterization (Gene expression)
• IPQC (in detail)
• Cryopreservation
• Reprocessing
• Results of sample data
• MFG QC
[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. [0092] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of

exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. [0093] In an embodiment of the present disclosure, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature of at least 35℃ for a time of at least 20 minutes to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer to obtain a filtrate; (h) centrifuging the filtrate to obtain a cell pellet; and (i) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate.
[0094] In an embodiment of the present disclosure, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to

1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate. In another embodiment of the present disclosure, chopping the clotted bone marrow is done in pieces ranging from 2 mm3 to 5 mm3. In yet another embodiment of the present disclosure, chopping the clotted bone marrow is done into pieces ranging from 2 mm3 to 3 mm3.
[0095] In an embodiment of the present disclosure, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time in a range of 20 to 30 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate.
[0096] In an embodiment of the present disclosure, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to

obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the at least one enzyme is selected from a group consisting of urokinase, collagenase, hyaluronidase, and combinations thereof. In another embodiment of the present disclosure, urokinase is present in a range of 10,000 units to 30,000 units, collagenase type I - II is present in a range of 200 units to 500 units, and hyaluronidase type I - IV is present in a range of 200 units to 1000 units, and chopping the clotted bone marrow is done into pieces ranging from 2 mm3 to 5 mm3, and incubating the clotted bone marrow reaction solution is done for a time in a range of 20-30 minutes.
[0097] In an embodiment of the present disclosure, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats;

(g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the growth medium is selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof. [0098] In another embodiment of the present disclosure, there is provided a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces in a range of 2 mm3 to 5 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time in a range of 20 to 30 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the growth medium is selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and wherein the at least one enzyme is selected from a group consisting of urokinase, collagenase, hyaluronidase, and combinations thereof, and urokinase is present in a range of 10,000 units to 30,000 units, collagenase type I -II is present in a range of 200 units to 500 units, and hyaluronidase type I - IV is present in a range of 200 units to 1000 units.

[0099] In an embodiment of the present disclosure, there is provided a method for
preparing mesenchymal stem cells suspension from a clotted bone marrow, said
method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow
sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into
pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the
chopped clotted bone marrow to at least one enzyme in presence of a buffer to
obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone
marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of
at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated
clotted bone marrow reaction solution; (e) contacting the incubated clotted bone
marrow reaction solution of step (d) to a growth medium to obtain a suspension,
wherein the growth medium; (f) mixing the suspension for a plurality of repeats;
(g) filtering the suspension of step (f) with a cell strainer with a pore size in a range
of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to
1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet;
and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal
stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a
platelet lysate, and wherein the nutrient medium comprises a medium selected from
a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations
thereof, and a plurality of factors selected from a group consisting of, FGF, TGF,
IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof.
[00100] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces in a range of 2 mm3 to 5 mm3, to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time in a range of 20 to 30 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e)

contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the nutrient medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof.
[00101] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the at least one enzyme is selected from a group consisting of urokinase, collagenase, hyaluronidase, and combinations

thereof, and wherein the nutrient medium comprises a medium selected from a
group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations
thereof, and a plurality of factors selected from a group consisting of, FGF, TGF,
IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof. In another
embodiment of the present disclosure, urokinase is present in a range of 10,000
units to 30,000 units, collagenase type I - II is present in a range of 200 units to 500
units, and hyaluronidase type I - IV is present in a range of 200 units to 1000 units.
[00102] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the growth medium is selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and wherein the nutrient medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof.

[00103] In another embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces in a range of 2 mm3 to 5 mm3, to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one or combinations of enzymes in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time in a range of 20 to 30 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm -100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the growth medium is selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and wherein the nutrient medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof.
[00104] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted

bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a
time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain
incubated clotted bone marrow reaction solution; (e) contacting the incubated
clotted bone marrow reaction solution of step (d) to a growth medium to obtain a
suspension, wherein the growth medium; (f) mixing the suspension for a plurality
of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore
size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at
1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a
cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a
mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to
20% of a platelet lysate, and wherein the at least one enzyme is selected from a
group consisting of urokinase, collagenase, hyaluronidase, and combinations
thereof, and wherein the growth medium is selected from a group consisting of α-
MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and wherein the
nutrient medium comprises a medium selected from a group consisting of α-MEM,
DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors
selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2,
and combinations thereof, and urokinase is present in a range of 10,000 units to
30,000 units, collagenase type I - II is present in a range of 200 units to 500 units,
and hyaluronidase type I - IV is present in a range of 200 units to 1000 units.
[00105] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a

suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which are culture flask-adherent and stained with crystal violet stain.
[00106] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces in a range of 2 mm3 to 5 mm3, to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time in a range of 20 to 30 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate. wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which are culture flask-adherent and stained with crystal violet stain.
[00107] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone

marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the
bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone
marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c)
contacting the chopped clotted bone marrow to at least one enzyme in presence of a
buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted
bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a
time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain
incubated clotted bone marrow reaction solution; (e) contacting the incubated
clotted bone marrow reaction solution of step (d) to a growth medium to obtain a
suspension, wherein the growth medium; (f) mixing the suspension for a plurality
of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore
size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at
1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a
cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a
mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to
20% of a platelet lysate, and wherein the at least one enzyme is selected from a
group consisting of urokinase, collagenase, hyaluronidase, and combinations
thereof, and wherein the growth medium is selected from a group consisting of α-
MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and wherein the
nutrient medium comprises a medium selected from a group consisting of α-MEM,
DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors
selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2,
and combinations thereof, and wherein the mesenchymal stem cell suspension
comprises mesenchymal stem cells which are culture flask-adherent and stained
with crystal violet stain, and urokinase is present in a range of 10,000 units to
30,000 units, collagenase type I - II is present in a range of 200 units to 500 units,
and hyaluronidase type I - IV is present in a range of 200 units to 1000 units.
[00108] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone

marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR.
[00109] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at

1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which are culture flask-adherent and stained with crystal violet stain, and wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR.
[00110] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma.
[00111] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone

marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma, and wherein the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml. In another embodiment of the present disclosure, the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 0.7×109 platelets/ml. In yet another embodiment of the present disclosure, the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 0.5×109 platelets/ml.
[00112] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c)

contacting the chopped clotted bone marrow to at least one enzyme in presence of a
buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted
bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a
time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain
incubated clotted bone marrow reaction solution; (e) contacting the incubated
clotted bone marrow reaction solution of step (d) to a growth medium to obtain a
suspension, wherein the growth medium; (f) mixing the suspension for a plurality
of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore
size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at
1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a
cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a
mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to
20% of a platelet lysate, and wherein the at least one enzyme is selected from a
group consisting of urokinase, collagenase, hyaluronidase, and combinations
thereof, and urokinase is present in a range of 10,000 units to 30,000 units,
collagenase type I - II is present in a range of 200 units to 500 units, and
hyaluronidase type I - IV is present in a range of 200 units to 1000 units, and
wherein the platelet lysate comprises a lysate obtained from a mixture of an
umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood
(MB) derived platelet-rich plasma and wherein the mixture of an umbilical cord
blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived
platelet-rich plasma comprises 0.3× 109 to 1×109 platelets/ml.
[00113] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain

incubated clotted bone marrow reaction solution; (e) contacting the incubated
clotted bone marrow reaction solution of step (d) to a growth medium to obtain a
suspension, wherein the growth medium; (f) mixing the suspension for a plurality
of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore
size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at
1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a
cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a
mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to
20% of a platelet lysate, and wherein the growth medium is selected from a group
consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and
wherein the platelet lysate comprises a lysate obtained from a mixture of an
umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood
(MB) derived platelet-rich plasma and wherein the mixture of an umbilical cord
blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived
platelet-rich plasma comprises 0.3× 109 to 1×109 platelets/ml.
[00114] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a

mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the at least one enzyme is selected from a group consisting of urokinase, collagenase, hyaluronidase, and combinations thereof, and wherein the growth medium is selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and urokinase is present in a range of 10,000 units to 30,000 units, collagenase type I - II is present in a range of 200 units to 500 units, and hyaluronidase type I - IV is present in a range of 200 units to 1000 units, and wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma, and wherein the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml.
[00115] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the nutrient medium comprises a medium

selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and
combinations thereof, and a plurality of factors selected from a group consisting of,
FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and
wherein the platelet lysate comprises a lysate obtained from a mixture of an
umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood
(MB) derived platelet-rich plasma, and wherein the mixture of an umbilical cord
blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived
platelet-rich plasma comprises 0.3× 109 to 1×109 platelets/ml.
[00116] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the growth medium is selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and wherein the nutrient medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the platelet lysate comprises a

lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma, and wherein the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml.
[00117] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the at least one enzyme is selected from a group consisting of urokinase, collagenase, hyaluronidase, and combinations thereof, and wherein the growth medium is selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and wherein the nutrient medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof. In another embodiment of the present disclosure, and urokinase is present in a range of 10,000 units to 30,000 units, collagenase type I -

