CN102978156A - Expansion in vitro purification culture method of mesenchymal stem cells and culture medium - Google Patents
Expansion in vitro purification culture method of mesenchymal stem cells and culture medium Download PDFInfo
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Abstract
The invention provides an expansion in vitro purification culture method of mesenchymal stem cells and a cell culture medium used in a culture process. According to the method, mesenchymal stem cells differentiate to osteogenic stem cells. The cell culture medium comprises the following components: 8-12mu g/L of bFGF (Basic Fibroblast Growth Factor), 8-12mu g/L of EGF (Epidermal Growth Factor), 8-12mu g/L of PDGF-BB (Platelet Derived Growth Factor-BB), 10 percent of fetal calf serum, 100U/mL of penicillin and 100mg of L of streptomycin; a solvent is a basic culture medium; and the basic culture medium is a DMEM (Dulbecco Modified Eagle Medium) culture solution, an F12 culture solution or a mixture of the DMEM culture solution and the F12 culture solution. The mesenchymal stem cells obtained by adopting a method comprising the steps of early replacement of the cell culture medium, cell adherence passage and combined addition of cell factors (bFGF, EGF and PDGF-BB) in the culture solution are high in purity and strong in vitro proliferation capacity, and have good capability of differentiation to osteogenic stem cells.
Description
Technical field
The claimed a kind of mesenchymal stem cells MSCs amplification in vitro purification cultivation method that is beneficial to bone mesenchymal stem cell to osteoblast differentiation of the present invention, and in culturing process used cell culture medium.
Background technology
Mesenchymal stem cells MSCs can be divided into the cell types such as scleroblast, chondrocyte, adipocyte under suitable conditioned stimulus, and be easy to separation and Culture, have the advantages such as stronger self, differentiation capability and immunosuppression, make it have important application prospect at the damaged property of histoorgan disease, histoorgan degenerative disease and cell therapy and field of tissue engineering technology.In-vitro separation and cultural method that numerous domestic and international research and probe mesenchymal stem cells MSCs is arranged recently, mainly contain the methods such as adherent separation, density gradient centrifugation and cell sorting, the result shows by the cell purity of the adherent separation of going down to posterity low, gradient centrifugation and cell sorting can obtain the cell of higher degree, and cell viability is relatively low, and required marrow amount is larger, affects follow-up research and application.And the mesenchymal stem cells MSCs that obtains by these methods loses the proliferation and differentiation ability gradually in the culturing process that goes down to posterity.Recently there is bibliographical information that some cytokines are applied in the relevant research of stem cell, the growth in vitro of mesenchymal stem cells MSCs is had certain biological effect.
Summary of the invention
The object of the invention is a kind of to be beneficial to the mesenchymal stem cells MSCs amplification in vitro purification cultivation method of bone mesenchymal stem cell to osteoblast differentiation, and in culturing process used cell culture medium.
The technical solution used in the present invention is:
A kind of amplification in vitro purification cultivation method of mesenchymal stem cells MSCs, described method comprises: extracting marrow cell, the single cell suspension behind screen filtration are inoculated in and carry out adherent culture, Initial seeding density 2 ~ 5 * 10 in the culture dish
6Individual/mL(preferably is about 5 * 10
6Individual/mL) cell culture medium, change cell culture medium (forming the same) after cultivating 3 ~ 5 hours (being preferably 3 hours), after this again change cell culture medium (forming the same) every 8 ~ 15 hours (preferred 12 hours), until after the inoculation 60 ~ 80 hours (being preferably 75 hours), and then changed cell culture medium (forming the same) every 3 days and continue to cultivate, until growing to 80% ~ 90%, cell merges, with trysinization, digestion time is no more than 2 minutes, passage continues to cultivate, the mesenchymal stem cells MSCs behind the acquisition purifying; Described cell culture medium final concentration is composed as follows: bFGF 8 ~ 12 μ g/L, and EGF 8 ~ 12 μ g/L, PDGF-BB 8 ~ 12 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is DMEM nutrient solution, F12 nutrient solution or its mixture.
Preferably, described cell culture medium final concentration is composed as follows: bFGF(mouse Prostatropin) 10 μ g/L, the EGF(mouse EGF) 10 μ g/L, the 10 μ g/L of PDGF-BB(mouse platelets derivative growth factor-BB), foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is the mixed solution of DMEM nutrient solution and F12 nutrient solution volume ratio 1:1.
