WO2008018684A1 - Culture medium for co-culturing of human stem cells and their feeder cells - Google Patents

Culture medium for co-culturing of human stem cells and their feeder cells Download PDF

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WO2008018684A1
WO2008018684A1 PCT/KR2007/002864 KR2007002864W WO2008018684A1 WO 2008018684 A1 WO2008018684 A1 WO 2008018684A1 KR 2007002864 W KR2007002864 W KR 2007002864W WO 2008018684 A1 WO2008018684 A1 WO 2008018684A1
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stem cells
cells
fbs
culture media
culture
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PCT/KR2007/002864
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French (fr)
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Hyun Sook Park
Sun Ray Lee
Jung Mi Lee
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Modern Cell & Tissue Technologies Inc.
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    • 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
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the present invention relates to a culture media composition for co-culturing human stem cells and feeder cells and for culturing a primary cell line, by which both stem cells and feeder cells can be cultivated under an optimized condition.
  • mice have been first cultured in a test tube.
  • this in vitro culture has been combined with a gene targeting technique so as to first make a transformed mouse having a targeted gene. Since that, this gene targeting technique of mice plays an important role to investigate genetic functions and to build up animal models of human diseases. Further, it drives life/medical sciences to develop a lot (Smith A. G., Annu. Rev. Cell Dev. Biol., 17: 435-62, 2001).
  • human embryonic stem cells started to be cultured in vitro, 17 years later I 1988. Dr. Thomson has first established human embryonic stem cells in University of Wisconsin (Thomson J. A., Science 282(5391): 1145-7, 1998).
  • the human embryonic stem cell is more difficult to be cultured and manipulated than the mouse stem cell. Either, it is improper to be mass-cultured, even if necessary to develop therapeutic agents by performing a gene manipulation or in vitro test. Therefore, it is required to complete a culture method that can proliferate stem cells effectively and control their quality easily.
  • the culture method popularly used is based upon the procedure Dr. Thompson established. Precisely, mouse embryonic fibroblasts are treated with mitomycin or irradiated in order to inhibit the cell growth. Then, the resulting fibroblasts are inoculated in order to previously express extra-cellular substrates and cytokines for embryonic stem cells' need. After that, embryonic stem cells are inoculated onto the resulting cells for feeder cell use.
  • the compositions of culture media are broadly classified to 2 kinds according to constituents added to basic medium.
  • the basic media is comprised of DMEM and/or DMEM/F12 (1 : 2) and may contain fibroblast growth factor, glutamine and ⁇ -mercaptoethanol.
  • 20% serum replacement(lnvitrogen Inc.) and/or 20% fetal bovine serum are added with insulin, transferrin and selenium.
  • the culture media that contains serum replacement instead of serum is improper to survive fibroblasts for feeder cell use.
  • the culture media that contains fetal bovine serum tends to differentiate stem cells a lot.
  • the culture medium of fibroblasts for feeder cell use often includes 10% fetal bovine serum, which plays an important role to survive fibroblasts. Therefore, it is clarified that fibroblasts may hardly function as feeder cells in the culture medium of embryonic stem cells containing fetal bovine serum instead of serum replacement(See FIG. 9 to 11 ; Experimental Result 3).
  • the composition of culture media for culturing embryonic stem cells should be optimized for both stem cells and feeder cells.
  • compositions of culture media for embryonic stem cells are provided.
  • compositions of culture media that comprises basic media containing fetal bovine serum(FBS), serum replacement(SR) and fibroblast growth factor(bFGF) in particular ratios. Then, we have identified that the compositions may optimize the growth of both stem cells and feeder cells and little differentiate stem cells and completed the present invention successfully.
  • FBS fetal bovine serum
  • SR serum replacement
  • bFGF fibroblast growth factor
  • the object of the present invention is to overcome conventional disadvantages of the method for culturing human embryonic stem cells.
  • the object of the present invention is to develop culture media that is optimized to survive both stem cells and feeder cells when culturing human embryonic stem cells and to maintain the capacity of undifferentiation by maximizing the function of feeder cells.
  • the object of the present invention is to improve the attachment and the growth of stem cells after restored from a freezer.
  • the culture media of the present invention may be optimized to cultivate different kinds of adult stem cells and primarily cultured cells, because it stimulate the growth of fibroblasts effectively in addition to the indifferent stem cells.
  • FIG. 1 depicts the cell numbers of feeder cells (CF1 , p5) after culturing for 60 hours by using several culture media, (a) seeding at 1,000 cells per unit area(cm 2 ); (b) at 2,000 cells per unit cm 2 .
  • FIG. 2 depicts the growth/division of feeder cells measured at various compositions of culture media when seeding at 2,000 cells per unit cm 2 according to culture periods, (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 3 depicts murine fetal fibroblasts (FIG. 2) cultured for 60 hours with several culture media by using a phase contrast microscopy (4OX magnified), (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 4 depicts the comparison of 3 kinds of culture media [general medium of fibroblasts(10% FBS), culture medium of the present invention(2% FBS, 10% SR, bFGF), culture medium for embryonic stem cell use(20% SR, bFGF)] as described in FIG. 2.
  • FIG. 5 depicts the growth/division of feeder cells measured at various compositions of culture media according to culture periods by observing the cell areas on culture plates with a CellScreen machine(lnnovatis Inc.) when seeding at 2,000 cells per unit cm 2 , (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 5 depicts the growth/division of feeder cells measured at various compositions of culture media according to culture periods by observing the cell areas on culture plates with a CellScreen machine(lnnovatis Inc.) when seeding at 2,000 cells per unit cm 2 , (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 6 depicts the growth of murine fetal fibroblasts cultured at various compositions of culture media by observing a CellScreen image of FIG. 5.
  • fibroblasts derived from CF1 mice and sub-cultured during 5 passages fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 7 depicts the comparison of 3 kinds of culture media [general medium of fibroblasts(10% FBS), culture medium of the present invention(2% FBS, 10% SR, bFGF), culture medium for embryonic stem cell use(20% SR, bFGF) as described in FIG. 5.
  • fibroblasts derived from CF1 mice and sub-cultured during 5 passages (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 8 depicts the growth of murine fetal fibroblasts cultured at various compositions of culture media by observing a CellScreen image of FIG. 7.
  • FIG. 9 depicts the cell survival of murine fetal fibroblasts according to culture media when treating mitomycin for one and a half hours to stop the cell growth by using a CellScreen image(lnnovatis Inc.).
  • fibroblasts derived from CF1 mice and sub- cultured during 5 passages fibroblasts derived from C57/Black mice and sub- cultured during 6 passages.
  • FIG. 10 depicts the comparison of 3 kinds of culture media [general medium of fibroblast ⁇ 10% FBS), culture medium of the present invention(2% FBS, 10% SR, bFGF), culture medium for embryonic stem cell use(20% SR, bFGF) as described in FIG. 9.
  • fibroblasts derived from CF1 mice and sub-cultured during 5 passages (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 11 depicts the cell survival after mitomycin treatment by observing a
  • FIG. 9 CellScreen image of FIG. 9. (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 12 depicts the comparison of cross-differentiation from murine fetal fibroblasts to muscle fibroblasts according to culture media by using an immunofluorescence staining onto ⁇ -smooth muscle actin (40X magnified), (a) and (b) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
  • FIG. 13 depicts the comparison of attachments and undifferentiations of human embryonic stem cells cultured with general media of embryonic stem cells(20% SR, bFGF) and the culture media of the present invention(2% FBS, 10% SR, bFGF) after stored in a freezer and restored, (a) HSF6 cell line; (b) Miz4 cell line.
  • FIG. 14 depicts the attachment and the undifferentiation of human embryonic stem cells cultured with the culture media of the present invention(2% FBS 1 10% SR, bFGF) after stored in a nitrogen tank and restored for at least one month.
  • FIG. 15 depicts the undifferentiate markers SSEA4, Tral-60 and Oct4 of human embryonic stem cells cultured with the culture media of the present invention(2% FBS, 10% SR, bFGF) after stored and restored repeatedly by using a fluorescence staining.
  • FIG. 16 depicts the comparison of attachments and undifferentiations of human embryonic stem cells sub-cultured with general media of embryonic stem cells(20% SR, bFGF) and the culture media of the present invention(2% FBS, 10% SR, bFGF).
  • FIG. 17 depicts the undifferentiation of human embryonic stem cells sub- cultured with conventional media of embryonic stem cells(20% SR, bFGF) and the culture media of the present invention(2% FBS, 10% SR, bFGF).
  • FIG. 18 depicts the cell surface expression of Tral-60 marker by using a flow cytometry.
  • FIG. 19 depicts the RNA levels of Nanog, Rex1 and SOX2 markers by performing a RT-PCR.
  • FIG. 20 depicts the chromosomal aberration of human embryonic stem cells cultured with the culture media of the present invention by using a karyotype analysis.
  • FIG. 21 depicts the chromosomal aberration of human embryonic stem cells that are cultured with the culture media of the present invention after stored in a freezer and restored by using a karyotype analysis.
  • FIG. 22 depicts the cell deaths of human embryonic stem cells cultured with general media and the culture media of the present invention by using a Tunel assay.
  • FIG. 23 depicts the embryonic bodies of human embryonic stem cells cultured with culture media of the present invention to identify a tridermic differentiation.
  • FIG. 24 depicts the RNA levels of endodermic marker aFP, mesodermic marker FLKI (KDR) and ectodermic marker NCAM1 by using a RT-PCR to identify the tridermic differentiation of embryoid bodies of human embryonic stem cells cultured with the culture media of the present invention.
  • FIG. 25 depicts the protein expressions of cytokeratin(tridermic hybrid marker and epithelium marker), aFP( ⁇ -fetoprotein, endodermic marker), SMA(smooth muscle actin, mesodermic marker) and Tuj1(blll-Tubulin, ectodermic marker) by using a fluorescence staining to identify the tridermic differentiation of embryoid bodies of human embryonic stem cells cultured with the culture media of the present invention.
  • FIG. 26 depicts the human embryonic stem cells treated with nicotine amide in the culture media of the present invention.
  • FIG. 27 depicts the result of RT-PCR of human embryonic stem cell lines HSF6 and Miz4 after nicotine amide treatment at 0, 10, 50 and 100 ⁇ g/ml of concentration with culture media of the present invention, in which Oct4, Rex1 and SOX2 are undifferentiation markers.
  • FIG. 28 depicts the phenotype of MSC(mesenchymal stem cells) cultured for 5 days after seeded at 8,000 cells per unit area.
