WO2007123363A1 - Culture media and methods for culturing mesenchymal stem cell - Google Patents
Culture media and methods for culturing mesenchymal stem cell Download PDFInfo
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- WO2007123363A1 WO2007123363A1 PCT/KR2007/002002 KR2007002002W WO2007123363A1 WO 2007123363 A1 WO2007123363 A1 WO 2007123363A1 KR 2007002002 W KR2007002002 W KR 2007002002W WO 2007123363 A1 WO2007123363 A1 WO 2007123363A1
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- mesenchymal stem
- stem cells
- medium
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- cells
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
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Abstract
The present invention relates to a culture medium for mesenchymal stem cells comprising a basal medium, and umbilical cord blood serum. The present invention further relates to a method for culturing mesenchymal stem cells using the same, and a method for obtaining mesenchymal stem cells using the same. Using the method, the mesenchymal stem cells can be massively obtained.
Description
Description
CULTURE MEDIA AND METHODS FOR CULTURING MESENCHYMAL STEM CELL
Technical Field
[1] The present invention relates to a culture medium for mesenchymal stem cells comprising a basal medium, and umbilical cord blood serum (which may be referred to human cord blood serum, hereinafter), a method for culturing mesenchymal stem cells using a culture medium, and a method for obtaining mesenchymal stem cells using the culturing method. Background Art
[2] Stem cells have properties such that the same cells are constantly produced in an undifferentiated state for a predetermined period of time, and can be differentiated into certain cells under suitable conditions. The stem cells can be classified into embryonic stem cells and adult stem cells according to their origins. Human embryonic stem cells are produced from an embryo which can be developed into a human, resulting in the dilemma of ethical issues. However, it is known that they have excellent cell proliferation property and differentiation abilities, as compared with the adult stem cells. The adult stem cells can be obtained from bone marrow, blood, brain, skin, or the like, without incurring ethical issues, but have limited differentiation abilities (multipotency), as compared with the embryonic stem cells. Of the adult stem cells, hematopoietic stem cells have been most developed, and recently the mesenchymal stem cells are actively being researched.
[3] Meanwhile in order to supply a medium for culturing animal cells in vitro rather than in vivo, it is necessary to sufficiently satisfy the conditions of the nutrients, temperatures, osmotic pressures, which are similar to the conditions in the body, based on blood plasma or lymph fluid. Accordingly, the composition of the culture medium is conventionally determined based on the body fluid, and the determined culture medium is selected, an applicable amount of serum is added to the medium, and the medium is used for cell culture. Thus, there have been disclosed many medium compositions for culturing human stem cells, for example, a method for culturing human stem cells using a medium containing animal serum (fetal bovine serum). However, this method has a problem in that even after washing the cells cultured with fetal bovine serum using serum free solution a significant amount of the fetal bovine serum remains within the cells to serve as a potent heterologous antigen, and moreover, the human body may be infected with a disease from cow, such as mad cow disease. That is, in order to use the stem cells clinically, the substances used in the culturing process
should be derived from human in respects to stability, or be approved on its stability.
[4] In regards to this, a medium comprising human serum instead of an animal- originated serum which has been conventionally used for cell culture is disclosed in Korean Patent Publication No. 2005-0099724. However, the cells cultured in the medium disclosed in this publication have a low expression rate of CD 105, which is an indicative marker of mesenchymal stem cells. Further, United States Patent Publication No. 2003/0232432 discloses a medium of a human umbilical cord blood serum, but it describes merely a method for preparing the umbilical cord blood serum.
[5] As such, the present inventors have extensively investigated with respect to a culture medium which increases the efficiency of culturing and proliferating mesenchymal stem cells in vitro, and has no problem in terms of the purpose of the study, and clinical application. As a result, we have found that a culture medium comprising umbilical cord blood serum is effective and specific in the culture of mesenchymal stem cells, thereby leading to the completion of the present invention. Disclosure of Invention Technical Solution
[6] It is an object of the present invention to provide a culture medium for mesenchymal stem cells, comprising a basal medium, and a medium containing umbilical cord blood serum.
[7] It is another object of the present invention to provide a method for culturing mesenchymal stem cells using the medium.
[8] It is still another object of the present invention to provide a method for obtaining the mesenchymal stem cells cultured using the medium. Brief Description of the Drawings
[9] Fig. 1 is a graph showing the growth of FACS (fibroblast- like adherent cells) after plating the mononuclear cells (MNCs) separated from the bone marrow in a T25 flask containing CG (CellGro™, CellGenix) and MSCGM™ (Cambrex) at a concentration of 5.28 x 10 cells, and culturing them for 15 days.
