WO2007123363A1 - Milieu de culture et procédés pour cultiver des cellules souches mésenchymateuses - Google Patents

Milieu de culture et procédés pour cultiver des cellules souches mésenchymateuses Download PDF

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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
amount
cells
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Jai Jun Choung
Kyung Suk Kim
Seung Hyun Kim
Hee Tae Kim
Young Gyu Chai
Mi Ran Choi
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Corestem Co., Ltd.
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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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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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
    • C—CHEMISTRY; METALLURGY
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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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    • 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]
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
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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

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • hematopoietic stem cells have been most developed, and recently the mesenchymal stem cells are actively being researched.
  • 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.
  • a method for culturing human stem cells using a medium containing animal serum fetal bovine serum
  • 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.
  • 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 (CellGroTM, CellGenix) and MSCGMTM (Cambrex) at a concentration of 5.28 x 10 cells, and culturing them for 15 days.
  • FACS fibroblast- like adherent cells
  • Fig. 2 is a graph showing the growth rate of MSC in a medium containing
  • MSCBMTM Mesenchymal stem cell basal medium, Cambrex
  • 10% CS a medium containing 20% CS (umbilical cord blood serum; cord serum)
  • CS umbilical cord blood serum
  • CellGroTM CellGenix
  • CellGenix a medium containing CellGroTM
  • 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 MSCBMTM medium with 10% cord serum, and a CellGroTM medium with 10% CS (umbilical cord blood serum; Cord serum), compared with the control group cells grown in MSCGMTM.
  • flow cytometry FACSCalibur, BD
  • Fig. 4 is a graph showing a histogram of surface markers of MSC which had been proliferated in an MSCGMTM medium.
  • Fig. 5 is a graph showing a histogram of surface markers of MSC which had been proliferated in an MSCBMTM medium with 10% CS (cord serum).
  • Fig. 6 is a graph showing a histogram of of surface markers of MSC which had been proliferated in a CellGroTM medium with 10% CS (cord serum).
  • 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 MSCGMTM.
  • CS artificial cord blood serum
  • 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 MSCGMTM.
  • MSCs mesenchymal stem cells
  • 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.
  • 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.
  • the present invention relates to a culture medium for mesenchymal stem cells comprising a basal medium, and a medium containing umbilical cord blood serum.
  • 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.
  • 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.
  • 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, ⁇ -MEM ( ⁇ -Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium, but are not limited thereto.
  • 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.
  • 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.
  • the blood is reacted at 56 0 C for 30 mins as for the serum components such as fetal bovine serum to inactivate the blood plasma components .
  • the serum components such as fetal bovine serum to inactivate the blood plasma components .
  • 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.
  • 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.
  • 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.
  • the culture medium of the present invention can further comprise mesenchymal stem cells growth factors which can affect the growth of mesenchymal stem cells.
  • 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).
  • EGF epidermal growth factor
  • LIF leukemia inhibitory factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • EPO erythropoietin
  • FGF fibroblast growth factor
  • IGF insulin-like growth factor
  • PDGF platelet-derived growth factor
  • SCF stem cell factor
  • 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
  • the amount of the LIF can be used.
  • 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).
  • 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.
  • 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.
  • the process for separating the mesenchymal stem cells of the present invention can be performed as known in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 MSCGMTM basal medium, and stored in -176 0 C 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.
  • 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.
  • 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.
  • 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 MSCGMTM, 10% fetal bovine serum (FBS), and 5% DMSO, and stored in liquid nitrogen at -176 0 C.
  • Example 2 Assay of proliferation of MSCs in CG (Cell Genix) medium
  • Example 1 using the media CG ®, ⁇ , and ⁇ which had been directly supplied from Cell Genix, and an MSCGMTM 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 37 0 C and 5% CO .
  • DMEM fetal bovine serum
  • 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.
  • Example 3 Analysis of effects of human serum (HS) and SSS in DMEM with control group being in MSCGMTM
  • the A -C group was used as a control group, which contains an MSCGMTM (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.
  • MSCs mesenchymal stem cells
  • FBS fetal bovine serum
  • DMSO fetal bovine serum
  • the medium was replaced with a fresh one, and the cells were observed.
  • MSCs in the B-D group, and the C-L group were relatively hardly observed by microscopy, as compared with the A-L group.
  • 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.
  • the B-D group, and the C-L group were inoculated into a DMEM containing 10% HS alone, or in combination of 5% SSS.
  • Example 4 Analysis of effects of fetal bovine serum (FBS), human serum (HS) and SSS on CG medium with control group being in MSCGMTM
  • MSCGMTM CellGroTM; CellGenix
  • 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.
  • FBS fetal bovine serum
  • HS human serum
  • SSS serum substitute supplement
  • Example 3 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 37 0 C and 5% CO 2 , and subcultured by replacing the medium every 3rd to 4th day.
  • 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.
  • Example 1 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 37 0 C and 5% CO , and subcultured by replacing the medium every 3rd to 4th day.
  • 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.
  • MSCs 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.
  • 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).
  • CD 105 was expressed in 95% or more of the cells cultured in the MSCGMTM as the control group, and the MSCBMTM medium group with 10% CS, but CD 105 was expressed in about 30% of the cells cultured in the CellGroTM 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.
  • an MSCGMTM (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.
  • a DMEM/F 12 (Gibco) medium ® with insulin (10 ng/ml), hydrocortisone (10 nM), EGF (10 ng/ml), and 10% CS were used.
  • Example 3 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 37 0 C 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.
  • 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).
  • BD flow cytometer from FACS Calibur
  • the MSCs cultured in the MSCGMTM 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).
  • 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).
  • 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.

