CN101407786A - Method for cultivating stereo proliferative cartilage cell - Google Patents
Method for cultivating stereo proliferative cartilage cell Download PDFInfo
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- CN101407786A CN101407786A CNA2008101984657A CN200810198465A CN101407786A CN 101407786 A CN101407786 A CN 101407786A CN A2008101984657 A CNA2008101984657 A CN A2008101984657A CN 200810198465 A CN200810198465 A CN 200810198465A CN 101407786 A CN101407786 A CN 101407786A
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Abstract
The invention discloses a culturing method for solidly proliferating chondrocyte, which includes the following steps: a single-layer cultured chondrocyte is firstly digested and then added into a sodium alginate-sodium chloride solution for mixing; then a suction nozzle is used for dropping the obtained mixture liquid into a calcium chloride solution for becoming gel; finally the obtained gel is washed by the sodium chloride solution and a complete medium; and then the gel is cultured on a compound DMEM/HamF12 culture medium. The compound DMEM/HamF12 culture medium includes 10ng/ml of basic fibroblast growth factors, 5Mu g/ml of hyaluronic acid, 0.2 Mu g/ml of fibronectin, 50 Mu g/ml of soluble chitosan and 1:1 of DMEM/HamF12. The culturing method can improve the number and the quality of the cultured cells in a short time, quickly reproduce the chondrocyte and can culture and preserve the cell phenotypes in a long period.
Description
Technical field
The present invention relates to the cellular biological technique field, especially relate to a kind of cultural method of stereo proliferative cartilage cell.
Background technology
The source of chondrocyte's tissue engineering seed cell is restricted, external monolayer adherence is prone to when cultivating and dedifferentes, the potential of anti-differentiation will fade away, make the chondrocyte change form, forfeiture phenotype and Expression of Related Genes gradually, can not produce distinctive glycosaminoglycan of cartilage and II Collagen Type VI, and producing I type and III Collagen Type VI, the main fibrous cartilage that forms in the back that implants can not be kept biomechanical property.
The reparation of articular cartilage damage is relevant with the mode of its weave construction characteristics and damage, persistence of repairing and good heavy burden articulum keep the quality that then depends on callus, but in fact repair normal imperfection, make the articulum deuterogenesis degenerative change of damage.
The healing of showing shallow damage depends on the Metabolic activity of articular chondrocytes, but it accurately rebuilds the limited in one's ability of structure, even articular chondrocytes can synthetic proteins polysaccharide and tropocollagen molecule, and can discharge epimatrix, these new synthetic molecules formation complex compound that do not mutually combine, therefore the structure that makes up is also imperfect, and the function of callus is also relatively poor.Behind the articular cartilage defect, the chondrocyte is because the blood vessel at its position is less, is difficult to regeneration or repairs very slowly, limited the ability of its reparation, and the later stage is easily caused degenerative osteoarthritis.
Organizational project provides new thinking and approach for articular cartilage defect reparation clinical.Cartilage tissue engineered basic line is at the vitro culture seed cell, and it is planted on the suitable holder with excellent biological compatibility degradation property with higher concentration, thereby form the cell-scaffold mixture, and mixture implantable bioartificial in-vivo tissue defect finally finished the reparation of tissue and reproduce.But seed cell, suitable holder material and suitable vitro culture system also wait clearly.
External chondrocyte's monolayer culture was prone to and dedifferentes to 5 generations, and form, extracellular matrix secreting function descend and phenotypic alternation, are difficult to long-term cultivation.At present the main method that solves is that bone marrow stroma stem cell (BMSCs) is induced chondroblast, short period of time fast breeding and cultivated at the suitable carriers neutral body.
Found that phenotype of chondrocytes can keep the long period in the Lalgine pearl in 1998, and the content of DNA, protein-polysaccharide (PG) keeps higher in 70 days.Transforming growth factor-1 (TGF-1) is added in chondrocyte's the Lalgine pearl, m-RNA is expressed increases by 400%, and precollagen genetic expression raises 180%.Lindenhayn found simple with sodium alginate pearl cultivation chondrocyte in 1999, its cell count has slightly and increases, and adds hyaluronic acid in sodium alginate, and its cell count can increase by 100% in 3 days, and on this basis, adding fibronectin, its cell count just can increase by 100% in 1 day.The more Lalgine pearl of professors such as Chubinskaya research in 2000 is used to preserve chondrocyte's survival and can reaches 8 months, the same year, the simple Lalgine of usefulness such as foreign scholar Xu Hu prepared gel, visible new capillary vessel and cartilage cavities form when being used in the animal body for 4 weeks with Protanal TXF 200/chondrocyte's mixture, illustrate the chondrocyte be compound in Protanal TXF 200 can be in the allosome animal body growing multiplication.Also have the scholar with the Protanal TXF 200 be carrier as the injectable cartilage, be used for cartilage tissue engineered research.But the amount that cell is bred in carrier not enough.
