CN106479971A - A kind of serum-free medium for cultivating mescenchymal stem cell and method - Google Patents

A kind of serum-free medium for cultivating mescenchymal stem cell and method Download PDF

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CN106479971A
CN106479971A CN201611237580.1A CN201611237580A CN106479971A CN 106479971 A CN106479971 A CN 106479971A CN 201611237580 A CN201611237580 A CN 201611237580A CN 106479971 A CN106479971 A CN 106479971A
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growth factor
mscs
serum
culture
free medium
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肖佳
朱江
韩荣飞
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Shenzhen Jiang Miao Medical Co Ltd
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Shenzhen Jiang Miao Medical Co Ltd
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Abstract

The application belongs to technical field of stem cell culture and in particular to a kind of serum-free medium for cultivating mescenchymal stem cell.Serum-free medium provided by the present invention is directed to MSCs In vitro culture to have carried out to basal medium L DMEM or DMEM/F12 and multiple adding ingredient compounding so that serving the effect of Synergistic between each group is divided in culture medium with customizing；The especially use of DKK 1, greatly improves stability and the success rate of In vitro culture MSCs, and additionally uses the formula of non-animal derived composition, improves the safety in clinical studies of institute's cultured cells.Compared with existing product, culture medium of the present invention drastically increases the increment efficiency of In vitro culture MSCs, the cell growth form and its cell phenotype that maintain MSCs are stable, provide the cultivating system of new efficient stable safety for the MSCs culture in clinical practice, there is very high scientific research and medical application is worth.

Description

A kind of serum-free medium for cultivating mescenchymal stem cell and method
Technical field
The invention belongs to technical field of stem cell culture is and in particular to a kind of serum-free for cultivating mescenchymal stem cell Culture medium and method.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that a kind of have the dry thin of height differentiation potential Born of the same parents, gain the name because being divided into stroma.MSCs is primarily present in connective tissue and organ interstitial, has the differentiation of height Function and immunoregulation function, can secrete cytokine profiles, be that cell therapy clinical practice is ground after hematopoietic stem cell Study carefully at most, be also a kind of comparatively ripe adult stem cell.MSCs can be to multiple mesoderms and neuroectodermal origin Histiocyte differentiation, such as osteoblast, chondrocyte, adipose cell, Tenocyte cell, smooth muscle cell, marrow stromal cell, Fibroblast and multiple vascular endothelial cell, or even the neuron of nervous system and neurogliocyte etc..
From nineteen ninety-five from the beginning of reported first MSCs is applied to clinical trial, MSCs is applied to clinical treatment both at home and abroad and moves Graft versus host disease (GVHD), congestive heart failure and heart infarction, spinal cord injury, type 2 diabetes mellitus, bone and cartilage injury, burn, class wind Wet arthritis, parkinson disease etc..But due to the MSCs limited amount directly obtaining from human body, the cell of clinical practice to be reached The order of magnitude is it is necessary to through external amplification cultivation.And during cultivating in vitro, suitable cultivating system is MSCs culture effect Rate and the key of safety.Select suitable cultivating system guarantee MSCs after Secondary Culture for several times, genotype with Phenotype keeps stable.Conventional MSCs culture system in vitro is made up of DMEM minimal medium and hyclone.But cultivating Cheng Zhong, serum albumin and other serum active compositions easily remain in the endochylema of cell, bring potential anaphylaxiss to patient Risk, therefore clinic must adopt serum-free medium with MSCs.
Serum-free medium (Serum-Free Media, SFM), as the term suggests it is simply that do not need to add in cell culture Increase serum, but somatomedin or cytokine may be added in some applications.Serum is with the addition of in serum-free medium Main component：Adhesion factor, somatomedin, necessary nutrient substance and hormone etc., can reduce the unfavorable factor that serum brings, Make the condition of cell culture more stable.Experienced natural medium, after synthetic medium, serum free culture system becomes thin now Born of the same parents cultivate a main trend in field.The program that purification can be simplified using serum-free culture and identify various cellular products, it is to avoid disease The harm that poison pollution causes.
