CN1676603A - A new method for in vitro expansion of human bone marrow mesenchymal stem cells - Google Patents
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Abstract
本发明公开了一种人骨髓间充质干细胞体外扩增的新方法。本发明通过适宜的低氧浓度对体外培养的人骨髓间充质干细胞进行调节，使这些细胞能够继续保持较高的增殖状态。通过本发明的方法，采用一种可调控的无损伤的物理手段，体外调节人骨髓间充质干细胞的增殖，实现少量取样、体外扩增、大量获取，进而实现应用人骨髓间充质干细胞治疗多种临床损伤疾病、退行性疾病、遗传缺陷疾病等，具有较好的经济效益和社会效益。
The invention discloses a new method for expanding human bone marrow mesenchymal stem cells in vitro. The invention regulates the human bone marrow mesenchymal stem cells cultured in vitro through appropriate low oxygen concentration, so that these cells can continue to maintain a higher proliferation state. Through the method of the present invention, an adjustable and non-damaging physical means is adopted to regulate the proliferation of human bone marrow mesenchymal stem cells in vitro, to realize a small amount of sampling, in vitro expansion, and a large amount of acquisition, and then realize the application of human bone marrow mesenchymal stem cells for treatment A variety of clinical injury diseases, degenerative diseases, genetic defect diseases, etc., have good economic and social benefits.
Description
Technical field
The present invention relates to a kind of method of regulating cell proliferation, relate in particular to a kind of novel method of regulating the human marrow mesenchymal stem cell propagation of vitro culture.
Background technology
By the tissue engineering technique in-vitro separation with cultivate cell or tissue replacement therapy that required target cell is used for relative disease in the body, be subjected to scholars' extensive concern and attention in recent years for the various clinical treatment of diseases provides new approach.But, limited carrying out and promoting of this technology because that multiple histiocytic in-vitro separation is cultivated difficulty is very big.The mesenchymal stem cells MSCs source is more extensive, it is more convenient to draw materials, can be divided into multiple histocytes such as bone, cartilage, tendon, muscle, ligament, blood vessel endothelium, liver, nerve, skin and fat, separation and Culture is more or less freely, and implanting a little less than the reaction, is a kind of target cell of good replacement therapy.Has important application prospects in fields such as histoorgan damage disease, histoorgan degenerative disease, hereditary defect diseases.Confirm that by experimentation on animals the independent application of mesenchymal stem cells MSCs can promote union of fracture and cartilaginous tissue injury repairing preferably, makes it be covered in ceramics bracket and carbon fibre web etc. by vitro culture and replants into the affected part effect more obvious at present.External also the development is used for the treatment of the hypogenetic clinical experiment of the bone in children that causes because of the type i collagen transgenation with mesenchymal stem cells MSCs.Bone Marrow Mesenchymal Stem Cells Transplantation also has immunological rejection or the lower advantage of degree does not take place, and is easy to control.Mesenchymal stem cells MSCs not only has multidirectional differentiation potential, also be easy to the importing and the expression of foreign gene, thereby, mesenchymal stem cells MSCs might become a kind of target cell of novel gene therapy, combine with its multidirectional differentiation potential so again, by importing goal gene, cell therapy and gene therapy are combined, undoubtedly more wide prospect will be arranged to above-mentioned treatment of diseases.
The importance of research human marrow mesenchymal stem cell mainly is its potential treatment using value, and at present, the human mesenchymal stem cell that is used for clinical treatment is mainly derived from marrow.Although the marrow sample easily obtains, wherein mescenchymal stem cell content is less, account for 100,000 of BMNC/; Particularly mesenchymal stem cells MSCs is under external serum-free culture condition, propagation slowly, and be in negative vegetative state along with the increase of passage number, therefore, acquisition abundance, safe and qualified mesenchymal stem cells MSCs are research and the precondition of using the bone mesenchymal stem cells treatment disease.Yet, also do not have a practicable physiology method to promote the in-vitro multiplication of human marrow mesenchymal stem cell at present.
