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Publication numberUS20040203142 A1
Publication typeApplication
Application numberUS 10/413,356
Publication dateOct 14, 2004
Filing dateApr 14, 2003
Priority dateApr 14, 2003
Also published asUS7897388, US20080124701
Publication number10413356, 413356, US 2004/0203142 A1, US 2004/203142 A1, US 20040203142 A1, US 20040203142A1, US 2004203142 A1, US 2004203142A1, US-A1-20040203142, US-A1-2004203142, US2004/0203142A1, US2004/203142A1, US20040203142 A1, US20040203142A1, US2004203142 A1, US2004203142A1
InventorsPrathibha Rai
Original AssigneeReliance Life Sciences Pvt. Ltd.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Growth of neural precursor cells using umbilical cord blood serum and a process for the preparation thereof for therapeutic purposes
US 20040203142 A1
Abstract
This invention is concerned with stem cells derived from umbilical cord blood serum and a method for growing human embryonic stem cells and adult cells comprising sera separated from clotted umbilical cord blood, including growing and differentiating cord blood stem cells into neural precursors, comprising transdifferentiating CD34+ stem cells from mononuclear cells derived from umbilical cord blood to neural precursors. The stem cells obtained from the umbilical cord include pluripotent stem and progenitor cell population of mononuclear cells, and separating pluripotent stem and progenitor cell population of mononuclear cells obtained from the umbilical cord blood. A magnetic cell separator is used to separate out cells which contain a CD marker and then expanding the cells in a growth medium containing retinoic acid and one or more growth factors BDNF, GDNF, NGF and FGF as a differentiating agent. The invention is also concerned with the transplantation and repair of nerve damage, stokes, spinal injury, Parkinson's and Alzheimer's, prepared in accordance with the aforesaid method and a media for culturing umbilical cord blood stem calls consisting essentially of cord blood stem cells derived from umbilical cord blood serum.
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Claims(24)
1. A method for growing human embryonic stem cells and adult cells comprising sera separated from clotted umbilical cord blood.
2. The method as claimed in claim 1, wherein the adult cells are neural precursor cells.
3. The method as claimed in claim 1 including transdifferentiation of CD+ 34 stem cells from mononuclear cells derived from the umbilical cord blood.
4. The method as claimed in claim 1 for growing and differentiating cord blood stem cells into neural precursors, comprising transdifferentiating CD34+ stem cells from mononuclear cells derived from umbilical cord blood to neural precursors.
5. The method according to claim 1 comprising growing and differentiating cord blood stem cells into neutral precursors.
6. The method as claimed in claim 5 wherein the stem cells obtained from the umbilical cord include pluripotent stem and progenitor cell population of mononuclear cells, and including the step of separating pluripotent stem and progenitor cell population of mononuclear cells obtained from the umbilical cord blood.
7. The method as claimed in claim 1 including a magnetic cell separator to separate out cells which contain a CD marker and then expanding these cells in a growth medium containing retinoic acid and one or more growth factors selected from the group consisting of BDNF, GDNF, NGF and FGF and mixtures thereof as a differentiating agent.
8. The method according to claim 7 wherein the differentiating agent is retinoic acid
9. The method according to claim 1 including a differentiating agent selected from the group consisting of 9-cis retinoic acid, all-trans retinoic and combinations thereof.
10. The method according to claim 9 including growth factor selected from the group consisting of BDNF, GDNF, NGF or FGF and combinations thereof.
11. The method according to claim 6 wherein pluripotent and progenitor cells are obtained from the group selected from fresh umbilical cord blood, reconstituted cryopreserved umbilical cord blood and a combination of a fresh or reconstituted cryopreserved mononuclear cell fraction thereof.
12. The method of growing mononuclear cells in a standard cellular media, comprising the steps of growing the cells in a neural proliferation medium, and then growing the cells in a differentiation medium.
13. The method according to claim 13, wherein the standard cellular media is a medium supplemented with non-essential amino acids, glutamine acid serum.
14. The method according to claim 12 wherein the growth factors are limited or removed.
15. The method according to claim 12 wherein the differentiation medium is similar to a neural proliferation medium containing DMEM/F12 1:1 cell medium supplementated with glutamine 2 mM, sodium bicarbonate 3 mM, EGF 20 ng/ml, bFGF10 ng/ml and NGF 100 ng/ml, the neural proliferation medium having neural differentiation agents added.