In recent years all have been reported that both at home and abroad, various clinical cell such as B cell, T cell, NK cell and DC cell etc. external Culture, all has different requirements, needs for different being customized of cell type to the special cells factor in cultivating system Culture.And In vitro culture clinical research when using MSCs, keeps the form of MSCs and function steady except need add special cytokine Fixed outer in addition it is also necessary to the special composition such as all kinds of adhesion anchoring factor such as fibronectins is to keep the abundant life of MSCs environment in vitro Long.Existing serum-free medium in market, such as MSCGM-CD, GIBICO of MESENCULT, LONZA of STEMCELL POWERSTEM MSC1 of STEMPRO MSC SFM CTS, PAN etc., commonly used a large amount of expensive restructuring fibronectins add Culture medium or be coated culture dish to ensure MSCs growth in vitro, the method is not only relatively costly, and operating process is complicated And biological safety is difficult to ensure.Simultaneously as commercially available universal serum-free medium is not customized with MSCs to clinic Optimize, therefore fail the maximized MSCs phenotype ensureing after In vitro culture number generation and the stablizing of function.
Content of the invention
In view of this, it is an object of the invention to provide a kind of serum-free medium for cultivating mescenchymal stem cell and Method, for solving the problems, such as in prior art that mescenchymal stem cell culture effect is undesirable, high cost.
The concrete technical scheme of the present invention is as follows：
The invention provides a kind of serum-free medium for cultivating mescenchymal stem cell, including：Basal medium, DKK-1, thrombin, fibronectin, human albumin, transferrinss, basic fibroblast growth factor, platelet-derived Somatomedin, insulin like growth factor, leukaemia inhibitory factor, epidermal growth factor, transforming growth factor, ferric nitrate, Asia Sodium selenate, copper sulfate and zinc sulfate, Vitamin E, reductive glutathione, hydrocortisone, progesterone, ascorbic acid phosphoric acid esters, Ethanolamine, linoleic acid, cholesterol and galactose.
Preferably, described basal medium is DMEM/F12 or L-DMEM.
Preferably, comprise in every milliliter of described basal medium in above-mentioned serum-free medium：DKK-1 10- 1000ng, thrombin 0.1-0.4IU, fibronectin 10-100ng, human albumin 1-50 μ g, transferrinss 10-200ng, Basic fibroblast growth factor 1-100ng, platelet derived growth factor 1-100ng, type-1 insulin like growth factor -100 μ g, leukaemia inhibitory factor 1-50ng, epidermal growth factor 1-10ng, transforming growth factor-beta 1-10ng, ferric nitrate 0.1-1 μ G, sodium selenite 0.01-0.1 μ g, copper sulfate 0.001-0.1 μ g, zinc sulfate 0.1-5 μ g, Vitamin E 1-10ng, reproducibility paddy Guang sweet peptide 1-10 μ g, hydrocortisone 1-100 μ g, progesterone 1-100nmol, ascorbic acid phosphoric acid esters 1-10 μ g, ethanolamine 1-10 μ G, linoleic acid 1-10 μ g, cholesterol 1-10 μ g and galactose 10-50 μ g.
It is furthermore preferred that comprising in every milliliter of described basal medium in above-mentioned serum-free medium：DKK-1 200ng、 Thrombin 0.2IU, fibronectin 50ng, human albumin 2 μ g, transferrinss 50ng, basic fibroblast growth factor 5ng, platelet derived growth factor 10ng, type-1 insulin like growth factor 0 μ g, leukaemia inhibitory factor 8ng, epidermal growth factor Sub- 2ng, transforming growth factor-beta 1 ng, ferric nitrate 0.1 μ g, sodium selenite 0.01 μ g, copper sulfate 0.001 μ g, zinc sulfate 0.5 μ g, Vitamin E 1ng, reductive glutathione 1.5 μ g, hydrocortisone 10 μ g, progesterone 20nmol, ascorbic acid phosphoric acid esters 2.5 μ G, ethanolamine 1 μ g, linoleic acid 0.2 μ g, cholesterol 2 μ g and galactose 20 μ g.
Present invention also offers a kind of method of culture mescenchymal stem cell, including：Mescenchymal stem cell is inoculated in State in serum-free medium and cultivated.
Preferably, the CO that the condition of described culture is 37 DEG C and volumetric concentration is 5%2.
The invention provides a kind of serum-free medium for cultivating mescenchymal stem cell, fixed for MSCs In vitro culture Inhibition and generation basal medium DMEM-L or DMEM/F12 and multiple adding ingredient carried out compounding so that each component in culture medium Between serve the effect of Synergistic；The especially use of DKK-1, greatly improves stability and the one-tenth of In vitro culture MSCs Power, and additionally use the formula of non-animal derived composition, improve the safety in clinical studies of institute's cultured cells.With now There is product to compare, culture medium of the present invention drastically increases the increment efficiency of In vitro culture MSCs, maintain the cell life of MSCs Long form and its cell phenotype are stable, are that the MSCs culture in clinical practice provides the safe culture body of new efficient stable System, has very high scientific research and medical application is worth.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing Have technology description in required use accompanying drawing be briefly described it should be apparent that, drawings in the following description be only this Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis The accompanying drawing providing obtains other accompanying drawings.
