CN104894064A - Culture medium for culturing mesenchymal stem cells - Google Patents
Culture medium for culturing mesenchymal stem cells Download PDFInfo
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- CN104894064A CN104894064A CN201510397087.5A CN201510397087A CN104894064A CN 104894064 A CN104894064 A CN 104894064A CN 201510397087 A CN201510397087 A CN 201510397087A CN 104894064 A CN104894064 A CN 104894064A
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- stem cell
- mescenchymal stem
- cell
- culture medium
- substratum
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of cell and tissue culture, in particular to a culture medium for culturing mesenchymal stem cells. Aiming at the problems that the mesenchymal stem cells exist in an existing bovine serum culture medium and a human serum culture medium, the invention provides the culture medium for culturing the mesenchymal stem cells. According to the technical scheme, the culture medium for culturing the mesenchymal stem cells provided by the invention is characterized by comprising two kinds of components, namely a basal culture medium and culture medium additives; the additives comprise a cell growth factor combination, insulin, trace elements, lipid materials, vitamin substances, hormone-like substances, protein molecules, amino acid substances and other compounds. According to the culture medium for culturing the mesenchymal stem cells, provided by the invention, the zoonosis and the risk of human immune responses in the cell therapy process can be avoided, and the possibility that the natures of the cultured mesenchymal stem cells are different due to the difference among culture system batches can be avoided; the culture medium is suitable for culturing all the mesenchymal stem cells and is broad in prospect.
Description
Technical field
The present invention relates to cell and tissue culture field, particularly a kind of substratum for cultivating mescenchymal stem cell.
Background technology
Mescenchymal stem cell, English name is mesenchymal stem cells (MSC), refer to that single or a group meets cell (the Dominici M of international uniform standard, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy, 2006, 8 (4): 315-7).
In marrow, MSC belongs to non-hematopoietic stem cell, possesses to skeletonization, the ability becoming cartilage and lipoblast differentiation, has immunomodulatory and Hematopoiesis Support affect.Nearest research shows, MSC is also present in other tissue, comprises fat, umbilical cord, placenta and muscle etc.The MSC of vitro culture can secrete various bioactivators, has and promotes that new vessel is formed, promote the effect of nervous tissue, osseous tissue, liver organization and islet tissue injury repairing.
Therefore, autologous or allosome MSC for clinical experimental study, to treat the various diseases such as graft versus host disease (GVH disease), hepatic fibrosis, Spinal injury, diabetes, peripheral vascular disease.In addition, seed cell important in building as tissue-engineered bone and cartilage, MSC is also for the treatment of nonunion and cartilage injury after necrosis of femoral head, fracture.
But the cell therapy relating to various diseases needs certain cell concentration.For carrying out the cell therapy based on MSC; people obtain a small amount of marrow usually; or obtain umbilical cord, fat or placenta tissue; screened by adherent growth in vitro and the MSC that increases; when cell concentration reaches treatment aequum; results MSC is used for the treatment of above disease, and amplification in vitro MSC is one of committed step implementing MSC cell therapy.
Usually, people utilize through screening, the bovine serum being suitable for people MSC growth, as the main stimulator of cell proliferation, and cultured and amplified in vitro MSC.But research shows, in culturing process, MSC can engulf the bovine protein in culture system, make xenogenesis-+
Albumen internalization, every 10
8in MSC, bovine protein content reaches 10-30mg (Spees JL, Gregory CA, Singh H, Tucker HA, Peister A, Lynch PJ, Hsu SC, Smith J, Prockop DJ.Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy.Mol Ther 2004; 9:747-56).
Therefore, use bovine serum cultivator MSC to be used for cell therapy, not only patient will be exposed to suffer from the risk of zoonosis, and also because transplanting MSC death release bovine protein, organism immune response may be caused, cause serum sickness or other immunological disease.
