FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENT RULES ,2003
PATENT OF ADDITION (See Section 54)
"PROCESS OF OBTAINING MESENCHYMAL CELLS FROM UMBILICAL CORD"


RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, NaviMumbai-400 701 Maharashtra India.

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-


CROSS REFERENCE TO RELATED APPLICATIONS
This application is a patent of addition to Indian Application number 532/MUM/2003 tiled on 26th May 2003.
FIELD OF THE INVENTION
The present disclosure relates to culture of mesenchymal cells from umbilical cord tissue (UCMSC in presence of cord blood serum and its potential to differentiate into various lineages.
BACKGROUND OF THE INVENTION
Current treatments for various degenerative disorders rely on surgical interventions and drugs that modulate the system, but these have their own limitations when it comes to regeneration of damaged tissues and cells. Cell-based therapies are gaining importance to address these shortcomings. The source of cells has become a critical issue for stem cell therapy or regenerative medicine. Tissue-specific cells represent the first choice because they possess the desired phenotype. However, the availability of these cells can be limited, and in the case of highly differentiated cells, their growth becomes a limiting factor. These limitations can be mitigated by the use of embryonic stem cells (ESCs). They are pluripotent, indefinitely self-renewing and thereby highly attractive for use in therapeutic applications. However, ethical controversies have posed serious problems associated with their use. Amongst the different types of adult stem cells studied, mesenchymal stem cells (MSCs) are gaining importance and has been widely used for their therapeutic applications. There are many reports of successful isolation and differentiation of MSCs from different sources. Adult bone marrow mesenchymal stem cells (MSCs) avoid these ethical problems, but they are much more limited than ESCs, both in their differentiation potential and in their longevity. Thus, there is a significant need for a source of stem cells that have great differentiation and at the same time avoids the ethical obstacles encountered by embryonic stem cells. MSCs are extensively studied with respect to their immunosuppressive properties, their differentiation to various mesodermal lineages, and their transdifferentiation potential to ectodermal lineage such as neural cells,
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BMMSCs {Bone Marrow Mesenchymal Stem Cells) have been used in several clinical trials in autologous settings. Allogenic BMMSCs are the subject of active research and are in advanced phase clinical trials for their application in cartilage injury and Crohn's disease, as BMMSCs express HLA DR, which makes it difficult for further use in allogenic transplantation.
As (he eithical related issue of the use of embryonic stem cells still remains un resolved Fetal tissues, such as the placenta and the umbilical cord, have been recently considered by researchers as a source of these alternative cells. As MSCs have the capcity for self renewal and differentiate into multiple lineages, these MSCs have generated immense attention. Further the MSCs are more "plastic" than hematopoetic stem cells (HSCs). Unlike HSCs, MSCs are found in large amounts in umbilical cord tissue, rather than cord blood or bone marrow. Hence to overcome this limitation there is a need to search for an alternative source that is easily available, non-controversial, and can generate higher number of MSCs. Umbilical cord blood and umbilical cord could replace bone marrow as a source for MSCs.
The current challenge is the availability of a non-controversial supply of starting material in adequate amounts to address the ever-increasing number of patients world over who require this mode of therapy. Umbilical cord overcomes the issue of availability as it an abundant and easily available source, and MSCs can be derived from umbilical cord and have high expansion potential. Since these MSCs do not express MHC class II antigens, they can be very useful as a ready source of stem cells for allogenic use.
The umbilical cord is a tube present in placental mammals that connects the fetus to the placenta. The umbilical cord is made of Wharton's jelly, not ordinary skin and connective tissue. It normally contains three vessels, two arteries and one vein, buried within Wharton's jelly, for the exchange of nutrient- and oxygen-rich blood between the embryo and placenta.. Cells from the Wharton's jelly of human umbilical cords contain high levels of transcription factors that are associated with pluripotent cells. As MSCs have the capcity for self renewal and differentiate into multiple lineages, these MSCs
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have generated immense attention. Wharton's jelly is a specialized tissue serving many purposes for the developing fetus. Its specialized cells contain gelatin-like mucus that encase fibers. These properties give it an elastic and cushion effect, which can tolerate the vibration, bending, stretching and twisting of an active fetus. In addition, it holds the vessels together, may regulate blood flow, plays a role in providing nutrition to the fetus, stores chemistry for the onset of labor, and protects the supply line,
Umbilical cords without much Wharton's jelly are more prone to compression, and complete absence is usually associated with fetal death. If an umbilical cord is twisted or knotted, it is more likely to lighten where there is less resistance, such as an area low in Wharton's jelly.
Presently, bone marrow has been a source of MSCs. Recently, research has been focused on the cord tissue as a source of MSCs. Although sscientists first suggested in 1991 that Wharton's jelly might contain precursor cells, there was no clarity as to where to look for such cells within the jelly,
There are various reports relating to stem cells isolated from umbilical cord tissue by various techniques are specific progenitor cells. For example Purchio, et al.t in United Slates Patent No. 5,919,702 have provided chondrogenic progenitor cells from Wharton's jelly of the umbilical cord tissue. Weiss, et «/., in United States Patent Application Publication Number 2003/0161818 have provided procedures for isolation of pluripotent cells from Wharton's jelly or non-blood umbilical cord matrix. These cells were CD34-and CD45-, indicating that these cells are non-hemalopoietic.