Phenotype of chondrocytes can keep the long period in simple Lalgine pearl, but the quantity of propagation is not enough, the simple TGF-1 somatomedin that adds, short period of time is used easy inactivation, long-time use easily brings still undetermined tumorigenesis chance, though the method simple possible of Lindenhayn, cell number propagation is very fast, but the cell carrier support lacks microkinetic, and suitable adjusting can make it more near the cells in vivo microenvironment.
Summary of the invention
Main purpose of the present invention is in order to solve the problems of the technologies described above, to reach short-term fast breeding chondrocyte, and long-term cultivation can preserve the purpose of cell phenotype, the invention provides a kind of cultural method of stereo proliferative cartilage cell.
In order to realize this purpose, adopt following technical scheme:
A kind of cultural method of stereo proliferative cartilage cell comprises the steps:
(1) single-layer cultured chondrocyte digestion back is added sodium alginate-sodium chloride solution for mixing;
(2) with inhaling the sharp mixing drop that step (1) is obtained in calcium chloride (CaCl
2) in the solution, become gel;
(3) gel that step (2) is obtained uses sodium-chlor (NaCl) solution and perfect medium to wash successively, cultivates on compound DMEM/HamF12 substratum again.
Wherein, described compound DMEM/HamF12 substratum comprises 10ng/ml Basic Fibroblast Growth Factor, 5 μ g/ml hyaluronic acids, 0.2 μ g/ml fibronectin, the DMEM/HamF12 of 50 μ g/ml soluble chitosans and 1: 1.
Preferably, the concentration of the single-layer cultured chondrocyte in the step (1) is 1-6 * 10
5/ mL.
Preferably, the concentration of the sodium alginate in the step (1) is 12g/L.
Preferably, the concentration of the NaCl solution in the step (1) is 0.15mol/L.
Preferably, the CaCl in the step (2)
2The concentration of solution is 102mmol/L.
Preferably, becoming the condition of gel in the step (2) is 37 ℃, 5%CO
2, 10min.
Preferably, the concentration of the NaCl solution in the step (3) is 0.15mol/L.
Compared with prior art, the present invention has following beneficial effect: the cultural method of stereo proliferative cartilage cell of the present invention can improve the quality and quantity of culturing cell at short notice, the fast breeding chondrocyte, and long-term cultivation can be preserved cell phenotype.
Adopt complex carrier that following advantage is arranged: 1. surface area/volume (S/V) is big, thereby the cell yield height of unit volume nutrient solution, 2. adopt uniform suspension culture, suspension culture and adherent culture are combined, have both advantages concurrently, 3. can observe the cell growth of complex carrier with simple simple microscope, 4. substratum utilization ratio height, 5. separate with primary cell and compare, the cell harvesting process is simple.
The present invention adopts the experiment of many factor design, orthogonal experimental design (Orthogonal experimentaldesign) is the multifactor multilevel a kind of method of design of research, it is to pick out the representational point of part according to orthogonality from comprehensive test to test, these representational points have possessed the characteristics of " homodisperse; neat comparable ", orthogonal experimental design is the main method of fraction factorial design, is a kind of high-level efficiency, quick, economic experimental design method.With setting up sodium alginate colloid stereoscopic culture chondrocyte system behind this method contrived experiment, and can improve quality and quantity at short notice, because it is easily to dedifferente that greatest problem is cultivated on the plane, the cell bottle easily comes in contact inhibition after covering with cell, and sodium alginate colloid stereoscopic culture is cultivated three-dimensional to the plane, and colloid has the minute-pressure force environment, cellular form is near in the body, a large amount of propagation form homologous cell group, and its peripheral expansion has reduced contact inhibition; Several extracellular matrix correlative factors of selecting for use are environment in the analog cell growth, help the cell growth.
