CN101984048A - Culture medium for culturing mesenchymal stem cells - Google Patents
Culture medium for culturing mesenchymal stem cells Download PDFInfo
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- CN101984048A CN101984048A CN2010105648741A CN201010564874A CN101984048A CN 101984048 A CN101984048 A CN 101984048A CN 2010105648741 A CN2010105648741 A CN 2010105648741A CN 201010564874 A CN201010564874 A CN 201010564874A CN 101984048 A CN101984048 A CN 101984048A
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Abstract
The invention discloses a culture medium for culturing mesenchymal stem cells. The culture medium comprises improved minimum medium IMDM, cell growth factor combination, insulin, trace elements, lipid substances, vitamin substances, hormone substances, protein molecules, amino acid substances and other compounds. The culture medium provided by the invention does not contain serum or plasma (comprising animal or human serum or plasma), thus, when the system is used for culturing the mesenchymal stem cells, zoonosis caused by the animal serum or plasma in the cell therapy process can be avoided, and the risk of human immunity reaction which is possibly caused in the cell therapy process can be avoided. The culture medium has specific chemical constituent, thus the possibility of different cultured mesenchymal stem cell properties caused by the difference of culture systems can be avoided. The culture medium is not only suitable for culturing the mesenchymal stem cells of human marrow and umbilical cord resources but also suitable for culturing the mesenchymal stem cells of other tissues or other animals, and has the advantage of wide application range.
Description
Technical field
The invention belongs to the culture medium prescription in cell and the field of tissue culture, relate to a kind of serum-free, chemical ingredients culture medium prescription clearly, this substratum is specially adapted to the vitro culture human mesenchymal stem cell.
Background technology
Mescenchymal stem cell, English name is mesenchymal stem cells (MSC), other title is also arranged, as marrow stromal cell, fat stem cell etc., be meant that single or a group meets the cell (DominiciM 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 multipotentmesenchymal stromal cells.The International Society for Cellular Therapyposition statement.Cytotherapy, 2006; 8 (4): 315-7).In marrow, MSC belongs to non-hematopoietic stem cell, possesses the ability to skeletonization, one-tenth cartilage and lipoblast differentiation, has immunomodulatory and hematopoiesis support effect.Nearest studies show that MSC also is present in other tissue, comprises fat, umbilical cord, placenta and muscle etc.The MSC of vitro culture can secrete multiple biologically active substance, has the new vessel of promotion and forms, and promotes the effect of nervous tissue, osseous tissue, liver organization and islet tissue injury repairing.Therefore, be used for clinical experimental study, with multiple diseases such as treatment graft versus host disease (GVH disease), hepatic fibrosis, Spinal injury, diabetes, peripheral vascular diseases from body or allosome MSC.In addition, as important seed cell in tissue-engineered bone and the cartilage structure, MSC also is used for the treatment of necrosis of femoral head, fracture back nonunion and cartilage injury.Yet, relate to the certain cell concentration of cell therapy needs of multiple disease.For carrying out cell therapy based on MSC; people obtain a spot of marrow usually, or obtain umbilical cord, fat or placenta tissue, screen and amplification MSC by adherent growth external; when treating that cell concentration reaches the treatment aequum, results MSC is used for above treatment of diseases.As seen, amplification in vitro MSC is one of committed step of implementing the MSC cell therapy.
Usually, people utilize through screening, are suitable for the bovine serum that people MSC grows, as the main stimulator of cell proliferation, cultured and amplified in vitro MSC.Yet, studies show that in culturing process, MSC can engulf the bovine protein in the culture system, makes the foreign protein internalization, per 10
8Bovine protein content reaches 10-30mg (Spees JL among the MSC, GregoryCA, Singh H, Tucker HA, Peister A, Lynch PJ, Hsu SC, Smith J, Prockop DJ.Internalized antigens must be removed to prepare hypoimmunogenic mesenchymalstem 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 will make patient be exposed to the risk of suffering from zoonosis, also may cause organism immune response, cause serum sickness or other immunological disease because of transplanting the dead bovine protein that discharges of MSC.In addition, the activity of every batch of serum is different, to batch between the stability of cell of results, certain influence is arranged.Therefore, utilizing a kind of substratum that does not have the animal serum composition, cultivate amplification MSC, to the security and the validity of treatment, all is very necessary.