  • FIG. 29 depicts the cell number of MSC cultured for 5 days.
  • the present invention provides compositions of culture media that help the growth and the self-renewal of human stem cells as remaining indifferent.
  • conventional culture media is modified to contain fetal bovine serum(FBS), serum replacement(SR) and fibroblast growth factor(bFGF) at proper ratios in basic media.
  • the present invention provides a culture medium composition for co-culturing stem cells and feeder cells, which comprises basic media containing 1 to 10% of fetal bovine serum(FBS), 1 to 20% of serum replacement(SR) and 0.1 to 4 ng/ml of fibroblast growth factor(bFGF).
  • the present invention provides a culture medium composition for culturing a primary cell line, which comprises basic media containing 1 to 10% of fetal bovine serum(FBS), 1 to 20% of serum replacement(SR) and 0.1 to 4 ng/ml of fibroblast growth factor(bFGF).
  • basic media containing 1 to 10% of fetal bovine serum(FBS), 1 to 20% of serum replacement(SR) and 0.1 to 4 ng/ml of fibroblast growth factor(bFGF).
  • the culture media compositions of the present invention are effective to culture fibroblasts primarily.
  • This compositions of culture media are also effective to cultivate MSC (mesenchymal stem cell) or SVF (stroma vascular fraction) and primarily-cultured skin fibroblasts.
  • MSC meenchymal stem cell
  • SVF stroma vascular fraction
  • the compositions of culture media for primary culture use are the same with those of stem cells, even if the glucose level is lower.
  • the basic medium can be selected from commercially available basic media, whatever it contains essential elements for animal cell culture such as amino acids, vitamins, minerals and the like.
  • this basic medium can be selected from DMEM (Dulbecco's modified eagle medium) and DMEM/F12 (DMEM + nutrient mixture).
  • the serum replacement is a product manufactured in order to replace animal serums.
  • the serum replacement is comprised of heat-treated bovine serum albumin, bovine transferrin, bovine insulin and the like.
  • the serum replacement used in the present invention can be selected from commercially available serum replacements.
  • the serum replacement can be selected from Omni Serum(Advanced Biotechnologies Inc., Columbia, Md.) and KnockoutTM Serum Replacement(lnvitrogen Inc.; See WO098/30679) described in following Examples.
  • the present invention provides a culture medium composition, which comprises basic media containing 1 to 5% of fetal bovine serum(FBS), 5 to 15% of serum replacement(SR) and 1 to 4 ng/ml of fibroblast growth factor(bFGF).
  • FBS fetal bovine serum
  • SR serum replacement
  • bFGF fibroblast growth factor
  • the stem cells and primarily-cultured cells can be derived from embryos and/or adult tissues. That is to say, the stem cells can be selected from embryonic stem cells derived from a fetus and adult stem cells derived from a mature tissue and differentiate toward various kinds of cells;
  • the stem cells can be stored in a freezer and restored before use.
  • conventional compositions of culture media are disadvantageous to attach stem cells on a culture plate and to remain indifferent after stored in a freezer and restored.
  • the culture medium composition of the present invention is optimized to attach stem cells on a culture plate and to retain the capacity of undifferentiation effectively, even if stored in a freezer and restored before use.
  • the feeder cells can be derived from animals and humans and also derived form embryos or adult tissues, whenever they can secrete various nutrients, cytokines and the like to help stem cells and primarily-cultured cells.
  • the feeder cells can be embryonic fibroblasts(MEF) described in following Examples.
  • the culture medium composition of the present invention may contain 3 to 30 ⁇ g/ml of nicotine amide additionally.
  • stem cells can remain indifferent more effectively than those without nicotine amide.
  • stem cells are rather negatively affected(See FIG. 27).
  • the present invention provides a method for optimizing the growth of stem cells and feeder cells, which comprises culturing the stem cells and feeder cells by using the culture medium composition of the present invention.
  • “optimization” means to stimulate not only stem cells but also feeder cells to proliferate effectively.
  • the present invention provides a method for optimizing the function of feeder cells along with the growth of feeder cells, which comprises culturing the feeder cells by using the culture medium composition of the present invention.
  • the present invention provides a method for culturing stem cells by using the culture medium composition of the present invention, which maintains inherent properties of stem cells and/or primarily-cultured cells.
  • "to maintains inherent properties of stem cells” means to retain the capacity of differentiation of stem cells toward tridermic cells until necessary, even if they usually remain indifferent.
  • the present invention provides a method for optimizing the cell attachment and the undifferentiation of stem cells after stored in a freezer and restored, which comprises culturing the stem cells by using the culture medium composition of the present invention.
  • “optimization” is to improve the cell attachment and the maintenance of undifferentiation after stem cells are stored in a freezer and restored.
  • the growth rate of fibroblasts is compared by measuring cell numbers and areas of cell colonies after cultured respectively with conventional media and the culture media of the present invention(See FIG. 1 to 8;
  • FIG. 12; Experimental Result 4 the survival ratios of fibroblasts are examined by measuring the areas of cell colonies after treating mitomycin as a cytostatic agent(See FIG. 9 to 11 ; Experimental Result 3).
  • mitomycin as a cytostatic agent
  • 2 kinds of fetal fibroblasts derived from mouse strains CF1 and C57/Black are selected after primary cultures, which are commonly used for feeder cells. The same result is observed from both mouse strains.
  • the initial attachment and the maintenance of undifferentiation of stem cells are measured in their ratios, after restored from a freezer with conventional media and the culture media of the present invention(See FIG. 13; Experimental Result 5).
  • the resulting stem cells are sub-cultured during more than 8 passages and observed to monitor the maintenance of undifferentiation(See FIG. 16; Experimental Result 6).
  • 3 kinds of human embryonic stem cell lines including of HSF6, Miz4 and Miz6 are used.
  • the morphological characteristics of cell colonies are observed by using an alkaline phosphatase staining(See FIG. 17).
  • Example 1 Cell culture and composition of culture media
  • Murine embryonic fibroblasts(MEF) were extracted from 13.5 day-pregnant mice (CFI, C57BL6) and cultured primarily with following culture media.
  • the basic media was comprised of DMEM/F12 media(GIBCO, USA, Cat.No. 12500-062) mixed with 3.069 g/l sodium bicarbonate (Sigma, USA, Cat.No. S5761), 2 mM L-glutamine (Sigma, Cat.No. S8540), penicillin(50 U/ml)(Sigma, Cat.No. P4687)/streptomycin (50 ⁇ g/ml)(Sigma, USA, Cat.No.S1277). Then, 10 % fetal bovine serum(FBS; Hyclone, Cat.No. SH30070.03) or Knock-Out serum replacement(SR; Invitrogen BRL,
  • Example 2 Measurement of cell numbers After stopping cell growth, the resulting cells were washed off with PBS buffer and treated with 0.25 % Trypsin(2.5 g/L)-EDTA(0.38 g/L)(lnvitrogen BRL,
  • Human embryonic stem cells were sorted by using alkaline phosphatase staining. Stained colonies of stem cells were judged to remain indifferent and counted.
  • Example 4 CellScreen analysis Murine embryonic fibroblasts were inoculated at 2,000 cells per unit cm 2 on a
  • Example 5 Mitomycin treatment In order to inhibit the cell growth of murine embryonic fibroblasts for feeder cell use, mitomycin C (Sigma, Cat.No.M-4287) was added to culture media at 10 ⁇ g/ml of concentration and reacted for one and a half hours. Then, the resulting cells were analyzed after 14, 28 and 48 hours by using a CelIScreen machine in order to identify whether mitomycin may affect cell survival and cell death of murine embryonic fibroblasts (See FIG. 9-11 ; Experimental Result 3).
  • mitomycin C Sigma, Cat.No.M-4287
  • ⁇ -smooth muscle actin(DAKO) as a monoclonal antibody was used to detect SSEA4 (Chemicon, Cat.No.90231), TRA1-60 (Chemicon, Cat.No. MAB4360) and OCT4 (Santa cruz, Cat.No.sc-5279) markers (See FIG. 17; Experimental Result 6).
  • SSEA4 Chemicon, Cat.No.90231
  • TRA1-60 Chemicon, Cat.No. MAB4360
  • OCT4 Oxera cruz, Cat.No.sc-5279
  • aFP as an endodermic marker(Zymed, Cat.No.18-0003)
  • SMA as mesodermic marker(DAKO, Cat.No.U7033)
  • Tuj1 as an ectodermic marker(Sigma, Cat.No. c- 4585) were selected for antibodies.
  • each cell was fixed with 4% formaldehyde and permeabilized with PBS-T buffer or 1 % PBS and 0.1% Tween-20 buffer in order to perform an immuno-fluorescence staining. Then, the resulting cell was treated with blocking solution (1% PBS, 0.1 bovine serum albumin) to block non- specific antibodies. The resultant was reacted with primary antibodies ( ⁇ -smooth muscle actin) at 4 0 C overnight and then, reacted with FITC-conjugated secondary antibodies (Vector Laboratories).
  • Example 7 Restoration and cultivation of embryonic stem cells
  • Embryonic stem cells frozen in a freezer were restored and then, treated with mitomycin.
  • the stem cells were inoculated on a culture plate in which murine embryonic fibroblasts were previously incubated for a day. Then, the resulting cells were sub-cultured by the same procedure described above. 2 kinds of compositions of culture media, general media for embryonic stem cells(20% SR) and the composition of culture media of the present invention were compared while freshly exchanged everyday (See FIGs. 13, 16; Experimental Result 5, 6).
  • Colonies of embryonic stem cells were cut to proper pieces after cultured confluent.
  • the resulting colons were floated on the culture medium comprising DMEM/F12 medium containing 10% Knockout Serum replacement without bFGF and 2% FBS.
  • the cell colons were cultured by using a hydrocell(Japan) so as to prepare embryoid bodies.
  • the hydrocell is specially-treated to prevent the attachment of stem cells.
  • RNAs were extracted by using Trizol® reagent (Invitrogen, Cat.No.15596-018). Then, the resulting RNAs were reacted with AMV reverse- transcriptase at 42°C for an hour to prepare cDNAs. The resulting cDNAs were amplified by performing a PCR by 30 cycles under 95°C, 45 sec; 55°C 45 sec; 72°C 45 sec. At this moment, ⁇ -Actin, Nanog, Rex1, SOX2, aFP, FLK1 and NCAM 1 primers were used.
