CN105316283B - A kind of clinical grade placenta mesenchyma stem cell preparation method - Google Patents
A kind of clinical grade placenta mesenchyma stem cell preparation method Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of clinical grade placenta mesenchyma stem cell, specifically, the method of the present invention includes: to the placenta tissue material through shredding, it is digested with combination digestive ferment, to obtain the tissue mixture through digesting, wherein the combination digestive ferment includes clostridiopetidase A IV and trypsase；Erythrocyte cracked liquid is added, to obtain the mixture handled through erythrocyte splitting；Centrifugation, is precipitated, as primary mescenchymal stem cell；Serum-free passage amplification is carried out to the primary mescenchymal stem cell of previous step, to obtain the clinical grade placenta mesenchyma stem cell through passing on.Its cell purity of the cell of the method for the present invention preparation is high, proliferative capacity is strong, can guarantee that cell clinical use needs.
Description
Technical field
The present invention relates to field of biomedicine technology, and specifically, the present invention provides clinical grade placenta mesenchyma stem cells
Preparation method.
Background technique
Human mesenchymal stem cell (mesenchymal stem cell, MSC) be it is a kind of it is tissue-derived extensively, including bone
Marrow, adipose tissue, deciduous teeth tissue, peripheral blood etc. and umbilical cord blood, umbilical cord, placenta tissue etc. have multi-lineage potential
And the adult tissue stem cell of very strong self-renewal capacity, it can be divided into osteoblast, cartilage cell, fat in vivo and in vitro carefully
Born of the same parents, myocyte, Tenocyte cell and liver cell or Deiter's cells secrete the pluripotent cell factor and improve microenvironment and lower
Immunogenicity；Based on the above feature, rapidly become it after discovery in the practical " kind of cell therapy, regenerative medicine field
Daughter cell ".
Placenta tissue Derived Stem Cells have typical mescenchymal stem cell characteristic, including good self-renewing, multidirectional
The features such as differentiation potential, low immunogenicity；And tissue sampling is convenient, it is noninvasive, without ethics limitation and utilization rate it is high, be clinical
The grade good tissue-derived type of mescenchymal stem cell.
Placenta mesenchyma stem cell separation method specifically includes that perfusion method, tissue adherent method and enzyme digestion at present.
Perfusion method needs repeatedly a large amount of flushings, and technique is cumbersome, reagent consumption is more and is not easy to rinse thoroughly, easily causes pollution
Etc. drawbacks be difficult to apply in industrialization production.
It organizes adherent method since primary cell expansion numerous period is longer, is difficult to obtain a large amount of cell quantity in a short time.
Enzymic digestion rule includes the modes such as single enzyme or combination enzyme sequential digestion.However, enzyme digestion enzyme selects
Numerous, digestion step is cumbersome and enzymic digestion is affected for ability of cell proliferation and cell activity.In addition, group after digestion
Knitting generally will could obtain single cell suspension, and a large amount of red blood cell mixes wherein, to a certain degree after filter screen filtration
On affect cell adherent growth and increase the period of originally culture.
In addition, for the amplification in vitro link of placenta mesenchyma stem cell, it is most at present still to use source of people containing low concentration
Or other animal sources serum free culture system systems, complexity and the ambiguity of serum composition limit it and are used for the amplification training of clinical grade cell
It supports.
Also it has tried to pass on the cytokine profiles replacement serum composition such as amplification stage addition bFGF in cell, and will
The various stromatin colloids of Tissue Culture Dish progress, such as Matrigel, gelatin special packet are processed, carry out serum-free amplification
Culture, although cell can be grown, cell state is inhomogenous, complicated for operation, coating link higher cost, cultivates amplification effect
Fruit lot stability is difficult to control, and is not suitable for preparing production on a large scale.
In conclusion there is an urgent need in the art to develop a kind of efficient placenta mesenchyma stem cell, low cost and be easy to flux
The separation method of programming operations, in order to cell industrialization.
Summary of the invention
It is an object of the present invention to provide a kind of for the efficient, easy of Human plactnta mescenchymal stem cell and is easy to
The preparation method of the clinical grade placenta mesenchyma stem cell of high-throughput industrialization preparation.
It is resulting another object of the present invention is to provide a kind of placenta mesenchyma stem cell prepared with the method
Placenta mesenchyma stem cell with good ability of cell proliferation, purity analysis up to international standard and have good skeletonization, at
Rouge and cartilage differentiation potential, and after multiple passage, still it is able to maintain normal caryogram.