Mitchell, et al? (Stem Cells 21:50-60, 2003) reported obtaining Wharton's jelly matrix cells from porcine umbilical cords. The undifferentiated cells were reported to be positive for telomerase, and a subpopulation was also reported to be positive for c-kit expression, i.e., telomerase-*-, CD! 17+, The cells were also reported to produce alpha-smooth muscle actin, indicative of their myofibroblast-like nature.
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Romanov, et al., (Stem Cells 21:105-110, 2003) reported a procedure to isolate mesenchymal stem cell (MSC)-like cells from human umbilical cord vein.
Mislry, et ai, in United States Patent Application Publication Number 2007/0036767, provide isolated umbilicus-derived cells having specific cell surface marker expression profiles, wherein particular cell surface marker proteins are produced. In particular, the cells produce one or more of CD10, CDI3, CD44, CD73, CD90, CD141, PDGFr-alpha, or HLA-A, -B, or -C. In addition, the cells do not produce one or more of CD3I, CD34, CD45, CD 1 17, CD 141, or HLA-DR, -DP, or-DQ, as detected by flow cytometry,
All of the above methods were involved with cells derived from Wharton's jelly or umbilical vein. It is noted that the Wharton's jelly is a gelatinous substance within the umbilical cord. To harvest the MSCs, one needs to open the umbilical cord and remove the blood vessels with their surrounding Wharton's jelly. The vessels are then sutured closed and suspended in collagenase, an enzyme that breaks down the Wharton's jelly around the vessels to release the cells inside. The cells are then isolated and cultivated in vitro. Hence the cell source is not clearly evident whether the MSCs are from the cord blood or from cord.
These isolated MSCs were expanded using fetal bovine serum (FBS) in the culture media used for such expansion. .The use of FBS during MSC propagation, however, carries the risk of transmission of known and unknown pathogens, as well as xenoimmunization. Attempts have been made by several groups to replace FBS with growth factors derived by mixing purified factors that are either isolated from FBS or a mixture of growth factors derived by recombinant methods. However, these culture media have their associated shortcomings and risks since they are derived from animal sources.
In patent application number 532/MUM/2003, the inventors of the applicant demonstrated that culluring of MSC isolated from human bone marrow aspirate in the presence of human umbilical cord blood serum instead of FBS promoted more effective
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expansion and retention of their differentiation capacity. The applicant further has shown the superiority of using cord blood serum as a xenofree alternative to FBS.. Under these circumstances, MSCs could be expanded with no undesirable effect whatsoever on transdiffcrentialion and stability in culture for more than 5 passages.
Looking into the need for improved methods of isolation of mesenchymal stem cells the present invention has developed a simple method of isolation of mesenchymal cells from the umbilical cord tissue (UCMSC) without the step of separation of Wharton's Jelly from cord without any loss of the cells during isolation process. Thus the present invention is rapid and less cumbersome with UCMSC cells being intact during the isolation process with no wastage of cells.
The present application further provides the use of CBS (Cord Blood Serum) for expansion of MSC derived from umbilical cord by approved validated protocols. CBS is processed as per available regulatory guidelines in controlled cGMP environment, using excipients that can satisfy quality parameters. Further, these cells has potential to differentiate not limited to osteogenic and chondrogenic lineages.
The present invention also intends to provide methods of banking of cells derived from cord tissue as well as banking of cord tissue for regenerative medicine. .
OBJECTIVES OF THE INVENTION
It is an objective of the present invention to provide culturing of mesenchymal stem cells
from umbilical cord tissue.
It is an objective of the present invention to provide methods of isolation of mesenchymal
stem cells from umbilical cord tissue directly without prior separation of Wharton's jelly
during the isolation process.
It is an objective of the present invention to characterize the mesenchymal stem cells
from umbilical cord tissue.
It is an objective of the present invention to provide process conditions for proliferation
of umbilical cord derived MSCs (UCMSCs) using xeno-free media,
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It is an objective of the present invention to determine the differentiation capacity of the
UCMSCs cultured in cord blood serum (CBS) in a regulated environment and assess their
in vitro and in vivo differential potential.
It is an objective of the present invention to study the genotypic, phenotypic and
functional properties of CBS cultured and expanded UCMSCs.
It is an objective of the present invention to provide a banking of cells, as well as cord
tissue, for regenerative cell therapy.
SUMMARY OF THE INVENTION
The present disclosure provides mesenchymal stem cells from umbilical cord tissue and methods of isolation. In one embodiment the present disclosure provides a method of isolation of mesenchymal stem cells from umbilical cord tissue, a process for expansion of UCMSC in the presence of media containing CBS, and their differentiation potential in vitro. In one aspect, the present disclosure provides methods for expanding the cord tissue derived MSCs under xeno-free conditions.