  • Example 10 Flow cytometry 2 kinds of human embryonic stem cells cultured respectively by using general culture media and the culture composition of the present invention were suspended to mono-cells.
  • the resulting cells were poured to PBS buffer containing BSA(bovine serum albumin) and reacted with Tral-60 (Chemicon, Cat.No.MAB4360) as a primary antibody at a low temperature for more than 3 hours.
  • Tral-60 Cemicon, Cat.No.MAB4360
  • the resultant was treated for 50 minutes with FITC anti-mouseCJacksonlmmunoResearch, Cat.No.315-095-003) as a secondary antibody and fixed with para-formaldehyde.
  • the stem cells were analyzed by performing a flow cytometry(BD).
  • cell karyotypes were analyzed. Above all, 2 kinds of human embryonic stem cells were cultured confluent by using the culture composition of the present invention. One cell kind had been sub-cultured to several passages(FIG. 20) and the other kind stored in a freezer and restored repeatedly(FIG. 21). Then, the resulting cells were treated with colcemide(0.1 ⁇ g/ml; Invitrogen) and suspended to mono-cells. After fixing the cells, G-band was observed on a slide glass. This karyotyping was conducted in at least 25 cases. As a result, it is identified that the culture composition of the present invention is so safe that embryonic stem cells do not have chromosomal aberrations.
  • Embryonic stem cells were cultured confluent and fixed with para-formaldehyde. Then, the resulting cells were treated with 0.1% Triton-X100 and permeated by Tunel reagent. At this moment, the Tunel reagent and its buffer solution(Roche, Cat.No.11- 684-795-910, Germany) were mixed in 1 : 9 of ratio and treated for more than one and a half hours at 37°C under a dark condition. The resulting cells were reacted with DAPI for 5 minutes in order to perform a nucleus staining. Then, they were observed under a fluorescence microscope to monitor the expression of fluorescence.
  • Example 12 Observation of embryonic stem cells after nicotine amide treatment
  • Human embryonic stem cells were cultured with the culture composition of the present invention in which nicotine amide was added at 10, 50 and 100 ⁇ g/ml of concentration.
  • the stem cells started to be affected remarkably by nicotine amide after more than 6-passages.
  • the stem cells remained most indifferent at 10 ⁇ g/ml of nicotine amide through a RT-PCR 1 .
  • fibroblasts were cultured by using the combinations of ITS, SR and bFGF and measured in the number after 60 hours. The initial number of cells was adjusted to 1 ,000 and 2,000 per unit cm 2 to compare differences according to cell numbers.
  • the initial number of cells was considered proper to be plated at 2,000 per unit cm 2 . Therefore, the initial number of cells was adjusted to 2,000 per unit cm 2 in following experiments.
  • the cell number did not increase sufficiently when using only serum, but increased highly when adding SR or/and ITS.
  • the fibroblasts proliferate most actively when cultured with the composition of culture media(NO. 5 in Table 2) comprising serum, SR and bFGF(See FIG. 1).
  • MEF cell growth at various compositions of culture media(FIG. 1) Cell number/cm 2 at 60 hr
  • FIG. 8 depicts the growth of murine fetal fibroblasts(CFI) by using CellScreen images according to time intervals.
  • 10% FBS was conventional feeder media for murine fibroblasts
  • 2% FBS + 10% SR + bFGF optimized media of the present invention
  • 20% SR + bFGF conventional media for human embryonic stem cell use.
  • the optimized media of the present invention become better to culture murine fibroblasts.
  • Experimental Result 3 Comparison of survival ratios of fibroblasts according to compositions of culture media after mitomycin treatment The cell numbers did not decrease only with culture media NO. 7 and NO. 8 until 46 hours, but decreased with the rest of media (See FIG. 9 to 11). Especially, the cell number of fibroblasts decreased so radically in conventional culture media for embryonic stem cell use after treated with mitomycin. As a result, it is considered that feeder cells may be difficult to survive under general culture media and hardly function due to this bad condition. The same result was observed from both mouse strains of
  • feeder cells are treated with mitomycin to inhibit the cell growth when co-cultured with human embryonic stem cells.
  • the fibroblasts for feeder cell use were observed to measure their maintenance with culture media after treated mitomycin.
  • FIG. 11 depicts CF1 cell survival after treating mitomycin for one and a half hour by using a CellScreen machine. As a result, it is identified that the composition of culture media of the present invention is also outstanding to culture feeder cells.
  • fibroblasts may cross-differentiate toward muscle fibroblasts due to the presence of TGF-b or serum, long-term culture and low-density. But, fibroblasts seldom cross-differentiate under a particular condition.
  • the cell state of fibroblasts was examined by estimating the ratios of cross-differentiation. As a result, it is judged that culture media NO. 7 to 11 may reduce the cross-differentiation(See FIG. 12). The same result was observed from both mouse strains of CR1 and C57Black.
  • human embryonic stem cell lines (HSF6 cub-cultured during 50 passages and Miz4 during 32 passages) were restored from a storage respectively with conventional media for embryonic stem cell use (20% SR + 4 ng/ml bFGF) and the culture media of the present invention (2% FBS + 10% SR + 2 ng/ml bFGF).
  • the resulting cells were seeded at 20 colonies per culture plate and stained by using alkaline phosphatase to measure degree of undifferentiation.
  • the culture media of the present invention become more outstanding to attach cells and increase the number of colonies remaining indifferent by more than 2-fold even after 5 days(See FIG. 13).
  • human embryonic stem cells cultured with the culture media of the present invention (10% SR + 2% FBS) were stored in a freezer and then, restored to measure whether they attach onto feeder cells and how they maintain undifferentiation.
  • FIG. 14 depicts the attachment and the undifferentiation of human embryonic stem cells cultured with the culture media of the present invention after stored in a nitrogen tank for at least one month and restored.
  • FIG. 13a and 13b depicts the cell attachments and cell undifferentiations of human embryonic stem cells that are continuously sub- cultured with the culture media of the present invention, then stored in a freezer and restored after a month. The resulting cells were labeled in undifferentiation markers by using a fluorescence staining. As a result, it is identified that human embryonic stem cells express the undifferentiation markers normally.
  • FIG. 13a and 13b depicts the cell attachments and cell undifferentiations of human embryonic stem cells that are continuously sub- cultured with the culture media of the present invention, then stored in a freezer and restored after a month.
  • the resulting cells were labeled in undifferentiation markers by using a fluorescence staining. As a result, it is identified that human embryonic stem cells express the undifferentiation markers normally.
  • FIG. 15 depicts the undifferentiation markers SSEA4, Tral-60 and Oct4 of human embryonic stem cells cultured with culture media of the present invention(2% FBS, 10% SR, bFGF) after repeatedly restored by using a fluorescence staining(HSF6; scale bar 10 ⁇ m).
  • the result of FIG. 15 illustrates fluorescence-stained markers of human embryonic stem cells that are continuously sub- cultured with the culture media of the present invention, then were stored in a freezer for a month and restored.
  • 3 kinds of human embryonic stem cell lines (HSF6 cub-cultured during 57 and 58 passages; Miz4 during 44 passages; and Miz6 during 35 passages) were cultured with conventional media for embryonic stem cell use (20% SR + 4 ng/ml bFGF) and the culture media of the present invention(2% FBS + 10% SR + 2 ng/ml bFGF).
  • the resulting cells were seeded at 20 colonies per culture plate. As a result, it is identified the after 24 hours, the culture media of the present invention become more outstanding to attach cells. They also increased the number of colonies remaining indifferent even after 5 days remarkably(See FIG. 16).
  • FIG. 17 depicts the undifferentiation markers of human embryonic stem cells by using a fluorescence staining. At this moment, SSEA4, Tral-60 and Oct4 are used as undifferentiation markers.
  • FIG. 17b is conventional media for human embryonic stem cell use(20% SR) and
  • FIG. 17c is the culture media of the present invention(2% FBS, 10% SR).
  • Tral-60 marker may be expressed normally with the culture media of the present invention. Further by performing a RT-PCR, the undifferentiation markers were detected.
  • FIG. 19 depicts the RNA levels of Nanog, Rex1 and SOX2 markers (expression of self- renewal markers) in 10% SR + 2% FBS medium. As a result, it is identified that Tral- 60 marker is expressed normally with the culture media of the present invention.
  • FIG. 20 depicts the chromosomal aberration of human embryonic stem cells cultured with the culture media of the present invention by using a karyotype analysis.
  • FIG. 21 depicts the chromosomal aberration of human embryonic stem cells cultured with the culture media of the present invention after stored in a freezer and restored by using a karyotype analysis.
  • FIG. 22 depicts the cell deaths of human embryonic stem cells cultured with conventional media and the culture media of the present invention by using a Tunel assay(scale bar 10 ⁇ m).
  • FIG. 23 depicts the embryonic bodies of human embryonic stem cells cultured with the culture media of the present invention in order to identify the capacity of tridermic differentiation(whole mount; scale bar 500 ⁇ m).
  • RNA levels of endodermic marker aFP, mesodermic marker FLKI(KDR) and ectodermic marker NCAM 1 by performing a RT-PCR in order to identify the capacity of tridermic differentiation of embryoid bodies of human embryonic stem cells cultured with the culture media of the present invention.
  • human embryonic stem cells were made toward embryoid bodies(EB) and cultured for a week. Then, the resulting cells were labeled in tridermic markers by using a fluorescence staining. As a result, it is identified that because all tridermic markers and all hybrid markers of endoderm, mesoderm and ectoderm are detected, human embryonic stem cells cultured with the culture media of the present invention may maintain all basic characteristics.
  • cytokeratin tridermic hybrid marker and epithelium marker
  • aFP ⁇ -fetoprotein, endodermic marker
  • SMA smooth muscle actin
  • mesodermic marker mesodermic marker
  • Tuj1 blll-Tubulin, ectodermic marker
  • FIG. 26 depicts the human embryonic stem cells treated with nicotine amide in the culture media of the present invention.
  • FIG. 27 depicts the result of RT-PCR of human embryonic stem cell lines HSF6 and Miz4 when treating 0, 10, 50 and 100 ⁇ g/ml of nicotine amide in culture media of the present invention.
  • Oct4, Rex1 and SOX2 are undifferentiation markers.
  • Experimental Result 11 Culture analysis of human adult embryonic stem cells It is identified that the compositions of culture media of the present invention are effective to culture MSC (mesenchymal stem cell), SVF (stroma vascular fraction) and the like.
  • basic media should have glucose in a low level.