For this reason, people are seeking the animal serum surrogate always, are used for the cultivation of people MSC.There is human serum substitute Ultroser G to substitute bovine serum, cultivator marrow MSC, obtained effect (Meuleman N preferably, TondreauT, Delforge A, Dejeneffe M, Massy M, Libertalis M, Bron D, Lagneaux L.Humanmarrow mesenchymal stem cell culture:serum-free medium allows betterexpansion than classical alpha-MEM medium.Eur J Haematol 2006; 76:309-16).Yet, itself just contains the bovine serum composition Ultroser G, and substituting of this mode can not fundamentally solve foreign protein pollution problem (Korhonen M.Culture of human mesenchymal stem cells inserum-free conditions:no breakthroughs yet.Eur J Haematol 2006; 78:167).Afterwards, the someone proposes to use the human blood platelets concentrated solution and substitutes bovine serum, and has obtained effect preferably.But whether the thrombocyte donor fully normal, the propagation that can this use-pattern produce communicable diseases, and can ensure the MSC that obtains batch between stability etc., all be the problem of necessary consideration.Therefore, the MSC substratum of development chemical ingredients clear and definite, no animal serum composition is necessary.
Someone once reported the serum-free system of a kind of human cord blood MSC, the blood serum substituting composition is Prostatropin (bFGF), human albumin, Regular Insulin-Transferrins,iron complexes-selenium-thanomin (ITSE) and hydrocortisone, can abbreviate FAITH as, it is reported better (Liu CH of effect, Wu ML, Hwang SM.Optimization of serumfree medium for cord blood mesenchymal stem cells.Biochemical EngineeringJournal, 2007; 33 1-9).Yet, utilize this system to carry out people's umbilical cord MSC and cultivate, find cell growth pattern (see figure 1) spherical in shape; And along with the prolongation of incubation time, spontaneous differentiating phenomenon appears in cell, and variation has taken place the essence of cell.This may be relevant with the hydrocortisone that adds high density.Corticoid, comprise dexamethasone and hydrocortisone, though under finite concentration, can stimulate MSC propagation, also can make MSC that non-specific differentiation (Williams JT takes place, Southerland SS, Souza J, Calcutt AF, Cartledge RG.Cellsisolated from adult human skeletal muscle capable of differentiating intomultiple mesodermal phenotypes.Am Surg.1999; 65:22-6).Therefore can think, carry out MSC based on the serum substitute of bFGF-ITSE and hydrocortisone etc. and cultivate that may there be many uncertain factors in itself stable and to the keeping of stem cell versatility.
Summary of the invention
The purpose of this invention is to provide a kind of serum-free, chemical ingredients and be applicable to the substratum of cultivating mescenchymal stem cell clearly.
For solving the problems of the technologies described above, the present invention takes following technical scheme: a kind of substratum that is used to cultivate mescenchymal stem cell, the called after serum free medium is abbreviated as SF-M (Serum-Free Medium), and this substratum is formed and comprised: (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.
Described (1) is to sell on the IMDM basis the merchant through the basic medium IMDM of improvement, adds to comprise that at least vitamin H (5-50 μ M, preferred 5-30 μ M) and glutamine (5-10 μ M, preferred 5-7.5 μ M) are at interior water-soluble cpds; Described water-soluble cpds can comprise that also iron(ic) chloride (50-1000ng/mL), 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) wait one or more.