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Abstract

La présente invention concerne un milieu de culture destiné aux cellules souches mésenchymateuses, qui comprend un milieu basal et un sérum sanguin du cordon ombilical. La présente invention concerne aussi un procédé pour cultiver des cellules souches mésenchymateuses utilisant ledit milieu de culture ainsi qu'un procédé pour obtenir des cellules souches mésenchymateuses utilisant ledit milieu de culture. Ce procédé permet d'obtenir des cellules souches mésenchymateuses en quantités massives.
PCT/KR2007/002002 2006-04-24 2007-04-24 Milieu de culture et procédés pour cultiver des cellules souches mésenchymateuses WO2007123363A1 (fr)

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KR1020060036754A KR100908481B1 (ko) 2006-04-24 2006-04-24 중간엽 줄기세포 배양 배지 및 이를 이용한 중간엽줄기세포의 배양 방법

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WO2011068792A2 (fr) * 2009-12-01 2011-06-09 The Regents Of The University Of California Compositions et procédés pour la fabrication et l'utilisation de cellules souches mésenchymateuses de moelle osseuse et de cellules progénitrices érythroïdes
WO2011087103A1 (fr) * 2010-01-15 2011-07-21 株式会社ジェイ・エム・エス Promoteur de prolifération pour les cellules souches mésenchymateuses, procédé de stimulation de la prolifération des cellules souches mésenchymateuses l'utilisant, et son procédé de production
EP2752484A1 (fr) * 2011-08-31 2014-07-09 Sewon Cellontech Co., Ltd Procédé de préparation de milieu de culture basique et milieu de culture basique pour cellules souches mésenchymateuses, et agent thérapeutique cellulaire mis en culture et différencié à l'aide de celui-ci
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US9828586B2 (en) 2010-10-08 2017-11-28 Mesoblast International Sárl Enhanced MSC preparations
US10104880B2 (en) 2008-08-20 2018-10-23 Celularity, Inc. Cell composition and methods of making the same
USD848022S1 (en) 2017-07-14 2019-05-07 Hope Biosciences, Llc Container for storing biological material
USD875967S1 (en) 2017-07-14 2020-02-18 Hope Biosciences, Llc Container for storing biological material
US10894947B1 (en) 2016-04-29 2021-01-19 Hope Biosciences, Llc Method for generating protein rich conditioned medium
WO2021020666A1 (fr) * 2019-07-26 2021-02-04 Brexogen Inc. Cellules précurseurs de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes induites et leur procédé de préparation
US10959425B2 (en) 2016-04-29 2021-03-30 Hope Biosciences, Llc Method of banking stem cells
US10988731B2 (en) 2016-04-29 2021-04-27 Hope Biosciences, Llc Formulation for storage, transportation, and delivery of protein rich conditioned medium
US11111480B2 (en) 2016-04-29 2021-09-07 Hope Biosctences, Llc Culture media for multipotent stem cells
CN114787340A (zh) * 2019-07-26 2022-07-22 布瑞克斯奥根株式会社 诱导多能干细胞来源间充质干细胞前体细胞及其制备方法
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US11821004B2 (en) 2006-01-13 2023-11-21 Mesoblast International Sárl Mesenchymal stem cells expressing TNF-α receptors
US10104880B2 (en) 2008-08-20 2018-10-23 Celularity, Inc. Cell composition and methods of making the same
WO2010043076A1 (fr) * 2008-10-17 2010-04-22 宁夏医科大学附属医院 Procédé d'élaboration d'une banque de cellules placentaires mésenchymateuses humaines à applications cliniques
