CN1563364A - Method for 3D cultivating and inducing stem cell of mesenchyme of bone marrow and chondroblast - Google Patents
Method for 3D cultivating and inducing stem cell of mesenchyme of bone marrow and chondroblast Download PDFInfo
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Abstract
A method for preparing chondroblast of bone marrow mesogalia stem cell with three dimensional cultivation and induction includes steps of placing bone marrow mesogalia stem cell into suspending culture device preset with culture medium and microcarrier for suspending cultivation; cloning abovesaid stem cells with agitation, adding induction culture medium of chondroblast in culture meidum containing harvested cytodex3 microcrrier and abovesaid stem cells gromn on it; and carrying out induction for chondroblast assembly from abovesaid stem cells.
Description
Technical field
The present invention relates to the method for a kind of cultivation and inducing bone mesenchymal stem cell chondroblast.
Background technology
Marrow is a kind of tissue of complexity, wherein contains the cell of various hematopoiesis systems and non-hematopoiesis system.Mesenchymal stem cells MSCs is a kind of non-hematopoiesis lineage stem cells that is present in the marrow, be easy to from marrow, separate, it can not only be at external fast breeding, and has a multidirectional differentiation potential, under specific physicochemical environment and cytokine induction, can generate various mescenchymal tissue cells, for example scleroblast, chondroblast, lipoblast, one-tenth Tenocyte cell, marrow stromal cell and neurocyte like cell etc.Based on above two big characteristics, mesenchymal stem cells MSCs can carry out the multiple autotelic operation of inducing after cultured and amplified in vitro, again in the defeated then ex vivo, carry out clinical treatment, as treat fracture, cartilaginous tissue damage etc., so mesenchymal stem cells MSCs has become the more a kind of stem cell of application in Tissue Engineering Study and the exploitation.
The traditional method of repairing cartilaginous tissue is to adopt from the body chondrocyte, and this method must cut the joint cartilage in non-heavy burden district under arthroscope, and wherein chondrocyte is increased, and the unique advantage of this operation is a no antigen, but causes body injury very big.The overwhelming majority is a matrix in the cartilaginous tissue in addition, the few and difficult separation of chondrocyte's quantity, the subculture in vitro separately number of times is limited, 3～4 generations of general maximum cultivation, so be difficult to obtain a large amount of cells (referring to Yang Zhiming. organizational project. Beijing: Chemical Industry Press, 2002).People such as Richardson are (referring to J.Bone ﹠amp; Jnt.Surg, 81-B:1064-1068,1999) reported 2 examples damaged back 12 months immunohistochemical analysis result of repairing articular cartilage in this way, find that the tissue regions of repairing is inhomogeneous, deep regional is the tissue of similar hyaline cartilage, upper layer like fibrous cartilage.The therefore present big more options mesenchymal stem cells MSCs of investigator is used for the treatment of experimentation on animals and preliminary clinical experiment well afoot that cartilaginous tissue damages.
At present, mesenchymal stem cells MSCs induces chondroblast generally to take the poly-group of high-density to induce.United States Patent (USP) 6,548, the structure that proposes reticular tissue in 734 needs highdensity seed cell, especially makes up cartilage.United States Patent (USP) 5,908, mention in 784 mesenchymal stem cells MSCs external can be after having possessed three-D space structure and chondroblast inductive substance along chondroblast approach differentiation, wherein three-dimensional structure is a key factor, must influence each other by the excretory material between the cell.Therefore in about the research of mesenchymal stem cells MSCs chondroblast inductive, the cell centrifugation after generally will increasing becomes cell mass, carries out micelle with the chondroblast inducing culture again and induces.In the research of repairing cartilage defect, then earlier highdensity mesenchymal stem cells MSCs is inoculated on the timbering material, the cell-scaffold mixture is inserted in the inducing culture induce then, the mixture after inducing continues to be implanted into the cartilage defect place.