In addition, the activity of every serum batches is different, to batch between the stability of cell of results, have certain influence.Therefore, utilizing a kind of substratum without animal serum composition, cultivate amplification MSC, to security and the validity for the treatment of, is all very necessary.
For this reason, people are finding animal serum surrogate always, for the cultivation of people MSC.
Someone substitutes bovine serum with serum substitute Ultroser G, cultivator marrow MSC, obtain good effect (Meuleman N, Tondreau T, Delforge A, Dejeneffe M, Massy M, Libertalis M, Bron D, Lagneaux L.Human marrow mesenchymal stem cell culture:serum-free medium allows better expansion than classical alpha-MEM medium.Eur J Haematol 2006; 76:309 – 16).
But, Ultroser G is inherently containing bovine serum composition, and substituting of this mode fundamentally can not solve foreign protein pollution problem (Korhonen M.Culture of human mesenchymal stem cells in serum-free conditions:no breakthroughs yet.Eur J Haematol 2006; 78:167).
Afterwards, someone proposes application human blood platelets concentrated solution and substitutes bovine serum, and obtains good effect.But whether Platelet donors completely normal, the propagation that can this use-pattern produce communicable diseases, and can ensure obtained MSC batch between stability etc., be all the problem that must consider.
Therefore, development chemical composition clear and definite, MSC substratum without animal serum composition are necessary.
Someone once reported the serum-free system of a kind of human cord blood MSC, blood serum substituting composition is Prostatropin (bFGF), human albumin, Insulin-Transferrin-selenium-thanomin (ITSE) and hydrocortisone, can referred to as FAITH, it is reported effect better (Liu CH, Wu ML, Hwang SM.Optimization of serum free medium for cord blood mesenchymal stem cells.Biochemical Engineering Journal, 2007; 331 – 9).
But, utilize this system to carry out people's umbilical cord MSC and cultivate, find cell growth pattern spherical in shape (see Fig. 1); And along with the prolongation of incubation time, there is Spontaneous Differentiation phenomenon in cell, and the essence of cell there occurs change.This may be relevant with the hydrocortisone adding high density.Corticoid, comprise dexamethasone and hydrocortisone, although MSC can be stimulated under finite concentration to breed, also can make MSC that non-specific differentiation (Williams JT occurs, Southerland SS, Souza J, Calcutt AF, Cartledge RG.Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes.Am Surg.1999; 65:22-6).Therefore can think, carry out MSC cultivation based on the serum substitute of bFGF-ITSE and hydrocortisone etc., may there is many uncertain factors in the stability of itself and the maintenance to stem cell versatility.
Summary of the invention
Cultivate mescenchymal stem cell Problems existing for existing bovine serum substratum and human serum, the object of this invention is to provide the substratum that a kind of serum-free, chemical composition are applicable to cultivate mescenchymal stem cell clearly.
Its technical scheme of dealing with problems is:
For cultivating a substratum for mescenchymal stem cell, this substratum composition comprises basic medium and culture medium additive two class component.
Described culture medium additive comprises cell growth factor sub-portfolio; Regular Insulin; Trace element; Lipid material; Vitamin substances; Hormonal substance; Protein molecule; Amino acids material; Other compound.
In above-mentioned substratum, every class material compositing characteristic is described below:
(1) basic medium IMDM, is on commercially IMDM basis, adds the water-soluble cpds at least comprising vitamin H (1-50 μM) and glutamine (1-50 μM); Described water-soluble cpds also can comprise chromium chloride (0.1-10 μ g/L), copper sulfate (0.1-1mg/L), Manganous chloride tetrahydrate 0.01-0.1mg/L and zinc sulfate 0.1-10mg/L.Carry out improveing rear mescenchymal stem cell rate of propagation to increase.