Microenvironment in the analog cell body, selected sodium alginate as cytoskeleton, the extracellular matrix materials that is fit to the cell growth, set up the vitro culture of compound DMEM/HamF12 with orthogonal experimental design, through orthogonal test and revision test, proof test, the effective concentration and the mix proportion scheme of each factor have been determined, in the sodium alginate carrier bracket of compound DMEM/F12 substratum, cultivate chondrocyte's energy fast breeding, cell count 48h just breeds more than 3 times, luxuriant red-O (Safranine O) carries out glycosaminoglycan (GAG) dyeing and shows that reaching P9 still produces a large amount of proteoglycan matrix.Serum lactic dehydrogenase (LDH) detects does not see toxic side effect.This complex carrier has good plasticity and histocompatibility.Be used for BMSCs inductive chondrocyte and also obtain effect same.
Description of drawings
Fig. 1 is the observation picture of a preferred embodiment of form of the sodium alginate complex carrier of rabbit cartilage cell;
Fig. 2 observes the picture that 7 days chondrocytes assemble a preferred embodiment of growth conditions with inverted microscope;
Fig. 3 is the picture with a preferred embodiment of 7 days individual cells states of phase microscope sem observation;
Fig. 4 is a picture of observing a preferred embodiment of monolayer cell state with laser confocal microscope;
Fig. 5 cuts and the picture of a preferred embodiment of the state of chondrocyte's homologous cell group in stereoscopic culture of the three dimensional viewing complex carrier of recombinating with the multiple light of laser confocal microscope;
Fig. 6 be luxuriant red-O carries out the picture of the painted preferred embodiment of extracellular matrix glycosaminoglycan.
Embodiment
Below in conjunction with accompanying drawing preferred embodiment of the present invention is described.
Embodiment one: the separation of rabbit cartilage cell and cultivation
Aseptic new zealand white rabbit (Guangdong Medical Lab Animal Center) knee cartilage of getting for 4～5 ages in week.Be cut into 1mm with scalper
3About small shreds, use Unidasa respectively, trypsinase, II Collagen Type VI enzymic digestion joint cartilage fragment obtains chondrocyte's suspension.25cm
3Culturing bottle inoculation chondrocyte adds and contains the DMEM/Ham ' s F12 nutrient solution that volume percent is 10% foetal calf serum, puts 37 ℃, and volume percent is 5%CO
2Incubator is cultivated, treats to change liquid every other day behind the cell attachment, and be that 0.02%EDTA wash with mass percent when the chondrocyte is paved with flat bottle at the end, mass percent is 0.25% trysinization, centrifugal collection chondrocyte is in order to go down to posterity or to test.
Embodiment two: the complex carrier components selection
The repopulating cell number is 5 * 10 in 96 orifice plates
4/ mL chondrocyte, add series mass concentration bFGF (0,1,5,10,50,100ng/mL), hyaluronic acid (0,12.5,25,50,100,200 μ g/mL) respectively, fibronectin (0,0.05,0.1,0.2,0.4,0.8 μ g/mL), soluble chitosan (0,12.5,25,50,100,200 μ g/mL) is used XTT
460nmThe propagation situation of colorimetric method for determining analysis of cells number is observed the influence of single component pair cell cartilage.
Embodiment three: the preparation of complex carrier
Single-layer culturing cell 2 * 10
5/ mL adds 12g/L sodium alginate-0.15mol/L NaCl, and mixed solution with inhaling point (50 μ L/ drip), is dripped in 102mmol/L CaCl
2, 37 ℃, 5%CO
2Incubator 10min becomes gel at a certain temperature, washes 3 times with 0.15mol/L NaCl, and perfect medium (CCM) is washed 1 time, and 10-15ml is at compound F12/DMEM culture medium culturing, routine observation.