In the first aspect of the present invention, a kind of preparation method of clinical grade placenta mesenchyma stem cell, the side are provided
Method comprising steps of
(a) it to the placenta tissue material containing mescenchymal stem cell of acquisition, carries out shredding processing, thus the tire through shredding
Disk organization material；
(b) the placenta tissue material through shredding obtained to previous step, is digested with combination digestive ferment, to obtain
The tissue mixture through digesting is obtained, wherein the combination digestive ferment includes clostridiopetidase A IV and trypsase；
(c) in the tissue mixture from described through digesting, supernatant suspension is taken；
(d) the supernatant suspension of previous step is centrifuged, is precipitated；
(e) to the precipitating of previous step, erythrocyte cracked liquid is added, thus what acquisition was handled through erythrocyte splitting
Mixture；
(f) it to the mixture handled through erythrocyte splitting of previous step, is centrifuged, is precipitated, it is as former
For mescenchymal stem cell；
(g) serum-free amplification passage is carried out to the primary mescenchymal stem cell of previous step, to face through what is passed on
Bed grade placenta mesenchyma stem cell.
In another preferred example, the placenta tissue material is feminine gender through viral diagnosis.
In another preferred example, the viral diagnosis including but not limited to detects one or more of infectiousness
Virus: hepatitis B, hepatitis C virus, inhibition of HIV, syphilis, cytomegalovirus etc..
In another preferred example, in step (a), the placenta tissue material through shredding, volume size be 0.2-2ml, one
As 0.5ml or so (may be generally disposed in the centrifuge tube of such as 15ml).
In another preferred example, in step (b), the digestive ferment includes that final concentration (working concentration) is 50U/ml-
100U/ml clostridiopetidase A IV and 0.005wt%-0.01wt% trypsase；Preferably 80U/ml clostridiopetidase A IV and 0.008wt%
Trypsase.
In another preferred example, in step (b), the digestive ferment is disappeared with the combination of IV containing clostridiopetidase A and trypsase
(such as 15ml centrifuge tube) is added in the container of the placenta tissue material containing described through shredding in the mode for changing enzyme solution.
In another preferred example, in step (b), digestion condition includes: to be digested at 37 ± 2 DEG C；Digest 6-24
Hour, it preferably digests 8-20 hours, more preferably digests 12-18 hours, preferably 16h.
In another preferred example, in step (b), clostridiopetidase A IV and trypsase can be added successively, successively or simultaneously；
Preferably, it is added simultaneously.
In another preferred example, the placenta tissue material include placenta amnion tissue, placental villi membrane tissue or its
Combination.
It in another preferred example, include: that tissue mixture to described through digesting carries out stewing process in step (c)
(- 3 minutes about 30 seconds, preferably 45 seconds), then take supernatant suspension.
In step (d), the condition of the centrifugation is 300-500g, and centrifugation time is 1-10 minutes.
In step (e), the condition of the erythrocyte splitting processing is 37 ± 2 DEG C, is handled 1-10 minutes.
In another preferred example, in step (e), after erythrocyte splitting processing, addition 1% penicillin of 1-10ml/
Streptomysin PBS liquid, after being mixed, to obtain the mixture handled through erythrocyte splitting.
In another preferred example, in step (f), the condition of the centrifugation is 300-500g, centrifugation time 1-10
Minute.
In another preferred example, in step (g), the number of the passage is 1-10 times, preferably 1-5 times.
In another preferred example, in step (g), the passage carries out in serum free medium.
In another preferred example, the serum free medium includesHMSC culture medium,
Without the stable culture medium of animal xenogeneic components, specific chemical components, batch or other serum free mediums.
In another preferred example, clinical grade placenta mesenchyma stem cell preparation meets following standard:
(i) serum-free, the cell culture passages system without heterologous animal component and specific chemical components are used；
(ii) cell surface marker CD73, CD90, CD105>95% or more, CD34, CD45<2%；
(iii) there is skeletonization, at rouge and cartilage differentiation potential；With
(iv) after external repeatedly passage operation, there is normal people's caryogram.
In another preferred example, in step (g), in secondary culture when growth of mesenchymal stem cells is melted to 80-90%
When right, culture solution is removed, the mescenchymal stem cell is cleaned, it is dry to the mesenchyma that trypsase is then added
Cell is handled, to obtain the mescenchymal stem cell handled through trypsin digestion, is used for later passages culture.
In the second aspect of the present invention, a kind of isolated clinical grade placenta mesenchyma stem cell group is provided, described is dry
Cell mass is prepared with first aspect present invention the method.
In another preferred example, the mescenchymal stem cell group is the mescenchymal stem cell in 1-3 generation.
In another preferred example, the mescenchymal stem cell group has the feature that
(a) >=95% cell (preferably >=97%) has surface antigen CD73；
(b) >=95% cell (preferably >=97%) has surface antigen CD90；With
(c) >=95% cell (preferably >=97%) has surface antigen CD105.
In another preferred example, the placenta mesenchyma stem cell is people's placenta mesenchyma stem cell.
In another preferred example, the population of stem cells also has the feature that
(d)≤2% (preferably≤1%) cell has surface antigen CD34；
(e)≤2% (preferably≤1%) cell has surface antigen CD45.
In the third aspect of the present invention, a kind of working solution for being used to prepare clinical grade placenta mesenchyma stem cell is provided,
The working solution contains a group synthase, and described group of synthase includes clostridiopetidase A IV type and trypsase, and
The working concentration of the clostridiopetidase A IV type and trypsase is respectively 50-100u/ml and 0.005-
0.01wt%.