II is present in a range of 200 units to 500 units, and hyaluronidase type I - IV is present in a range of 200 units to 1000 units, and wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma, andwherein the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml.
[00118] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension, wherein the growth medium; (f) mixing the suspension for a plurality of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, and wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which are culture flask-adherent and stained with crystal violet stain, and wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma, and wherein the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml.

[00119] In an embodiment of the present disclosure, there is provided a
method for preparing mesenchymal stem cells suspension from a clotted bone
marrow, said method comprising: (a) obtaining a bone marrow sample, wherein the
bone marrow sample comprises clotted bone marrow; (b) chopping the clotted bone
marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (c)
contacting the chopped clotted bone marrow to at least one enzyme in presence of a
buffer to obtain a clotted bone marrow reaction solution; (d) incubating the clotted
bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a
time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain
incubated clotted bone marrow reaction solution; (e) contacting the incubated
clotted bone marrow reaction solution of step (d) to a growth medium to obtain a
suspension, wherein the growth medium; (f) mixing the suspension for a plurality
of repeats; (g) filtering the suspension of step (f) with a cell strainer with a pore
size in a range of 50μm - 100μm to obtain a filtrate; (g) centrifuging the filtrate at
1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a
cell pellet; and (h) dissolving the cell pellet with a nutrient medium to obtain a
mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to
20% of a platelet lysate, and wherein the mesenchymal stem cell suspension
comprises mesenchymal stem cells which test positive in flow cytometry cell
surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and
HLA-DR, and wherein the platelet lysate comprises a lysate obtained from a
mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a
maternal blood (MB) derived platelet-rich plasma, andwherein the mixture of an
umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood
(MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml.
[00120] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the

adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; and (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells.
[00121] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (i) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (ii) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (iii) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (iv) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (v) contacting the incubated clotted bone marrow reaction solution of step (iv) to a growth medium to obtain a suspension; (vi) mixing the suspension for a plurality of repeats; (vii) filtering the suspension of step (vi) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (viii) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (ix) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate, in a nutrient medium

comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells.
[00122] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of

α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof.
[00123] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof. In another embodiment of the present disclosure, the differentiation medium comprises a medium selected from a group consisting of α-MEM, IMDM, and combinations thereof, and the differentiation medium

comprises plurality of factors selected from a group consisting of FGF, TFG, IFG,
L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, and combinations thereof.
[00124] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof. In another embodiment of the present disclosure, the differentiation medium comprises a medium selected from a group consisting of α-MEM, IMDM, and combinations thereof, and the differentiation medium comprises plurality of factors selected from a group consisting of FGF, TFG, IFG, L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, and combinations thereof.
[00125] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a

mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the expansion medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of Genistein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof. In another embodiment of the present disclosure, the expansion medium comprises EMEM, F12, and combinations thereof, and the expansion medium comprises a plurality of factors selected from a group consisting of Geinstein, FGF, TGF, IGF, and combinations thereof.
[00126] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with

fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the expansion medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of Geinstein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof.
[00127] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready

osteoblast cells, wherein the nutrient medium for culturing the adhered
mesenchymal stem cells comprises a medium selected from a group consisting of
α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality
of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF,
BMP-2, and combinations thereof, and wherein the differentiation medium
comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM,
F12, EMEM and combinations thereof; and a plurality of factors selected from a
group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine,
Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and
combinations thereof, and wherein the expansion medium comprises a medium
selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and
combinations thereof; and a plurality of factors selected from a group consisting of
Geinstein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof.
[00128] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method for preparing mesenchymal stem cells suspension from a clotted bone marrow, said method comprising: (i) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow; (ii) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow; (iii) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution; (iv) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution; (v) contacting the incubated clotted bone marrow reaction solution of step (iv) to a growth medium to obtain a suspension; (vi) mixing the suspension for a plurality of repeats; (vii) filtering the suspension of step (vi) with a cell strainer with a pore size in a range of 50μm - 100μm to obtain a filtrate; (viii) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and (ix) dissolving the cell pellet

