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: PLATELET LYSATE, AND METHOD OF PREPARATION
THEREOF
2. Applicant(s)
NAME NATIONALITY ADDRESS
REGROW BIOSCIENCES Indian 2-ABC, ACME Plaza, Andheri-Kurla
PRIVATE LIMITED 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 culture media and cell culture media supplements. Particularly, the present disclosure relates to an animal-product free cell culture media and cell culture media supplements. More particularly, the present disclosure relates to platelet lysate obtained from platelet-rich plasma, said platelet-rich plasma comprises a mixture of umbilical cord blood (UCB) and maternal blood (MB). Further, the present disclosure relates to a method of preparing said platelet lysate.
BACKGROUND OF INVENTION
[002] Cell and tissue culture media are typically composed of mineral salts, amino acids and vitamins to aid cultivation and propagation of cells under ex-vivo conditions. Generally, these media are supplemented with non-human, animal-derived additives such as non-human animal-derived Fetal Bovine Serum (FBS), which has become a routine and well-accepted practice. An alternative means of supplementing cell culture media is by means of feeder layer cells that aid the primary culture of cells from animal tissues. These non-human animal-derived supplements and feeder layer cells provide an important means of growth factors, hormones, lipids, trace elements, vitamins, protease inhibitors, adhesion molecules, chelators and/or uncharacterized factors, that may support cell expansion, proliferation, survival, propagation and differentiation.
[003] However, the use of FBS is marred with challenges that include: (a) a cocktail of undefined qualitative and quantitative composition, which is added to a chemically defined basal medium; (b) a considerable ethical concern in terms of replacement, reduction, refinement; were raised regarding the mode of blood collection and FBS harvest, respectively, from bovine foetuses; (c) FBS being a by¬product of the beef industry, global supply and availability of FBS is dependent on many factors and can be highly fluctuating; and (d) the fraudulent blending of FBS batches with adult bovine serum albumin, water and cell growth promoting additives result in batch-to-batch variation.

[004] The risk with using FBS in propagating and culturing stem cells and differentiated cells meant for further therapeutic applications is a major and undesirable risk for patients in clinical settings. Non-human animal-derived products such as FBS may cause immune-reactions towards foreign factors as well as cross-species pathogen infections; possible allergic reactions caused by FBS proteins internalized in the stem cells and risks of transmitting bovine-viral and bacterial contamination, notably the mycoplasma infections. Additionally, clinical trials and any future therapeutic applications of stem cells recommend avoiding any usage of animal serum in in-vitro cell expansion. The use of fetal bovine serum (FBS) as a cell culture supplement is also discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. [005] Thus, there is a need to find an appropriate replacement to the aforementioned traditional culture media supplements, which would possess the required efficacy and efficiency for culturing and survival of sensitive and tricky cells such as stem cells and cells for therapeutic applications. One such growth supplement that has been explored as an FBS alternative in recent research has focused on the application of platelet-derived products, e.g., platelet lysates that can be produced from regular platelet transfusion units by lysis. [006] Platelet are known to contribute to homeostasis and wound healing. Due to their role in different biological processes, research focused on various therapeutic applications of platelets by exploiting their growth factor secretion and varied proteins for potential clinical treatments has been fueled recently. Platelet-based biomaterials such as platelet gel, platelet glue and platelet rich plasma have been reported to be used in the treatment of chronic ulcers and are a potential source in orthopaedics to facilitate healing and enhancing bone-grafting following implantation (refer Shaheen A "Platelet Rich Plasma (PRP) for Treatment Non-Healing Ulcers: A Review Study," Austin J Dermatolog, 2018, 5(1):1085-1094, Wellington K Hsu et al. "Platelet-rich Plasma in Orthopaedic Applications: Evidence-based Recommendations for Treatment,' J Am Acad Orthop Surg 2013;21:739-748, and Mlynarek RA et al. "Platenet-Rich Plasma (PRP) in Orthopedic Sports Medicine,} Am J Orthop. 2016 May;45(4):290-294, 326).

[007] The methods for isolation and enrichment of platelet fraction from blood and further preparation of platelet lysates have relied upon centrifugation as a means to segregate platelet-rich plasma fraction from a blood sample, such as peripheral blood from a human subject from the cell pellet and platelet-poor plasma fraction, for example.
[008] CN106236779 discloses a method for preparing platelet-rich plasma (PRP) from cord blood comprises the steps of: centrifuging fresh umbilical cord blood at a low speed to remove the lower layer of cells, and ultra-high-speed centrifugation of the upper platelet-containing plasma, the liquid is divided into two layers, and the upper layer is a plasma layer. Here, the platelet lysate is used in treating cartilage damage, relieving pain and restoring function, and particularly effective in treating senile osteoarthrosis. However, this document also discusses that due to different methods, different concentrations, and different treatment options, the obtained PRP varies greatly in treatment effect.
[009] Bernardi et al. ('The production method affects the efficacy of platelet derivatives to expand Mesenchymal stromal cells in vitro" Journal of translational medicine, 2017, vol. 15,1 90. 1, doi:10.1186/s12967-017-1185-9), also discloses that the production method used to release platelet factors significantly affects the enrichment in growth factors and overall product performance. The standardization of the production process of platelet derivatives and the definition of their release criteria requires further investigation. Further, this document provides that platelet derivatives are valid FBS substitutes to support the ex-vivo expansion of bone-marrow derived mesenchymal stromal cells (BM-MSC).
[0010] EP2757879A1 discloses a method for producing a platelet lysate with a maximum level of safety by subjecting platelets to a pathogen inactivation procedure such as by a photochemical agent and UV radiation, followed by at least one freeze/thaw cycle for platelet lysis followed by centrifugation to isolate platelet lysate containing liquid phase, which is capable of acting as an adjuvant for the isolation and/or growth and/or expansion of stem cells, fibroblasts or dendritic cells. [0011] US9700583B2 discloses a method of autologous mesenchymal stem cells (MSCs) isolation from a patient, expansion of said MSCs in presence of autologous

growth factors located on patient's platelets for subsequent implantation into said patient. Here again, by reference (US5198357A), the said document teaches preparation of platelet lysate from plasma derived from animal whole blood prepared by centrifuging plasma to produce platelet rich paste, adding calcium to paste to lyse the platelets therein to obtain the platelet lysate to be useful as FBS replacement. [0012] Shirzad et al. ("Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells." Cell journal, 2017; 19(3):403-414.), discloses culture supplements devoid of animal-derived products and provides umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for FBS and human peripheral blood-PL (PB-PL). Interestingly, this document found that PB-PL promoted osteoblastic differentiation, while UCB-PL induced chondrogenic differentiation. Further, this disclosure teaches pooling various UCB samples to obtain UCB-PL by means of centrifugation and freeze/thaw cycle lead to UCB-PL. [0013] Kwok et al. ("Maternal plasma or human serum albumin in wash buffer enhances enrichment and ex vivo expansion of human umbilical cord blood CD34+ cells" Br J Haematol. 2007; 137(5):468-74), discloses that the presence of maternal plasma/human serum albumin (HSA) in the purification and culture conditions of CD34+ cells derived from human umbilical cord blood increases the purity and fold expansion of said CD34+ cells, which was over and above such expansion in the presence of FBS, while HSA and maternal plasma were equivalent and HAS was proposed to be responsible in maternal plasma for the effect of expansion of CD34+ cells.
[0014] US20110123503A1 discloses platelet fractions which can be obtained from placental blood, with high concentrations of platelet factors as well as gels and lysates deriving therefrom as well as methods for preparing said platelet fractions and uses thereof as platelet gels or as lysates. This document points out the drawbacks and problems associated with usual sources of media supplements such as peripheral blood from a human (allogenic blood of non-autologous origin) which cannot be used since immune system of a human does not recognize said platelet fraction as autologous as well as problems associated with FBS as have been alluded to herein above. Further, this document teaches platelet fraction is derived from

placental blood, preferably umbilical cord blood, wherein the source preferably is human. The process for preparation again teaches centrifugation methods. Further, the document teaches that said platelet fraction is characterized by a higher concentration of platelet factors than human peripheral blood. [0015] The above described methods for preparation of platelet fractions, including platelet lysates have drawbacks and problems in terms of loss of logistics, costs and amount of cellular (platelet) damage due to the use of centrifugation/apheresis manufacturing procedures on blood products. Further, the loss of discarded umbilical cord blood (UCB) or discarded maternal blood (MB) isolated for testing or otherwise, which potentially comprises a plethora of growth factors and differentiation factors that may be exploited for therapeutic applications in cell and tissue cultures meant for implantation into patients and beyond has potential economic benefits.
[0016] In line with the problems in the art highlighted above, Gifford et al. (|A portable system for processing donated whole blood into high quality components without centrifugation} 1/07+ PLoS ONE 13(1): e0190827), discloses that the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. This document discloses a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation. The process disclosed involves (i) a blood-bag compression apparatus designed to maximize the speed and efficiency of passive RBC sedimentation for separating RBCs and platelet-rich plasma (PRP) from WB at normal gravity (i.e., 1×g), and (ii) a flow-through microfluidic concentrator for continuous high-throughput enrichment of platelets from the PRP fraction, to produce plasma cells and purified plasma. [0017] Hence, there remains a problem of finding an appropriate method for platelet fraction isolation, providing a viable and economical source for isolation, which is not associated with problems of long distance transport from the collection facilities, is logistically viable in different regions and facilities thereby avoiding centrifugation/apheresis manufacturing equipment and therefore, can overcome the problems disclosed above, keeping and even increasing its application potential.