  • MSC cells were seeded at 8,000 cells per unit area(cm 2 ) on a culture plate and cultured for 5 days with conventional media and the culture media of the present invention respectively. Then, the resulting cells were observed in their shapes(FIG. 28), estimated in their growth rates(FIG. 29) and calculated in their numbers by using a trypan blue staining (Sigma, Cat.No.T8-154).
  • FIG. 28 depicts the phenotype of MSC(mesenchymal stem cells) cultured for 5 days after seeded at 8,000 cells per unit area.
  • FIG. 29 depicts the cell number of MSC cultured for 5 days.
  • the present invention relates to compositions of culture media for co-culturing stem cells and feeder cells and for a primary culture of cells, by which both stem cells and feeder cells can be cultivated under an optimized condition.
  • compositions of culture media are effective to cultivate fibroblasts for feeder cell use. They are also useful to culture MSC (mesenchymal stem cell) and SVF (stroma vascular fraction) and primarily-cultured skin fibroblasts.
  • MSC mesenchymal stem cell
  • SVF stroma vascular fraction
  • the culture media of the present invention is cheaper and more conveniently optimized for both stem cells and feeder cells than conventional media.
  • this culture media is highly effective to attach stem cells and undifferentiate stem cells right after restored from a freezer. Therefore, the composition of culture media of the present invention is very useful to culture stem cells as remaining indifferent and to produce stem cells in a large scale. Furthermore, this composition of culture media can be widely applied for all kinds of cells, because it is optimized for both stem cells and primarily-cultured cells.

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Abstract

The present invention relates to a culture media composition for co-culturing human stem cells and feeder cells and for culturing a primary cell line. The culture media of the present invention is cheaper and more conveniently optimized for both stem cells and feeder cells than conventional media. Especially, this culture media is highly effective to attach stem cells and undifferentiate stem cells right after restored from a freezer. Therefore, the composition of culture media of the present invention is very useful to culture stem cells as remaining indifferent and to produce stem cells in a large scale. Furthermore, this composition of culture media can be widely applied for all kinds of primarily-cultured cells, because it is optimized for both stem cells and primarily-cultured fibroblasts.

Description

CULTURE MEDIUM FOR CO-CULTURING OF HUMAN STEM CELLS AND THEIR
FEEDER CELLS
Technical Field
The present invention relates to a culture media composition for co-culturing human stem cells and feeder cells and for culturing a primary cell line, by which both stem cells and feeder cells can be cultivated under an optimized condition.
Background Art
In 1981, mouse embryonic stem cells have been first cultured in a test tube. In 1988, this in vitro culture has been combined with a gene targeting technique so as to first make a transformed mouse having a targeted gene. Since that, this gene targeting technique of mice plays an important role to investigate genetic functions and to build up animal models of human diseases. Further, it drives life/medical sciences to develop a lot (Smith A. G., Annu. Rev. Cell Dev. Biol., 17: 435-62, 2001). In contrast to the mice case, human embryonic stem cells started to be cultured in vitro, 17 years later I 1988. Dr. Thomson has first established human embryonic stem cells in University of Wisconsin (Thomson J. A., Science 282(5391): 1145-7, 1998). The human embryonic stem cell is more difficult to be cultured and manipulated than the mouse stem cell. Either, it is improper to be mass-cultured, even if necessary to develop therapeutic agents by performing a gene manipulation or in vitro test. Therefore, it is required to complete a culture method that can proliferate stem cells effectively and control their quality easily.
The culture method popularly used is based upon the procedure Dr. Thompson established. Precisely, mouse embryonic fibroblasts are treated with mitomycin or irradiated in order to inhibit the cell growth. Then, the resulting fibroblasts are inoculated in order to previously express extra-cellular substrates and cytokines for embryonic stem cells' need. After that, embryonic stem cells are inoculated onto the resulting cells for feeder cell use.
As described in Table 1, the compositions of culture media are broadly classified to 2 kinds according to constituents added to basic medium. In particular, the basic media is comprised of DMEM and/or DMEM/F12 (1 : 2) and may contain fibroblast growth factor, glutamine and β-mercaptoethanol. Then into the basic media, 20% serum replacement(lnvitrogen Inc.) and/or 20% fetal bovine serum are added with insulin, transferrin and selenium. The culture media that contains serum replacement instead of serum is improper to survive fibroblasts for feeder cell use. In contrast, the culture media that contains fetal bovine serum tends to differentiate stem cells a lot. The culture medium of fibroblasts for feeder cell use often includes 10% fetal bovine serum, which plays an important role to survive fibroblasts. Therefore, it is clarified that fibroblasts may hardly function as feeder cells in the culture medium of embryonic stem cells containing fetal bovine serum instead of serum replacement(See FIG. 9 to 11 ; Experimental Result 3). Hence, the composition of culture media for culturing embryonic stem cells should be optimized for both stem cells and feeder cells. <Table 1 >
Compositions of culture media for embryonic stem cells
Figure imgf000003_0001
In order to settle above-mentioned problems, the present inventors have tried to develop compositions of culture media that comprises basic media containing fetal bovine serum(FBS), serum replacement(SR) and fibroblast growth factor(bFGF) in particular ratios. Then, we have identified that the compositions may optimize the growth of both stem cells and feeder cells and little differentiate stem cells and completed the present invention successfully.
Disclosure of Invention
The object of the present invention is to overcome conventional disadvantages of the method for culturing human embryonic stem cells. In particular, the object of the present invention is to develop culture media that is optimized to survive both stem cells and feeder cells when culturing human embryonic stem cells and to maintain the capacity of undifferentiation by maximizing the function of feeder cells. Further, the object of the present invention is to improve the attachment and the growth of stem cells after restored from a freezer.
The culture media of the present invention may be optimized to cultivate different kinds of adult stem cells and primarily cultured cells, because it stimulate the growth of fibroblasts effectively in addition to the indifferent stem cells.
Brief Description of the Drawings
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;
FIG. 1 depicts the cell numbers of feeder cells (CF1 , p5) after culturing for 60 hours by using several culture media, (a) seeding at 1,000 cells per unit area(cm2); (b) at 2,000 cells per unit cm2. FIG. 2 depicts the growth/division of feeder cells measured at various compositions of culture media when seeding at 2,000 cells per unit cm2 according to culture periods, (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
FIG. 3 depicts murine fetal fibroblasts (FIG. 2) cultured for 60 hours with several culture media by using a phase contrast microscopy (4OX magnified), (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages. FIG. 4 depicts the comparison of 3 kinds of culture media [general medium of fibroblasts(10% FBS), culture medium of the present invention(2% FBS, 10% SR, bFGF), culture medium for embryonic stem cell use(20% SR, bFGF)] as described in FIG. 2. (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages. FIG. 5 depicts the growth/division of feeder cells measured at various compositions of culture media according to culture periods by observing the cell areas on culture plates with a CellScreen machine(lnnovatis Inc.) when seeding at 2,000 cells per unit cm2, (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages. FIG. 6 depicts the growth of murine fetal fibroblasts cultured at various compositions of culture media by observing a CellScreen image of FIG. 5. (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
FIG. 7 depicts the comparison of 3 kinds of culture media [general medium of fibroblasts(10% FBS), culture medium of the present invention(2% FBS, 10% SR, bFGF), culture medium for embryonic stem cell use(20% SR, bFGF) as described in FIG. 5. (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages. FIG. 8 depicts the growth of murine fetal fibroblasts cultured at various compositions of culture media by observing a CellScreen image of FIG. 7. (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages. FIG. 9 depicts the cell survival of murine fetal fibroblasts according to culture media when treating mitomycin for one and a half hours to stop the cell growth by using a CellScreen image(lnnovatis Inc.). (a) fibroblasts derived from CF1 mice and sub- cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub- cultured during 6 passages. FIG. 10 depicts the comparison of 3 kinds of culture media [general medium of fibroblast^ 10% FBS), culture medium of the present invention(2% FBS, 10% SR, bFGF), culture medium for embryonic stem cell use(20% SR, bFGF) as described in FIG. 9. (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages. FIG. 11 depicts the cell survival after mitomycin treatment by observing a
CellScreen image of FIG. 9. (a) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
FIG. 12 depicts the comparison of cross-differentiation from murine fetal fibroblasts to muscle fibroblasts according to culture media by using an immunofluorescence staining onto α-smooth muscle actin (40X magnified), (a) and (b) fibroblasts derived from CF1 mice and sub-cultured during 5 passages; (b) fibroblasts derived from C57/Black mice and sub-cultured during 6 passages.
FIG. 13 depicts the comparison of attachments and undifferentiations of human embryonic stem cells cultured with general media of embryonic stem cells(20% SR, bFGF) and the culture media of the present invention(2% FBS, 10% SR, bFGF) after stored in a freezer and restored, (a) HSF6 cell line; (b) Miz4 cell line.
FIG. 14 depicts the attachment and the undifferentiation of human embryonic stem cells cultured with the culture media of the present invention(2% FBS1 10% SR, bFGF) after stored in a nitrogen tank and restored for at least one month.
FIG. 15 depicts the undifferentiate markers SSEA4, Tral-60 and Oct4 of human embryonic stem cells cultured with the culture media of the present invention(2% FBS, 10% SR, bFGF) after stored and restored repeatedly by using a fluorescence staining.
FIG. 16 depicts the comparison of attachments and undifferentiations of human embryonic stem cells sub-cultured with general media of embryonic stem cells(20% SR, bFGF) and the culture media of the present invention(2% FBS, 10% SR, bFGF). (a) HSF6 cell line; (b) Miz4 cell line.
FIG. 17 depicts the undifferentiation of human embryonic stem cells sub- cultured with conventional media of embryonic stem cells(20% SR, bFGF) and the culture media of the present invention(2% FBS, 10% SR, bFGF). (a) alkaline phosphatase staining; (b) immuno-fluorescence staining of SSEA4, Tral-60 and Oct4 markers in general media for embryonic stem cell use; (c) immuno-fluorescence staining of SSEA4, Tral-60 and Oct4 markers in the culture media of the present invention.
FIG. 18 depicts the cell surface expression of Tral-60 marker by using a flow cytometry. FIG. 19 depicts the RNA levels of Nanog, Rex1 and SOX2 markers by performing a RT-PCR.
FIG. 20 depicts the chromosomal aberration of human embryonic stem cells cultured with the culture media of the present invention by using a karyotype analysis.