16. The method according to claim 15 including using a magnetic cell separator (MACS) for removing cells which contain a CD marker, and then expanding the remaining cells in a growth medium or differentiating the remaining cells in a growth medium having a differentiating agent, and co-culturing with adult mesenchymal stem cells or accessory cells from other portions of fetal and mature central nervous system.
17. The method according to claim 12 including enriching mononuclear cells comprising depleting red blood cell using 3% v/v Dextran (high molecular weight) in the ratio of 1:1 with respect to the volume of blood, collecting and centrifuging Leucocyte rich plasma, washing and layering the cells of Histopaque™ 1077, centrifuging tubes containing the sample at 400 g for 30 minutes, washing and separating mononuclear cells separated from the interface using pipettes, and washed mononuclear cells are then suspended neural proliferation medium.
18. The method according to claim 12, including straining the cells with fluorochrome conjugated antibodies for flow cytometry, removing specified numbers of cells in polystyrene round bottom tubes, staining the anti-CD34-FITC, anti-CD133-PE and anti-CD45-PerCP antibodies, and analyzing the stained cells on a FACSCalibur flow cytometer.
19. The method as claimed in claim 12 including plating mononuclear cells from cord blood in Nunc T75 culture flasks in a neural cells proliferation medium containing DMEM/F123 (1:1) supplemented with FBS or CBS, seeding the cells at a density of 1106 to cells/ml, harvesting the non-adherent a fixed culture period of 1 week, counting and analyzing the non-adherent cells for the expression of CD133, CD45 and CD34 markers, whereby the proliferation kinetics of these cells in this medium shows no significant difference (p>0.3) as measured by the paired T test in the numbers of non adherent cells, as observed in cells cultured in the presence of CBS or FBS, thereby including that cord blood serum supports growth of non adherent cells in said cultures with equal efficacy as compared to fetal bovine serum.
20. The method as claimed in claim 12, including plating mononuclear cells from umbilical cord blood in neural cell proliferation medium, feeding the cultures every 3-4 days and allowing the cultures to proceed to 90% confluency, detaching the adherent cells using cell dissociation buffer and seeding them into the next passage with cell dissociation buffer in both these cultures.
21. The method as claimed in claim 19, including using cultures having FBS or CBS.
22. The method as claimed in claim 26, including using trypsin-EDTA for detachment of the cells.
23. Stem cells derived from umbilical cord blood serum for use in connection with transplantation and repair of nerve damage, stokes, spinal injury, Parkinson's and Alzheimer's, prepared in accordance with the method as claimed in claim 16.
24. A media for culturing umbilical cord blood stem calls consisting essentially of cord blood stem cells derived from umbilical cord blood serum.
Description
    TECHNICAL FIELD
  • [0001]
    The present invention provides the use of sera separated from the clotted umbilical cord blood for growing human embryonic stem cells and adult cells such as Neural precursor cells, for therapeutic purposes in regenerative medicine. In particular, the present invention relates to transdifferentiation of CD+ 34 stem cells from Mononuclear cells derived from Umbilical Cord Blood to Neural precursors and such cells may be used in transplantation and repair of nerve damage, stroke, spinal injury, Parkinson's and Alzheimer's.
  • BACKGROUND OF THE INVENTION
  • [0002]
    Stem cell technology is an emerging field that may yield many promising therapies. Stem cells are special cells that have the ability to develop into many different types of tissue: bone, muscle, nerve, etc. In theory, they could be grown into replacements for almost any part of the human body. Stem cells are typically found in the embryo and umbilical cord of an organism, and in reservoirs within the human body. Researchers hope that stem cells will provide a solution to cure diseases caused by cell failure, and for repairing tissues that do not repair themselves. Heart damage, spinal cord injuries, Parkinson's disease, leukemia, and diabetes are among diseases named in relation to stem cell research. Hence, researchers are of the opinion, if these stem cells are controlled, they could cure a variety of debilitating diseases in the years to come. Stem cells are separated into 3 distinct categories viz. Totipotent, Pluripotent, and Multipotent. Stem cells are best described in relation to normal human development. Thus, a fertilized egg is totipotent. It produces an entire organism. After several cycles of cell division, these totipotent cells begin to specialize, becoming pluripotent. As the embryo begins to develop, these pluripotent cells become multipotent, specifically producing blood, skin, nerve, or other types of body cells. Multipotent stem cells are envisioned to potentially treat a variety of muscular-skeletal and neural disorders. While stem cells are extraordinarily important in early human development, multipotent stem cells are also found in children and adults. For example, one of the best understood stem cells are the blood stem cells. Blood stem cells reside in the bone marrow of every child and adult, and in fact, they can be found in very small numbers circulating in the blood stream. Blood stem cells perform the critical role of continually replenishing supply of blood cells—red blood cells, white blood cells, and platelets throughout the life span.