SUMMARY OF THE INVENTION
[0018] In an aspect of the present invention, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets. [0019] In an aspect of the present invention, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;

(g) storing the UCB+MB platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen UCB+MB platelet-rich plasma mixture; and (h) subjecting the frozen UCB+MB platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
[0020] In another aspect of the present invention, there is provided an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelets; and (b) a platelet lysate obtained from maternal blood (MB) derived platelets, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml. [0021] 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
[0022] 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.
[0023] Figure 1 illustrates a flow-chart of the process summary of obtaining the
unique umbilical cord blood and maternal blood (UCB+MB) platelet lysate (PL), in
accordance with an embodiment of the present disclosure.
[0024] Figure 2 illustrates SDS-PAGE results from aliquots of platelet lysates from
USB+MB, UCB, and FBS, in accordance with an embodiment of the present
disclosure. Different lanes represented are: Lane 1 - Marker, Lane 2 - Standard

Human Albumin Protein, Lane 3 - FBS Protein, Lane 4 - UCB PL Protein, and Lane
5 - (UCB+MB) PL Protein.
[0025] Figure 3 illustrates a Chromatogram for UCB+MB PL, in accordance with
an embodiment of the present disclosure.
[0026] Figure 4 illustrates a Chromatogram for FBS, in accordance with an
embodiment of the present disclosure.
[0027] Figure 5 illustrates a Chromatogram for UCB PL, in accordance with an
embodiment of the present disclosure.
[0028] Figure 6 illustrates a Chromatogram for MB PL, in accordance with an
embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0029] 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.
Definitions
[0030] 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.
[0031] 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.
[0032] 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".

[0033] 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.
[0034] The term "including" as used herein means "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0035] The term "at least" as used herein means not less than the following amount.
[0036] 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.
[0037] 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.
[0038] The term "platelet lysate" as used herein means cell lysates produced from
regular platelet transfusion units by lysis.
[0039] 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.
[0040] The term "subject" as used herein refers to any vertebrate animal and does
not merely cover human. Human subjects have been used to exemplify the invention
but, said exemplification should not be considered in any way limiting to the scope
of the subject matter as covered under the term subject.
[0041] 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.

[0042] 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.
[0043] 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.
[0044] The term "umbillical 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.
[0045] The "sedimentation" as used herein refers to the tendency for heavier
particles in suspension to settle out of the fluid in which they are entrained and come
to rest against a barrier like bottom of a container. Such a motion through the fluid
may be in response to the forces acting on them, which may
include, gravity, centrifugal acceleration, or electromagnetism.
[0046] term "atleast one sedimentation" as used herein refers to minimum of one
sedimentation.
[0047] The term "at least two sedimentation" as used herein refers to a minimum
of two sedimentations.

[0048] The term "sedimentation mixture" as used herein means any mix of substances that leads to and/or aids a sedimentation process, where heavier components of the mixture settle on the bottom, due to gravity, leaving the lighter components amenable for separation such as by decantation or pipetting. [0049] The term "sedimentation reagent" as used herein means any substance that aids coagulation or aggregation of particles or components in suspension to form heavier particles that then come to rest against a barrier like bottom of a container due to the forces acting on them such as gravity. As used herein, a sedimentation reagent such as Ficoll-Hipaque, Hespan®, Pentastarch, and combinations thereof. [0050] The term "frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture" as used herein means the mixture obtained by freezing said mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours.
[0051] The term "freeze-thaw cycle" as used herein refers to a process of rapid freeze (bringing to low temperature) and slow thaw (bringing to high temperature) that results in cell death and/or cell lysis. Often repeated a set number of times according to the size and nature of cells being treated. As used herein the freeze-thaw cycles may range from 1 to 5, and the temperature for freezing ranges from -70°C to -86°C and the temperature for thawing ranges from 30°C to 40°C. [0052] The term "platelets/ml" as used herein refers to the number of platelets per millilitre of a fluid. Preferably said fluid is plasma.
[0053] 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, and preferably, 10:1 to 26:1.
[0054] The term "infectious diseases" as used herein refers to diseases caused by pathogens such as microbes including HIV I and II, HBs, HCV, Syphilis, Mycoplasma etc.
[0055] The term "enriched" as used herein refers to enhancement in concentration of an amount of a component per volume containing said component following

processing steps. For platelet-rich plasma as used herein, to obtain umbilical cord blood and maternal blood (UCB+MB) 'enriched' platelet lysate, the platelets in platelet-rich plasma obtained by combination of umbilical cord blood and maternal blood (UCB+MB) are in a range of 0.3×109 to 1×109 platelets/ml. [0056] The term "culturing" as used herein refers to maintaining (tissues, cells, bacteria, etc.) in conditions suitable for growth, propagation, expansion, differentiation and the like, in a specially prepared nutrient medium under supervised conditions.
[0057] The term "cells" as used herein refers to any type of stem cells, progenitor cells and differentiated cells. Preferably, the cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, and cardiomyocytes. The cells are preferably human by origin. [0058] The term "therapeutic applications" means applications including, but not limited to, mesenchymal stem cells derived from human umbilical cord tissue, osteoblasts differentiated from bone marrow derived mesenchymal stem cells, cardiomyocytes differentiated from human umbilical cord tissue derived mesenchymal stem cells, islets cells of pancreas differentiated from human umbilical cord tissue derived mesenchymal stem cells, chondrocytes from human cartilage biopsy and buccal biopsy derived dermal fibroblasts. The therapeutic applications are further incorporated with but not limiting to, certain biomaterials or cell-gel techniques or methods thereof.
[0059] 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.
[0060] To address the problems encountered with use of FBS, the present disclosure provides an optimized non-animal origin cell and tissue culture supplement in the form of an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate. Further, the present disclosure provides a method for preparing a lysate,

wherein the lysate is the umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate.
[0061] 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.
[0062] 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. [0063] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets. [0064] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood

(UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least two sedimentations to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets. [0065] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of

umbilical cord blood derived platelets and maternal blood derived platelets, wherein storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture. In another embodiment of the present disclosure, storing is done at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture.
[0066] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least two sedimentations to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture. In another embodiment of the present disclosure, storing is done at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture.

[0067] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate.
[0068] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least two sedimentations to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical

cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate.
[0069] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate, and wherein storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture. In another embodiment of the present disclosure, the storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture.
[0070] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least

one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least two sedimentations to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate, and wherein storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture. In another embodiment of the present disclosure, the storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture.
[0071] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and

the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
[0072] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C tro -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate. [0073] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood

(UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1.
[0074] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation

mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least two sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate, and wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1.
[0075] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-

rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate, and wherein the at least one sedimentation is carried out at a temperature in a range of -70°C to -86°C, and wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1. In another embodiment of the present disclosure, the at least one sedimentation is carried out at a temperature in a range of -70 to -86°C for a time in a range of 24 hours to 96 hours. [0076] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at

least two sedimentations to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate, and wherein storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture, and wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1. In another embodiment of the present disclosure, the at least one sedimentation is carried out at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours.
[0077] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least

one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1.
[0078] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least two sedimentation to obtain an umbilical cord blood (UCB) derived platelet-

rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1.
[0079] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB)

derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture, and wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1. In another embodiment of the present disclosure, the at least one sedimentation is carried out at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours. [0080] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least two sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood

(MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein storing is done at a temperature in a range of -70°C to -86°C to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture, and wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1. In another embodiment of the present disclosure, the at least one sedimentation is carried out at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours. [0081] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation

to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1.
[0082] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation

to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the first, the second and the third sedimentation are carried out for 25-55 minutes. In another embodiment of the present disclosure, the first, the second and the third sedimentation are carried out for 30-50 minutes. In yet another embodiment of the present disclosure, the first, the second and the third sedimentation are carried out for 32-42 minutes. In an alternate embodiment of the present disclosure, the first, the second and the third sedimentation are carried out for 35 minutes.
[0083] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an

umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sample is at least 100ml and the maternal blood (MB) sample is in a range of 15ml to 20ml. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sample is in a range of 80ml to 120ml and the maternal blood (MB) sample is in a range of 15ml to 20ml.
[0084] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature of -86°C for a time of 48 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a

combination of umbilical cord blood derived platelets and maternal blood derived platelets.
[0085] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature of -86°C for a time of 48 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sample and maternal blood (MB) sample are independently subjected to an initial platelet count by withdrawing aliquot of 100-200 µl.
[0086] In an embodiment of the present disclosure, therein is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation

to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature of -86°C for a time of 48 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the human umbilical cord blood (UCB) and maternal blood (MB) platelets are obtained from plasma which is considered as a bio-waste obtained after processing the cord blood. Said umbilical cord blood (UCB) is generally considered as bio-waste which are discarded after delivery, and similarly, said maternal blood (MB) taken for testing purposes is also discarded after tests. Further, said umbilical cord blood (UCB) and maternal blood (MB) are further checked for infectious disease testing and sample free of contaminations and free from infectious diseases are selected for the preparation of platelet lysate.
[0087] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and

the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is selected from a group consisting of cell lysate, platelet lysate, plasma and combinations thereof. [0088] In an embodiment of the present disclosure there is provided a method for preparing a platelet lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma;
(e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma;
(f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;
(g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a platelet lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.

