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
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, 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]
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 | |
| US20120178159A1 (en) | Mesenchymal stem cells (msc) expansion methods and materials | |
| 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 | |
| CN105238751A (en) | Umbilical cord tissue mesenchymal stem cell isolated culture method | |
| CN105331580A (en) | Method for enhancing mesenchymal stem cell chemotactic capacity and chemotactic factor CCL5 expression | |
| CN107557331A (en) | A kind of method for separating and cultivating human adipose-derived stem cell | |
| CN107267453A (en) | A kind of culture medium and its application for being used to cultivate fat mesenchymal stem cell | |
| CN103966159A (en) | Sub-totipotent stem cell of human placenta and stem cell bank construction method thereof | |
| CN107418930A (en) | A kind of preparation method purified with amplification human marrow mesenchymal stem cell | |
| Liu et al. | Tendon-derived stem cell differentiation in the degenerative tendon microenvironment | |
| 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 | |
| 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 | |
| CN102174573A (en) | Porcine trachea epithelial cell line and building method thereof | |
| CN103865873A (en) | Exosome secreted by sub-totipotent stem cells and application thereof | |
| Jun-Jiang et al. | Vascular endothelial growth factor 165-transfected adipose-derived mesenchymal stem cells promote vascularization-assisted fat transplantation | |
| CN106754650B (en) | A kind of endothelial progenitor cells cultural method of derived from bone marrow |
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 |