CN101412985A - Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells - Google Patents
Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells Download PDFInfo
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- CN101412985A CN101412985A CNA2007100470350A CN200710047035A CN101412985A CN 101412985 A CN101412985 A CN 101412985A CN A2007100470350 A CNA2007100470350 A CN A2007100470350A CN 200710047035 A CN200710047035 A CN 200710047035A CN 101412985 A CN101412985 A CN 101412985A
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Abstract
The present invention belongs to the field of biotechnology, and discloses a serum-free culture medium with specific chemical compositions for in vitro culture and amplification of bone marrow mesenchymal stem cells. By adding insulin, transferrin, ethanolamine, sodium selenite, growth factors, adherent factors, hormone, putrescine, inorganic salt, vitamin, albumin and antioxidant into a basic culture medium, the bone marrow mesenchymal stem cells can attach to the culture medium under a serum free condition, so the in vitro culture and amplification are realized, the potential of multi-directional differentiation is maintained, and the amplified cells can be induced to be osteoblast and lipocyte in vitro. The serum-free culture medium has the advantages that the clinic level cell products for human produced by the serum free culture medium can effectively avoid the potential risk of producing cell products by serum culture medium. The drawing appended is a photo of the confluence of the bone marrow mesenchymal stem cells cultured by the serum-free culture medium.
Description
[technical field]
The present invention relates to a kind of cell culture medium of biological technical field, especially a kind of serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification.
[background technology]
Mesenchymal stem cells MSCs (MSCs) is the major ingredient of bone marrow matrix, for the hemopoietic function of marrow provides support on the 26S Proteasome Structure and Function.Present studies show that, mesenchymal stem cells MSCs can promote the reconstruction of hemopoietic system, has effective immunoloregulation function, can be at vitro inhibition T-lymphopoiesis, delay the immunological rejection that occurs after the transplanted tissue, can also be used for the graft versus host disease (GVHD) that prevention and treatment autologous stem cell transplantation (ASCT) produce, avoid in organ transplantation, producing immunological rejection, and the treatment autoimmune disorder, as rheumatic arthritis etc.Mesenchymal stem cells MSCs has the potential of multidirectional differentiation, can be divided into the various kinds of cell pedigree, as scleroblast, chondrocyte, myocyte, adipocyte, liver cell, nephrocyte, myocardial cell and neurocyte or the like, thereby can carry out tissue repair and regeneration by tissue engineering technique.Because mesenchymal stem cells MSCs can be divided into the various kinds of cell type, and a little less than the immunogenicity, thereby can be used as organizational project ideal seed cell source.In addition, mesenchymal stem cells MSCs is easy to the transfection and the expression of foreign gene, can be widely used in cell therapy and gene therapy, has very wide application prospect.But mesenchymal stem cells MSCs content in marrow is few, about 10
4～10
5Only contain a mesenchymal stem cells MSCs in the individual myelomonocyte, therefore, only, need actively carry out, to satisfy people's demand on using at vitro culture and amplification mesenchymal stem cells MSCs by obviously satisfying clinical demand from body or allosome separation mesenchymal stem cells MSCs.
Carry out vitro culture and amplification mesenchymal stem cells MSCs (MSCs) need add the foetal calf serum of a certain amount of (10%～20%) usually in substratum, this once was considered to cultivate the top condition of mesenchymal stem cells MSCs, and can keep its biological characteristics preferably.
But use animal serum to have dual character: on the one hand, animal serum is the growth of sustenticular cell well: the one, and, a large amount of albumen that contains in the serum can play a protective role by pair cell; The 2nd,, the trace element that contains and a large amount of nutritive ingredients can be enriched the growth of substratum with sustenticular cell; The 3rd,, the various kinds of cell factor that contains can promote cell proliferation; The 4th,, can also provide anchoring factor for wall dependent cells.
