CN107475188A - A kind of cultural method of cell culture medium and embryonic stem cell - Google Patents
A kind of cultural method of cell culture medium and embryonic stem cell Download PDFInfo
- Publication number
- CN107475188A CN107475188A CN201710875128.6A CN201710875128A CN107475188A CN 107475188 A CN107475188 A CN 107475188A CN 201710875128 A CN201710875128 A CN 201710875128A CN 107475188 A CN107475188 A CN 107475188A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- cell
- culture
- concentration
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001671 embryonic stem cell Anatomy 0.000 title claims abstract description 34
- 239000006143 cell culture medium Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 10
- 210000000130 stem cell Anatomy 0.000 claims abstract description 80
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 34
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims abstract description 34
- 239000003636 conditioned culture medium Substances 0.000 claims abstract description 17
- 239000012679 serum free medium Substances 0.000 claims abstract description 9
- 239000012228 culture supernatant Substances 0.000 claims abstract description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 13
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 8
- 239000002777 nucleoside Substances 0.000 claims description 7
- 125000003835 nucleoside group Chemical group 0.000 claims description 7
- 238000002965 ELISA Methods 0.000 claims description 6
- 239000012930 cell culture fluid Substances 0.000 claims description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 3
- 239000003797 essential amino acid Substances 0.000 claims 1
- 235000020776 essential amino acid Nutrition 0.000 claims 1
- 230000012010 growth Effects 0.000 abstract description 11
- 230000004069 differentiation Effects 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 28
- 210000001161 mammalian embryo Anatomy 0.000 description 13
- 101150021185 FGF gene Proteins 0.000 description 6
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 5
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 5
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 5
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 5
- 229920002971 Heparan sulfate Polymers 0.000 description 4
- 108010019160 Pancreatin Proteins 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- 229940055695 pancreatin Drugs 0.000 description 4
- 108091016585 CD44 antigen Proteins 0.000 description 3
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 3
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 3
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 108090000054 Syndecan-2 Proteins 0.000 description 3
- 239000011149 active material Substances 0.000 description 3
- 210000000577 adipose tissue Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 102100022464 5'-nucleotidase Human genes 0.000 description 2
- 102000004266 Collagen Type IV Human genes 0.000 description 2
- 108010042086 Collagen Type IV Proteins 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 102100037241 Endoglin Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 2
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 2
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000008730 Nestin Human genes 0.000 description 2
- 108010088225 Nestin Proteins 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000005055 nestin Anatomy 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- -1 Acyl heparin sulfate Chemical compound 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 description 1
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 1
- 101000868152 Homo sapiens Son of sevenless homolog 1 Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 102100020880 Kit ligand Human genes 0.000 description 1
- 101710177504 Kit ligand Proteins 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 1
- 101710088580 Stromal cell-derived factor 1 Proteins 0.000 description 1
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/40—Nucleotides, nucleosides or bases
-
- 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
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Rheumatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application belongs to stem cells technology field, and in particular to a kind of cultural method of cell culture medium and embryonic stem cell.Cell culture medium provided by the present invention includes：Fat stem cell conditioned medium；Fat stem cell conditioned medium is：Fat stem cell cultivates obtained culture supernatant in the serum free medium containing basic fibroblast growth factor；Material containing various active in culture supernatant, it can promote to promote growth and the propagation of embryonic stem cell.Above-mentioned cell culture medium is applied to culture embryonic stem cell, the propagation of embryonic stem cell can be promoted, suppress the technical problems such as its differentiation.
Description
Technical field
The invention belongs to stem cells technology field, and in particular to a kind of culture side of cell culture medium and embryonic stem cell
Method.
Background technology
Fat stem cell is that one kind is present in adipose tissue, being capable of self-renewing, the adult with multi-lineage potential
Stem cell.Fat does fine wide material sources, is easily obtained, and have powerful secreting function, can break up substantial amounts of cell because
Son.
