CN107022521A - Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell - Google Patents
Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell Download PDFInfo
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Abstract
Decidua vera tissue freezing of the present invention, the method recovered and be separately cultured mescenchymal stem cell, comprise the following steps：(1) pretreatment of placenta tissue；(2) separation and processing of placenta decidua vera tissue；(3) placenta decidua vera tissue freezes；(4) recovery of placenta decidua vera tissue；(5) the separating mesenchymal stem cell from placenta decidua vera tissue；(6) Secondary Culture of decidua vera stem cell.The present invention is to shred the decidua vera tissue after stripping, the method directly frozen.The cell culture after processing and separation per a tissue is required for regular hour and the consumption of personnel.Therefore recovery carries out the way of stem cell separation and more meets cost benefit relatively by decidua vera tissue freezing and in need when.
Description
Technical field
The invention belongs to cell biology, and in particular to a kind of decidua vera tissue freezing, recover and be separately cultured between
The method of mesenchymal stem cells.
Background technology
With the rise of regenerative medicine, cell therapy is just continued to develop, and mescenchymal stem cell divides because wide material sources
Easy from culture, propagation and Multidirectional Differentiation ability are strong, it has also become the study hotspot in cell therapy field.
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) originates from mesoderm, is adult stem cell
It is a kind of.Because have wide material sources, obtain easily, amplification ability is strong, immunogenicity is low, non-tumorigenesis, can go back to the nest, without Medicine Ethics
The advantages of dispute and as the main selection cell in regenerative medicine, cell therapy.It has been found that this cell can be divided into middle embryo
A variety of mature tissue's cells of layer, such as bone, fat, muscle, cartilage and blood vessel endothelium, discovered in recent years mescenchymal stem cell
Plasticity, can be across differentiation of germinal layers
The non-mesoblastema such as nerve cell, liver cell, renal cells, pneumonocyte, beta Cell of islet.Mesenchyma
Stem cell in organizational project, organ because with Multidirectional Differentiation and itself renewal, low immunogenicity and immunological regulation inhibitory action, moving
There is very wide application prospect in the field of plant and treatment immunologically mediated disease.
The source of mescenchymal stem cell has a lot, including placenta, umbilical cord, Cord blood, marrow, fat, dental pulp, skin etc..
Placenta derived mesenchymal stem cell is compared with mesenchymal stem cells MSCs, with source is sufficient, immunogenicity is low, viral pollution rate
It is relatively low, and without the advantage in terms of social forest dispute, make it have more preferable potential applicability in clinical practice.From a variety of groups in placenta
Knit and can extract mescenchymal stem cell, such as amnion, chorion, decidua, placental lobules and placenta.
Decidua vera is as one of parent part, therefrom isolated decidua mescenchymal stem cell (Decidua-
Derived Mesenchymal Stem Cells, DMSCs) it can be used for the transplanting of mother's own cells or freeze, with pin
To property.Mescenchymal stem cell of the decidua mescenchymal stem cell in terms of phenotype, propagation and cytokine-expressing with other sources
There is difference, with unique property, such as low immunogenicity has caused numerous scientists to pay close attention to, filled between decidua in recent years
The tissue repairs such as the reconstruction of matter stem cell endometrium, pelvic organ prolapse, bladder body reparation have broad application prospects.
But the methods and techniques for separating stem cell from placenta decidua vera tissue are currently not full maturity, it is more difficult to obtain pure
Net mother source mescenchymal stem cell, and it is required for consumption one per processing, separation and the culture of a placenta decidua vera tissue
Fixed human and material resources and time cost.Therefore recovery carries out stem cell by decidua vera tissue freezing and in need when
The way of separation more meets cost benefit relatively.
The content of the invention
Instant invention overcomes shortcoming of the prior art there is provided a kind of decidua vera tissue freezing, recover and be separately cultured
The method of mescenchymal stem cell, can be applied to the preservation of the decidua vera tissue in mother source, and decidua vera tissue can be deep in liquid nitrogen
Low temperature environment is preserved for a long time, it is to avoid cell survival rate is lost in resuscitation process.
