CN105316283A - Clinical-grade placenta mesenchymal stem cell preparation method - Google Patents
Clinical-grade placenta mesenchymal stem cell preparation method Download PDFInfo
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Abstract
The invention provides a clinical-grade placenta mesenchymal stem cell preparation method. Specifically, the method includes: using a combined digestive enzyme to digest a placenta tissue material after being cut up to obtain a digested tissue mixture, wherein the combined digestive enzyme comprises collagenase IV and trypsase; adding a red blood cell lysis buffer to obtain a mixture after going through red blood cell lysis treatment; centrifuging to obtain precipitate, namely primary-generation mesenchymal stem cells; performing serum-free passage amplification on the primary-generation mesenchymal stem cells obtained in the previous step to obtain clinical-grade placenta mesenchymal stem cells after going through passage. The cells prepared by the method are high in purity and proliferation capability and can guarantee needs on clinical use of the cells.
Description
Technical field
The present invention relates to field of biomedicine technology, particularly, the invention provides the preparation method of clinical grade placenta mesenchyma stem cell.
Background technology
Human mesenchymal stem cell (mesenchymalstemcell, MSC) be class loading wide material sources, comprise the adult tissue stem cell that marrow, fatty tissue, deciduous teeth tissue, peripheral blood etc. and umbilical cord blood, umbilical cord, placenta tissue etc. have multi-lineage potential and very strong self-renewal capacity, scleroblast, chondrocyte, adipocyte, myocyte, Tenocyte cell and liver cell or neurogliocyte can be divided in vivo and in vitro, secrete the pluripotent cell factor and improve microenvironment and lower immunogenicity; Based on above feature, it is made to become rapidly afterwards at cell therapy, regenerative medicine field practical " seed cell " in discovery.
Placenta tissue Derived Stem Cells has typical mescenchymal stem cell characteristic, comprises the features such as good self, multi-lineage potential, immunogenicity be low; And tissue sampling is convenient, without wound, to limit without ethics and utilization ratio is high, it is the tissue-derived type that clinical grade mescenchymal stem cell is good.
Current placenta mesenchyma stem cell separation method mainly comprises: perfusion method, organize adherent method and enzyme digestion.
Perfusion method needs repeatedly to rinse in a large number, and technique is loaded down with trivial details, reagent consumption is many and not easily rinse thoroughly, the drawback such as easily to pollute and is difficult to application in industrialization is produced.
Organize adherent method longer because primary cell expands numerous cycle, be difficult in a short time obtain a large amount of cell quantity.
Enzymic digestion rule comprises the mode such as single enzyme or combination enzyme sequential digestion.But enzyme digestion enzyme is selected numerous, digestion step is loaded down with trivial details and enzymic digestion affects larger for ability of cell proliferation and cytoactive.In addition, after digestion, tissue generally after filtration after net filtration, could obtain single cell suspension, and a large amount of red corpuscle will mix wherein, have impact on the cycle that cell attachment grows and increases original cuiture to a certain extent.
In addition, for the amplification in vitro link of placenta mesenchyma stem cell, most or use at present contains lower concentration people source or other animal source serum free culture system systems, and the complexity of serum composition and indefinite property limit it and cultivate for clinical grade cell amplification.
Also someone attempts adding the cytokine profiles such as bFGF in the passage amplification stage and replaces serum composition, and Tissue Culture Dish is carried out various stromatin colloid, as the special packet such as Matrigel, gelatin is processed, carry out serum-free amplification cultivation, although cell can grow, but cell state heterogeneity, complicated operation, bag are higher by link cost, cultivate expanding effect lot stability is difficult to control, and is not suitable for preparing production on a large scale.
In sum, in the urgent need to developing, a kind of placenta mesenchyma stem cell is efficient in this area, low cost and be easy to the separation method of flux programming operations, so that cell industrialization.
Summary of the invention
One object of the present invention is to provide a kind of and is easy to the preparation method of clinical grade placenta mesenchyma stem cell prepared by high-throughput industrialization for the efficient, easy of Human plactnta mescenchymal stem cell.
Another object of the present invention is to provide a kind of placenta mesenchyma stem cell prepared by described method, the placenta mesenchyma stem cell of gained has good ability of cell proliferation, purity check reaches international standard and have good skeletonization, become fat and cartilage differentiation potential, and after repeatedly going down to posterity, still can keep normal caryogram.
In a first aspect of the present invention, provide a kind of preparation method of clinical grade placenta mesenchyma stem cell, described method comprises step:
A (), to the placenta tissue material containing mescenchymal stem cell obtained, carries out shredding process, thus the placenta tissue material through shredding;
The b placenta tissue material through shredding that () obtains previous step, digests with combination digestive ferment, thus acquisition organizes mixture through what digest, and wherein said combination digestive ferment comprises collagenase IV and trypsinase;
C () organizes mixture from described through what digest, get supernatant suspension;
D () described supernatant suspension to previous step carries out centrifugal, obtain precipitation;
E (), to the described precipitation of previous step, adds erythrocyte cracked liquid, thus obtain the mixture through erythrocyte splitting process;
F (), to the described mixture through erythrocyte splitting process of previous step, carries out centrifugal, obtain precipitation, be primary mescenchymal stem cell;
G () described primary mescenchymal stem cell to previous step carries out serum-free amplification and goes down to posterity, thus the clinical grade placenta mesenchyma stem cell through going down to posterity.