with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the
nutrient medium comprises 5% to 20% of a platelet lysate, in a nutrient medium
comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture
flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal
stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the
nutrient medium; (c) supplementing the nutrient medium of step (b) with
differentiation factors and growth factors to obtain a differentiation medium; (d)
complementing the differentiation nutrient medium of step (c) with fresh
differentiation nutrient medium comprising 5% to 20% of a platelet lysate,
differentiation factors and growth factors to obtain a population of pre-osteoblast
cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time
in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-
osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a
platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready
osteoblast cells, wherein the nutrient medium for culturing the adhered
mesenchymal stem cells comprises a medium selected from a group consisting of
α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality
of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF,
BMP-2, and combinations thereof, and wherein the differentiation medium
comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM,
F12, EMEM and combinations thereof; and a plurality of factors selected from a
group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine,
Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and
combinations thereof, and wherein the expansion medium comprises a medium
selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and
combinations thereof; and a plurality of factors selected from a group consisting of
Geinstein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof
[00129] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in

a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which are culture flask-adherent and stain with crystal violet stain.
[00130] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready

osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which are culture flask-adherent and stain with crystal violet stain.
[00131] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine,

Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof, and wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain.
[00132] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the expansion medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of Genistein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof, and wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain.
[00133] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the

adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain, and wherein the mesenchymal stem cells test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR.
[00134] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered

mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the expansion medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of Genistein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof, and wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain, and wherein the mesenchymal stem cells test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR.
[00135] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF,

BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof, and wherein the expansion medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of Genistein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof, and wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain, and wherein the mesenchymal stem cells test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR.
[00136] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain, and wherein the mesenchymal stem cells test positive

in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and
negative for CD34 and HLA-DR, and wherein wherein the mesenchymal stem cells
test positive for OCT-4, Nanog, and Sox-2 markers in RT-PCR analysis.
[00137] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain, and wherein the mesenchymal stem cells test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR, and wherein the mesenchymal stem cells test positive for OCT-4, Nanog, and Sox-2 markers in RT-PCR analysis.
[00138] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal

stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the expansion medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of Genistein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof, and wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain, and wherein the mesenchymal stem cells test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR, and wherein wherein the mesenchymal stem cells test positive for OCT-4, Nanog, and Sox-2 markers in RT-PCR analysis.
[00139] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of

step (b) with differentiation factors and growth factors to obtain a differentiation
medium; (d) complementing the differentiation nutrient medium of step (c) with
fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate,
differentiation factors and growth factors to obtain a population of pre-osteoblast
cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time
in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-
osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a
platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready
osteoblast cells, wherein the nutrient medium for culturing the adhered
mesenchymal stem cells comprises a medium selected from a group consisting of
α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality
of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF,
BMP-2, and combinations thereof, and wherein the expansion medium comprises a
medium selected from a group consisting of α-MEM, DMEM, IMDM, F12,
EMEM and combinations thereof; and a plurality of factors selected from a group
consisting of Genistein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations
thereof, and wherein the mesenchymal stem cells are culture flask-adherent and
stain with crystal violet stain, and wherein the mesenchymal stem cells test positive
in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and
negative for CD34 and HLA-DR, and wherein wherein the mesenchymal stem cells
test positive for OCT-4, Nanog, and Sox-2 markers in RT-PCR analysis.
[00140] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with

fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the pre-osteoblast cells test positive in flow cytometry cell surface marker analysis for bone ALP. In another embodiment of the present disclosure, the pre-osteoblast cells test positive for ALP, collagen-1, osterix, and runx2 and negative for ephrinB4 markers in RT-PCR analysis. In yet another embodiment of the present disclosure, the pre-osteoblast cells test positive for bone ALP by histochemical analysis.
[00141] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality

of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof, and wherein the pre-osteoblast cells test positive in flow cytometry cell surface marker analysis for bone ALP.
[00142] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a

group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof, and wherein pre-osteoblast cells test positive for ALP, collagen-1, osterix, and runx2 and negative for ephrinB4 markers in RT-PCR analysis.
[00143] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the transplantation-ready osteoblast cells test positive in flow cytometry cell surface marker analysis for ALP.
[00144] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of