In one aspect, the present disclosure provides methods of isolation of mesenchymal stem cells from umbilical cord tissue. In one embodiment, the present disclosure provides methods for isolation of mesenchymal stem cells from umbilical cord tissue without prior separation of Wharton's jelly. In one aspect, the present disclosure provides UCMSCs isolated directly from cord tissue explants without any enzyme treatment.
hi one aspect, the present disclosure provides methods for culturing and expansion of mesenchymal stem cells from the umbilical cord tissue. In another aspect, the present disclosure provides methods for expansion of cells derived from umbilical cord tissue in the presence of CBS.
In yet another aspect, the present disclosure provides methods for characterization of the cord tissue-derived cells cultured in CBS. In one aspect, the present disclosure provides the genotypic, phenotypic, and functional characterization of the cells.
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In one aspect the present disclosure provides methods of assessing the differentiation capacity of the xeno-frec cultured UCMSCs.
In one aspect the present disclosure provides methods of banking cells derived from cord tissue, as well as banking of cord tissue for regenerative medicine.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Fijjure 1: (A) Umbilical cord is decontaminated with PBS; (B) Umbilical cord is laterally cut to remove blood vessels; (C) Rxplants seeded in culture media; (D) UCMSCs migrate from theexplant by day 10-15; (E) UCMSCs form a monolayer of cells by day 25.
Figure 2: UCMSC show an exponential growth potential. Approximately 2.7xl016 cells could be obtained after passage 11.
Figure 3: Undifferentiated UCMSCs were negative for hematopoietic markers and expressed mesenchymal markers. Immunophenotype expressed by the UCMSCs was CD73+/CD105+/SSEA4+/HI,A ABC+/CD44+/CD29+/CD45-/CD31-/HLA DR-/CD14-
/vWF-.
Figure 4: Expression of osteogenic genes BMP4 and osteopontin after (A) Day 7; (B) Day 14; and (C) Day 21 of osteogenic differentiation.
Figure 5: Immunohistochemical staining of chondrogenic pellets.
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DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "umbilical cord blood" or "cord blood" is used throughout the specification to refer to blood obtained from a neonate or fetus, most preferably a neonate and preferably refers to blood that is obtained from the umbilical cord or placenta of neonate. The use of cord or placental blood as a source of mononuclear cells is advantageous because it can be obtained relatively easily and without trauma to the donor. Cord blood cells can be used for autologous or allogenic transplantation when and if needed. Cord blood is preferably obtained by direct drainage from the umbilical vein.
The term "umbilical cord tissue," as used herein, refers to the cords obtained after normal or caesarian deliveries with informed consent from the mother.
As used herein, the term "confluent" indicates that the cells have formed a coherent cellular monolayer on the surface so that virtually all available surface is used, leading to inhibition of cell proliferation.
The term "umbilical cord tissue derived mesenchymal stem cells" or "UCMSC," as used herein, refers to mesenchymal stem cells derived from umbilical cord tissue and is characterized by the expression of CD markers CD73, CD 105, CD29, CD44, and further positive for SSI-A4, HLA ABC. The UCMSC are negative for CD45, CD14, vWF, HLA DR., and CD31. In certain aspects, the USMSC are characterized by being at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% (or greater) positive for the MSC markers CD73, CD 105, CD29, and CD44. In other words, in certain embodiments the UCMSC are at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% (or greater) pure based on the expression of'the MSC markers CD73, CD105, CD29, and CD44,
The parent application (532/MUM/2003) of the applicant , which is incorporated herein by reference in its entirety, showed that mesenchymal stem could be cultured and
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expanded in medium containing CBS. The same culture conditions have proven to be beneficial for the expansion of MSCs from umbilical cord tissue.
The umbilical cord tissue has been obtained from voluntarily donors with informed consent. The present disclosure provides herein a process wherein the mesenchymal stem cells have been directly obtained from the cord tissue without the need of first isolating Wharton's jelly, thus obviating the need of enzymatic treatment,
Further the present disclosure also addresses the risk of xenofactors by using cord blood scrum. The cord blood serum has been also shown to be efficacious in expanding and maintaining the proliferative potential of mesenchymal stem cells derived from umbilical cord tissue.
The present disclosure provides the use of CBS (cord blood serum) for expansion of MSCs derived from umbilical cord by approved validated protocols, CBS is processed as per available regulatory guidelines in a controlled cGMP environment, using excipients that can satisfy quality parameters. The present disclosure also details that umbilical cord-derived MSCs cultured under xeno free conditions continue to maintain the mesenchymal surface marker expression, and preferably display a typical mesenchymal phenotype, for example the cells are positive for one or more early stem cell markers and MHC class 1 antigens, bm not MHC class II antigens. In other embodiments the UCMSCs are CD73+/CDI05+/CD44+/CD29+/SSEA4+/ HLA ABC+/CD45-/CD31-/vWF-/CD14-/HLA DK-.
Further, the present disclosure details that these cells can be efficiently induced to differentiate into osteogenic and chondrogenic lineages in vitro,
MSCs were successfully isolated from three different sources. The isolated MSCs were expanded and characterized for a panel of MSC markers. MSCs could be easily isolated from all 5 bone marrow and 4 umbilical cord samples as compared to cord blood. Of the 70 cord blood samples processed, only 4 samples (i.e., 6%) of the samples yielded MSCs. In sharp contrast, 100% of the umbilical cord and bone marrow samples processed yielded MSCs. MSCs from all these different sources were similar morphologically and
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phenotypically, except for ULA OR expression, which was observed only in BMMSCs. It was observed that the BMMSCs showed a decline in the fold expansion on subsequent passaging. MSCs derived from cord blood initially showed lower fold expansion at passage 2 which increased in the third and fourth passage and later declined and remained steady after the fifth passage.