In another preference, described placenta tissue material is negative through Viral diagnosis.
In another preference, described Viral diagnosis includes, but is not limited to detect one or more infective virus following: hepatitis B virus, hepatitis C virus, HIV virus, syphilis, cytomegalovirus etc.
In another preference, in step (a), the placenta tissue material through shredding, its volume size is 0.2-2ml, general about 0.5ml (generally can be placed in the centrifuge tube of such as 15ml).
In another preference, in step (b), described digestive ferment comprises final concentration (working concentration) for 50U/ml-100U/ml collagenase IV and 0.005wt%-0.01wt% trypsinase; Preferably 80U/ml collagenase IV and 0.008wt%.
In another preference, in step (b), described digestive ferment is to add containing the mode of collagenase IV and tryptic combination digestive ferment liquid in the container containing the described placenta tissue material through shredding (such as 15ml centrifuge tube).
In another preference, in step (b), digestion condition comprises: digest at 37 ± 2 DEG C; Digestion 6-24 hour, preferably digests 8-20 hour, more preferably digests 12-18 hour, preferred 16h.
In another preference, in step (b), collagenase IV and trypsinase can add successively, successively or simultaneously; Preferably, add simultaneously.
In another preference, described placenta tissue material comprises placenta amnion tissue, placental villi membrane tissue or its combination.
In another preference, comprising in step (c): carry out leaving standstill process (about 30 seconds-3 minutes, preferably 45 seconds) to the described mixture of organizing through digestion, then get supernatant suspension.
In step (d), described centrifugal condition is 300-500g, and centrifugation time is 1-10 minute.
In step (e), the condition of described erythrocyte splitting process is 37 ± 2 DEG C, process 1-10 minute.
In another preference, in step (e), after erythrocyte splitting process, add 1-10ml1% penicillin/streptomycin PBS liquid, after mixing, thus obtain the mixture through erythrocyte splitting process.
In another preference, in step (f), described centrifugal condition is 300-500g, and centrifugation time is 1-10 minute.
In another preference, in step (g), described in the number of times that goes down to posterity be 1-10 time, preferably 1-5 time.
In another preference, in step (g), described going down to posterity is carried out in serum free medium.
In another preference, described serum free medium comprises
hMSC substratum, without animal xenogeneic components, specific chemical components, batch stable substratum, or other serum free medium.
In another preference, described clinical grade placenta mesenchyma stem cell preparation meets following standard:
I () uses serum-free, cell culture passages system without allos animal component and specific chemical components;
(ii) cell surface marker CD73, CD90, more than CD105>95%, CD34, CD45<2%;
(iii) there is skeletonization, become fat and cartilage differentiation potential; With
(iv), after external operation of repeatedly going down to posterity, there is normal people's caryogram.
In another preference, in step (g), when Secondary Culture when growth of mesenchymal stem cells is to 80-90% degrees of fusion, remove nutrient solution, described mescenchymal stem cell is cleaned, then add trypsinase to process described mescenchymal stem cell, thus obtain the mescenchymal stem cell through tryptic digestion process, cultivate for later passages.
In a second aspect of the present invention, provide a kind of clinical grade placenta mesenchyma stem cell group of separation, described in described population of stem cells first aspect present invention prepared by method.
In another preference, described mescenchymal stem cell group is the mescenchymal stem cell in 1-3 generation.
In another preference, described mescenchymal stem cell group has following characteristics:
A the cell of () >=95% (preferably >=97%) has surface antigen CD73;
B the cell of () >=95% (preferably >=97%) has surface antigen CD90; With
C the cell of () >=95% (preferably >=97%) has surface antigen CD105.
In another preference, described placenta mesenchyma stem cell behaviour placenta mesenchyma stem cell.
In another preference, described population of stem cells also has following characteristics:
D ()≤2% (preferably≤1%) cell has surface antigen CD34;
E ()≤2% (preferably≤1%) cell has surface antigen CD45.
In a third aspect of the present invention, provide a kind of working fluid for the preparation of clinical grade placenta mesenchyma stem cell, described working fluid contains combination enzyme, and described combination enzyme comprises collagenase IV type and trypsinase, and
Described collagenase IV type and tryptic working concentration are respectively 50-100u/ml and 0.005-0.01wt%.
In another preference, collagenase IV type and tryptic working concentration are respectively 60-90u/ml (as 80U/ml) and 0.006-0.009wt% (as 0.008wt%).
In another preference, the enzyme contained by described working fluid is made up of collagenase IV type and trypsinase.
In another preference, described working fluid is that the collagenase IV type Digestive system of 1-2:2-1 and tryptic digestive juice mix by volume ratio.
In another preference, the ratio of described collagenase IV type and tryptic content is: 50-100U collagenase IV type: the trypsinase of 5-10mg.
In another preference, described working fluid uses with the ratio of 2-4g:1ml volume ratio placenta tissue material.