In another preferred example, the working concentration of clostridiopetidase A IV type and trypsase is respectively 60-90u/ml (such as 80U/
) and 0.006-0.009wt% (such as 0.008wt%) ml.
In another preferred example, enzyme contained by the working solution is made of clostridiopetidase A IV type and trypsase.
In another preferred example, clostridiopetidase A IV type digestive juice and pancreas that the working solution is 1-2:2-1 by volume ratio
Protease digestion liquid mixes.
In another preferred example, the ratio between the clostridiopetidase A IV type and the content of trypsase are as follows: 50-100U clostridiopetidase A IV
Type: the trypsase of 5-10mg.
In another preferred example, the working solution is made with the ratio of 2-4g:1ml volume ratio placenta tissue material
With.
In the fourth aspect of the present invention, a kind of preparation method of clinical grade placenta mesenchyma stem cell, the side are provided
Method includes:
(i) placenta tissue material is digested with combination digestive ferment, so that the tissue mixture through digesting is obtained,
Described in combination digestive ferment include clostridiopetidase A IV and trypsase, and the work of the clostridiopetidase A IV type and trypsase
It is respectively 50-100u/ml and 0.005-0.01wt% as concentration；
(ii) separation obtains primary placenta mesenchyma stem cell in the tissue mixture from described through digesting；With
(iii) serum-free amplification passage is carried out to the primary mescenchymal stem cell, thus the clinical grade placenta through passing on
Mescenchymal stem cell.
In another preferred example, the working concentration of clostridiopetidase A IV type and trypsase is respectively 60-90u/ml (such as 80U/
) and 0.006-0.009wt% (such as 0.008wt%) ml.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited,
Not repeated them here.
Detailed description of the invention
Fig. 1 shows mescenchymal stem cell form that is prepared in an example of the invention and passing on through free serum culture.
As it can be seen that the mescenchymal stem cell is that spindle shape, size, form are uniform in figure.
Fig. 2 is shown to be marked with surface of the flow cytometry analysis to placenta mesenchyma stem cell in an example of the invention
The testing result of will object.
Fig. 3 shows the differentiation capability of placenta mesenchyma stem cell in an example of the invention.Wherein, Fig. 3 A is shown into
Bone breaks up situation；Fig. 3 B shows into rouge differentiation situation；Fig. 3 C shows cartilage differentiation situation.
Fig. 4 shows in an example of the invention, the placenta mesenchyma stem cell repeatedly passed on through free serum culture
(the 8th generation), cell still has normal caryogram.
Fig. 5 shows that serum-free amplification method and traditional fetal calf serum are prepared in the embodiment of the present invention 2 and embodiment 9 to expand
Increase the cell volume size comparative analysis situation of obtained placenta mesenchyma stem cell.Serum free culture system obtains as the result is shown
It is significantly less than serum free culture system group to placenta mesenchyma stem cell volume, wherein A indicates the placenta that amplification method containing serum obtains
Mescenchymal stem cell；B indicates the placenta mesenchyma stem cell that serum-free amplification method obtains.
Specific embodiment
Inventor after extensive and in-depth study, it was unexpectedly found that, by using the spy of extremely low concentration
Surely digestive ferment (i.e. trypsase of the collagen IV enzyme of low concentration with the use of low concentration) is combined, to placenta tissue material
After carrying out prolonged digestion process, and cooperate free serum culture and passage, it is dry can efficiently, easily to prepare placenta mesenchyma
Cell.The present invention is completed on this basis.
Specifically, in the methods of the invention, the placenta tissue material shredded is carried out at above-mentioned specific combination enzymic digestion
It after reason, then handles through erythrocyte splitting, resulting placenta mesenchyma stem cell is subjected to passage amplification with serum free medium,
To obtain clinical grade placenta mesenchyma stem cell.
Term
As used herein, " clinical grade " refers to the rank of suitable clinical use.In general, being filled between the clinical grade placenta
Matter stem cell meets following standard:
(i) serum-free, the cell culture passages system without heterologous animal component and specific chemical components are used；
(ii) cell surface marker CD73, CD90, CD105>95% or more, CD34, CD45<2%；
(iii) there is skeletonization, at rouge and cartilage differentiation potential；With
(iv) after external repeatedly passage operation, there is normal people's caryogram.
As used herein, term " cell of the present invention ", " mescenchymal stem cell of the invention " or " between placenta of the invention
Mesenchymal stem cells " refer to the placenta mesenchyma stem cell separated with the method for the present invention.It should be understood that these terms include primary
And the placenta mesenchyma stem cell through (such as passage 1-10 times) passed on.
Combine digestive ferment and working solution
In the present invention, an important feature is specific combination digestive ferment (the i.e. collagen egg of low concentration with extremely low concentration
White IV enzyme is used cooperatively the trypsase of low concentration) placenta tissue material is digested.