In order to solve the above-mentioned technical problem, the present invention is achieved by the following technical solutions：
Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, comprise the following steps：
(1) pretreatment of placenta tissue：Collection placenta and female blood, deposit in placenta tissue collection suit in an aseptic environment
It is interior, label is posted, maintains temperature at 4-22 DEG C, is sent to laboratory and is handled；Take out after placenta, 75% alcohol spray disinfectant tire
After the outer surface 2min of disk tissue, the rapid remained blood and blood clot that flushing tissue outer surface is cleaned with organization protection's liquid, and
The project of detection, which includes glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST), to be detected to placenta and female blood sample；Blister sore
Malicious (EB)；Cytomegalovirus (CMV)；AIDS virus (HIV)；Microspironema pallidum (TP)；Hepatitis B (HBV)；Hepatitis C virus
(HCV)；Thermophilic T cell viral (HTLV), bacterium and fungi etc.；
(2) separation and processing of placenta decidua vera tissue：After the placenta for separating acquisition is cleaned with physiological saline, wall is peeled off
Decidua, and the decidua vera separated is cut into 2mm3Fritter is placed in centrifuge tube；
(3) placenta decidua vera tissue freezes：Deepfreeze under the conditions of decidua vera tissue freezing solution, 4 DEG C is prepared, is then pressed
Often pipe 1mL is dispensed into cryopreservation tube, cooling, is most preserved overnight after -196 DEG C, and liquid nitrogen is transferred to after microorganism detection is feminine gender
It is medium-term and long-term to preserve；
(4) recovery of placenta decidua vera tissue：The placenta decidua vera tissue that step (3) is freezed takes out from liquid nitrogen, and 37
DEG C condition quick-thawing, utilizes mescenchymal stem cell culture medium clean wall decidua tissue, filtering removes waste liquid, so that the wall frozen
Decidua tissue is recovered；
(5) the separating mesenchymal stem cell from placenta decidua vera tissue：Use AccutaseTMCell dissociation buffer is to decidua vera
Tissue is digested, and postdigestive decidua vera is filtered, and mescenchymal stem cell is obtained after washing, mescenchymal stem cell is being faced
Cultivated in bed level serum free medium；Hereafter liquid is changed once within every 3 days, it is dry to mesenchyma during to cell fusion degree up to more than 80%
Cell is passed on, and uses AccutaseTMAfter cell dissociation buffer digestion, with clinical grade free serum culture Secondary Culture；
(6) Secondary Culture of decidua vera stem cell：It is available when attached cell fusion rate reaches 80% or so
AccutaseTMAttached cell is departed from plate bottom by cell dissociation buffer, supernatant is removed after centrifugation, and add mescenchymal stem cell
Culture medium suspension cell again, is inoculated in T25 Tissue Culture Flasks and is passed on, and carry out amplification cultivation；Hereafter liquid one is changed within every 3 days
It is secondary until fusion rate reaches 80% after, produce, passed on again if necessary.
Further, the antibiotic that organization protection's liquid described in step (1) is DMEM/F12 and 1% is constituted；Antibiotic is
Penicillin, streptomysin or amphotericin B.
Further, the step of decidua vera is peeled off described in step (2) peels off the amnion on placenta surface for operating theater instruments, completely
The fine hair membrane tissue of separation placenta tissue edge connection and the decidua vera tissue for being attached to surface, blood is cleaned with organization protection's liquid
And blood clot, decidua vera tissue is scraped, is collected in after cleaning in 50ml centrifuge tubes.
Further, the cooling process described in step (3) is as follows：Prior to 4 DEG C balance 30min, then with 1 DEG C/min speed
Degree is down to -5 DEG C, is then down to -54 DEG C with 21 DEG C/min speed, then be warming up to -21 DEG C with 10 DEG C/min speed, then with
2 DEG C/min speed is down to -40 DEG C, is then down to -80 DEG C with 10 DEG C/min speed, is placed in afterwards in liquid nitrogen container, depth storage
Deposit.
Further, to contain trehalose, DMSO, human serum albumin and serum-free complete for the tissue freezing solution described in step (3)
Full culture medium.
Further, the composition of the clinical grade serum free medium described in step (5) is：Basal medium DMEM/F12,
20-50ng/mL platelet derived growth factors (PDGF-BB), 10-20ng/mL basic fibroblast growth factors (bFGF),
5-10ng/mL stem cell factors (SCF), 10-20ng/mL epithelical cell growth factors (EGF), 3-5mg/mL turn iron egg
In vain, 20-100ug/mL vitamin Cs, 1~5mmol/ml Glus, 5-30g/L human serum albumins (HSA), 5-10 μ g/ml
Insulin, 30-40ng/ml FTNs, 1-2x10-6Mol/L hydrocortisones, 100U/ml penicillin and 100mg/ml chains
Mycin.