(2) described cell growth factor combination at least comprises Prostatropin (bFGF, 1-100ng/mL) with Urogastron (EGF, 1-100ng/mL), also can comprise activin A (activin A, 1-100ng/ml), transforming growth factor-beta (TGF-β, 1-100ng/mL), bone morphogenetic protein (BMP, 1-100ng/mL), leukaemia inhibitory factor (LIF, 1-100ng/mL), stem cell factor (SCF, 1-100ng/mL), pHGF (HGF, 1-100ng/mL), insulin-like growth factor-i (IGF-1, 1-100ng/mL), erythropoietin (EPO, 0.1-50U/mL), thrombotonin (1-100nM/L), platelet derived growth factor BB (PDGF-BB, 1-100ng/mL), interleukin-6 (IL-6, 1-100ng/mL).These cytokines and somatomedin are the important component parts of initiating mescenchymal stem cell propagation.
(3) described Regular Insulin addition is 5-500 μ g/mL, can be clinical grade medication or recombinant protein.
Regular Insulin participates in cell glucose metabolism.
(4) described trace element at least comprises selenium (10-100nM), there is provided with Sodium Selenite or other compound form, described trace and REE elements also can comprise chromium chloride (0.1-10 μ g/L), cupric chloride (0.1-1mg/L), Manganous chloride tetrahydrate 0.01-0.1mg/L, zinc chloride 0.1-10 μ g/L and Sodium orthomolybdate (100-1000ng/mL).
Trace element not only promotes cell proliferation, is also the composition that cellularstructure composition is necessary.
(5) described lipid material can be cholesterol (10-100 μ g/mL), also can be linolic acid (1-50mg/L), linolenic acid (1-50 μ g/mL), medium chain triglyceride (1-500 μ g/mL), Yelkin TTS (0.1-20 μ g/mL) and injection soybean oil (1-500 μ g/mL).
Lipid is the main component of membrane structure.
(6) described vitamin substances comprises fat-soluble and water-soluble vitamins two kinds, comprises vitamins B group as Vit-B5 (1-100 μM), vitamin A (1-200 μM), vitamins C (50 μ g/mL), vitamin-E (10-100 μ g/mL).
The object of adding VITAMIN is that in basic medium, vitamin contents is on the low side, can promote cell proliferation after interpolation.
(7) described hormonal substance is dexamethasone (lower than 10-9M) or hydrocortisone (lower than 5 μ g/mL).Corticoid is one of composition of short cell proliferation in serum, adds, can promote that mescenchymal stem cell is bred in serum-free system.
(8) described protein molecule is mainly injection human albumin, and addition is 5-50mg/mL, also can comprise other molecule, as acetyl-CoA (0.1-10U/mL), nadide (10-1000ng/mL).
As a kind of carrier, albumin is the key molecule of cellular material transhipment.
(9) described amino acids material refers to multiple amino acids mixture, can comprise L-PROLINE, Serine, ALANINE, ILE, L-ASPARTIC ACID, TYR, Pidolidone, L-Phe, arginine, Methionin, α-amino-isovaleric acid, Threonine, Histidine, methionine(Met), Gelucystine and glycine, addition is 1-100 μM.Can provide by the form of the cell cultures aminoacids complex be purchased.Amino acid is the required composition of cellular protein metabolism.
(10) other compound described: also can include 3-mercaptoethanol (1 × 10 in described substratum
-6m-1 × 10
-4and other compound M); comprise reduced glutathion (0.1-10mg/L; as the infringement of a kind of reductive agent Cell protection from oxyradical), collagen or fibronectin or gelatin (10 μ g-10mg/mL) are to promote that cell attachment, wnt-3a (1-100ng/mL) and Bio (5-250nM) are to regulate cell proliferation.
Substratum provided by the present invention, not containing serum or blood plasma, uses this system to cultivate mescenchymal stem cell, will avoid the zoonosis because animal serum or blood plasma cause in cell therapy procedures, and the risk of the Human immune responses caused because of cell therapy.In addition, this substratum specific chemical components is clear, avoids because of difference between culture system batch, causes the possibility of cultivated mescenchymal stem cell different in kind.Substratum of the present invention is not only applicable to the cultivation of the mescenchymal stem cell in people's marrow and people's umbilical cord source, is applicable to the cultivation of other tissue or other animal mescenchymal stem cell yet, has a extensive future.