Oxygen is the prerequisite that life exists, and also is a kind of important regulatory factor of cell physiological function.And hypoxemia is the basic environment that life is grown, and is development growth in low-oxygen environment as mammiferous embryo; The in-house local oxygen levels of adult mammal brain also has only 1%～5%, and the oxygen concn in the marrow is 4%～7%, all is a kind of physiological low-oxygen environments.Yet the standard conditions of mammalian cells in vitro cultivation at present are under 37 ℃, 5% carbonic acid gas and 95% Air mixing gas (oxygen level is 20%).About hypoxemia the regulating effect research of stem cells hyperplasia had only odd report in recent years, SeanJ.Morrison and Studer Lorenz studied report respectively in 2000: hypoxemia (3%) can promote to derive from peripheral nerve ridge stem cell (the Culture in reduced levels of oxygen promotes clonogenicsympathoadrenal differentiation by isolated neural crest stem cells.JNeurosciencce of tire mouse, 2000,20:7370-7376) with nervus centralis precursor cell (Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.JNeurosciencce, 2000, propagation 20:7377-7383); 5% oxygen can promote rat bone marrow mesenchymal stem cells propagation (Lennon DP, et al.Cultivation of rat marrow-derived mesenchymal stemcell in reduced oxygen tension:effects in vitro and in vivo osteochondrogenesis.J Cell Physiol, 2001,187:345-355).Yet, do not see the report of relevant hypoxemia so far to the influence of vitro culture human marrow mesenchymal stem cell propagation.
Summary of the invention
The invention discloses a kind of novel method of regulating the human marrow mesenchymal stem cell propagation of vitro culture.
The present inventor is unexpected in experiment to find that suitable low oxygen concentration has its proliferation function of promotion to the human marrow mesenchymal stem cell of vitro culture, makes these cells can continue to keep vegetative state.
Method of the present invention comprises following content:
The separation and Culture of human marrow mesenchymal stem cell and evaluation
Extract fetal femur marrow, immigration contains in the centrifuge tube of DMEM-LG substratum, and centrifugal degrease and supernatant add the Percoll parting liquid to cell mass, and be centrifugal.Get interface cloud cellular layer and add DMEM-LG, blow and beat into single cell suspension, the counting karyocyte in the 25ml culturing bottle, carries out cell inoculation former being commissioned to train of routine and supports and the cultivation of going down to posterity.
Identify mesenchymal stem cells MSCs with immunocytochemical method, about 50% the time when the cytogamy on the cover glass, take out cover glass, with PBS flushing, room temperature natural air drying, alcohol fixation, distillation washing; Handle with TritonX-100 and hydrogen peroxide; Drip the sealing of normal sheep serum room temperature; It is anti-to drip an anti-working fluid and biotinylation two respectively; With DAB test kit color development at room temperature; Phenodin is slightly redyed, dehydration, transparent, neutral gum mounting.
Hypoxemia is to the short proliferation function of human marrow mesenchymal stem cell
It is that 20% normal oxygen and oxygen level are respectively in 3% and 10% the low-oxygen environment and cultivate that the human marrow mesenchymal stem cell (hMSCs) that goes down to posterity is placed on oxygen level respectively, and microscopically is observed the mesenchymal stem cells MSCs of cultivating in low-oxygen environment more.The cell counting result shows that compare with normal oxygen control group, 3% hypoxia group and 10% hypoxia group hMSCs quantity all significantly increase (P＜0.001), and 3% hypoxia group hMSCs quantity is than 10% hypoxia group showed increased (P＜0.01).With the dna content of cells were tested by flow cytometry hMSCs, find to have only cell seldom to be in division stage among the hMSCs of normal oxygen control group, the proliferation index average is 5.27%, has illustrated that 5.27% cell is in division stage, has nearly 95% cell to be in G
0/ G
1Phase.Compare with normal oxygen control group, 3% hypoxia group and 10% hypoxia group hMSCs proliferation index all significantly increase (P＜0.001), and 3% hypoxia group hMSCs proliferation index obviously increases (P＜0.01) than 10% hypoxia group.
In sum, hypoxemia can promote the in-vitro multiplication of human marrow mesenchymal stem cell, makes these cells can continue to keep vegetative state.The propagation of utilizing the hypoxemia in the environment to regulate the human marrow mesenchymal stem cell of vitro culture, it is simple to have an experiment flow, and repeatability is strong, reliable results, at external easy-regulating, to mesenchymal stem cells MSCs not damaged and toxic side effect, characteristics such as practical.
Description of drawings
Fig. 1. be the human marrow mesenchymal stem cell aspect graph (* 100) of vitro culture near fusion.
Fig. 2. for the mesenchymal stem cells MSCs immunocytochemistry is identified figure.