Complex carrier morphological observation result: the sodium alginate complex carrier morphologic observation gross examination of skeletal muscle of rabbit cartilage cell: the about 5mm of carrier diameter, circle, oyster white, translucent, flexible, suspension growth in substratum is seen Fig. 1; The visible chondrocyte of 7d assembles growth under the inverted microscope, has colony of one's own, sees Fig. 2; The visible individual cells circle of phase microscope mirror 7d, karyon is clear, and after birth is complete, sees Fig. 3; Laser confocal microscope is observed: monolayer cell is cultivated with complex medium, Acridine orange (AO) dyeing, and the cell growth is vigorous, and cell is the fusiformis shape, and nuclear DNA is bright green, and endochylema RNA is orange, and visible colony and nuclear fission are seen Fig. 4 mutually; The chondrocyte of complex carrier is rounded or polygon in stereoscopic culture, cuts and the 3D that recombinates observes with the multiple light of laser confocal microscope, and the visible cell division growth, 30d forms homologous cell group, sees Fig. 5.
Four: two kinds of factors of embodiment influence the chondrocyte
With bFGF be main factor series concentration (0,1,5,10,50,100ng/mL) respectively with hyaluronic acid (100 μ g/mL), fibronectin (0.1 μ g/mL), soluble chitosan (100 μ g/mL) acts on 5 * 10
4/ mL chondrocyte compares, and chooses the optimum synergistic point, gets rid of negative collaborative point.Use XTT
460nmThe propagation situation of colorimetric method for determining analysis of cells number, observation of cell is to the reaction of two kinds of materials.
Embodiment five: the design of orthogonal test
Select bFGF, hyaluronic acid (Hyaluronic), fibronectin (Fibronectin), 4 factors of soluble chitosan (Chitosan), 3 levels of each factor are according to SPSS 13.0 software designs, Oryhogonal Design 3-Way interactive analysis, level of factor sees Table 1, and table 1 is the design factor water-glass of orthogonal test, does 81 experiments altogether.After preliminary screening, establish A, B, C, D data splitting duplicate detection for therefrom preferred 24 groups.The orthogonal experimental design interpretation of result sees Table 2.
Table 1
Table 2
△ and control group (12,24 groups) relatively have statistical significance, F=36.35 P=0.000
△ △ result and control group relatively cell count 48h increase more than 2 times
According to SPSS 13.0 software designs, Oryhogonal Design 3-Way interactive analysis according to 81 groups of experiment interaction results mean values, preferably repeats 24 groups, comprises 2 groups of control groups.There are 18 groups of results and control group that statistical significance is relatively arranged, are forward propagation, F=36.36, P＜0.01, wherein having 6 groups of preferred result and control group to compare cell count 48h increases more than 2 times, sees Table 2.
Embodiment six: proof test
Checking divides four groups, 10ng/ml bFGF; 5 μ g/ml Hyaluronic; 10ng/ml bFGF+5 μ g/mlHyaluronic; 10ng/ml bFGF+5 μ g/ml Hyaluronic+0.2 μ g/ml Fibronectin+50 μ g/ml Chitosan, each group is a control group with the chondrocyte who does not add intervention factor, adds respectively in 96 orifice plates, with 5 * 10
4/ ml chondrocyte mixed culture, 4 multiple holes, the checking cell through the orthogonal test preferred ingredients under identical conditions to single and reaction matrix material, use XTT
460nmThe propagation of colorimetric method for determining analysis of cells.Carry cell with complex carrier again, be placed on the complex culture medium of orthogonal test checking and cultivate 48h after the time, with separating pearl liquid (55mmol/L Trisodium Citrate, 0.15mmol/LNaCl) the capable cell counting of dissolving colloid.Results of statistical analysis sees Table 3.
Table 3
△ and control group (0) relatively have statistical significance, P＜0.01
A：bFGF(ng/mL)F＝14.968，P＜0.005
B：bFGF(ng/mL)+Fibronectin?0.12μg/mL?F＝31.267，P＜0.005
C：bFGF(ng/mL)+Hyaluronic?50μg/mL?F＝43.300，P＜0.005
D：bFGF(ng/mL)+Chitosan?50μg/mL?F＝33.935，P＜0.005
Table 3 for bFGF be main factor series concentration respectively with fibronectin, hyaluronic acid and soluble chitosan are to chondrocyte's the result that influences.Design the full interactive analysis table of 4 factor 3-Way according to SPSS13.0; With bFGF is the interaction of series concentration and three kinds of factors of main factor, uses the ANOVAOne-way statistical analysis; The multifactor factor analysis of collaborative analysis between factor.BFGF has the effect of short cell proliferation with other 3 kinds of factor keying actions in the chondrocyte respectively, the ANOVA statistical analysis, and P＜0.05, bFGF plays the main factor effect, sees Table 3.