The passage concrete steps comprise: PBS damping fluid washed cell, add 0.25% pancreatin (containing 0.02%EDTA) room temperature peptic cell, add perfect medium and stop digestion, cell suspension is removed supernatant liquid through centrifugation, add again the perfect medium suspension cell, be inoculated in and continue in the new culture dish to cultivate, the cell inoculum density is 5 * 10
5/ mL.Perfect medium is to remove the somatomedin composition in the cell culture medium, and it is composed as follows: foetal calf serum 10%, and penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is DMEM nutrient solution, F12 nutrient solution or its mixture.
The invention still further relates to a kind of amplification in vitro purifying cultured cells substratum for mesenchymal stem cells MSCs, its final concentration is composed as follows: bFGF 8 ~ 12 μ g/L, EGF 8 ~ 12 μ g/L, PDGF-BB 8 ~ 12 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is DMEM nutrient solution, F12 nutrient solution or its mixture.
Preferably, described cell culture medium final concentration is composed as follows: bFGF 10 μ g/L, and EGF 10 μ g/L, PDGF-BB 10 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is the mixed solution of DMEM nutrient solution and F12 nutrient solution volume ratio 1:1.
The present invention is on Research foundation in the past, adopt cell attachment to go down to posterity and nutrient solution in add the method that cytokine (bFGF, EGF and PDGF-BB) combines, the bone marrow stem cell purity that obtains is high, and the in-vitro multiplication ability is strong, and has preferably the ability to osteoblast differentiation.
Cytokine has important impact to in-vitro multiplication and the differentiation of mesenchymal stem cells MSCs.PDGF, FGF signal path are two crucial paths that affect growth of mesenchymal stem cells and differentiation, and bFGF and EGF can suppress the growth of hemopoietic stem cell, are conducive to purifying and the growth of mesenchymal stem cells MSCs.The in-vitro separation of mesenchymal stem cells MSCs provided by the invention and the cultural method of amplification, inoculate by low density, the early stage nutrient solution of changing, unite and add cytokine and pancreatin cell attachment that digestion the combines cultural method that goes down to posterity that goes down to posterity, thereby reach the purification rate that improves mesenchymal stem cells MSCs and the purpose of proliferation and differentiation ability.
The method that mesenchymal stem cells MSCs of the present invention separates and cultivates has following advantage:
(1) different according to cell attachment speed, the early stage nutrient solution of changing can be removed the cell relic that exists in the cell of hemopoietic stem cell in the marrow, other type and the supernatant liquor after the low density inoculation, is beneficial to the early stage purifying of cell;
(2) different to the susceptibility of pancreatin according to cell, digestion is no more than 2 minutes under the room temperature condition, the mescenchymal stem cell to the pancreatin sensitivity can be digested first, is further purified cell; And short period of time digestion do not affect the vigor of cell, is conducive to the amplification of cell;
The cytokine of (3) adding in the nutrient solution is conducive to the propagation of mesenchymal stem cells MSCs, can suppress the adherent growth of hematopoiesis lineage stem cells, is beneficial to the purifying of cell; The undifferentiated state that can also keep cell simultaneously is conducive to the differentiation research of cell.
Description of drawings
Fig. 1 is 72 hour cell forms after the initial inoculation.
Fig. 2 is 1 pericyte form after the initial inoculation.
Fig. 3 is 15 days cellular form after the initial inoculation.
Fig. 4 is the cellular form that reached for the 3rd generation.
Fig. 5 be expanded to the 3rd generation cell flow cytometric analysis figure (FL1-H is the homotype IgG contrast of NA group cell antibody, and FL2-H is the homotype IgG contrast of A group cell antibody).
Fig. 6 is the scleroblast expression of specific gene figure of osteogenic induction different time.
Fig. 7 is the alkaline phosphatase staining figure in 2 weeks of osteogenic induction.
Fig. 8 is the Alizarin Red S colored graph that scleroblast induced for 3 weeks.
Wherein, NA represents to add the Comparative Examples of cytokine EGF and PDGF-BB, and A represents to add cytokine bFGF, the embodiment of EGF and PDGF-BB.
Embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
Agents useful for same: foetal calf serum (Hyclone), penicillin, Streptomycin sulphate, DMEM nutrient solution, F12 nutrient solution (Gibco) bFGF, EGF, PDGF-BB(PeproTech):
1, get the male adult mouse of ICR (buying in the Zhejiang Academy of Medical Sciences Experimental Animal Center) about 6 weeks, the cervical vertebra dislocation is put to death, and takes out femur and shin bone under the aseptic condition;
2, sterile scissors is cut off the two ends epiphysis, draw perfect medium flushing medullary space, perfect medium component: 10%(v/v) foetal calf serum, penicillin 100U/mL with syringe, Streptomycin sulphate 100mg/L, solvent are DMEM/F12 nutrient solution (being DMEM and F12 volume ratio 1:1 mixed solution);
3, the bone marrow cell suspension that obtains filters through 200 eye mesh screens, is inoculated in the culture dish of 10 cm, and Initial seeding density is about 5 * 10
6Individual/mL, cell culture medium is that perfect medium is united interpolation cytokine bFGF, and EGF and PDGF-BB, final concentration are 10 μ g/L, and the culture volume of initial inoculation cell is 2mL, can cover at the bottom of the culture dish;
4, initial inoculation is placed on 37 ℃, and saturated humidity contains 5% CO
2Incubator in cultivated 3 hours, then take out culture dish, change cell culture medium, volume is 2 mL;
5, first replaced medium is after 12 hours, again change cell culture medium, volume still is 2 mL, after 12 hours, again changes cell culture medium, to the primary vaccination 75 hours, change the cell culture medium volume and increase to 8 mL, changed a cell culture medium in per 3 days afterwards, volume is 8 mL, treat to carry out had digestive transfer culture when cell grows to 80% fusion, under inverted microscope, observe the cellular form of cultivating different time;
6, passage: behind twice of the PBS damping fluid washed cell, add 0.25%(w/v) pancreatin (containing 0.02%(w/v) EDTA) 1mL room temperature peptic cell 2 min, the 2mL perfect medium stops digestion, cell suspension is behind centrifugal 5 min of 300g, the perfect medium suspension cell, be inoculated in and continue in the new culture dish to cultivate, the cell inoculum density is 5 * 10
5/ mL;
Comparative Examples:
Step is identical with step described in the embodiment, and the two difference is cytokine EGF and the PDGF-BB that the nutrient solution in the present embodiment only adds same concentrations.
The phenotypic characteristic of mesenchymal stem cells MSCs:
Get the Marrow Mesenchymal Stem Cells that reached for the 3rd generation, pancreatin room temperature 0.25%(w/v) digests 2 min, centrifugal 5 min of 300g after perfect medium stops, add again 500 μ l PBS Eddy diffusion cells after the PBS washing, anti-mouse mesenchymal cell surface antigen antibody CD45 and CD90 and corresponding each 1 μ L of of the same race fluorescently-labeled non-specific homotype antibody of adding respectively PE and FITC mark, the room temperature lucifuge is hatched 30 min, the centrifugal 5min of 800 r/min, add again 200 μ l PBS damping fluid suspension cells after discarding antibody incubation liquid, adopt BD FACSCalibur flow cytometer to detect the expression of cell-surface antigens.
The scleroblast of mesenchymal stem cells MSCs is induced differentiation and is identified:
Get the Marrow Mesenchymal Stem Cells that reached for the 3rd generation and be inoculated in 12 orifice plates, when treating that cell reaches 80% fusion, change the skeletonization inducing culture: the substratum DMEM/F12+10% foetal calf serum of factor-containing, 1 * 10
-8The mol/L dexamethasone, 50 μ mol/L xitix, 10 mmol/L Sodium Glycerophosphates, per 3 days replacing 1 subcultures.3,7 and 14 days the total RNA of Marrow Mesenchymal Stem Cells of differentiation is induced in extraction, the SYBGreen fluorescence quantitative PCR detection is induced the scleroblast specific gene Collagen I of differentiation and the expression of Osteocalcin, the Marrow Mesenchymal Stem Cells of select respectively to induce differentiation 14 days and 28 days carries out the osteoblast differentiation result of alkaline phosphatase staining and alizarin red S staining examine Marrow Mesenchymal Stem Cells after 4% Paraformaldehyde 96 is fixed 30 minutes.
Result such as accompanying drawing 1,2 are shown in 3, the medullary cell of initial inoculation is through changing in early days liquid, before trysinization was gone down to posterity, the cellular form of embodiment and Comparative Examples was consistent, and the primary cell form that microscopically is observed acquisition is single, be long shuttle shape Polygons, refractivity is strong, clear-cut margin, and present colony formula growth pattern, compare with Comparative Examples, the primary cell rate of propagation among the embodiment is relatively very fast.
