CN101490243A - Cell growth medium - Google Patents

Cell growth medium Download PDF

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CN101490243A
CN101490243A CNA2007800249934A CN200780024993A CN101490243A CN 101490243 A CN101490243 A CN 101490243A CN A2007800249934 A CNA2007800249934 A CN A2007800249934A CN 200780024993 A CN200780024993 A CN 200780024993A CN 101490243 A CN101490243 A CN 101490243A
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cell
cell culture
embryonic stem
stem cells
culture medium
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古江美保
彼得·安德鲁斯
冈本哲治
丹瑞·J·佐藤
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University of Sheffield
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University of Sheffield
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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    • 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
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

Abstract

The invention relates to a method to culture primate embryonic stem cells in feeder and serum free conditions.

Description

Cell growth medium
The present invention relates to primate embryonic stem cells, preferred human embryo stem cell (human embryonicstem cells, hES) keeping in the cultivation of no feeder layer cells and serum-free.
The cultivation for example embryonic cell of some mammalian cell has become conventional method, and allows the cell culture condition of some cell proliferation that detailed description is also arranged.Usually, the cell cultures of the mammalian cell sterile chamber and the growth medium that need be made of plastics usually.For example, the growth needs feeder layer cells of embryonic stem cell and the existence of serum.The function of feeder layer cells is not also clearly understood.But by inference, the effect of feeder layer cells may provide the mitogenesis signal, the undifferentiated state of this token stimulus cell proliferation and/or maintenance cell.Feeder layer cells is the inoblast through handling (as mitomycin or radiotreatment) so that can not breeding normally.Usually, the feeder layer inoblast is the mouse source, but also can be from other species.
Term " stem cell " is the general name of a group undifferentiated cell, and described undifferentiated cell has the ability of self, has kept simultaneously to form the cell of differentiation and the diversified potential of tissue.Stem cell can be (multipotent) of all-round (totipotent), polyenergic (pluripotent) or special energy.The derived stem cells that also has the forfeiture differentiation capability is called as " incompetent (ullipotent) " stem cell.Myeloid-lymphoid stem cell is to can be formed in all cells and the tissue found in the complete life entity to comprise that the embryo organizes the cell of (being placenta) outward.Myeloid-lymphoid stem cell comprises utmost point body early embryo (8 cells), and can form complete life entity.Although multipotential stem cell can not form complete life entity, multipotential stem cell be have be formed on find in the complete life entity the cell of ability in a organized way.Specially can have the cell of the differentiation of forming and the limited capacity of tissue by stem cell.Usually, adult stem cell is specially can stem cell, and is that the precursor stem cell or the ancestral that can form some cell or tissue and replenish aging or damaged cells/tissue is qualification stem cell (lineage restricted stem cells).Although for this type of " adult " stem cell, some report is claimed the potential that it is stronger than having of initial expectation, they can not form usually find in the life entity the institute in a organized way.
Pluripotent embryonic stem cells is mainly from two kinds of embryo sources.The cell that is located away from inner cell mass is called the embryo and does (ES) cell.In experiment mice, similarly cell can obtain from genitaloid culture, and primordial germ cells separate mesentery or the gonocrista from 8.5 to 12.5 days embryo of post-coitum.They are called as embryonic genital cell (EG cell).Each of the pluripotent cell of these types all has the potentiality of development that similarly is divided into other cell types, but possible difference makes these cells differ from one another again in the habit (as the marking).But term " pluripotent embryonic stem cells " comprises cell and primordial germ cells from inner cell mass.
Prove that the vitro culture thing of setting up primate embryonic stem cells remains difficulty.Put down in writing definite permission among the WO96/22362 and set up the index of the condition of the hES cell of cultivating.WO96/22362 has described permission primate es cell continuous proliferating cells system and growth conditions, and described primate es cell has shown a series of characteristic or the marks relevant with the stem cell of tool multipotency characteristic.When these include, but not limited in being maintained at the inoblast feeder layer, in culture, kept at least 20 generations; Generation is called as the cell of the cluster of embryoid body; In the time of in being incubated at suspension, can in cultivating, monolayer be divided into the various kinds of cell type; In the time of in being injected into immunodeficient mouse, forming the xenotransplantation teratoma of cell type and express embryonic stem cell specificity marker thing, particularly SSEA3, SSEA4, TRA-1-60, TRA-1-81, alkaline phosphatase and Oct4 with multiple differentiation.WO96/22362 discloses under the situation of l cell feeder layer cells and serum existence, and the primate es cell of keeping cultivation is in the method for undifferentiated state.