step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the transplantation-ready osteoblast cells test positive for alizarin red staining.
[00145] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal
stem cell suspension, said method comprising: (a) seeding in a culture flask, a
mesenchymal stem cell suspension obtained from a method as described herein, in
a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient
medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the
adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a
platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of
step (b) with differentiation factors and growth factors to obtain a differentiation
medium; (d) complementing the differentiation nutrient medium of step (c) with
fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate,
differentiation factors and growth factors to obtain a population of pre-osteoblast
cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time
in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-
osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a
platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready
osteoblast cells, wherein the transplantation-ready osteoblast cells test positive for
bone ALP, collagen-1, osterix, runx2 and ephrinB4 markers in RT-PCR analysis.
[00146] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a

mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the transplantation-ready osteoblast cells are in a range of 12X106 cells to 60x106 cells. In another embodiment of the present disclosure, the transplantation-ready osteoblast cells are in a range of 48X106 cells to 60x106 cells. In yet another embodiment of the present disclosure, the transplantation-ready osteoblast cells are not less than 48X106 cells.
[00147] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time

in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-
osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a
platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready
osteoblast cells, wherein the platelet lysate comprises a lysate obtained from a
mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a
maternal blood (MB) derived platelet-rich plasma. In another embodiment of the
present disclosure, the mixture of an umbilical cord blood (UCB) derived platelet-
rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises
0.3×109 to 1×109 platelets/ml. In yet another embodiment of the present disclosure,
the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a
maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 0.7×109
platelets/ml. In an alternate embodiment of the present disclosure, the mixture of an
umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood
(MB) derived platelet-rich plasma comprises 0.3×109 to 0.5×109 platelets/ml.
[00148] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered

mesenchymal stem cells comprises a medium selected from a group consisting of
α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality
of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF,
BMP-2, and combinations thereof, and wherein the differentiation medium
comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM,
F12, EMEM and combinations thereof; and a plurality of factors selected from a
group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine,
Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and
combinations thereof, and wherein the transplantation-ready osteoblast cells test
positive in flow cytometry cell surface marker analysis for ALP.
[00149] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium

comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof, and wherein the transplantation-ready osteoblast cells test positive for alizarin red staining.
[00150] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and

combinations thereof, and wherein the transplantation-ready osteoblast cells test positive for bone ALP, collagen-1, osterix, runx2 and ephrinB4 markers in RT-PCR analysis.
[00151] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof, and wherein the transplantation-ready osteoblast cells are in a range of 12X106 cells to 60x106 cells. In another embodiment of the present disclosure, the transplantation-ready osteoblast cells are in a range of 48X106 cells

to 60x106 cells. In yet another embodiment of the present disclosure, the
transplantation-ready osteoblast cells are not less than 48X106 cells.
[00152] In an embodiment of the present disclosure, there is provided a
method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising: (a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as described herein, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells; (b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium; (c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium; (d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells; (e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells; (f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells, wherein the nutrient medium for culturing the adhered mesenchymal stem cells comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof, and wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitrol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof, and wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma. In another embodiment of the present disclosure, the mixture of an umbilical cord blood (UCB) derived platelet-

rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises
0.3×109 to 1×109 platelets/ml. In yet another embodiment of the present disclosure,
the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a
maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 0.7×109
platelets/ml. In an alternate embodiment of the present disclosure, the mixture of an
umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood
(MB) derived platelet-rich plasma comprises 0.3×109 to 0.5×109 platelets/ml.
[00153] Although the subject matter has been described in considerable
detail with reference to certain examples and implementations thereof, other implementations are possible.
EXAMPLES
[00154] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply. [00155] The examples as presented herein describe the best working process of the present disclosure.
EXAMPLE 1
COLLECTION OF BIOPSIES
[00156] Patient enrolled for biopsy who had undergone the required physical and
medical examination and found fit therefrom, was selected for bone therapy.
Patient under medical supervision of surgeon and under required hospital
conditions underwent biopsy collection under anaesthetic conditions. Using

