CN102899287A - Method for inducing differentiation from mesenchymal stem cells to cartilage cells and application of mesenchymal stem cells in osteoarthritis - Google Patents
Method for inducing differentiation from mesenchymal stem cells to cartilage cells and application of mesenchymal stem cells in osteoarthritis Download PDFInfo
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Abstract
The invention relates to a method for inducing differentiation from mesenchymal stem cells to cartilage cells and application of the mesenchymal stem cells in osteoarthritis, and provides a method for preparing umbilical cord mesenchymal stem cells differentiated to the cartilage cells, mesenchymal stem cells differentiated to the cartilage cells and application of the prepared drug prepared by the mesenchymal stem cells in treatment of the osteoarthritis. The method comprises the following steps of: inducing and culturing the mesenchymal stem cell in a 15ml taper-bottom centrifuge tube by using a globule method; and slightly shaking the centrifuge tube for one time every 24 hours to separate cell sphere from the bottom of the tube, wherein the differentiation inducing culture medium is prepared by adding 100mu g/ml of sodium pyruvate, 10ng/ml TGFbeta3, 100nM of dexamethasone, 25mu g/ml of vitamin C, 40mu g/ml of proline and 1*ITS and 1 of premix into a high-glucose dulbecco modified eagle medium (DMEM). A non-bracket cartilage built by the method can be used for repairing damaged articular cartilages.
Description
Technical field
The present invention relates to the stem cells technology field, specifically, relate to the preparation method of the UC-MSCs of chondrocyte induction and the application in osteoarthritis thereof.
Background technology
Joint cartilage is a kind of heavy burden reticular tissue, often because of tumour, and motion, degeneration or geriatric disease injury; Yet joint cartilage self repair ability is limited, has caused very large difficulty for the clinical cure cartilage defect.The method that multiple treatment cartilage defect occurred in recent years comprises from body chondrocyte cell transplantation, microfracture and inlays plasty, but these methods have its limitation separately.In recent years, tissue engineering bone/cartilage becomes the new focus of repair of cartilage research, and mescenchymal stem cell (Mesenchymal stem cells, MSCs) is its most promising current seed cell.MSCs derives to grow early stage mesoderm and an ectodermic class multipotential stem cell, finds in marrow at first.At present the main source of MSCs is Adult Human Bone Marrow, but Adult Human Bone Marrow source MSCs cell quantity and proliferation and differentiation potential descends with the increase at age, and viral infection rate is higher, and the collection of donor MSCs must the row bone marrow puncture, and the source is restricted.Research finds that MSCs Various Tissues after human fetal and birth extensively exists, and comprises periosteum, fat, amniotic fluid, corium, tooth, skeletal muscle, tire lung, tire liver and bleeding of the umbilicus etc.Under the subsidy of previous National Nature fund " umbilical cord mesenchymal stem cells biological nature, hematopoiesis support research and the application in transplanting " (30570905), the collagenase of our application enhancements and the direct digestion method of enzyme that pancreatin combines, by the magnetic bead screening, from umbilical cord, obtained a large amount of MSCs.And set up a kind of method that can separate amplification umbilical cord MSCs, and operation is simple (ZL200910242375.8).According to present method, 90% mature umbilical cord can be isolated MSCs, obtains 1 * 10
9The MSCs cell fully satisfies the needs of Clinical and experimental study.
Prior art is not still with the trial of Mesenchymal Stem Cells from Umbilical Cord to the chondrocyte induction differentiation, we are with the seed cell of Mesenchymal Stem Cells from Umbilical Cord as us, it is induced to differentiate into without the support cartilage, and its ability of repairing impaired joint cartilage is studied, have important Clinical significance of MG.
Summary of the invention
The present invention provides the method for a kind of inducing mesenchymal stem cell to Chondrocyte Differentiation in first aspect, wherein, in the 15ml conical centrifuge tube, use bead method inducing culture mescenchymal stem cell, and rocked gently centrifuge tube once every about 24 hours, cell ball was separated with the pipe end.