Fig. 1 is the cell growth curve of MSCs in embodiment 3；
Fig. 2 is the cellular morphology measurement result of MSCs in embodiment 3, and wherein, left figure is the optical microphotograph of experimental group MSCs Mirror photo, right figure is the optical microscope photograph of matched group 2MSCs；
Fig. 3 is the testing result that in embodiment 3, MSCs cell ROS generates, and left figure is that the fluorescence microscopy of experimental group MSCs shines Piece, right figure is the fluorescence micrograph of MSCs in matched group 2.
Specific embodiment
The invention provides a kind of serum-free medium for cultivating mescenchymal stem cell, including：Basal medium, DKK-1, thrombin, fibronectin, human albumin, transferrinss, basic fibroblast growth factor, platelet-derived Somatomedin, insulin like growth factor, leukaemia inhibitory factor, epidermal growth factor, transforming growth factor, ferric nitrate, Asia Sodium selenate, copper sulfate and zinc sulfate, Vitamin E, reductive glutathione, hydrocortisone, progesterone, ascorbic acid phosphoric acid esters, Ethanolamine, linoleic acid, cholesterol and galactose.
In culture medium of the present invention, no any animal serum, will not bring any mycoplasma and virus.In the present invention, basis Culture medium is preferably DMEM/F12 or L-DMEM, more preferably DMEM/F12.Wherein, L-DMEM is the DMEM culture of low-sugar type Base.The present invention does not have special restriction to the source of basal medium, using basal medium well known to those skilled in the art , such as using its commercial goods.
DKK (Dickkopf) is a kind of secreting type glycoprotein, including DKK-1～4 four member.In the present invention, DKK-1 Preferably for people restructuring DKK-1.The present invention does not have special restriction to the source of DKK-1, using well known to those skilled in the art DKK-1, such as using its commercial goods.
Thrombin is a kind of multi-functional serine protease, can not only promote blood coagulation by adjusting platelet aggregation Journey is moreover it is possible to excite various kinds of cell to produce many different biologicallies by thrombin receptor, such as chemotaxiss, rush cell increases Grow, promote extracellular matrix synthesis and release cytokine.In the present invention, thrombin can promote MSCs secretion fibronectin, promotees Enter MSCs itself extracellular matrix secretion and propagation, promote MSCs propagation and its adherent growth.The source to thrombin for the present invention There is no special restriction, using thrombin well known to those skilled in the art, such as using its commercial goods.
In the present invention, fibronectin, human albumin and transferrinss are both preferably human recombination protein.The present invention trains More comprehensive combination of cytokines, preferably basic fibroblast growth factor, platelet-derived life is with the addition of in foster base The long factor, insulin like growth factor, leukaemia inhibitory factor, epidermal growth factor and transforming growth factor.In the present invention, The combinations of. growth factors added is preferably people's recombinant cytokine.Trace and REE elements and fat is also comprised in culture medium of the present invention Class, trace and REE elements are preferably ferric nitrate, sodium selenite, copper sulfate and zinc sulfate, and lipid is preferably cholesterol and linoleic acid. DKK-1, thrombin, albumen, cell growth factor sub-portfolio, trace and REE elements, lipid, ethanolamine, progesterone, sodium ascorbyl phosphate Ester, Vitamin E, galactose, reductive glutathione and hydrocortisone Synergistic, collectively promote the propagation of MSCs.This Bright to albumen, cell growth factor sub-portfolio, trace and REE elements, lipid, ethanolamine, progesterone, ascorbic acid phosphoric acid esters, vitamin The source of E, galactose, reductive glutathione and hydrocortisone does not have special restriction, ripe using those skilled in the art Know, such as using its commercial goods.
The present inventor is found by research：Using more comprehensively combination of cytokines, trace and REE elements etc., And breakthrough interpolation has the DKK-1 of important function to maintenance MSCs phenotype and function, and promote the fine even egg of MSCs secretion White thrombin, can greatly improve stability and safety etc. present in existing MSCs serum-free Process of in vitro all Many problems, make cultivated MSCs be more suitable for using in clinic.