Accompanying drawing explanation
Fig. 1 is people's umbilical cord MSC cellular form that different culture media is cultivated.
Fig. 2 is people's marrow MSC phenotype analytical result that different culture media is cultivated.
Fig. 3 is different culture system people umbilical cord and marrow MSC proliferative properties.
Fig. 4 is different system culture condition servant marrow MSC differentiation characteristics.
Embodiment
Be described in detail below in conjunction with the content of embodiment to invention:
Embodiment 1,
For cultivating a substratum for mescenchymal stem cell, this substratum has following one-tenth to be grouped into comprise basic medium and culture medium additive two class component,
Culture medium additive comprises cell growth factor sub-portfolio; Regular Insulin; Trace element; Lipid material; Vitamin substances; Hormonal substance; Protein molecule; Amino acids material; Other compound.
The formula of serum free medium
Embodiment 2,
For cultivating a substratum for mescenchymal stem cell, this substratum composition comprises basic medium and culture medium additive two class component.
Culture medium additive comprises cell growth factor sub-portfolio; Regular Insulin; Trace element; Lipid material; Vitamin substances; Hormonal substance; Protein molecule; Amino acids material; Other compound.
The formula of serum free medium
Embodiment 3,
For cultivating a substratum for mescenchymal stem cell, this substratum composition comprises basic medium and culture medium additive two class component.
Culture medium additive comprises cell growth factor sub-portfolio; Regular Insulin; Trace element; Lipid material; Vitamin substances; Hormonal substance; Protein molecule; Amino acids material; Other compound.
The formula of serum free medium
Embodiment 4,
For cultivating a substratum for mescenchymal stem cell, this substratum composition comprises basic medium and culture medium additive two class component.
Culture medium additive comprises cell growth factor sub-portfolio; Regular Insulin; Trace element; Lipid material; Vitamin substances; Hormonal substance; Protein molecule; Amino acids material; Other compound.
The formula of serum free medium
Embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Following embodiment and test in method therefor be ordinary method if no special instructions.
Experiment one,
The cultivation of people's umbilical cord MSC in different culture media
1, test materials
Sample: people's umbilical cord MSC takes from the postnatal fetus of normal labor.
Reagent: Collagenase I, trypsinase, α-MEM and IMDM are purchased from Invitrogen company; Foetal calf serum (FCS) is Canadian StemCell Products; The many available from Sigma of reagent of preparation SF-M and FAITH, pure for cultivating.Human albumin is injection, is recombinant protein, is produced by North China Pharmaceutical Factory.
2, test method
1) people's umbilical cord MSC cultivates and carries out according to a conventional method, and primary cell culture system is the alpha-MEM containing 10%FCS.Get first-generation cell for following test.
2) cell cultures and going down to posterity: after leapfrog structure is formed, digest with 0.05% trypsinase/2mM EDTA, cell counting.Cell is suspended in respectively the alpha-MEM containing 10%FCS, the IMDM containing SF-M or containing in the IMDM of FAITH, then presses 3000 cells/cm
2carry out Secondary Culture.In passage process, photomicrography, record cellular form.
3, test-results
Result is as Fig. 1
Fig. 1. the form of different culture system servant umbilical cord MSC.People's umbilical cord MSC is respectively at 10%FCS (A, B), serum-free system (the FAITH of bibliographical information, C and D) and the middle cultivation of system of the present invention (SF-M, E and F), (A during cell low density, C, E) and high-density time (B, D, F) form feature.