Existing great mass of data has proved embryonic stem cell, particularly people ES (hES) cell potential effectiveness in the therapeutic type organizational project.The multipotency characteristic of these cells allows to select the hES cell and make the hES cytodifferentiation become any cell/tissue type.Yet, when use is exposed to the cell of foetal calf serum or mouse feeder layer cells in the treatment, have potential risk such as the exogenous factor PI acceptor of protein virus or virus.Therefore, must handle the cell cultures of hES cell, so that this risk is reduced to is minimum.The research and development of the condition of no feeder layer and serum-free will help to reduce this risk.
In addition, be divided into the hES cell of particular cell types derivative because it is identical on gene type, stable and originate known, so can be used for identifying the gene target of novel drugs and existing medicine.Use the ES clone of different genotype also to provide possible approach as drug screening and toxicology at the pharmacogenomics related aspect.
The progress of the serum-free culture condition of primate es cell is known.For example, WO01/66697 discloses the serum-free growth of primate es cell, wherein, uses fibroblast growth factor, normally rh-bFGF (bFGF, 4ng/ml) alternative serum.Cell culture medium comprises the KnockOut SR that has replenished bFGF Tm(be described in WO98/30679, its full content is incorporated by reference).Yet cell culture comprises through radiating mouse fibroblast cell feeder cell.
The serum-free that is used for hES cell growth and the progress of no feeder layer cultural method are disclosed in WO2006/029198.BFGF (40-100ng/ml), supplement that these growth conditions working concentrations improve, and comprise amino acid, fat, VITAMIN and glucose, described supplement comprise γ-An Jidingsuan, nipecotic acid and lithium.WO2006/029198 also discloses the purposes that contains such as fibronectin, vitronectin (vitronectin) and the ln proteic cell culture substrates of people such as (laminin).
In addition, Furue et al (In vitro Cell Dev.Biol.Animal 41:19-28,2005) discloses under the situation that leukaemia inhibitory factor (LIF) exists, the serum-free of mouse embryo stem cell and the growth of no feeder layer.This is also on the books in WO2005/063968.
It is favourable setting up simple cell culture condition, it does not need to add such as xenobiotic materials such as foetal calf serum or mouse feeder layer cells, because their use can increase infectious factor (as, the particularly virus of dairy products and protein virus, and the Murivirus of mouse feeder layer cells) infect the possibility of growing mammalian cells in the culture.The invention provides selectable simple cell substratum, it can keep the hES cell under the condition of serum-free and feeder layer.
One aspect of the present invention provides the method for keeping primate embryonic stem cells under the cell culture condition of acellular feeder layer and serum-free, it comprises: form primate embryonic stem cells prepared product and the undifferentiated state of keeping primate embryonic stem cells in containing the cell culture container of cell culture medium.Described cell culture medium comprises fibroblast growth factor, heparin and xitix, or ascorbyl phosphate, or derivatives thereof.
One aspect of the present invention provides the method for keeping primate embryonic stem cells under the cell culture condition of acellular feeder layer and serum-free, and it comprises the steps:
I) in by cell culture container, form the primate embryonic stem cells prepared product based on proteinic cell cultures upholder bag quilt, wherein, with described cell cultures in cell culture medium, described cell culture medium comprise Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix, or ascorbyl phosphate, or derivatives thereof, and
Ii) keep primate embryonic stem cells and be in undifferentiated state.
The present invention includes particularly people's pluripotent embryonic stem cells of primates, and also comprise the teratocarcinoma stem cell, be also referred to as embryonal carcinoma (EC) cell." pluripotent embryonic stem cells " refer to from the cell of inner cell mass and primordial germ cells (EG) both.The stem cell reprogrammed that also has the cell of the outer differentiation of somatocyte or embryo or more limit is got back to and possibility from the similar multipotency state of the ES cell of body early embryo.A kind of mode that realizes above-mentioned possibility is, in the mode of SCNT, will move on to non-nucleus egg mother cell from the consideration convey of this type of noble cells, stimulates this ovocyte to grow to the blastocyst stage as plumule subsequently, then obtains ES clone from blastocyst.The experiment of cytogamy shows that also the tenuigenin of EC and ES cell also can make the cell reprogrammed of somatocyte or other types get back to the state of ES sample.