  • [0003]
    Stem cells are the building blocks of blood and immune systems. They form the white cells that fight infection, the red cells that carry oxygen and platelets that promote clotting. Stem cells are normally found in bone marrow where they continue to generate new blood cells throughout the live span of an individual. The presence of these stem cells in the bone marrow has made marrow transplantation an important therapeutic modality in the treatment of variety of malignant and non-malignant diseases. This is because of the realization that permanent clinical benefit from transfused blood cells can come from transplantation of multipotent haematopoietic stem cells. Besides bone marrow, Mobilized Peripheral Blood (MPB), and Umbilical Cord Blood (UCB) have also been used successfully for transplantation. In recent years although significant advances have been made in bone marrow transplantation (BMT), the basic problem of finding a suitable matching donor still remains. This is because a group of antigens expressed by the leukocytes called the human leukocyte antigens (HLA) need to match between the donor and the recipient. Further bone marrow harvesting is a painful and invasive procedure and many donors are unwilling to donate marrow. Therefore the search for alternate sources of stem cells has led to the development of stem cell transplant protocols from different tissues like liver (Kochupillai 1991), mobilized peripheral blood (Benboubker 1995), and cord blood (Mayani 1998). Of these, cord blood has significant advantages over the others. Increasingly, experts say cord blood transplants have distinct advantages over more traditional bone marrow transplants in stimulating the growth of healthy white blood cells. Stem cells can be collected from the bone marrow. However, the collection procedure is invasive, time-consuming, requires an anaesthetic and is painful for the donor. Also, cord blood is easily available, involves a non-invasive collection procedure and is better tolerated in transplants across the HLA barrier.
  • [0004]
    Like bone marrow, umbilical cord blood is rich in stem cells. Umbilical cord blood is the blood that remains in the placenta and umbilical cord following birth. Until recently the placenta and umbilical cord were discarded after delivery as medical waste, but now research has shown that cord blood is a rich source of blood (haematopoetic) stem cells, which can be collected, processed and frozen for potential future use. An experimental procedure to use umbilical cord blood instead of bone marrow to treat immune diseases is gaining attention from doctors and patients.
  • [0005]
    Research in human developmental biology has led to the discovery of human stem cells (precursor cells that can give rise to multiple tissue types), including embryonic stem (ES) cells, embryonic germ (EG) cells, fetal stem cells, and adult stem cells. Recently, techniques have been developed for the in vitro culture of stem cells, providing unprecedented opportunities for studying and understanding human embryology. As a result, scientists can now carry out experiments aimed at determining the mechanisms underlying the conversion of a single, undifferentiated cell, the fertilized egg, into the different cells comprising the organs and tissues of the human body. Although it is impossible to predict the outcomes, scientists and the public will gain immense new knowledge in the biology of human development that will likely hold remarkable potential for therapies and cures.
  • [0006]
    Using cell replacement therapy, to cure diseases may prove to be one of the most significant advances in medicine. Unlike all current treatments that rely on surgical interventions or drugs that modulate cell activities, stem cells provide a replacement for dysfunctional or degenerating tissue.
  • [0007]
    Cells are regarded as stem cells if they retain the capacity to renew themselves as well as more specialized progeny stem cells can be obtained from early embryo, fetal tissues, adult blood, and umbilical cord blood. Identification of the full term umbilical cord blood (which is discarded at birth), as a source has made haematopoietic stem cells more accessible for study and clinical use. Cord blood stem cells are multipotent. These stem cells in addition to production of blood cells, have the ability to differentiate into cells of other tissue or organs. This ability has made cord blood stem cells more accessible for study and clinical use.