(A: umbilical cord MSC (P1) is cultivating containing in 10%FCS (StemCell Co.Canada) alpha-MEM, form when cell does not merge completely; B: umbilical cord MSC (P1) is cultivating containing in 10%FCS (StemCell Co.Canada) alpha-MEM, form when cell merges completely; The form of cell when C: umbilical cord MSC (P1) cultivates in the IMDM containing FAITH; Cellular form when D: umbilical cord MSC (P1) cultivates in the IMDM containing FAITH; E: umbilical cord MSC (P1) cultivates in containing the IMDM of SF-M, form when cell does not merge completely; F: umbilical cord MSC (P1) cultivates in containing the IMDM of SF-M, form when cell merges completely) shown in, relatively can be found by A/B and E/F, the MSC cell refractivity that SF-M cultivates is strong, and cell is more elongated; Cell shown in C/D is agglomerate growth pattern.
Test two,
People's marrow MSC phenotype analytical that different culture media is cultivated
1, test materials
Sample: people's heparinization marrow 5mL, takes from the person that normally offers marrow.
Reagent: PE marks mouse anti human CD14, CD31, CD44, CD45, CD73 are BD company (U.S.) product, and other products is as tested described by one.
2, test method
1) cell cultures: people's marrow MSC cultivates and carries out (Jin JD by the method recorded in document, Wang HX, Xiao FJ, Wang JS, Lou X, Wang LS, Guo ZK.A novel source of human mesenchymal stem cells from the debris of bone marrow sample.Biochem Biophys Res Comm 2008; 376:191-5), primary cell culture system is the alpha-MEM containing 10%FCS.Get first-generation cell for following test.
People's marrow MSC is suspended in respectively in the alpha-MEM containing 10%FCS or the IMDM containing SF-M, then by 3000 cells/cm
2carry out Secondary Culture.When cell reaches the third generation, digest with 0.05% trypsinase/2mM EDTA, cell counting.
2) cell marking: with the MSC of PBS washing results, add monoclonal antibody room temperature lucifuge and react 30 minutes.After PBS washes twice, with flow cytomery, 10000 point data are at least collected in each reaction.
3) interpretation of result: WinMdi2.9 data that software analysis is gathered in the crops, establishes a removal cell debris in analytic process.
3, test-results
Result is as Fig. 2
Fig. 2: cell is phenotypic characteristic under different system is cultivated.Ordinate zou: cell count; X-coordinate: relative intensity of fluorescence.
(ordinate zou: cell count; X-coordinate: relative intensity of fluorescence) shown in, people's marrow MSC (P3) is at 10%FCS (StemCell Co, Canada) and in SF-M system cultivate, cell phenotype is consistent, all express CD44, CD73, do not express CD31 (endothelial cell marker), CD14 (Monocytes/Macrophages mark), CD45 (hemocyte mark), show the phenotype homogeneity of the marrow MSC that two kinds of systems are cultivated.
Test three,
MSC multiplication characteristic under different system culture condition
1, test materials
Sample: people's marrow MSC and umbilical cord MSC (P3) is by test one and test two method results, and P3 is used for following experiment for cell.
Reagent: cell fluorescence marker CFSE is Sigma product.Other products is as tested described by one.
2, test method
1) method that cell marking: CFSE mark MSC reports by this group carries out (Jin JD, Wang HX, Xiao FJ, Wang JS, Lou X, Wang LS, Guo ZK.A novel source of human mesenchymal stem cells from the debris of bone marrow sample.Biochem Biophys Res Comm 2008; 376:191-5).
2) cell cultures: the cell after mark is suspended in respectively in the alpha-MEM containing 10%FCS or the IMDM containing SF-M or the IMDM containing FAITH, then by 3000 cells/cm
2cultivate.Digest with 0.05% trypsinase/2mM EDTA after 72 hours, cell counting.
2) flow cytometer obtains data and interpretation of result: with the MSC of PBS washing results, with flow cytomery, 10000 point data are at least collected in each reaction.WinMdi2.9 data that software analysis is gathered in the crops, establish a removal cell debris in analytic process.