[0089] In an embodiment of the present disclosure there is provided a method for preparing a platelet lysate from a combination of umbilical cord blood (UCB) derived platelets and maternal blood (MB) derived platelets, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a platelet lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
[0090] In an embodiment of the present disclosure there is provided a method for preparing a platelet lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB)

sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma;
(f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;
(g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature of -86°C for a time of 48 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a platelet lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
[0091] In an embodiment of the present disclosure there is provided a method for preparing a platelet lysate from a combination of umbilical cord blood (UCB) derived platelets and maternal blood (MB) derived platelets, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a

platelet lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
[0092] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture comprises platelets in a range of 0.3×109 to 1×109 platelets/ml. In another embodiment of the present disclosure, the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture comprises platelets in a range of 0.3×109 to 0.7×109 platelets/ml. In yet another embodiment of the present disclosure, the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture comprises platelets in a range of 0.3×109 to 0.5×109 platelets/ml.
[0093] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood

(UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the mixing of umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) is done in a volume ratio having a range of 10:1 to 30:1. In another embodiment of the present disclosure, the mixing of umbilical cord blood (UCB) derived platelet-rich plasma and the maternal blood (MB) derived platelet-rich plasma is done in a volume ratio having a range of 20:1 to 30:1.
[0094] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation

to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the freeze-thaw cycles range from 1-5. In another embodiment of the present disclosure, the freeze-thaw cycles range from 2-3.
[0095] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood

(UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the at least one sedimentation reagent is selected from a group consisting of Ficoll-Hipaque, Hespan®, Pentastarch, and combinations thereof.
[0096] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, and wherein subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets is done for at least 30 minutes each, and wherein subjecting the plasma to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma is done for at least 30 minutes. In another embodiment of the present disclosure, subjecting the

umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets is done for a time period in a range of 30-45 minutes, and subjecting the plasma to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma is done for a time period in a range of 30-45 minutes. [0097] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein subjecting the maternal blood sample (MB) sample to sedimentation is done for a time in a range of 50-70 minutes. [0098] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a

first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the platelet lysate obtained in step (h) is filtered using a 0.22µ filter.
[0099] In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of

24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood
(UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical
cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-
thaw cycles to obtain a lysate from a combination of umbilical cord blood derived
platelets and maternal blood derived platelets, wherein the lysate obtained in step (h)
is subjected to screening for presence of infectious diseases (ID).
[00100] In an embodiment of the present disclosure, there is provided a
method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma;
(e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma;
(f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;
(g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate obtained in step (h) is subjected to screening for presence of infectious diseases (ID), wherein the screening involves testing for microbial testing, mycoplasma testing, and infectious diseases (ID) viz., HIV I and II antibodies, HBs Ag (surface antigen), HCV antibodies and Syphilis antibodies.

[00101] In an embodiment of the present disclosure, there is provided a
method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma;
(e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma;
(f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;
(g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate obtained in step (h) is subjected to screening for presence of infectious diseases (ID), wherein the screening involves testing for microbial testing, mycoplasma testing, and infectious diseases (ID) viz., HIV I and II antibodies, HBs Ag (surface antigen), HCV antibodies and Syphilis antibodies, and wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation

reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1.
[00102] In an embodiment of the present disclosure, there is provided a
method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma;
(e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma;
(f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;
(g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate obtained in step (h) is subjected to screening for presence of infectious diseases (ID), wherein the screening involves testing for microbial testing, mycoplasma testing, and infectious diseases (ID) viz., HIV I and II antibodies, HBs Ag (surface antigen), HCV antibodies and Syphilis antibodies, and wherein the first, the second and the third sedimentation are carried out for 25-55 minutes. In another embodiment of the present disclosure, the first, the second and the third sedimentation are carried out for 30-50 minutes. In yet another embodiment of the present disclosure, the first, the

second and the third sedimentation are carried out for 32-42 minutes. In an alternate embodiment of the present disclosure, the first, the second and the third sedimentation are carried out for 35 minutes.
[00103] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml. In another embodiment of the present disclosure, the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 0.7×109 platelets/ml. In yet another embodiment of the present disclosure, the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 0.5×109 platelets/ml.
[00104] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml, for use in culturing cells.
[00105] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml, for use in culturing cells, wherein the cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, and cardiomyocytes.

[00106] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate
comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived
platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB)
derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and
the maternal blood derived platelets have a combined platelet count in a range of
0.3×109 to 1×109 platelets/ml, for use in therapeutic applications.
[00107] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate
comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived
platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB)
derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and
the maternal blood derived platelets have a combined platelet count in a range of
0.3×109 to 1×109 platelets/ml, wherein said umbilical cord blood and maternal blood
(UCB+MB) enriched platelet lysate is cryopreserved for a time in a range of 1 day
to 12 months at a temperature in a range of -70°C to -86°C for use in culturing cells.
[00108] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml, wherein said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time of 6 months at a temperature in a range of -86°C for use in culturing cells.
[00109] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of

0.3×109 to 1×109 platelets/ml, wherein said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time of 12 months at a temperature in a range of -86°C for use in culturing cells.
[00110] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml, wherein said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time in a range of 1 day to 12 months at a temperature in a range of -70°C to -86°C for use in culturing cells, and wherein the cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, and cardiomyocytes.
[00111] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml, wherein said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time in a range of 1 day to 12 months at a temperature in a range of -70°C to -86°C for use in therapeutic applications.
[00112] In an embodiment of the present disclosure, there is provided an
umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of

0.3×109 to 1×109 platelets/ml, wherein, the enriched platelet lysate is obtained by a method comprising: (i) obtaining an umbilical cord blood (UCB) sample; (ii) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (iii) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (iv) subjecting the plasma of step (iii) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (v) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (vi) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (iv) and the maternal blood (MB) derived platelet-rich plasma of step (v) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (vii) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of -70°C to -86°C for a time in a range for 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (viii) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4:1 to 8:1. In yet another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 4.5:1 to 6:1. In an alternate embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent in a volume ratio of 5:1.

[00113] Although the subject matter has been described in considerable detail
with reference to certain examples and implementations thereof, other implementations are possible.
EXAMPLES
[00114] 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.
[00115] The examples as presented herein describe the best working process of the present disclosure.
EXAMPLE 1
Umbilical Cord blood collection
[00116] Umbilical cord blood (UCB) was collected from an identified subject. Before collection, the segment of the umbilical cord of said subject was cleaned with 10% povidone Iodine and 70% alcohol (Ethyl Alcohol /Isopropyl Alcohol) thrice alternately, swabbing away from the collection area. After the spirit evaporated, the outer gloves were removed to prevent contamination. Then the umbilical cord blood (UCB) collection bag was held with sterile inner gloves and a sterile needle was inserted into the umbilical cord vein near the cord clamp and said needle was connected to the umbilical cord blood (UCB) collection bag that held a sterile anticoagulant such as citrate-phosphate-dextrose solution. The flowing cord blood through the needle into the umbilical cord blood (UCB) collection bag was gently and properly mixed with the anticoagulant in the umbilical cord blood (UCB)

collection bag. After the collection of maximal amounts of cord blood, till the umbilical cord ran empty and appeared whitish, multiple knots were tied at the opening end of the umbilical cord blood (UCB) collection bag after cutting the input from the needle with care and the collection bag was checked for leakage. The umbilical cord blood (UCB) collection bag was finally cleaned with sterile gauge pieces and used for transport to a cell processing centre. The collected umbilical cord blood (UCB) was transported to the cell processing centre at 18°C to 28°C within a period of less than 72hours post-collection. After receiving the sample, umbilical cord blood (UCB) bag was cleaned with 70% IPA and then it was sealed and labelled.
EXAMPLE 2
Maternal blood collection
[00117] The accuracy and identity of the subject mother was confirmed. Maternal blood (MB) sample was collected in a sterile vacutainer tube with a sterile anticoagulant such as EDTA. The collection of maternal blood (MB) sample was carried out at the time of admission for delivery (after initiation of labour) or before transfusion/infusion of any intravenous fluid (colloids/crystalloids/blood products) and immediately before/after cord blood collection. The collected maternal blood (MB) was transported to the cell processing centre at 18°C to 28°C within a period of less than 72hours post-collection. After receiving the sample, maternal blood (MB) vacutainer tube was cleaned with 70% IPA and then it was sealed and labelled.
EXAMPLE 3
Process for preparation of platelet lysate
[00118] Steps for preparation of platelet lysate from umbilical cord blood (UCB) and maternal blood (MB) samples is represented in a flow-chart (refer Figure 1). To begin, at the cell processing centre, an aliquot of 100-200 µl were withdrawn for initial platelet count from the labelled umbilical cord blood (UCB) bag and maternal blood (MB) vacutainer tube.
[00119] For processing the umbilical cord blood (UCB) bag, a calculated volume of sedimentation reagent, such as Ficoll-Hipaque, Hespan®, Pentastarch, etc., was