In a preferred method of the invention, described cell has stable caryogram.
In another preferable methods of the present invention, xitix is an ascorbyl phosphate.
The functional derivatives of xitix and ascorbyl phosphate is known in this area.For example, EP 1666484 (its full content is incorporated by reference) has described the stable derivatives of xitix, and this derivative shows that the stability to heat or oxidation increases.
In a preferred method of the invention, described primate embryonic stem cells is the pluripotent human embryonic stem cell.
In the preferred embodiment of the invention, described primate embryonic stem cells has kept in whole cell cultivation process and has been divided into the characteristic of entoderm, mesoderm and ectoderm tissue at least.
In another preferable methods of the present invention, fibroblast growth factor (FGF) is selected from bFGF/FGF-2, hereinafter acid FGF/FGF-1, bFGF, FGF-4, FGF-9, FGF-17 or FGF-18.
In a preferred method of the invention, described fibroblast growth factor is bFGF.Preferably, the concentration of the bFGF that provides is 1-50ng/ml; Preferred about 10ng/ml.
Preferably, fibroblast growth factor is recombinated.
In the other preferable methods of the present invention, the concentration of the ascorbyl phosphate that provides is 10-300 μ g/ml; Preferred about 100 μ g/ml.
In the other preferable methods of the present invention, the concentration of the 2-thanomin that provides is 0.05-2.0 μ g/ml; Preferred about 0.6 μ g/ml.
In the other preferable methods of the present invention, the oleic concentration that provides is 3-15 μ g/ml; Preferred about 9.5 μ g/ml.
In the other preferable methods of the present invention, the concentration of the heparin that provides is 10-500ng/ml; Preferred about 100ng/ml; Preferably, heparin is vitriol heparin (heparin sulphate salt).
In a preferred method of the invention, described protein cell cultivation upholder is based on collagen protein.
In a preferred method of the invention, the cell cultures upholder based on collagen protein comprises type i collagen albumen; The type i collagen albumen of preferred reorganization.
In the optional preferred method of the present invention, described cell cultures upholder comprises people's albumen of reorganization, and people's albumen of described reorganization is selected from type i collagen albumen, IV collagen type, fibronectin, ln and vitronectin.
In the optional preferred method of the present invention, described cell cultures upholder comprises at least two kinds of recombinant proteins, and described recombinant protein is selected from type i collagen albumen, IV collagen type, fibronectin, ln and vitronectin.
In a preferred method of the invention, described cell upholder comprises type i collagen albumen, IV collagen type, fibronectin, ln and the vitronectin of reorganization.
In a preferred method of the invention, described cell cultures upholder is Matrigel Tm
In a preferred method of the invention, after in cell culture container, adding EDTA, described primate embryonic stem cells is gone down to posterity.
In the optional preferred method of the present invention, after adding collagenase, preferably adding collagenase IV, described primate embryonic stem cells is gone down to posterity.
In the optional preferred method of the present invention, after adding Dispase, described primate embryonic stem cells is gone down to posterity.
In the optional preferred method of the present invention, after adding trypsinase/EDTA, preferably adding recombinant trypsin, described primate embryonic stem cells is gone down to posterity.
In the other preferable methods of the present invention, clone described primate embryonic stem cells.
In a preferred method of the invention, cell culture medium does not comprise buffer reagent HEPES.
Another aspect of the present invention provides the method that primate embryonic stem cells is divided at least a cell type under the cell culture condition of acellular feeder layer and serum-free, and it comprises the steps:
I) in by cell culture container, form the embryonic stem cell prepared product based on proteinic cell cultures upholder bag quilt, wherein with described cell cultures in cell culture medium, described cell culture medium comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor and heparin, and
Ii) adding induces primate embryonic stem cells to be divided into the reagent of at least a cell type.
In a preferred method of the invention, primate embryonic stem cells is a human pluripotent embryonic stem cell.