FIG. 21 depicts the chromosomal aberration of human embryonic stem cells that are cultured with the culture media of the present invention after stored in a freezer and restored by using a karyotype analysis.
FIG. 22 depicts the cell deaths of human embryonic stem cells cultured with general media and the culture media of the present invention by using a Tunel assay. FIG. 23 depicts the embryonic bodies of human embryonic stem cells cultured with culture media of the present invention to identify a tridermic differentiation.
FIG. 24 depicts the RNA levels of endodermic marker aFP, mesodermic marker FLKI (KDR) and ectodermic marker NCAM1 by using a RT-PCR to identify the tridermic differentiation of embryoid bodies of human embryonic stem cells cultured with the culture media of the present invention.
FIG. 25 depicts the protein expressions of cytokeratin(tridermic hybrid marker and epithelium marker), aFP(α-fetoprotein, endodermic marker), SMA(smooth muscle actin, mesodermic marker) and Tuj1(blll-Tubulin, ectodermic marker) by using a fluorescence staining to identify the tridermic differentiation of embryoid bodies of human embryonic stem cells cultured with the culture media of the present invention.
FIG. 26 depicts the human embryonic stem cells treated with nicotine amide in the culture media of the present invention.
FIG. 27 depicts the result of RT-PCR of human embryonic stem cell lines HSF6 and Miz4 after nicotine amide treatment at 0, 10, 50 and 100 μg/ml of concentration with culture media of the present invention, in which Oct4, Rex1 and SOX2 are undifferentiation markers.
FIG. 28 depicts the phenotype of MSC(mesenchymal stem cells) cultured for 5 days after seeded at 8,000 cells per unit area. FIG. 29 depicts the cell number of MSC cultured for 5 days.
Best Mode for Carrying Out The Invention
In order to attain the above-mentioned objects, the present invention provides compositions of culture media that help the growth and the self-renewal of human stem cells as remaining indifferent. In the present invention, conventional culture media is modified to contain fetal bovine serum(FBS), serum replacement(SR) and fibroblast growth factor(bFGF) at proper ratios in basic media. In a preferred embodiment, the present invention provides a culture medium composition for co-culturing stem cells and feeder cells, which comprises basic media containing 1 to 10% of fetal bovine serum(FBS), 1 to 20% of serum replacement(SR) and 0.1 to 4 ng/ml of fibroblast growth factor(bFGF). In another preferred embodiment, the present invention provides a culture medium composition for culturing a primary cell line, which comprises basic media containing 1 to 10% of fetal bovine serum(FBS), 1 to 20% of serum replacement(SR) and 0.1 to 4 ng/ml of fibroblast growth factor(bFGF).
It is identified that the culture media compositions of the present invention are effective to culture fibroblasts primarily. This compositions of culture media are also effective to cultivate MSC (mesenchymal stem cell) or SVF (stroma vascular fraction) and primarily-cultured skin fibroblasts. The compositions of culture media for primary culture use are the same with those of stem cells, even if the glucose level is lower.
In the culture media compositions of the present invention, the basic medium can be selected from commercially available basic media, whatever it contains essential elements for animal cell culture such as amino acids, vitamins, minerals and the like. Preferably, this basic medium can be selected from DMEM (Dulbecco's modified eagle medium) and DMEM/F12 (DMEM + nutrient mixture).
In addition, the serum replacement is a product manufactured in order to replace animal serums. The serum replacement is comprised of heat-treated bovine serum albumin, bovine transferrin, bovine insulin and the like. The serum replacement used in the present invention can be selected from commercially available serum replacements. Preferably, the serum replacement can be selected from Omni Serum(Advanced Biotechnologies Inc., Columbia, Md.) and Knockout™ Serum Replacement(lnvitrogen Inc.; See WO098/30679) described in following Examples.
Preferably, the present invention provides a culture medium composition, which comprises basic media containing 1 to 5% of fetal bovine serum(FBS), 5 to 15% of serum replacement(SR) and 1 to 4 ng/ml of fibroblast growth factor(bFGF). Within the ratio of compositions, the culture media increase outstandingly the growth rate of stem cells and to maintain their undifferentiation.
In the culture medium composition of the present invention, the stem cells and primarily-cultured cells can be derived from embryos and/or adult tissues. That is to say, the stem cells can be selected from embryonic stem cells derived from a fetus and adult stem cells derived from a mature tissue and differentiate toward various kinds of cells;
In the culture medium composition of the present invention, the stem cells can be stored in a freezer and restored before use. In contrast, conventional compositions of culture media are disadvantageous to attach stem cells on a culture plate and to remain indifferent after stored in a freezer and restored. The culture medium composition of the present invention is optimized to attach stem cells on a culture plate and to retain the capacity of undifferentiation effectively, even if stored in a freezer and restored before use.
In the culture medium composition of the present invention, the feeder cells can be derived from animals and humans and also derived form embryos or adult tissues, whenever they can secrete various nutrients, cytokines and the like to help stem cells and primarily-cultured cells. Preferably, the feeder cells can be embryonic fibroblasts(MEF) described in following Examples.
Preferably, the culture medium composition of the present invention may contain 3 to 30 μg/ml of nicotine amide additionally. When 10 μg/ml of nicotine amide is added, stem cells can remain indifferent more effectively than those without nicotine amide. In contrast, when more than 50 μg/ml of nicotine amide is added, stem cells are rather negatively affected(See FIG. 27).
In another preferred embodiment, the present invention provides a method for optimizing the growth of stem cells and feeder cells, which comprises culturing the stem cells and feeder cells by using the culture medium composition of the present invention. In this case, "optimization" means to stimulate not only stem cells but also feeder cells to proliferate effectively.
In another preferred embodiment, the present invention provides a method for optimizing the function of feeder cells along with the growth of feeder cells, which comprises culturing the feeder cells by using the culture medium composition of the present invention.
In another preferred embodiment, the present invention provides a method for culturing stem cells by using the culture medium composition of the present invention, which maintains inherent properties of stem cells and/or primarily-cultured cells. In this case, "to maintains inherent properties of stem cells" means to retain the capacity of differentiation of stem cells toward tridermic cells until necessary, even if they usually remain indifferent.
In the other preferred embodiment, the present invention provides a method for optimizing the cell attachment and the undifferentiation of stem cells after stored in a freezer and restored, which comprises culturing the stem cells by using the culture medium composition of the present invention. In this case, "optimization" is to improve the cell attachment and the maintenance of undifferentiation after stem cells are stored in a freezer and restored.
In the present invention, the growth rate of fibroblasts is compared by measuring cell numbers and areas of cell colonies after cultured respectively with conventional media and the culture media of the present invention(See FIG. 1 to 8;
Experimental Result 1 and 2). The ratios of cross-differentiation toward muscle fibroblasts are compared by using an immuno-staining on α-smooth muscle actin(See
FIG. 12; Experimental Result 4). Then, the survival ratios of fibroblasts are examined by measuring the areas of cell colonies after treating mitomycin as a cytostatic agent(See FIG. 9 to 11 ; Experimental Result 3). For this purpose, 2 kinds of fetal fibroblasts derived from mouse strains CF1 and C57/Black are selected after primary cultures, which are commonly used for feeder cells. The same result is observed from both mouse strains. In order to investigate the effects of culture media upon human embryonic stem cells, the initial attachment and the maintenance of undifferentiation of stem cells are measured in their ratios, after restored from a freezer with conventional media and the culture media of the present invention(See FIG. 13; Experimental Result 5). Then, the resulting stem cells are sub-cultured during more than 8 passages and observed to monitor the maintenance of undifferentiation(See FIG. 16; Experimental Result 6). In this case, 3 kinds of human embryonic stem cell lines including of HSF6, Miz4 and Miz6 are used. In order to identify degrees of differentiation, the morphological characteristics of cell colonies are observed by using an alkaline phosphatase staining(See FIG. 17).
EXAMPLES
Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1 : Cell culture and composition of culture media
Murine embryonic fibroblasts(MEF) were extracted from 13.5 day-pregnant mice (CFI, C57BL6) and cultured primarily with following culture media. The basic media was comprised of DMEM/F12 media(GIBCO, USA, Cat.No. 12500-062) mixed with 3.069 g/l sodium bicarbonate (Sigma, USA, Cat.No. S5761), 2 mM L-glutamine (Sigma, Cat.No. S8540), penicillin(50 U/ml)(Sigma, Cat.No. P4687)/streptomycin (50 μg/ml)(Sigma, USA, Cat.No.S1277). Then, 10 % fetal bovine serum(FBS; Hyclone, Cat.No. SH30070.03) or Knock-Out serum replacement(SR; Invitrogen BRL,
Cat.No.10828-028), Basic Fibroblasts Growth Factor (bFGF; Invitrogen BRL, Cat.No. 13256-029) and Insulin-Transferrin-Sodium Selenite Media Supplement(ITS; Sigma, Cat. No. 1-1884) were blended additionally at particular ratios. The resulting cells were cultured at 37°C in 5% CO2 incubator (See Table 2 and 3a) while changing culture media freshly every other day. In order to examine the proper cell number, the inoculated cells were adjusted to 1 ,000 and 2,000 per unit cm2 (See Experimental Result 1). As a result, it is clarified that 2,000 per unit cm2 of the cell number is more reasonable.
Example 2: Measurement of cell numbers After stopping cell growth, the resulting cells were washed off with PBS buffer and treated with 0.25 % Trypsin(2.5 g/L)-EDTA(0.38 g/L)(lnvitrogen BRL,
Cat.No.25200-114). Then, the cells were collected and stained with 0.4% Trypan blue
(Sigma, Cat.No. T8154) solution to measure cell numbers with a haematocytometer
(See FIG. 1 , 2, 4, 5, 7, 9 and 10; Experimental Result 1 and 2).
Example 3: Alkaline phosphatase staining
Human embryonic stem cells were sorted by using alkaline phosphatase staining. Stained colonies of stem cells were judged to remain indifferent and counted.
Clear colonies without staining were judged to differentiate and calculated (See FIG. 13 and 14; Experimental Result 5 and 6). In order to conduct alkaline phosphatase staining, NBT/BCIP(Roche, Germany, Cat. No. 1 681 451) solution was added to Tris-
CI(pH 9.5) buffer in 99 : 1 of ratio and reacted to monitor coloring reactions.