WO2011068792A2 (fr) * 2009-12-01 2011-06-09 The Regents Of The University Of California Compositions et procédés pour la fabrication et l'utilisation de cellules souches mésenchymateuses de moelle osseuse et de cellules progénitrices érythroïdes
WO2011068792A3 (fr) * 2009-12-01 2011-09-29 The Regents Of The University Of California Compositions et procédés pour la fabrication et l'utilisation de cellules souches mésenchymateuses de moelle osseuse et de cellules progénitrices érythroïdes
US8703487B2 (en) 2009-12-01 2014-04-22 The Regents Of The University Of California Compositions and methods for making and using bone marrow mesenchymal stem cells and erythroid progenitor cells
JP2011160799A (ja) * 2010-01-15 2011-08-25 Jms Co Ltd 間葉系幹細胞の増殖促進剤、それを用いた間葉系幹細胞の増殖促進方法および製造方法
WO2011087103A1 (fr) * 2010-01-15 2011-07-21 株式会社ジェイ・エム・エス Promoteur de prolifération pour les cellules souches mésenchymateuses, procédé de stimulation de la prolifération des cellules souches mésenchymateuses l'utilisant, et son procédé de production
US10550369B2 (en) 2010-10-08 2020-02-04 Mesoblast International Sarl Enhanced MSC preparation
US11708560B2 (en) 2010-10-08 2023-07-25 Mesoblast International Sarl Enhanced MSC preparations
EP3679939A1 (fr) 2010-10-08 2020-07-15 Mesoblast International Sàrl Préparations de msc améliorées
US9828586B2 (en) 2010-10-08 2017-11-28 Mesoblast International Sárl Enhanced MSC preparations
US9963678B2 (en) 2010-10-08 2018-05-08 Mesoblast International Sàrl Enhanced MSC preparations
EP2752484A1 (fr) * 2011-08-31 2014-07-09 Sewon Cellontech Co., Ltd Procédé de préparation de milieu de culture basique et milieu de culture basique pour cellules souches mésenchymateuses, et agent thérapeutique cellulaire mis en culture et différencié à l'aide de celui-ci
EP2752484A4 (fr) * 2011-08-31 2015-04-15 Sewon Cellontech Co Ltd Procédé de préparation de milieu de culture basique et milieu de culture basique pour cellules souches mésenchymateuses, et agent thérapeutique cellulaire mis en culture et différencié à l'aide de celui-ci
JP2014525262A (ja) * 2011-08-31 2014-09-29 セウォン セロンテック カンパニー リミテッド 間葉系幹細胞の基本培養培地の調製方法、間葉系幹細胞の基本培養培地及びこれを利用して培養分化した細胞治療剤
WO2016181114A1 (fr) * 2015-05-08 2016-11-17 Imagen Therapeutics Limited Milieu personnalisé
US10894947B1 (en) 2016-04-29 2021-01-19 Hope Biosciences, Llc Method for generating protein rich conditioned medium
US10959425B2 (en) 2016-04-29 2021-03-30 Hope Biosciences, Llc Method of banking stem cells
US10988731B2 (en) 2016-04-29 2021-04-27 Hope Biosciences, Llc Formulation for storage, transportation, and delivery of protein rich conditioned medium
US11111480B2 (en) 2016-04-29 2021-09-07 Hope Biosctences, Llc Culture media for multipotent stem cells
USD848022S1 (en) 2017-07-14 2019-05-07 Hope Biosciences, Llc Container for storing biological material
USD875967S1 (en) 2017-07-14 2020-02-18 Hope Biosciences, Llc Container for storing biological material
WO2021020666A1 (fr) * 2019-07-26 2021-02-04 Brexogen Inc. Cellules précurseurs de cellules souches mésenchymateuses dérivées de cellules souches pluripotentes induites et leur procédé de préparation
CN114787340A (zh) * 2019-07-26 2022-07-22 布瑞克斯奥根株式会社 诱导多能干细胞来源间充质干细胞前体细胞及其制备方法

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