In a fourth aspect of the present invention, provide a kind of preparation method of clinical grade placenta mesenchyma stem cell, described method comprises:
I () digests placenta tissue material combination digestive ferment, thus acquisition organizes mixture through what digest, wherein said combination digestive ferment comprises collagenase IV and trypsinase, and described collagenase IV type and tryptic working concentration are respectively 50-100u/ml and 0.005-0.01wt%;
(ii) primary placenta mesenchyma stem cell is obtained from the described separation mixture of organizing through digestion; With
(iii) carry out serum-free amplification to described primary mescenchymal stem cell to go down to posterity, thus the clinical grade placenta mesenchyma stem cell through going down to posterity.
In another preference, collagenase IV type and tryptic working concentration are respectively 60-90u/ml (as 80U/ml) and 0.006-0.009wt% (as 0.008wt%).
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 to show in the present invention's example the prepared and mescenchymal stem cell form gone down to posterity through serum-free culture.Visible in figure, described mescenchymal stem cell is that spindle shape, size, form are homogeneous.
Fig. 2 shows by the detected result of flow cytometry analysis to the surface marker of placenta mesenchyma stem cell in the present invention's example.
Fig. 3 shows the differentiation capability of placenta mesenchyma stem cell in the present invention's example.Wherein, Fig. 3 A shows Osteoblast Differentiation situation; Fig. 3 B shows into fat differentiation situation; Fig. 3 C shows cartilage differentiation situation.
Fig. 4 shows in the present invention's example, and the placenta mesenchyma stem cell (the 8th generation) repeatedly gone down to posterity through serum-free culture, cell still has normal caryogram.
Fig. 5 shows in the embodiment of the present invention 2 and embodiment 9 and prepares serum-free amplification method and traditional foetal calf serum and to increase the cell volume size comparative analysis situation of the placenta mesenchyma stem cell obtained.Result display serum free culture system obtains placenta mesenchyma stem cell volume and is significantly less than serum free culture system group, and wherein, A represents the placenta mesenchyma stem cell obtained containing serum amplification method; B represents the placenta mesenchyma stem cell that serum-free amplification method obtains.
Embodiment
Inventor is through extensive and deep research, find unexpectedly, by adopting the particular combination digestive ferment (namely the collagen protein IV enzyme of lower concentration is with the use of the trypsinase of lower concentration) of extremely low concentration, after long digestion process is carried out to placenta tissue material, and coordinate serum-free culture and go down to posterity, efficiently, easily can prepare placenta mesenchyma stem cell.Complete the present invention on this basis.
Particularly, in the methods of the invention, after above-mentioned specific combination collagenase treatment is carried out to the placenta tissue material shredded, then through erythrocyte splitting process, the placenta mesenchyma stem cell serum free medium of gained is carried out amplification of going down to posterity, thus obtains clinical grade placenta mesenchyma stem cell.
Term
As used herein, " clinical grade " refers to the rank of applicable Clinical practice.Usually, described clinical grade placenta mesenchyma stem cell meets following standard:
I () uses serum-free, cell culture passages system without allos animal component and specific chemical components;
(ii) cell surface marker CD73, CD90, more than CD105>95%, CD34, CD45<2%;
(iii) there is skeletonization, become fat and cartilage differentiation potential; With
(iv), after external operation of repeatedly going down to posterity, there is normal people's caryogram.
As used herein, term " cell of the present invention ", " mescenchymal stem cell of the present invention " or " placenta mesenchyma stem cell of the present invention " refer to the placenta mesenchyma stem cell be separated by the inventive method.Should be understood that these terms comprise the placenta mesenchyma stem cell of primary and through going down to posterity (as going down to posterity 1-10 time).
Combination digestive ferment and working fluid
In the present invention, key character is that particular combination digestive ferment with extremely low concentration (namely the collagen protein IV enzyme of lower concentration is with the use of the trypsinase of lower concentration) digests placenta tissue material.
In the present invention, combine digestive ferment and comprise collagenase IV and trypsinase.Combination Digestive system of the present invention contains (a) described collagenase IV and trypsinase, (b) water, the buffer composition that (c) is optional; (d) other optional composition.
In the present invention, the concentration (by working concentration) of collagenase IV type is generally 50U/ml-100u/ml, preferred 80U/ml.
In the present invention, tryptic concentration (by working concentration) is generally 0.005wt%-0.01wt%, preferred 0.008wt% (mass percent).
In the present invention, described buffer composition can select the buffer composition of this area routine, is especially applicable to the buffer composition of collagenase IV, and is applicable to tryptic buffer composition.
In the present invention, described optional member comprises: basic medium, as l-DMEM.
The present inventor is shown by research, adopt the particular combination digestive ferment of extremely low concentration to digest placenta tissue material, contribute to keeping cytoactive, and long digestion time has ensured tissue digestion thoroughness, eliminate the operation links such as filtration, be suitable for flux operation.
Preparation method
The invention provides a kind of clinical grade placenta mesenchyma stem cell preparation method, comprise cellular segregation, primary with the steps such as serum-free amplification that go down to posterity.