  • [0008]
    Cord blood stem cells express CD34 antigen. CD 34 antigen has been commonly used as a marker for the enrichment and isolation of candidate stem cells. Cord blood stem cells can be isolated on the basis of the presence of this marker. CD34 positive cells can be isolated from mononuclear cells of cord blood. Cord blood mononuclear cells constitute about 1-2% of CD34 positive cells. On exposure to a novel environment, cord blood stem cells are known to transdifferentiate to various cells like neural cells, liver cells, bone, cartilage etc.
  • [0009]
    Transdifferentiation is the ability of the adult stem cells from one tissue or organ, which can overcome their intrinsic restrictions upon exposure to novel environment perhaps via genomic reprogramming to cells of other organs either in vitro, or after transplantation in vivo.
  • [0010]
    Neural stem cell research is still in its early stages, is intriguing because scientists believe that the primitive cells can transform into virtually any cell type in the body and could be a source of tissue or organs to cure diseases such as repair of nerve damage, strokes, spinal injury, Parkinson's and Alzheimer's. For years, researchers studying stem cells have been intrigued by the possibility that these cells might be use to treat brain diseases. Recent studies have suggested neural stem cells transplanted into the brain can migrate throughout the brain and develop into other types of cells.
  • [0011]
    Up to the present, Stem Cells (Embryonic/Adult) are being cultured in animal serum such as Fetal Bovine Serum (FBS), or complex mixture of growth factors derived by mixing purified factors which are either isolated from FBS or Human Adult blood serum or a mixture of growth factors derived from recombinant methods.
  • [0012]
    However, these conventional culture media are associated with shortcomings and risks. Stem cells from adult/fetal as well as other sources are being widely used to regenerate tissues in patients after they have degenerated. For this purpose, these cells have to be grown in the tissue culture for varying periods of time using defined media, the principle constituent of which is animal serum such as Fetal Bovine Serum (FBS).
  • [0013]
    FBS is the most widely used serum in the culturing of cells, tissues and organs in vitro, in industry, medicine, and science. FBS has been shown to be essential for adhesion, proliferation and differentiation of the cells. However, animal serum such as FBS can be infected with several pathogens such as prions. Several known and unknown viruses may be present in the serum. Therefore cells/tissue cultured in the presence of FBS get infected and transmit these pathogens to the patient on transplantation. As stated FBS may have known and unknown pathogens, which may be transmitted to the human transplant subject if these cells are grown in FBS. The pathogens present in FBS are difficult to screen for likely causative agents of diseases in humans. Hence, using such cells in a human can be life threatening as there is every chance of a pathogen getting transmitted along with these cells. Human cells grown in FBS constitute a xenograft, if used for cells based therapies in humans.
  • [0014]
    Human adult blood serum also supports growth of several cells, however, it cannot substitute for FBS, since it does not provide growth factors, present in FBS. Hence, it is not used for culturing of stem cells in vitro.
  • [0015]
    Several investigators have tried to use a combination of complex mixture of growth factors, which are known to influence growth and differentiation of stem cells. However, the success is limited and it has been shown conclusively that 2% v/v of the tissue culture media should be made up of FBS for optimal growth of the cells.
  • [0016]
    There is a dire need to find an adequate substitute for conventional culture media for growing neural precursor cells. Looking to the need of the hour, the present inventors have resolved the above issue of concern and have come out with a solution, which will be of utmost importance in the field of regenerative medicine. The inventors of the present invention, have come out with a unique media for culturing cord blood stem cells which comprises of umbilical cord blood serum as a substitute for FBS and such cells may be used in transplantation and repair of nerve damage, strokes, spinal injury, Parkinson's and Alzheimer's. Cord blood being a natural substance, is found to be rich in growth factors. Taking this factor in mind, the inventors of the present invention have investigated a method of growing and differentiating cord blood stem cells into neural precursors. The present invention is advantageous over the prior art as it obviates the problems associated with the conventional culture media for growing stem cells for human use.