In a preferred method of the invention, cell type is a neurone.
In the optional method of the present invention, cell type is an epithelial cell.
In a preferred method of the invention, described is ln based on proteinic cell cultures upholder.
The present invention provides cell culture on the other hand, it comprises: based on primate embryonic stem cells and the cell culture medium on the proteinic cell cultures upholder, described cell culture medium comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix, or ascorbyl phosphate, or derivatives thereof.
In the preferred embodiment of the invention, primate embryonic stem cells is the pluripotent human embryonic stem cell.
Additional aspects of the present invention provide cell culture, it comprises: based on primate embryonic stem cells and cell culture medium on the proteinic cell cultures upholder, described cell culture medium comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, bovine serum albumin bonded oleic acid with FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor and heparin, it is characterized in that described cell culture also comprises at least a reagent of inducing primate embryonic stem cells to be divided at least a cell type.
In the preferred embodiment of the invention, primate embryonic stem cells is the pluripotent human embryonic stem cell.
The present invention provides the cell culture container that contains cell culture medium on the other hand, described cell culture medium contains: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix, or ascorbyl phosphate, or derivatives thereof.
In the preferred embodiment of the invention, described cell culture container also contains primate embryonic stem cells; Preferred pluripotent human embryonic stem cell.
In the other preferred embodiment of the present invention, described container is selected from petri diss (petri-dish), Tissue Culture Flask or flask, porous plate." container " can be regarded as any suitable device that holds primate embryonic stem cell cultures.
The present invention provides the cell culture medium container that comprises cell culture medium on the other hand, described cell culture medium contains: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix, or ascorbyl phosphate, or derivatives thereof.
The present invention provides the cell culture medium container that contains cell culture medium on the other hand, described cell culture medium contains: Regular Insulin, transferrin, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor and heparin.
In the description and claims of whole specification sheets, word " comprise (comprise) " and " containing (contain) " with and variant, for example comprise (comprising) and comprise (comprises), mean " including but not limited to ", and be not that intention (and not) is got rid of other parts (moieties), additive, component, integral body or step.
In the description and claims of whole specification sheets, the odd number speech comprises plural, except as otherwise noted.Particularly, if used indefinite article, then specification sheets should be understood that to have considered plural number and odd number, except as otherwise noted.
Characteristic, integral body, feature, compound, chemical part or the cohort described in conjunction with concrete aspect of the present invention, embodiment or embodiment are interpreted as being applicable to any other aspect as herein described, embodiment or embodiment, unless there is contradiction.
Only, embodiment of the present invention are described by embodiment and with reference to following accompanying drawing.
Fig. 1 shows the influence of bFGF to human embryo stem cell propagation;
Fig. 2 shows that bFGF and heparin are bred human embryo stem cell and the influence of form;
Fig. 3 shows the expression in the cultured cells under no feeder layer condition of human embryo stem cell mark;
Fig. 4 shows the growth of human embryo stem cell in various substratum;
Fig. 5 shows the growth curve of human embryonic stem cell HUES under no feeder layer condition; And
Fig. 6 shows the growth curve of human embryonic stem cell Shef1 under no feeder layer condition; And
Table 1 has shown the cell culture medium component guide look of cultivator embryonic stem cell under the condition of no feeder layer.
Human embryo stem cell does not have material and the method that feeder layer is cultivated
Table 1 defines hESF9.The Hesf5 substratum is identical with the Hesf9 substratum, but does not add and bovine serum albumin bonded oleic acid, ascorbyl phosphate, bFGF and heparin sulfate (heparin sulphate).
A. reagent
1. the T25 flask of the undifferentiated embryonic stem cell of people
2.hESF9 substratum: the ESF basic medium of no HEPES, it is supplemented with 9 kinds of factors: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, 2 mercapto ethanol, 2-thanomin, with bovine serum albumin bonded oleic acid, ascorbyl phosphate, bFGF and the heparin sulfate of FAF.
3.EDTA solution
4.I collagen type (Nitta Gelatine, Co., Osaka, Japan)
B. step
1. with 100 μ g/cm 2Type i collagen albumen bag by T25 (Corning).