In the present invention, combination digestive ferment includes clostridiopetidase A IV and trypsase.Combination digestive juice of the invention contains
(a) the clostridiopetidase A IV and trypsase, (b) water, (c) optional buffer composition；(d) optional other ingredients.
In the present invention, the concentration (based on working concentration) of clostridiopetidase A IV type is usually 50U/ml-100u/ml, preferably
80U/ml。
In the present invention, the concentration (based on working concentration) of trypsase is usually 0.005wt%-0.01wt%, preferably
0.008wt% (mass percent).
In the present invention, the buffer composition of this field routine can be selected in the buffer composition, is especially adapted for use in clostridiopetidase A
The buffer composition of IV, and the buffer composition suitable for trypsase.
In the present invention, the optional member includes: basal medium, such as l-DMEM.
The present inventor passes through studies have shown that the specific combination digestive ferment using extremely low concentration carries out placenta tissue material
Digestion helps to maintain cell activity, and long digestion time has ensured tissue digestion completeness, eliminates the behaviour such as filtering
Make link, is suitable for flux operation.
Preparation method
The present invention provides a kind of clinical grade placenta mesenchyma stem cell preparation methods, including cell separation, primary and biography
For serum-free amplification.
In the present invention, suitable material includes placenta tissue material, usually optional Human plactnta amnion or chorion group
It knits.
In the methods of the invention, the volume of the placenta tissue material as raw material is not particularly limited.Typically, body
Product is 0.1-5ml, preferably 0.2-2ml, or by weight, is 0.1-5g, preferably 0.2-2g.
Placenta amnion or villus membrane tissue of the invention combines digestive juice volume ratio with the present invention and is not particularly limited, and leads to
Often it is 1g:2-4ml (weight/volume).
In the present invention, carrying out digestion condition with group synthase includes: to be digested at 37 ± 2 DEG C；And/or digestion 6-
It 24 hours, preferably digests 8-20 hours, more preferably digests 12-18 hours (such as 16 hours).
In the present invention, the placenta tissue material for above-mentioned through digestion process can further be split by adding red blood cell
It solves liquid and carries out erythrocyte splitting processing, and (be preferably centrifuged) through further separation, be can be obtained without filtration treatment primary
Placenta mesenchyma stem cell.
In the present invention, the mescenchymal stem cell primary by the present invention prepared by the above method, is particularly suitable for using nothing
Blood serum medium is cultivated and is passed on.
In the present invention, applicable serum free medium can be conventional serum free medium, a kind of particularly preferred
Serum free medium isHMSC Medium, the culture medium without animal xenogeneic components, chemistry at
It is clearly demarcated true.
In the present invention, secondary culture, a kind of particularly preferred cell training can be carried out in any suitable culture vessel
Feeding container isCellBind surface culture dish or culture bottle.
In a preference of the invention, the placenta mesenchyma stem cell separation method includes:
1. clip placenta tissue shreds after thoroughly being cleaned with PBS liquid, with scissors machinery under hundred grades of clean environments,
Certain volume/volume ratio low concentration clostridiopetidase A IV type, tryptic digestive juice is added；After 37 DEG C sufficiently digest, sufficiently dispel
It mixes, supernatant suspension 300-500g, 5min centrifugation harvests cell precipitation.
2. the cell of above-mentioned steps harvest, is added and removes erythrocyte cracked liquid 1-2ml, after cracking 3-5min, sufficiently dispel mixed
Even, 300-500g, 5min centrifugation harvest cell precipitation.
3. above-mentioned steps 2 are harvested cell precipitation, after serum free medium resuspension is added, it is inoculated in culture dish or culture
In bottle, it is placed in ordinary cells incubator, the later half amount of 24-72h changes liquid, and full dose changes the liquid once within 3-4 days later, 7-10 days cells
Degrees of fusion reaches 80%-90%.
4. the cell that 80%-90% in above-mentioned steps 3 is merged carries out passage amplification operation, 0.05% tryptose is added
Enzymic digestion 2-3 minutes, after trypsin inhibitor 1-2ml is added, 300-500g, 5min centrifugation abandoned supernatant, serum-free are added
Culture medium is resuspended, and passes on and expands by 1:3-6.
5. the cell of above-mentioned steps harvest was carried out 2-3 passage at 14 days or so, it can be obtained 107The order of magnitude it is thin
It is spare to carry out -196 DEG C of low temperature storages by born of the same parents.Or can to cell carry out fluidic cell surface marker analysis, cell skeletonization, at
Rouge, cartilage direction induction differentiation identification and karyotyping.
Placenta mesenchyma stem cell
Human mesenchymal stem cell (mesenchymal stem cell, MSC) be it is a kind of it is tissue-derived extensively, including bone
Marrow, adipose tissue, deciduous teeth tissue, peripheral blood etc. and umbilical cord blood, umbilical cord, placenta tissue etc. have multi-lineage potential
And the adult tissue stem cell of very strong self-renewal capacity, it can be divided into osteoblast, cartilage cell, fat in vivo and in vitro carefully
Born of the same parents, myocyte, Tenocyte cell and liver cell or Deiter's cells secrete the pluripotent cell factor and improve microenvironment and lower
Immunogenicity；Based on the above feature, rapidly become it after discovery in the practical " kind of cell therapy, regenerative medicine field
Daughter cell ".