Further, comprise in every milliliter of described basal medium in serum-free medium provided by the present invention： DKK-1 10-1000ng, thrombin 0.1-0.4IU, fibronectin 10-100ng, human albumin 1-50 μ g, transferrinss 10-200ng, basic fibroblast growth factor 1-100ng, platelet derived growth factor 1-100ng, insulin-like growth Factor 1-100 μ g, leukaemia inhibitory factor 1-50ng, epidermal growth factor 1-10ng, transforming growth factor-beta 1-10ng, nitric acid Ferrum 0.1-1 μ g, sodium selenite 0.01-0.1 μ g, copper sulfate 0.001-0.1 μ g, zinc sulfate 0.1-5 μ g, Vitamin E 1-10ng, Reductive glutathione 1-10 μ g, hydrocortisone 1-100 μ g, progesterone 1-100nmol, ascorbic acid phosphoric acid esters 1-10 μ g, second Hydramine 1-10 μ g, linoleic acid 1-10 μ g, cholesterol 1-10 μ g and galactose 10-50 μ g.
Further, wrap in every milliliter of described basal medium in above-mentioned mesenchymal stem cell serum-free culture medium Contain：DKK-1 200ng, thrombin 0.2IU, fibronectin 50ng, human albumin 2 μ g, transferrinss 50ng, alkalescence become fine Dimension cell growth factor 5ng, platelet derived growth factor 10ng, type-1 insulin like growth factor 0 μ g, leukaemia inhibitory factor 8ng, epidermal growth factor 2ng, transforming growth factor-beta 1 ng, ferric nitrate 0.1 μ g, sodium selenite 0.01 μ g, copper sulfate 0.001 μ G, zinc sulfate 0.5 μ g, Vitamin E 1ng, reductive glutathione 1.5 μ g, hydrocortisone 10 μ g, progesterone 20nmol, anti-bad Hematic acid phosphate ester 2.5 μ g, ethanolamine 1 μ g, linoleic acid 0.2 μ g, cholesterol 2 μ g and galactose 20 μ g.
The preparation of serum-free medium provided by the present invention can be using basal medium DMEM-L or DMEM/F12 and many Plant adding ingredient to be compounded, all configuration process can be carried out according to the preparation condition of existing routine complete medium and method Prepare.
In some embodiments of the invention, described serum-free medium is preferably obtained in accordance with the following methods：
By L-DMEM or DMEM/F12 dry powder, it is dissolved to 800-900mL using ultra-pure water, weigh other components by formula, Add successively to the basal medium having dissolved, and be sufficiently stirred for dissolving in room temperature, finally will be cultivated completely using ultra-pure water Base is settled to 1000mL, and using 0.22-0.1 μm of membrane filtration, 4 DEG C save backup.
The invention provides a kind of method of culture mescenchymal stem cell, step is：Mescenchymal stem cell is inoculated in State in serum-free medium and cultivated.
The present invention does not have special restriction to the source of described mescenchymal stem cell, using well known to those skilled in the art Mescenchymal stem cell, including fat mesenchymal stem cell and mesenchymal stem cells MSCs.If commercial goods can be adopted, The well known to those skilled in the art technical scheme of preparing mescenchymal stem cell may also be employed voluntarily prepare.
Below in conjunction with detailed description accompanying drawing of the present invention, technical scheme is clearly and completely described, Obviously, described embodiment is a part of embodiment of the present invention, rather than whole embodiments.Those skilled in the art should Work as understanding, the specific embodiment of the present invention is modified or some technical characteristics are replaced on an equal basis, without deviating from this The spirit of inventive technique scheme, all should cover in the scope of protection of the invention.
Embodiment 1
1st, formula
2nd, prepare
Weigh each component, 10g L-DMEM dry powder is dissolved in 800mL ultra-pure water, then add remaining each group thereto Point, add ultra-pure water to be settled to 1L after so that it is fully dissolved using magnetic stirring apparatuss under room temperature, then with 0.22 μm of aperture filter membrane mistake Filter, 4 DEG C save backup.
Embodiment 2
1st, formula
2nd, prepare
Weigh each component, 10g DMEM/F12 dry powder is dissolved in 800mL ultra-pure water, then add remaining thereto each Component, adds ultra-pure water to be settled to 1L under room temperature after so that it is fully dissolved using magnetic stirring apparatuss, then with 0.22 μm of aperture filter membrane Filter, 4 DEG C save backup.