[10] Fig. 2 is a graph showing the growth rate of MSC in a medium containing
MSCBM™ (Mesenchymal stem cell basal medium, Cambrex) added with 10% CS, a medium containing 20% CS (umbilical cord blood serum; cord serum), a medium containing CellGro™ (CellGenix) added with 10% CS, and a medium containing CellGro™ (CellGenix) added with 20% CS, with a control group cells grown in MSCGM™ (Cambrex).
[11] Fig. 3 is a graph showing the change in the size and phenotype of the cells(SSC-H; granular content within cell, FSC-H; cell size), as determined by flow cytometry (FACSCalibur, BD) of MSC, which had been proliferated in MSCBM™ medium with
10% cord serum, and a CellGro™ medium with 10% CS (umbilical cord blood serum; Cord serum), compared with the control group cells grown in MSCGM™.
[12]
[13] Fig. 4 is a graph showing a histogram of surface markers of MSC which had been proliferated in an MSCGM™ medium.
[14] Fig. 5 is a graph showing a histogram of surface markers of MSC which had been proliferated in an MSCBM™ medium with 10% CS (cord serum).
[15] Fig. 6 is a graph showing a histogram of of surface markers of MSC which had been proliferated in a CellGro™ medium with 10% CS (cord serum).
[16] Fig. 7 is a graph showing the growth and proliferation rate of MSC which had been proliferated in a medium obtained by incorporating CS (umbilical cord blood serum), and growth factors of insulin, hydrocortisone, EGF, and LIF into a DMEM/F12 medium, compared with the control group cells grown in MSCGM™.
[17] Fig. 8 is a graph showing the change in the size and phenotype of the mesenchymal stem cells (MSCs) which had been proliferated in a medium obtained by incorporating 5% or 10% CS (umbilical cord blood serum), and insulin, hydrocortisone, EGF, and LIF into a DMEM/F12 medium, compared with the control group cells grown in MSCGM™. (SSC-H; granular content within cell, FSC-H; cell size)
[18] Fig. 9 is a graph showing a histogram of of surface markers, of MSC which had been proliferated in a medium obtained by incorporating 5% umbilical cord blood serum, and growth factors of insulin, hydrocortisone, EGF, and LIF into a DMEM/F12 medium.
[19] Fig. 10 is a graph showing a histogram of of surface markers of MSC which had been proliferated in a medium obtained by incorporating 10% umbilical cord blood serum, and growth factors of insulin, hydrocortisone, EGF, and LIF into a DMEM/F12 medium.
[20]
Best Mode for Carrying Out the Invention
[21] In one embodiment, the present invention relates to a culture medium for mesenchymal stem cells comprising a basal medium, and a medium containing umbilical cord blood serum.
[22] As used herein, the term "medium" refers to a mixture comprising the elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, sera, growth factors, and minerals, which is useful for the growth and proliferation of cells such as stem cells in vitro. In particular, the medium of the present invention is a medium for culturing the mesenchymal stem cells. The "mesenchymal stem cells" as described above are cells separated from the embryonic stem cells or
adult stem cells which had been derived from mammals including human. The cells have an ability to proliferate infinitely, and an ability of differentiating into various cell types (for example, fat cells, cartilage cells, muscle cells, and bone cells). The "culture" means the growth and proliferation of the mesenchymal stem cells.
[23] The "basal medium" of the present invention is a mixture comprising sugars, amino acids, water, and the like, which are essential for cell survival, and excluding sera, nutrients, and various mesenchymal stem cell growth factors. The basal medium of the present invention can be artificially synthesized and prepared, or commercially prepared. The commercially prepared medium is, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-IO, F- 12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium, but are not limited thereto.
[24] The serum is a supernatant obtained by centrifuging the animal or human blood.
The serum comprises, in addition to nutrients essential for cell growth, trace amounts of a variety of factors including various inorganic salts which are essential, but are not still definitely identified, polypeptidic growth factors, and polypeptidic hormones.