In the present invention, suitable material comprises placenta tissue material, usual optional employment placenta amnion or chorion tissue.
In the methods of the invention, the volume for the placenta tissue material as raw material is not particularly limited.Typically, volume is 0.1-5ml, preferably 0.2-2ml, or by weight, is 0.1-5g, is preferably 0.2-2g.
Placenta amnion of the present invention or chorion tissue and the present invention combine Digestive system volume ratio and are not particularly limited, and are generally 1g:2-4ml (weight/volume).
In the present invention, carry out digestion condition with combination enzyme to comprise: digest at 37 ± 2 DEG C; And/or digestion 6-24 hour, preferably digest 8-20 hour, more preferably digest 12-18 hour (as 16 hours).
In the present invention, for the above-mentioned placenta tissue material through digestion process, erythrocyte splitting process can be carried out further by interpolation erythrocyte cracked liquid, and through being separated (preferably centrifugal) further, thus primary placenta mesenchyma stem cell can be obtained without the need to filtration treatment.
In the present invention, by the mescenchymal stem cell that the present invention prepared by aforesaid method is primary, be particularly suitable for carrying out cultivating and going down to posterity with serum free medium.
In the present invention, the serum free medium be suitable for can be conventional serum free medium, and a kind of particularly preferred serum free medium is
hMSCMedium, this substratum is without animal xenogeneic components, specific chemical components.
In the present invention, can carry out Secondary Culture in any suitable culture vessel, a kind of particularly preferred cell culture container is
cellBindsurface culture dish or culturing bottle.
In a preference of the present invention, described placenta mesenchyma stem cell separation method comprises:
1., under hundred grades of clean environments, clip placenta tissue, after thoroughly cleaning, shreds with scissors machinery, add lower concentration collagenase IV type, the tryptic digestive juice of certain volume/volume ratio with PBS liquid; After 37 DEG C of abundant digestion, fully dispel mixing, supernatant suspension 300-500g, 5min is centrifugal, and harvested cell precipitates.
2. the cell of above-mentioned steps results, add except erythrocyte cracked liquid 1-2ml, after cracking 3-5min, fully dispel mixing, 300-500g, 5min are centrifugal, and harvested cell precipitates.
3. by above-mentioned steps 2 harvested cell precipitate, add serum free medium resuspended after, be inoculated in culture dish or culturing bottle, be placed in ordinary cells incubator, the later half amount of 24-72h changes liquid, and within 3-4 days afterwards, full dose changes liquid once, and within 7-10 days, cytogamy degree reaches 80%-90%.
4., by the cell that 80%-90% in above-mentioned steps 3 merges, carry out amplification operation of going down to posterity, add 0.05% tryptic digestion 2-3 minute, after adding trypsin inhibitor 1-2ml, 300-500g, 5min are centrifugal, abandon supernatant, add serum free medium resuspended, to go down to posterity amplification by 1:3-6.
5. the cell that above-mentioned steps is gathered in the crops was carried out going down to posterity for 2-3 time at about 14 days, can 10 be obtained
7the cell of the order of magnitude, carries out-196 DEG C of low temperature and stores for subsequent use.Maybe can carry out fluidic cell surface marker analysis, cell skeletonization to cell, become fat, cartilage direction induction differentiation qualification, and karyotyping.
Placenta mesenchyma stem cell
Human mesenchymal stem cell (mesenchymalstemcell, MSC) be class loading wide material sources, comprise the adult tissue stem cell that marrow, fatty tissue, deciduous teeth tissue, peripheral blood etc. and umbilical cord blood, umbilical cord, placenta tissue etc. have multi-lineage potential and very strong self-renewal capacity, scleroblast, chondrocyte, adipocyte, myocyte, Tenocyte cell and liver cell or neurogliocyte can be divided in vivo and in vitro, secrete the pluripotent cell factor and improve microenvironment and lower immunogenicity; Based on above feature, it is made to become rapidly afterwards at cell therapy, regenerative medicine field practical " seed cell " in discovery.
Placenta tissue Derived Stem Cells has typical mescenchymal stem cell characteristic, comprises the features such as good self, multi-lineage potential, immunogenicity be low; And tissue sampling is convenient, without wound, to limit without ethics and utilization ratio is high, it is the tissue-derived type that clinical grade mescenchymal stem cell is good.
Present invention also offers placenta mesenchyma stem cell prepared by a kind of the inventive method.Requirement that placenta mesenchyma stem cell of the present invention has good ability of cell proliferation, purity check meets international standards, there is good skeletonization, become fat and cartilage differentiation potential, after repeatedly going down to posterity, still keep normal caryogram.
Have very high purity with mescenchymal stem cell prepared by the inventive method, it is substantially devoid of cell or the stem cell of other types, also not containing red corpuscle.This detection by cell-surface antigens is verified.
Mescenchymal stem cell has multiple specific antigens and acceptor, mainly contains CD34, CD45, CD73, CD90, CD105 etc.CD34 antigen is a kind of high glycosylation I type transmembrane protein, it is optionally expressed in mankind hemopoietic stem cell (HSC), progenitor cell (PC) and vascular endothelial cell (EC) surface, mescenchymal stem cell with CD34 is preferably≤2% in the ratio of total stem cell, more preferably ,≤0.09%.