The combination of described (2) cell growth factor comprises Prostatropin (bFGF at least, 1-100ng/mL, preferred 5-50ng/ml) and Urogastron (EGF, 1-150ng/mL, preferred 25-100ng/ml), also can comprise activator A (activin A, 1-100ng/ml, preferred 5-50ng/ml), transforming growth factor-beta (TGF-β, 1-100ng/mL, preferred 5-50ng/ml), bone morphogenetic protein (BMP, 1-100ng/mL, preferred 5-50ng/ml), leukaemia inhibitory factor (LIF, 1-100ng/mL, preferred 5-50ng/ml), stem cell factor (SCF, 1-100ng/mL, preferred 50-10ng/ml), pHGF (HGF, 1-100ng/mL, preferred 10-50ng/ml), insulin-like growth factor-i (IGF-1,1-150ng/mL, preferred 25-100ng/ml), erythropoietin (EPO, 0.1-50U/mL, preferred 0.5-5U/ml), thrombotonin (1-100nM/L, preferred 5-50nM/l), platelet derived growth factor BB (PDGF-BB, 1-150ng/mL, 10-50ng/ml), interleukin-6 (IL-6,1-100ng/mL, preferred 5-50ng/ml) etc. wherein one or more.
Described (3) Regular Insulin addition is 1-100 μ g/mL (preferred 2.5-20 μ g/ml), can be clinical grade medication or recombinant protein;
Described (4) trace element comprises selenium (10-100nM, preferred 30nM) at least, provides with Sodium Selenite or other compound form;
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) etc. wherein one or more.
The described vitamin substances of described (6) vitamin substances (6) comprises in two kinds of the fat-soluble and water-soluble vitaminss at least a, comprises wherein one or more such as vitamins B group such as Vit-B5 (1-100 μ M), vitamin A (1-200 μ M), vitamins C (50 μ g/mL), vitamin-E (10-100 μ g/mL).
Described (7) hormonal substance can be dexamethasone and (is lower than 10
-9M) or hydrocortisone (being lower than 5 μ g/mL).
Described (8) protein molecule is mainly the injection human albumin, addition is 1-50mg/mL (preferred 2-20mg/ml), also can comprise other molecule, as Transferrins,iron complexes (10-100 μ g/ml, preferred 20-50 μ g/ml), acetyl-CoA (0.1-10U/mL, preferred 1-5U/ml), nadide (10-1000ng/ml, preferred 25-200ng/ml) etc.
Described (9) amino acids material refers to the multiple amino acids mixture, comprise in L-proline(Pro), L-Serine, L-L-Ala, L-Isoleucine, L-Aspartic Acid, L-tyrosine, L-L-glutamic acid, L-phenylalanine, arginine, Methionin, Xie Ansuan, Threonine, Histidine, methionine(Met), Gelucystine and the glycine etc. one or more, addition is 1-100 μ M.
Described (10) other compound includes 3-mercaptoethanol (1 * 10
-6M-1 * 10
-4M) and other compound, comprise reduced glutathion (0.1-10mg/L), collagen or fibronectin or gelatin (10 μ g-10mg/mL), wnt-3a (1-100ng/mL, preferred 5-50ng/ml) one or more and among the Bio (5-250nM preferably is lower than 100nM).
The application of above-mentioned substratum in cultivating mescenchymal stem cell also belongs to content of the present invention.Described mescenchymal stem cell comprises people's marrow and people's umbilical cord source, also comprises the mescenchymal stem cell of other tissue or other animal.
The invention provides a kind of serum free medium, be used for mescenchymal stem cell, especially the vitro culture of human mesenchymal stem cell.Substratum provided by the present invention does not contain serum or blood plasma (comprising animal or human's serum or blood plasma), therefore, use this system to cultivate mescenchymal stem cell, in the cell therapy process, will avoid the zoonosis that causes because of animal serum or blood plasma, and the risk of the human immunity reaction that causes because of cell therapy.In addition, this substratum specific chemical components is clear, has avoided causing the possibility of the mescenchymal stem cell different in kind of being cultivated because of culture system batch differences.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, applicable to the cultivation of other tissue or other animal mescenchymal stem cell, has a extensive future yet.
Description of drawings
Fig. 1 is people's umbilical cord MSC cellular form of different culture medium culturing
Fig. 2 is people's marrow MSC phenotype analytical result of different culture medium culturing
Fig. 3 is different culture system people umbilical cords and marrow MSC propagation characteristics
Fig. 4 is different system culture condition servant marrow MSC differentiation characteristics
Embodiment
The invention provides a kind of substratum that is used to cultivate mescenchymal stem cell, the called after serum free medium, be abbreviated as SF-M (Serum-Free Medium), this substratum is formed and is comprised: the basic medium IMDM of (1) process improvement (Iscove ' s Modified Dulbecco ' s Medium); (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.