[25] The "umbilical cord blood serum" of the present invention refers to a serum separated from a fetal umbilical cord. The umbilical cord blood serum of the present invention can be preferably obtained from the umbilical cord which had been discarded upon a fetal birth or loss. The umbilical cord blood can be collected by inserting a needle that is connected with a blood bag into the umbilical cord vein. Such blood collection is performed at room temperature, and the blood that can be obtained is at most 100 cc per umbilical cord. The entire process can be performed in a clean room free from contamination. From the collected umbilical cord blood, only blood plasma components can be collected by blood coagulation. The blood coagulation occurs by means of a blood bag without anti-coagulating agent, or a coagulating agent can be further added to promote coagulation, the former being preferable, which induces natural coagulation. The time required for coagulation may be 5 mins, 30 mins 1 hour, 5 hours, 8 hours, or longer, and the coagulated blood can be centrifuged to separate the hemocytes and the blood plasma components from each other. The vessels used throughout the process should be sterilized, and tested for its biological stability against microorganism contamination. Further, the blood is reacted at 560C for 30 mins as for the serum components such as fetal bovine serum to inactivate the blood plasma components .When the mesenchymal stem cells are cultured in a medium comprising the umbilical cord blood serum of the present invention, and as a result, it was found that the fetal bovine serum is better than the human serum, and the umbilical cord blood serum is better than the fetal bovine serum, as shown from the
relative ratios, and it was further found that better culture effect is attained, as compared with conventionally known culture media for mesenchymal stem cells (see Table 1, Figs. 1, and 2).
[26] The umbilical cord blood serum of the present invention can originate from any animal having an umbilical cord, such as a human, a cow, a goat, a pig, and a horse, preferably from a human, and a cow, and more preferably a human.
[27] The concentration of the umbilical cord blood serum used in the medium of the present invention is 1 to 30%, preferably 3 to 25%, more preferably 3 to 20 %, most preferably 5 to 10%, based on the total medium composition.
[28] In one specific embodiment, the culture medium of the present invention can further comprise a nutrient mixture. The nutrient mixture is a mixture comprising various amino acids, vitamins, inorganic salts, and the like, which are generally used for cell culture, and it can be prepared by mixing the amino acids, vitamins, inorganic salts, and the like, or can be commercially prepared. As the commercially prepared nutrient mixture, for example, F- 12, M199, MCDBI lO, MCDB202, and MCDB302, and preferably an F- 12 nutrient mixture, M199, and MCDB media can be used. The nutrient mixture can be used after dilution with a ratio of 1 to 10: 1, preferably with a ratio of 1 to 5:1.
[29] In specific embodiments, the culture medium of the present invention can further comprise mesenchymal stem cells growth factors which can affect the growth of mesenchymal stem cells. Examples of the growth factors for the mesenchymal stem cells include insulin, hydrocortisone, an epidermal growth factor (EGF), a leukemia inhibitory factor (LIF), a granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), a stem cell factor (SCF), and a transforming growth factor (TGF). In specific practices of the present invention, insulin, hydrocortisone, EGF, LIF, and the like are used. Among the mesenchymal stem cells growth factors, for example, the amount of insulin can be at a concentration of 0.1 to 500 ng/ml, preferably 0.5 to 100 ng/ml, more preferably 0.5 to 50 ng/ml, and most preferably 1 to 30 ng/ml in the basal medium of the present invention; the amount of the hydrocortisone can be at a concentration of 0.1 to 500 nM, preferably 0.5 to 100 nM, more preferably 0.5 to 50 nM, and most preferably 1 to 30 nM in the basal medium of the present invention; the amount of the EGF can be at a concentration of 0.1 to 500 ng/ml, preferably 0.5 to 100 ng/ml, more preferably 0.5 to 50 ng/ml, and most preferably 1 to 30 ng/ml in the basal medium of the present invention; and the amount of the LIF can be at a concentration of 0.01 to 500 ng/ml, preferably 0.05 to 100 ng/ml, more preferably 0.1 to 50 ng/ml, and most preferably 0.5 to 30 ng/ml in the basal medium of the present invention. The present inventors observed that the stem
cell grew in a medium obtained by incorporating insulin, hydrocortisone, EGF, and LIF into various basal media and umbilical cord blood sera at concentrations of 10 ng/ ml, 10 nM, 10 ng/ml, and 10 ng/ml, respectively. As a result, they found that the growth rates in the above media were relatively higher that those in the non-added medium (Figs. 2, and 7).
[30] In another embodiment, the present invention relates to a method for culturing mesenchymal stem cells using the culture medium for mesenchymal stem cells. Specifically, the present invention relates to a method for culturing mesenchymal stem cells, comprising: a step of separating the mesenchymal stem cells, and a step of culturing the separated mesenchymal stem cells in the medium of the present invention.
[31] In the culturing method of the present invention, the mesenchymal stem cells are pluripotent cells derived from the embryonic stem cells or adult stem cells of a human or an animal, preferably from human embryonic stem cells, or adult stem cells.