Former generation purifying mesenchymal stem cells MSCs go down to posterity through trysinization after, the shuttle shape shape the when mesenchymal stem cells MSCs of Comparative Examples loses former culture gradually becomes roomy flat form, edge blurry, propagation slowly presents ageing state (Fig. 4 is shown in the NA).Form did not occur obviously to change (Fig. 4 is shown in the A) when the mesenchymal stem cells MSCs of embodiment reached for the 3rd generation, and rate of propagation is very fast.Through flow cytometer detection, mesenchymal stem cells MSCs among the embodiment is not expressed CD45 substantially, CD90 expresses positive cell proportion and surpasses 90%(Fig. 5, shown in the A), still have part CD45 to express positive cell in the cell of Comparative Examples, the cell proportion of expressing CD90 is no more than 70%(Fig. 5, shown in the NA).As seen, unite amplification in vitro and the cell purification that three kinds of cytokines of interpolation more are conducive to mesenchymal stem cells MSCs in the nutrient solution.
Such as Fig. 6, shown in 7,8, the mesenchymal stem cells MSCs of cultivating through going down to posterity is through behind the osteogenic induction of different time, the expression of the scleroblast specific gene among the embodiment and the expression of alkaline phosphatase are all apparently higher than Comparative Examples, and the formation volume of calcified material is more.The cell expansion ex vivo purification cultivation method that the present invention adopts is conducive to the bone mesenchymal stem cell to osteoblast differentiation.
Claims (4)
1. the amplification in vitro purification cultivation method of a mesenchymal stem cells MSCs, described method comprises: extracting marrow cell, the single cell suspension behind screen filtration are inoculated in and carry out adherent culture, Initial seeding density 2 ~ 5 * 10 in the culture dish
6Individual/the mL cell culture medium, cultivate and change first cell culture medium after 3 ~ 5 hours, after this again changed cell culture medium every 8 ~ 15 hours, until inoculate rear 60 ~ 80 hours, and then changed cell culture medium every 3 days and continue to cultivate, until cell grows to 80% ~ 90% fusion, with trysinization, digestion time is no more than 2 minutes, and passage continues to cultivate, and obtains the mesenchymal stem cells MSCs of purifying; Described cell culture medium final concentration is composed as follows: bFGF 8 ~ 12 μ g/L, and EGF 8 ~ 12 μ g/L, PDGF-BB 8 ~ 12 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is DMEM nutrient solution, F12 nutrient solution or its mixture.
2. the method for claim 1, it is characterized in that: described cell culture medium final concentration is composed as follows: bFGF 10 μ g/L, EGF 10 μ g/L, PDGF-BB 10 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is the mixed solution of DMEM nutrient solution and F12 nutrient solution volume ratio 1:1.
3. amplification in vitro purifying cultured cells substratum that is used for mesenchymal stem cells MSCs, its final concentration is composed as follows: bFGF 8 ~ 12 μ g/L, EGF 8 ~ 12 μ g/L, PDGF-BB 8 ~ 12 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is DMEM nutrient solution, F12 nutrient solution or its mixture.
4. cell culture medium as claimed in claim 3 is characterized in that described cell culture medium final concentration is composed as follows: bFGF 10 μ g/L, EGF 10 μ g/L, PDGF-BB 10 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is the mixed solution of DMEM nutrient solution and F12 nutrient solution volume ratio 1:1.