withdrawn under sterile conditions and injected into the umbilical cord blood (UCB) collection bag and kept on a rocking shaker for 5-10 minutes. A processing bag was labelled and attached to the said umbilical cord blood (UCB) collection bag. The said umbilical cord blood (UCB) collection bag was subjected to a first and then a second sedimentation for 35 minutes each so as to allow Rouleax formation of the red blood cells (RBCs) for better separation of the RBCs from the plasma. After said sedimentations, with the use of auto volume expresser, umbilical cord blood (UCB) platelet-rich plasma was collected in the umbilical cord blood (UCB) processing bag. Further, the umbilical cord blood (UCB) platelet-rich plasma in the umbilical cord blood (UCB) processing bag was subjected to a third sedimentation for another 35 minutes to separate out hematopoietic stem cells (HSCs) from the plasma with the use of auto volume expresser and the final umbilical cord blood (UCB) derived platelet-rich plasma was collected in a 50ml conical tube.
[00120] At the same time as the processing of umbilical cord blood (UCB), the maternal blood (MB) sample was kept for sedimentation for 60minutes and then, the supernatant was collected to procure maternal blood (MB) derived platelet-rich plasma into a 50ml conical tube.
[00121] The isolated umbilical cord blood (UCB) derived platelet-rich plasma and maternal blood (MB) derived platelet-rich plasma were mixed together and stored at -86°C for 48 hours and further this mixture of platenet-rich plasma was taken through multiple cycles of freeze-thaw cycles ranging from 1 - 3 times to get to good quality platelet lysate (PL) called umbilical cord blood (UCB) plus maternal blood (MB) platelet lysate or UCB+MB platelet lysate. Said UCB+MB platelet lysate was filtered using a 0.22 µ filter and tested for microbial sterility, mycoplasma and infectious diseases viz., HIV I and II antibodies, HBs Ag (surface antigen), HCV antibodies and Syphilis antibodies.
[00122] Using this method, from around 100 ml of umbilical cord blood (UCB), approximately 40ml to 60ml of umbilical cord blood (UCB) derived platelet lysate may be obtained and from around 15ml to 20ml of maternal blood, approximately 10 ml of maternal blood (MB) derived platelet lysate may be obtained.

EXAMPLE 4
Validation of sedimentation time for isolation of platelets from umbilical cord blood
[00123] Example 4 analyzed three different time points used for isolation of platelets from human umbilical cord blood (UCB) samples, namely, 20 minutes, 35 minutes and 60 minutes. Sample size for the experiment was 5 umbilical cord blood (UCB) samples. Samples 1 to 5 refer to the UCB obtained from five subjects. [00124] Table 1 presents umbilical cord blood (UCB) samples initial and final platelet counts (platelets/ml) recovered with different time periods of sedimentation with a sedimentation reagent (Hespan® which is 6% hetastarch in 0.9% sodium chloride solution). A total of 5 sample data have been represented in triplicates in said table. Herein, quantification having average and standard deviation were calculated of platelet counts of UCB.

Table 1: Results of Platelet count derived from UCB through Sedimentation
process

S. no
1 2 3
Sam ple no
Sam ple 1
Sam ple 2
Sam ple 3
Volu me
98.ml
115m l
86ml Before
Sediment
action After Sedimentation

Initial
Count
(Platelets/
ml) Final Count (Platelets/ml)

20mi ns
0.19 X109
0.22 X109
0.16 X109 Recov ery
100%
100%
94.11 % 35mi ns
0.18 X109
0.20 X109
0.16 X109 Recov ery
94.73 %
90.90 %
94.11 % 60mi ns
0.15 X109
0.17 X109
0.14 X109 Recov ery
78% 77% 82%

0.19X109

0.22X109

0.17X109

4 5 Sam ple 4
Sam ple 5 121m l
92ml 0.26X109 0.25 X109
0.20 X109 96.15 %
100% 0.24 X109
0.20 X109 92.30 %
100% 0.19 X109
0.16 X109 73% 80%

0.20X109

*Inference: At different time points Platelet recovery was carried out.
[00125] From the table above, one can deduce that at 35 minutes of sedimentation platelet recovery was more than 90%, while at 20 mins recovery was high but mixed population along with platelets and RBC, WBCs and at 60mins platelet recovery was very less (less than 85%).
EXAMPLE 5
Validation for Volume of sedimentation reagent used for isolation of platelets from umbilical cord blood
[00126] Example 5 analyzed volume ratio of cord blood along with sedimentation reagent used for isolation of platelets from human umbilical cord blood samples 2.5ml UCB: 1ml Sedimentation reagent(SDR)-Ratio-1; 5ml UCB:1ml Sedimentation reagent (SDR)- Ratio-2; and 10ml UCB:1ml Sedimentation reagent (SDR)- Ratio-3. Sample size for the experiment was 5 umbilical cord blood (UCB) samples. Each sample was divided into three parts.
[00127] Table 2 presents umbilical cord blood (UCB) samples initial and final platelet counts (platelets/ml) recovered with sedimentation using different volume ratios of UCB (ml) to sedimentation reagent (Hespan® which is 6% hetastarch in 0.9% sodium chloride solution). A total of 5 sample data have been represented in triplicates in said table. Herein, quantification having average and standard deviation were calculated of platelet counts of UCB.

Table 2: Results of Platelet count derived from UCB through Sedimentation
process
Before Sedimentation After Sedimentation

S. n o
1 2 3 4 5 Sam ple no
Sam ple 1
Sam ple 2
Sam ple 3
Sam ple 4
Sam ple 5 Initial Count (Platelets/ml) Final Count (Platelets/ml)

Ratio-1 Reco very
56% 60% 57% 40% 62% Rati o-2
0.45 X108
0.38 X108
0.65 X108
0.48 X108
0.78 X108 Reco very
90.00 %
95.00 %
92.85 %
96.00 %
97.50 % Rati o-3
0.20 X108
0.20 X108
0.32 X108
0.21 X108
0.45 X108 Reco very
40.00 %
50.00 %
45.71 %
42.00 %
56.25 %

0.50X108 0.28 X108

0.40X108 0.30 X108

0.70X108 0.40 X108

0.50X108 0.20 X108

0.80X108 0.50 X108

*Inference: At different volume ratios Platelet recovery was carried out. [00128] From the table above, one can deduce that at 5:1 ratio of UCB: sedimentation reagent, platelet recovery was more than 90%, while at 2.5:1 and 10:1 ratios, the recovery was less than 60%. Therefore, the ratio of UCB to sedimentation reagent plays a critical role in platelet recovery. The present Example highlights the experimental efforts to arrive at a specific ratio.
EXAMPLE 6
Comparison of processing of umbilical cord blood (UCB) and maternal blood
(MB) from
[00129] Blood sample Collection:
[00130] Umbilical cord blood (UCB, about 150 ml) was drawn from the umbilical
cord tissue of the five healthy subjects into the collection bag containing citrate{
phosphate{dextrose as an anticoagulant. The umbilical cord blood (UCB) collection
bag was then transported to the cell processing centre (CPC) at 18-17â along with
10 ml of maternal blood in (MB) vacutainer tube.

[00131] Isolation of platelet lysate from UCB and MB using sedimentation: [00132] The method described in Example 3 was employed for isolation of platelets from UCB and MB to obtain UCB, MB and UCB+MB platelet lysates.
[00133] Isolation of platelet lysate from UCB using centrifugation: [00134] The procedure followed has been taken from Shaheen A et al, Wellington K Hsu et al., and Mlynarek RA et al. (refer Shaheen A "Platelet Rich Plasma (PRP) for Treatment Non-Healing Ulcers: A Review Study," Austin J Dermatolog, 2018, 5(1):1085-1094, Wellington K Hsu et al. "Platelet-rich Plasma in Orthopaedic Applications: Evidence-based Recommendations for Treatment," J Am Acad Orthop Surg 2013;21:739-748, and Mlynarek RA et al. "Platelet-Rich Plasma (PRP) in Orthopedic Sports Medicine," Am J Orthop. 2016 May;45(4):290-294, 326). [00135] The aliquots from platelet-rich plasma from UCB and MB before and after sedimentation process showed that the average platelet count remained almost the same, even with the decrease in plasma volume.
[00136] Table 3 presents (a) the umbilical cord blood (UCB) derived platelet count before and after sedimentation; and (b) the maternal blood (MB) derived platelet count before and after sedimentation. A total of 5 sample data have been represented in triplicates in said table. Herein, quantification having average and standard deviation were calculated of platelet counts of UCB and MB respectively.