Placenta tissue Derived Stem Cells have typical mescenchymal stem cell characteristic, including good self-renewing, multidirectional
The features such as differentiation potential, low immunogenicity；And tissue sampling is convenient, it is noninvasive, without ethics limitation and utilization rate it is high, be clinical
The grade good tissue-derived type of mescenchymal stem cell.
The present invention also provides a kind of placenta mesenchyma stem cells prepared with the method for the present invention.Between placenta of the invention
Mesenchymal stem cells with good ability of cell proliferation, purity analysis meet international standards requirement, have good skeletonization, at rouge
With cartilage differentiation potential, normal caryogram is still repeatedly kept after passage.
There is very high purity with mescenchymal stem cell prepared by the method for the present invention, essentially free of other types
Cell or stem cell, also be free of red blood cell.This can be verified by the detection of cell surface antigen.
Mescenchymal stem cell has a variety of specific antigens and receptor, mainly there is CD34, CD45, CD73, CD90, CD105
Deng.CD34 antigen is a kind of I type transmembrane protein of high glycosylation, its selectivity is expressed in mankind hemopoietic stem cell (HSC),
Progenitor cells (PC) and the surface vascular endothelial cell (EC), the mescenchymal stem cell with CD34 are preferred in the ratio of total stem cell
It is≤2%, more preferably ,≤0.09%.
CD45 is present in the surface of all hematopoietic cells, including candidate stem cell and osteoclast.With being filled between CD45
Matter stem cell is preferably≤2%, more preferably ,≤0.06% in the ratio of total stem cell.
CD73, CD90, CD105 etc. are primarily present in placenta mesenchyma stem cell surface.
Usually have the characteristics that following one or more in mescenchymal stem cell of the present invention:
Mescenchymal stem cell with CD73 is preferably >=95% in the ratio of total stem cell, more preferably >=98%, most preferably
Ground >=99.93%.
Mescenchymal stem cell with CD105 is preferably >=95% in the ratio of total stem cell, more preferably >=96%, most
Goodly >=96.37%.
Mescenchymal stem cell with CD90 is preferably >=95% in the ratio of total stem cell, more preferably >=98%, most preferably
Ground >=98.90%.
The purity and differentiation degree of general method detection mescenchymal stem cell can be used in those skilled in that art,
Such as Flow cytometry.When detection, it is added different with targeted specific antibody, antibody can be complete monoclonal
Or polyclonal antibody, it is also possible to that there is immunocompetent antibody fragment, such as Fab ' or (Fab) 2 segment；Heavy chain of antibody；Antibody
Light chain；Genetically engineered Single Chain Fv Molecule A (Ladner et al., United States Patent (USP) No.4,946,778)；Or chimeric antibody, such as
With mouse antibody binding specificity but still retain the antibody moiety from people antibody.The antigen of antibody and cell surface is added
In conjunction with certain time, cell is automatically analyzed and sorted with flow cytometer.
Adipogenic induction and detection
Since mescenchymal stem cell has Multidirectional Differentiation ability, mescenchymal stem cell is divided under certain conditions
Change induction, the differentiated cell of specific function can be obtained.
Those skilled in that art can be used general method and carry out adipogenic induction to mescenchymal stem cell.One kind is logical
Abductive approach is that dexamethasone is added into culture solution.The condition for inducing into rouge mainly has 3 kinds, including dexamethasone plus
1- methyl -3- isobutyl group xanthine (IBMX), dexamethasone add insulin or dexamethasone to add antinfan
(indomethacin, indocin), 1- methyl -3- isobutyl group xanthine and insulin.Most important one exactly fills in rice
Pine, the dexamethasone of low concentration is one of the essential component of serum-free or low serum free culture system mescenchymal stem cell, between capable of promoting
The external fast breeding of mesenchymal stem cells；The dexamethasone of higher concentration then can be with inducing mesenchymal stem cell to fat cell
Differentiation.
General method and dyestuff (such as Oil Red, tonyred 5B and solvent red can be used in those skilled in that art
27 etc.) mescenchymal stem cell is induced and is detected at rouge.Most common dyestuff is Oil Red (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, is a kind of
Red powder, Oil Soluble Azo Dyes are soluble in benzene, ethyl alcohol and acetone.During Adipogenic induction, cell is in endochylema
Constantly there is the accumulation of oil droplet, and constantly increase and become larger, is all oil droplet in the endochylema of last entire cell.Oil red O is as biology
Coloring agent, easily in conjunction with grease, but it is poor with the structure tinting strength, tinting power of cell itself.Rouge can be clearly carried out under the microscope
Dyeing observation.
Osteogenic induction and detection
Due to mescenchymal stem cell have Multidirectional Differentiation ability, under certain conditions to mescenchymal stem cell carry out at
Bone induction can obtain osteoblast.