2. at no Ca 2+And Mg 2+Da Erbaikeshi (Dulbecco ' s) phosphoric acid buffer liquor in separate the ES cell colony with the EDTA4Na (Sigma) of 0.45mM to 0.5mM.(cellular segregation should not become individual cells, the concentration of EDTA depends on clone)
3. with hESF9 substratum collecting cell.
4. cell suspending liquid was made it with the 800rpm rotation in 3 minutes.
5. cell is resuspended in the hESF9 substratum.
6. cell suspending liquid was made it with the 800rpm rotation in 3 minutes.
7. cell is resuspended in the hESF9 substratum.
With cell inoculation in by 100 μ g/cm 2The 25cm of type i collagen albumen bag quilt 2In the flask, place the hESF9 substratum.
9. containing 10% CO 2Wet air in hatch in 37 ℃.
The neurone differentiation method
A. reagent
1. the T25 flask of the undifferentiated embryonic stem cell of people
2.hESF9 substratum
2.hESF5 substratum: the ESF basic medium of no HEPES, it is supplemented with 5 kinds of factors: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, 2 mercapto ethanol and 2-thanomin.
3.EDTA solution
4. ln (Sigma)
5.bFGF and heparin
B. step
1. with 5 μ g/cm 2The ln bag by plastic culture dish.
2. collect undifferentiated ES cell with EDTA solution.
With this cell inoculation on the culture dish of ln bag quilt, place the hESF5 that is supplemented with 10ng/ml bFGF and 100ng/ml heparin.
Option: seed cells on the culture dish of ln bag quilt, place the hESF9 substratum, second day, this substratum is changed into the hESF5 substratum that is supplemented with 10ng/ml bFGF and 100ng/ml heparin.
Under 37 ℃, contain 5% CO 2Wet air in cultivated 1 day.
5. second day, 10ng/ml bFGF is added in the culture.
6. the 2nd～4 cultivate day, change substratum into the hESF5 substratum.
7. per 2 days, change substratum into fresh hESF5 substratum.
8. the 7th～10 cultivate day, begin to occur neuronal cell.
Be divided into the epithelioid cell
A. reagent
1. the T25 flask of the undifferentiated embryonic stem cell of people
2.hESF9 substratum
2. be supplemented with the hESF5 substratum of FA-BSA: the EFS basic medium of no HEPES, it is supplemented with 5 kinds of factors: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, 2 mercapto ethanol, 2-thanomin, and the bovine serum albumin of 0.5mg/ml FAF (FA-BSA)
3.EDTA solution
4. ln (Sigma)
5.BMP4
B. step
1. with 5 μ g/cm 2The ln bag by plastic culture dish.
2. collect undifferentiated ES cell with EDTA solution.
With cell inoculation on the culture dish of ln bag quilt, place the hESF5 that is supplemented with FA-BSA and 10ng/ml BMP4.
4. per 2 days, change substratum into the fresh hESF5 substratum that is supplemented with FA-BSA and 10ng/ml BMP4.
8. from the 3rd day of cultivation, the epithelioid cell appears.