Embodiment 3
1st, formula
2nd, prepare
Weigh each component, 10g L-DMEM dry powder is dissolved in 800mL ultra-pure water, then add remaining each group thereto Point, add ultra-pure water to be settled to 1L after so that it is fully dissolved using magnetic stirring apparatuss under room temperature, then with 0.22 μm of aperture filter membrane mistake Filter, 4 DEG C save backup.
Embodiment 4
1st, formula
2nd, prepare
Weigh each component, 10g DMEM/F12 dry powder is dissolved in 800mL ultra-pure water, then add remaining thereto each Component, adds ultra-pure water to be settled to 1L under room temperature after so that it is fully dissolved using magnetic stirring apparatuss, then with 0.22 μm of aperture filter membrane Filter, 4 DEG C save backup.
Embodiment 5
1st, formula
2nd, prepare
Weigh each component, 10g L-DMEM dry powder is dissolved in 800mL ultra-pure water, then add remaining each group thereto Point, add ultra-pure water to be settled to 1L after so that it is fully dissolved using magnetic stirring apparatuss under room temperature, then with 0.22 μm of aperture filter membrane mistake Filter, 4 DEG C save backup.
Embodiment 6
1st, formula
2nd, prepare
Weigh each component, 10g DMEM/F12 dry powder is dissolved in 800mL ultra-pure water, then add remaining thereto each Component, adds ultra-pure water to be settled to 1L under room temperature after so that it is fully dissolved using magnetic stirring apparatuss, then with 0.22 μm of aperture filter membrane Filter, 4 DEG C save backup.
Embodiment 7
1st, tested culture medium
The culture medium that experiment group selection embodiment 1 is provided；Matched group 1 selects to the addition of the DMEM/ of 10% hyclone F12 culture medium；Matched group 2 selects to the addition of the DMEM/F12 culture medium of the Ultroser G serum substitute being purchased from PALL.
2nd, the selection of mescenchymal stem cell (MSCs) and its culture
The people umbilical cord MSCs choosing for the 3rd generation is recovered, and cultivates in transport medium, and culture is thin using the T75 of standard Born of the same parents' culture bottle, is placed in 37 DEG C and 5%CO2Cell culture incubator in cultivated.Recovery carried out changing liquid, afterwards after 2 days first Carry out within every 3 days changing a not good liquor until cell fusion degree reaches and carries out passage when about 70%.
3rd, the determination of cell cycle
Collect the 5th generation MSCs and the 10th generation MSCs respectively, rinse the pre-cooled ethanol using 70% after 3 times through digestion and PBS It is fixed, and adds PI (propidium iodide) and RNaseA after 16 hours in 4 DEG C of standings, after 4 DEG C of incubations 30 minutes, upper machine is carried out FCM analysis, every group sets 5 repetitions, the ratio of record G0/G1 phase, G2/M phase and S phase, wherein focusing on comparative G0/G1 phase The ratio of cell.Table 1 is FCM analysis result, and result shows that the MSCs being cultivated using culture medium of the present invention is had relatively High G0/G1 phase cell proportion, shows to apply the MSCs of present invention culture can keep the cell growth of long period and higher Differentiation capability.
Table 1
4th, the mensure of cell growth curve
Collect culture to the MSCs in the 5th generation, after digestion and counting, be inoculated in 96 porocyte culture plates, every hole contains 1000 cells, every group sets 5 holes, subsequently every 24 hours, each group cell is carried out changing liquid, changes every hole during liquid and adds 100 μ L DMEM/F12 basal medium and 10 μ L MTT (5mg/ml), are then replaced in culture in 37 DEG C of incubators, take out, often after 4h Hole adds 100 μ L SDS-HCL solution and mixes, and measures 570nm's using spectrophotometric plate reading machine after room temperature avoid light place 4h Readings is simultaneously mapped.Fig. 1 is the MSCs In vitro culture growth curve of first day to the 8th day, as shown in Fig. 1 result, using the present invention The MSCs speed of growth that culture medium is cultivated is substantially better than matched group.
5th, the mensure of cellular morphology
Collect and after the 5th generation MSCs continues culture 24 hours, carry out microscope observation, the MSCs of contrast experiment's group and matched group 2 Cellular morphology and its adhered state.Fig. 2 is the cellular morphology measurement result for MSCs for the P5, and wherein left figure is the light of experimental group MSCs Learn microphotograph, right figure is the optical microscope photograph of MSCs in matched group 2, result is shown and carried out using culture medium of the present invention The MSCs cellular morphology of culture and adherent degree are substantially better than matched group 2.