[32] The process for separating the mesenchymal stem cells of the present invention can be performed as known in the art. For example, the cells obtained from a donated tissue are diluted in a basal medium for mesenchymal stem cells, and centrifuged to collect the cells only. The cells are cultured at a concentration of 1 to 500,000 monocytes/cm , preferably 1,000 to 50,000 monocytes/cm for 1 to 30 days, preferably 5 to 15 days in a flask for cell culture, and for 1 to 10 days, preferably 2 to 4 days in a replaced medium, thereby obtaining the adherent cells, and the proliferated mesenchymal stem cells. The above-described donated tissue can include bone marrow, blood, brain, skin, umbilical cord blood, embryos and various tissues, preferably umbilical cord blood, bone marrow, or blood having excellent cell proliferation and differentiation abilities. More preferably, the mesenchymal stem cells can be easily obtained from bone marrow in plenty, which minimizes the immune response because the mesenchymal stem cells are separated from their own tissue. The mesenchymal stem cells are easily differentiated to various tissues and cells, and can be used as a cell therapy products as the cells themselves.
[33] The separated mesenchymal stem cells are cultured in the culture medium of the present invention. The separated and cultured mesenchymal stem cells express the positive surface markers such as CD105 (SH2), CD29, CD44, CD73, and CD166 at 90% or more, preferably 95% or more, particularly express the positive surface markers such as CD44 and CD 105 (SH2) at 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more. Further, the separated and cultured mesenchymal stem cells express the negative surface markers such as CD34, CD45, HLA-DR, CDIa, CD14, CD31, and CD80 at 50% or less, preferably 10% or less, and particularly express the negative surface marker CD34 at
50% or less, preferably 10% or less, more preferably 5% or less.
[34] In another embodiment, the present invention relates to a method for obtaining mesenchymal stem cells, comprising: a step of culturing the mesenchymal stem cells in the medium of the present invention, and a step of separating the culture medium from the centrifuge to obtain the mesenchymal stem cells.
[35] The method for obtaining mesenchymal stem cells comprises a step of culturing the mesenchymal stem cells in the culture medium of the present invention, and a step of separating the mesenchymal stem cells from the culture medium. The separation of the mesenchymal stem cells can be performed in a process known in the art. By way of an example, the mesenchymal stem cells are cultured at a concentration of 1 to 10,000 cells/cm , preferably 50 to 6000 cells/cm for 3 to 40 days, preferably 6 to 20 days in a flask for cell culture comprising the culture medium of the present invention, and for 1 to 10 days, preferably 2 to 4 days in a replaced medium, thereby obtaining the adherent cells, and the proliferated mesenchymal stem cells. The adherent cells obtained by the above-described method can be further subcultured, or dispersed in a culture medium and then stored using an ultra- freezing method. The ultra- freezing method can be further modified using a common method which is apparent from a skilled person in the art, and for example, the cells can be dispersed and stored in a frozen storage solution, obtained by incorporating 10 to 20% serum and 5 to 10 % DMSO into a basal medium for stem cells, and more preferably dispersed in a frozen storage solution, obtained by incorporating 10% fetal bovine serum (FBS) and 5% DMSO into an MSCGM™ basal medium, and stored in -1760C liquid nitrogen. The obtained mesenchymal stem cells can be, if desired, differentiated into various tissues and nerve cells, and can be used as regenerating and treating agents for bone, cartilage, fat, liver, nerve cells, and various organs.
[36] The method for cell culture of the present invention can be usefully employed for mass culture of the mesenchymal stem cells, and storage of the mesenchymal stem cells to be used as a cell therapy reagent. If the method is used for a cell therapy reagent for treating diseases in a human stability of the culture medium should be confirmed.
[37] Hereinbelow, the present invention will be described in detail with reference to
Examples. These Examples are provided only for the purpose of illustrating the present invention, but it is not intended that Examples limit the scope of the present invention according to the subject matters of the present invention. Mode for the Invention
[38] Example 1: Isolation and culture of MSC
[39] 11 ml of bone marrow donated from a normal healthy man or a patient himself was
diluted with a DMEM medium with a ratio of 1:2, and dropped on 10 ml of a histopaque layer (Sigma, density 1.077 g/ml), previously prepared, and centrifuged at 400xg for 30 mins at room temperature. The mononuclear cells were separated, a DMEM medium was added thereto, and centrifuged at 400xg for 5 mins at room temperature. The obtained mononuclear cells were washed with the DMEM medium twice, and then cultured in an MSCGM™ medium while maintaining the conditions at 370C and 5% CO . After culturing for 48 hours, the cells which were not adhered on the bottom of the flask were removed by replacing the medium with a fresh medium, and the cells which were adhered on the bottom of the flask were cultured with replacing the medium every 3rd to 4th day. When the cultured cells were grown to about 80%, a part of the cells were subcultured to a fresh flask, and continuously subcultured, and the other part was dispersed in a frozen storage solution comprising MSCGM™, 10% fetal bovine serum (FBS), and 5% DMSO, and stored in liquid nitrogen at -1760C.