Jamshidi needle, 5 ml of bone marrow biopsy from iliac crest/sternum of patient was collected in transport medium containing 90% α-MEM with 10% platelet lysate (PL) or 10% FBS. The collected sample properly labelled and packed and details of the patient recorded in biopsy collection form.
EXAMPLE 2
TRANSPORTATION:
[00157] The transportation kit was validated at different temperatures before collecting the bone marrow biopsy such as 2-8℃ for a minimum of 72 hours before transportation of actual biopsy sample. The collected bone marrow biopsy along with patient details was transported in transport kit box under controlled temperature (2-8℃) to processing facility precaution has been taken that sample should reached and processed before 72 hours from the time if collection.
EXAMPLE 3
ISOLATION OF OSTEOPROGENITOR/MSCS FROM CLOTTED BONE MARROW:
[00158] Bone marrow biopsy was collected in transport vial containing transport medium (90% α-MEM+10% UCB+MB PL). Clot formation was due to proteins in the blood and platelets of bone marrow sample and improper mixing of bone marrow. The clotted bone marrow sample (see Figures 1 and 2) was not suitable for isolation of osteoprogenitor cells/MSCs and would have resulted in discarding of the bone marrow sample. To overcome this problem clotted bone marrow sample was placed on 100 μm cell strainer (see Figure 3) on top of a sterile 50 ml centrifuge tube. Carefully the bone marrow aspirate was transferred from the transport kit onto the cell strainer, tilting the strainer or moving around the clot, using a sterile pipet tip for better flow through the filter mesh. Clotted bone marrow which was collected on cell strainer was then taken into an empty cell culture Petri dish using sterile forceps. Clot was chopped into small pieces of approximately 2-3

mm3 using a sterile scalpel and sterile needle. Small pieces of the clot were then transferred into a 50 ml centrifuge tube using the sterile forceps.
By using enzymatic method along with above chopping method: part of isolation [00159] To the above-mentioned small chopped pieces of bone clots, 10 ml of freshly prepared enzymes (Urokinase mixed with enzymes including but not limiting to collagenase enzymes (either Type I or Type II) and Hyaluronidase suspended in Hank's balanced salt solution (1X-HBSS)) and incubated for 20-30 min at 37°C in a shaking incubator at 150 rpm. Further, 10 ml of growth medium in the clotted biopsy-enzyme solution was added and vigorously mixed to form a suspension using 25 ml sterile pipette. The above vigorous mixing procedure was repeated for 3-4 times. Growth medium containing bone marrow sample was filtered with 50-100 µM cell strainer followed by centrifugation at 1300-1800 rpm for 10-15 min. After centrifugation, the supernatant was discarded, and cell pellet was dissolved with growth medium (DMEM with 10% PL/FBS). Different tests viz., cell count, cell viability (refer Table 1) and other QC tests viz., sterility and mycoplasma were performed. 1ml each of the nucleated cells of bone marrow cell suspension were seeded in four 35 mm Petri plates/ culture flasks for confirmation of CFU test and cell characterization was conducted through flow cytometry. Remaining cell suspension was seeded in T75 tissue culture grade cell culture flasks for further culture, differentiation into osteoblasts and their expansion with different combination of medium viz., DMEM, F12, RPMI and α-MEM along with 10% umbilical cord blood (UCB) and maternal blood (MB) derived Platelet lysate (PL) or with FBS, along with growth factor, FGF.

Table 1: Isolation of bone marrow nucleated cells from Clotted bone marrow
Sample ID Cell count at isolation stage Cell viability
at isolation
stage Sterility Mycoplasma
Sample-01 5.1X106 98.2 No growth Negative
Sample-02 4.4 X106 97.5 No growth Negative

Sample-03 3.6 X106 96.4 No growth Negative
Sample-04 4.8 X106 95.7 No growth Negative
Sample-05 5.6 X106 90.8 No growth Negative
Sample-06 5.2 X106 89.6 No growth Negative
Sample-07 5.9 X106 93.8 No growth Negative
Sample-08 6.4 X106 97.5 No growth Negative
Sample-09 4.9 X106 92.2 No growth Negative
Sample-10 5.3 X106 90.8 No growth Negative
Sample-11 6.2 X106 91.6 No growth Negative
Sample-12 6.4 X106 94.5 No growth Negative
Sample-13 3.9 X106 90.8 No growth Negative
Sample-14 4.6 X106 89.9 No growth Negative
Sample-15 4.1 X106 96.6 No growth Negative
[00160] One in the art would appreciate from the data above, the efficacious isolation of MSCs from clotted bone marrow using the procedure of the present disclosure.
EXAMPLE 4
INITIAL CHARACTERIZATION OF OSTEOPROGENITOR/MSCS BY USING FLOW CYTOMETRY AND MSC-CFU ASSAY:
[00161] Cells that were seeded for MSC-CFU assay and flow cytometry for initial cell characterization in four 90 mm cell culture Petri plates, were used. Bone