3, test-results
Result is as Fig. 3
Fig. 3. people's umbilical cord and the proliferative conditions of marrow MSC under different system is cultivated.Third generation people marrow or umbilical cord MSC, cultivate 72 hours after fluorescein based dye CFSE mark, flow cytometer collects data, winmdi29 software analysis.Histogram: CFSE relative intensity of fluorescence; Scatter diagram: X-coordinate represents relative intensity of fluorescence, ordinate zou represents SSC, represents cell granulations degree.
(ordinate zou: cell count; X-coordinate: relative intensity of fluorescence) shown in, application CFSE marks third generation marrow MSC (BM-MSC) and umbilical cord MSC (UC-MSC), cultivates after 72 hours, flow cytomery cell fluorescence intensity.Histogram: X-coordinate represents FL1 relative intensity of fluorescence; Ordinate zou represents events.Scatter diagram: X-coordinate represents FL1 relative intensity of fluorescence; Ordinate zou represents SSC.BM-MSC and UC-MSC is under 10%FCS and SF-M culture condition in result prompting, and two kinds of cells speed of growth in two kinds of systems is suitable, without significant difference.Cell grows poor in FAITH.
Test four,
Different system culture condition servant marrow MSC differentiation characteristic
1, test materials
Sample: people's marrow MSC (P1) is by test two method results, and P1 is used for following test for cell.
Reagent: induction reagent comprises vitamins C, phospho-glycerol, dexamethasone, INDOMETHACIN, IMBX, Regular Insulin, is Sigma product.
2, test method
1) passage is cultivated: be suspended in the alpha-MEM containing 10%FCS or the IMDM containing SF-M respectively, by 3000 cells/cm by P1 for marrow MSC
2cultivate.For after following experiment when being passaged to the third generation.
2) differentiation-inducing:
By the method recorded in document (Sun S, Guo Z, Xiao X, et al.Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method.Stem Cells 2003; 21:527-35) (Jin JD, Wang HX, Xiao FJ, Wang JS, Lou X, Wang LS, Guo ZK.A novel source of human mesenchymal stem cells from the debris of bone marrow sample.Biochem Biophys Res Comm 2008; 376:191-5) carry out the differentiation-inducing of gained MSC cell.
3) histochemical stain qualification
Not add the cell of induction system for contrast, utilize NBT-BCIP and oil red O stain, identify scleroblast and adipocyte respectively.
3, test-results
Fig. 4. people's marrow MSC when different system is cultured to the third generation cell skeletonization (ALP) with become fat (Oil-Red) capability analysis.
Result as shown in Figure 4, through the third generation people marrow MSC that different system is cultivated, external evoked to skeletonization (ALP) with become fat (Oil-Red) cytodifferentiation, histochemical stain is carried out after 10 days, the people's marrow MSC showing two kinds of systems cultivations has skeletonization and adipogenic differentiate ability, obtained MSC cell is described, meets generally acknowledged MSC standard.
Claims (13)
1. cultivate a substratum for mescenchymal stem cell, it is characterized in that: this substratum composition comprises: (1) is through the basic medium IMDM of improvement; (2) cell growth factor sub-portfolio; (3) Regular Insulin; (4) trace element; (5) lipid material; (6) vitamin substances; (7) hormonal substance; (8) protein molecule; (9) amino acids material; (10) other compound.
2. a kind of substratum cultivating mescenchymal stem cell according to claim 1, it is characterized in that: described (1) is through the basic medium IMDM of improvement, be on commercially IMDM basis, add the water-soluble cpds at least comprising vitamin H (1-50 μM) and glutamine (1-50 μM); Described soluble compound also can comprise chromium chloride (0.1-10 μ g/L), cupric chloride (0.1-1mg/L), Manganous chloride tetrahydrate 0.01-0.1mg/L and zinc chloride 0.1-10mg/L.