But on the other hand, for the cellular product that animal serum is produced, its biological safety is subjected to people's generally query: the one, and, because the component difference between each batch serum is bigger, thereby the stability of pair cell product exerts an influence; The 2nd,, composition is indeterminate, and a large amount of agnoproteins brings great interference to research work; The 3rd,, even more serious is, may contain the infectious agent and the unknown zoonosis that are made of protein in the animal serum, when others uses cellular product in the production clinical grade, can cause the propagation of these infective agents in theory; The 4th,, albumen in the serum or peptide may be integrated by mesenchymal stem cells MSCs in culturing process, like this, will cause the host to produce immune response when the bone marrow mescenchymal stem cell.
In order to overcome the defective that animal serum may exist when (MSCs) at vitro culture and amplification mesenchymal stem cells MSCs, since 20th century the nineties people just the research of serum-free culture mesenchymal stem cells MSCs.Nineteen ninety-five, people such as Donald have developed a kind of serum free medium RDM-F of specific chemical components, are used for the amplification in vitro rat bone marrow mesenchymal stem cells.It is basic medium that this substratum adds 40%MCDB-201 with 60%DMEM-LG, add Regular Insulin, 0.1% (w/v) LA-BSA, 10ng/mL PDGF-β β and the 1ng/mL bFGF of 5 μ g/mL, rat bone marrow mesenchymal stem cells in blood serum medium is arranged adherent fully after, be replaced with serum free medium RDM-F and cultivate [Lennon, D.P., et al., Exp Cell Res, 219, p.211-222 (1995)].
People such as Gronthos and Simmons is a basic medium with α-MEM, 10 μ g/mL Sigma I8405s, 2% (w/v) BSA, 4 μ g/mL human ldls, the saturated human transferrin of 200 μ g/mL, 2mmol/L L-glutaminate, 10nM dexamethasone sodium phosphate, 100 μ M xitix (L-ascorbicacid2-phosphate have been added, ASAP), 50 μ M beta-mercaptoethanol [Gronthos, S., Simmons, P.J.Blood, 85, p.929-940 (1995)].
The people such as Liu Jixian of Taiwan Changgeng Univ. have developed the serum free medium that is used for the amplification in vitro umbilical cord blood mesenchymal stem cells, this substratum is basic medium with IMDM, added human serum albumin, hydrocortisone, (SITE is a trade(brand)name to SITE, be the abbreviation of four kinds of materials: Selenite selenate, Insulin Regular Insulin, Transferrin Transferrins,iron complexes, Ethanolamine thanomin) and bFGF (Prostatropin) [Chi-Hsien Liu, et al., Biochem Eng J, 33, p.1-9 (2007)].
In addition, U.S. Pat P 5,908,782 is basic medium with IMDM, has added BSA, human lipoprotein, saturated human transferrin, recombinant human insulin, VITAMIN, amino acid, trace element and recombinant human PDGF-β β (Thr6 PDGF BB) and serotonin as the serum free medium of cultivating mesenchymal stem cells MSCs.U.S. Pat P 7,109,032 based on F12 (Coon ' s modifiedHam ' s F12) substratum, has added HAS, Transferrins,iron complexes, xitix, beta-mercaptoethanol, cholesterol, selenium, vitamin H, pantetheine sodium, dexamethasone and EGF (Urogastron)/PDGF (Thr6 PDGF BB)/FGF-2 (fibroblast growth factor)/IGF-I (insulin like growth factor-1)/LIF (leukaemia inhibitory factor)/SCF cytokines such as (STEM CELL FACTOR).
More than Yan Jiuhekaifa serum free medium all is materials such as the albumen that adds purifying on basic medium, hormone, somatomedin, trace element, VITAMIN, be characterized in specific chemical components, can keep the growth of mesenchymal stem cells MSCs preferably, keep the multidirectional differentiation potential of mesenchymal stem cells MSCs.But these serum free mediums can not promote that all mesenchymal stem cells MSCs is adherent.When vitro culture, mesenchymal stem cells MSCs need be in containing blood serum medium adherent fully after, being replaced with serum free medium again cultivates, can cause residual a certain amount of animal serum in substratum like this, on the meaning of strictness, say, do not reach real serum-free culture, in clinical application, still may have very big risk.