Fig. 3. be the action diagram of hypoxemia to mesenchymal stem cells MSCs quantity.(p＜0.001)
Fig. 4. be the action diagram of hypoxemia to the mesenchymal stem cells MSCs proliferation index.(p＜0.001)
Embodiment
The separation and Culture and the evaluation of embodiment one human marrow mesenchymal stem cell
1. the separation and Culture of human marrow mesenchymal stem cell
Aseptic extraction fetus (6 months induction of labor with water bag fetuses, pluripara's informed consent provides) the about 1ml of femur bone marrow, immigration contains in the aseptic centrifuge tube of 3ml DMEM-LG substratum (Hyclone company product) the centrifugal 5min of 900g.Degrease and supernatant add 70%Percoll parting liquid (Pharmacia company product), the centrifugal 30min of 900g to cell mass.Get interface cloud cellular layer and add the DMEM-LG that contains 20% foetal calf serum, blow and beat into single cell suspension, the counting karyocyte, with cell with 10
5The density of individual/ml is inoculated in the 25ml culturing bottle, at 37 ℃, 5%CO
2, saturated humidity CO
2Cultivate in the incubator.Change fresh medium behind the 24h, remove not attached cell, later per 3～4d changes liquid once.When cell merges when reaching about 90% growth surface (see figure 1) mutually, with 0.25% tryptic digestion cell dispersion, with the cultivation of going down to posterity of 1: 3 ratio.
2. the evaluation of human marrow mesenchymal stem cell
About 50% the time when the cytogamy on the cover glass in the culturing bottle, take out cover glass, 0.01M PBS flushing, room temperature natural air drying, 95% alcohol fixation 30min, distillation washing; 0.2%TritonX-100 handles 10min, 0.5% hydrogen peroxide 10min; Drip normal sheep serum, room temperature sealing 20min gets rid of unnecessary liquid; Drip an anti-working fluid (mouse anti human Vimentin is available from Beijing Zhong Shan biotech firm), 4 ℃ are spent the night, and 0.01M PBS washes; It is anti-to drip biotinylation two, 37 ℃ of 20min, and 0.01M PBS washes; Drip SABC, 37 ℃ of 20min, 0.01M PBS washes; With DAB test kit color development at room temperature; Phenodin is slightly redyed, dehydration, transparent, neutral gum mounting.Show the positive (see figure 2) of 98% cell Vimentin in the microscopically count results.
Embodiment two low-oxygen environments are to the short proliferation function of human marrow mesenchymal stem cell
1. experiment grouping and hypoxia condition determines
Experiment divides 3 groups, is respectively normal oxygen control group (20%O
2) 3% hypoxia group (3%O
2) and 10% hypoxia group (10%O
2).Self-control cell hypoxemia incubator feeds respectively in closed cabinet and contains 3% or 10%O
2, 5%CO
2With the mixed gas of nitrogen balance, beginning air flow 1L/min, continue ventilation 30min after, be adjusted to maintenance dose 0.1L/min and continue ventilation, keep oxygen level in the encloses container to be respectively 3% or 10%O
2
2. the detection method of mesenchymal stem cells MSCs propagation
1) cell counting
Mesenchymal stem cells MSCs is placed on respectively in the environment of different oxygen concentrations and cultivates after 3 days, use 0.25% trypsin digestion and cell, and with the nutrient solution termination that contains foetal calf serum, make the individual cells suspension, employing blood counting chamber counting process is carried out cell counting.Every group 8 ware calculates its mean value, relatively uses non-matching Student ' s T-test between group.
2) mensuration of cell cycle
To cultivate the hMSCs digestion of 3d, volume fraction is fixedly 24h of 4 ℃ of 75% cold ethanol; Centrifugal removal ethanol, 0.1M PBS washed cell 2 times in 0.5ml PBS, adds 10mg/mlRNase A 10 μ l with cell suspension, after 30min is hatched in 37 ℃ of water-baths, puts into ice bath immediately; Add 5mg/100ml ethidium bromide (PI, Sigma company product) dyeing (4 ℃, lucifuge 30min), detect and analytical results with flow cytometer (FACSCalibur type, Becton Dickinson product).
3. low-oxygen environment is to the short proliferation function of human marrow mesenchymal stem cell
Mesenchymal stem cells MSCs is placed on respectively in normal oxygen (oxygen level is 20%) and hypoxemia (oxygen level the is respectively 3% and 10%) environment cultivated 3 days, microscopically is observed the mesenchymal stem cells MSCs of cultivating and is increased in low-oxygen environment.Analyze by statistics: the cell counting result compares with normal oxygen control group, 3% hypoxia group and 10% hypoxia group hMSCs quantity all significantly increase (P＜0.001), and 3% hypoxia group hMSCs quantity is than 10% hypoxia group showed increased (P＜0.01) (see Table 1 and Fig. 3).With the dna content of cells were tested by flow cytometry hMSCs, find to have only cell seldom to be in division stage among the hMSCs of normal oxygen control group, the proliferation index average is 5.27%, has illustrated that 5.27% cell is in division stage, has nearly 95% cell to be in G
0/ G
1Phase.Compare with normal oxygen control group, 3% hypoxia group and 10% hypoxia group hMSCs proliferation index all significantly increase (P＜0.001), and 3% hypoxia group hMSCs proliferation index obviously increases (P＜0.01) (see Table 2 and Fig. 4) than 10% hypoxia group.