Table 3: Results of Platelet count derived from UCB and maternal Blood
through Sedimentation process
Sample
Numbe
r Tripl ets UCB derived Platelet Maternal Blood derived Platelet

Before Sedimentation After
Sedimentati
on Before Sedimentation After Sedimentation

Initia
l
Volu
me
(ml) Initial
Count
(Platelets
/ml) Final
Volu
me
(ml) Final Coun
t
(Plat
elets/
ml) Initia
l
Volu
me
(ml) Initial
Count
(Platelets
/ml) Final
Volu
me
(ml) Final Count (Platelets/ml)

S.1.1 S.1.2 S.1.3
S.2.1 S.2.2 S.2.3
S.3.1 S.3.2 S.3.3
S.4.1 S.4.2 S.4.3 87.7
143
138. 6
138 0.16X109 50 80 80 82 0.19 X109
0.14 X109
0.18 X109
0.17 X109
±0.0 2
0.24 X109
0.28 X109
0.26 X109
0.26 X109
±0.0 2
0.34 X109
0.29 X109
0.33 X109
0.32 X109
±0.0 2
0.23 X109
0.28 X109
0.27 X109
0.26 X109 10 10 6 10 0.60X109 5 5 3 5 0.61X109
Sample 1

0.18X109


0.64X109
0.63X109


0.17X109


0.65X109
0.62X109
Averag e

0.17X109


0.63X109
0.62X109
STDE V

±0.01


±0.02
±0.01


0.26X109


0.18X109
0.20X109
Sample 2

0.29X109


0.14X109
0.12X109


0.26X109


0.16X109
0.13X109
Averag e

0.27X109


0.16X109
0.15X109
STDE V

±0.01


±0.02
±0.04


0.36X109


0.24X109
0.26X109
Sample 3

0.33X109


0.26X109
0.22X109


0.30X109


0.22X109
0.24X109
Averag e

0.33X109


0.24X109
0.24X109
STDE V

±0.03


±0.02
±0.02


0.25X109


0.08X109
0.10X109
Sample 4

0.28X109


0.10X109
0.07X109


0.25X109


0.09X109
0.10X109
Averag e

0.26X109


0.09X109
0.09X109

STDE V S.5.1 S.5.2 S.5.3 88 ±0.01 60 ±0.0 2
0.19 X109
0.22 X109
0.18 X109
0.20 X109
±0.0 2 8 ±0.01 4 ±0.01


0.18X109


0.22X109
0.15X109
Sample 5

0.22X109


0.14X109
0.18X109


0.2X109


0.15X109
0.15X109
Averag e

0.20X109


0.17X109
0.16X109
STDE V

±0.02


±0.04
±0.01
[00137] The average platelet counts and platelet recovery before and after sedimentation was calculated and presented in Table 4. From Table 4, one can infer that the average recovery of platelets from UCB and maternal blood by sedimentation method ranges from 94% - 100%. It can be appreciated from the Table 4 that most platelets were recovered during the sedimentation process without major loss.

Table 4: Average Platelet count derived from UCB and maternal Blood (MB)
through Sedimentation process
Average values of Sedimentation method
A B
S.no UCB Platelets Maternal blood
Before sedimentati
on
(Platelets/m
l) After sedimentati
on
(Platelets/m
l) Recover y Before sedimentati
on
(Platelets/m
l) After sedimentati
on
(Platelets/m
l) Recover y
Sample-01 0.17X109 0.17X109 100% 0.63X109 0.62X109 98%
Sample-02 0.27X109 0.26X109 96% 0.16X109 0.15X109 94%
Sample-03 0.33X109 0.32X109 97% 0.24X109 0.24X109 100%
Sample-04 0.26X109 0.26X109 100% 0.09X109 0.09X109 100%
Sample-05 0.20X109 0.20X109 100% 0.17X109 0.16X109 94%

[00138] The platelet counts obtained from UCB through the centrifugation process was measured for comparison with the sedimentation process. [00139] Table 5 shows the UCB derived platelet counts before and after centrifugation. A total of 5 sample data have been represented in triplicates. Quantification having average and standard deviation were calculated of Platelet counts of UCB.

Table 5: Results of Platelet count derived from UCB through Centrifugation
process
Sample Number Triplets UCB derived Platelet
Before Centrifugation After Centrifugation
Initial
Volume
(ml) Initial Count (Platelets/ml) Final
Volume
(ml) Final Count (Platelets/ml)
S.1.1 96.43 0.16X109 45 0.08X109
Sample 1 S.1.2
0.18X109
0.09X109
S.1.3
0.17X109
0.08X109
Average 0.17X109 0.08X109
STDEV ±0.01 ±0.005
S.2.1 91.54 0.19X109 35 0.11X109
Sample 2 S.2.2
0.19X109
0.11X109
S.2.3
0.20X109
0.11X109
Average 0.19X109 0.11X109
STDEV ±0.005 0
S.3.1 90.08 0.24X109 38 0.16X109
Sample 3 S.3.2
0.23X109
0.16X109
S.3.3
0.23X109
0.16X109
Average 0.23X109 0.16X109
STDEV 0 0
S.4.1 109.72 0.25X109 42 0.15X109
Sample 4 S.4.2
0.24X109
0.15X109

S.4.3 0.24X109 0.15X109
Average 0.24X109 0.15X109
STDEV ±0.005 0
S.5.1 99.56 0.21X109 40 0.14X109
Sample 5 S.5.2
0.21X109
0.15X109
S.5.3
0.21X109
0.15X109
Average 0.21X109 0.14X109
STDEV 0 ±0.005
[00140] Average platelet counts and platelet recovery before and after centrifugation of umbilical cord blood (UCB) samples were calculated and have been shown in Table 6. From Table 6, one can infer that the average recovery of platelets from UCB by centrifugation method ranges from 47% - 70%. This states that most platelets were not recovered during the standard centrifugation process.

Table 6: Average Platelet count derived from UCB through Centrifugation
process
Average values
Sr. No UCB Platelets
Before centrifugation (Platelets/ml) After centrifugation (Platelets/ml) Recovery
Sample-01 0.17X109 0.08X109 47%
Sample-02 0.19X109 0.11X109 58%
Sample-03 0.23X109 0.16X109 70%
Sample-04 0.24X109 0.15X109 63%
Sample-05 0.21X109 0.14X109 67%
[00141] Table 7 provides a comparison of sedimentation versus centrifugation method of (UCB+MB) derived platelets and UCB derived platelets respectively.
Table 7: Comparison of sedimentation and centrifugation method

SEDIMENTATION CENTRIFUGATION
STEPS Multiple sedimentation (thrice) spin@1261rpmX10MINS
Ficoll-Hypaque (a mixture of Ficoll (polysucrose) and Hypaque (sodium diatrizoate)), Hespan®
REAGENTS which is 6% hetastarch NIL
NEEDED in 0.9% sodium chloride solution), and Pentastarch (artificial colloid (hydroxyethyl starch derivative) -Sedimentation reagent
PLATELET YIELD ~0.35X109/ml platelets/ml for 1 UCB+MB sample 0.12 x 109 platelets/ml for 1 sample
RECOVERY 94 -100 (%) 47-70 (%)
FINAL VOLUME 40-60 ml/100 ml UCB 35-40ml/100 ml UCB
FREEZE THAW 1-3 times 1-3 times
Addition, if any UCB PL mixed with MATERNAL PL Only UCB PL only
TESTING HIV I & II, HBsAg, Anti HCV, Syphilis, MYCOPLASMA, STERILITY HIV I & II, HBsAg, Anti HCV, Syphilis, MYCOPLASMA, STERILITY
[00142] It can be appreciated that the present process as disclosed herein provides significantly higher recovery than centrifugation method.
EXAMPLE 7

ID TESTING: HIV I and II, HBsAg, Anti HBc, Anti HCV, CMV IgM, CMV IgG Anti HTLV I and II and Syphilis ID testing for HIV:
[00143] HIV I /II ELISA was employed herein, which involved an immunosorbent enzyme assay which consists of recombinant protein for gp120, gp41 of HIV-I and gp36 of HIV-II bound to wells of microplate. During the course of assay, diluted controls and diluted specimens were added to the wells and incubated. HIV specific antibody (Ab), if present, binds to the antigens. After a thorough washing of the wells to remove unbound Ab and other serum components, standardized preparation of horse radish peroxidase - conjugate was added to each well. The conjugate preparation was then allowed to react with antibodies which bind to the assay wells on the basis of the specificity for antigenic determinants present within HIV antigens. After second thorough washing of the wells to remove unbound horseradish peroxidase-conjugated Ab, a substrate solution containing hydrogen peroxide and TMB was added to each well. A blue colour developed in proportion to the amount of HIV specific antibodies present, if any, in serum or plasma samples tested. This enzyme-substrate reaction was terminated by addition of sulfuric acid. The colour changes to yellow that have occurred in each well were then measured spectrophotometrically at a wavelength of 450nm/630 nm. The sample was considered negative when OD value of the sample is less than the value of positive control.
ID testing for HCV:
[00144] An indirect antibody EIA assay for HCV ELISA was performed for detection of antibodies to HCV in human serum and plasma. It employed an immunosorbent enzyme assay which consists of recombinant protein for core and Ns3 protein and synthetic peptides corresponding to highly antigenic segments, Ns4 and Ns5 regions of the hepatitis C virus bound to wells of a microplate. During the course of the assay, diluted controls and diluted specimens were added to the wells and incubated. HCV specific Ab, if present, binds to the antigens. After a thorough washing of the wells to remove unbound Ab and other serum components, standardized preparation of horse radish peroxidase-conjugate was added to each

well. The conjugate preparation was then allowed to react with antibodies which bind to the assay wells on the basis of the specificity for antigenic determinants present within HCV antigens. After second thorough washing of the wells to remove unbound horseradish peroxidase-conjugated Ab, a substrate solution containing hydrogen peroxide and TMB was added to each well. A blue colour develops in proportion to the amount of HCV specific antibodies present, if any, in serum or plasma samples tested. This enzyme-substrate reaction was terminated by addition of sulfuric acid. The colour changes to yellow was measured in each well spectrophotometrically at a wavelength of 450nm/630 nm. The sample was considered negative when OD value of the sample was less than the value of positive control.
ID testing for HBsAg:
[00145] A solid phase ELISA for HBsAg detection based on sandwich capture principle was performed. During the course of the assay, diluted controls and diluted specimens were added to the wells and incubated. HBs specific Ab, if present, binds to the antigens. When patient serum containing HBsAg was added, it combined with the goat anti-HBsAg attached to polystyrene surface of the microwells and simultaneously bound with the horse radish peroxidase conjugated monoclonal anti-HBsAg. Wells were washed and a colorless enzyme substrate (H2O2) and chromogen (TMB, tetramethylbenzidine) were added. The enzyme acts on substrate/chromogen and produced a blue colored end product. This enzyme-substrate reaction was terminated by addition of sulfuric acid. The color changes to yellow that have occurred in each well are then measured spectrophotometrically at a wavelength of 450nm/630 nm. The yellow color intensity was directly related to concentration of Hepatitis B surface antigen in the patient sample. The sample was considered negative when OD value of the sample was less than the value of positive control.
ID testing for HBc:
[00146] An indirect antibody EIA assay for HBc ELISA was performed for the simultaneous detection of antibodies of total antibodies to hepatitis B virus core in human serum or plasma. It was based upon the use of a solid phase prepared with recombinant HBc antigen. The sera to be tested and the control sera were added to