[summary of the invention]
The objective of the invention is, overcome the deficiency that prior art exists, for vitro culture and amplification mesenchymal stem cells MSCs (MSCs) provide a kind of serum free medium of specific chemical components, avoid containing the risk that the product of blood serum medium production may exist in clinical application.
The serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification is by various nutritive ingredients, comprises what numerous one-tenth such as amino acid, carbohydrate, inorganic salt, VITAMIN, hormone, somatomedin, trace element were grouped into.Must be concerned about in the research of nutrient media components: one, the exhaustion of any nutritive substance all can influence the growth of cell in culturing process, and serious meeting causes the death of cell; Conversely, if the nutritive substance surplus also can accumulate a large amount of by products in culture environment, the pair cell toxigenicity, serious meeting causes the death of cell; Therefore, various nutritive ingredients want rationally balanced in the substratum, to satisfy cell growth, metabolism and to keep the demand of function.Two, mesenchymal stem cells MSCs has the potential of multidirectional differentiation, the potential that in the amplification in vitro process, will keep its multidirectional differentiation, thereby in substratum, to add Prostatropin, Urogastron etc., when promoting propagation, keep the potential of the multidirectional differentiation of mesenchymal stem cells MSCs.Three, mesenchymal stem cells MSCs is an attached cell, need add anchoring factor to promote it adherent in serum free medium, makes directly adherent growth in serum free medium of mesenchymal stem cells MSCs.
For achieving the above object, the present invention is used for the serum free medium of mesenchymal stem cells MSCs vitro culture and amplification, it is characterized in that, is a kind of aqueous solution, and its component (comprising basic medium) is as follows:
Regular Insulin 1～20 μ g/mL
Transferrins,iron complexes 1～20 μ g/mL
Thanomin 0.01～0.05mM
Putrescine 200 μ M
Dexamethasone 10nM
FeSO
4·7H
2O 0.834mg/mL
CuSO
4·5H
2O 0.0025mg/mL
ZnSO
4·7H
2O 0.863mg/mL
Antioxidant: mercaptoethanol 0.01～0.05mM
Anchoring factor: fibronectin 5～10 μ g/cm
2
Described basic medium is that α-MEM, DMEM, IMDM, DMEM and Ham ' s F12 press a kind of in the 1:1 blended substratum.
Described antioxidant is one or more of mercaptoethanol, catalase, superoxide-dismutase, Sodium Selenite.The mercaptoethanol of 0.01～0.05mM in the described component can use the hydrogen oxide enzyme of 50-150U/mL or the superoxide-dismutase of 50-150U/mL or the Sodium Selenite of 0.01～0.05mM to substitute, or multiple being used in combination.
Described anchoring factor be fibronectin, gelatin, type i collagen proteic one or more.5～10 μ g/cm in the described component
2Fibronectin can be with 50～100 μ g/cm
2Gelatin or 100～200 μ g/cm
2Type i collagen albumen substitute or multiple being used in combination.
Vitamins C in the described component is L-ascorbic acid, or available L-ascorbic acid2-phosphate (vitamins C phosphoric acid salt) substitutes.
Anchoring factor in the described component need be laid on developing medium (as culture dish, culturing bottle etc.) surface in advance, perhaps is added directly in the substratum.
Described α-MEM, DMEM, IMDM, component existing open report in existing document of pressing 1:1 blended DMEM and Ham ' s F12 basic medium repeat no more here.
Utilize serum free medium of the present invention to carry out the vitro culture and the amplification of mesenchymal stem cells MSCs, owing to added VITAMIN, hormone, somatomedin, trace element, antioxidant and anchoring factor, therefore do not need in containing blood serum medium, to carry out earlier in advance adherent, then change serum free medium, reduced the residual of serum most possibly, keep simultaneously the growth of mesenchymal stem cells MSCs preferably, kept the multidirectional differentiation potential of mesenchymal stem cells MSCs.