In a specific embodiment, the inductive differentiation medium that uses adds 100 μ g/ml Sodium.alpha.-ketopropionates as DMEM in high glucose, 10 ng/ml TGF β 3,100 nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1 premix; Inducing the differentiation culture time is 21 days.
Preferably, described mescenchymal stem cell is Mesenchymal Stem Cells from Umbilical Cord.
Aspect second of the present invention, provide the mescenchymal stem cell of prepared according to the methods of the invention to Chondrocyte Differentiation.
Preferably, described mescenchymal stem cell is Mesenchymal Stem Cells from Umbilical Cord.
Aspect the 3rd of the present invention, provide the application of mescenchymal stem cell in the medicine of preparation treatment osteoarthritis to Chondrocyte Differentiation.
Preferably, described mescenchymal stem cell is Mesenchymal Stem Cells from Umbilical Cord.
The present invention has following advantage:
1. adopt bead method inducing culture mescenchymal stem cell, can make without the support cartilage, rock gently centrifuge tube once every about 24 hours, cell ball was separated with the pipe end, guaranteed that cell ball fully contacts with inductive differentiation medium, the Derived from Mesenchymal Stem Cells state homogeneous in the cell ball.
2. use TGF β 3 to replace TGF β 1 commonly used in the inductive differentiation medium, reached and better induced effect.
3. Mesenchymal Stem Cells from Umbilical Cord is easy with respect to the bone marrow mescenchymal stem cell separation preparation of routine, and the source is abundant.
Description of drawings
Fig. 1 is to the Toluidine blue staining result of the UC-MSCs bead of chondrocyte induction differentiation after 21 days, and red-purple proves the chondrocyte.
HE coloration result after the UC-MSCs cell suspension treatment of Fig. 2 rat bone arthritis model warp-wise chondrocyte induction.
The A Normal group; The B model group; C joint cavity inner cell suspension treatment group 1; D joint cavity inner cell suspension treatment group 2; E tail vein injection cell suspension treatment group 3; F tail vein injection cell suspension treatment group 4.
Embodiment
Embodiment 1 Mesenchymal Stem Cells from Umbilical Cord is induced to the chondrocyte
1. MSC growth medium individual layer amplification Mesenchymal Stem Cells from Umbilical Cord:
The MSC growth medium (adds 13.5ml DF12 in per 75 square centimeters of culturing bottles, 10% human serum, 20 ng/ml EGF, 5ng/ml FGF2) cultivates P3 for Mesenchymal Stem Cells from Umbilical Cord (UC-MSCs), Growth of Cells reaches 70-80% and merges, after using D-Hank ' s washing lotion to wash twice, adding 1.5ml pancreas enzyme-EDTA (0.25% pancreatin-0.02% EDTA) digests, microscopically is observed, and when cell presents the ball state, stops digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, get 200 microlitres counting and determine cell quantity, all the other cell suspensions are centrifugal, 1000rpm, 10min.Outwell the supernatant after centrifugal, go down to posterity with the ratio of 1:3, be cultured to P4 generation, Growth of Cells reaches 70-80% and merges, use D-Hank ' s washing lotion to wash twice after, adding 1.5ml pancreas enzyme-EDTA digests, microscopically is observed, when cell presents the ball state, stop digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, centrifugal, 1000rpm, 10min.Outwell the supernatant after centrifugal, add 30ml D-hank ' s washing lotion, recentrifuge behind the mixing is outwelled supernatant, again adds 30ml D-hank ' s washing lotion, behind the mixing, takes out 200 microlitres counting, and all the other cell suspensions are centrifugal, 1000rpm, 10min.Remove supernatant after centrifugal.
2. MSC growth medium re-suspended cell is adjusted cell concn to 1 * 10
6/ ml cell.