the wells. If antibodies to HBc were present, they will bind to the antigen fixed on solid phase. The peroxidase- labeled antibodies to human IgG and IgM are added after a washing step. They in turn bind to the specific antibodies captures on solid phase. After removal of unbound enzymatic conjugate, the antigen-antibody complex is revealed by addition of substrate. This enzyme- substrate reaction is terminated by addition of sulfuric acid. The colour changes to yellow that have occurred in each well are then measured spectrophotometrically at a wavelength of 450nm/630 nm. The absorbance measured for a sample allows the presence and absence of antibodies to HBc to be determined. The colour intensity is proportional to the quantity of anti HBc antibodies bound to the solid phase. The sample is considered negative when OD value of the sample is less than the value of positive control.
ID testing for CMV IgG:
[00147] Microplates were coated with the native Cytomegalovirus antigens, highly purified by sucrose gradient centrifugation and inactivated. The solid phase was first treated with the diluted sample and IgG to Cytomegalovirus are captured, if present, by the antigens. The peroxidase- labeled conjugated polyclonal antibodies to human IgG are added after a washing step. They in turn bind to the specific antibodies captures on solid phase. The enzyme captured on solid phase, acting on substrate and chromogen mixture, generate an optical signal that is proportional to the amount of anti-Cytomegalovirus IgG antibodies present in sample at 450/630nm. The sample was considered negative when OD value of the sample is less than the value of positive control.
ID testing for CMV IgM:
[00148] The assay is based on the principle of IgM capture where IgM class antibodies in sample are first captured by solid phase coated with hIgM antibody. A complex composed of biotinylated CMV antigen and Streptavidin, labeled with peroxidase were added after a washing step. They in turn bind to the specific antibodies captures on solid phase. The enzyme captured on solid phase, acting on substrate and chromogen mixture, generate an optical signal that is proportional to the amount of anti-Cytomegalovirus IgM antibodies present in sample at 450/630nm.

The sample is considered negative when OD value of the sample is less than the value of positive control.
ID testing for HTLV I & II Ab:
[00149] Microplates were coated with the HTLV I & II specific synthetic immunodominant antigens, derived from gp46-I, gp46II and gp21-I. The solid phase was first treated with the sample and Anti HTLV I & II are captured, if present, by the antigens coated to the microplate. The peroxidase- labeled specific synthetic antigens derived from gp46-I, gp46-II and gp21 were added after a washing step. They in turn bind to the specific antibodies captures on solid phase. The enzyme captured on solid phase, acting on substrate and chromogen mixture, generate an optical signal that is proportional to the amount of anti HTLV I & II antibodies present in sample at 450/630nm. The sample was considered negative when OD value of the sample is less than the value of positive control.

[00150] Table 8: ID testing:
Manufacturing Time (Zero) Six Month One Year
Details Sedimenta
tion
method
(UCB+M B) PL Centrifu
gation
method
UCB PL FBS
Negat ive
Non-reacti ve
Non-reacti ve Sedimenta
tion
method
(UCB+M B) PL Centrif ugation method
UCB PL FBS
Negati ve
Non-reactiv e
Non-reactiv e Sediment
ation
method
(UCB+M B) PL Centrifu
gation
method
UCB PL FBS
Negati ve
Non-reactiv e
Non-reactiv e
ID Testing

HIV I&II Negative Negative
Negative Negativ e
Negative Negative

HBsAg Non-reactive Non-reactive
Non-reactive Non-reactive
Non-reactive Non-reactive

Anti HBc Non-reactive Non-reactive
Non-reactive Non-reactive
Non-reactive Non-reactive

HCV Non-reactive Non-reactive Non-reacti ve Non-reactive Non-reactive Non-reactiv e Non-reactive Non-reactive Non-reactiv e
CMV IgM Negative Negative Negat ive Negative Negativ e Negati ve Negative Negative Negati ve
CMV IgG Negative Negativ e Nega tive Negative Negati ve Negati ve Negative Negativ e Negat ive
Anti HTLV I &II Negative Negative Negat ive Negative Negativ e Negati ve Negative Negative Negati ve
Syphili s. Non-reactive Non-reactive Non-reacti ve Non-reactive Non-reactive Non-reactiv e Non-reactive Non-reactive Non-reactiv e
[00151] Tables 8 enlist lists of infectious disease (ID) testing which were performed at initial stage, after six months and after a year. The results were found free from all infectious diseases.
EXAMPLE 8
Albumin levels in (UCB+MB) PL, UCB PL and FBS
[00152] A primary function of albumin is to bind and stabilize a range of small molecules and ions. In in vitro, albumin acts as a multifaceted antioxidant. Its total antioxidant activity is a composite of many individual antioxidant activities. Albumin binds fatty acids and protects them from oxidation; binds copper and keeps it from participating in oxidation reactions. Albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival.

[00153] Method:
[00154] Albumin was estimated quantitatively by colorimetric based method. Albumin standards were prepared at different concentrations such as 0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0 g/dL. The samples were diluted by 2 folds for testing.5µl each of diluted standards and diluted samples were transferred into wells of a clear bottom plate. 200µl of Bromocresol green (BCG) reagent were added and incubated for 5 min at room temperature. The reagent, Bromocresol green forms a colored complex with albumin when tested positive. The intensity of the color was measured at 620nm which is directly proportional to the albumin concentration in the sample.

[00155] Table 9: Albumin Assay
Manufacturing Time (Zero) Six Month One Year
Sr .
N o
B. Details
Album in
Assay Level Sedimentatio n method
(UCB+MB) PL Centrifuga
tion
method
UCB PL FBS
2.17 g/dL Sedimenta
tion
method
(UCB+M B) PL Centrifuga
tion
method
UCB PL FB S
2.1 5
g/d L Sedimen
tation
method
(UCB+ MB) PL Centrifug
ation method
UCB PL FBS
2.15g/ dL

2.4 g/dL 2.1 g/dL
2.3 g/dL 1.95 g/dL
2.25 g/dL 1.9g/dL

[00156] Albumin was estimated by colorimetric method at initial stage, after six months and after a year. Table 9 infers that the level of albumin is higher in the UCB+MB PL when compared to standard UCB PL and FBS.
EXAMPLE 9
Presence of Protein using SDS-PAGE in (UCB+MB) PL, UCB PL and FBS
[00157] The plates were assembled for casting gel as per manufacturer's instructions. [00158] Add 60 µI of APS (ammonium persulfate), 2.4 µI of TEMED solution and 2.4ml of 30% Acrylamide to 3.6ml of SDS separating gel mix and pour the gel solution between then plates till the level is 2cm below the top edge of notched plate. Add 200 to 250 µl of water to make the surface even. After the gel are set

(approximately 30-40 min), wash the top of the separating gel with distilled water and drain off the water completely. Add 40 µI of APS, 4 µl of TEMED solution and 0.67ml of 30% Acrylamide to 3.4 ml of stacking gel mix and pour directly onto the polymerized separating gel. Insert the comb into the gel solution carefully without trapping any bubbles, about 1 cm above the separating gel. The stacking gel will set in approximately 20min.
[00159] Take 2, 3, 4, 5 µI of provided Protein sample in 4 microfuge vials. Label them as 1, 2, 3, 4 respectively. Add 20 µI of sample loading buffer to protein samples. Place the samples in water bath for 5 min. After the stacking gel has set, carefully remove the comb and the bottom spacer. Wash the wells immediately with distil led water to remove non-polymerized acrylamide. Assemble the gel set in the Gel running apparatus as per manufacturer's intruction. Fill the top and bottom reservoir with 1X Gel running buffer. Load 20 µI protein markers in the first well and then load prepared Protein samples in well 2,3,4,5. Note down the order of loading. Connect the cords to the power supply according to the convention. Set voltage at 1OOV and switch on the power supply. When the dye front comes to 0.5cm above the bottom of the gel, turn off the power. This will take approximately 1 to 1:30 hours. Remove the gel plates and gently open the plates apart using a spatula or similar tool, don't try to separate the plates at the notch as it might damage the notch. [00160] Transfer the gel to a tray containing water; wash the gel for 1-2 minutes at room temperature. Decant water, cut the gel along lane 4. Transfer lanes 1- 5, i.e. protein sample in 10 ml of blotting buffer taken in a Petri dish. Keep at room temperature for 10 minutes. Following incubation, proceed for electroblotting. To the gel piece add minimum of 20ml water. Wash the gel by rotating gently. Decant the water; add 20 ml of Ezee Blue Stain. Stain at room temperature for 1-2 hours. For uniform staining and washing, place the tray on a rocker intermittently every 10 to 15 minutes. Decant the staining solution add minimum quantity of water to cover the gel. Cover the tray and leave it overnight at room temperature. [00161] Figure 2 shows the SDS PAGE gel picture having markers and tested samples in different lanes.