The mesenchymal stem cells MSCs that the chondroblast derived need is a large amount of, in order to obtain a large amount of mesenchymal stem cells MSCs, must be in the external cultivation of going down to posterity for a long time, and along with the prolongation of cell cultures time, procedural losing can appear in the multidirectional differentiation potential of stem cell, and the pancreatin that uses in the digestive process also can cause the reduction of cytoactive.In the application process of mesenchymal stem cells MSCs-timbering material, should consider whether this material has excellent biological compatibility, whether help contacting and the release of bioactive molecules of cell and nutritive substance, whether the aperture of material and space geometry structure help the growth expansion of cell.Several cartilage tissue engineered solid support material commonly used at present, for example poly(lactic acid) and polyglycolic acid, acid product after the degradation in vivo can influence the growth of tissue and cell, natural biomaterial such as collagen, decalcification bone, fibrin gel etc., because the biological characteristics of itself, increased interfering factors, in the histology of newborn cartilage and biochemical identification, be difficult to obtain correct evaluation (referring to defending late autumn etc., the 3rd the academic meeting of national organizational project and first Chinese international organization engineering conference paper compilation: 144-146).
Summary of the invention
The technical issues that need to address of the present invention provide the method for a kind of 3 D stereo cultivation and inducing bone mesenchymal stem cell chondroblast, to overcome a series of problems that prior art exists, satisfy the needs of scientific research and the development of relevant field.
Technical conceive of the present invention is such:
The present invention induces the chondroblast of mesenchymal stem cells MSCs with the microcarrier culture technique and combines, the chondroblast of both can having increased is in a large number fast induced needed seed cell, and be the three-dimensional environment that cytoskeleton is built into chondrocyte induction with the microcarrier, reduced damage and degeneration that mesenchymal stem cells MSCs may be subjected in various operation steps.
Method of the present invention comprises the steps:
(1) with mesenchymal stem cells MSCs suspension culture in presetting the suspension culture device of substratum and microcarrier, mesenchymal stem cells MSCs increases under the stirring suspension state;
Said suspension culture device comprises rolling bottle or bio-reactor;
Said substratum is the α-MEM that contains 2%～20% volume percent foetal calf serum, and α-MEM forms for disclosed substratum can adopt the commercially available prod, as the product of Gibco or Sigma company;
Said microcarrier is Cytodex 3 microcarriers, and microcarrier content is benchmark with the microcarrier quality that is contained in the unit volume nutrient solution, is 1～10mg/ml, and preferred content is 3mg/ml;
Culture environment is 37 ℃, 5%CO
2, saturated humidity, incubation time is 3～15 days, amount was changed substratum in per 1～3 day half, mixing speed is 10～80rpm.
(2) then the chondroblast inducing culture is added in the culture apparatus of the mesenchymal stem cells MSCs that contains Cytodex 3 microcarriers of results and grow thereon, mesenchymal stem cells MSCs is gathered a group chondroblast induce;
The method of mesenchymal stem cells MSCs of wherein gathering in the crops Cytodex 3 microcarriers and growth thereon is all nutrient solutions in the sucking-off culture apparatus under the aseptic condition, the chondroblast inducing culture is based on DMEM, adding concentration is the Regular Insulin of 1～10 μ g/ml, the transforming growth factor-beta 3 of 1～20ng/ml, the dexamethasone of 10～150nmol/L, the ascorbic acid phosphoric acid esters of 10～100mg/ml, the proline(Pro) of 10～100mg/ml, 0.5 the Sodium.alpha.-ketopropionate of～5mmol/L, the DMEM substratum is formed for disclosed substratum, can adopt the commercially available prod, as the product of Gibco or Sigma company;
Poly-the chondroblast of being addressed induced to refer to the chondroblast inducing culture slowly added in the culture apparatus, and microcarrier and cell keep the poly-bulk attitude of late stage of culture, and the inducing culture environment is 37 ℃, 5%CO
2, saturated humidity, the inducing culture time is 14～21 days, amount was changed inducing culture in per 3 days half.