The defined medium (hESF9) of no feeder layer of table 1 and serum-free growth
Component Concentration (mg/L) Component Concentration (mg/L)
The L-L-Ala 2.225 Vitamins B 12 0.34125
The L-arginine 50 Xanthoglobulin 1.02
The L-arginine monohydrochloride 94.75 Oleic acid 9.4
The altheine hydrate 16.2525 Linolic acid 0.021
The L-aspartic acid 8.325 Thioctic Acid 0.0525
The L-cysteine hydrochloride 7.88 The putrescine dihydrochloride 0.04025
L-Gelucystine dihydrochloride 47.5725 Thymine deoxyriboside 0.1825
L-L-glutamic acid 8.675 Sodium-chlor 6599.75
L-glutaminate 549.65 Repone K 355.9
Glycine 19.375 Calcium chloride (anhydrous) 108.305
The L-Histidine 23.165 Four water-calcium nitrate 25
The L-oxyproline 5 Magnesium chloride (anhydrous) 14.305
The L-Isoleucine 65.935 Sal epsom (anhydrous) 61.055
The L-leucine 68.225 Nine water iron nitrates 0.05
L lysine HCL 92.175 Cupric sulfate pentahydrate 0.000625
The L-methionine(Met) 19.87 Iron vitriol 0.2085
The L-phenylalanine 37.99 Zinc Sulphate Heptahydrate 0.216
The L-proline(Pro) 13.625 Sodium Selenite 0.0034588
The L-Serine 31.125 SODIUM PHOSPHATE, MONOBASIC (anhydrous) 54.35
The L-Threonine 55.525 Mono phosphoric acid ester disodium hydrogen (anhydrous) 235.51
The L-tryptophane 9.76 Sodium.alpha.-ketopropionate 110
L-tyrosine 42.36 2 mercapto ethanol 0.7813
The L-Xie Ansuan 54.825 The 2-thanomin 0.6108
Gsh 0.25 Regular Insulin 10
Para-amino benzoic acid 0.25 Apotransferrin 5
Vitamin H 0.05185 Heparin sodium 0.1
Calcium pantothenate 2.1825 Albumin 1000
Choline chloride 60 6.24 Glucose (anhydrous) 2500
Folic acid 2.575 NaHCO 3 2000
Inositol 16.85 Phenol red 6.56
Niacinamide 2.25925 Fibroblast growth factor-2 0.01
Pyridoxal hydrochloride 2
Vitamin B6 hydrochloride 0.2655
Ascorbyl phosphate 100
Riboflavin 0.2595
Hydrochloric tiamide 2.335

Claims (48)

1. under the cell culture condition of acellular feeder layer and serum-free, keep the method for primate embryonic stem cells, described method comprises: form the primate embryonic stem cells prepared product in comprising the cell culture container of cell culture medium, and keep primate embryonic stem cells and be in undifferentiated state, described cell culture medium contains fibroblast growth factor, heparin and xitix or ascorbyl phosphate or derivatives thereof.
2. keep the method for primate embryonic stem cells under the cell culture condition of acellular feeder layer and serum-free, it comprises following steps:
I) in by cell culture container, form the primate embryonic stem cells prepared product based on proteinic cell cultures upholder bag quilt, wherein with described cell cultures in cell culture medium, described cell culture medium comprise Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix or ascorbyl phosphate or derivatives thereof; And
Ii) keep primate embryonic stem cells and be in undifferentiated state.
3. method as claimed in claim 1 or 2, wherein said primate embryonic stem cells has stable caryogram.
4. as each described method among the claim 1-3, wherein xitix is an ascorbyl phosphate.
5. as each described method among the claim 1-4, wherein said primate embryonic stem cells is the pluripotent human embryonic stem cell.
6. as each described method among the claim 1-5, wherein said primate embryonic stem cells has kept in whole cell cultivation process and has been divided into the characteristic of entoderm, mesoderm and ectoderm tissue at least.
7. as each described method among the claim 1-6, wherein fibroblast growth factor (FGF) is selected from aFGF, bFGF, FGF-4, FGF-9, FGF-17 or FGF-18.
8. method as claimed in claim 7, wherein the concentration with 1-100ng/ml is provided as fibroblast growth factor.
9. method as claimed in claim 8, wherein the concentration with about 10ng/ml is provided as fibroblast growth factor.
10. as each described method among the claim 1-9, wherein said fibroblast growth factor is bFGF.
11. as each described method among the claim 1-10, wherein fibroblast growth factor is recombinated.
12. as each described method among the claim 1-11, wherein the concentration with 0.01-0.2mg/ml provides xitix or ascorbyl phosphate or derivatives thereof.
13. method as claimed in claim 12, wherein the concentration with about 0.1mg/ml provides xitix or ascorbyl phosphate or derivatives thereof.
14. as each described method among the claim 1-13, wherein the concentration with 0.1-1.0mg/ml provides 2-thanomin.
15. method as claimed in claim 14, wherein the concentration with about 0.6mg/ml provides 2-thanomin.
16. as each described method among the claim 1-15, wherein the concentration with 3-15 μ g/ml provides oleic acid.
17. method as claimed in claim 16, wherein the concentration with about 9.5 μ g/ml provides oleic acid.
18. as each described method among the claim 1-17, wherein the concentration with 10-500ng/ml provides heparin.
19. as the method for claim 18, wherein the concentration with about 100ng/ml provides heparin.