CD45 is present in the surface of all hematopoietic cells, comprises hemopoietic stem cell and osteoclast.Mescenchymal stem cell with CD45 is preferably≤2% in the ratio of total stem cell, more preferably, and≤0.06%.
CD73, CD90, CD105 etc. are mainly present in placenta mesenchyma stem cell surface.
At mescenchymal stem cell of the present invention, there is following one or more feature usually:
Mescenchymal stem cell with CD73 is preferably >=95%, more preferably >=98%, best >=99.93% in the ratio of total stem cell.
Mescenchymal stem cell with CD105 is preferably >=95%, more preferably >=96%, best >=96.37% in the ratio of total stem cell.
Mescenchymal stem cell with CD90 is preferably >=95%, more preferably >=98%, best >=98.90% in the ratio of total stem cell.
Those skilled in that art can use general method to detect purity and the differentiation degree of mescenchymal stem cell, as Flow cytometry.During detection, add different from specific antibody targetedly, antibody can be complete mono-clonal or polyclonal antibody, also can be have immunocompetent antibody fragment, as Fab ' or (Fab) 2 fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered Single Chain Fv Molecule A (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as there is murine antibody binding specificity but still retaining the antibody from the antibody moiety of people.The antigen adding antibody and cell surface, in conjunction with certain hour, carries out automatic analysis and sorting with flow cytometer to cell.
Adipogenic induction and detection
Because mescenchymal stem cell has Multidirectional Differentiation ability, under certain conditions induction is carried out to mescenchymal stem cell, the cell broken up of specific function can be obtained.
Those skilled in that art can use general method to carry out adipogenic induction to mescenchymal stem cell.A kind of general induction method adds dexamethasone in nutrient solution.The condition of inducing into fat mainly contains 3 kinds, comprise dexamethasone and add 1-methyl-3-isobutyl-xanthine (IBMX), dexamethasone adds Regular Insulin, or dexamethasone adds indomethacin (indomethacin, INDOMETHACIN), 1-methyl-3-isobutyl-xanthine and Regular Insulin.Wherein most important is exactly dexamethasone, and the dexamethasone of lower concentration is one of required composition of serum-free or low serum free culture system mescenchymal stem cell, can promote the external fast breeding of mescenchymal stem cell; The dexamethasone of higher concentration then can inducing mesenchymal stem cell to Adipocyte Differentiation.
Those skilled in that art can use general method to induce into fat with dyestuff (as OilRed, Sudan red 5B and solvent red 27 etc.) to mescenchymal stem cell and detect.The most frequently used dyestuff is OilRed (O), i.e. oil red O.
The structure of oil red O is 1-[2,5-dimethyl-4-(2,5-p-dimethylamino) benzeneazo]-2 naphthols, and be a kind of red powder, Oil Soluble Azo Dyes, is soluble in benzene, ethanol and acetone.In the process of Adipogenic induction, cell constantly has the accumulation of oil droplet in endochylema, and it is large constantly to increase change, is all oil droplet in the endochylema of last whole cell.Oil red O, as biological stain, is easily combined with grease, but poor with the structure tinting strength of cell itself.Clearly can carry out into fat dyeing under the microscope to observe.
Osteogenic induction and detection
Because mescenchymal stem cell has Multidirectional Differentiation ability, under certain conditions Osteoblast Differentiation induction is carried out to mescenchymal stem cell, can scleroblast be obtained.
Those skilled in that art can use general method to carry out osteogenic induction to mescenchymal stem cell.Classical chemical osteogenic induction liquid formula is: DMEM nutrient solution, Sodium Glycerophosphate, vitamins C and dexamethasone.The process of osteogenic induction is that calcium ion is precipitated in the mode of calcium salt, i.e. " calcium tubercle ".
The dyestuff conventional " sodium alizarinsulfonate " of qualification calcium tubercle.Alizarin red aqueous solution comprises: sodium alizarin sulfonate, alizarin S, alizarin red S, alizarin carmine, 1,2-dihydroxyanthraquinone-3-sodium sulfonate, 1,2-dihydroxyanthraquinone-3-sulfonate sodium.Sodium alizarinsulfonate is orange-yellow or yellowish brown powder, soluble in water, is slightly soluble in ethanol, is insoluble to benzene and chloroform and can generates with many metal ions and be with look compound, can with the color reaction of zirconium, thorium, aluminium, titanium and beryllium and calcium.The principle of Alizarin red staining is exactly sodium alizarinsulfonate and calcium generation color reaction, and produce a kind of wine-colored band look compound, the calcium tubercle deposited outside the cell of such osteogenic induction has also just been dyed to scarlet.
Chondrocyte induction and detection
Because mescenchymal stem cell has Multidirectional Differentiation ability, under certain conditions cartilage differentiation induction is carried out to mescenchymal stem cell, can chondrocyte be obtained.