Those skilled in that art can be used general method and carry out osteogenic induction to mescenchymal stem cell.Classical
Chemical osteogenic induction formula of liquid are as follows: DMEM culture solution, sodium glycero-phosphate, vitamin C and dexamethasone.The process of osteogenic induction
It is that calcium ion is enable to precipitate in a manner of calcium salt, i.e., " calcium tubercle ".
Identify that the dyestuff of calcium tubercle commonly uses " alizarin red ".Alizarin red aqueous solution includes: sodium alizarine sulphonate, alizarin S, alizarin red
S, alizarin carmine, 1,2- dihydroxy anthraquinone -3- sodium sulfonate, 1,2- dihydroxy anthraquinone -3- sulfonate sodium.Alizarin red is orange
Color or yellowish-brown powder, it is soluble easily in water, it is slightly soluble in ethyl alcohol, coloredization can be generated with many metal ions insoluble in benzene and chloroform
Object is closed, it can be with the chromogenic reaction of zirconium, thorium, aluminium, titanium and beryllium and calcium.The principle of Alizarin red staining is exactly that alizarin red and calcium occur to show
Colour response, generates a kind of wine-colored colored compound, and the calcium tubercle deposited outside the cell of such osteogenic induction is also just contaminated
At peony.
Chondrocyte induction and detection
Since mescenchymal stem cell has Multidirectional Differentiation ability, mescenchymal stem cell is carried out under certain conditions soft
Bone induction can obtain cartilage cell.
Those skilled in that art can be used general method and carry out chondrocyte induction to mescenchymal stem cell, available
Traditional chemical formula: dexamethasone, vitamin C and TGF-B etc.；Or commodity dress cartilage differentiation kit carries out chondrocyte induction
Differentiation, after induction 3 weeks, discovery cell forms the sphere of ellipse.It is combined using alcian blue (Alcian blue) dyestuff soft
The content situation of acid glycosaminoglycan in bone noble cells epimatrix is blue in conjunction with rear positive cell, to evaluate between placenta
The ability and efficiency situation of mesenchymal stem cells cartilage differentiation.
The present invention has the advantages that
(a) cell isolation method uses low concentration clostridiopetidase A IV type, trypsase to combine digestion method in the method for the present invention,
This helps to improve that cell purity, proliferative capacity are strong, shorten primary proliferation time, ensure that the timeliness of cell clinical use.
(b) in the methods of the invention, the cell after tissue digestion does not have to filter operation, and manual steps are few, are easy to produce
Industry normalizing operation.
(c) the method for the present invention uses complete serum-free amplification technique, avoids the risk of serum composition infection.
(d) the method for the present invention is not necessarily to carry out coating processing to Tissue Culture Dish, and operating method is simple, effect stability.
(e) placenta mesenchyma stem cell of the method for the present invention preparation has good differentiation potential, and cellular morphology is big
It is small uniform, after small in size, multiple passage operation, normal caryogram is still kept, the clinical application in later period is conducive to.
(f) it the preparation of general 2-3 weeks or so achievable cell and freezes, method is easy to operate, is easy to program flux grasps
Make, prepares clinical grade placenta mesenchyma stem cell more suitable for mechanisms such as industry cell banks than existing conventional method.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine
Condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated,
Otherwise percentage and number are weight percent and parts by weight.
Embodiment 1
The preparation of primary placenta mesenchyma stem cell
1. Placenta samples are by stringent infective virus detection (hepatitis B, hepatitis C virus, AIDS virus, syphilis and huge
Cell virus) result feminine gender is qualified sample, link is separated into cell.
2. in superclean bench, clip placenta amnion or chorion are organized in 1% mould under hundred grades of clean environments
In element/streptomysin PBS liquid, remaining bloodstain is removed, after being shredded with scissors machinery, is collected into 15ml centrifuge tube, 1:3- is added
4ml volume ratio (clostridiopetidase A IV type, the trypsase of 80U/ml, 0.008wt% working concentration) combines digestive juice；37 DEG C of digestion
After 12-18h, preferably 16h sufficiently dispels mixing, shifts supernatant suspension into new 15ml centrifuge tube, 300-500g, centrifugation
5min harvests cell precipitation.
3. the cell of above-mentioned steps harvest is added 1-2ml erythrocyte cracked liquid, 37 DEG C, after 3-5min, 5ml 1% is added
Penicillin/streptomycin PBS liquid, sufficiently dispels mixing, and 300-500g, centrifugation 5min harvest cell precipitation.
4. above-mentioned steps 2 are harvested cell precipitation, after serum free medium 2ml resuspension is added, it is inoculated in CORNING
In CellBind surface 100mm culture dish, it is placed in 37 DEG C, 5%CO2It is cultivated in cell incubator, the later half amount of 24-48h
Liquid is changed, full dose changes the liquid once within 3-4 days later, and cell fusion degree can reach 80%-90% within 7-10 days.