[description of drawings]
Accompanying drawing 1 is to adopt serum free medium of the present invention to cultivate mesenchymal stem cells MSCs to the photo that converges;
Accompanying drawing 2 is to adopt the mesenchymal stem cells MSCs of α-MEM+10%FBS culture medium culturing to the photo that converges;
Accompanying drawing 3 is that the rabbit bone marrow mescenchymal stem cell is at serum free medium of the present invention and the comparison diagram that contains growth curve in the 10% foetal calf serum substratum;
Accompanying drawing 4 are rabbit bone marrow mescenchymal stem cells serum free medium of the present invention with contain amplification in the 10% foetal calf serum substratum after the comparison diagram of calcium apposition outside the induced osteogenesis differentiation back born of the same parents;
Accompanying drawing 5 are rabbit bone marrow mescenchymal stem cells serum free medium of the present invention with contain 10% foetal calf serum substratum in induce the comparison diagram of fat differentiation back differentiation degree after the amplification.
[embodiment]
Below provide the embodiment that the present invention is used for the serum free medium of mesenchymal stem cells MSCs vitro culture and amplification with 6 embodiment, but the invention is not restricted to these embodiment.
With α-MEM is basic medium, and its component is as follows:
α-MEM 10.2g/L
Prostatropin 10ng/mL
Urogastron 10ng/mL
Bovine serum albumin 2mg/mL
Sigma I8405 10 μ g/mL
Transferrins,iron complexes 5.5 μ g/mL
Thanomin 0.02mM
Putrescine 200 μ M
Vitamins C 100 μ M
Dexamethasone 10nM
FeSO
4·7H
2O 0.834mg/mL
CuSO
4·5H
2O 0.0025mg/mL
ZnSO
4·7H
2O 0.863mg/mL
Mercaptoethanol 0.05mM
Sodium Selenite 0.01mM
Said components is dissolved in tri-distilled water or the ultrapure water at normal temperatures, and stirring and dissolving is the said serum free medium of the present invention; After 0.2 μ m filtering membrane filtration sterilization, promptly can be used for mesenchymal stem cells MSCs vitro culture and amplification.
In the acetum of 0.02N, collagen concentration is 50mg/mL with I type mouse tail collagenolysis.With 10 times of this collagen solution dilutions, after 0.2 μ m filtering membrane filtration sterilization, with 100 μ g/cm
2Be laid in the aseptic culture dish, and room temperature is air-dry in gnotobasis, has overlay the plate twice of collagen with the PBS rinse, this plate promptly can be used for the mesenchymal stem cells MSCs serum-free culture.
Put to death 1 monthly age new zealand white rabbit with the air embolism method, dissect, isolate femur, shin bone, ulna, scratch bone, and be soaked in 75% ethanol 5 minutes.PBS flushing 3～5 times shreds bone with osteophore, changes in two centrifuge tubes, adds 15～30mL PBS, and supernatant is got in concussion.Under the 1000rpm condition centrifugal 5 minutes, abandon supernatant.Add 30mL PBS, after mixing, under the 1000rpm condition centrifugal 5 minutes, abandon supernatant.Add 10mL PBS and mix, filter through 150 eye mesh screens, collect filtrate, centrifugal, the α of serum-free-MEM substratum suspends again.This marrow extract is slowly added on the equal-volume Ficoll parting liquid along centrifugal tube wall, and level is centrifugal 30 minutes under the 3000rpm condition.Draw middle mist layer with sharp suction pipe, add the PBS centrifuge washing.With cell suspension, the trypan blue exclusion method is counted with α-MEM+10%FBS nutrient solution.Then cell suspension is diluted to 1 * 10
6Cells/mL is seeded in the 10cm culture dish, in CO
2Cultivate in the incubator, observation of cell is adherent after 24～48 hours, changes liquid and removes suspension cell.Changed substratum every 3～4 days.Primary cell after overgrowing with the culture dish bottom in 7～10 days, the cultivation of can going down to posterity.