[40] Example 2: Assay of proliferation of MSCs in CG (Cell Genix) medium
[41] For an experiment for the assay of the proliferation ability of the MSCs of CG (Cell
Genix), using the media CG ®, ©, and © which had been directly supplied from Cell Genix, and an MSCGM™ medium, which had been directly supplied from Cambrex, and a DMEM (Gibco) medium, comprising 10% fetal bovine serum (FBS, Hyclone), the mononuclear cells which had been separated and washed in Example 1 were inoculated in the T25 flasks each having five kinds of media at 5.28 x 10 cells/flask, and cultured at 370C and 5% CO . After culturing for 48 hours, the cells which were not adhered on the bottom of the flask were removed by replacing the medium with a fresh medium, and the cells which were adhered on the bottom of the flask were further cultured by replacing the medium every 3rd to 4th day.
[42] As a result, cells which were aliquoted into the CG ®, ©, and © formed the drift bands within 24 hours. After first replacement of the medium, there was substantially no cell which was adhered on the bottom of the flask, and then the culture was maintained by exchanging the media for 22 days. The culture was stopped at day 22 after starting the culture. In the flask where the culture was performed using the MSCGM™ medium, ten or more cell colonies were observed, and on day 15 after starting the culture, 3x10 fibroblast- like adherent cells (FACs) were harvested. In the flask where the culture was performed using the DMEM medium containing 10% fetal bovine serum (FBS), five or more cell colonies were observed, and on day 15 after starting the culture, 1.5x10 fibroblast-like adherent cells (FACs) were harvested (Fig. 1). Therefore, it was confirmed that in the culture of mesenchymal stem cells, the basal medium of CG comprising sera was not as effective, as compared with MSCGM™ purchased from Cambrex.
[43]
[44] Example 3: Analysis of effects of human serum (HS) and SSS in DMEM with control group being in MSCGM™
[45] In order to examine the proliferation ability of MSC by a DMEM medium as a basal medium added with human serum (HS), and SSS, an MSCGM™ (Cambrex) medium was used as a control group, and the DMEM (Gibco) was dissolved under various conditions, and cells were cultured in the DMEM medium (Gibco) added with various sera for analysis of MSC proliferation efficiency. The A -C group was used as a control group, which contains an MSCGM™ (Cambrex) medium; the B-D group contained a DMEM (Gibco) medium dissolved in distilled water, containing 10% HS (Cambrex) alone, or in combination with 5% SSS (Irvine Scientific); the C-L group contained a DMEM (Gibco) dissolved in distilled water (LOVIS) (distributed by Kyongwon Enterprise), containing 10% HS (Cambrex) alone, or in combination with 5% SSS (Irvine Scientific). Thereafter, the experiments were performed.
[46] 2.0 x 10 mesenchymal stem cells (MSCs) which had been stored in the frozen storage solution containing MSCGM™, 10% fetal bovine serum (FBS) and 5% DMSO of Example 1 were quickly thawed in a water bath at 370C, washed with an MSCGM™ medium twice, and then mixed with 5 ml of an MSCGM™ medium. The cells were aliquoted into T25 flasks, each of which comprises A-C group, as the control group, the B-D and C-L groups, each of which is a DMEM media added with 10% HS, at 6.6 x 105 cells/flask, and cultured at 370C and 5% CO . On the day 4 after starting the culture, the medium was replaced with a fresh one, and the cells were observed. As a result, MSCs in the B-D group, and the C-L group were relatively hardly observed by microscopy, as compared with the A-L group. Then, on day 7 after starting the culture, the cells were treated with 0.125% trypsin-EDTA, and then detached, and thereafter, a half of the cells cultured in the T75 flasks were subcultured. At this time, the B-D group, and the C-L group were inoculated into a DMEM containing 10% HS alone, or in combination of 5% SSS.
[47] As a result, MSCs, which were aliquoted into both the media added with 10% HS alone, B-D, and C-L, and the media added with 10% HS and 5% SSS, were observed to be detached on the bottom of the flask and died within 48 hours, whereas only the mesenchymal stem cells (MSCs) which had been cultured in the control group, the A-L medium, grew (Table 1). This indicates that 10% HS and 10% SSS do not substantially contribute to the growth of the mesenchymal stem cells.