  • [0017]
    Use of umbilical cord blood stem cells in haematopoietic reconstitution has been around since 1970. However, no work has been done in using umbilical cord blood as a source for growing and differentiating stem cells in neural precursors. The inventors of the present invention have been successful in discovering this novel process for growing cord blood stem cells and differentiating into neural precursors.
  • OBJECTS OF THE INVENTION
  • [0018]
    It is an object of the present invention to grow human embryonic stem cells and adult stem cells such as cord blood stem cells for therapeutic purposes in regenerative medicine.
  • [0019]
    It is further object of the present invention to develop a method for growing and differentiating stem cells into neural precursors using a novel media consisting of cord blood serum and such cells may be used in transplantation and repair of nerve damage, stroke, spinal injury, Parkinson's and Alzheimer's.
  • [0020]
    It is still further object of the present invention to transdifferentiate CD34+stem cells from Mononuclear cells derived from Umbilical Cord blood to neural precursors.
  • SUMMARY OF THE INVENTION
  • [0021]
    In accordance with one aspect of this invention, there is provided the use umbilical cord blood sera for growing human embryonic stem cells and adult cells, like cord blood stem cells for therapeutic purposes in regenerative medicine.
  • [0022]
    To overcome the major obstacle described above, the inventors of the present invention have conducted research on human umbilical cord blood and have replaced conventionally used Fetal Bovine Serum (FBS) by cord blood serum, for culturing cord blood stem cells for neural transdifferentiation.
  • [0023]
    As stated, Human umbilical cord blood is a fetal product and a waste product during childbirth. During the gestation of the child in the mother's womb, the placenta and the blood present in the placenta nourish the developing fetus and are therefore rich in several growth promoting factors. The inventors of the present invention have taken advantage of this property of Human umbilical cord blood and have substituted it for FBS. The procedure by which Human umbilical cord blood is collected is given below. It is this cord blood that is used as a serum for growing cord blood stem cells of the present invention.
  • [0024]
    The method of the present invention may include the step of separating the pluripotent stem and progenitor cell population of mononuclear cells obtained from umbilical cord blood using a magnetic cell separator to separate out cells which contain a CD marker and then expanding these cells in a growth medium containing a differentiation agent such as retinoic acid and growth factors such as BDNF, GDNF, NGF, FGF or mixtures thereof. Preferably the a mixture of retinoic acid and at least one growth factor for example nerve growth factor is used as the differentiation agent. The retinoic acid may be 9-cis retinoic acid, all-trans retinoic acid or mixtures thereof. The separation and incubation steps maybe interchanged.
  • [0025]
    The umbilical cord blood sample from which the pluripotent stem/progenitor cells are obtained may be fresh umbilical cord blood, reconstituted cryopreserved umbilical cord blood or a fresh or reconstituted cryopreserved mononuclear cell fraction thereof.
  • BRIEF DESCRIPTION OF THE FIGURES
  • [0026]
    [0026]FIG. 1: Morphology of adherent cells grown in the presence of FBS and CBS. In the first passage (P1) higher numbers of adherent cells are observed in the CBS culture. The FBS culture shows a higher number of rounded cells, but lower number of adherent cells. A similar difference is observed in cultures in the second passage (P2). The adherent cells in CBS also appear to be larger in size.
  • [0027]
    [0027]FIG. 2: Flow cytomteric analysis of cord blood cells cultured in the presence of FBS and CBS. In P1 and P2 cultures >90% of the cells exhibit a CD133−/CD45+ phenotype. The regions in these dot plots are drawn on the basis of isotype controls.
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0028]
    The following terms are used throughout the specification to describe the present invention.
  • [0029]
    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 which is obtained from the umbilical cord or placenta of newborns. 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 auologous or allogenic transplantation when and if needed. Cord blood is preferably obtained by direct drainage from the umbilical vein.
  • [0030]
    The term “cell medium” or “cell media” is used to describe a cellular growth medium in which mononuclear cells and/or neural cells are grown. Cellular media are well known in the art and comprise at least of minimum essential medium plus optional agents such as growth factors, glucose, non-essential amino acids, insulin, transferring and other agents well known in the art. In certain preferred embodiments at least one differentiation agent is added to the cell media in which a mononuclear fraction is grown in order to promote differentiation of certain cells within the mononuclear fraction into neural cells.
