CN101735980B - Complete culture medium for in vitro culture of bone marrow mesenchymal stem cells - Google Patents
Complete culture medium for in vitro culture of bone marrow mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a complete culture medium for in vitro culture of bone marrow mesenchymal stem cells. A basal culture medium is DMEM-L or DMEM/F12; and the complete culture medium is also added with insulin, albumins, transferrin, sodium selenite, hydrocortisone, alkali fibroblast growth factors, progesterone, reduced glutathione and fetal calf serum. In the complete culture medium, the low-concentration fetal calf serum is mixed with other adding materials and all components are synergetic, so that the bone marrow mesenchymal stem cells are successfully cultured, the same effect achieved by the high-concentration serum is achieved by the complete culture medium in an aspect of enforcing the cell amplification, and the complete culture medium is better than the fetal calf serum in an aspect of maintaining the dryness of the mesenchymal stem cells. Because the used serum is low in concentration, the defects of unstability between batches, cytotoxicity, a great number of heterologous proteins and the like existing in the use of the high-concentration serum are radically overcome, and the invention provides a quite good tool for fundamental research and clinical treatment of the bone marrow mesenchymal stem cells.
Description
Technical field
The present invention relates to a kind of substratum, specifically a kind of perfect medium that is used for the external mesenchymal stem cells MSCs of high-efficient culture.
Background technology
Mescenchymal stem cell is to derive from a mesoblastic class adult stem cell, can from multiple tissues such as marrow, periosteum, synovial membrane, muscle, fat, separate, and experiment confirm is under different inductive conditions, it has the ability to mesoderm and the differentiation of neuroderm histocyte, as the ability to differentiation such as scleroblast, chondroblast, adipocyte, inoblast, endotheliocytes.Marrow is considered to the main inhabitation place of MSCs.In whole vital process, histoorgan still is that environment change and being upset all needs the MSCs of these derived from bone marrow constantly to replenish, upgrade in steady state.Because marrow has: it is convenient and harmless to body 1. to draw materials; 2. can cultivate amplification in a large number external; 3. induce by it and come be organized in the problem that does not have tissue matching and immunological rejection when transplanting; 4. change the characteristic that does not influence MSCs behind the allogenic gene over to, therefore, marrow MSC becomes cell therapy and organizational project ideal seed cell.Yet mescenchymal stem cell comparatively small amt in the marrow is difficult to satisfy the demand of clinical treatment and research aspect, so need increases in a large number to mescenchymal stem cell external.
At present, all added a certain proportion of animal serum in the conventional culture system of MSCs, relatively more commonly used is foetal calf serum.The very complicated mixed thing that serum is made up of the biomolecules that much varies in size, the main effect of its pair cell when vitro culture provides somatomedin, hormone, conjugated protein, and the effect of protecting is provided.But it also contains some supressor or toxicants of being unfavorable for the cell growth, has the effect of potential cellularity.The known unknown pathogenic agent that animal serum may exist animal to carry constitutes a threat to possible clinical application later on, has increased difficulty to being used for the mass-producing culturing cell.And there are differences between batches between the serum of each batch, make the cultivation results that obtains difference occur.Although the researchist has attempted the scheme of a lot of serum free mediums, serum free medium ubiquity poor growth even negative value-added problem cause the cell that is in RS to proceed to the SR state and lose the ability of differentiation.Therefore, serum free medium is used for the research that mescenchymal stem cell cultivates and does not also obtain important breakthrough so far.Along with bone marrow stem cell research is further goed deep into, Application of stem cells research has also had progressively development, and then how safe problem, stable, quick, the high quality amplification of mesenchymal stem cells be also more and more outstanding, needs badly between a kind of marrow of exploitation and fill the stem cell suitable medium.
Summary of the invention
The objective of the invention is to, a kind of problem of having avoided the high density foetal calf serum to bring is provided, and have the perfect medium of the external mesenchymal stem cells MSCs of good promotion cell proliferation effect.
The inventor has finally determined perfect medium composition of the present invention through secular experiment and research, the present invention is foetal calf serum and the Prostatropin that adds lower concentration on the basis of existing serum free medium, it is not high to the mescenchymal stem cell amplification efficiency to have remedied serum-free, and there is the defective on using in the high density foetal calf serum.Be in basic medium, to have added cell growth factor subconstiuents such as Regular Insulin, Transferrins,iron complexes, albumin, third selenic acid are received, hydrocortisone, progesterone, reduced glutathion concretely, add the foetal calf serum of lower concentration again.