5. the cell that above-mentioned steps 380%-90% is merged removes culture solution and is added after the cleaning of 4ml PBS liquid
0.05% trypsase 1ml digest 2-3 minute, addition 1-2ml trypsin inhibitor after, dispel mixing move back to 15ml from
In heart pipe, 300-500g, centrifugation 5min abandon supernatant, for primary placenta mesenchyma stem cell of the invention to be cultivated.
Embodiment 2
Serum-free secondary culture, harvest and the passage of primary cell
Serum free medium is added in the primary placenta mesenchyma stem cell obtained in embodiment 1 to be resuspended, it is (thin by 1:3-6
Born of the same parents' quantity dilution ratio) or 3000-6000 cell/cm2Density passage amplification.
After carrying out 1-2 amplification passage, low generation, 10 can be obtained7The above order of magnitude cell concentration carries out -196 DEG C
Low temperature storage, cell carry out fluidic cell surface marker analysis.
Cellular morphology qualification result
Morphological observation is carried out to obtained cell is expanded, as a result as follows:
The placenta mesenchyma stem cell obtained using this separation, serum-free amplification method.Form under inverted microscope is such as
Shown in attached drawing 1, P5 is in spindle shape, polygonal for attached cell, and cellular morphology size is uniform and ability of cell proliferation is strong.
Fluidic cell surface marker analysis
Fluidic cell surface marker analysis is carried out to resulting cell is cultivated in above-mentioned experiment, concrete operations are as follows:
The cell (P5 is for cell) for taking serum-free amplification to obtain, is distributed into 7 pipes, every solencyte is 106More than, respectively plus
Enter (CD73, CD105, CD90, CD34, CD45, FITC Mouse IgG1 and PE Mouse IgG1), be protected from light and be incubated for 15min,
After 0.4ml paraformaldehyde is added, as a result flow cytometer (FACS Calibur) analysis is detailed in Fig. 2 and the following table 1.
Fluidic cell surface marker analysis result of 1 P5 of table for 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%
As the result is shown: placenta mesenchyma stem cell meets international mescenchymal stem cell standard.
Embodiment 3
Cell skeletonization, at rouge and chondrocyte induction Analytical Chemical Experiment
Cell skeletonization breaks up at rouge, cartilage direction induction:
The cell of serum-free amplification in the embodiment of the present invention 2 is taken to pass on to 24 orifice plates, cell confluency degree reaches 80%-
When 90%, suck serum free medium and dead cell, be separately added into skeletonization, at rouge, chondrocyte induction differential medium (Osteogenesis Differentiation Kit、 Adipogenesis
Differentiation Kit、Chondrogenesis Differentiation Kit), full dose is changed within 3-4 days
It is observed under liquid and inverted microscope
As a result:
3.1 adipogenic induction
At fat induction differentiation the 10th day or so, the round fat drips of visible a large amount of yellow were piled up in nucleus week under light microscopic
It encloses, cellular morphology is rounded, and after oil red O stain, fat drips take on a red color, and extends certain induction time, and fat drips constantly become larger.It is attached
Fig. 3 B shows the visible cell containing a large amount of fat drips after oil red O stain.
The result shows that mescenchymal stem cell of the invention has the differentiation potential for being divided into fat cell and tissue.
3.2 osteogenic induction
In Osteoinductive differentiation experiment, after a week, three-dimensional sense is presented in cellular morphology for induction, is constantly formed after proliferation more
Confluent monolayer cells after induction 3 weeks, suck culture medium, after 4% paraformaldehyde fixes 20min, alizarin red S solution are added and dyes 30min,
It can be seen that a large amount of Ca accumulations, are detailed in Fig. 3 A；
The result shows that mescenchymal stem cell of the invention has the differentiation potential for being divided into osteocyte and tissue.
3.3 chondrocyte induction
After chondrocyte induction breaks up liquid induction 2 weeks, discovery cell forms the sphere of ellipse, alcian blue (Alcian
Blue) stained positive is detailed in Fig. 3 C.
The result shows that mescenchymal stem cell of the invention has the differentiation potential for being divided into cartilage cell and tissue.
Embodiment 4
Passage test
In the present embodiment, the placenta mesenchyma stem cell in the 8th generation prepared to embodiment 2 carries out karyotyping.Method
It is as follows:
To the cell in logarithmic growth phase of the 8th generation cell, colchicine processing 2-3h is added and collects broken thin
Born of the same parents fix 60min at 37 DEG C of addition fixer (formaldehyde: glacial acetic acid=3:1), repeat after fixing, and it is aobvious at that drop piece simultaneously carries out G-
Reason and microscopy.
As the result is shown: having normal human karyotype (being detailed in Fig. 4), show to be prepared with the method for the present invention and repeatedly expanded
The cell of harvest has good differentiation potential, and cellular morphology size is uniform, after small in size, multiple passage operation, cell
Normal caryogram is still kept, the clinical application in later period is conducive to.
Embodiment 5-8
Examples 1 and 2 are repeated, the difference lies in that using single enzyme shown in the following table 2.
It for the mescenchymal stem cell of preparation, is detected with the same procedure of embodiment 3 and 4, is as a result listed in table 2.