Proof test: cell under identical conditions to the single and influence matrix material chondrocyte proliferation.Detection single and matrix material under identical conditions adopts multifactor factor analysis to the influence of chondrocyte proliferation, and prompting bFGF has common synergy with hyaluronic acid, and bFGF can promote chondrocyte's growth, and hyaluronic acid does not influence the chondrocyte.BFGF (10ng/ml) associating hyaluronic acid (50～100 μ g/ml) use effect to chondrocyte proliferation greater than single with bFGF (10ng/ml), bFGF and HA interaction have statistical significance (P＜0.01), when bFGF is in different levels (50-500ng/ml), the effect of HA (10-50 μ g/ml) factor is different, the common result of use of two medicines is better, and synergy is arranged.Prepare complex culture medium with 4 kinds of factors with optimal proportions simultaneously, cell count 48h just breeds more than 3 times, sees Table 4.Table 4 is the collaborative analytical data of complex factors to chondrocyte proliferation.
Table 4
△ and control group relatively have statistical significance, F=1142.91 P＜0.005
Embodiment seven: chondrocyte's functional examination
With above-mentioned culture medium culturing chondrocyte, get respectively the 5th, 7,9 generation cell climbing sheet, adopt SafranineO to carry out the dyeing of extracellular matrix glycosaminoglycan, the glycosaminoglycan of visible cell synthesis secretion is deposited in the cell peripheral matrix and dyes redness, showing a large amount of proteoglycan matrix produces, in the 9th generation, still had colony to produce, and sees Fig. 6.
In the sodium alginate carrier of DMEM/HAM`F12 complex medium, microenvironment in the analog cell body, selected 1.2% sodium alginate as cytoskeleton, the extracellular matrix materials that is fit to the cell growth, set up the vitro culture of compound DMEM/HamF12 with orthogonal experimental design, comprise 10ng/ml bFGF, 5 μ g/ml Hyaluronic, 0.2 μ g/ml Fibronectin (U.S. Sigma company), 50 μ g/ml solubility Chitosan, 1: 1 main components such as (U.S. Gibco companies) of (biological company limited is very won in Shanghai) DMEM/HamF12, through orthogonal test and revision test, proof test, the effective concentration and the mix proportion scheme of each factor have been determined, at compound DMEM/F
12Cultivate chondrocyte's energy fast breeding in the sodium alginate carrier bracket of substratum, cell count 48h just breeds more than 3 times, and Safranine O GAG dyeing shows that reaching P9 still produces a large amount of proteoglycan matrix.LDH detects and does not see toxic side effect.This complex carrier has good plasticity and histocompatibility.Fast breeding also helps keeping the expression of chondrocyte's phenotype and function.Be used for MSCs inductive chondrocyte and also obtain effect same.
Embodiment eight: a preferred version of the present invention and K.Lindenhayn method cell proliferation quantity are relatively
K.Lindenhayn cultivates the chondrocyte with 1.2% sodium alginate pearl merely, its cell count has slightly and increases, in sodium alginate, add 0.35% hyaluronic acid, its cell count can increase by 100% in 3 days, and on this basis, adding 1.25% fibronectin, its cell count just can increase by 100% in 1 day.
This programme 1.2% sodium alginate adds 102mmol/LCaCl behind the mixed cell suspension
2Make colloid, again in order to DMEM/F12 1: 1 as basic medium, add the 10ng/ml Basic Fibroblast Growth Factor of fresh configuration, 5 μ g/ml hyaluronic acids, 0.2 μ g/ml fibronectin, 50 μ g/ml soluble chitosans are cultivated with method.The most important thing is to have utilized Basic Fibroblast Growth Factor and hyaluronic synergy altogether, add fibronectin almost with all types of tropocollagen molecules can both in conjunction with, but and have higher avidity between chondrocyte's excretory II tropocollagen molecule, impel cell proliferating number 48h just to breed more than 3 times.
The preparation of K.Lindenhayn method is in the 1.2% sodium alginate process, uses 102mmol/LCaCl
2In the crosslinked fluid, hatch the generation scleroproein with 55NIH fibrillarin enzyme unit+46IU/ml zymoplasm through 15-20min, scleroproein is included in the stock support, this programme is directly used pure product fibronectin, water soluble, and the pair cell action effect is obvious.