Further, the Accutase described in step (5)TMCell dissociation buffer is that one kind includes proteolytic enzyme and collagen
A kind of cell dissociation buffer of enzymatic activity.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) present invention is to shred the decidua vera tissue after stripping, the method directly frozen.Processing per a tissue and
Cell culture after separation is required for regular hour and the consumption of personnel.Therefore by decidua vera tissue freezing and in need
When recovery carry out stem cell separation way more meet cost benefit relatively.
(2) the inventive method can be applied to the preservation of the decidua vera tissue in mother source, and decidua vera tissue can be in liquid nitrogen
Profound hypothermia environment is preserved for a long time, it is to avoid cell survival rate is lost in resuscitation process, and recovery can obtain mescenchymal stem cell, cell life
Long status is good, multiplication capacity is strong, cell survival rate more than 96%, Multidirectional Differentiation performance is good, with thin to Gegenbaur's cell, fat
Born of the same parents, the ability of Chondrocyte Differentiation.
(3) Accutase that the present invention is usedTMCell dissociation buffer, is pancreatin/EDTA with protease and collagenase activity
Replacement products, can gently effective decidua vera tissue and mescenchymal stem cell, freeze rear cell and have higher anabiosis rate.Simultaneously should
During digestive juice vitellophag, cleaning or neutralization reaction are not required to, the passage time is saved.
(4) present invention uses serum free medium, and adds the cell factor of different component, to promote mescenchymal stem cell
Propagation and suppress differentiation, meanwhile, the variation of quality between serum batch can be avoided, it is to avoid toxic action and serum of the serum to cell
Source contact scar, the amplification cultivation for being adapted to clinical grade mescenchymal stem cell is used.
(5) it is mother source that the inventive method, which obtains mescenchymal stem cell, and the fetus originated different from umbilical cord and placenta is dry thin
Born of the same parents, the mescenchymal stem cell of preparation can feed back the treatment for a variety of diseases of puerpera itself as autologous stem cells, while can be with
Customizing a variety of stem cell class beauty and anti-ageing product by the mescenchymal stem cell is used for puerpera itself.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, together with embodiments of the present invention for explaining the present invention,
It is not construed as limiting the invention, in the accompanying drawings：
Fig. 1 difference generation decidua vera mescenchymal stem cell aspect graphs, A:P1 generations；B：P3 generations；C:P5 generations；D：P7 generations
Fig. 2 decidua vera growth of mesenchymal stem cells curve maps
Fig. 3 decidua vera mescenchymal stem cell flow cytometer detection figures
Fig. 4 decidua veras mescenchymal stem cell is into fat Osteoblast Differentiation figure
Fig. 5 decidua vera mescenchymal stem cells STR is identified
Embodiment
The preferred embodiments of the present invention are illustrated below in conjunction with accompanying drawing, it will be appreciated that preferred reality described herein
Apply example to be merely to illustrate and explain the present invention, be not intended to limit the present invention.
The method of decidua vera tissue freezing
(1) collection and pretreatment of placenta tissue
Collection placenta and female blood, deposit in placenta tissue collection suit, post label, maintain temperature in an aseptic environment
At 4-22 DEG C, it is sent to laboratory and is handled；Take out after placenta, the outer surface 2min of 75% alcohol spray disinfectant placenta tissue
Afterwards, it is rapid that the remained blood and blood clot for rinsing tissue outer surface are cleaned with organization protection's liquid.And placenta and female blood sample are entered
Row detection, the project of detection includes glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST)；Herpesviral (EB)；Cytomegalovirus
(CMV)；AIDS virus (HIV)；Microspironema pallidum (TP)；Hepatitis B (HBV)；Hepatitis C virus (HCV)；Thermophilic T cell virus
(HTLV), bacterium and fungi etc..