20. as claim 18 or 19 described methods, wherein heparin is a heparin sodium.
21. as each described method among the claim 1-20, wherein said protein cell is cultivated upholder and is based on collagen protein.
22. method as claimed in claim 21, wherein said cell cultures upholder based on collagen protein comprises type i collagen albumen.
23. method as claimed in claim 22, wherein type i collagen albumen is the type i collagen albumen of reorganization.
24. as each described method among the claim 1-20, wherein said cell cultures upholder comprises the recombinant human protein who is selected from type i collagen albumen, IV collagen type, fibronectin, ln and vitronectin.
25. method as claimed in claim 24, wherein said cell upholder comprises at least two kinds of recombinant proteins that are selected from IV collagen type, fibronectin, ln and vitronectin.
26. method as claimed in claim 24, wherein said cell upholder comprises IV collagen type, fibronectin, ln and the vitronectin of reorganization.
27. as each described method among the claim 1-20, it is Matrigel that wherein said protein cell is cultivated upholder Tm
28., wherein, after in cell culture container, adding EDTA, described primate embryonic stem cells is gone down to posterity as each described method among the claim 1-27.
29., wherein, behind the collagenase that adds preferred collagenase IV, described primate embryonic stem cells is gone down to posterity as each described method among the claim 1-27.
30., wherein, after adding Dispase, described primate embryonic stem cells is gone down to posterity as each described method among the claim 1-27.
31., wherein, after adding trypsinase/EDTA, preferably adding recombinant trypsin, described primate embryonic stem cells is gone down to posterity as each described method among the claim 1-27.
32., wherein clone described primate embryonic stem cells as each described method among the claim 1-31.
33. as each described method among the claim 1-32, wherein said cell culture medium does not comprise buffer reagent HEPES.
34. primate embryonic stem cells is divided into the method for at least a cell type under the cell culture condition of acellular feeder layer and serum-free, it comprises following steps:
I) in by cell culture container, form the primate embryonic stem cells prepared product based on proteinic cell cultures upholder bag quilt, wherein with described cell cultures in cell culture medium, described cell culture medium comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor and heparin, and
Ii) adding induces primate embryonic stem cells to be divided into the reagent of at least a cell type.
35. method as claimed in claim 34, wherein said primate embryonic stem cells are human pluripotent embryonic stem cell.
36. as claim 34 or 35 described methods, wherein said cell type is a neurone.
37. as claim 34 or 35 described methods, wherein said cell type is an epithelial cell.
38. as each described method among the claim 34-37, wherein said is ln based on proteinic cell cultures upholder.
39. cell culture, it comprises primate embryonic stem cells and the cell culture medium that is positioned at based on the proteinic cell cultures upholder, described cell culture medium comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix or ascorbyl phosphate or derivatives thereof.
40. cell culture as claimed in claim 39, wherein said primate embryonic stem cells are the pluripotent human embryonic stem cell.
41. cell culture, it comprises primate embryonic stem cells and the cell culture medium that is positioned at based on the proteinic cell cultures upholder, described cell culture medium comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor and heparin, and described cell culture also comprises at least a reagent of inducing primate embryonic stem cells to be divided at least a cell type.
42. cell culture as claimed in claim 41, wherein said primate embryonic stem cells are the pluripotent human embryonic stem cell.
43. cell culture container, it comprises cell culture medium, described cell culture medium contains: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix, or ascorbyl phosphate, or derivatives thereof.
44. as the described cell culture container of claim 43, wherein said cell culture container also comprises primate embryonic stem cells.
45. as claim 43 or 44 described cell culture containers, wherein said primate embryonic stem cells is the pluripotent human embryonic stem cell.
46. as each described cell culture container among the claim 43-45, wherein said container is selected from petri diss, Tissue Culture Flask or flask, porous plate.
47. cell culture medium container, it comprises cell culture medium, described cell culture medium contains: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor, heparin and xitix, or ascorbyl phosphate, or derivatives thereof.
48. cell culture medium container, it comprises cell culture medium, described cell culture medium contains: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, thanomin, 2 mercapto ethanol, with the bovine serum albumin bonded oleic acid of FAF, and wherein said cell culture medium also is supplemented with fibroblast growth factor and heparin.
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