Every class material is formed and characteristics are described below in the above-mentioned substratum, and listed concentration behind each material wherein is the final concentration in overall broth:
(1) described basic medium IMDM through improvement is to sell on the IMDM basis the merchant, adds to comprise that at least vitamin H (5-50 μ M, preferred 5-30 μ M) and glutamine (5-10 μ M, preferred 5-7.5 μ M) are at interior water-soluble cpds; Described water-soluble cpds can comprise that also iron(ic) chloride (50-1000ng/mL), 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) wait one or more.Basic medium after improveing itself can impel the mescenchymal stem cell rate of propagation to increase.
(2) described cell growth factor combination comprises Prostatropin (bFGF at least, 1-100ng/mL, preferred 5-50ng/ml) and Urogastron (EGF, 1-150ng/mL, preferred 25-100ng/ml), also can comprise activator A (activin A, 1-100ng/ml, preferred 5-50ng/ml), transforming growth factor-beta (TGF-β, 1-100ng/mL, preferred 5-50ng/ml), bone morphogenetic protein (BMP, 1-100ng/mL, preferred 5-50ng/ml), leukaemia inhibitory factor (LIF, 1-100ng/mL, preferred 5-50ng/ml), stem cell factor (SCF, 1-100ng/mL, preferred 50-10ng/ml), pHGF (HGF, 1-100ng/mL, preferred 10-50ng/ml), insulin-like growth factor-i (IGF-1,1-150ng/mL, preferred 25-100ng/ml), erythropoietin (EPO, 0.1-50U/mL, preferred 0.5-5U/ml), thrombotonin (1-100nM/L, preferred 5-50nM/l), platelet derived growth factor BB (PDGF-BB, 1-150ng/mL, 10-50ng/ml), interleukin-6 (IL-6,1-100ng/mL, preferred 5-50ng/ml) etc. wherein one or more.These cytokines and somatomedin are the important component parts of initiating mescenchymal stem cell propagation.
(3) described Regular Insulin addition is 1-100 μ g/mL (preferred 2.5-20 μ g/ml), can be clinical grade medication or recombinant protein.Regular Insulin participates in cell glucose metabolism, also is other serum free medium composition commonly used.
(4) described trace element comprises selenium (10-100nM) at least, provides with Sodium Selenite or other compound form.Selenium not only promotes cell proliferation, also is that cellularstructure is formed necessary composition.
(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) etc. wherein one or more.Lipid is the main component of membrane structure.
(6) described vitamin substances comprises in two kinds of the fat-soluble and water-soluble vitaminss at least aly, comprises vitamins B group such as Vit-B5 (1-100 μ M), vitamin A (1-200 μ M), vitamins C (50 μ g/mL), vitamin-E (10-100 μ g/mL) etc.The purpose of adding VITAMIN is that vitamin contents is on the low side in the basic medium, can promote cell proliferation after the interpolation.
(7) described hormonal substance can be dexamethasone (being lower than 10-9M) or hydrocortisone (being lower than 5 μ g/mL).Corticoid is one of composition of short cell proliferation in the serum, adds in the serum-free system, can promote mescenchymal stem cell propagation.
(8) described protein molecule is mainly the injection human albumin, addition is 1-50mg/mL (preferred 2-20mg/ml), also can comprise other molecule, as Transferrins,iron complexes (10-100 μ g/ml, preferred 20-50 μ g/ml), acetyl-CoA (0.1-10U/mL, preferred 1-5U/ml), nadide (10-1000ng/ml, preferred 25-200ng/ml) etc.As a kind of carrier, albumin is the key molecule of cellular material transhipment.
(9) described amino acids material refers to the multiple amino acids mixture, can comprise one or more combination such as L-proline(Pro), L-Serine, L-L-Ala, L-Isoleucine, L-Aspartic Acid, L-tyrosine, L-L-glutamic acid, L-phenylalanine, arginine, Methionin, Xie Ansuan, Threonine, Histidine, methionine(Met), Gelucystine and glycine, addition is 1-100 μ M.Can provide with the form of the cell cultures that is purchased with aminoacids complex.Amino acid is the metabolic essential composition of cell protein.