[48] Table 1
Cell numbers according to culture media and subculturing numbers
[49] [50] Example 4: Analysis of effects of fetal bovine serum (FBS), human serum (HS) and SSS on CG medium with control group being in MSCGM™
[51] In order to examine the proliferation ability of MSC by a CG medium as a basal medium added with fetal bovine serum, human serum, and SSS, an MSCGM™ (Cambrex) medium was used as a control group, and the culture was performed in the CG (CellGro™; CellGenix) to observe the proliferation of MSC.
[52] The conditions of the CG media were as follows: a CG medium with 10% fetal bovine serum (FBS), a CG medium with 10% human serum (HS; Cambrex), a CG medium with 10% HS and 5% SSS (serum substitute supplement; Irvine Scientific), and a CG medium alone.
[53] The MSCs which had been cryo-preserved in Example 1 was thawed in the same manner as in Example 3, and 3x10 cells were aliquoted into a T25 flask having each of the above-described media, cultured at 370C and 5% CO 2 , and subcultured by replacing the medium every 3rd to 4th day.
[54] As a result, on day 6 after starting the culture, the growths of the MSCs were observed only in the CG medium (added) with the MSCGM™ (Cambrex) and 10% fetal bovine serum (FBS), and not observed in the other media.
[55] [56] Example 5: Analysis of proliferation ability and surface markers by addition of human cord serum to MSC basal medium
[57] [58] 5-1. Analysis of the proliferation ability by addition of human cord serum [59] In order to examine the proliferation ability by addition of human cord serum (CS), MSCBM™(Mesenchymal stem cell basal medium, Cambrex) and CellGro™(CellGenix) as the basal media with CS at varying concentrations were used as the culture media, and MSCGM™was used as a control group. The conditions of the media used for the experiment were as follows: a MSCBM
[60] medium with 10% CS, a MSCBM medium with 20% CS, a CG medium added with
10% CS, and a CG medium with 20% CS. For the CS, umbilical cord blood was taken from the umbilical cord obtained from a byproduct of delivery, transferred into a sterilized tube, stored in the tube at room temperature for 20 min for coagulation, and centrifuged at 700xg for 10 min, thereby only the serum in the supernatant is separated and used.
[61] The MSCs which had been cryo-preserved in Example 1 was thawed in the same manner as in Example 3, and 2.4x10 cells were aliquoted into a T25 flask having each of the above-described media, cultured at 370C and 5% CO , and subcultured by replacing the medium every 3rd to 4th day.
[62] Further, when 80% or more of the area of the bottom were occupied with cells, subculture was performed.
[63] As a result, in the 2nd and 3rd passages, the growth rates of the MSCs in the four kinds of media with CSs were higher than that of the control group cultured in the MSCGM™ medium in the subculture process. Particularly, it was observed that the growth of the MSCs increased as 2 times as much or more in the MSCGM™ medium group added with CS. These results indicate that addition of CS to the medium for culturing and proliferating the MSCs is very useful (Fig. 2).
[64]
[65] 5-2. Analysis of cell surface markers in the culture of MSCs by addition of human cord serum
[66] After 3rd subculturing of the cells in the culture medium in Example 5-1, the cultured MSCs were treated with 0.125% trypsin-EDTA and detached, and centrifuged at 400xg for 5 mins to obtain cells. The cells were washed two times with Mg + and Ca +-free PBS (Gibco) containing 2% FBS, and the MSCs obtained by washing were aliquoted into each of nine tubes (BD) for FACS at 100 D, and added 10 ul of each of nine kinds of antibodies. They were reacted at room temperature for 30 mins and the MSCs were washed with Mg + and Ca +-free PBS (Gibco) containing 2% FBS, and centrifuged at 400xg at room temperature for 5 mins, in which this process was repeated two times. After washing, 50 D of 2% FBS-PBS was added into the resultants, and analyzed by flow cytometry. The cell surface markers of MSCs were analyzed to observe the change in the phenotypes thereof as the stem cells. As used herein, the antibodies were an IgGl negative control group (Serotec) bonded with fluorescein isothiocyanate (FITC); CD29 (Serotec), CD44 (Dako), CD105 (Serotec), HLA-DR (Serotec), and an IgGl negative control group (Serotec) bonded with phycoerythrin (PE); and CD34 (Serotec), CD45 (Dako), and D73 (BD Biosciences).