(2) separation and processing of placenta decidua vera tissue
Operating theater instruments peels off the amnion on placenta surface, the fine hair membrane tissue of complete separation placenta tissue edge connection and attachment
Decidua vera tissue in surface, blood and blood clot are cleaned with organization protection's liquid, are scraped decidua vera tissue, are collected in after cleaning
In 50ml centrifuge tubes.
(3) placenta decidua vera tissue freezes
Prepare decidua vera tissue freezing solution：In the umbilical cord tissue frozen stock solution containing 2mmol/ml trehaloses, 15%DMSO,
2.5% human serum albumin+Serum-free complete medium, the cold liquid storage prepared is placed on 4 DEG C of refrigerators and kept in.
The decidua vera tissue shredded and the frozen stock solution prepared are added in cryopreservation tube, cryopreservation tube is then put into program drop
Wen Yizhong carries out Programmed freezing, and cooling process is as follows：Prior to 4 DEG C balance 30min, are then down to -5 DEG C with 1 DEG C/min speed,
Then -54 DEG C are down to 21 DEG C/min speed, then -21 DEG C is warming up to 10 DEG C/min speed, then with 2 DEG C/min speed
Degree is down to -40 DEG C, is then down to -80 DEG C with 10 DEG C/min speed, is placed in afterwards in liquid nitrogen container, depth storage.
Cooled by programmed cooling instrument, gradient cooling freezes decidua vera tissue, during decidua vera tissue freezing
Heating and cooling rule setting freeze speed accordingly, can to greatest extent protective bulkhead decidua tissue cell it is injury-free.It is logical
More than decidua vera tissue 15-20 can be preserved by crossing the decidua vera tissue freezing method of the invention used, be entered according to service condition
Row resuscitation team block is used for cell culture.
The method of decidua vera tissue recovery
The decidua vera tissue of freezing is taken out from liquid nitrogen, is placed in water bath with thermostatic control to thaw to half frozen stock solution and starts to melt
Change, using serum free medium (wherein comprising basal medium DMEM/F12,50ng/mL platelet derived growth factor
(PDGF-BB), 20ng/mL basic fibroblast growth factors (bFGF), 5ng/mL stem cell factors (SCF), 10ng/
ML epithelical cell growth factors (EGF), 3mg/mL transferrins, 50ug/mL vitamin Cs, 5mmol/ml Glus,
20g/L human serum albumins (HSA), 50 μ g/ml insulin, 340ng/ml FTNs, 2x10-6mol/L hydrocortisones,
100U/ml penicillin and 100mg/ml streptomysins) clean wall decidua tissue is carried out, waste liquid is removed using 100um filters, weight
Clean 3 times again.
Decidua vera mescenchymal stem cell is separated and cultural method
Cumulative volume according to decidua vera tissue is washed adds the Accutase of 1 times of volumeTMDigestive juice, 37 DEG C of digestion tissues of solution
Block 0.5h, entirely digests and is carried out in constant temperature gas phase shaking table, after the completion of digestion, digestive juice screen filtration, with the filter screen of 200 mesh
Filtering, removes indigested tissue, and 1500g centrifuges 10min and obtains mescenchymal stem cell after washing.
Mescenchymal stem cell is obtained by 2 × 104/ cm2 is inoculated in cell material culture dish, using serum free medium, puts 37
DEG C, 5%CO2 incubator cultures change liquid after 3 days, discard non-adherent cell, and later every 3 days half amounts change liquid.Reached to cell fusion degree
When more than 80%, mescenchymal stem cell is passed on, Accutase is usedTMAfter cell dissociation buffer digestion, with clinical grade without blood
Clear subculture culture.
AccutaseTM cell dissociation buffers are a kind of a kind of cell dissociations for including proteolytic enzyme and collagenase activity
Liquid, is the perfect replacement products of pancreatin/EDTA digestive juices, gentleer to stem cell digestion, can increase cell yield and survival
Rate.
Utilize AccutaseTMAttached cell is departed from plate bottom by cell dissociation buffer, supernatant is removed after centrifugation, and add
Mescenchymal stem cell culture medium suspension cell again, is inoculated in T75 Tissue Culture Flasks and is passed on, and carry out amplification cultivation；This
Liquid is changed within every 3 days afterwards once until after fusion rate reaches 80%, producing, being passed on again if necessary.