Embryonic stem cell is a kind of cell that body early embryo is separated, it have in vitro culture infinite multiplication, self more
New and Multidirectional Differentiation characteristic.No matter in vitro or vivo environment, embryonic stem cell can be induced to differentiate into body almost
All cell types, have a wide utilization prospect, but at present the cultivating system of embryonic stem cell also exist cell propagation compared with
Slowly, state labile, the problems such as easily breaking up.Therefore, the humanization cultivating system of human embryo stem cell is further researched and developed, it is long
Phase maintains the biological characteristics and multi-lineage potential of embryonic stem cell, and clinical practice is eventually striking to embryonic stem cell and seems outstanding
To be important.
The content of the invention
In order to solve the relatively slow cultivating system cell of current embryonic stem cell propagation, easily state labile, the technology such as differentiation
Problem, it is an object of the invention to provide a kind of cell culture medium, with applied to culture embryonic stem cell.
The concrete technical scheme of the present invention is as follows：
A kind of cell culture medium, comprising：Fat stem cell conditioned medium；
The fat stem cell conditioned medium is：Fat stem cell containing basic fibroblast growth factor without blood
Obtained culture supernatant is cultivated in clear culture medium.
In fat stem cell incubation, fat stem cell secretion various active material, wherein cell factor is main
One of active material, this kind of active material is to maintaining stem cell dryness and promoting cell propagation to have positive role.
Preferably, in the serum free medium containing basic fibroblast growth factor, the basic fibroblast
The concentration of Porcine HGF is 5~10ng/mL.
Preferably, the time of the culture is 20~30h.
Preferably, the fat stem cell is fat stem cell of the third generation to the tenth generation.
In the present invention, the serum free medium includes：Serum substitute, Glu, penicillin, streptomysin and
DMEM/F12 culture mediums.
Preferably, the cell culture medium also includes：Serum substitute, nonessential amino acid, nucleosides, Glu,
Beta -mercaptoethanol, LIF ELISA, basic fibroblast growth factor, stem cell factor and DMEM culture mediums.
Preferably, cell culture fluid described in every 1L includes：
720~770mL of the fat stem cell conditioned medium；
110~the 180mL of serum substitute；
1~the 5g of nonessential amino acid；
1~the 5g of nucleosides；
The molar concentration of the Glu is 1~5mM；
The molar concentration of the beta -mercaptoethanol is 0.1~1mM；
The concentration of the LIF ELISA is 5~10 μ g/L；
The concentration of the basic fibroblast growth factor is 5~10 μ g/L；
The concentration of the stem cell factor is 5~10 μ g/L；
The DMEM is supplied.
It is highly preferred that cell culture fluid described in per 1L includes：
The fat stem cell conditioned medium 750mL；
The serum substitute 150mL；
The nonessential amino acid 1g；
The nucleosides 1g；
The molar concentration of the Glu is 2mM；
The molar concentration of the beta -mercaptoethanol is 0.1mM；
The concentration of the LIF ELISA is 5~10 μ g/L；
The concentration of the basic fibroblast growth factor is 5~10 μ g/L；
The concentration of the stem cell factor is 5~10 μ g/L；
The DMEM 100mL.
Present invention also offers a kind of cultural method of embryonic stem cell, and embryonic stem cell is seeded into above-mentioned cell culture
Cultivated in base.
Preferably, the culture is cultivated in the coated Tissue Culture Dish of Matrixgel basilar memebranes.
In the present invention, the Matrixgel is preferably solubilized basement membrane prepared product, and its main component is by layer adhesion egg
In vain, type Ⅳ collagen, the composition such as heparan sulfate proteoglycan (HSPG) and nestin, also comprising growth factor such as TGF-
Other growth factors that beta, EGF, IGF, FGF, tissue plasminogen activator and EHS tumours contain itself, raising can be replaced
Confluent monolayer cells are in embryonic stem cell or the upper use of inducing pluripotent stem cells culture.
Preferably, the temperature of the culture is 37 DEG C.
Preferably, the fat stem cell is the third generation to the tenth generation.