In general, the following steps are involved in the processes for derivation of
mesenchymal stem cells from umbilical cord tissue, although not every step is necessarily
required for isolating these cells: (I) collection of cord tissue; (2) plating of the tissue for
expansion in a culture media; (3) expansion of the cells; (4) passaging/harvesting of the
cells; (5) characterization of the cells; (6) analysis of the differentiation potential into
neural cells in vitro and in vivo; and (7) banking of cells/cord tissue. These steps are
detailed below,
1. Cord tissue collection
The umbilical cord tissue was obtained from hospitals and was collected with informed consent. The cord samples were collected in a medium containing DMEM supplemented with antibiotics. The cord tissue can either be obtained from mothers within 48 hours of delivery or can be used from cryopreserved cord tissue banks. At the time of birth the umbilical cord tissue is removed, suspended in DMEM solution, and then transported to a cord tissue bank or used for the isolation of cells within 48 hours or can be used within 15 days if stored in refereigerator.
2H Plating of the tissue for expansion in a culture media
The cord tissue samples were washed with PBS supplemented with antibiotics and then plated in tissue culture dishes with culture medium. Fresh culture medium was added to the dishes after 3-4 days.
3. Expansion of the cells.
The adherent cells were allowed to expand with regular change of the culture media. UCMSCs could be successfully isolated from all 4 cords processed. UCMSCs started to migrate from the explant after 10 to 15 days of culture (Figure ID), The isolated UCMSCs grew as adherent fibroblast-like cells and formed a monolayer of spindle shaped cells after 25 days in culture (Figure 1E).
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4. Passaging/harvesting of the cells
The cells were harvested at 80-90% confluence using TrypLE™ Select (Invitrogen Corporation, Carlsbad, CA, USA) and replated into T75 tissue culture flasks at a density ol 5 x 103 cells/cm2 in cell culture media. UCMSCs could be expanded in culture by repealed harvesting and replating of cells every 7 to 8 days up to passage 11.
5. Characterization
No change in morphology or immunophenotype was observed in cells that were cultured up to passage II. . After every passage (PI to Pll) the cells were harvested using TrypLE™ Select and approximately 2x I06 cells were used for immunophenotyping studies (Figure 2). Flow cytometry studies showed that cultured UCMSC displayed no expression of hematopoietic markers {i.e., CD45 and CD14), or markers for endothelial cells (i.e., von Willehrand Factor (vWF) and CD31). No expression of HLA DR was observed throughout the culture period (from PI to P10). More than 90% of UCMSCs expressed the MSC-marker proteins CD73, CD105, CD29, and CD44. UCMSCs were also positive for SSEA 4 and HLA ABC (Figure 3).
6. Differentiation potential
The UCMSC cultured in CBS were then evaluated for its differential potential under differentiation medium. The UCMSC showed differential potential for chondrogenic and osteogenic lineage. For osteogenic differentiation Alkaline Phosphatase marker is used. Von Kossa staining can also be done to detect calcium deposition. Cell banking In the present invention, the mesenchymal stem cells isolated from cord tissue is banked/cryopreserved for transplantation. Alternatively cord tissue is cropreserved depending on the need of the recipient. The banking procedure includes suitable protocols of informed consent, and testing the donor of the tissue for absence of infectious diseases markers (e.g., HIV I & 2, Hepatitis B core antibodies, Hepatitis B surface antigens, human T lymphoma virus (HTLV 1 &2), syphilis, cytomegalovirus (CMV), Immunophenotyping can be conducted to characterize the MSCs before freezing. Once these cells are characterized and tested for viability, appropriate numbers are suspended in a cryoprotectant and stored at -196°C in liquid nitrogen.
Therapeutic potential
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Whenever any transplantation of cells is required, the cells are usually thawed and cultured for transfer to the recipient. The average number of cells can be adjusted according to the needs of the patient. Alternatively, the banked cord tissue can be thawed, and mesenchymal stem cells derived, expanded and differentiated according to the requirements of the recipient.
Thus the present disclosure has shown that the UCMSC derived from cord tissue without enzymatic treatment nor previous isolation of Wharton's jelly expanded and proliferated well in culture medium comprising cord blood serum. Thus the cells disclosed herein may be used for therapeutic application without the risk of xenofactors.
Further the present disclosure provides an excellent source of MSC from cord tissue directly, without the need of isolation of Wharton's jelly. This is beneficial in cryopreservation of cord tissue samples (banking), since the issue of preservation or initial isolation of Wharton's jelly is not crucial for derivation of MSC.
Thus the present disclosure details that MSC derived from umbilical cord tissue (either freshly collected or cryopreserved) can be expanded under xeno-free conditions while maintaining the differential capacity of the cells.