(1) microcarrier of mesenchymal stem cells MSCs is cultivated
Prepare microcarrier: a certain amount of Cytodex 3 (Pharmacia) microcarrier is put into culture apparatus, soak into 2 times, each 30 minutes, be immersed in the phosphate buffered saline buffer autoclaving (120 ℃) 30 minutes at last with phosphate buffer soln.Sucking-off phosphate buffered saline buffer under aseptic condition adds the α-MEM nutrient solution that contains 2%～20% volume percent foetal calf serum, and stirring is spent the night;
Prepare mesenchymal stem cells MSCs: with the mesenchymal stem cells MSCs seed cell cultivated with after containing 0.02% ethylenediamine tetraacetic acid (EDTA) and 0.25% tryptic Digestive system and digesting, change in the culture apparatus that contains Cytodex 3, the content of adjusting Cytodex 3 is 1～10mg/ml;
The microcarrier of mesenchymal stem cells MSCs is cultivated: after mesenchymal stem cells MSCs inserted in the culture apparatus that contains Cytodex 3, the adjustment mixing speed was 10～80rpm, in 37,5%CO
2The guarantor and humidity under cultivate, incubation time is 3～15 days, amount was changed nutrient solution in per 1～3 day half.
(2) results mesenchymal stem cells MSCs microcarrier culture
With the careful sucking-off of the nutrient solution in the culture apparatus, and the poly-bulk attitude of maintenance culture.
(3) induce based on the chondroblast of microcarrier culture
Preparation chondrocyte inducing culture: in the DMEM substratum, add 1～10 μ g/ml Regular Insulin, 1～20ng/ml transforming growth factor-beta
3, 10～150nmol/L dexamethasone, 10～100mg/ml ascorbic acid phosphoric acid esters, 10～100mg/ml proline(Pro), 0.5～5mmol/L Sodium.alpha.-ketopropionate;
Chondroblast is induced: the chondroblast inducing culture for preparing slowly added in the culture apparatus, and inducing culture 14～28 days, amount was changed inducing culture liquid in per 3 days half.After finishing, cultivation takes out the inducing culture product, but the fractional analysis of progressive type collagen immunization group, and it is damaged or provide competent seed cell source for scientific research to be used in the body transplantation treatment cartilaginous tissue.
Carry out the mesenchymal stem cells MSCs amplification according to the inventive method, can once obtain a large amount of cells at short notice, reduced in the conventional static cultivation and lost, reduced the probability that pollutes, reduced cell injury owing to passage number increases the multidirectional differentiation potential of cell that causes.The one-tenth chondrocyte induction of mesenchymal stem cells MSCs needs the high-density micelle to cultivate, after the nutrient solution sucking-off in the device, the poly-bulk attitude that keeps culture, slowly add inducing culture liquid, can very realize this purpose easily, and the mesenchymal stem cells MSCs form and the function of growing are good on microcarrier.The differentiation of cell depends on the nutritive substance concentration in the environment and the interaction of iuntercellular excretory cytokine in the chondroblast process, with general high-density micelle induction phase ratio, therefore this three-dimensional inducing culture more helps the transmission of iuntercellular nutritive substance owing to have certain hole between the poly-group of stirring and microcarrier culture.In the cell growth later stage, microcarrier can clustering phenomena occur owing to the raising of cell density, this is favourable for becoming chondrocyte induction, it is this that the method for formation cell-biomaterial composites is simple naturally by the cell bridge formation, omitted the conventional step that seeds cells on the timbering material, reduced owing to the not high loss cell that causes of rate of vaccination, and Cytodex 3 microcarriers of Shi Yonging are made by the dextran material herein, outer bag is by covalently bound sex change inactivation collagen, have excellent biological compatibility, induce directly this mixture to be implanted into after finishing and carry out the cartilage defect reparation in the body or provide competent seed cell source for scientific research.
Description of drawings
Fig. 1 shows the primary growth situation of mesenchymal stem cells MSCs on microcarrier.
Fig. 2 shows the clustering phenomena of growing mesenchymal stem cells MSCs on microcarrier the later stage beginning to occur.