In summary, cell culture medium provided by the present invention includes：Fat stem cell conditioned medium；Fat stem cell
Various kinds of cell active factors (such as VEGF, SCF, HGF, bFGF, IGF-I, SDF-1 α etc.), these factors are rich in conditioned medium
The self-renewal capacity of embryonic stem cell is able to maintain that, and is made by finite concentration basic fibroblast growth factor (bFGF)
For fat stem cell, the fat stem cell conditioned medium of generation is possessed suppression eosinophil differentiation, maintain it
The ability of normal growth；Moreover, bFGF can also promote the propagation and secretion capacity of fat stem cell, and then it can preferably promote embryo
The growth of tire stem cell and propagation.Therefore, cell culture medium provided by the present invention is applied to culture embryonic stem cell, can be with
Promote the propagation of embryonic stem cell, suppress the technical problems such as its differentiation.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
The embodiment of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 a are that fat stem cell shows antigen CD4 5 and CD34 expression of results in embodiment 1；
Fig. 1 b are the expression of results that fat stem cell shows antigens c D90 in embodiment 1；
Fig. 1 c are the expression of results that fat stem cell shows antigens c D73 in embodiment 1；
Fig. 1 d are the expression of results that fat stem cell shows antigens c D105 in embodiment 1；
The growth curve for the fat stem cell that Fig. 2 embodiments 1 are cultivated；
The detection for the fat stem cell secrete cytokines that Fig. 3 embodiments 1 are cultivated；
The aspect graph of the embryonic stem cell of cell culture medium culture of the present invention is used in Fig. 4 embodiments 2；
The growth curve of the embryonic stem cell of cell culture medium culture of the present invention is used in Fig. 5 embodiments 2.
Embodiment
In order to solve the relatively slow cultivating system cell of current embryonic stem cell propagation, easily state labile, the technology such as differentiation
Problem, the embodiment of the present invention provide a kind of cultural method of cell culture medium and embryonic stem cell.
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Pancreatin used, clostridiopetidase A, DMEM/F12, KnockOut in following examplesTMDMEM, serum substitute (KSR), L-
Glutamine, beta -mercaptoethanol, penicillin, streptomysin are purchased from GIBCO companies；
RNA extraction agents box, DNA Marker, AMV reverse transcription reagent box, Ex-Taq enzymes are purchased from TaKaRa companies；
Matrixgel basilar memebranes, FIHC-CD45 antibody, FIHC-CD105 antibody, PE-CD34 antibody, PE-CD90 antibody
BD companies are purchased from PE-CD73 antibody；
BFGF, SCF, bFGF cell factor are purchased from PeproTech companies；
Tissue Culture Plate, Tissue Culture Flask, pipette are purchased from Corning companies.
Embodiment 1
1. fat stem cell is separately cultured
(1) under aseptic condition, subcutaneus adipose tissue 5-8g is gathered, is fully rinsed 3 times, picked with containing dual anti-physiological saline
Except the blood vessel and connective tissue in adipose tissue, it is fully shredded with eye scissors, then rinsed repeatedly with physiological saline, is removed red
Cell；
(2) 0.2% NTx enzyme of 2 times of volumes is added, after vibration mixes, digests 60min in 37 DEG C of warm water bath cabinets,
1000r/min centrifuges 10min, discards upper-layer fat and supernatant, retains sinking cell precipitation；
(3) fat stem cell complete medium (basal medium containing 90%DMEM/F12,10% are added
KnockOutTMSerum Replacement, 1mmol/mL Glus, 100U/ml penicillin and 100U/ml streptomysins)
Cell precipitation is resuspended, adjustment cell concentration is 1 × 106It is seeded in 100mm culture dishes, under the conditions of 37 DEG C, containing 5%CO2Training
Support and cultivated in case；
(4) after finding to have cell colony growth, attached cell fusion 80%, (0.2% is contained with 0.25% pancreatin
EDTA) had digestive transfer culture, passage bottle number is determined according to cell number situation, is designated as P1 for cell.After attached cell fusion 80%, pancreas
Enzymic digestion is passed on, and amplification passage once, is designated as P2 generations, and by that analogy, the third generation may be selected to the fat in the tenth generation in conditioned medium
Fat stem cell.
2. the identification of fat stem cell
P3-P5 fat subsitutes stem cells, digestion is taken to add CD34, CD45, CD73, CD90 and CD105 monoclonal antibody after being collected by centrifugation
Fluidic cell dyeing is carried out, and is detected with flow cytometry analysis.
As shown in Fig. 1 a to Fig. 1 d, data are shown fat stem cell streaming qualification result, and fat stem cell does not express CD34
And CD45, and high expression CD73, CD90, CD105.