Example 4: CellScreen analysis Murine embryonic fibroblasts were inoculated at 2,000 cells per unit cm2 on a
96-well plate and estimated by the area % occupied according to time intervals(14, 26, 40, 52, 65 and 84 hours after starting cell culture)(See FIG. 5, 7, 9 and 10; Experimental
Result 2 and 3). Then, cell distribution was measured by using an image analysis with a CelIScreen machine(lnnovatis Inc.) at each time interval (See FIG. 6, 8 and 11; Experimental Result 2 and 3).
Example 5: Mitomycin treatment In order to inhibit the cell growth of murine embryonic fibroblasts for feeder cell use, mitomycin C (Sigma, Cat.No.M-4287) was added to culture media at 10 μg/ml of concentration and reacted for one and a half hours. Then, the resulting cells were analyzed after 14, 28 and 48 hours by using a CelIScreen machine in order to identify whether mitomycin may affect cell survival and cell death of murine embryonic fibroblasts (See FIG. 9-11 ; Experimental Result 3).
Example 5: Immunofluorescence staining
In order to examine the cross-differentiation from fibroblasts to muscle fibroblasts(See FIG. 12; Experimental Result 4), α-smooth muscle actin(DAKO) as a monoclonal antibody was used to detect SSEA4 (Chemicon, Cat.No.90231), TRA1-60 (Chemicon, Cat.No. MAB4360) and OCT4 (Santa cruz, Cat.No.sc-5279) markers (See FIG. 17; Experimental Result 6). In order to identify tridermic differentiation, human embryonic stem cells were made to embryoid bodies. Then, several markers including aFP as an endodermic marker(Zymed, Cat.No.18-0003), SMA as mesodermic marker(DAKO, Cat.No.U7033) and Tuj1 as an ectodermic marker(Sigma, Cat.No. c- 4585) were selected for antibodies. Above all, each cell was fixed with 4% formaldehyde and permeabilized with PBS-T buffer or 1 % PBS and 0.1% Tween-20 buffer in order to perform an immuno-fluorescence staining. Then, the resulting cell was treated with blocking solution (1% PBS, 0.1 bovine serum albumin) to block non- specific antibodies. The resultant was reacted with primary antibodies (α-smooth muscle actin) at 40C overnight and then, reacted with FITC-conjugated secondary antibodies (Vector Laboratories). Example 7: Restoration and cultivation of embryonic stem cells
Embryonic stem cells frozen in a freezer were restored and then, treated with mitomycin. The stem cells were inoculated on a culture plate in which murine embryonic fibroblasts were previously incubated for a day. Then, the resulting cells were sub-cultured by the same procedure described above. 2 kinds of compositions of culture media, general media for embryonic stem cells(20% SR) and the composition of culture media of the present invention were compared while freshly exchanged everyday (See FIGs. 13, 16; Experimental Result 5, 6).
Example 8: Formation of embryoid body
Colonies of embryonic stem cells were cut to proper pieces after cultured confluent. The resulting colons were floated on the culture medium comprising DMEM/F12 medium containing 10% Knockout Serum replacement without bFGF and 2% FBS. Then the cell colons were cultured by using a hydrocell(Japan) so as to prepare embryoid bodies. The hydrocell is specially-treated to prevent the attachment of stem cells.
Example 9: RT-PCR
From human embryonic stem cells cultured with the culture compositions of the present invention and embryoid bodies prepared above, RNAs were extracted by using Trizol® reagent (Invitrogen, Cat.No.15596-018). Then, the resulting RNAs were reacted with AMV reverse- transcriptase at 42°C for an hour to prepare cDNAs. The resulting cDNAs were amplified by performing a PCR by 30 cycles under 95°C, 45 sec; 55°C 45 sec; 72°C 45 sec. At this moment, β-Actin, Nanog, Rex1, SOX2, aFP, FLK1 and NCAM 1 primers were used.
Example 10: Flow cytometry 2 kinds of human embryonic stem cells cultured respectively by using general culture media and the culture composition of the present invention were suspended to mono-cells. The resulting cells were poured to PBS buffer containing BSA(bovine serum albumin) and reacted with Tral-60 (Chemicon, Cat.No.MAB4360) as a primary antibody at a low temperature for more than 3 hours. Then, the resultant was treated for 50 minutes with FITC anti-mouseCJacksonlmmunoResearch, Cat.No.315-095-003) as a secondary antibody and fixed with para-formaldehyde. The stem cells were analyzed by performing a flow cytometry(BD).
Example 11: Cytogenetic analysis
In order to observe chromosomal aberrations, cell karyotypes were analyzed. Above all, 2 kinds of human embryonic stem cells were cultured confluent by using the culture composition of the present invention. One cell kind had been sub-cultured to several passages(FIG. 20) and the other kind stored in a freezer and restored repeatedly(FIG. 21). Then, the resulting cells were treated with colcemide(0.1 μg/ml; Invitrogen) and suspended to mono-cells. After fixing the cells, G-band was observed on a slide glass. This karyotyping was conducted in at least 25 cases. As a result, it is identified that the culture composition of the present invention is so safe that embryonic stem cells do not have chromosomal aberrations.
Example 11 : Tunel assay
Embryonic stem cells were cultured confluent and fixed with para-formaldehyde. Then, the resulting cells were treated with 0.1% Triton-X100 and permeated by Tunel reagent. At this moment, the Tunel reagent and its buffer solution(Roche, Cat.No.11- 684-795-910, Germany) were mixed in 1 : 9 of ratio and treated for more than one and a half hours at 37°C under a dark condition. The resulting cells were reacted with DAPI for 5 minutes in order to perform a nucleus staining. Then, they were observed under a fluorescence microscope to monitor the expression of fluorescence. Example 12: Observation of embryonic stem cells after nicotine amide treatment
Human embryonic stem cells were cultured with the culture composition of the present invention in which nicotine amide was added at 10, 50 and 100 μg/ml of concentration. When observing phenotypes, the stem cells started to be affected remarkably by nicotine amide after more than 6-passages. As a result, it is identified that the stem cells remained most indifferent at 10 μg/ml of nicotine amide through a RT-PCR1.
Experimental Result 1 : Components of culture media suitable for cell growth and division of fibroblasts
In order to examine effects of culture media upon cell growth/division, fibroblasts were cultured by using the combinations of ITS, SR and bFGF and measured in the number after 60 hours. The initial number of cells was adjusted to 1 ,000 and 2,000 per unit cm2 to compare differences according to cell numbers.
When seeding at 2,000 per unit cm2, the cell number increased by more than 4- fold after 60 hours. In contrast when seeding at 1 ,000 per unit cm2, the cell number increased by only 2-fold after 60 hours. As a result, the initial number of cells was considered proper to be plated at 2,000 per unit cm2. Therefore, the initial number of cells was adjusted to 2,000 per unit cm2 in following experiments.
As depicted in FIG. 1 , the cell number did not increase sufficiently when using only serum, but increased highly when adding SR or/and ITS. Hence, it is identified that the fibroblasts proliferate most actively when cultured with the composition of culture media(NO. 5 in Table 2) comprising serum, SR and bFGF(See FIG. 1). <Table 2>
MEF (CF1) cell growth at various compositions of culture media(FIG. 1) Cell number/cm2 at 60 hr
Media components
Plated at 1 ,000cells/cm2 Plated at 2,000cells/cm2
1 10%FBS 2,363 8,273
2 10%FBS+ITS 4,727 1 1 ,818
20%SR
3 4ng/ml bFGF 4,255 7,092
20%SR
4 3,545 8,273 4ng/ml bFGF+ITS
10%SR+5%FBS
5 2ng/ml bFGF 5,909 16,545
1Q%SR+5%FBS
3,545 14, 182 2ng/ml bFGF+ITS
Experimental Result 2: Composition of culture media suitable for cell growth and division of fibroblasts In order to examine a proper composition of culture media, 2 to 10% of fetal bovine serum was added and mixed with various ratios of SR, ITS and bFGF. Then, several SR media containing bFGF were compared how affecting the growth of fibroblasts(FIG. 2 to 6).
As a result, the fibroblast proliferated most actively when cultured with fetal bovine serum containing SR and bFGF(NO. 7 and 8 in Table 3a), but most weakly when cultured with simple SR media. In addition, this data is observed to correspond to the data of cell areas measured with a CellScreen machine. Also, the same result was obtained from both mouse strains of CR1 and C57Black.