Thus, in certain aspects the present disclosure encompasses methods of treating, managing, and/or preventing a chondrogenic or osteogenic disorder, which comprise administering to a patient in need of such treatment, management, or prevention a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising UCMSCs that have been differentiated into chondrogenic or osteogenic cells. As used herein, and unless otherwise indicated, the terms "treat," "treating," and "treatment'1 contemplate an action that occurs while a patient is suffering from a chondrogenic or osteogenic disorder, that reduces the severity of one or more symptoms or effects of the chondrogenic or osteogenic disorder, or a related disease or disorder. As used herein, and unless otherwise indicated, the terms "prevent," "preventing," and "prevention" contemplate an action that occurs before a patient begins to suffer from a
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chondrogenic or osteogenic disorder, that prolongs the onset of, and/or inhibits or reduces the severity of, the chondrogenic or osteogenic disorder.
As used herein, and unless otherwise indicated, the terms "manage," "managing," and "management" encompass preventing, delaying, or reducing the severity of a recurrence of a chondrogenic or osteogenic disorder in a patient who has already suffered from the chondrogenic or osteogenic disorder. The terms encompass modulating the threshold, development, and/or duration of a chondrogenic or osteogenic disorder, or changing the way that a patient responds to a chondrogenic or osteogenic disorder.
As used herein, and unless otherwise specified, a "therapeutically effective amount" of differentiated UCMSCs is an amount sufficient to provide any therapeutic benefit in the treatment or management of a chondrogenic or osteogenic disorder, or to delay or minimize one or more symptoms associated with a chondrogenic or osteogenic disorder. A therapeutically effective amount of differentiated UCMSCs means an amount of differentiated UCMSCs, alone or in combination with one or more other therapy and/or therapeutic agent, that provides any therapeutic benefit in the treatment or management of a chondrogenic or osteogenic disorder, or related diseases or disorders. The term "therapeutically effective amount" can encompass an amount that cures a chondrogenic or osteogenic disorder, improves or reduces a chondrogenic or osteogenic disorder, reduces or avoids symptoms or causes of a chondrogenic or osteogenic disorder, improves overall therapy, or enhances the therapeutic efficacy of another therapeutic a^ent.
As used herein, and unless otherwise specified, a "prophylactically effective amount" of differentiated UCMSCs is an amount sufficient to prevent or delay the onset of a chondrogenic or osteogenic disorder, or one or more symptoms associated with a chondrogenic or osteogenic disorder, or prevent or delay its recurrence. A prophylactically effective amount of differentiated UCMSCs means an amount of differentiated UCMSCs, alone or in combination with one or more other treatment and/or prophylactic agent, that provides a prophylactic benefit in the prevention of a
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chondrogenic or osteogenic disorder such as Osteoarthritis and osteogenesis imperfecta, The term "prophylactically effective amount" can encompass an amount that prevents a chondrogenic or osteogenic disorder, improves overall prophylaxis, or enhances the prophylactic efficacy of another prophylactic agent.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: Processing of Umbilical Cord for Isolation of Mesenchymal Stem Cells
Umbilical cords were collected after normal or caesarian deliveries into tubes containing sterile media with antibiotics. Informed consent was obtained for collection of cords. The cord was then washed with sterile PBS containing antibiotics (Figure 1). An approximately 1-2 cm piece of the cord was taken for further processing. The cord was cut open longitudinally (Figure IB) and washed with PBS (Figure 1),
EXAMPLE 2: Culture and Expansion of the Mesenchymal Stem Cells Cell Culture:
Cells were allowed to adhere for four days. The first media change was done after 4 days and thereafter every alternate day. Once the cells were confluent the cultures were detached using TrypLE™ Select, and washed with PBS by centrifugation at 1300 rpm for 5 minutes. The cells were replated into tissue culture flasks at a density of 0.2 x 106 for 25cm2 flasks and 0,4 x \0b for 75cm2 flasks. Cells grew as monolayer of adherent fibroblast-like cells (Figure IE). Umbilical cords (gestational ages 31 to 37 weeks) were obtained from the maternity hospitals after normal or caesarian deliveries. The cords were collected after obtaining informed consent from the mother. Approximately 10 cms of the
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cord was collected into tubes containing DMEM supplemented with routine antibiotics and were transported to the lab within 48hrs of collection. Cord samples were transported at 20 - 22°C in a validated cold chain. Processing was done in a GMP compliant environment. The cord was washed with phosphate buffered saline (PBS) supplemented with routine antibiotics. The cord was cut open longitudinally and was cleaned to remove the blood clots. Cord explants of about 2mm were placed in 100mm tissue culture dishes with culture medium. The dishes were left undisturbed in a 5% C02 incubator maintained al 37°C for 4-5 days after which fresh cell culture media was added to the dishes. Cord samples were divided into two groups; group A samples (n=4) were cultured in DMEM/F12 (1:1) containing 10% fetal bovine serum (FBS) supplemented with l-2ng/ml fibroblast growth factor and group B (n-4) were cultured in DMEM/F12 (1:1) containing 10% cord blood serum (CBS) supplemented with l-2ng/ml fibroblast growth factor.