For realizing purpose of the present invention, the present invention adopts following technical scheme:
A kind of perfect medium of vitro culture mesenchymal stem cells MSCs, its basic medium are DMEM-L or DMEM/F12; Also be added with Regular Insulin, albumin, Transferrins,iron complexes, Sodium Selenite, hydrocortisone, Prostatropin, progesterone, reduced glutathion and foetal calf serum in the described substratum.
The consumption of described each added ingredients is recommended as follows:
Regular Insulin 1～100 μ g/ml
Transferrins,iron complexes 1～50ng/ml
Sodium Selenite 1～100ng/ml
Reduced glutathion 1～10 μ g/ml
Prostatropin 1～100ng/ml
The consumption of described each added ingredients is preferably as follows:
Regular Insulin 1～10 μ g/ml
Transferrins,iron complexes 1～10ng/ml
Sodium Selenite 1～30ng/ml
Reduced glutathion 1～5 μ g/ml
The optimum consumption of described each added ingredients is as follows:
Regular Insulin 5 μ g/ml
Albumin 5mg/ml
Transferrins,iron complexes 5ng/ml
Sodium Selenite 10ng/ml
Progesterone 20nmol/L
Reduced glutathion 1.5 μ g/ml
Prostatropin 5ng/ml
Foetal calf serum 10ul/ml.
The purposes of substratum of the present invention in the vitro culture mesenchymal stem cells MSCs.
The main inventive point of the present invention is culture medium prescription, substratum preparation of the present invention can be undertaken by existing ordinary method, recommend as follows: shown in prescription, in described amount ranges, select institute's expense, take by weighing DMEM (L) dry powder, Regular Insulin, albumin, Transferrins,iron complexes, Sodium Selenite, hydrocortisone, foetal calf serum, Prostatropin, progesterone, reduced glutathion, ultrapure water adds to 1000ml, stirring and dissolving, 0.22 the degerming of μ m membrane filtration, 4 ℃ of preservations are standby.
The present invention is raw materials used all can enough to be can buy by commercially available.
Advantage of the present invention and beneficial effect:
Substratum of the present invention adopts foetal calf serum and other added ingredientss of lower concentration to carry out composite, each composition plays synergy, successfully cultivated mesenchymal stem cells MSCs, from promoting the cell proliferation aspect to reach and high density serum same effect, be better than the high density foetal calf serum aspect the mescenchymal stem cell dryness keeping.Cultivation speed is fast owing to adopted the concentration of low serum, avoided greatly that high density serum exists batch between instability, cytotoxicity, defective such as heterologous proteins in a large number, for the fundamental research and the clinical treatment of mesenchymal stem cells MSCs provides good instrument.
The present invention will be further described below in conjunction with accompanying drawing and preferred forms, so that the public has whole to summary of the invention and understand fully, and is not qualification to protection domain of the present invention.Aforementioned part fully discloses the protection domain that the present invention can implement, and therefore allly any well known in the artly is equal to replacement according to what the disclosure of invention was carried out, all belongs to infringement of the present invention.
Description of drawings
Fig. 1 is the morphological specificity of human marrow mesenchymal stem cell
(a: cultivate 2d, b: cultivate 7d, c: cultivate 12d);
Fig. 2 human marrow mesenchymal stem cell growth curve;
Fig. 3 human marrow mesenchymal stem cell cell growth cycle;
Fig. 4 human marrow mesenchymal stem cell is induced differentiation to adipocyte
(a: induce 10d, b: induce 20d, c: induce 20d) through oil red O stain;
The morphological specificity of Fig. 5 rat bone marrow mesenchymal stem cells (a: cultivate 3d, b: cultivate 8d);
Fig. 6 rat bone marrow mesenchymal stem cells growth curve;
Fig. 7 rat bone marrow mesenchymal stem cells cell growth cycle;
Fig. 8 rat bone marrow mesenchymal stem cells is induced differentiation to adipocyte
(a: induce 10d, b: induce 20d, c: induce 20d) through oil red O stain.
Embodiment
Raw material sources:
DMEM (L) dry powder: U.S. Hyclone company
DMEM/F12 dry powder: Hyclone company
Regular Insulin: Sigma company
Albumin: Sigma company
Transferrins,iron complexes: Sigma company
Sodium Selenite: Amresco company
Hydrocortisone: Sigma company
Progesterone: Sigma company
Reduced glutathion: Sigma company
Prostatropin: PeproTech company
Foetal calf serum: Gibco company
Following mesenchymal stem cells MSCs nutrient solution is prepared used basic medium and can is DMEM (L), also can be DMEM/F12.