Former mesenchymal stem cells MSCs of being commissioned to train after supporting adds 3mL 0.025% pancreatin/0.02% EDTA to be digested, when cell rounding, take off and fall behind under the 1200rpm condition centrifugal 5 minutes, abandon Digestive system, add in 2～3mL 10mg/mL soybean pancreatin inhibitor and the pancreatin activity.Removed supernatant after under the 1200rpm condition centrifugal 5 minutes, PBS cleans twice, adds embodiment 1 described serum free medium suspension cell, with 1 * 10
4Cells/cm
2Be inoculated in embodiment 2 described having overlay in the proteic culture dish of type i collagen.
Referring to accompanying drawing 1, adopt serum free medium of the present invention to cultivate mesenchymal stem cells MSCs to the photo that converges; Referring to accompanying drawing 2, the mesenchymal stem cells MSCs that adopts α-MEM+10%FBS culture medium culturing is to the photo that converges.
Serum-free culture to the mesenchymal stem cells MSCs that converges is added 3mL 0.025% pancreatin/0.02% EDTA to be digested, when cell rounding, take off and fall behind under the 1200rpm condition centrifugal 5 minutes, abandon Digestive system, add in 2～3mL10mg/mL soybean pancreatin inhibitor and the pancreatin activity.Removed supernatant after under the 1200rpm condition centrifugal 5 minutes, PBS cleans twice, and the serum free medium that adds embodiment 1 suspends, with 0.5 * 10
4The cells/ hole is inoculated in and has overlay in proteic 24 orifice plates of type i collagen, and each hole adds 1mL serum free medium of the present invention, sampling every day counting.
Referring to accompanying drawing 3.Accompanying drawing 3 has compared the rabbit bone marrow mescenchymal stem cell at embodiment 1 described serum free medium and the growth curve that contains in the 10% foetal calf serum substratum.
To use the rabbit bone marrow mescenchymal stem cell of embodiment 1 described serum free medium amplification with 1 * 10
5The cells/ hole is inoculated in 6 orifice plates, replenishes 10 μ g/cm in embodiment 1 described serum free medium
2Fibronectin and 50 μ g/cm
2Gelatin, add in 6 orifice plates, to cultivate mesenchymal stem cells MSCs.When cell grows to 50% when converging, be replaced with the osteogenic induction substratum and induce.The osteogenic induction substratum is that DMEM-LG adds 10%FBS, 0.1 μ M dexamethasone, 50 μ g/mL xitix (L-ascorbic acid 2-phosphate), 10mM sodium.
Induce 2 week backs with control cells and osteogenic induction cells in the PBS rinse orifice plates, add the 0.1M hydrochloric acid of 200 μ L afterwards, react after 20 minutes hydrochloric acid reaction liquid is changed in the frozen centrifugation pipe frozen under-20 ℃ condition.Take out centrifuge tube during mensuration and thaw, the mixing that fully vibrates on eddy mixer is measured calcium ion concn with the calcium determinating reagent box.
Referring to accompanying drawing 4.Accompanying drawing 4 contrasted with embodiment 1 described serum free medium with contain rabbit bone marrow mescenchymal stem cell after the 10% foetal calf serum substratum amplification deposition of calcium matrix outside the born of the same parents after the induced osteogenesis differentiation.
To use the rabbit bone marrow mescenchymal stem cell of embodiment 1 described serum free medium amplification with 1 * 10
5The cells/ hole is inoculated in described 6 orifice plates that overlay collagen of embodiment 2, adds embodiment 1 described serum free medium and cultivates, and when cell grows to 80% when converging, is replaced with fatty inducing culture and induces.The fat inducing culture is that DMEM-LG adds 10%FBS, 1 μ M dexamethasone, 10 μ g/mL Regular Insulin, 0.5mM IBMX, 200 μ M INDOMETHACIN.After inducing for 2 weeks, use 10% formaldehyde fixed, the dyeing of 0.5% oil red.Fat in the adipocyte drips and will be dyed redness.With Virahol the oil red in the born of the same parents is extracted, survey absorbancy at wavelength 510nm place, can reflect fatty differentiation degree with spectrophotometer.