<Table 3a> MEF(CFI) cell growth at various compositions of culture media plated at 2,000 cells/cm2 (FIG. 2a) Culturing Time
Media components Ohr 30hr 60hr
1 10%FBS 2,000 2,272 4,563
2 5%FBS 2,000 1,993 3,484
3 2%FBS 2,000 2,204 3,396
4 2%FBS+ITS 2,000 2,873 3,662
5 2%FBS+FGF 2,000 2,777 3,484
6 2%FBS+10%SR 2.000 3,790 4,080
7 2%FBS+10%SR+FGF 2,000 4.373 5,368
8 2%FBS+5%SR+FGF 2,000 4,676 5.537
9 20%SR+FGF 2,000 2,400 3,244
10 10%SR+FGF 2,000 2,608 2,680
11 5%SR+FGF 2,000 2,044 1 , 360
<Table 3b>
MEF(C57Black) cell growth at various compositions of culture media, plated at 2,000 cells/cm2 (FIG.2b)
Culturing Time
Media components Ohr 30hr 60hf
1 10%FBS 2,000 2,932 5,035
2 5%FBS 2.000 2,200 2 765
3 2%F8S 2,000 2,542 2,285
4 2%FBS+ITS 2,000 2,328 2 840
5 2%FBS+bFGF 2,000 2,790 3.617
6 2%FBS + 1 0%SR 2,000 2,305 2,907
7 2%FBS+10%SR+bFGF 2,000 2,907 4,649
8 2%FBS+5%SR+bFGF 2,000 2,837 5, 128
9 20%SR+bFGF 2,000 1 ,477 2,205
10 10%SR+bFGF 2,000 1 ,773 2,577
1 1 5%SR+bFGF 2,000 1 ,832 2.050
<Table 4a> MEF(CFI) cell growth in conventional feeder media(10% FBS), optimized media(2% FBS + 10% SR + bFGF) and ES media(20% SR + bFGF), plated at 2,000 cells/cm2 (FIG. 4a)
Culturing Time
Ohr 30hr 60hr
10XFBS 2,000 2,272 4,563
2%FBS+ 10%SR+bFGF 2,000 4,378 5, 368
20%SR + bFGF 2,000 2,400 3, 244 <Table 4b>
MEF(C57Black) cell growth in conventional feeder media(10% FBS), optimized media(2% FBS + 10% SR + bFGF) and ES media(20% SR + bFGF), plated at 2,000 cells/cm2 (FIG.4b)
Culturing Time
Media components Ohr 30hr 60hr
1 10%FBS 2,000 2,932 5,035
7 2%FBS+10%SR + bFGF 2,000 2,907 4,649
9 20%SR + bFGF 2,000 1,773 2,577
<Table 5a>
MEF(CFI) cell growth measured by CellScreen (Innovatis inc.)(FIG.5a)
1 2 3 4 5 6 7 8 9 10 11
2% 2%
Culture ,ftβ, ™ ™ 2% 2% 2% 20% 10% 5%
T Tiimmee 10% 5% 2% F3S + FBS+ Fes FBS FBS FBS+ FBS FBS SR+ SR+ SR+
1D%SR 5%SR+ bFGF bFGF bFGF
+bFGF bFGF
(hj
0 51.80 54,32 55.02 56.17 59. 12 59 .38 57,77 65.73 50,81 54,71 52,08
14 64.79 64.14 6201 62.84 66. 80 72 .57 73.82 82.63 64.61 69.73 61.53
26 6612 64.96 64.81 63.45 71. 17 68 .03 68.75 79.79 66.11 69.28 59.97
40 77.95 69,95 69.07 66.16 73. 41 75 .15 84.52 87,49 63,64 65,05 49,09
52 73.63 70.86 55,12 65.36 75. 88 77 .84 90,49 92.16 74.72 76,89 49,39
65 76.62 68.28 52,91 5640 61, 71 72 .38 86,51 87.16 70.69 71,44 8.99
78.78 75.13 6024 53.71 71.92 80.87 87.94 87.42 75.09 70.17 7,05 <Table 5b>
MEF(C57Black) cell growth measured by CellScreen (Innovatis lnc.)(FIG. 5b)
1 2 3 4 5 6 7 8 9 10 11
Culture 2%FBS+ 2%FBS
2%FBS 2%FBS 2%FBS+ 20%SR 10%SR 5%SR+ Time 10%FBS 5%FBS 2%FBS 10%ΛSR +5%SR +ITS ÷bFGF 10%SR +bFGF +bFGF bFGF +bFGF +bFGF
0 36 31 38.08 45.71 44 1 1 36 61 36.59 38.38 39.46 33.14 32.51 36 46
14 50,33 55.34 61.26 61.53 46.34 48.78 50.08 51 ,44 43.69 44.25 39 91
26 56.07 54.39 64. 15 60.78 46.24 45.85 48,49 48,96 42.56 45, 87 36 37
40 58.49 53.70 58.88 56.43 43.41 46.51 58,31 53.60 41.65 44, 40 22 73
52 68.29 61 07 62.48 59 91 45 37 55.96 75 82 67 94 53.64 58.03 18 52
65 70 67 65. 16 49.39 47. 17 43. 15 54.71 87.67 71.68 57.38 62.91 7.26
84 82 96 73,69 53, 50 51 78 45, 00 58.42 93.09 84,90 69.93 71.63 4, 27
<Table 6a>
MEF(CFI) cell growth measured by CellScreen (Innovatis Inc.) in conventional feeder media, optimized media and ES media, plated at 2,000 cells/cm2 (FIG. 7a)
1 7 9
Culture TimeCh) 10%FBS 2%FBS+ 10%SR+bFGF 20%SR+bFGF
0 51.8 57, 77 50.81
14 64.79 73.82 64, 61
26 66. 12 68,75 66.1 1
40 77,95 84, 52 63.64
52 73,63 90.49 74.72
65 76,62 86, 51 70,69
84 78.78 87,94 75.09
<Table 6b> MEF(C57Black) cell growth measured by CellScreen (Innovatis Inc.) in conventional feeder media, optimized media and ES media, plated at 2,000 cells/cm2 (FIG. 7b)
1 7 9
Culture 2%FBS+ 10%SR+bFG 20%SR+bFG
10%FBS Time (h) F F
0 36.31 38.38 33. 1 4
14 50.33 50.08 43.69
26 56, 07 48.49 42.56
40 58.49 58, 31 41 .65
52 68, 29 75.82 53.64
65 70.67 87.67 57.38
84 82.96 93.09 69.93
It is identified that the composition of culture media of the present invention is outstanding to culture murine fibroblasts for feeder cell use. FIG. 8 depicts the growth of murine fetal fibroblasts(CFI) by using CellScreen images according to time intervals. At this moment, 10% FBS was conventional feeder media for murine fibroblasts; 2% FBS + 10% SR + bFGF, optimized media of the present invention; and 20% SR + bFGF, conventional media for human embryonic stem cell use. As a result, it is proved that the optimized media of the present invention become better to culture murine fibroblasts.
Experimental Result 3: Comparison of survival ratios of fibroblasts according to compositions of culture media after mitomycin treatment The cell numbers did not decrease only with culture media NO. 7 and NO. 8 until 46 hours, but decreased with the rest of media (See FIG. 9 to 11). Especially, the cell number of fibroblasts decreased so radically in conventional culture media for embryonic stem cell use after treated with mitomycin. As a result, it is considered that feeder cells may be difficult to survive under general culture media and hardly function due to this bad condition. The same result was observed from both mouse strains of
CR1 and C57Black.
<Table 7a>
MEF(CFI) cell number change at 1.5 hours after MMC treatment measured by
CellScreen (Innovatis lnc.)(FIG. 9a)
1 2 3 4 5 6 7 8 9 10 11
Culture 2% 2%
2% 2% 2% 20% 10% 5%
5% Time 10% FBS+ FBS+
FBS 2% S FBS FBS+ SR+ SR+ SP+ FBS FBS FB 10%SR 5%SR+ (h) +ITS +bF6F 10%SR bFGF bFGF bFGF +bFGF bFGF
0 55 , 589 59.378 55 .916 55.734 53. 19 51.371 55. 197 59 627 55.086 54.983 56.757
14 54 875 59 316 57 194 52.271 50.689 53.619 57 739 62.046 55.882 50 308 71 855
28 51 71 1 59 041 57 .058 50, 795 47.636 51.962 72.325 76.873 61.721 55.996 62 29
46 49 367 53.092 57 .994 48.59 47 132 50.32 73.842 73.985 47.465 39.041 44. 104
<Table 7b>
MEF(C57Black) cell number change at 1.5 hours after MMC treatment measured by CellScreen (Innovatis InC)(FIG. 9b)
1 2 3 4 5 6 7 8 9 10 11
2%FBS 2%FBS
Culture 10%FBS 2%FBS 2%FBS 2%FBS 20%SR 10%SR 5%SR
5%FBS 2%FBS + 10%SR +5%SR Time (h) +ITS +bFGF + 10%SR +bFGF +bFGF +bFGF
+bFGF +bFGF
0 25,678 29,717 24.164 22.456 23.472 24.496 17,988 21 ,471 18,641 21.79 13.4
14 40,078 38.633 31.681 31.507 32.061 22.649 27,904 32,349 26.171 24.634 17 159
28 49,452 41 ,594 29.506 25,954 29, 186 23,841 30 597 32,31 22.076 15.465 9,529
46 48,573 46,382 26.232 25.518 23.489 24,082 31.33 28,286 16.8 9,8009 6 1 124
<Table 8a> MEF(CFI) cell number change at 1.5 hours after MMC treatment measured by CellScreen (Innovatis Inc.) in conventional feeder media, optimized media and ES media. (FIG. 10a)
1 7 9
2%FBS+ 10%SR+
Culture Time (h) 10%FBS 20%SR+bFGF bFGF
Ohr 55.589 55. 197 55.086
14hr 54.875 57.739 55.882
28hr 51 ,711 72.325 61 , 721
46hr 49.367 73.842 47, 465
<Table 8b>
MEF(C57Black) cell number change at 1.5 hours after MMC treatment measured by CellScreen (Innovatis Inc.) in conventional feeder media, optimized media and ES media. (FIG. 10b)
1 7 9
Culture Time (h) 10%FBS 2%FBS+10%SR+bFGF 20%SR+bFGF
0 25.678 17, 988 18.641
14 40.078 27, 904 26. 171
28 49.452 30.597 22.076
46 48.573 31 ,33 16.8
In general, feeder cells are treated with mitomycin to inhibit the cell growth when co-cultured with human embryonic stem cells. The fibroblasts for feeder cell use were observed to measure their maintenance with culture media after treated mitomycin. FIG. 11 depicts CF1 cell survival after treating mitomycin for one and a half hour by using a CellScreen machine. As a result, it is identified that the composition of culture media of the present invention is also outstanding to culture feeder cells.
Experimental Result 4: Maintenance of characteristics of fibroblast according to composition of culture media
In reference, fibroblasts may cross-differentiate toward muscle fibroblasts due to the presence of TGF-b or serum, long-term culture and low-density. But, fibroblasts seldom cross-differentiate under a particular condition. The cell state of fibroblasts was examined by estimating the ratios of cross-differentiation. As a result, it is judged that culture media NO. 7 to 11 may reduce the cross-differentiation(See FIG. 12). The same result was observed from both mouse strains of CR1 and C57Black.
Experimental Result 5: Maintenance of cell attachment and undifferentiation of human embryonic stem cells after restoration according to compositions of culture media
2 kinds of human embryonic stem cell lines(HSF6 cub-cultured during 50 passages and Miz4 during 32 passages) were restored from a storage respectively with conventional media for embryonic stem cell use (20% SR + 4 ng/ml bFGF) and the culture media of the present invention (2% FBS + 10% SR + 2 ng/ml bFGF). The resulting cells were seeded at 20 colonies per culture plate and stained by using alkaline phosphatase to measure degree of undifferentiation. As a result, it is identified that after restored for 24 hours, the culture media of the present invention become more outstanding to attach cells and increase the number of colonies remaining indifferent by more than 2-fold even after 5 days(See FIG. 13). In addition, human embryonic stem cells cultured with the culture media of the present invention(10% SR + 2% FBS) were stored in a freezer and then, restored to measure whether they attach onto feeder cells and how they maintain undifferentiation.