3. the culture of fat stem cell and the collection of conditioned medium that are handled through bFGF
P3 fat subsitutes stem cells are taken, when cell growth is fused to 80%, culture supernatant is sucked, 2 is washed with physiological salt liquid
It is secondary, the special serum free medium of fat stem cell of the bFGF containing 10ng/mL is added, is put into 37 DEG C, 5%CO2In incubator
Cultivate, culture supernatant, as fat stem cell conditioned medium are collected after 24h.
4. the drafting of fat stem cell growth curve
The fat stem cell in the 3rd generation is taken, (inoculum density is 1.0 × 10 for inoculation after pancreatin digests4Individual/mL) in 24 orifice plates
In, it is placed in 37.5 DEG C, containing 5%CO2Incubator in cultivated.Randomly select 3 holes daily to be digested, counted with pancreatin, often
Hole is counted 3 numbers and averaged, and continues 7d.Growth curve is drawn, abscissa is incubation time, and ordinate is cell number.Experiment is set
Two groups, including normal group and bFGF treatment groups are put, bFGF treatment groups use the fat stem cell for the bFGF that with the addition of 10ng/mL
Special serum free medium culture, normal group use the no added bFGF special serum free medium culture of fat stem cell.
The growth curve of the fat stem cell of normal group and bFGF treatment groups is as shown in Fig. 2 result is shown two after cultivating 7 days
The fat stem cell quantity of group dramatically increases, and bFGF treatment groups are more compared with the fat stem cell number of normal group, illustrate culture medium
Middle addition bFGF can promote the propagation of fat stem cell.
5. the detection of cell factor in fat stem cell culture solution
The fat stem cell culture solution after culture 24h is collected, concentrate is formed after efficient filter filters, and to culture
Cell factor in base concentrate is detected, using cytokine detection kits detect nutrient solution in VEGF, TGF-β,
EGF, FGFs, KGF content.Experiment sets two groups, including normal group and bFGF treatment groups, and bFGF treatment groups are used and with the addition of
The 10ng/mL bFGF special serum free medium culture of fat stem cell, normal group are dry thin using no added bFGF fat
The special serum free medium culture of born of the same parents.VEGF in the nutrient solution of normal group and bFGF treatment groups, TGF-β, EGF, FGFs, KGF
Content detection result as shown in figure 3, compared with normal group, do not send out by cell factor EGF, KGF and VEGF content of bFGF treatment groups
Raw significant change, and the content of cell factor FGFs and TGF-β significantly raises, and illustrates that fat stem cell can be stimulated by adding bFGF
The expression of cell factor TGF-β and FGFs.
Embodiment 2
1. Tissue Culture Plate is coated with
Use Matrix Gel basilar memebrane coated cell culture plates.1g/L Matrix Gel are diluted to the PBS of precooling
Final concentration of 10mg/L.Take 1mL Matrix Gel solution to be uniformly laid on 4 orifice plates, 4 DEG C of overnight incubations, then suction out, DMEM/
F12 is cleaned 1 time, is spontaneously dried, standby.
Matrixgel is solubilized basement membrane prepared product, and its main component is by laminin, type Ⅳ collagen, sulfuric acid second
Acyl heparin sulfate proteoglycans (HSPG) and nestin etc. form, also comprising growth factor such as TGF-beta, EGF, IGF, FGF, tissue
Other growth factors that activator of plasminogen and EHS tumours contain itself, feeder cells can be replaced in embryonic stem cell or
The upper use of inducing pluripotent stem cells culture.
2. the preparation of cell culture medium
Formula：
Fat stem cell conditioned medium：75%vol；
KnockOutTMSerum Replacement：15%vol；
Nonessential amino acid：1g/100mL；
Nucleosides：1g/100mL；
Glu：2mM；
Beta -mercaptoethanol：0.1mM；
LIF ELISA：10μg/L；
bFGF：10μg/L；
Stem cell factor：10μg/L；
KnockOutTMDMEM is supplied.
The each component listed in being formulated is mixed successively, you can preparation obtains the cell culture for cultivating embryonic stem cell
Base.