6th, the mensure that cell ROS generates
The MSCs collecting culture to the 10th generation passes on and is inoculated in 24 orifice plates of preset circle coverslip, cultivates to about Carry out immunofluorescence dyeing when 70% cell fusion is spent.According to the conventional method of attached cell immunofluorescence dyeing, lid will be affixed on The cell of slide is carried out using PBS and fixes 10 minutes using 4% formaldehyde in room temperature, subsequently using PBS 3 times and make Closed 1 hour in room temperature with blocking antibody.Coverslip after cleaning subsequently will use green using ROS antibody in 4 DEG C of stained over night Color fluorescence two resists in room temperature lucifuge incubation 1 hour.Cell one side is affixed on microscope slide using glimmering for coverslip once purged reversion Light microscope is observed.The death and morphological function that lead to mescenchymal stem cell are changed by the great expression due to ROS, Therefore it is more excellent to produce one group of relatively low cell of ROS.Fig. 3 is the testing result that MSCs cell ROS generates, and left figure is experimental group MSCs's Fluorescence micrograph, right figure is the fluorescence micrograph of MSCs in matched group 2, and result shows to be trained using culture medium of the present invention The ROS content that foster MSCs cell produces is significantly lower than matched group 2.
The above is only the preferred embodiment of the present invention it is noted that coming for those of ordinary skill in the art Say, under the premise without departing from the principles of the invention, some improvement can also be made and modify, these improve and modify and also should be regarded as Protection scope of the present invention.

Claims (6)

1. a kind of serum-free medium for cultivating mescenchymal stem cell is it is characterised in that include：Basal medium, DKK- 1st, thrombin, fibronectin, human albumin, transferrinss, basic fibroblast growth factor, platelet derived growth The factor, insulin like growth factor, leukaemia inhibitory factor, epidermal growth factor, transforming growth factor, ferric nitrate, Monohydrated selenium dioxide Sodium, copper sulfate and zinc sulfate, Vitamin E, reductive glutathione, hydrocortisone, progesterone, ascorbic acid phosphoric acid esters, ethanol Amine, linoleic acid, cholesterol and galactose.
2. serum-free medium according to claim 1 is it is characterised in that described basal medium is DMEM/F12 or L- DMEM.
3. serum-free medium according to claim 1 is it is characterised in that comprise in every milliliter of described basal medium： DKK-1 10-1000ng, thrombin 0.1-0.4IU, fibronectin 10-100ng, human albumin 1-50 μ g, transferrinss 10-200ng, basic fibroblast growth factor 1-100ng, platelet derived growth factor 1-100ng, insulin-like growth Factor 1-100 μ g, leukaemia inhibitory factor 1-50ng, epidermal growth factor 1-10ng, transforming growth factor-beta 1-10ng, nitric acid Ferrum 0.1-1 μ g, sodium selenite 0.01-0.1 μ g, copper sulfate 0.001-0.1 μ g, zinc sulfate 0.1-5 μ g, Vitamin E 1-10ng, Reductive glutathione 1-10 μ g, hydrocortisone 1-100 μ g, progesterone 1-100nmol, ascorbic acid phosphoric acid esters 1-10 μ g, second Hydramine 1-10 μ g, linoleic acid 1-10 μ g, cholesterol 1-10 μ g and galactose 10-50 μ g.
4. serum-free medium according to claim 1 is it is characterised in that comprise in every milliliter of described basal medium： DKK-1 200ng, thrombin 0.2IU, fibronectin 50ng, human albumin 2 μ g, transferrinss 50ng, basic fibroblast Cell growth factor 5ng, platelet derived growth factor 10ng, type-1 insulin like growth factor 0 μ g, leukaemia inhibitory factor 8ng, epidermal growth factor 2ng, transforming growth factor-beta 1 ng, ferric nitrate 0.1 μ g, sodium selenite 0.01 μ g, copper sulfate 0.001 μ G, zinc sulfate 0.5 μ g, Vitamin E 1ng, reductive glutathione 1.5 μ g, hydrocortisone 10 μ g, progesterone 20nmol, anti-bad Hematic acid phosphate ester 2.5 μ g, ethanolamine 1 μ g, linoleic acid 0.2 μ g, cholesterol 2 μ g and galactose 20 μ g.
5. a kind of method of culture mescenchymal stem cell is it is characterised in that be inoculated in Claims 1-4 by mescenchymal stem cell Cultivated in serum-free medium described in any one.
6. method according to claim 5 is it is characterised in that the condition of described culture is 37 DEG C and volumetric concentration is 5% CO2.
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Application publication date: 20170308