(10) other compound: also can include 3-mercaptoethanol (1 * 10 in the described substratum
-6M-1 * 10
-4M) and other compound wherein one or more; reduced glutathion (0.1-10mg/L wherein; avoid the infringement of oxyradical as a kind of reductive agent protection cell); collagen or fibronectin or gelatin (10 μ g-10mg/mL) are to promote cell attachment, wnt-3a (1-100ng/mL; preferred 5-50ng/ml) and Bio (5-250nM preferably is lower than 100nM) to regulate cell proliferation.
Embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Method therefor is ordinary method if no special instructions in following embodiment and the test.
The prescription of embodiment 1, serum free medium SF-M
The prescription of embodiment 2, serum free medium SF-M
The prescription of embodiment 3, serum free medium SF-M
The prescription of embodiment 4, serum free medium SF-M
Test one, the cultivation of people's umbilical cord MSC in different substratum
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 available from Invitrogen company; Foetal calf serum (FCS) is a Canadian StemCell company product; The reagent of preparation SF-M and FAITH is many available from Sigma company, and is pure for cultivating.Human albumin is an 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 the primary cell culture system is the alpha-MEM that contains 10%FCS.Get first-generation cell and be used for following test.
2) cell cultures and going down to posterity: after adherent layer forms, digest cell counting with 0.05% trypsinase/2mM EDTA.Cell is suspended in the alpha-MEM that contains 10%FCS respectively, contains the IMDM of SF-M or contain among the IMDM of FAITH, then by 3000 cells/cm
2The cultivation of going down to posterity.In the passage process, photomicrography, record cellular form.
3, test-results
(A: umbilical cord MSC (P1) cultivates the form when cell does not merge fully in containing 10%FCS (StemCell Co.Canada) alpha-MEM for result such as Fig. 1; B: umbilical cord MSC (P1) cultivates the form when cell merges fully in containing 10%FCS (StemCell Co.Canada) alpha-MEM; The form of cell when C: umbilical cord MSC (P1) cultivates in containing the IMDM of FAITH; Cellular form when D: umbilical cord MSC (P1) cultivates in containing the IMDM of FAITH; E: umbilical cord MSC (P1) cultivates in containing the IMDM of SF-M, the form when cell does not merge fully; F: umbilical cord MSC (P1) cultivates in containing the IMDM of SF-M, the form when cell merges fully) shown in, can find relatively that by A/B and E/F the MSC cell refractivity that SF-M cultivates is strong, cell is more elongated; Cell shown in the C/D is the agglomerate growth pattern.
People's marrow MSC phenotype analytical of test two, different culture medium culturing
1, test materials
Sample: people's heparinization marrow 5mL, take from the person that normally offers the marrow.
Reagent: PE mark mouse anti human CD14, CD31, CD44, CD45, CD73 are BD company (U.S.) product, and other products is as testing a description.
2, test method
1) cell cultures: go into marrow MSC cultivation and carry out (Jin JD by the method for putting down in writing in the document, Wang HX, XiaoFJ, Wang JS, Lou X, Wang LS, Guo ZK.A novel source of human mesenchymal stemcells from the debris of bone marrow sample.Biochem Biophys Res Comm 2008; 376:191-5), the primary cell culture system is the alpha-MEM that contains 10%FCS.Get first-generation cell and be used for following test.
People's marrow MSC is suspended in the alpha-MEM that contains 10%FCS respectively or contains among the IMDM of SF-M, then by 3000 cells/cm
2The cultivation of going down to posterity.When treating that cell reaches the third generation, with 0.05% trypsinase/2mM EDTA digestion, cell counting.
2) cell marking:, add monoclonal antibody room temperature lucifuge reaction 30 minutes with the MSC of PBS washing results.After the PBS washed twice, detect with flow cytometer, 10000 point data are collected in each reaction at least.
3) interpretation of result: WinMdi2.9 data that software analysis is gathered in the crops, establish a removal cell debris in the analytic process.