Adherent cells from both groups were allowed to expand with regular media changes. The cells were harvested at 80-90% confluence using Tryple Select and replated into 75 cm2 tissue culture flasks at a density of 5x103 cells/cm2 in appropriate cell culture media. Colls were expanded upto a minimum of 8 passages,
Cell Culture Media:
Preparation of Cord Blood Serum. The cord blood that is normally discarded was collected after delivery from pre-screened mothers after an informed consent. These women were screened for mandatory infectious diseases markers. No collection was made if there were any complications during delivery. The blood was collected by allowing it to flow into a sterile Schott glass bottle from the cut end after the baby is separated. 10 - 25ml of blood was collected and transported to the facility at 20 - 25°C within 10-12 hrs. The clotted blood was then centrifuged at 1000 g for 30mins at 20°C and the clear serum was collected into sterile containers. Serum from a minimum of three donors was pooled to eliminate batch variations that would arise from different donors. The pooled serum was filter sterilized by passing through a 0.22D filter. Complement was inactivated by keeping the serum at 56°C for Vi hour. Serum was labeled, aliquoted and stored at -20"C for upto 4 months for further use in research.
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DMEM/P12 (1:1) containing 10% CBS supplemented with 2 ng/ml of basic fibroblast growth factor was used to culture the cells.
Expansion:
No change in morphology or immunophenotype was observed in cells that were cultured up to passage 11. UCMSC showed exponential growth, with approximately 2.7 x 1016 UCMSCs obtained after passage 11 (Figure 2).
EXAMPLE 3: Characterization of the Cells Immunophcnotyping of Cells
Cell surface antigen phenotyping was done at every passage using flow cytometry. The cells were harvested and incubated for 20 minutes at 4°C with the following flurochrome labeled antibodies; CD45-PerCP, CD73-PE, CD105-PE, SSEA4-PE, HLA^DR~PE, HLA-ABC-PE, CD31-PE, CD44-PE, CD29-PE, CD14-PE, and vWF-FITC. Appropriate isotype controls were used to determine non-specific fluorescence. These cells were acquired on a FACS Calibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with a 488 nm argon laser. Approximately 5,000 -10,000 events were acquired and analyzed using Cell Quest Software. For viability determination cells were stained with 7-Amino Actinomycin D (7-AAD), (BD Biosciences, San Jose, CA, USA) and acquired on the (low cytometer.
Flow cytometry studies showed that the majority of the cells were negative for haematopoietic (CD45 and CD 14) and endothelial (CD3I and vWF) markers, and positive for MSC markers (CD73, CD105). These cells also expressed markers for adhesion molecules (CD44 and CD29). The cells were also positive for SSEA4, which is an early stern cell marker. The cells showed no expression of MHC class II antigen (HLA DR), but expressed MHC class I antigen (HLA-ABC) (Figure 3).
EXAMPLE 4: In vitro Differentiation Potential
Differentiation potential of the isolated cells into osteocytes and chondrocytes was evaluated at passage 11.
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Osteogenic Potential
For osteogenic differentiation cells were cultured in cell culture medium for 24 hours in a 37"C incubator with 5% CO2. After 24hrs Osteogenesis was induced by replacing DMEM media with osteogenic induction medium (Cambrex Corporation, East Rutherford, NJ, USA). The cells were fed regularly with osteogenic induction medium for 3-4 weeks. Osteogenesis was confirmed by the presence osteogenic markers such as osLeopontin, osteocalcin and osteonectin as detected by flow cytometry. Osteogenic-spccific genes osteopontin and BMP4 were detected by RT PCR. The cell pellets of both induced and uninduced cells were used for total RNA extraction. Total RNA was isolated from I x 106 cells using RNeasy. 5 ug of RNA was used for cDNA synthesis. The cDNA was synthesized using Superscript reverse-transcriptase II (Invitrogen Corporation). 1 \x\ of cDNA was amplified by polymerase chain reaction using ABgene 2x master mix (ABgene, Rochester, NY, USA) with appropriate primers. Cycling parameters arc as follows: Initial denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 55-65cC for 30 seconds depending on the primer, and elongation for 1 minute, and the number of cycles varied between 25 and 40. Final elongation was carried out at 72°C for 7 minutes. The osteogenic medium comprised basal medium with growth factors such as dexamethasone, L-glutamine ascorbate, Pen/Strep, MCGS, and beta-glycerophosphate.
For flow cytometry studies approximately 1 x 106 cells were fixed and permeabilized using the 3D Cytofix/Cytaporm Fixation/PermeabiIization kit (BD Biosciences). Cells were then incubated with mouse anti-human osteocalcin, mouse anti-human SPARC and mouse anti-human osteopontin for 20 minutes at 4°C. All antibodies were procured from R&D Systems (Minneapolis, M.N, USA), After incubation the ceils were washed with PBS and acquired on FACS Calibur Flow Cytometer,
Expression of osteonectin and osteocalcin increased up to day 14 of differentiation, after which a decrease in expression was seen at day 21. Osteopontin expression did not show any significant change (Figure 4).
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RT PCR showed expression of osteogenic specific gene osteopontin and BMP4 (Figure 3). No expression of HLA DR was observed by flow cytometry or RT PCR even in differentiated UCMSCs.