  • [0031]
    The term “non adherent cells” is used to describe cells remaining in suspension in the tissue culture flask at the end of the culture period. The term “adherent cells” is used to describe cells that are attached to the tissue culture plastic, but are detached from the flask by addition of enzyme free cell dissociation buffer from Gibco-BRL or by addition of trypsin-EDTA.
  • [0032]
    In a preferred aspect of the present invention, mononuclear cells grown in standard cellular media (preferably, at least a minimum essential medium supplemented with non-essential amino acids, glutamine and serum) are grown in a “neural proliferation medium” (i.e. a medium which efficiently grows neural cells) followed by growth in a “differentiation medium”, generally which is similar to neural proliferation medium with the exception that specific neural differentiation agents are added to the medium and in other cases, certain growth factors are limited or removed. A particularly preferred neural proliferation medium is a medium which contains DMEM/F12 1:1 cell medium supplemented with glutamine 2 mM, sodium biocarbonate 3 mM, EGF 20 ng/ml, bFGF 10 ng/ml and NGF 100 ng/ml. One of ordinary skill will readily recognize that any number of cellular media maybe used to grow mononuclear cell fractions of umbilical cord blood or to provide appropriate neural proliferation media and/or differentiation media.
  • [0033]
    Umbilical cord blood serum is prepared in the following manner. Umbilical cord blood is collected at the time to birth from pre-screened mothers for infectious disease causing organisms, such as HIV 1 and 2, Hbs and HCV and sexually transmitted diseases. The collection is made after the baby is separated from the clamped cord, and therefore there is no harm to the baby. Blood is collected from an umbilical vein using the conventional blood bag containing no anticoagulants. The needle of the bag is inserted into the vein and blood is allowed to flow into the blood bag. A good collection can exceed 100 ml. This blood is now allowed to clot at room temperature and transported to the processing area, which is a cGMP clean room. The clotting process is allowed to take place from 8-16 hours. The blood is then centrifuged at 1000 g in a blood bag centrifuge and the clear serum is collected into sterile containers. The serum is now tested for sterility by microbiological assays for aerobic or anaerobic microorganisms. The complement is inactivated by keeping sera at 56 C. for hour. Serum is aliquoted into 10 ml sterile vials and capped. Lot number and Batch number are fixed on it.
  • [0034]
    Selecting for umbilical cord blood pluripotential stem/progenitor cells according to the present invention can be done in a number of ways. For example, the cells maybe selected using, for example a magnetic cell separator (MACS) or other system which removes all the cells which contain a CD marker and then the remaining cells maybe expanded in growth medium or differentiated in growth medium which includes a differentiation agent.
  • [0035]
    Additional in vitro differentiation techniques can be adapted through the use of various cell growth factors and co-culturing techniques known in the art. Besides co-culturing with adult mesenchymal stem cells, a variety of other cells can be used, including but not limited to accessory cells and cells from other portions of the fetal and mature central nervous system.
  • [0036]
    The following written description provides exemplary methodology and guidance for carrying out many of the varying aspects of the present invention.
  • [0037]
    Enrichment of mononuclear cells is well known to the practitioners of the art. Briefly, red blood cell depletion is carried out using 3% v/v Dextran (high molecular weight) in the ratio of 1:1 with respect to the volume of blood. Leucocyte rich plasma is collected carefully and centrifuged. Cells are washed and layered on Histopaque™ 1077. The tubes containing the sample are centrifuged at 400 g for 30 minutes. Mononuclear cells are separated from the interface using pipettes. These cells are washed and counted. The mononuclear cells are then suspended neural proliferation medium.
  • [0038]
    The cells are also stained with fluorochrome conjugated antibodies for flow cytometry. Specified numbers of cells are taken in polystyrene round bottom tubes. These cells are then stained with anti-CD34-FITC, anti-CD133-PE and anti-CD45-PerCP antibodies. The stained cells are then acquired and analyzed on a FACSCalibur flow cytometer.