And take part cell to carry out bacterium, fungi and viral pathogenic microorganism inspection, cell viability detection, ability of cell proliferation
Detection, streaming antibody test, Multidirectional Differentiation detection, cell STR identification detection etc..
The measure of cell growth curve
Taking the mescenchymal stem cell pancreatin in the 3rd generation to be inoculated with after digesting, (density is 1.0 × 104Individual/mL) in 24 orifice plates, put
Cultivated in 37.5 DEG C, the incubator containing 5%CO2.Randomly select 3 hole pancreatin daily to digest, count, the meter 3 per hole
Number of times is averaged, and continues 7d.The tetrazolium bromide (MTT) that the concentration for taking every hole plus 20 μ l is 5mg/mL, continues to cultivate；After 4 hours
Carefully remove culture supernatant；100 μ l dimethyl sulfoxide (DMSO) is added per hole, micro oscillator shakes 5 minutes；Put and surveyed on ELIASA
Growth curve is drawn out after absorbance value under 490nm, statistical analysis.P3 is for growth of mesenchymal stem cells curve such as Fig. 2 institutes
Show.Fig. 1 difference generation decidua vera mescenchymal stem cell aspect graphs.
FCM analysis
5 × 106 mescenchymal stem cells are digested to single cell suspension；Concentration is adjusted after being washed twice with PBS to 1 × 105/
Ml, flow cytomery cell surface marker positive indication CD29, CD90, CD73 and CD105, negative indication CD19, CD34,
CD45, HLA-DR, positive cell rate is more than 99%, and negative indication meets less than 1%, meets the feature of mescenchymal stem cell,
As shown in Figure 3.
Multidirectional Differentiation ability is detected
The P5 of 60mm culture dish cultures is taken respectively for mescenchymal stem cell, it is to be generated when growing to 80%-90% degree of converging, remove
Culture medium, is washed 3 times with PBS.Lipoblast induction liquid is added (to say into fat differential medium (ADM) with reference to mescenchymal stem cell
Bright book is prepared) and Gegenbaur's cell induction liquid (mescenchymal stem cell is compareed into fat differential medium (ODM) specification Fiber differentiation
Group adds conventional complete culture solution, and a nutrient solution is changed per 2-3d.Daily observation cell growth state and the form of cell become
Change, carrying out alizarin red, oil red O stain to the Gegenbaur's cell after induction, lipoblast in good time identifies.It is visible after oil red O stain
The fat drips of the intracellular generation of induction group can dye red, be found after alizarin red S dyeing, cell shows bright at the tubercle of induced synthesis
Red, shows that mescenchymal stem cell prepared by this method has Multidirectional Differentiation ability, as a result as shown in Figure 4.
Cell STR identification
The decidua vera mescenchymal stem cell 5 × 105 and fetal cord tissue in same individual source, the dolantin from are taken respectively
Connection AGCU EX22 fluorescence detection reagent kits are expanded to sample, and automatic laser is carried out on the type genetic analyzers of ABI 3730
Fluorescent capillary electrophoresis tube detected, testing result is free of as shown in figure 5, result shows and can obtain by the inventive method pure
Mother source mescenchymal stem cell of foetal DNA pollution.
Finally it should be noted that：The preferred embodiments of the present invention are these are only, are not intended to limit the invention, although
The present invention is described in detail with reference to embodiment, for those skilled in the art, it still can be to foregoing
Technical scheme described in each embodiment is modified, or to which part technical characteristic carry out equivalent substitution, but it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc. should be included in the protection of the present invention
Within the scope of.