Those skilled in that art can use general method to carry out chondrocyte induction to mescenchymal stem cell, can utilize traditional chemical formula: dexamethasone, vitamins C and TGF-B etc.; Or commodity dress cartilage differentiation test kit carries out chondrocyte induction differentiation, after inducing 3 weeks, finds the spheroid of cell stars ellipse.Utilize alcian blue (Alcianblue) dyestuff in conjunction with the content situation of the acid glycosaminoglycan in cartilage differentiation extracellular matrix, be blue in conjunction with rear positive cell, evaluate ability and the efficiency situation of placenta mesenchyma stem cell cartilage differentiation.
The invention has the advantages that:
A in () the inventive method, cell isolation method adopts lower concentration collagenase IV type, trypsinase combination digestion method, this contribute to improving cell purity, multiplication capacity strong, shorten primary proliferation time, ensure that the promptness of cell Clinical practice.
B () in the methods of the invention, the cell after tissue digestion is without filter operation, and manual steps is few, be easy to industrialization normalizing operation.
C () the inventive method adopts complete serum-free amplification technology, avoid the risk that serum composition infects.
D () the inventive method is processed without the need to carrying out bag to Tissue Culture Dish, working method is simple, effect stability.
E placenta mesenchyma stem cell prepared by () the inventive method possesses good differentiation potential, and cellular form size is homogeneous, and volume is little, after operation of repeatedly going down to posterity, still keep normal caryogram, be conducive to the clinical application in later stage.
F () can complete the preparation of cell and frozen about general 2-3 week, method is simple to operate, be easy to program throughput, is more suitable for the mechanisms such as industry cell bank prepares clinical grade placenta mesenchyma stem cell than existing general method.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number are weight percent and parts by weight.
Embodiment 1
The preparation of primary placenta mesenchyma stem cell
1. Placenta samples detects (hepatitis B virus, hepatitis C virus, virus of AIDS, syphilis and cytomegalovirus) result feminine gender through strict infective virus is qualified sample, enters cellular segregation link.
2. under hundred grades of clean environments, as in Bechtop, clip placenta amnion or chorion are organized in 1% penicillin/streptomycin PBS liquid, remove residual bloodstain, after shredding with scissors machinery, collect in 15ml centrifuge tube, add 1:3-4ml volume ratio (the collagenase IV type of 80U/ml, 0.008wt% working concentration, trypsinase) and combine Digestive system; After 37 DEG C of digestion 12-18h, preferred 16h, fully dispels mixing, and transfer supernatant suspension is in new 15ml centrifuge tube, and 300-500g, centrifugal 5min, harvested cell precipitates.
3. the cell of above-mentioned steps results, adds 1-2ml erythrocyte cracked liquid, 37 DEG C, after 3-5min, adds 5ml1% penicillin/streptomycin PBS liquid, fully dispel mixing, 300-500g, centrifugal 5min, and harvested cell precipitates.
4. by above-mentioned steps 2 harvested cell precipitate, add serum free medium 2ml resuspended after, be inoculated in CORNINGCellBindsurface100mm culture dish, be placed in 37 DEG C, 5%CO
2cultivate in cell culture incubator, the later half amount of 24-48h changes liquid, and within 3-4 days afterwards, full dose changes liquid once, and within 7-10 days, cytogamy degree can reach 80%-90%.
5. by the cell of above-mentioned steps 380%-90% fusion, remove nutrient solution, after the cleaning of 4mlPBS liquid, add 0.05% trypsinase 1ml and digest 2-3 minute, after adding 1-2ml trypsin inhibitor, move in 15ml centrifuge tube after dispelling mixing, 300-500g, centrifugal 5min, abandoning supernatant, is of the present invention primary placenta mesenchyma stem cell to be cultivated.
Embodiment 2
The serum-free Secondary Culture of primary cell, gather in the crops and go down to posterity
The primary placenta mesenchyma stem cell obtained in embodiment 1 is added serum free medium resuspended, by 1:3-6 (cell quantity Dilution ratio) or 3000-6000 cell/cm
2density goes down to posterity amplification.
Carry out 1-2 time amplification go down to posterity after, low generation, 10 can be obtained
7above order of magnitude cell concentration, carry out-196 DEG C of low temperature and store, cell carries out fluidic cell surface marker analysis.
cellular form qualification result
To increasing, the cell obtained carries out morphological observation, and result is as follows:
Adopt the placenta mesenchyma stem cell of this separation, the acquisition of serum-free amplification method.As shown in Figure 1, P5 is spindle shape, polygon for attached cell to form under inverted microscope, and cellular form size is homogeneous and ability of cell proliferation is strong.
fluidic cell surface marker analysis
Carry out fluidic cell surface marker analysis to the cell cultivating gained in above-mentioned experiment, concrete operations are as follows:
Get the cell (P5 is for cell) that serum-free amplification obtains, be distributed into 7 pipes, every tube cell is 10
6above, (CD73, CD105, CD90 is added respectively, CD34, CD45, FITCMouseIgG1 and PEMouseIgG1), lucifuge hatches 15min, after adding 0.4ml paraformaldehyde, flow cytometer (FACSCalibur) is analyzed, the results detailed in Fig. 2 and following table 1.