Wherein, the nonessential amino acid used in the present embodiment be MEM nonessential amino acid solutions (100 ×, Sigma
Article No. M7145), composition includes：Glutamic acid, alanine, glycine, asparatate, cystine, proline, serine and junket
Propylhomoserin.
3. the culture of embryonic stem cell
Conventional recovery human embryo stem cell strain BG02, embryonic stem cell is transferred to containing 10mL KnockOutTMDMEM's
In centrifuge tube, 800g centrifugation 5min, the cell culture medium prepared using step 2 is resuspended embryonic stem cell BG02, and by embryo
Stem cell is seeded on the coated Tissue Culture Dish of Matrixgel basilar memebranes, is placed under the conditions of 37 DEG C, containing 5%CO2Incubator
Middle culture, changes liquid daily.Passed on once per 4-5 days, and 1 is pressed according to cell quantity:(2~3) ratio passes on.
In succeeding generations, find continuously to cultivate for 15 generations using the human embryo stem cell of the cell culture medium culture of the present embodiment
Afterwards, remain able to maintain typical embryonic stem cell sample colony morphology well, as shown in figure 4, it clones sharpness of border, cell
Closely, kernel is obvious.Compared with using the human embryo stem cell of traditional cell culture medium culture, the inventive method obtains thin
Born of the same parents' form is consistent with its, but the plastidogenetic clone of the present invention is bigger.
4. the growth curve of embryonic stem cell
After the adherent 24h of human embryo stem cell colony of passage, 10 colonies are randomly selected every 24h, are digested to be unicellular,
Regular growth is counted, and experiment is repeated 6 times.Experiment sets two groups, including experimental group 1 and control group 1, the Human embryo of experimental group 1
Stem cell uses cell culture medium culture of the present invention, and the human embryo stem cell of control group 1 uses conventional medium culture.
The drafting curve of the growth curve of human embryo stem cell is as shown in figure 5, after culture in 8 days, human embryo stem cell number
The obvious increase of amount, and the human embryo stem cell quantity of experimental group 1 is more compared with the stem cell population of control group 1, shows institute of the present invention
The cell culture medium of offer can promote the propagation of embryonic stem cell.
Claims (10)
1. a kind of cell culture medium, it is characterised in that include：Fat stem cell conditioned medium；
The fat stem cell conditioned medium is：Fat stem cell is trained in the serum-free containing basic fibroblast growth factor
Support the culture supernatant for cultivating to obtain in base.
2. cell culture medium according to claim 1, it is characterised in that contain basic fibroblast growth factor described
Serum free medium in, the concentration of the basic fibroblast growth factor is 5~10ng/mL.
3. cell culture medium according to claim 1, it is characterised in that the time of the culture is 20~30h.
4. cell culture medium according to claim 1, it is characterised in that the fat stem cell is the third generation to the tenth generation
Fat stem cell.
5. cell culture medium according to claim 1, it is characterised in that also include：Serum substitute, non-essential amino
Acid, nucleosides, Glu, beta -mercaptoethanol, LIF ELISA, basic fibroblast growth factor, stem cell because
Son and DMEM culture mediums.
6. cell culture medium according to claim 5, it is characterised in that cell culture fluid described in per 1L includes：
720~770mL of the fat stem cell conditioned medium；
110~the 180mL of serum substitute；
10~the 50g of nonessential amino acid；
10~the 50g of nucleosides；
The molar concentration of the Glu is 1~5mM；
The molar concentration of the beta -mercaptoethanol is 0.1~1mM；
The concentration of the LIF ELISA is 5~10 μ g/L；
The concentration of the basic fibroblast growth factor is 5~10 μ g/L；
The concentration of the stem cell factor is 5~10 μ g/L；
The DMEM is supplied.
7. cell culture medium according to claim 5, it is characterised in that cell culture fluid described in per 1L includes：
The fat stem cell conditioned medium 750mL；
The serum substitute 150mL；
The nonessential amino acid 10g；
The nucleosides 10g；
The molar concentration of the Glu is 2mM；
The molar concentration of the beta -mercaptoethanol is 0.1mM；
The concentration of the LIF ELISA is 5~10 μ g/L；
The concentration of the basic fibroblast growth factor is 5~10 μ g/L；
The concentration of the stem cell factor is 5~10 μ g/L；
The DMEM 100mL.