marrow samples (2ml each) were seeded in 90 mm Petri plate cultured with α-MEM and 10% UCB+MB PL or FBS, 2mM L‐Glutamine and 1X pen-strep antibiotics (growth medium) along with growth factors, such as 1-10 ng/ml FGF. These Petri plates were further incubated in CO2 incubator with 5% CO2 at 37℃. [00162] Medium changes (α-MEM and 10% UCB+MB PL or FBS) were given at subsequence intervals of 2-4 days up to 12-14 days in order to replenish the media which got depleted during cell growth and multiplication; which otherwise would have caused depletion of media or provided acidic nature to the culture media leading to improper growth. Therefore, media was changed at regular intervals.
MSC-CFU assay:
[00163] IDENTIFICATION OF MSCs USING CFU-F Assay
[00164] CFU‐F assay was used to enumerate or identify the number of MSCs
within a given heterogeneous population of cells isolated from bone marrow. It
was carried out by using freshly isolated primary cells from bone marrow.
[00165] Use of Crystal Violet Staining for CFU-F Assay:
[00166] Preparation of Early Passage MSCs for the CFU‐F assay:
[00167] Harvested bone marrow derived primary cells were seeded in 90 mm Petri
plate with growth medium as described above. Medium changes were given at
alternate day. Cells were incubated in CO2 incubator at 37℃ at 5% CO2 under
humidified conditions. On day 14 ± 3, cultured cells were taken out from incubator
and observed under microscope for growth of the cells. After confirmation of
growth, the cells were analyzed for crystal violet staining.
Procedure for Staining the CFU‐F:
[00168] After 14 ± 3 days of culture, petri plates were removed from the CO2
incubator, cell culture medium was pipetted out and cells were washed with buffer
for three times. Cells were stained with 0.5% of crystal violet solution freshly
prepared in methanol and incubated at room temperature for 30 minutes. Stain was
discarded and then plate washed with buffer followed by air drying.
Counting Colonies:

I/We Claim:
1. A method for preparing mesenchymal stem cells suspension from a clotted
bone marrow, said method comprising:
a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow;
b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow;
c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution;
d) incubating the clotted bone marrow reaction solution at a temperature of at least 35℃ for a time of at least 20 minutes to obtain incubated clotted bone marrow reaction solution;
e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension;
f) mixing the suspension for a plurality of repeats;
g) filtering the suspension of step (f) with a cell strainer to obtain a
filtrate;
h) centrifuging the filtrate to obtain a cell pellet; and
i) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate.
2. The method for preparing mesenchymal stem cells suspension from a
clotted bone marrow as claimed in claim 1, said method comprising:
a) obtaining a bone marrow sample, wherein the bone marrow sample comprises clotted bone marrow;
b) chopping the clotted bone marrow into pieces of at least 2 mm3 to obtain chopped clotted bone marrow;
c) contacting the chopped clotted bone marrow to at least one enzyme in presence of a buffer to obtain a clotted bone marrow reaction solution;

d) incubating the clotted bone marrow reaction solution at a temperature in a range of 35℃ to 39℃ for a time of at least 20 minutes at a speed in a range of 100 rpm to 200 rpm to obtain incubated clotted bone marrow reaction solution;
e) contacting the incubated clotted bone marrow reaction solution of step (d) to a growth medium to obtain a suspension;
f) mixing the suspension for a plurality of repeats;
g) filtering the suspension of step (f) with a cell strainer with a pore
size in a range of 50μm - 100μm to obtain a filtrate;
h) centrifuging the filtrate at 1300 rpm to 1800 rpm for a time in a range of 10 minutes to 15 minutes to obtain a cell pellet; and
i) dissolving the cell pellet with a nutrient medium to obtain a mesenchymal stem cell suspension, wherein the nutrient medium comprises 5% to 20% of a platelet lysate.
3. The method as claimed in claim 1, wherein the at least one enzyme is selected from a group consisting of urokinase, collagenase, hyaluronidase, and combinations thereof.
4. The method as claimed in claim 1, wherein the growth medium is selected from a group consisting of α Minimum Essential Medium (α-MEM), Dulbecco’s Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), F12, Eagle’s Minimum Essential Medium (EMEM) and combinations thereof.
5. The method as claimed in claim 1, wherein the nutrient medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof, and a plurality of factors selected from a group consisting of, Fibroblast Growth Factor (FGF), Transforming Growth Factor (TGF), Insulin-like growth factor 1 (IGF), Vascular endothelial growth factor (VEGF), Platelet-derived growth factor (PDGF), Bone Morphogenic Protein-2 (BMP-2), and combinations thereof.
6. The method as claimed in claim 3, wherein urokinase is present in a range of 10,000 units to 30,000 units, collagenase type I - II is present in a range of 200 units to 500 units, and hyaluronidase type I - IV is present in a range of 200 units to 1000 units.