3. a kind of substratum cultivating mescenchymal stem cell according to claim 1, it is characterized in that: the combination of described (2) cell growth factor at least comprises Prostatropin (bFGF, 1-100ng/mL) with Urogastron (EGF, 1-100ng/mL), also can comprise activin A (1-100ng/ml), transforming growth factor-beta (TGF-β, 1-100ng/mL), bone morphogenetic protein (BMP, 1-100ng/mL), leukaemia inhibitory factor (LIF, 1-100ng/mL), stem cell factor (SCF, 1-100ng/mL), pHGF (HGF, 1-100ng/mL), insulin-like growth factor-i (IGF-1, 1-100ng/mL), erythropoietin (EPO, 0.1-50U/mL), thrombotonin (1-100nM/L), platelet derived growth factor BB (PDGF-BB, 1-100ng/mL), interleukin-6 (IL-6, 1-100ng/mL).
4. a kind of substratum cultivating mescenchymal stem cell according to claim 1, is characterized in that: described (3) Regular Insulin addition is 5-500 μ g/mL, can be clinical grade medication or recombinant protein.
5. a kind of substratum cultivating mescenchymal stem cell according to claim 1, it is characterized in that: described (4) trace element at least comprises selenium (10-100nM), there is provided with Sodium Selenite or other compound form, described trace and REE elements also can comprise iron(ic) chloride (50-1000ng/mL), Manganous chloride tetrahydrate (1-100ng/mL), cupric chloride (50-1000ng/mL), chromium chloride and Sodium orthomolybdate (100-1000ng/mL).
6. a kind of substratum cultivating mescenchymal stem cell according to claim 1, it is characterized in that: described (5) lipid material can be cholesterol (10-100 μ g/mL), also can be linolic acid (1-50mg/L), linolenic acid (1-50 μ g/mL), medium chain triglyceride (1-500 μ g/mL), Yelkin TTS (0.1-20 μ g/mL) and injection soybean oil (1-500 μ g/mL).
7. a kind of substratum cultivating mescenchymal stem cell according to claim 1, it is characterized in that: described (6) vitamin substances comprises fat-soluble and water-soluble vitamins two kinds, comprises vitamins B group as Vit-B5 (1-100 μM), vitamin A (1-200 μM), vitamins C (50 μ g/mL), vitamin-E (10-100 μ g/mL).
8. a kind of substratum cultivating mescenchymal stem cell according to claim 1, is characterized in that: described (7) hormonal substance can be dexamethasone (lower than 10
-9or hydrocortisone (lower than 5 μ g/mL) M).
9. a kind of substratum cultivating mescenchymal stem cell according to claim 1, is characterized in that: described (8) protein molecule is mainly injection human albumin, and addition is 5-50mg/mL; Also other molecule can be comprised, as acetylcoenzyme (0.1-10U/mL), nadide (10-1000ng/mL).
10. a kind of substratum cultivating mescenchymal stem cell according to claim 1, it is characterized in that: described (9) amino acids material refers to multiple amino acids mixture, comprise L-PROLINE, Serine, ALANINE, ILE, L-ASPARTIC ACID, TYR, Pidolidone, L-Phe, arginine, Methionin, α-amino-isovaleric acid, Threonine, Histidine, methionine(Met), Gelucystine and glycine etc., addition is 1-100 μM.
11. a kind of substratum cultivating mescenchymal stem cell according to claim 1, is characterized in that: described (10) other compound includes 3-mercaptoethanol (1 × 10
-6m-1 × 10
-4m) and other compound, reduced glutathion (0.1-10mg/L), collagen or fibronectin or gelatin (10 μ g-10mg/mL), wnt-3a (1-100ng/mL) and Bio (5-250nM) is comprised.
12. a kind of substratum cultivating mescenchymal stem cell described in any one of claim 1-11 is cultivating the application in mescenchymal stem cell.
13. application according to claim 12, is characterized in that: described mescenchymal stem cell comprise people's marrow and people's umbilical cord source, also comprise the mescenchymal stem cell of other tissue or other animal.
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