8. a kind of cultural method of embryonic stem cell, it is characterised in that it is any that embryonic stem cell is seeded to claim 1 to 7
Cultivated in cell culture medium described in one.
9. cultural method according to claim 8, it is characterised in that the culture is coated in Matrixgel basilar memebranes
Cultivated in Tissue Culture Dish.
10. cultural method according to claim 8, it is characterised in that the temperature of the culture is 37 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710875128.6A CN107475188A (en) | 2017-09-25 | 2017-09-25 | A kind of cultural method of cell culture medium and embryonic stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710875128.6A CN107475188A (en) | 2017-09-25 | 2017-09-25 | A kind of cultural method of cell culture medium and embryonic stem cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107475188A true CN107475188A (en) | 2017-12-15 |
Family
ID=60587308
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710875128.6A Pending CN107475188A (en) | 2017-09-25 | 2017-09-25 | A kind of cultural method of cell culture medium and embryonic stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475188A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220230A (en) * | 2018-02-01 | 2018-06-29 | 上海莱馥生命科学技术有限公司 | A kind of separation of human adipose-derived stem cell and cultural method |
| CN108486045A (en) * | 2018-01-23 | 2018-09-04 | 广东颜值科技有限公司 | A kind of culture composition of human fibroblasts |
| CN110438076A (en) * | 2018-05-02 | 2019-11-12 | 强普生技股份有限公司 | T cell culture medium and cultural method |
| CN110699318A (en) * | 2018-07-09 | 2020-01-17 | 广西慧宝源健康产业有限公司 | T cell culture medium and culture method |
| CN111944743A (en) * | 2020-08-17 | 2020-11-17 | 广州同康生物科技有限公司 | Human embryonic stem cell culture medium and preparation method thereof |
| WO2020241132A1 (en) * | 2019-05-29 | 2020-12-03 | パナジー株式会社 | Cell proliferation method, cell proliferation agent, and medium for cell proliferation |
| CN112626012A (en) * | 2019-09-20 | 2021-04-09 | 深圳市未来细胞生命科技有限公司 | Culture solution for promoting stem cell growth and preparation method thereof |
| CN113755423A (en) * | 2021-10-12 | 2021-12-07 | 徐海 | Application of embryonal cancerous cells in preparation of 3D cell culture substrate, preparation method of embryonal cancerous cells and 3D cell culture substrate |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2433943B (en) * | 2004-09-28 | 2010-06-16 | Wicell Res Inst Inc | Cultivation of primate embryonic stem cells |
| CN105062958A (en) * | 2015-08-26 | 2015-11-18 | 兰诺生物技术无锡有限公司 | Composition for embryonic stem cell culture and application thereof |
| CN105586311A (en) * | 2016-03-06 | 2016-05-18 | 李倩 | Culture medium for culturing human adipose-derived stem cells |
| CN106635979A (en) * | 2017-01-16 | 2017-05-10 | 广东万海细胞生物科技有限公司 | Serum-free culture medium for adipose tissue-derived stromal cells |
-
2017
- 2017-09-25 CN CN201710875128.6A patent/CN107475188A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2433943B (en) * | 2004-09-28 | 2010-06-16 | Wicell Res Inst Inc | Cultivation of primate embryonic stem cells |
| CN105062958A (en) * | 2015-08-26 | 2015-11-18 | 兰诺生物技术无锡有限公司 | Composition for embryonic stem cell culture and application thereof |
| CN105586311A (en) * | 2016-03-06 | 2016-05-18 | 李倩 | Culture medium for culturing human adipose-derived stem cells |
| CN106635979A (en) * | 2017-01-16 | 2017-05-10 | 广东万海细胞生物科技有限公司 | Serum-free culture medium for adipose tissue-derived stromal cells |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486045A (en) * | 2018-01-23 | 2018-09-04 | 广东颜值科技有限公司 | A kind of culture composition of human fibroblasts |
| CN108220230A (en) * | 2018-02-01 | 2018-06-29 | 上海莱馥生命科学技术有限公司 | A kind of separation of human adipose-derived stem cell and cultural method |
| CN108220230B (en) * | 2018-02-01 | 2022-01-18 | 上海莱馥医疗科技有限公司 | Method for separating and culturing human adipose-derived stem cells |
| CN110438076A (en) * | 2018-05-02 | 2019-11-12 | 强普生技股份有限公司 | T cell culture medium and cultural method |