  • EXAMPLES
  • [0039]
    Effect of cord blood serum on the proliferation of non-adherent cells: Mononuclear cells from cord blood are plated in Nunc T75 culture flasks in neural cell proliferation medium containing DMEM/F12 (1:1) supplemented with FBS or CBS. The cells are seeded at a density of 1106 to cells/ml. After a fixed culture period of 1 week, the non-adherent cells are harvested, counted and analyzed for the expression of CD133, CD45 and CD34 markers. Table 1 shows the proliferation kinetics of these cells in this medium. No significant difference (p>0.3) as measured by the paired T test in the numbers of non adherent cells, is observed in cells cultured in the presence of CBS or FBS. This shows that cord blood serum supports growth of non-adherent cells in these cultures with equal efficacy as compared to fetal bovine serum.
    TABLE 1
    Cell count of non adherent cells from cord blood
    grown in FBS and CBS
    Sample Cell number (106) at P1
    no CBS FBS
    1 2.5 2.6
    2 2.0 4.0
    3 3.6 4.3
    4 16.0 11.8
    5 0.5 0.5
    6 1.5 1.7
    7 8.0 1.1
    Mean 4.9 3.7
  • [0040]
    Effect of cord blood serum on the proliferation of adherent cells: Mononuclear cells from umbilical cord blood were plated in neural cell proliferation medium as described above. The cultures were fed every 3-4 days and were allowed to proceed to 90% confluency, which was determined by visual examination of the flask under an inverted microscope. The adherent cells were initially detached using cell dissociation buffer and seeded into the next passage. Table-2 shows the numbers of cells detached with cell dissociation buffer in both these cultures. Cultures containing FBS showed a 1.7 fold higher number of adherent cells (p<0.05) as compared to cultures containing CBS. However since an appreciable number of cells were still adherent to the CBS tissue culture flask, it was decided to use trypsin-EDTA instead of cell dissociation buffer for detachment of cells. In 1 experiment where cells were detached using trypsin-EDTA, cultures containing CBS showed a 2 fold higher number of adherent cells compared to the culture containing FBS. This is also clear from FIG. 1. At each passage an aliquot of the cells was phenotyped by flow cytometry. FIG. 1 shows the morphology of these cells in culture. Although equal numbers of cells were seeded in these cultures the cells in the flask containing CBS appear to be growing at a higher cell density as compared to the flask containing FBS.
    TABLE 2
    Cell count and phenotype of adhered cord blood cells grown
    in FBS and CBS
    % CD133−/CD34−/CD45+
    Cell number (106) (P1)
    Sample No. CBS FBS CBS FBS
    1 0.3 0.5
    2 0.2 0.6 94.9 87.4
    3 0.3 0.7 93.3 92.5
    4 2.2 2.5 93.9 90.7
    5 0.4 1.5 93.8 91.9
    Mean 0.7 1.2 91.8 89.2
  • [0041]
    The morphology of the cells grown in these two cultures is also very different. Cells grown in the presence of CBS have a flattened morphology and are larger in size as compared to cells grown in the presence of FBS. These cells grown in the presence of CBS also attach firmly to the tissue culture flask and are difficult to detach using cell dissociation buffer. FIG. 2 shows the flow cytometric dot plot of the cells cultured in the presence of FBS and CBS at passage 2. It is clear that >90% of the cells in both cultures at the first passage (P1) are CD45+ indicating a hematopoietic cell phenotype. The numbers of hematopoietic cells are in both these cultures are not significantly different (p>0.2 by the paired T test). Therefore it is clear that CBS is as effective as FBS in supporting growth of adherent cells.
  • [0042]
    At the third passage (P3), these cells gradually loose the CD45 antigen (FIG. 2), indicating that they are converting to a non-hematopoietic phenotype. 60% of the cells cultured with FBS were CD45−, whereas 80% of the cells cultured with CBS exhibited a similar phenotype.
  • [0043]
    While there has been shown and described what is considered to be the preferred embodiments of the invention, it will be readily obvious to those skilled in this art that various changes and modifications may be made without departing from the scope of the invention.
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Classifications
U.S. Classification435/368, 435/372
International ClassificationC12N5/0789
Cooperative ClassificationC12N2501/11, C12N2501/115, C12N2501/13, C12N5/0647
European ClassificationC12N5/06B11P
Legal Events
DateCodeEventDescription
Aug 8, 2003ASAssignment
Owner name: RELIANCE LIFE SCIENCES PYT. LTD., INDIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:RAI, PRATHIBHA;REEL/FRAME:015059/0824
Effective date: 20030802