FIG. 14 depicts the attachment and the undifferentiation of human embryonic stem cells cultured with the culture media of the present invention after stored in a nitrogen tank for at least one month and restored. FIG. 13a and 13b depicts the cell attachments and cell undifferentiations of human embryonic stem cells that are continuously sub- cultured with the culture media of the present invention, then stored in a freezer and restored after a month. The resulting cells were labeled in undifferentiation markers by using a fluorescence staining. As a result, it is identified that human embryonic stem cells express the undifferentiation markers normally. FIG. 15 depicts the undifferentiation markers SSEA4, Tral-60 and Oct4 of human embryonic stem cells cultured with culture media of the present invention(2% FBS, 10% SR, bFGF) after repeatedly restored by using a fluorescence staining(HSF6; scale bar 10 μm). Compared to the result of FIG. 17b and 17c, the result of FIG. 15 illustrates fluorescence-stained markers of human embryonic stem cells that are continuously sub- cultured with the culture media of the present invention, then were stored in a freezer for a month and restored.
Experimental Result 6: Maintenance of cell attachment and undifferentiation of human embryonic stem cells according to compositions of culture media
3 kinds of human embryonic stem cell lines(HSF6 cub-cultured during 57 and 58 passages; Miz4 during 44 passages; and Miz6 during 35 passages) were cultured with conventional media for embryonic stem cell use (20% SR + 4 ng/ml bFGF) and the culture media of the present invention(2% FBS + 10% SR + 2 ng/ml bFGF). The resulting cells were seeded at 20 colonies per culture plate. As a result, it is identified the after 24 hours, the culture media of the present invention become more outstanding to attach cells. They also increased the number of colonies remaining indifferent even after 5 days remarkably(See FIG. 16). This result was obtained during 8- passages by using an immuno-staining and an alkaline phosphatase staining(See FIG. 17). It is also clarified that the indifferent stem cells may be sub-cultured until more than 13 passages. FIG. 17 depicts the undifferentiation markers of human embryonic stem cells by using a fluorescence staining. At this moment, SSEA4, Tral-60 and Oct4 are used as undifferentiation markers. FIG. 17b is conventional media for human embryonic stem cell use(20% SR) and FIG. 17c is the culture media of the present invention(2% FBS, 10% SR). By performing a flow cytometric analysis, the undifferentiation marker Tral-60 was detected expressed onto the cell surface. FIG. 18 depicts the cell surface expression of Tral-60 marker by using a flow cytometry. As a result, it is identified that Tral-60 marker may be expressed normally with the culture media of the present invention. Further by performing a RT-PCR, the undifferentiation markers were detected.
FIG. 19 depicts the RNA levels of Nanog, Rex1 and SOX2 markers (expression of self- renewal markers) in 10% SR + 2% FBS medium. As a result, it is identified that Tral- 60 marker is expressed normally with the culture media of the present invention.
Experimental Result 7: Karyotyping of human embryonic stem cells
Human embryonic stem cells cultured with the culture media of the present invention were examined by using a karyotype analysis. As a result, it is proved that the culture media of the present invention is so safe not to have chromosomal aberrations. FIG. 20 depicts the chromosomal aberration of human embryonic stem cells cultured with the culture media of the present invention by using a karyotype analysis.
Also, human embryonic stem cells cultured with the culture media of the present invention were examined by using a karyotype analysis, after stored in a freezer and restored. FIG. 21 depicts the chromosomal aberration of human embryonic stem cells cultured with the culture media of the present invention after stored in a freezer and restored by using a karyotype analysis. As a result, it is identified that the culture media of the present invention be harmless without chromosomal aberrations Therefore, the culture media of the present invention is proved safe. Experimental Result 8: Tunel assay of human embryonic stem cells
In order to examine the death of human embryonic stem cells according to culture media, Tunel assay was performed to measure degrees of fluorescence under a fluorescence microscope. As a result, it is observed that the stem cells cultured with culture media of the present invention appeared more highly florescent than those cultured with conventional media. FIG. 22 depicts the cell deaths of human embryonic stem cells cultured with conventional media and the culture media of the present invention by using a Tunel assay(scale bar 10 μm).
Experimental Result 9: Examination of tridermic differentiation of human embryonic stem cells
In order to examine the capacity of tridermic differentiation, human embryonic stem cells were made toward embryoid bodies(EB) and RT-PCR was conducted. As a result, it is identified that because all tridermic markers are expressed in RNA levels, the culture media of the present invention is proper to culture human embryonic stem cells and maintains basic characteristics. At this moment, β-actin is control; afP, endodermic marker; FLKI(KDR), mesodermic marker; and NCAM1 , ectodermic marker. FIG. 23 depicts the embryonic bodies of human embryonic stem cells cultured with the culture media of the present invention in order to identify the capacity of tridermic differentiation(whole mount; scale bar 500μm). FIG. 24 depicts the RNA levels of endodermic marker aFP, mesodermic marker FLKI(KDR) and ectodermic marker NCAM 1 by performing a RT-PCR in order to identify the capacity of tridermic differentiation of embryoid bodies of human embryonic stem cells cultured with the culture media of the present invention.
In order to examine the capacity of tridermic differentiation, human embryonic stem cells were made toward embryoid bodies(EB) and cultured for a week. Then, the resulting cells were labeled in tridermic markers by using a fluorescence staining. As a result, it is identified that because all tridermic markers and all hybrid markers of endoderm, mesoderm and ectoderm are detected, human embryonic stem cells cultured with the culture media of the present invention may maintain all basic characteristics. FIG. 25 depicts the protein expressions of cytokeratin(tridermic hybrid marker and epithelium marker), aFP(α-fetoprotein, endodermic marker), SMA(smooth muscle actin, mesodermic marker) and Tuj1 (blll-Tubulin, ectodermic marker) in order to identify the tridermic differentiation after forming embryoid bodies of human embryonic stem cells cultured with culture media of the present invention.
Experimental Result 10: Observation of embryonic stem cells after nicotine amide treatment
2 kinds of human embryonic stem cells, HSF6 and Miz4 were treated with nicotine amide and their phenotypes were observed. At 10 to 50 μg/ml of nicotine amide, the stem cells appeared good and especially at 50 μg/ml of nicotine amide, became better in respect of their phenotypes. But, the resulting cells tend to differentiate highly at 100 μg/ml of nicotine amide, as continuously sub-cultured. At this moment, the control group was cultured with the culture media of the present invention without nicotine amide. In contrast, when Oct4, Rex1 and SOX2 markers were detected by using a RT-PCR, human embryonic stem cells remained indifferent in the control group and at 10 μg/ml of nicotine amide. Especially, the stem cells appeared best at 10 μg/ml of nicotine amide. FIG. 26 depicts the human embryonic stem cells treated with nicotine amide in the culture media of the present invention. FIG. 27 depicts the result of RT-PCR of human embryonic stem cell lines HSF6 and Miz4 when treating 0, 10, 50 and 100 μg/ml of nicotine amide in culture media of the present invention. At this moment, Oct4, Rex1 and SOX2 are undifferentiation markers.
Experimental Result 11 : Culture analysis of human adult embryonic stem cells It is identified that the compositions of culture media of the present invention are effective to culture MSC (mesenchymal stem cell), SVF (stroma vascular fraction) and the like. For this purpose, basic media should have glucose in a low level. MSC cells were seeded at 8,000 cells per unit area(cm2) on a culture plate and cultured for 5 days with conventional media and the culture media of the present invention respectively. Then, the resulting cells were observed in their shapes(FIG. 28), estimated in their growth rates(FIG. 29) and calculated in their numbers by using a trypan blue staining (Sigma, Cat.No.T8-154). As a result, it is clarified that the culture media of the present invention increase the growth rate more highly. FIG. 28 depicts the phenotype of MSC(mesenchymal stem cells) cultured for 5 days after seeded at 8,000 cells per unit area. FIG. 29 depicts the cell number of MSC cultured for 5 days.
Industrial Applicability
As illustrated and confirmed above, the present invention relates to compositions of culture media for co-culturing stem cells and feeder cells and for a primary culture of cells, by which both stem cells and feeder cells can be cultivated under an optimized condition.
When using the culture media of the present invention, human embryonic stem cells are observed to become healthier, maintain the capacity of undifferentiation effectively and seldom have any chromosomal aberration. In addition, this compositions of culture media are effective to cultivate fibroblasts for feeder cell use. They are also useful to culture MSC (mesenchymal stem cell) and SVF (stroma vascular fraction) and primarily-cultured skin fibroblasts. Besides, the culture media of the present invention is cheaper and more conveniently optimized for both stem cells and feeder cells than conventional media. Especially, this culture media is highly effective to attach stem cells and undifferentiate stem cells right after restored from a freezer. Therefore, the composition of culture media of the present invention is very useful to culture stem cells as remaining indifferent and to produce stem cells in a large scale. Furthermore, this composition of culture media can be widely applied for all kinds of cells, because it is optimized for both stem cells and primarily-cultured cells.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention.
Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

Claims
1. A culture medium composition for co-culturing stem cells and feeder cells, which comprises basic media containing 1 to 10% of fetal bovine serum(FBS), 1 to 20% of serum replacement(SR) and 0.1 to 4 ng/ml of fibroblast growth factor(bFGF).
2. A culture medium composition for culturing a primary cell line, which comprises basic media containing 1 to 10% of fetal bovine serum(FBS), 1 to 20% of serum replacement(SR) and 0.1 to 4 ng/ml of fibroblast growth factor(bFGF).
3. The culture medium composition according to claim 1 or 2, in which the basic medium is selected from DMEM and DMEM/F12.
4. The culture medium composition according to claim 1 or 2, which comprises basic media containing 1 to 5% of fetal bovine serum(FBS), 5 to 15% of serum replacement(SR) and 1 to 4 ng/ml of fibroblast growth factor(bFGF).
5. The culture medium composition for co-culturing stem cells and feeder cells according to claim 1 , in which the stem cells are embryonic stem cells or adult stem cells.
6. The culture medium composition for co-culturing stem cells and feeder cells according to claim 1 , in which the stem cells are stored in a freezer and restored.
7. The culture medium composition for co-culturing stem cells and feeder cells according to claim 1 , which contains 3 to 30 μg/ml of nicotine amide additionally.
8. A method for optimizing the growth of stem cells and feeder cells, which comprises culturing the stem cells and feeder cells by using the culture medium composition of claim 1.
9. A method for optimizing the function of feeder cells along with the growth of feeder cells, which comprises culturing the feeder cells by using the culture medium composition of claim 1.
10. A method for culturing stem cells by using the culture medium composition of claim 1 , which maintains inherent properties of stem cells.
11. A method for optimizing the cell attachment and the undifferentiation of stem cells after stored in a freezer and restored, which comprises culturing the stem cells by using the culture medium composition of claim 1.
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