Embodiment 9
Examples 1 and 2 are repeated, the difference lies in that being cultivated and being passed on using serum-containing media shown in the following table 2.
It for the mescenchymal stem cell of preparation, is detected with the same procedure of embodiment 3 and 4, is as a result listed in table 2.
Embodiment 10-13
Examples 1 and 2 are repeated, the difference lies in that using the clostridiopetidase A IV and tryptose of working concentration shown in the following table 2
Enzyme.
It for the mescenchymal stem cell of preparation, is detected with the same procedure of embodiment 3 and 4, is as a result listed in table 2.
2 embodiment 5-13 of table and result
The above results, which show no matter to use, is applied alone a kind of enzyme, including clostridiopetidase A IV, trypsase or clostridiopetidase A I etc.；
Or improve on the digestion conditions such as enzyme concentration, although can isolated placenta mesenchyma stem cell, in primary cell number
On amount, purity and/or ability of cell proliferation, all it is not so good as the method for the present invention.
In passage link: using serum-containing media and culture method in serum-free of the present invention, though in vitro growth rates and
Cell surface marker is expressed on upper no significant difference, but the placenta mesenchyma stem cell volume that system containing serum free culture system obtains is wanted
Stem cell is filled between the placenta that serum free culture system significantly greater than of the invention obtains.Fig. 5 is shown in the embodiment of the present invention
The placenta mesenchyma stem cell volume size that serum-free amplification method obtains is prepared, in figure as it can be seen that close to 2 peaks of coordinate origin
Figure will be significantly less than tradition containing serum free culture system and obtain cell volume.
It discusses
In order to meet clinical requirement, the preparation of usual placenta mesenchyma stem cell is needed in the separation of sample cell, amplification
Etc. meet the requirement standard of clinical application product in important links.The present invention provides one kind can efficiently, easily separate placenta
It mescenchymal stem cell and based on free serum culture and passes on and the clinical grade placenta mesenchyma stem cell that can produce with high throughput
Preparation method.Even if mescenchymal stem cell cell purity prepared by method of the invention is high, proliferative capacity is strong, through multiple
After passage operation, normal caryogram is still kept.In addition, method provided by the invention is the application neck such as organizational project, cell therapy
Domain provides good seed cell resource, can be widely applied to industrialization cellular resources preservation library or other use mechanism etc..
All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet
It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Claims (3)
1. a kind of preparation method of clinical grade placenta mesenchyma stem cell, which is characterized in that the method includes the steps:
(a) it to the placenta tissue material containing mescenchymal stem cell of acquisition, carries out shredding processing, to obtain the placenta through shredding
Organization material；
(b) the placenta tissue material through shredding obtained to previous step, is digested with combination digestive ferment, to obtain through disappearing
The tissue mixture of change, wherein the combination digestive ferment include final concentration of 50U/ml-100U/ml clostridiopetidase A IV and
0.005wt%-0.01wt% trypsase；
(c) in the tissue mixture from described through digesting, supernatant suspension is taken；
(d) the supernatant suspension of previous step is centrifuged, is precipitated；
(e) to the precipitating of previous step, 1-2ml erythrocyte cracked liquid is added, thus what acquisition was handled through erythrocyte splitting
Mixture；
(f) it to the mixture handled through erythrocyte splitting of previous step, is centrifuged, is precipitated, as primary
Mesenchymal stem cells；
(g) serum-free amplification passage is carried out to the primary mescenchymal stem cell of previous step, faced to obtain through what is passed on
Bed grade placenta mesenchyma stem cell；
Wherein, in step (b), clostridiopetidase A IV and trypsase are added simultaneously；And digestion condition include: at 37 ± 2 DEG C into
Row digestion；Digestion 12-18 hours.
2. the method as described in claim 1, which is characterized in that in step (b), the digestive ferment includes final concentration of
80U/ml clostridiopetidase A IV and 0.008wt% trypsase.
3. the method as described in claim 1, which is characterized in that the placenta tissue material includes placenta amnion tissue, tire
Disk villus membrane tissue or combinations thereof.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005084691A1 (en) * | 2004-03-04 | 2005-09-15 | Susan Oriole Meakin | Method of proliferating precursor cells |
| CN1961069A (en) * | 2004-04-09 | 2007-05-09 | 亚历山大·S·台普利亚申 | Method for obtaining mesenchyme stem cells |
| CN101720354A (en) * | 2006-08-04 | 2010-06-02 | 人类起源公司 | Utilize placenta stem-cell to suppress tumour |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005084691A1 (en) * | 2004-03-04 | 2005-09-15 | Susan Oriole Meakin | Method of proliferating precursor cells |
| CN1961069A (en) * | 2004-04-09 | 2007-05-09 | 亚历山大·S·台普利亚申 | Method for obtaining mesenchyme stem cells |
| CN101720354A (en) * | 2006-08-04 | 2010-06-02 | 人类起源公司 | Utilize placenta stem-cell to suppress tumour |
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