The preparation of 1000ml mesenchymal stem cells MSCs nutrient solution:
(1) takes by weighing 10g DMEM (L) dry powder, 1mg Regular Insulin, 10g albumin, 25ug Transferrins,iron complexes, 30ug Sodium Selenite, 20mg hydrocortisone, 1ml foetal calf serum, 30ug Prostatropin, 10mg reduced glutathion, 20nmol/L progesterone, ultrapure water adds to 1000ml, stirring and dissolving, 0.22 the degerming of μ m membrane filtration is deposited for 4 ℃.
(2) take by weighing 10g DMEM (L) dry powder, 5mg Regular Insulin, 5g albumin, 5ug Transferrins,iron complexes, 10ug Sodium Selenite, 10mg hydrocortisone, 10ml foetal calf serum, 5ug Prostatropin, 1.5mg reduced glutathion, 20nmol/L progesterone, ultrapure water adds to 1000ml, stirring and dissolving, 0.22 the degerming of μ m membrane filtration is deposited for 4 ℃.
(3) take by weighing 10g DMEM/F12 dry powder, 50mg Regular Insulin, 50g albumin, 50ug Transferrins,iron complexes, 100ug Sodium Selenite, 80mg hydrocortisone, 10ml foetal calf serum, 50ug Prostatropin, 10mg reduced glutathion, 100nmol/L progesterone, ultrapure water adds to 1000ml, stirring and dissolving, 0.22 the degerming of μ m membrane filtration is deposited for 4 ℃.
(1) separation of human marrow mesenchymal stem cell, cultivation and amplification
Extract marrow 5ml with medullo-puncture needle sour jujube on behind the bone, the 1ml that packs into contains in the aseptic centrifuge tube of PBS of 500u/ml heparin, and equivalent PBS dilution is added on the lymphocyte separation medium that isopyknic proportion is 1.073g/ml.2000 commentaries on classics/min, centrifugal 30min, nebulous mononuclearcell layer in the middle of getting is with DMEM (L) medium centrifugal washing 2 times, each 1000 commentaries on classics/min, centrifugal 5min, abandoning supernatant.
With the mescenchymal stem cell substratum suspendible and the counting cells of embodiment 1 (2) preparation, by 5 * 10
6In the culturing bottle that/ml is inoculated in, put 37 ℃ of 5%CO
2The CO of saturated humidity
2Cultivate in the incubator.Change liquid after 2 days first, discard not attached cell, the replacing nutrient solution was 1 time in later per 3 days.Go down to posterity for the first time when cytogamy reaches 50%-60% behind 8～10d, with 0.25% trypsinase-37 ℃ of digestion of 0.02%EDTA 2-2.5min.Collect cell suspension, 1000 commentaries on classics/min, centrifugal 5min.Press 1 * 10 with embodiment 1 (2) described substratum suspension cell again after abandoning supernatant
4/ ml goes down to posterity and cultivates as Fig. 1.
(2) cell growth curve
Get the culturing cell in 3 generations, the preparation single cell suspension, counting, adjusting cell concn is 5 * 10
3/ ml is inoculated in the 24 porocyte culture plates, with cell and counting in 3 holes in the trysinization orifice plate, gets 3 holes every day continuously behind the 24h, stops counting when cell covers with the hole, and (d) be X-coordinate with the time, is ordinate zou with the cell count, the drafting growth curve.Result such as Fig. 2 do not have obvious variation postvaccinal the 1st, 2 day cell count, and from the 3rd day, cell rolled up, and peak to the 6th day cell concentration, and later cell enlargement speed slows down.
(3) cell cycle is detected
The MSCs that cultivates is after the trysinization, PBS liquid rinsing 2-3 time, 4 ℃ of 70% ethanol are 16h fixedly, adds 50 μ g/ml iodate third ingots and 50 μ g/ml rnases, hatches 30min for 4 ℃, get the 100ul cell suspension to streaming pipe bottom, flow cytometer detects (counting 104 cells).Result such as Fig. 3, the G0/G1 phase is 54.53%, and the G2-M phase is 16.31%, and the S phase is 29.16%.A high proportion of G0/G1 phase cell is showing that mesenchymal stem cells MSCs has the differentiation potential of height.
(4) induce differentiation to adipocyte
Collect postdigestive the 4th generation BM-MSC, by 2 * 10
4/ cm
2Be inoculated in 6 orifice plates, after treating that cytogamy reaches more than 50%,, add fat and induce system (dexamethasone 1 μ mol/L, insulin human 5mg/L, IBMX 0.5mmol/L, INDOMETHACIN 100 μ mol/L) with the low sugar DMEM nutrient solution that contains 10%FCS, full dose was changed liquid in per 3 days, kept for 3 weeks.Cell is through oil red O stain, and the interior lipid droplet of observation of cell forms situation under the mirror.Result such as Fig. 4 add fat and induced after the system about 7 days, just occur small bright fat granule in a few cell.Along with induction time prolongs, the cytosis that fat drips appears, and cell also becomes square or dihedral.Be cultured to for 3 weeks, the fat granule of fusion almost is full of whole cell, and it is bright orange red that oil red O stain is.