Referring to accompanying drawing 5.Accompanying drawing 5 contrasted with embodiment 1 described serum free medium with contain 10% foetal calf serum substratum amplification after the rabbit bone marrow mescenchymal stem cell induce differentiation degree after the fat differentiation.
Claims (10)
1, a kind of serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification is characterized in that, is a kind of aqueous solution, its component, and it is as follows to comprise basic medium:
Basic medium 10～18g/L
Prostatropin 1～20ng/mL
Urogastron 1～10ng/mL
Serum albumin 1～20mg/mL
Regular Insulin 1～20 μ g/mL
Transferrins,iron complexes 1～20 μ g/mL
Thanomin 0.01～0.05mM
Putrescine 200 μ M
Vitamins C 10～100 μ M
Hydrocortisone 10 μ g/mL
Dexamethasone 10nM
FeSO
4·7H
2O 0.834mg/mL
CuSO
4·5H
2O 0.0025mg/mL
ZnSO
4·7H
2O 0.863mg/mL
Antioxidant: mercaptoethanol 0.01～0.05mM
Anchoring factor: fibronectin 5～10 μ g/cm
2
2, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1, it is characterized in that described basic medium is that α-MEM, DMEM, IMDM, DMEM and Ham ' s F12 press a kind of in the 1:1 blended substratum.
3, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1 is characterized in that, described antioxidant is one or more of mercaptoethanol, catalase, superoxide-dismutase, Sodium Selenite.
4, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1, it is characterized in that, the mercaptoethanol of 0.01～0.05mM in the described component can substitute with the superoxide-dismutase of the hydrogen oxide enzyme of 50～150U/mL or 50～150U/mL or the Sodium Selenite of 0.01～0.05mM, or multiple being used in combination.
5, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1 is characterized in that, described anchoring factor be fibronectin, gelatin, type i collagen proteic one or more.
6, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1 is characterized in that, 5～10 μ g/cm in the described component
2Fibronectin can be with 50～100 μ g/cm
2Gelatin or 100～200 μ g/cm
2Type i collagen albumen substitute or multiple being used in combination.
7, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1 is characterized in that, the vitamins C in the described component can substitute with vitamins C phosphoric acid salt.
8, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1 is characterized in that, the anchoring factor in the described component need be laid on the surface of developing medium in advance, or is added directly in the substratum.
9, the serum free medium that is used for mesenchymal stem cells MSCs vitro culture and amplification according to claim 1 is characterized in that, is basic medium with α-MEM, and its component is as follows:
α-MEM 10.2g/L
Prostatropin 10ng/mL
Urogastron 10ng/mL
Bovine serum albumin 2mg/mL
Sigma I8405 10 μ g/mL
Transferrins,iron complexes 5.5 μ g/mL
Thanomin 0.02mM
Putrescine 200 μ M
Vitamins C 100 μ M
Hydrocortisone 10 μ g/mL
Dexamethasone 10nM
FeSO
4·7H
2O 0.834mg/mL
CuSO
4·5H
2O 0.0025mg/mL
ZnSO
4·7H
2O 0.863mg/mL
Mercaptoethanol 0.05mM
Sodium Selenite 0.01mM
10, according to claim 1, or the 9 described serum free mediums that are used for mesenchymal stem cells MSCs vitro culture and amplification, it is characterized in that in the acetum of 0.02N, collagen concentration is 50mg/mL with I type mouse tail collagenolysis; With 10 times of this collagen solution dilutions, after 0.2 μ m filtering membrane filtration sterilization, with 100 μ g/cm
2Be laid in the aseptic culture dish, and room temperature is air-dry in gnotobasis, has overlay the plate twice of collagen with the PBS rinse, this plate promptly can be used for the serum-free culture of mesenchymal stem cells MSCs.
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