3. Pellet(bead method) culturing cell: 1ml is contained 1 * 10
6The cell suspension of individual cell adds in the 15ml conical centrifuge tube, and centrifugal (240g, 5 minutes) put into 37 ℃ with centrifuge tube, 5% CO
2In the cell culture incubator, after about 24 hours, rock gently centrifuge tube, cell ball was separated with the pipe end, (DMEM in high glucose adds 100 μ g/ml Sodium.alpha.-ketopropionates to change afterwards the adding inductive differentiation medium, 10 ng/ml TGF β, 3,100 nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1 premix (10 μ g/ml Insulin, 5.5 μ g/ml transferrin, 5ng/ml selenium, 5.35 μ g/ml linoleic acid), rocked gently centrifuge tube once every 24 hours, cell ball is separated with tube wall, cartilage differentiation substratum of replacing in per two days.After 21 days, take out bead, Toluidine blue staining, as shown in Figure 1, red-purple proves the chondrocyte.
Embodiment 2 is to the application of UC-MSCs in osteoarthritis of chondrocyte induction
With obtain among the embodiment 1 to the cell ball of chondrocyte induction in centrifuge tube after the II Collagenase Type digestion half an hour with 3ml 2% (w/v), 1000rpm, after 5min was centrifugal, being inoculated in floorage after usefulness inductive differentiation medium similarly to Example 1 is resuspended was 25 cm
2Culturing bottle in amplification cultivation, cover with afterwards with the digestion of 0.5ml pancreas enzyme-EDTA, make the UC-MSCs cell suspension to chondrocyte induction.
Select healthy male Wistar rat, body weight 200 ± 50 g, 60.Be divided at random the ⑴ intraarticular injection to the UC-MSCs cell suspension treatment group 1 of chondrocyte induction; ⑵ intraarticular injection is to the UC-MSCs cell suspension treatment group 2 of chondrocyte induction; ⑶ tail vein injection is to the UC-MSCs cell suspension treatment group 3 of chondrocyte induction; ⑷ tail vein injection is to the UC-MSCs cell suspension treatment group 4 of chondrocyte induction; ⑸ control group (intraarticular injection cell suspension solvent); ⑹ model group; ⑺ normal group, wherein front four groups every group 10, rear three groups every group 6.Modeling method is the Chloral Hydrate by 0.3 ml/100 g abdominal injection, 10 %, and after the anesthesia, the rats with bilateral knee joint is injected 4 % papoid poloxamer phosphate buffer solutions, and lower limb are placed 2h in 50 ℃ of self-control baking boxs, namely finishes modeling after the end.The 1st week after modeling is finished was 0 week, respectively at 0,2,4 weeks injection cell suspension.Intraarticular injection group 1 and tail vein injection group 3 every injection 100 μ l 4 * 10
7Individual/ml, intraarticular injection group 2 and tail vein injection group 4 every injection 100 μ l 2 * 10
7Individual/ml, control group is respectively at 0,2,4 weeks injection, 100 μ l cell suspension solvents.All treated animals were put to death in the 6th week, drew materials.
After treating for 6 weeks, anaesthetize by the Chloral Hydrate of 0.3 ml/100 g body weight abdominal injection, 10 %.Abdominal aortic blood, centrifugal 10 minutes of 3000 r/min, separation of serum, 4 ℃ of preservations, for subsequent use.