[67] As a result, the cell sizes and volumes were similar of the MSCs cultured in the
MSCGM™ as the control group, and the MSCB M™ medium group with 10% CS, but the cell sizes were smaller and the SSC-H distributions were wider in the CellGro™
medium with 10% CS (Fig. 3). For the serological types, CD 105 was expressed in 95% or more of the cells cultured in the MSCGM™ as the control group, and the MSCBM™ medium group with 10% CS, but CD 105 was expressed in about 30% of the cells cultured in the CellGro™ medium (added with 10% CS) (Figs. 4, 5, and 6). Further, generally the expressions of CD29+, CD44+, CD73+, CD105+, CD34-, CD45-, HLA-DR-, which were used as the cell surface markers of the MSCs, are shown in Table 2.
[68] Table 2 Expression rates of surface markers of the MSCs according to culture media
[69] Example 6: Analysis of the proliferation ability of MSC and surface markers in medium during the development
[70] [71] 6-1. Analysis of the proliferation ability of MSC in the medium under the development
[72] In order to examine the proliferation ability of MSC in the medium under the development, an MSCGM™ (Cambrex) was used as a control group; a DMEM /F 12 (Gibco) medium ® with insulin (10 ng/ml), hydrocortisone (10 nM), EGF (10 ng/ml), and 5% CS (cord serum); and a DMEM/F12 medium ® with insulin (10 ng/ml), hydrocortisone (10 nM), EGF (10 ng/ml), and 10% CS were used.
[73] The MSCs which had been cryo-preserved in Example 1 was thawed in the same manner as in Example 3, and 2x10 cells were aliquoted into a T25 flask having each of the above-described media, cultured at 370C and 5% CO , and cultured by replacing the medium every 3rd to 4th day. Further, when 80% or more of the area of the bottom were occupied with cells, subculture was performed.
[74] As a result, it was observed that the proliferation rates of the MSCs cultured in the 1st subculture was substantially similar to that of the MSCGM™ as the control group, but the proliferation rates of the MSCs cultured in the DMEM/F12 ® medium with a 5% CS medium was two times more or higher than those of the MSCGM™. Further, it was observed that the proliferation rates of the MSCs cultured in the 2nd subculture
were significantly higher than those of the DMEM/F12 ® medium with a 5% CS medium, a DMEM/F12 © medium with a 10% CS medium, and the MSCGM™ medium as the control group (Fig. 7).
[75]
[76] 6-2. Analysis of surface markers of MSC cultured in medium under the development
[77] In order to examine the cell surface markers of the MSCs cells cultured in Example
6-1, the cells were treated in the same manner as in Example 5-2, and the change in the phenotypes thereof was observed by using a flow cytometer from FACS Calibur (BD). As a result, it was found that the MSCs cultured in the MSCGM™ as the control group had various cell sizes and volumes, but the MSC cells cultured in the DMEM/F12 medium with a human umbilical cord blood and insulin, EGF, hydrocortisone, LIF, and the like had constant cell sizes and volumes (Fig. 8). Based on these results, the analysis of the phenotypes of MSCs grown in the media, each 5% and 10% umbilical cord bloods indicates that in the MSCs cultured in the DMEM/F12 medium with 5% umbilical cord blood serum and various growth factors, and the DMEM/F12 medium with 10% umbilical cord blood serum and various growth factors, the graph of the CD45 expression shows right shift, as compared with the negative control group, but the expressions were all interpreted to be negative, and 98% or more of the positive cells were obtained in CD29, CD44, CD 105, and CD73, which are known as the mesenchymal stem cell surface markers (Figs. 9 and 10). Further, the cell surface markers of the MSCs were analyzed to observe the change in the phenotypes thereof, and as a result, it was found that the phenotypes of the proliferated MSCs showed some difference, but not significant difference in their distribution according to CD34, CD45, and HLA-DR labels (Table 2). Industrial Applicability
[78] The culture medium for mesenchymal stem cells comprising the umbilical cord blood serum of the present invention have better effects of growth and proliferation, while maintaining the pluripotency of the mesenchymal stem cells, as compared with the conventional stem cell culture media, and therefore, they can be used to massively culture the mesenchymal stem cells, and obtain pure mesenchymal stem cells therefrom.
[79]
Claims
[I] A culture medium for mesenchymal stem cells, comprising a basal medium and umbilical cord blood serum.
[2] The culture medium according to claim 1, wherein the umbilical cord blood serum is contained in the amount of 1 to 30%, based on the total medium composition.