Table 1P5 is for the fluidic cell surface marker analysis result of cell
| Surface antigen | CD73 | CD105 | CD90 | CD34 | CD45 |
| Cell proportion | 99.93％ | 96.37％ | 98.90％ | 0.09％ | 0.06％ |
| International standard | >95％ | >95％ | >95％ | <2％ | <2％ |
Note: mescenchymal stem cell international standard is: CD73>95%, CD90>95%, CD105>95%; CD34<2%, CD45<2%
Result shows: placenta mesenchyma stem cell meets international mesenchymal stem cells standard.
Embodiment 3
Cell skeletonization, one-tenth fat and chondrocyte induction Analytical Chemical Experiment
Cell skeletonization, one-tenth fat, the differentiation of cartilage direction induction:
Get the passage of serum-free amplification in the embodiment of the present invention 2 to 24 orifice plates, when cell confluency degree reaches 80%-90%, suck serum free medium and dead cell, add respectively skeletonization, become fat, chondrocyte induction division culture medium (
osteogenesisDifferentiationKit,
adipogenesisDifferentiationKit,
chondrogenesisDifferentiationKit), 3-4 days full doses are changed liquid and observe under inverted microscope
Result:
3.1 adipogenic induction
At fat differentiation-inducing about the 10th day, under light microscopic, visible a large amount of yellow circular fat dripped and piles up around nucleus, and cellular form is rounded, and after oil red O stain, fat drips and takes on a red color, and extends certain induction time, and fat drips and constantly becomes large.The visible cell dripped containing a large amount of fat after figure 3 a illustrates oil red O stain.
Result shows, mescenchymal stem cell of the present invention has the differentiation potential being divided into adipocyte and tissue.
3.2 osteogenic induction
In Osteoinductive differentiation experiment, after inducing one week, cellular form presents stereoscopic sensation, constantly forms multi-layer cellular after propagation, after inducing 3 weeks, suck substratum, after 4% paraformaldehyde fixes 20min, add alizarin red S solution-dyed 30min, visible a large amount of Ca piles up, and refers to Fig. 3 B;
Result shows, mescenchymal stem cell of the present invention has the differentiation potential being divided into osteocyte and tissue.
3.3 chondrocyte induction
After chondrocyte induction differentiation liquid induces 2 weeks, find the spheroid of cell stars ellipse, alcian blue (Alcianblue) stained positive, refers to Fig. 3 C.
Result shows, mescenchymal stem cell of the present invention has the differentiation potential being divided into chondrocyte and tissue.
Embodiment 4
To go down to posterity test
In the present embodiment, karyotyping is carried out to the placenta mesenchyma stem cell in the 8th generation prepared by embodiment 2.Method is as follows:
To the 8th generation cell the cell being in logarithmic phase, add colchicine process 2-3h and collect smudge cells, fix 60min at adding stationary liquid (formaldehyde: Glacial acetic acid=3:1) 37 DEG C, repeat fixing after, drip sheet and carry out G-and show tape handling and microscopy.
Result shows: have normal human karyotype (referring to Fig. 4), show to possess good differentiation potential with the cell of the inventive method preparation and the results that repeatedly increase, and cellular form size is homogeneous, volume is little, after operation of repeatedly going down to posterity, cell still keeps normal caryogram, is conducive to the clinical application in later stage.
Embodiment 5-8
Repeat embodiment 1 and 2, difference is, adopts the single enzyme shown in following table 2.
For the mescenchymal stem cell of preparation, detect by the same procedure of embodiment 3 and 4, the results are shown in table 2.
Embodiment 9
Repeat embodiment 1 and 2, difference is, adopts the blood serum medium that contains shown in following table 2 to carry out cultivating and going down to posterity.
For the mescenchymal stem cell of preparation, detect by the same procedure of embodiment 3 and 4, the results are shown in table 2.
Embodiment 10-13
Repeat embodiment 1 and 2, difference is, adopts collagenase IV and the trypsinase of the working concentration shown in following table 2.
For the mescenchymal stem cell of preparation, detect by the same procedure of embodiment 3 and 4, the results are shown in table 2.
Table 2 embodiment 5-13 and result
The above results shows, no matter adopts alone a kind of enzyme, comprises collagenase IV, trypsinase or collagenase I etc.; Or improve on the digestion conditions such as enzyme concn, obtain placenta mesenchyma stem cell although can be separated, on primary cell quantity, purity and/or ability of cell proliferation, be all not so good as the inventive method.
In the link that goes down to posterity: use containing blood serum medium and culture method in serum-free of the present invention, though on vitro growth rates and cell surface marker are expressed on no significant difference, the placenta mesenchyma stem cell volume obtained containing serum free culture system system obviously will be greater than between placenta that serum free culture system of the present invention obtains and fills stem cell.Fig. 5 shows in embodiments of the invention the placenta mesenchyma stem cell volume size prepared serum-free amplification method and obtain, visible in figure, near true origin 2 peak figure, be significantly less than tradition and obtain cell volume containing serum free culture system.