3, test-results
Result such as Fig. 2 (ordinate zou: cell count; X-coordinate: relative intensity of fluorescence), people's marrow MSC (P3) is at 10%FCS (StemCell Co, Canada) and in the SF-M system cultivate, the cell phenotype unanimity, all express CD44, CD73, do not express CD31 (endothelial cell marker), CD14 (Monocytes mark), CD45 (hemocyte mark), show the phenotype homogeneity of the marrow MSC that two kinds of systems are cultivated.
MSC multiplication characteristic under test three, the different system culture condition
1, test materials
Sample: people's marrow MSC and umbilical cord MSC (P3) are by test one and test two methods results, and P3 is used for following experiment for cell.
Reagent: cell fluorescence marker CFSE is the Sigma product.Other products is as testing a description.
2, test method
1) cell marking: CFSE mark MSC carries out (Jin JD by this group reported method, Wang HX, XiaoFJ, Wang JS, Lou X, Wang LS, Guo ZK.A novel source of human mesenchymal stemcells from the debris of bone marrow sample.Biochem Biophys Res Comm 2008; 376:191-5).
2) cell cultures: the cell behind the mark is suspended in the alpha-MEM that contains 10%FCS respectively or contains the IMDM of SF-M or contain among the IMDM of FAITH, then by 3000 cells/cm
2Cultivate.Digest cell counting after 72 hours with 0.05% trypsinase/2mM EDTA.
2) flow cytometer obtains data and interpretation of result: the MSC with PBS washing results, detect with flow cytometer, and 10000 point data are collected in each reaction at least.WinMdi2.9 data that software analysis is gathered in the crops are established a removal cell debris in the analytic process.
3, test-results
Result such as Fig. 3 (ordinate zou: cell count; X-coordinate: relative intensity of fluorescence), use CFSE mark third generation marrow MSC (BM-MSC) and umbilical cord MSC (UC-MSC), cultivate after 72 hours, flow cytometer detects cell fluorescence intensity.Histogram: X-coordinate is represented the FL1 relative intensity of fluorescence; Ordinate zou is represented events.Scatter diagram: X-coordinate is represented the FL1 relative intensity of fluorescence; Ordinate zou is represented SSC.Results suggest BM-MSC and UC-MSC are under 10%FCS and SF-M culture condition, and two kinds of cells speed of growth in two kinds of systems is suitable, no significant difference.Cell is grown relatively 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 methods results, and P1 is used for following test for cell.
Reagent: induction reagent comprises vitamins C, phospho-glycerol, dexamethasone, INDOMETHACIN, IMBX, Regular Insulin, is the Sigma product.
2, test method
1) passage is cultivated: P1 is suspended in the alpha-MEM that contains 10%FCS respectively for marrow MSC or contains among the IMDM of SF-M, by 3000 cells/cm
2Cultivate.After being used for following experiment when being passaged to the third generation.
2) induce differentiation:
By the method for putting down in writing in the document (Sun S, Guo Z, Xiao X, et al.Isolation of mouse marrowmesenchymal 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 novelsource of human mesenchymal stem cells from the debris of bone marrow sample.Biochem Biophys Res Comm 2008; 376:191-5) carry out the differentiation of inducing of gained MSC cell.
3) histochemical stain is identified
Not add the cell of inducing system is contrast, utilizes NBT-BCIP and oil red O stain, identifies scleroblast and adipocyte respectively.
3, test-results
The result as shown in Figure 4, third generation people marrow MSC through different systems cultivations, external evoked to skeletonization (ALP) with become fat (Oil-Red) cytodifferentiation, carry out histochemical stain after 10 days, show that people's marrow MSC that two kinds of systems are cultivated has skeletonization and becomes fatty differentiation capability, resulting MSC cell is described, meets generally acknowledged MSC standard.
Claims (10)
1. substratum of cultivating mescenchymal stem cell, this substratum is formed and comprised: (1) is through 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. substratum according to claim 1, it is characterized in that: described (1) is through the basic medium IMDM of improvement, be to sell on the IMDM basis the merchant, add and comprise vitamin H (5-50 μ M at least, preferred 5-30 μ M) and glutamine (5-10 μ M, preferably 5-7.5 μ M) at interior water-soluble cpds; Described water-soluble cpds can comprise that also iron(ic) chloride (50-1000ng/mL), 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) wait one or more.