Chondrogenic Potential
To induce chondrogenic differentiation the cells were seeded at 0.5 x 106 cells per 15 ml polypropelene tube and were maintained in chondrogenic medium (Cambrex Corporation) supplemented with TGF|33. The tubes were kept in 5% C02 incubator at 37"(\ The cell cultures grew as pellets and were fed with chondrogenic medium regularly. After 19 days the pellets were fixed with 10% buffered formalin and embedded in paraffin. 4-10 urn sections were made for immiinohistochemical studies, The composition of chondrogenic medium comprised basal media including growth factors such as dexaracttvasoue, ascotbate, ITS + Supplement, Pen/Steep, sodium* pyruvate, proline, and L-glutamine.
Immunohistochemical studies for chondrogenic differentiation
For Alcian Blue and Safranin O staining the sections were deparaffinised and hydrated to water. Pellets were then stained with 1% Alcian Blue prepared in 3% acetic acid for 30 minutes, and with Safranin 0 for 10 minutes.
For chondrogenic specific markers (e.g., collagen type I, collagen type II and aggrecan), the deparaffinized and hydrated sections were fixed with 4% paraformaldehyde. Blocking and permeabilization was done using PBS containing 1% BSA and 0.1% Triton X 100. Pellets were incubated overnight with 1:100 dilution of primary antibody (collagen type I, collagen type II and aggracan). Biotinylated streptavidin horseradish peroxidase was used as the secondary antibody. DAB (3,3t-Diaminobenzidine) was used as the substrate to produce brown color reaction.
The cells formed a pellet within I day and the size increased over a period of 2-3 weeks. After 2-3 weeks of culture a spheroid cell mass was formed, which was fixed in buffered formalin and paraffin embedded. Thin sections (4-5 u.m) were made, which were stained using Alcian blue and Safranin O. The sections stained positive with Alcian blue and
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Safranin O, indicating the presence of cartilage matrix. Differentiated UCMSC also expressed the chondrocyte specific markers collagen type II and aggrecan. Collagen type I was also expressed by the differentiated UCMSCs (Figure 5).
EXAMPLE 5: Banking of Cells and Tissue
The banking of the umbilical cord involved two steps; (I) storage of umbilical cord tissue; and (2) storage of umbilical cord-derived mesenchymal stem cells. The cord was received in the specific transport media and approximately 1-2 cm piece of the cord was taken for further processing. The cord was cut open longitudinally and washed with PBS.
Storage of the cord tissue: The 1-2 cm cut cord tissue was cryopreserved in the freezing mix having 90% FBS and 10% DMSO, Arouad I ml of the freezing mix was added to a 1.5 ml cryovial with a single piece of the cord tissue, which was washed in PBS having antibiotics, A single cord tissue of 1-2 cm was stored in one cryovial, This tissue was then subjected Lo slow rate freezing and once it reached -80°C it was transferred to liquid nitrogen having a temperature of -196°C. The cord tissue in this manner can be stored for a very long time and can be an immortal source of stem cells. When the cord of around 25 cm is received 5cm of the cord is cryopreserved. This is done in five 1.5 ml cryovials. Each vial has I cm of the cord preserved and stored in liquid nitrogen. The remaining 20 cm of the cord was utilized for isolation of the mesenchymal stem cells. Storage of the umbilical cord derived mesenchymal stem cells: This process is further involved the creation of a Master Cell Bank and a Working Cell Bank. The 20 cm of the cord was cut into small explants, which were plated. After 21 days, a minimum of two million cells fat P0) was obtained. These cells were then plated on five 75 cm2 flasks, each flask having an initial plating density of 0.4 million cells. After 7 days of plating these flasks attained confluency, and were then harvested. A cell count of 25 million cells was achieved of which 23 million cells were frozen and 2 million cells were again plated for the creation of a Working Cell Bank. The cells were frozen in freezing mix, which comprised 90% FBS and 10% DMSO. The 23 million cells were frozen in five 1.5 ml cryovials. Pour cryovials have 5 million cells frozen in each, and one cryovial has 3
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million cells frozen in it. These vials were then subjected to slow rate freezing and once it leached -80°C it was transferred to liquid nitrogen having a temperature of -196°C. The 5 vials frozen at passage I constituted the Master Cell Bank (MCB). One or two vials of the MCB were allocated for quality control, providing confirmation that the cell count and viability of the bank is acceptable, and that the bank is free of bacteria/fungi and mycoplasma.
After the completion of tests, the vials from the Master Cell Bank were expanded and cultured to produce a Working Cell Bank. Quality control tests (cell count and viability and the absence of microbial contaminants) were again required prior to using the cultures for production. It is also important at this stage to confirm that the Master and Working Cell Banks are genetically identical by DNA profiling techniques. Cells from Passage 2 onwards were used as a Working Cell Bank,
The 2 million cells at passage 1 were again plated in to five 75 cm2 flasks. These attained eonfhiency on the 7lh day after seeding and were then harvested. A cell count of 25 million was obtained. The cells were frozen in freezing mix which contained 90% FBS and 10% DMSO. Five cryoviais were frozen, each having 5 million cells. These vials were then subjected to slow rate freezing and once it reached -80°C it was transferred to liquid nitrogen having a temperature of -I96°C, These 25 million cells frozen in 5 cryoviais constituted the Working Cell Bank.