Be to serve as main crosslinked forming with 1.2% sodium alginate in the stock support of K.Lindenhayn method, biomechanics is not enough, and this programme is to add soluble chitosan to be improved.
The above only is preferred embodiment of the present invention, and is in order to restriction the present invention, within the spirit and principles in the present invention not all, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1, a kind of cultural method of stereo proliferative cartilage cell is characterized in that, comprises the steps:
(1) single-layer cultured chondrocyte digestion back is added sodium alginate-sodium chloride solution for mixing;
(2) with inhaling mixing drop that point obtains step (1) in calcium chloride solution, become gel;
(3) gel that step (2) is obtained is washed with sodium chloride solution and perfect medium successively, cultivates on compound DMEM/HamF12 substratum again;
Wherein, described compound DMEM/HamF12 substratum comprises 10ng/ml Basic Fibroblast Growth Factor, 5 μ g/ml hyaluronic acids, 0.2 μ g/ml fibronectin, the DMEM/HamF12 of 50 μ g/ml soluble chitosans and 1: 1.
2, the cultural method of a kind of stereo proliferative cartilage cell as claimed in claim 1 is characterized in that, the concentration of the single-layer cultured chondrocyte in the described step (1) is 1～6 * 10
5/ mL.
3, the cultural method of a kind of stereo proliferative cartilage cell as claimed in claim 1 is characterized in that, the concentration of the sodium alginate in the described step (1) is 12g/L.
4, the cultural method of a kind of stereo proliferative cartilage cell as claimed in claim 1 is characterized in that, the concentration of the sodium-chlor in the described step (1) is 0.15mol/L.
5, the cultural method of a kind of stereo proliferative cartilage cell as claimed in claim 1 is characterized in that, the concentration of the calcium chloride in the described step (2) is 102mmol/L.
6, the cultural method of a kind of stereo proliferative cartilage cell as claimed in claim 1 is characterized in that, becoming the condition of gel in the described step (2) is 37 ℃, 5%CO
2, 10min.
7, the cultural method of a kind of stereo proliferative cartilage cell as claimed in claim 1 is characterized in that, the concentration of the sodium-chlor in the described step (3) is 0.15mol/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344905A (en) * | 2010-08-04 | 2012-02-08 | 北京大学 | Bionic culture model for chondrocyte, and preparation method thereof |
| CN104667348A (en) * | 2015-02-04 | 2015-06-03 | 北京大学第三医院 | Pharmaceutical composition containing sodium alginate and preparation method of pharmaceutical composition |
| CN105132365A (en) * | 2015-08-17 | 2015-12-09 | 深圳华毓造血干细胞研究有限公司 | Three-dimensional induction culture method of mesenchymal stem cells |
| CN109221088A (en) * | 2018-09-30 | 2019-01-18 | 成都远山原力生物科技有限公司 | A kind of cell cryopreservation solution, sodium alginate gel, Cellular gels, preparation method, application method and its application |
-
2008
- 2008-09-10 CN CNA2008101984657A patent/CN101407786A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344905A (en) * | 2010-08-04 | 2012-02-08 | 北京大学 | Bionic culture model for chondrocyte, and preparation method thereof |
| CN102344905B (en) * | 2010-08-04 | 2014-04-23 | 北京大学 | Bionic culture model for chondrocyte, and preparation method thereof |
| CN104667348A (en) * | 2015-02-04 | 2015-06-03 | 北京大学第三医院 | Pharmaceutical composition containing sodium alginate and preparation method of pharmaceutical composition |
| CN104667348B (en) * | 2015-02-04 | 2016-03-02 | 北京大学第三医院 | A kind of Pharmaceutical composition containing sodium alginate and preparation method thereof |
| CN105132365A (en) * | 2015-08-17 | 2015-12-09 | 深圳华毓造血干细胞研究有限公司 | Three-dimensional induction culture method of mesenchymal stem cells |
| CN109221088A (en) * | 2018-09-30 | 2019-01-18 | 成都远山原力生物科技有限公司 | A kind of cell cryopreservation solution, sodium alginate gel, Cellular gels, preparation method, application method and its application |
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Open date: 20090415 |