Adopt the mescenchymal stem cell substratum of embodiment 1 preparation, successfully be used for human marrow mesenchymal stem cell is increased, see the growth characteristics that meet mescenchymal stem cell from growth curve.It has the height differentiation potential that stem cell had of a high proportion of G0/G1 phase Cell cycles showed.The mesenchymal stem cells MSCs that obtains has the differentiation potential to adipocyte.
(1) separation of rat bone marrow mesenchymal stem cells and cultivation
The disconnected neck of SD rat is put to death sufficiently sterilised.Separate under the aseptic condition and cut femur and shin bone, cut off femur, shin bone two ends marrow, appear medullary space.With 5ml needle tubing puncture medullary space, wash medullary space repeatedly with the DMEM substratum that has added heparin, collect medullary cell suspension.Suspension is adherent to join in the centrifuge tube of the lymphocyte separation medium that presets equal-volume 1.077g/L the centrifugal 20min of 2000r/min gently.Milky interfacial layer in the middle of drawing, the DMEM substratum of 5 times of volumes of adding, the centrifugal 10min of 1000r/min behind the mixing.Abandon supernatant, leave and take centrifuge tube bottom white floss, add (3) mescenchymal stem cell substratum of embodiment 1 preparation, behind the mixing with 2.0 * 10
6The density of/ml is inoculated in the culturing bottle, puts into 37 ℃, 5%CO
2Cultivate in the incubator.Change liquid behind the 48h, remove not attached cell.Later every 3d changes liquid 1 time.During 60-70%, outwell nutrient solution at the bottom of cell is paved with bottle, with 0.25% trypsinase+0.02%EDTA attached cell is digested 2-3min, see the cell retraction, form becomes circle, when the gap increases, and termination reaction.Add nutrient solution piping and druming and make cell suspension, by the cultivation of going down to posterity in 1: 3, the result as shown in Figure 5.
(2) cell growth curve
Get the culturing cell in 3 generations, the preparation single cell suspension, counting, adjusting cell concn is 5 * 10
3/ ml is inoculated in the 24 porocyte culture plates, with cell and counting in 3 holes in the trysinization orifice plate, gets 3 holes every day continuously behind the 24h, stops counting when cell covers with the hole, and (d) be X-coordinate with the time, is ordinate zou with the cell count, the drafting growth curve.Result such as Fig. 6 do not have obvious variation postvaccinal the 1st, 2 day cell count, and from the 3rd day, cell rolled up, and peak to the 6th day cell concentration.
(3) cell cycle is detected
The MSCs that cultivates is after the trysinization, PBS liquid rinsing 2-3 time, 4 ℃ of 70% ethanol are 16h fixedly, adds 50 μ g/ml iodate third ingots and 50 μ g/ml rnases, hatches 30min for 4 ℃, get the 100ul cell suspension to streaming pipe bottom, flow cytometer detects (counting 104 cells).Result such as Fig. 7, the G0/G1 phase is 58.07%, and the G2-M phase is 26.44%, and the S phase is 36.36%.A high proportion of G0/G1 phase cell is showing that mesenchymal stem cells MSCs has the differentiation potential of height.
(4) induce differentiation to adipocyte
Collect postdigestive the 4th generation BM-MSC, by 2 * 10
4/ cm
2Be inoculated in 6 orifice plates, after treating that cytogamy reaches more than 50%,, add fat and induce system (dexamethasone 1 μ mol/L, insulin human 5mg/L, IBMX 0.5mmol/L, INDOMETHACIN 100 μ mol/L) with the low sugar DMEM nutrient solution that contains 10%FCS, full dose was changed liquid in per 3 days, kept for 3 weeks.Cell is through oil red O stain, and the interior lipid droplet of observation of cell forms situation under the mirror.Result such as Fig. 8 add fat and induced after the system about 7 days, just occur small bright fat granule in a few cell.Along with induction time prolongs, the cytosis that fat drips appears, and cell also becomes square or dihedral.Be cultured to for 3 weeks, the fat granule of fusion almost is full of whole cell, and it is bright orange red that oil red O stain is.