Discuss
In order to meet clinical requirement, the preparation of usual placenta mesenchyma stem cell needs in the requirement standard meeting clinical application goods in the important steps such as sample cellular segregation, amplification.The invention provides a kind of can efficiently, isolation of human placenta mesenchymal stem and based on serum-free culture with go down to posterity and the preparation method of clinical grade placenta mesenchyma stem cell that can produce easily high-throughput.Even if the mescenchymal stem cell cell purity prepared by method of the present invention is high, multiplication capacity after operation of repeatedly going down to posterity, still keeps normal caryogram by force.In addition, method provided by the invention provides good seed cell resource for the Application Areas such as organizational project, cell therapy, can be widely used in industrialization cellular resources and preserve storehouse or other use mechanisms etc.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a preparation method for clinical grade placenta mesenchyma stem cell, is characterized in that, described method comprises step:
A (), to the placenta tissue material containing mescenchymal stem cell obtained, carries out shredding process, thus the placenta tissue material through shredding;
The b placenta tissue material through shredding that () obtains previous step, digests with combination digestive ferment, thus acquisition organizes mixture through what digest, and wherein said combination digestive ferment comprises collagenase IV and trypsinase;
C () organizes mixture from described through what digest, get supernatant suspension;
D () described supernatant suspension to previous step carries out centrifugal, obtain precipitation;
E (), to the described precipitation of previous step, adds erythrocyte cracked liquid, thus obtain the mixture through erythrocyte splitting process;
F (), to the described mixture through erythrocyte splitting process of previous step, carries out centrifugal, obtain precipitation, be primary mescenchymal stem cell;
G () described primary mescenchymal stem cell to previous step carries out serum-free amplification and goes down to posterity, thus the clinical grade placenta mesenchyma stem cell through going down to posterity.
2. the method for claim 1, is characterized in that, in step (b), described digestive ferment comprises final concentration (working concentration) for 50U/ml-100U/ml collagenase IV and 0.005wt%-0.01wt% trypsinase; Preferably 80U/ml collagenase IV and 0.008wt%.
3. the method for claim 1, is characterized in that, in step (b), digestion condition comprises: digest at 37 ± 2 DEG C; Digestion 6-24 hour, preferably digests 8-20 hour, more preferably digests 12-18 hour, preferred 16h.
4. the method for claim 1, is characterized in that, in step (b), collagenase IV and trypsinase can add successively, successively or simultaneously; Preferably, add simultaneously.
5. the method for claim 1, is characterized in that, described placenta tissue material comprises placenta amnion tissue, placental villi membrane tissue or its combination.
6. the clinical grade placenta mesenchyma stem cell group be separated, it is characterized in that, described in described population of stem cells claim 1 prepared by method.
7. cell mass as claimed in claim 6, it is characterized in that, described mescenchymal stem cell group has following characteristics:
A the cell of () >=95% (preferably >=97%) has surface antigen CD73;
B the cell of () >=95% (preferably >=97%) has surface antigen CD90; With
C the cell of () >=95% (preferably >=97%) has surface antigen CD105.
8. mescenchymal stem cell group as claimed in claim 7, it is characterized in that, described population of stem cells also has following characteristics:
D ()≤2% (preferably≤1%) cell has surface antigen CD34;
E ()≤2% (preferably≤1%) cell has surface antigen CD45.
9. for the preparation of a working fluid for clinical grade placenta mesenchyma stem cell, it is characterized in that, described working fluid contains combination enzyme, and described combination enzyme comprises collagenase IV type and trypsinase, and
Described collagenase IV type and tryptic working concentration are respectively 50-100u/ml and 0.005-0.01wt%.
10. a preparation method for clinical grade placenta mesenchyma stem cell, described method comprises:
I () digests placenta tissue material combination digestive ferment, thus acquisition organizes mixture through what digest, wherein said combination digestive ferment comprises collagenase IV and trypsinase, and described collagenase IV type and tryptic working concentration are respectively 50-100u/ml and 0.005-0.01wt%;
(ii) primary placenta mesenchyma stem cell is obtained from the described separation mixture of organizing through digestion; With
(iii) carry out serum-free amplification to described primary mescenchymal stem cell to go down to posterity, thus the clinical grade placenta mesenchyma stem cell through going down to posterity.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106032529A (en) * | 2016-07-06 | 2016-10-19 | 章毅 | Method for in-vitro separation, amplification and induced differentiation of placenta-derived mesenchymal stem cells |
| CN107385517A (en) * | 2017-08-07 | 2017-11-24 | 章毅 | The construction method of mesenchyma stem cell |
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| CA2558436A1 (en) * | 2004-03-04 | 2005-09-15 | Susan Oriole Meakin | Method of proliferating precursor cells |
| RU2252252C1 (en) * | 2004-04-09 | 2005-05-20 | Тепляшин Александр Сергеевич | Method for isolation of mesenchymal stem cells |
| US7993918B2 (en) * | 2006-08-04 | 2011-08-09 | Anthrogenesis Corporation | Tumor suppression using placental stem cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106032529A (en) * | 2016-07-06 | 2016-10-19 | 章毅 | Method for in-vitro separation, amplification and induced differentiation of placenta-derived mesenchymal stem cells |
| CN107385517A (en) * | 2017-08-07 | 2017-11-24 | 章毅 | The construction method of mesenchyma stem cell |
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