Pathologic sampling: take whole right knee joint, remove surrounding skin, muscle, 10 % formalin are fixed, the routine paraffin wax embedding, and HE dyeing, the histopathology of observing the joint under the light microscopic changes, as shown in Figure 2.The A Normal group, diagram normal cartilage structure, cartilage surface is more smooth, and continuity is good, and top layer, the layer of dividing a word with a hyphen at the end of a line, radiating layer, 4 layers of clear in structure of calcification layer can be distinguished, chondrocyte's marshalling, the matrix even dyeing, damp line is complete; The B model group, articular cartilage surface is coarse, chondrocyte and the arrangement disorder of visible dehydration pyknosis necrosis around the visible more crack, top layer, crack, each layer structure is difficult for differentiating; C joint cavity inner cell suspension treatment group 1, articular cartilage surface is smooth than model group, and reduce in the crack, top layer, but the chondrocyte fills the air and increases, and damp line owes complete; D joint cavity inner cell suspension treatment group 2, coloration of substrates shoals, and dyeing is owed evenly, and recovery situation is weaker than joint cavity inner cell suspension treatment group 1; E tail vein injection cell suspension treatment group 3, the joint cartilage structure develops to normal cartilage, but matrix dyeing still owes even; F tail vein injection cell suspension treatment group 4, articular chondrocytes is downright bad, and recovery situation is inferior to tail vein injection cell suspension treatment group 3.
Through above-mentioned to the joint cartilage PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM, the joint cartilage for the treatment of group than model group be improved significantly, and the cartilage injury most pronounced effects of high dosage tail vein injection cell suspension treatment osteoarthritis, to sum up, the effect that has the impaired joint cartilage of reparation to a certain degree to the UC-MSCs cell of chondrocyte induction.
Claims (8)
1. an inducing mesenchymal stem cell wherein, uses bead method inducing culture mescenchymal stem cell, and rocked gently centrifuge tube once every about 24 hours to the method for Chondrocyte Differentiation in the 15ml conical centrifuge tube, and cell ball was separated with the pipe end.
2. method claimed in claim 1, wherein, the inductive differentiation medium that uses adds 100 μ g/ml Sodium.alpha.-ketopropionates as DMEM in high glucose, 10ng/ml TGF β 3,100nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1premix.
3. claim 1 or 2 described methods, wherein, inducing the differentiation culture time is 21 days.
4. claim 1 or 2 described methods, wherein, described mescenchymal stem cell is Mesenchymal Stem Cells from Umbilical Cord.
5. method claimed in claim 4, its step comprises:
1) at each 75cm
2Add 13.5ml growth of mesenchymal stem cells culture medium culturing P3 in the culturing bottle for Mesenchymal Stem Cells from Umbilical Cord, Growth of Cells reaches 70-80% and merges, use D-Hank ' s washing lotion to wash twice after, adding the 1.5ml pancreas enzyme-EDTA digests, microscopically is observed, and when cell presents the ball state, stops digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, get 200 microlitres counting and determine cell quantity, all the other cell suspensions are centrifugal, 1000rpm, 10min; Outwell the supernatant after centrifugal, go down to posterity with the ratio of 1:3, be cultured to P4 generation, Growth of Cells reaches 70-80% and merges, use D-Hank ' s washing lotion to wash twice after, adding 1.5ml pancreas enzyme-EDTA digests, microscopically is observed, when cell presents the ball state, stop digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, centrifugal, 1000rpm, 10min; Outwell the supernatant after centrifugal, add 30ml D-hank ' s washing lotion, recentrifuge behind the mixing is outwelled supernatant, again adds 30ml D-hank ' s washing lotion, behind the mixing, takes out 200 microlitres counting, and all the other cell suspensions are centrifugal, 1000rpm, 10min; Remove supernatant after centrifugal;
Wherein, described growth of mesenchymal stem cells substratum is that DMEM/F12 adds 10% human serum, 20ng/ml EGF, 5ng/ml FGF2;
2) with growth of mesenchymal stem cells substratum re-suspended cell, adjust cell concn to 1 * 10
6Cell/ml;
3) bead method inducing culture cell: 1ml is contained 1 * 10
6The cell suspension of individual cell adds in the 15ml conical centrifuge tube, and centrifugal 5 minutes of 240g puts into 37 ℃ with centrifuge tube, 5%CO
2In the cell culture incubator, after about 24 hours, rock gently centrifuge tube, cell ball was separated with the pipe end, change afterwards the adding inductive differentiation medium; Rocked gently centrifuge tube once every 24 hours, cell ball is separated, inductive differentiation medium of replacing in per two days with the pipe end; After 21 days, take out bead;
Wherein, described inductive differentiation medium is that DMEM in high glucose adds 100 μ g/ml Sodium.alpha.-ketopropionates, 10ng/mlTGF β 3,100nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1premix.