[00162] In lane one, there is a ladder with marker ranging from 10kDa to 220kDa. These are used to determine the size of proteins in the gel. Each band in the ladder is a known molecular weight. The samples can be determined from these known weights.
[00163] In lane two there is standard Human Albumin protein of 49kDa. Further lane 3, 4 and 5 shows the results of tested sample.
[00164] From the gel, it is determined that standard Human Albumin protein and FBS Protein, UCB PL Protein and UCB+MB PL Protein are relatively pure proteins. It was found that a standard Human Albumin protein will have the same molecular weight regardless of the concentration of the tested sample as the albumin levels tested were in the range of 1.9-2.5 g/dL (Table 9). Further, Figure 2 and Table 9 shows that UCB+MB PL sample has more of the albumin protein compared to FBS and UCB PL.
EXAMPLE 10
Identification of proteins using LCMS in (UCB+MB) PL, UCB PL and FBS
Tryptic digestion of proteins
[00165] Proteins were estimated by UV spectrophotometer using Bradford protein quantification protocol. 50 µg proteins were denatured and digested by Proteomic Grade trypsin (Sigma T6567). In brief, 50μg of protein was dissolved in 50mM ammonium bicarbonate buffer containing 0.1% RapiGest (Waters Corporation, MA, USA). Proteins were reduced and alkylated by treating with 100mM dithiothreitol for 15 min at 60 °C and 200mM iodoacetamide for 30 min at room temperature respectively. Denatured proteins were treated with trypsin (1:25) at 37 °C for 18 h and the reaction was stopped by addition of 0.1% formic acid. Digested peptides were desalted by using C18 Zip tips (Millipore, Billerica, MA) and the eluted peptides were concentrated by using vacuum concentrator. The peptides were reconstituted in 3% ACN with 0.1% formic Acid and used for mass spectrometric analysis.
LC Separation

[00166] Peptide digests (2.5 µg) were separated by using Accela 1250 UHPLC (Thermo Fisher Scientific) equipped with a Hypersil Gold C18-reverse phase column (150*2.1 mm, 1.9 µm). The sample was loaded onto the column with 98% of mobile phase A (100% water, 0.1% formic acid (FA)) and 2% of mobile phase B (100% ACN, 0.1% FA) at 350 µl/min flow rate. Peptides were eluted with a 45 min linear gradient of 2 to 40% mobile phase B. In case of plasma samples, the LC method was extended to 120 min with a linear gradient of 2 to 50% of mobile phase B. The column temperature was set to 40 °C and auto sampler at 8 °C. All samples were analyzed on hybrid quadruple Q-Exactive Orbitrap MS. The instrument tune parameters were optimized for the better results as: spray voltage 4,200 V, capillary temperature 320 °C, heater temperature 200 °C, S-lens RF value 55, sheath and auxiliary gases pressure were 30 and 8 psi, respectively. The samples were acquired in positive ionization mode in data-dependent manner using a top-five method with scan range from 350-1,800 m/z. MS spectra were acquired at a resolution of 70,000 with maximum injection time (IT) of 120 ms and automatic gaincontrol (AGC) value of 1 e6 ions; MS/MS spectra were acquired at 17,500 resolution with maximum IT of 120 ms and AGC value of le5 ions. Precursor's selectivity was performed at an isolation width of 3 m/z, under fill ratio of 0.3%, and dynamic exclusion time of 15 s. The peptide fragmentation was performed in high energy collision induced dissociation (HCD) cell using normalized HCD at 30 eV. Protein identification:
[00167] Proteins were searched against UniProt reviewed human protein database by using Proteome Discoverer software with following parameters, 1% FDR. The precursor and fragment initial mass error tolerance was set to 0.05 and 0.1 Da, respectively. Search parameters also included carbamidomethylation of cysteine residues as fixed modifications and methionine oxidation as variable modification.

Sample ID No. of Total Proteins No. of common proteins with Resemblance with sample 1 (%)

identified by LC- respect to sample MS 1
Sample 1 (UCB Platelets + maternal PL) 206 206 100 %
Sample 2 (Fetal Bovine serum) 93 31 15.04 %
Sample 3 (Umbilical Cord blood PL) 171 108 52.42 %
[00169] Umbilical Cord blood platelets with maternal blood platelets (Sample 1)
showed a total of 206 proteins whereas Umbilical Cord platelets (Sample 3) showed
only a total of 171 proteins and commercially available serum supplement such as
FBS (Sample 2) shows a total of 93 proteins (Table 10).
[00170] Thus, it can be observed that the mixture of UCB and MB platelets resulted
in an addition of 35 proteins compared to the Standard Umbilical Cord blood
platelets and an addition of 113 proteins compared to commercial FBS.
[00171] Conclusively, it can be stated that the optimized combination of UCB and
MB results in higher protein identification and confirmation when compared to
commercial or standard serum supplements.
[00172] Masses are graphed according to their relative abundance against time (See
Figures 3 (Graph 1: Sample-01 (UCB+MB PL) Chromatogram), 4 (Graph 2:
Sample-02(FBS) Chromatogram), 5 (Graph 3: Sample-02(UCB PL)
Chromatogram)), 6 (Graph 4: Sample-02(MB PL) Chromatogram)). The relative
abundance corresponds to the different identified proteins as mentioned in Table 11.

Sample ID No. of Total Proteins No. of common Resemblance with proteins with sample 1(%)

identified by LC-MS respect to sample 1
Sample 1 (UCB Platelets + maternal PL) 206 206 100 %
Sample 2 (Fetal Bovine serum) 93 31 15.04 %
Sample 3 (Umbilical Cord blood PL) 171 108 52.42 %
Sample 4 (Maternal blood PL) 137 69 33.5%
[00174] Thus, the mixture of UCB+MB platelet lysates gives the highest amount and
number of proteins.
[00175] The list of identified proteins in the optimized (UCB + MB) PL of
significant value for cell culture are as follows:
[00176] Vimentin [VIME_HUMAN] (SEQ ID NO: 1) which is an intermediate
filament (IF) protein that is the predominant IF in cells of mesenchymal origin such
as vascular endothelium and blood cells. It facilitates cell migration and motility by
recycling internalized trailing edge integrins back to the cell surface at the leading
edge.
[00177] Vascular endothelial growth factor receptor 1 [VGFR1_HUMAN] (SEQ ID
NO: 2) which mediates signals for differentiation. Expression of this receptor is
found in vascular endothelial cells, placental trophoblast cells and peripheral blood
monocytes. This protein binds to VEGFR-A, VEGFR-B and placental growth factor
and plays an important role in angiogenesis and vasculogenesis. Expression of this
receptor is found in vascular endothelial cells, placental trophoblast cells and
peripheral blood monocytes.
[00178] Glyceraldehyde-3-phosphate dehydrogenase [E7EUT5_HUMAN] (SEQ ID
NO: 3) which is an enzyme of ~37kDa that catalyzes the sixth step of glycolysis and
thus serves to break down glucose for energy and carbon molecules. GAPDH has
been implicated in several non-metabolic processes, including transcription
activation, initiation of apoptosis, ER to Golgi vesicle shuttling, and fast axonal, or

axoplasmic transport. In sperm, a testis-Specific isoenzyme GAPDHS is expressed. GAPDH act as an oxidoreductase, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor.
[00179] Pyruvate kinase PKM [KPYM_HUMAN] (SEQ ID NO: 4) which encodes a protein involved in glycolysis. The encoded protein is a pyruvate kinase that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP and pyruvate. This protein has been shown to interact with thyroid hormone and may mediate cellular metabolic effects induced by thyroid hormones. [00180] Mesoderm posterior protein 1 [MESP1_HUMAN] (SEQ ID NO: 5) which plays a role in the epithelialization of somatic mesoderm and in the development of cardiac mesoderm.
[00181] Isoform 3 of N-alpha-acetyltransferase 60 [NAA60_HUMAN] (SEQ ID NO: 6) which is a protein that was located on the Golgi apparatus and mainly catalyze the N-acetylation of transmembrane proteins. Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation1, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm.
[00182] Isoform 3 of Serine/threonine-protein kinase Chk1 [CHK1_HUMAN] (SEQ ID NO: 7) is encoded by this gene belongs to the Ser/Thr protein kinase family; checkpoint mediated cell cycle arrest in response to DNA damage or the presence of unreplicated DNA.DNA damage induces activation of Chk1, which then transduces the checkpoint signal and facilitates cell cycle arrest and DNA damage repair. [00183] Fructose-bisphosphate aldolase A [ALDOA_HUMAN] (SEQ ID NO: 8) encodes a protein which is a glycolytic enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-biphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde-3-phosphate or glyceraldehyde, respectively. [00184] Isoform 4 of Triosephosphate isomerase [TPIS_HUMAN] (SEQ ID NO: 9) enhances triose-phosphate isomerase activity and ubiquitin protein ligase binding. [00185] Trinucleotide repeat-containing gene 6C protein [TNR6C_HUMAN] (SEQ ID NO: 10) is a gene that plays a role in RNA-mediated gene silencing by micro-