[3] The culture medium according to claim 2, wherein the umbilical cord blood serum is contained in the amount of 3 to 20%, based on the total medium composition.
[4] The culture medium according to claim 1, further comprising mesenchymal stem cell growth factors.
[5] The culture medium according to claim 4, wherein the mesenchymal stem cell growth factors further comprising one or more of insulin, hydrocortisone, EGF, and FGF.
[6] The culture medium according to claim 5, wherein the amount of the insulin is at a concentration of 1 to 500 ng/ml, the amount of the hydrocortisone is at a concentration of 0.1 to 500 nM, the amount of the EGF is at a concentration of 0.1 to 500 ng/ml, or the amount of the LIF is at a concentration of 0.01 to 500 ng/ml, in the total medium composition.
[7] The method according to claim 1, wherein the mesenchymal stem cells are derived from a human.
[8] A culture medium for mesenchymal stem cells comprising a basal medium, and umbilical cord blood serum, wherein positive surface markers, CD 105 and CD44, are expressed at 95% or more in the cultured mesenchymal stem cells.
[9] A culture medium for mesenchymal stem cells comprising a basal medium, and umbilical cord blood serum, wherein a negative surface marker CD34 is expressed at 10% or less in the cultured mesenchymal stem cells.
[10] A culture medium for mesenchymal stem cells comprising a basal medium, and umbilical cord blood serum, wherein positive surface markers, CD 105 and CD44, are expressed at 95% or more, and a negative surface marker CD34 is expressed at 10% or less in the cultured mesenchymal stem cells.
[I I] The culture medium for mesenchymal stem cells according to claim 10, wherein the positive surface markers, CD 105 and CD44, are expressed at 98% or more, and the negative surface marker CD34 is expressed at 5% or less in the cultured mesenchymal stem cells.
[12] The culture medium for mesenchymal stem cells according to claim 10, wherein the positive surface markers, CD105, CD29, CD44, CD73 and CD166, are
expressed at 95% or more, and the negative surface markers, CD34, CD45, HLA-DR, CDIa, CD14, CD31 and CD80, are expressed at 5% or less in the cultured mesenchymal stem cells.
[13] The culture medium according to claim 10, wherein the umbilical cord blood serum is contained in the amount of 1 to 30%, based on the total medium composition.
[14] The culture medium according to claim 10, further comprising mesenchymal stem cell growth factors.
[15] The culture medium according to claim 14, wherein the mesenchymal stem cell growth factors further comprise one or more of insulin, hydrocortisone, EGF, and FGF.
[16] The culture medium according to claim 15, wherein the amount of the insulin is at a concentration of 1 to 500 ng/ml, the amount of the hydrocortisone is at a concentration of 0.1 to 500 nM, the amount of the EGF is at a concentration of 0.1 to 500 ng/ml, or the amount of the LIF is at a concentration of 0.01 to 500 ng/ml, in the total medium composition.
[17] A method for culturing mesenchymal stem cells using the culture medium according to claim 1.
[18] The method according to claim 17, wherein the umbilical cord blood serum is contained in the amount of 1 to 30%, the amount of the insulin is at a concentration of 1 to 500 ng/ml, the amount of the hydrocortisone is at a concentration of 0.1 to 500 nM, the amount of the EGF is at a concentration of 0.1 to 500 ng/ml, and the amount of the LIF is at a concentration of 0.01 to 500 ng/ml, in the total medium composition.
[19] The method according to claim 17, wherein the mesenchymal stem cells are derived from a human.
[20] A method for obtaining mesenchymal stem cells, comprising the steps of: culturing mesenchymal stem cells in the culture medium according to claim 1 ; centrifuging the cultured mesenchymal stem cells; and obtaining the mesenchymal stem cells.
[21] The method according to claim 20, wherein the umbilical cord blood serum is contained in the amount of 1 to 30%, the amount of the insulin is at a concentration of 1 to 500 ng/ml, the amount of the hydrocortisone is at a concentration of 0.1 to 500 nM, the amount of the EGF is at a concentration of 0.1 to 500 ng/ml, and the amount of the LIF is at a concentration of 0.01 to 500 ng/ml, in the culture medium.
[22] The method according to claim 21, wherein the mesenchymal stem cells are derived from a human.
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| KR10-2006-0036754 | 2006-04-24 | ||
| KR1020060036754A KR100908481B1 (en) | 2006-04-24 | 2006-04-24 | Mesenchymal stem cell culture medium and culture method of mesenchymal stem cells using the same |
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| KR100908481B1 (en) | 2009-07-21 |
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