Claims (7)
1. decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, it is characterised in that including following step
Suddenly：
(1) pretreatment of placenta tissue：Collection placenta and female blood, are deposited in placenta tissue collection suit in an aseptic environment,
Label is posted, maintains temperature at 4-22 DEG C, is sent to laboratory and is handled；Take out after placenta, 75% alcohol spray disinfectant placenta
It is rapid that the remained blood and blood clot for rinsing tissue outer surface are cleaned with organization protection's liquid and right after the outer surface 2min of tissue
Placenta and female blood sample are detected that the project of detection includes glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST)；Herpesviral
(EB)；Cytomegalovirus (CMV)；AIDS virus (HIV)；Microspironema pallidum (TP)；Hepatitis B (HBV)；Hepatitis C virus
(HCV)；Thermophilic T cell viral (HTLV), bacterium and fungi etc.；
(2) separation and processing of placenta decidua vera tissue：After the placenta for separating acquisition is cleaned with physiological saline, peel off wall and slough off
Film, and the decidua vera separated is cut into 2mm3Fritter is placed in centrifuge tube；
(3) placenta decidua vera tissue freezes：Decidua vera tissue freezing solution is prepared, deepfreeze under the conditions of 4 DEG C, then by often managing
1mL is dispensed into cryopreservation tube, cooling, is most preserved overnight after -196 DEG C, is transferred to after microorganism detection is feminine gender in liquid nitrogen long
Phase preserves；
(4) recovery of placenta decidua vera tissue：The placenta decidua vera tissue that step (3) is freezed takes out from liquid nitrogen, 37 DEG C of bars
Part quick-thawing, using mescenchymal stem cell culture medium clean wall decidua tissue, filtering removes waste liquid, so that the decidua vera frozen
Tissue recovery；
(5) the separating mesenchymal stem cell from placenta decidua vera tissue：Use AccutaseTMCell dissociation buffer is to decidua vera tissue
Digested, postdigestive decidua vera is filtered, mescenchymal stem cell is obtained after washing, by mescenchymal stem cell in clinical grade
Cultivated in serum free medium；Hereafter liquid is changed once within every 3 days, during to cell fusion degree up to more than 80%, to mescenchymal stem cell
Passed on, use AccutaseTMAfter cell dissociation buffer digestion, with clinical grade free serum culture Secondary Culture；
(6) Secondary Culture of decidua vera stem cell：When attached cell fusion rate reaches 80% or so, using AccutaseTMCarefully
Attached cell is departed from plate bottom by born of the same parents' digestive juice, supernatant is removed after centrifugation, and add mescenchymal stem cell culture medium again
Suspension cell, is inoculated in T25 Tissue Culture Flasks and is passed on, and carry out amplification cultivation；Hereafter liquid is changed within every 3 days once until fusion
After rate reaches 80%, produce, passed on again if necessary.
2. decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, its feature according to claim 1
It is, organization protection's liquid described in step (1), the antibiotic for being DMEM/F12 and 1% is constituted；Antibiotic is penicillin, chain
Mycin or amphotericin B.
3. decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, its feature according to claim 1
It is, the step of decidua vera is peeled off described in step (2) peels off the amnion on placenta surface, complete separation placenta group for operating theater instruments
The fine hair membrane tissue of selvedge edge connection and the decidua vera tissue for being attached to surface, blood and blood clot are cleaned with organization protection's liquid,
Decidua vera tissue is scraped, is collected in after cleaning in 50ml centrifuge tubes.
4. decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, its feature according to claim 1
It is, the cooling process described in step (3) is as follows：Prior to 4 DEG C balance 30min, are then down to -5 with 1 DEG C/min speed
DEG C, -54 DEG C then are down to 21 DEG C/min speed, then -21 DEG C are warming up to 10 DEG C/min speed, then with 2 DEG C/min
Speed be down to -40 DEG C, be then down to -80 DEG C with 10 DEG C/min speed, be placed in afterwards in liquid nitrogen container, depth storage.
5. decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, its feature according to claim 1
It is, the tissue freezing solution described in step (3) contains trehalose, DMSO, human serum albumin and Serum-free complete medium.
6. decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, its feature according to claim 1
It is, the composition of the clinical grade serum free medium described in step (5) is：Basal medium DMEM/F12,20-50ng/mL
Platelet derived growth factor (PDGF-BB), 10-20ng/mL basic fibroblast growth factors (bFGF), 5-10ng/mL
Stem cell factor (SCF), 10-20ng/mL epithelical cell growth factors (EGF), 3-5mg/mL transferrins, 20-
100ug/mL vitamin Cs, 1~5mmol/ml Glus, 5-30g/L human serum albumins (HSA), 5-10 μ g/ml pancreas islet
Element, 30-40ng/ml FTNs, 1-2x10-6mol/L hydrocortisones, 100U/ml penicillin and 100mg/ml strepto-s
Element.
7. decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell, its feature according to claim 1
It is, the Accutase described in step (5)TMCell dissociation buffer is a kind of to include the one of proteolytic enzyme and collagenase activity
Plant cell dissociation buffer.
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