6. the mescenchymal stem cell to Chondrocyte Differentiation of each method preparation according to claim 1-5.
7. the mescenchymal stem cell to Chondrocyte Differentiation claimed in claim 6, wherein said mescenchymal stem cell is Mesenchymal Stem Cells from Umbilical Cord.
8. claim 6 or the 7 described application of mescenchymal stem cell in the medicine of preparation treatment osteoarthritis to Chondrocyte Differentiation.
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| 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 |
| 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 |
| CN104771413A (en) * | 2014-12-09 | 2015-07-15 | 周治宇 | Application of arthroscopic flushing fluid sourced mesenchymal stem cells in regenerative repair |
| CN104958320A (en) * | 2015-07-29 | 2015-10-07 | 西安芙金细胞科技有限公司 | Cell preparation for treating osteoarthritis and preparation method thereof |
| CN104983742A (en) * | 2015-04-22 | 2015-10-21 | 南京康雅生物科技有限公司 | Stem cell preparation for treating degenerative osteoarthropathy and preparation method of stem cell preparation |
| CN104988117A (en) * | 2015-07-03 | 2015-10-21 | 深圳中基恒润投资有限公司 | Method for separating and culturing mesenchymal stem cells from umbilical cord and inducing and differentiating mesenchymal stem cells into cartilage cells |
| 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 |
| CN106635968A (en) * | 2016-10-14 | 2017-05-10 | 中卫华医（北京）生物科技有限公司 | Method of inducing and differentiating human umbilical cord derived mesenchymal stem cells into cartilage cells |
| CN106834223A (en) * | 2017-04-05 | 2017-06-13 | 上海逍鹏生物科技有限公司 | Method from inducing umbilical cord mesenchymal stem to Chondrocyte Differentiation |
| CN107338218A (en) * | 2017-07-28 | 2017-11-10 | 中国人民解放军总医院第附属医院 | Derivant and method of a kind of induced lipolysis stem cell to Chondrocyte Differentiation |
| CN109456939A (en) * | 2018-11-23 | 2019-03-12 | 北京太东生物科技有限公司 | Culture medium used in cultural method and this method of the inducing umbilical cord mesenchymal stem at cartilage differentiation |
| CN109825469A (en) * | 2019-03-09 | 2019-05-31 | 和携科技(北京)有限公司 | A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation |
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| 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 |
| CN106635968A (en) * | 2016-10-14 | 2017-05-10 | 中卫华医（北京）生物科技有限公司 | Method of inducing and differentiating human umbilical cord derived mesenchymal stem cells into cartilage cells |
| CN106834223A (en) * | 2017-04-05 | 2017-06-13 | 上海逍鹏生物科技有限公司 | Method from inducing umbilical cord mesenchymal stem to Chondrocyte Differentiation |
| CN106834223B (en) * | 2017-04-05 | 2020-03-27 | 上海逍鹏生物科技有限公司 | Method for inducing differentiation of umbilical cord mesenchymal stem cells into chondrocytes |
| CN107338218A (en) * | 2017-07-28 | 2017-11-10 | 中国人民解放军总医院第附属医院 | Derivant and method of a kind of induced lipolysis stem cell to Chondrocyte Differentiation |
| CN109456939A (en) * | 2018-11-23 | 2019-03-12 | 北京太东生物科技有限公司 | Culture medium used in cultural method and this method of the inducing umbilical cord mesenchymal stem at cartilage differentiation |
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| CN110747165A (en) * | 2019-11-19 | 2020-02-04 | 山东省齐鲁细胞治疗工程技术有限公司 | Preparation method and application of mesenchymal stem cell scaffold-free three-dimensional gel |
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