Embodiment
Embodiment 1
150mg Cytodex 3 microcarriers are put into rolling bottle, soak into 2 times with phosphate buffer soln, each 30 minutes, be immersed at last in the phosphate buffered saline buffer, put into 120 ℃ of sterilizations of high-pressure sterilizing pot 30 minutes.Rolling bottle takes out back sucking-off phosphate buffered saline buffer in Bechtop, adds α-MEM nutrient solution that 30ml contains 10% volume percent foetal calf serum, and rolling bottle is put into to stir on the Rotary Machine and spent the night.With the mesenchymal stem cells MSCs seed cell cultivated with after containing 0.02% ethylenediamine tetraacetic acid (EDTA) and 0.25% tryptic Digestive system and digesting, change in the rolling bottle that contains Cytodex 3, the content of adjusting Cytodex 3 is 3mg/ml, and mixing speed is 50rpm, in 37 ℃, 5%CO
2, cultivate under the saturated humidity, incubation time is 4 days, every day half, amount was changed nutrient solution.After finishing, cultivation, keeps poly-culture in the rolling bottle with the slow sucking-off of the nutrient solution in the rolling bottle.(the DMEM substratum adds 6.25 μ g/ml Regular Insulin, 10ng/ml transforming growth factor-beta to the one-tenth chondrocyte induction substratum that will prepare then
3, 100nmol/L dexamethasone, 50mg/ml ascorbic acid phosphoric acid esters, 40mg/ml proline(Pro), 1mmol/L Sodium.alpha.-ketopropionate) slowly add in the rolling bottle, in case break up the microcarrier and the cell of poly-group, the adjustment mixing speed is 20rpm, in 37 ℃, 5%CO
2, inducing culture 14～28 days under the saturated humidity, amount was changed inducing culture liquid in per 3 days half.Induce and take out the inducing culture product after finishing, it is damaged to be directly used in the body transplantation treatment cartilaginous tissue, or is applied to the other field studying and use.
Claims (9)
1. a 3 D stereo is cultivated and the method for inducing bone mesenchymal stem cell chondroblast, it is characterized in that, comprises the steps:
(1) with mesenchymal stem cells MSCs suspension culture in presetting the suspension culture device of substratum and microcarrier, mesenchymal stem cells MSCs under agitation increases;
Said substratum is the α-MEM that contains 2%～20% volume percent foetal calf serum;
Said microcarrier is Cytodex 3 microcarriers;
(2) then the chondroblast inducing culture is added in the culture apparatus of the mesenchymal stem cells MSCs that contains Cytodex 3 microcarriers of results and grow thereon, mesenchymal stem cells MSCs is gathered a group chondroblast induce;
The chondroblast inducing culture is based on DMEM, and adding concentration is the Regular Insulin of 1～10 μ g/ml, the transforming growth factor-beta 3 of 1～20ng/ml, the dexamethasone of 10～150nmol/L, the ascorbic acid phosphoric acid esters of 10～100mg/ml, the proline(Pro) of 10～100mg/ml, the Sodium.alpha.-ketopropionate of 0.5～5mmol/L.
2. method according to claim 1 is characterized in that, said suspension culture device comprises rolling bottle or bio-reactor.
3. method according to claim 1 is characterized in that microcarrier content is benchmark with the microcarrier quality that is contained in the unit volume nutrient solution, is 1-～10mg/ml.
4. method according to claim 3 is characterized in that, microcarrier content is 3mg/ml.
5. method according to claim 1 is characterized in that, culture environment is 37 ℃, 5%CO
2, saturated humidity, incubation time is 3-～15 day, every 1-～3 day half amount is changed substratum.
6. method according to claim 1 is characterized in that, mixing speed is 10-～80rpm.
7. method according to claim 1 is characterized in that, the method for mesenchymal stem cells MSCs of wherein gathering in the crops Cytodex 3 microcarriers and growth thereon is all nutrient solutions in the sucking-off culture apparatus under the aseptic condition.
8. method according to claim 1 is characterized in that, poly-the chondroblast of being addressed induced to refer to the chondroblast inducing culture is slowly added in the culture apparatus, and microcarrier and cell keep the poly-bulk attitude of late stage of culture.