Table 1. hypoxemia and CoCl
2To hMSCs (5 * 10
3) influence of quantity
| Preparation | ?Control | ?Oxygen?concentration | |
| ????10％ * | ????3％ * | ||
| ????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 | ????74 ????62 ????67 ????84 ????68 ????88 ????63 ????64 | ????92 ????87 ????81 ????102 ????97 ????96 ????106 ????95 | ????110 ????144 ????106 ????128 ????123 ????98 ????91 ????138 |
| ????mean ????SD | ????71.25 ????9.896 | ????94.5 ????7.946 | ????117.25 ????19.024 |
*Significantly?higher(P＜0.001)than?cell?yield?for?hMSCs
maintained?in?control?oxygen?when?data?are?analyzed
with?the?paired?t-test.
Table 2. hypoxemia and CoCl
2Influence to hMSCs proliferation index (%)
| ??Preparation | ??Control | ???O 2?concentration | |
| ????10％ * | ????3％ * | ||
| ????1 ????2 ????3 ????4 | ????5.63 ????4.49 ????6.12 ????4.83 | ????11.75 ????10.01 ????11.28 ????13.31 | ????19.38 ????21.28 ????21.31 ????28.05 |
| ????mean ????SD | ????5.27 ????0.743 | ????11.59 ????1.363 | ????20.66 ????3.805 |
*P＜0.001
Claims (5)
1, a kind of new method of myeloid stem cell external expansion is characterized in that adopting low-oxygen environment, promotes the propagation of the human marrow mesenchymal stem cell of vitro culture.
2, method according to claim 1 is characterized in that said low-oxygen environment is that oxygen concn is lower than 20% environment.
3, method according to claim 1 and 2, the concentration that it is characterized in that oxygen in the low-oxygen environment is 3% to 10%.
4, method according to claim 3 is characterized in that the oxygen concn in the low-oxygen environment is 3%.
5, method according to claim 3 is characterized in that the oxygen concn in the low-oxygen environment is 10%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103180435A (en) * | 2010-08-31 | 2013-06-26 | 库克通用生物技术有限责任公司 | Systemic, allogenic stem cell therapies for treatment of diseases in animals |
| CN105617462A (en) * | 2016-01-27 | 2016-06-01 | 浙江大学 | Bionics-based preparation method for tissue-engineered bone |
| WO2016184427A1 (en) * | 2015-05-21 | 2016-11-24 | 中国科学院上海生命科学研究院 | Low-oxygen-treated mesenchymal stem cell and use thereof |
| CN107849533A (en) * | 2015-02-20 | 2018-03-27 | 洪士杰 | Use of mesenchymal stem cells for the treatment of osteoarthritis |
| CN110205289A (en) * | 2019-04-23 | 2019-09-06 | 华南生物医药研究院 | Expand method, system and the computer-readable medium of mesenchymal stem cell |
| CN111363720A (en) * | 2020-03-28 | 2020-07-03 | 海门生原干细胞科技有限公司 | Bone marrow mesenchymal stem cell for treating cerebral ischemia and preparation method and application thereof |
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2004
- 2004-03-30 CN CNA2004100298411A patent/CN1676603A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103180435A (en) * | 2010-08-31 | 2013-06-26 | 库克通用生物技术有限责任公司 | Systemic, allogenic stem cell therapies for treatment of diseases in animals |
| CN107849533A (en) * | 2015-02-20 | 2018-03-27 | 洪士杰 | Use of mesenchymal stem cells for the treatment of osteoarthritis |
| WO2016184427A1 (en) * | 2015-05-21 | 2016-11-24 | 中国科学院上海生命科学研究院 | Low-oxygen-treated mesenchymal stem cell and use thereof |
| CN105617462A (en) * | 2016-01-27 | 2016-06-01 | 浙江大学 | Bionics-based preparation method for tissue-engineered bone |
| CN110205289A (en) * | 2019-04-23 | 2019-09-06 | 华南生物医药研究院 | Expand method, system and the computer-readable medium of mesenchymal stem cell |
| CN111363720A (en) * | 2020-03-28 | 2020-07-03 | 海门生原干细胞科技有限公司 | Bone marrow mesenchymal stem cell for treating cerebral ischemia and preparation method and application thereof |
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