Adopt the mescenchymal stem cell substratum of embodiment 1 preparation, successfully be used for rat bone marrow mesenchymal stem cells is increased, see the growth characteristics that meet mescenchymal stem cell from growth curve.It has the height differentiation potential that stem cell had of a high proportion of G0/G1 phase Cell cycles showed.The mesenchymal stem cells MSCs that obtains has the differentiation potential to adipocyte.
Claims (4)
1. the perfect medium of a vitro culture mesenchymal stem cells MSCs, it is also to be added with Regular Insulin, albumin, Transferrins,iron complexes, Sodium Selenite, hydrocortisone, Prostatropin, progesterone, reduced glutathion and foetal calf serum in basic medium;
Described basic medium is DMEM-L substratum or DMEM/F12 substratum;
The consumption of described each added ingredients is as follows:
Regular Insulin 1～100 μ g/ml
Albumin 1～50mg/ml
Transferrins,iron complexes 1～50ng/ml
Sodium Selenite 1～100ng/ml
Hydrocortisone 1～100 μ g/ml
Progesterone 1～100nmol/L
Reduced glutathion 1～10 μ g/ml
Prostatropin 1～100ng/ml
Foetal calf serum 1～50ul/ml.
2. the perfect medium of vitro culture mesenchymal stem cells MSCs according to claim 1 is characterized in that, the consumption of described each added ingredients is as follows:
Regular Insulin 1～10 μ g/ml
Albumin 1～10mg/ml
Transferrins,iron complexes 1～10ng/ml
Sodium Selenite 1～30ng/ml
Hydrocortisone 1～40 μ g/ml
Progesterone 1～50nmol/L
Reduced glutathion 1～5 μ g/ml
Prostatropin 1～50ng/ml
Foetal calf serum 5～20ul/ml.
3. the perfect medium of vitro culture mesenchymal stem cells MSCs according to claim 2 is characterized in that, the consumption of described each added ingredients is as follows:
Regular Insulin 5 μ g/ml
Albumin 5mg/ml
Transferrins,iron complexes 5ng/ml
Sodium Selenite 10ng/ml
Hydrocortisone 10 μ g/ml
Progesterone 20nmol/L
Reduced glutathion 1.5 μ g/ml
Prostatropin 5ng/ml
Foetal calf serum 10ul/ml.
4. the purposes of any described perfect medium in the vitro culture mesenchymal stem cells MSCs in the claim 1 to 3.
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| CA2856764C (en) | 2011-12-01 | 2018-04-03 | K-Stemcell Co., Ltd. | Medium composition for rejuvenating stem cells |
| US8497124B2 (en) * | 2011-12-05 | 2013-07-30 | Factor Bioscience Inc. | Methods and products for reprogramming cells to a less differentiated state |
| KR101588394B1 (en) | 2013-05-09 | 2016-01-25 | 라정찬 | Media Composition for Improving Renewal Ability and Method for Culturing Stem Cells Using the Same |
| JP6494756B2 (en) * | 2014-10-29 | 2019-04-03 | アール バイオ カンパニー リミテッドＲ Ｂｉｏ Ｃｏ．， Ｌｔｄ． | Medium composition for stem cell culture |
| CN105112366B (en) * | 2015-08-18 | 2019-02-01 | 广东美赛尔细胞生物科技有限公司 | A kind of mesenchymal stem cell serum-free culture medium containing jamaicin |
| CN107475184A (en) * | 2016-06-07 | 2017-12-15 | 广州美萨生物科技有限公司 | A kind of low blood serum medium for being used to cultivate human mesenchymal stem cell |
| CN106754677A (en) * | 2016-12-24 | 2017-05-31 | 严志海 | A kind of external mesenchymal stem cells MSCs culture medium |
| CN110331130B (en) * | 2019-07-03 | 2021-02-05 | 依科赛生物科技（太仓）有限公司 | Mesenchymal stem cell serum-free medium and application thereof |
| CN110317780B (en) * | 2019-07-17 | 2021-06-01 | 福建省海西细胞生物工程有限公司 | Preparation method of placenta membrane mesenchymal stem cells |
| CN113106059B (en) * | 2021-04-07 | 2023-08-15 | 清华大学深圳国际研究生院 | High-migration mesenchymal stem cells, and preparation method and application thereof |
| CN114958734B (en) * | 2021-10-22 | 2023-03-24 | 中国医学科学院生物医学工程研究所 | Human mesenchymal stem cell culture medium |
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| CN101525594A (en) * | 2009-04-17 | 2009-09-09 | 中国医学科学院血液学研究所 | Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same |
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| 崔红强.骨髓间充质干细胞研究进展.《西南军医》.2009,第11卷(第4期),715-717. * |
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