9. according to each described method of claim 1～8, it is characterized in that the inducing culture environment is 37 ℃, 5%CO
2, saturated humidity, the inducing culture time is 14-～21 day, amount was changed inducing culture in per 3 days half.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477700B (en) * | 2009-02-06 | 2011-04-27 | 南京师范大学 | Real tri-dimension display method oriented to Google Earth and Sketch Up |
| CN102985534A (en) * | 2010-07-22 | 2013-03-20 | 吉林大学 | Culture method for amplifying large numbers of hair follicle stem cells in vitro |
| CN103146645A (en) * | 2013-03-14 | 2013-06-12 | 深圳市博泰生物医学科技发展有限公司 | Method for inducing mesenchymal stem cells (MSCs) into chondrocytes |
| CN103361308A (en) * | 2012-03-30 | 2013-10-23 | 华东理工大学 | Method for amplifying mesenchymal stem cells on large scale |
| CN103849596A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Large-scale production process of uterine membrane stem cells |
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
| CN105695402A (en) * | 2016-04-14 | 2016-06-22 | 安沂华 | Composition and method for inducing mesenchymal stem cells to be differentiated to cartilage cells |
| CN106414722A (en) * | 2014-04-07 | 2017-02-15 | 汉阳大学校产学协力团 | In-vitro expansion of erythroid cells |
| CN111206015A (en) * | 2020-04-21 | 2020-05-29 | 广东省生物资源应用研究所 | Three-dimensional dynamic culture method for amplifying spermatogonial stem cells in vitro by using FACT III microcarriers |
| CN115089614A (en) * | 2022-06-28 | 2022-09-23 | 中国人民解放军军事科学院军事医学研究院 | Method for enhancing performance of skeletal stem cells and application of method in treatment of osteoarthritis |
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- 2004-04-09 CN CNB2004100175888A patent/CN100529062C/en not_active Expired - Fee Related
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101477700B (en) * | 2009-02-06 | 2011-04-27 | 南京师范大学 | Real tri-dimension display method oriented to Google Earth and Sketch Up |
| CN102985534A (en) * | 2010-07-22 | 2013-03-20 | 吉林大学 | Culture method for amplifying large numbers of hair follicle stem cells in vitro |
| CN103361308B (en) * | 2012-03-30 | 2016-02-10 | 华东理工大学 | A kind of method of mass-producing amplification mesenchymal stem cells MSCs |
| CN103361308A (en) * | 2012-03-30 | 2013-10-23 | 华东理工大学 | Method for amplifying mesenchymal stem cells on large scale |
| CN103849596A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Large-scale production process of uterine membrane stem cells |
| CN103146645B (en) * | 2013-03-14 | 2015-04-15 | 深圳市博泰生物医学科技发展有限公司 | Method for inducing mesenchymal stem cells (MSCs) into chondrocytes |
| CN103146645A (en) * | 2013-03-14 | 2013-06-12 | 深圳市博泰生物医学科技发展有限公司 | Method for inducing mesenchymal stem cells (MSCs) into chondrocytes |
| CN106414722A (en) * | 2014-04-07 | 2017-02-15 | 汉阳大学校产学协力团 | In-vitro expansion of erythroid cells |
| CN106414722B (en) * | 2014-04-07 | 2020-01-03 | 汉阳大学校产学协力团 | In vitro expansion of erythroid cells |
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
| CN104096266B (en) * | 2014-07-25 | 2015-12-02 | 中国人民解放军第三军医大学 | Based on tissue engineered bone and the construction process thereof of entochondrostosis system |
| CN105695402A (en) * | 2016-04-14 | 2016-06-22 | 安沂华 | Composition and method for inducing mesenchymal stem cells to be differentiated to cartilage cells |
| CN111206015A (en) * | 2020-04-21 | 2020-05-29 | 广东省生物资源应用研究所 | Three-dimensional dynamic culture method for amplifying spermatogonial stem cells in vitro by using FACT III microcarriers |
| CN115089614A (en) * | 2022-06-28 | 2022-09-23 | 中国人民解放军军事科学院军事医学研究院 | Method for enhancing performance of skeletal stem cells and application of method in treatment of osteoarthritis |
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