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Cited By (14)
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| CN104232573A (en) * | 2014-09-11 | 2014-12-24 | 安沂华 | Growth medium and use thereof, and method for cultivating umbilical cord mesenchymal stem cells |
| CN104762259A (en) * | 2015-04-21 | 2015-07-08 | 广州赛莱拉干细胞科技股份有限公司 | Culture medium for mesenchymal stem cells and large-scale culture method thereof |
| CN106957814A (en) * | 2017-05-19 | 2017-07-18 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of amnion mesenchymal stem cell culture medium and culture amnion mesenchymal stem cell |
| CN106967677A (en) * | 2017-05-19 | 2017-07-21 | 苏州大学附属第医院 | A kind of method that use hydrogen peroxide promotes mesenchymal stem cells MSCs Osteoblast Differentiation |
| CN107090430A (en) * | 2017-03-27 | 2017-08-25 | 山西医科大学 | A kind of preparation of Secoiridoid Glycosides compound and its application for improving mesenchymal stem cells MSCs vigor |
| CN109706115A (en) * | 2017-10-26 | 2019-05-03 | 中国医科大学 | A kind of construction method of Marrow Mesenchymal Stem Cells cell line |
| CN110172445A (en) * | 2019-04-15 | 2019-08-27 | 华南生物医药研究院 | Expand method, culture medium and the application of mesenchymal stem cell |
| CN110438076A (en) * | 2018-05-02 | 2019-11-12 | 强普生技股份有限公司 | T cell culture medium and cultural method |
| CN110468101A (en) * | 2019-09-18 | 2019-11-19 | 安徽科门生物科技有限公司 | A kind of Multiplying culture method of mesenchymal stem cell |
| CN112111448A (en) * | 2020-08-21 | 2020-12-22 | 深圳市中科广瑞生物技术有限公司 | Improved mesenchymal stem cell culture medium, bone marrow mesenchymal stem cell and culture method and application thereof |
| CN113005079A (en) * | 2021-05-08 | 2021-06-22 | 河北驰熙科技发展有限公司 | Additive for human bone marrow mesenchymal stem cell in vitro amplification and amplification method |
| CN113755433A (en) * | 2021-08-09 | 2021-12-07 | 合肥滴碧云生物科技有限公司 | Synovial mesenchymal stem cell suspension and preparation method thereof |
| CN114317421A (en) * | 2021-12-16 | 2022-04-12 | 北京科技大学 | Method, composition and application for enhancing mesenchymal stem cells to promote angiogenesis |
| CN114591898A (en) * | 2022-02-21 | 2022-06-07 | 赛尔生命科技(深圳)有限公司 | Preparation method of bone marrow mesenchymal stem cell culture inducing composition |
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| CN104232573B (en) * | 2014-09-11 | 2017-01-11 | 安沂华 | Growth medium and use thereof, and method for cultivating umbilical cord mesenchymal stem cells |
| CN104232573A (en) * | 2014-09-11 | 2014-12-24 | 安沂华 | Growth medium and use thereof, and method for cultivating umbilical cord mesenchymal stem cells |
| CN104762259A (en) * | 2015-04-21 | 2015-07-08 | 广州赛莱拉干细胞科技股份有限公司 | Culture medium for mesenchymal stem cells and large-scale culture method thereof |
| CN107090430B (en) * | 2017-03-27 | 2021-03-23 | 山西医科大学 | Preparation of secoiridoid glycoside compound and application of compound in improving activity of mesenchymal stem cells |
| CN107090430A (en) * | 2017-03-27 | 2017-08-25 | 山西医科大学 | A kind of preparation of Secoiridoid Glycosides compound and its application for improving mesenchymal stem cells MSCs vigor |
| CN106957814A (en) * | 2017-05-19 | 2017-07-18 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of amnion mesenchymal stem cell culture medium and culture amnion mesenchymal stem cell |
| CN106967677A (en) * | 2017-05-19 | 2017-07-21 | 苏州大学附属第医院 | A kind of method that use hydrogen peroxide promotes mesenchymal stem cells MSCs Osteoblast Differentiation |
| CN109706115A (en) * | 2017-10-26 | 2019-05-03 | 中国医科大学 | A kind of construction method of Marrow Mesenchymal Stem Cells cell line |
| CN109706115B (en) * | 2017-10-26 | 2023-05-30 | 中国医科大学 | Construction method of mouse bone marrow mesenchymal stem cell line |
| CN110438076A (en) * | 2018-05-02 | 2019-11-12 | 强普生技股份有限公司 | T cell culture medium and cultural method |
| CN110172445A (en) * | 2019-04-15 | 2019-08-27 | 华南生物医药研究院 | Expand method, culture medium and the application of mesenchymal stem cell |
| CN110468101A (en) * | 2019-09-18 | 2019-11-19 | 安徽科门生物科技有限公司 | A kind of Multiplying culture method of mesenchymal stem cell |
| CN112111448A (en) * | 2020-08-21 | 2020-12-22 | 深圳市中科广瑞生物技术有限公司 | Improved mesenchymal stem cell culture medium, bone marrow mesenchymal stem cell and culture method and application thereof |
| CN113005079A (en) * | 2021-05-08 | 2021-06-22 | 河北驰熙科技发展有限公司 | Additive for human bone marrow mesenchymal stem cell in vitro amplification and amplification method |
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| CN114317421A (en) * | 2021-12-16 | 2022-04-12 | 北京科技大学 | Method, composition and application for enhancing mesenchymal stem cells to promote angiogenesis |
| CN114591898A (en) * | 2022-02-21 | 2022-06-07 | 赛尔生命科技(深圳)有限公司 | Preparation method of bone marrow mesenchymal stem cell culture inducing composition |
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Application publication date: 20130320 |