To summarize the banking process, it takes 35- 40 days for the complete banking of the umbilical cord. Five vials of cord tissue are stored, and five vials of Master Cell Bank and five vials of Working Cell Bank are stored, which constitute a total of 48 million
cells.
The quality testing for endotoxin, bioburden, mycoplasma, and sterility was performed at two points: (1) at P2, when the batch was being closed for banking; and (2) when the cells are being used for therapy purposes. The characterization of MSCs was done by Flow cytometry using CD markers (e.g., CD 73, CD 90, CD 105, CD 34, and CD 45).
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Storage of Banked Cells
The cells were stored in liquid nitrogen freezers, which provide a secure storage environment that maintains temperatures below -150°C. The cells were labeled with cryogenic bar code labels, and the freezing records were maintained. The cells were analyzed for mycoplasma and endotoxin, and tested for sterility. As an added precaution, when cells were being banked, each bank was divided equally and stored in separate liquid nitrogen freezers so that in the rare event of a complete freezer malfunction, a second grouping of vials was still available for subsequent use.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Dated this 26 day of May ,2009
For Reliance Life Sciences Pvt, Ltd
K. V, Subramaniam President
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CLAIMS:
1. A method of culturing mesenchymal stem cells derived from umbilical cord tissue
(UCMSC) comprising
a. collecting umbilical cord tissue;
b. plating a piece of umbilical cord tissue into a tissue culture flask comprising a
culture medium along with cord blood serum for about 3-4 days to produce
adhered cell cultures;
c. incubating the adhered cell cultures of step b) for at least 10-15 days;
d. analyzing the cultured cells for expression of one or more CD markers; and
e. replating or passaging the cells.
2. The method of claim 1, further comprising analyzing the differentiation potential of the UCMSC in vitro and in vivo.
3. The method of claim I, wherein the cord blood serum is about 1-50%.
4. The method of claim 1, wherein the adhered cell cultures of step b) is incubated at 37°C in 5% CO? air incubator.
5. The method of claim 1, wherein the piece of umbilical cord tissue is about 1-2 cm.
f>. The method of claim I t wherein The umbilical cord tissue is freshly collected within 48 hours of delivery or from a cryopreserved cord tissue bank.
7. The method of claim 1, wherein the CD marker is selected from CD73, CDI05, CD44, CD29, SSEA4, CD45, CD31, vWF, and CD 14.
8. The method of claim I, wherein the mesenchymal stem cells obtained from umbilical cord tissue are positive for CD73, CD 105, CD44, CD29, and SSEA4 markers.
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9. The method of claim 1, wherein the mesenchymal stem cells obtained from umbilical cord tissue are negative for CD45, CD31, vWF, and CD14 markers.
10. The method of claim 1, wherein the mesenchymal stem cells obtained express MHC class II and do not express MHC class I,
11. The method of claim 1 wherein the cells are about 90% pure in terms of MSC antigen expression.
12. A method of differentiating the mesenchymal stem cells of claim 1 into osteogenic cells, comprising the steps of:
a. culturing the umbilical cord derived MSCs of claim 1 in osteogenic induction
medium for about 3-4 weeks; and
b, characterizing the cells for expression of osteogenic-specific genes by RT-PCR or
flow cytometry.
13. Thu method of claim 12, wherein the osteogenic induction medium comprises basal medium and one or more of dexamethasone, L-glutamine, ascorbate, Pen/Strep, MCGS, and p-glycerophosphate.
14. The method of claim 12, wherein the osteogenic-specific genes characterized by flow cytometry are osteocalcin, osteopontin, and osteonectin.
15. The method of claim 12, wherein the osteogenic-specific genes characterized by RT PCR are osleopontin and BMP4.
16. The method of claim 12, wherein the UCMSCs are used in regenerative medicine in an osteogenic disorder.
17. A method of differentiating the mesenchymal stem cells of claim 1 into chondrogenic cells comprising the steps of:
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a. culturing the umbilical cord derived MSCs of claim I in chondrogenic induction
medium for about 3-4 weeks; and
b. characterizing the cells by immunohistochemical studies.
18. The method of claim 17, wherein the chondrogenic induction medium comprises basal media and at least one of dexamethasone, ascorbate, ITS + Supplement, Pen/Strep, sodium pyruvate, proline, and L-glulamine.
19. The method of claim 17, wherein the cells are characterized for chondrogenic-specific markers selected from collagen type I, collagen type II, and aggrecan.
20. The method of claim 17, wherein the mesenchymal stem cells are used in regenerative medicine in a chondrogenic disorder.
21. The method of claim 17, wherein the mesenchymal stem cells are used for therapeutic regenerative medicine.
22. The method of claim 1, wherein the mesenchymal stem cells or the umbilical cord tissue are cryopreserved for banking of cells.
23. The method of claim 22, wherein the cryopreservation is done in a cryoprotectant.
24. A method of culturing mesenchymal stem cells (UCMSC) from umbilical cord tissue, as claimed above exemplified herein substantially in the examples and figures.
Dated this 26 day of May 2009
For Reliance Life Sciences Pvt. Ltd.

KA. Subramaniam
President
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