| CN110699318A (en) * | 2018-07-09 | 2020-01-17 | 广西慧宝源健康产业有限公司 | T cell culture medium and culture method |
| WO2020241132A1 (en) * | 2019-05-29 | 2020-12-03 | パナジー株式会社 | Cell proliferation method, cell proliferation agent, and medium for cell proliferation |
| JP2020195363A (en) * | 2019-05-29 | 2020-12-10 | パナジー株式会社 | Cell proliferation method, cell proliferation agent, and medium for cell proliferation |
| CN112626012A (en) * | 2019-09-20 | 2021-04-09 | 深圳市未来细胞生命科技有限公司 | Culture solution for promoting stem cell growth and preparation method thereof |
| CN111944743A (en) * | 2020-08-17 | 2020-11-17 | 广州同康生物科技有限公司 | Human embryonic stem cell culture medium and preparation method thereof |
| CN113755423A (en) * | 2021-10-12 | 2021-12-07 | 徐海 | Application of embryonal cancerous cells in preparation of 3D cell culture substrate, preparation method of embryonal cancerous cells and 3D cell culture substrate |
| CN113755423B (en) * | 2021-10-12 | 2024-04-26 | 徐海 | Application of embryonic cancerous cells in preparation of 3D cell culture matrix, preparation method and 3D cell culture matrix |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475188A (en) | A kind of cultural method of cell culture medium and embryonic stem cell | |
| Grayson et al. | Human mesenchymal stem cells tissue development in 3D PET matrices | |
| CN104263697B (en) | A kind of method that inducing culture and induction human adipose mesenchymal stem cells generate insulin secretory cell | |
| CN103805562B (en) | Cultivate the serum free medium of placenta mesenchyma stem cell | |
| CN102618491B (en) | Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution | |
| CN101864395B (en) | In-vitro inducing differentiation of umbilical cord mesenchymal stem cells into tissue engineering skin seed cells | |
| CN102757936B (en) | Proliferation accelerator for human adipose-derived stem cells and application method thereof | |
| Saleh et al. | Turning round: multipotent stromal cells, a three-dimensional revolution? | |
| CN104884468A (en) | Modified laminin having collagen-binding molecule attached thereto, and use of same | |
| CN102533659A (en) | Method for inducing human umbilical cord mesenchymal stem cells into testicular interstitial cells and application thereof | |
| CN104805054A (en) | Serum-free medium of stem cell | |
| CN105331580A (en) | Method for enhancing mesenchymal stem cell chemotactic capacity and chemotactic factor CCL5 expression | |
| CN103966159B (en) | Human plactnta Subaerial blue green algae and stem cell bank construction process thereof | |
| CN107217028A (en) | A kind of organization engineering skin containing appendicle and preparation method thereof | |
| CN105039248B (en) | Tree shrew mesenchymal stem cell culture systems | |
| CN105132360A (en) | Method for inducing placenta-derived mesenchymal stem cells to be differentiated into islet-like cells | |
| CN107418930A (en) | A kind of preparation method purified with amplification human marrow mesenchymal stem cell | |
| CN104774808A (en) | Method for inducible differentiation of umbilical cord mesenchymal stem cells into gamma-aminobutyric acid-ergic neuron | |
| CN107083359A (en) | Stem cell media and method for separating stem cell | |
| Jun-Jiang et al. | Vascular endothelial growth factor 165-transfected adipose-derived mesenchymal stem cells promote vascularization-assisted fat transplantation | |
| CN103881972B (en) | A kind of method being applicable to mesenchymal stem cell serum-free and cultivating | |
| CN107937346A (en) | A kind of method by the use of human urine cell as feeder layer culture induced multi-potent stem cell | |
| CN103865873A (en) | Exosome secreted by sub-totipotent stem cells and application thereof | |
| CN106754650B (en) | A kind of endothelial progenitor cells cultural method of derived from bone marrow | |
| Cheng et al. | Cell-derived matrices (CDM)—Methods, challenges and applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171215 |