3. substratum according to claim 1 and 2, it is characterized in that: the combination of described (2) cell growth factor comprises Prostatropin (bFGF at least, 1-100ng/mL, preferred 5-50ng/ml) and Urogastron (EGF, 1-150ng/mL, preferred 25-100ng/ml), also can comprise activator A (activin A, 1-100ng/ml, preferred 5-50ng/ml), transforming growth factor-beta (TGF-β, 1-100ng/mL, preferred 5-50ng/ml), bone morphogenetic protein (BMP, 1-100ng/mL, preferred 5-50ng/ml), leukaemia inhibitory factor (LIF, 1-100ng/mL, preferred 5-50ng/ml), stem cell factor (SCF, 1-100ng/mL, preferred 50-10ng/ml), pHGF (HGF, 1-100ng/mL, preferred 10-50ng/ml), insulin-like growth factor-i (IGF-1,1-150ng/mL, preferred 25-100ng/ml), erythropoietin (EPO, 0.1-50U/mL, preferred 0.5-5U/ml), thrombotonin (1-100nM/L, preferred 5-50nM/l), platelet derived growth factor BB (PDGF-BB, 1-150ng/mL, 10-50ng/ml), interleukin-6 (IL-6,1-100ng/mL, preferred 5-50ng/ml) etc. wherein one or more.
4. according to claim 1 or 2 or 3 described substratum, it is characterized in that: described (3) Regular Insulin addition is 1-100 μ g/mL (preferred 2.5-20 μ g/ml), can be clinical grade medication or recombinant protein; Described (4) trace element comprises selenium (10-100nM, preferred 30nM) at least, provides with Sodium Selenite or other compound form; Described (7) hormonal substance can be dexamethasone and (is lower than 10
-9M) or hydrocortisone (being lower than 5 μ g/mL).
5. according to the described substratum of one of aforementioned claim 1 to 4, 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) etc. wherein one or more.
6. according to the described substratum of one of aforementioned claim 1 to 5, it is characterized in that: the described vitamin substances of described (6) vitamin substances (6) comprises in two kinds of the fat-soluble and water-soluble vitaminss at least a, comprises wherein one or more such as vitamins B group such as Vit-B5 (1-100 μ M), vitamin A (1-200 μ M), vitamins C (50 μ g/mL), vitamin-E (10-100 μ g/mL).
7. according to the described substratum of one of aforementioned claim 1 to 6, it is characterized in that: described (8) protein molecule is mainly the injection human albumin, addition is 1-50mg/mL (preferred 2-20mg/ml), also can comprise other molecule, as Transferrins,iron complexes (10-100 μ g/ml, preferred 20-50 μ g/ml), acetyl-CoA (0.1-10U/mL, preferred 1-5U/ml), nadide (10-1000ng/ml, preferred 25-200ng/ml) etc.
8. according to the described substratum of one of aforementioned claim 1 to 7, it is characterized in that: described (9) amino acids material refers to the multiple amino acids mixture, comprise in L-proline(Pro), L-Serine, L-L-Ala, L-Isoleucine, L-Aspartic Acid, L-tyrosine, L-L-glutamic acid, L-phenylalanine, arginine, Methionin, Xie Ansuan, Threonine, Histidine, methionine(Met), Gelucystine and the glycine etc. one or more, addition is 1-100 μ M.
9. according to the described substratum of one of aforementioned claim 1 to 8, it is characterized in that: described (10) other compound includes 3-mercaptoethanol (1 * 10
-6M-1 * 10
-4M) and other compound, comprise reduced glutathion (0.1-10mg/L), collagen or fibronectin or gelatin (10 μ g-10mg/mL), wnt-3a (1-100ng/mL, preferred 5-50ng/ml) one or more and among the Bio (5-250nM preferably is lower than 100nM).
10. the application of each described substratum of claim 1-9 in cultivating mescenchymal stem cell, 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-origin.
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