RNAs (miRNAs). Required for miRNA-dependent translational repression of complementary mRNAs by argonaute family proteins.
[00186] Histone H4 [H4_HUMAN] (SEQ ID NO: 11) display a peak in transcription in early S phase and are ideal models for cell cycle-regulated gene expression. [00187] Centromere protein F [CENPF_HUMAN] (SEQ ID NO: 12) is required for kinetochore function and chromosome segregation in mitosis. Regulates recycling of the plasma membrane by acting as a link between recycling vesicles and the microtubule network.
[00188] Neutrophil defensin 1 [DEF1_HUMAN] (SEQ ID NO: 13) is a part of defensins, which are a family of antimicrobial and cytotoxic peptides thought to be involved in host defense. The protein encoded by this gene, defensin, alpha 1, is found in the microbicidal granules of neutrophils and likely plays a role in phagocyte-mediated host defense.
[00189] Haptoglobin [HPT_HUMAN] (SEQ ID NO: 14) is a hemoglobin-binding protein synthesized in the liver and released into the circulation. Haptoglobin is an acute phase protein capable of binding hemoglobin, thus preventing iron loss and renal damage. Haptoglobin also acts as an antioxidant, has antibacterial activity and plays a role in modulating many aspects of the acute phase response. [00190] Isoform 2 of Heat shock cognate 71 kDa protein [HSP7C_HUMAN] (SEQ ID NO: 15) binds to nascent polypeptides to facilitate correct protein folding. Its role in protein folding contributes to its function in signal transduction, apoptosis, protein homeostasis, and cell growth and differentiation.
[00191] Mannose-binding protein C [MBL2_HUMAN] (SEQ ID NO: 16) is a pattern recognition molecule of the innate immune system. This provides the host with a first-line of defense before the adaptive immune system becomes operative. [00192] Protein S100-A8 [S10A8_HUMAN] (SEQ ID NO: 17) encodes a vitamin K-dependent plasma protein that functions as a cofactor for the anticoagulant protease, activated protein C (APC) to inhibit blood coagulation. It is found in plasma in both a free, functionally active form and also in an inactive form complexed with C4b-binding protein. Mutations in this gene result in autosomal dominant hereditary thrombophilia. An inactive pseudogene of this locus is located

at an adjacent region on chromosome 3. Alternative splicing results in multiple
transcript variants encoding different isoforms that may undergo similar processing
to generate mature protein.
[00193] Serpin A12 [SPA12_HUMAN] (SEQ ID NO: 18) belongs to a family of
serpins, which are a broadly distributed family of protease inhibitors that use a
conformational change to inhibit target enzymes. They are central in controlling
many important proteolytic cascades, including the mammalian coagulation
pathways.
[00194] Synaptotagmin-13 [SYT13_HUMAN] (SEQ ID NO: 19) based on their
brain/endocrine distribution and biochemical properties, in particular C2 domains of
certain synaptotagmins bound to calcium. synaptotagmins were proposed to function
as calcium sensors in the regulation of neurotransmitter release and hormone
secretion.
[00195] Isoform 2 of Tubulin alpha-1B chain [TBA1B_HUMAN] (SEQ ID NO: 20)
is part of microtubules, which are built from a basic a/p-tubulin building block, yet
subpopulations of microtubules can be differentially marked by a number of post-
translational modifications. Tubulin modifications play an important role in
regulating microtubule properties, such as stability and structure, as well as
microtubule-based functions, such as ciliary beating, cell division, and intracellular
trafficking.)
[00196] Profilin-1 [PROF1_HUMAN] (SEQ ID NO: 21) encodes a member of the
profilin family of small actin-binding proteins. The encoded protein plays an
important role in actin dynamics by regulating actin polymerization in response to
extracellular signals.
[00197] Adenylyl cyclase-associated protein [B4DNW7_HUMAN] (SEQ ID NO:
22) is a Receptor for Human Resistin and Mediates Inflammatory Actions of Human
Monocytes.
[00198] C-myc promoter-binding protein 1 [E2DRY6_HUMAN] (SEQ ID NO: 23)
encodes a DENN domain-containing protein that may function as a guanine
nucleotide exchange factor that specifically activates Ras-related protein Rab-10.
This protein also contains an interferon stimulated response element-binding domain

and may be involved in regulating the v-myc avian myelocytomatosis viral (MYC) oncogene.)
[00199] Mitochondrial heat shock 60kD protein 1 variant 1 [B3GQS7_HUMAN] (SEQ ID NO: 24) are generally responsible for preventing damage to proteins in response to high levels of heat. It may facilitate the correct folding of imported proteins and may also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix.
[00200] Protein S100 [B2R4M6_HUMAN] (SEQ ID NO: 25) are a family of low-molecular-weight proteins characterized by two calcium-binding sites that have helix-loop-helix ("EF-hand type") conformation. They are also considered as Damage-associated molecular pattern molecules (DAMPs) and knockdown of AHR down regulates the expression of S100 proteins in THP-1 cell. [00201] Beta tropomyosin isoform [A7XZE4_HUMAN] (SEQ ID NO: 26) reduction associated with transformation, regulates anoikis. Associated with establishing focal adhesions. Restores stress fibers in transformed cells. [00202] Peroxiredoxin-1 [A0A0A0MRQ5_HUMAN] (SEQ ID NO: 27) encodes a protein that may play an antioxidant protective role in cells and may contribute to the antiviral activity of CD8(+) T-cells. This protein may have a proliferative effect and play a role in cancer development or progression.
EXAMPLE 11
Final product sterility, mycoplasma and endotoxin testing
Sterility:
[00203] The sample to be tested is inoculated into the vial which is entered into the BACTEC (BD BACTE FX 400 blood culture system) instrument for incubation and periodic reading. Each vial contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms or the consumption of oxygen needed for the growth of microorganisms. The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the

increasing amount of CO2 or the decreasing amount of O2 present in the vial. A positive reading indicates the presumptive presence of viable microorganisms in the vial.
Mycoplasma:
[00204] Mycoplasma contamination is detected using PCR technique. In this method, DNA extraction of given sample is carried out and PCR is performed on the samples, using primers specific for mycoplasma DNA along with dNTPs and another DNA synthesis enzyme. Running the PCR product on a gel shows the presence/absence of mycoplasma DNA by band(s) of distinct sizes.
Endotoxin:
[00205] Bacterial endotoxin level is determined using Endosafe PTs reader method. The test is a rapid, point-of-use handheld spectrophotometer that uses disposable cartridge for accurate convenient and realtime endotoxin testing, glucan identification and gram identification.

Manufacturing Time (Zero) Six Month One Year
S r . Deta ils Sedime ntation method Centrif ugation method F B S Sedime ntation method Centrif F ugation B method S Sedime ntation method Centrif ugation method F B S
N o (UCB+ MB) PL UCB PL (UCB+ MB) PL UCB PL (UCB+ MB) PL UCB PL
E . Final
Prod
uct
Safet
y
1 Sterili ty No
Gro
wth No
Gro
wth No
Gro
wth No
Growt
h No
Growt
h No
Growt
h No
Growt
h No
Growt
h No
Grow
th
2 Myco plasm a Neg ativ e Neg ativ e Neg ativ e Negati ve Negati ve Negati ve Negati ve Negati ve Negat ive

I/We Claim:
1. A method for preparing a lysate, said method comprising:
a) obtaining an umbilical cord blood (UCB) sample;
b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture;
c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma;
d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma;
e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;
f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and
g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
2. The method as claimed in claim 1, wherein the lysate is selected from a group consisting of platelet lysate, cell lysate, plasma, and combinations thereof.

3. The method as claimed in claim 1, wherein the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture comprises platelets in a range of 0.3×109 to 1×109 platelets/ml.
4. The method as claimed in claim 1, wherein mixing of umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) is done in a volume ratio having a range of 10:1 to 30:1.
5. The method as claimed in claim 1, wherein the freeze-thaw cycles range from 1 to 5.
6. The method as claimed in claim 1, wherein the at least one sedimentation reagent is selected from a group consisting of Ficoll-Hypaque, Hespan®, Pentastarch, and combinations thereof.
7. The method as claimed in claim 1, wherein the at least one sedimentation to obtain the umbilical cord blood (UCB) derived platelet-rich plasma comprises sedimentation having a time range of at least 30 minutes each.
8. The method as claimed in claim 1, wherein subjecting the maternal blood (MB) sample to sedimentation is done for a time in a range of 50-70 minutes.
9. The method as claimed in claim 1, wherein the lysate obtained in step (g) is filtered using a 0.22µ filter.
10. The method as claimed in claim 1, wherein the lysate obtained in step (g) is subjected to screening for presence of infectious diseases (ID).
11. The method as claimed in claim 1, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1.
12. An umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma, said UCB derived platelet-rich plasma

comprises UCB derived platelets; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, said MB derived platelet-rich plasma comprises MB derived platelets, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1×109 platelets/ml.
13. The umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate as claimed in claim 12 for use in culturing cells.
14. The umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate as claimed in claim 13, wherein the cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, and cardiomyocytes.
15. The umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate as claimed in claim 12 for use in cell and tissue culture.
16. The umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate as claimed in claim 12 for use in therapeutic applications.
17. The umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate as claimed in claim 12, wherein said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time in a range of 1 day to 12 months at a temperature in a range of -70°C to -86°C for use in culturing cells.
18. The umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate as claimed in claim 17, wherein the cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, and cardiomyocytes.
19. The umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate as claimed in claim 17 for use in therapeutic applications.