7. The method as claimed in claim 1, wherein the mesenchymal stem cell suspension comprises mesenchymal stem cells which are culture flask-adherent and stained with crystal violet stain.
8. The method as claimed in claim 1, wherein the mesenchymal stem cells test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR.
9. The method as claimed in claim 1, wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma.
10. The method as claimed in claim 9, wherein the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml.
11. A method for preparing transplantation-ready osteoblast cells from mesenchymal stem cell suspension, said method comprising:

a) seeding in a culture flask, a mesenchymal stem cell suspension obtained from a method as claimed in claim 1, in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium, to obtain culture flask-adhered mesenchymal stem cells;
b) culturing the adhered mesenchymal stem cells in a nutrient medium comprising 5% to 20% of a platelet lysate in the nutrient medium;
c) supplementing the nutrient medium of step (b) with differentiation factors and growth factors to obtain a differentiation medium;
d) complementing the differentiation nutrient medium of step (c) with fresh differentiation nutrient medium comprising 5% to 20% of a platelet lysate, differentiation factors and growth factors to obtain a population of pre-osteoblast cells;
e) sub-culturing the population of pre-osteoblast cells of step (d) for a time in a range of 6 to 10 days to obtain a pre-osteoblast cells;
f) expanding the pre-osteoblast cells in an expansion nutrient medium comprising 5% to 20% of a platelet lysate, for a time in a range of 10 to 20 days to obtain transplantation-ready osteoblast cells.
12. The method as claimed in claim 11, wherein the nutrient medium for
culturing the adhered mesenchymal stem cells comprises a medium selected

from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, VEGF, PDGF, IGF, BMP-2, and combinations thereof.
13. The method as claimed in claim 11, wherein the differentiation medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of, FGF, TGF, IGF, VEGF, PDGF, BMP-2 L-Thyroxine, Calcitriol, Stanozolol, Dexamethasone, β-glycerophosphate, L-Ascorbic acid, and combinations thereof.
14. The method as claimed in claim 11, wherein the expansion medium comprises a medium selected from a group consisting of α-MEM, DMEM, IMDM, F12, EMEM and combinations thereof; and a plurality of factors selected from a group consisting of Genistein, FGF, TGF, IGF, VEGF, PDGF, BMP-2, and combinations thereof.
15. The method as claimed in claim 11, wherein the mesenchymal stem cells are culture flask-adherent and stain with crystal violet stain.
16. The method as claimed in claim 11, wherein the mesenchymal stem cells test positive in flow cytometry cell surface marker analysis for CD90, CD73 and CD105, and negative for CD34 and HLA-DR.
17. The method as claimed in claim 11, wherein the mesenchymal stem cells test positive for OCT-4, Nanog, and Sox-2 markers in RT-PCR analysis.
18. The method as claimed in claim 11, wherein the pre-osteoblast cells test positive in flow cytometry cell surface marker analysis for bone ALP.
19. The method as claimed in claim 11, wherein the pre-osteoblast cells test positive for ALP, collagen-1, osterix, and runx2 and negative for ephrinB4 markers in RT-PCR analysis.
20. The method as claimed in claim 11, wherein the pre-osteoblast cells test positive for bone ALP by histochemical analysis.
21. The method as claimed in claim 11, wherein the transplantation-ready osteoblast cells test positive in flow cytometry cell surface marker analysis for ALP.
22. The method as claimed in claim 11, wherein the transplantation-ready osteoblast cells test positive for alizarin red staining.

23. The method as claimed in claim 11, wherein the transplantation-ready osteoblast cells test positive for bone ALP, collagen-1, osterix, runx2 and ephrinB4 markers in RT-PCR analysis.
24. The method as claimed in claim 11, wherein the transplantation-ready osteoblast cells are in a range of 12X106 cells to 60x106 cells.
25. The method as claimed in claim 11, wherein the platelet lysate comprises a lysate obtained from a mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma.
26. The method as claimed in claim 25, wherein the mixture of an umbilical cord blood (UCB) derived platelet-rich plasma and a maternal blood (MB) derived platelet-rich plasma comprises 0.3×109 to 1×109 platelets/ml.
27. The transplantation-ready osteoblast cells obtained using the method as claimed in claim 11 for use in transplantation of the osteoblast cells into a subject.