CN106434544A - Method for acquiring chorion mesenchymal stem cells and kit - Google Patents
Method for acquiring chorion mesenchymal stem cells and kit Download PDFInfo
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- CN106434544A CN106434544A CN201610983609.4A CN201610983609A CN106434544A CN 106434544 A CN106434544 A CN 106434544A CN 201610983609 A CN201610983609 A CN 201610983609A CN 106434544 A CN106434544 A CN 106434544A
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention discloses a method for acquiring chorion mesenchymal stem cells and a kit. The method for acquiring the chorion mesenchymal stem cells includes subjecting chorion to digestive treatment with a mixed enzyme preparation so as to obtain a digestive product; isolating the chorion mesenchymal stem cells from the digestive product, wherein the mixed enzyme preparation comprises pancreatin, collagenase IV and collagenase II. The method for acquiring the chorion mesenchymal stem cells is capable of acquiring a great number of high-purity and high-activity chorion mesenchymal stem cells.
Description
Technical field
The present invention relates to bioengineering field.In particular it relates to the method obtaining chorion mescenchymal stem cell
And kit.
Background technology
Chorion mescenchymal stem cell is closely related with the distribution of fine hair, be distributed in that fine hair is subendothelial more and fine hair near
Interstitial.Chorion mescenchymal stem cell because draw materials conveniently, operation no invasive, multi-lineage potential is big, multiplication capacity is strong, exempt from
Epidemic focus is low, pathogen contamination risk low advantage and become important cells source and the most potential applicability in clinical practice of regenerative medicine
Function stem cell.
However, the method for currently acquired chorion mescenchymal stem cell still has much room for improvement.
Content of the invention
It is contemplated that at least solving at least one technical problem present in prior art to a certain extent.
It should be noted that the present invention is to be completed based on the following discovery of inventor：
The chorion mescenchymal stem cell separation method commonly used at present is to shred chorion directly to be trained with tissue block method afterwards
Support, or adopt enzyme digestion, but enzymic digestion method is complicated, the multiple enzymic digestion of need it is easy to cause cell damage it is difficult to
Obtain a large amount of and higher chorion mescenchymal stem cell of purity.
Inventor finds, during collagenase treatment, the factor such as the species of enzyme, consumption, digestion time significantly affects enzyme and disappears
Change treatment effect, the especially species of enzyme, and then affect yield and the activity of chorion mescenchymal stem cell.Inventor is through deeply
Research finds, the digestion effect of single enzyme not good it is difficult to obtain a large amount of and higher chorion mescenchymal stem cell of purity, and mix
Synthase can digest to chorion effectively, thus obtaining the chorion mesenchymas a large amount of, purity is higher and activity is higher
Stem cell.Further, it is surprisingly found by the inventors that, the digestion effect of the mixed enzyme of pancreatin, clostridiopetidase A IV and clostridiopetidase A II
Most preferably.
For this reason, in one aspect of the invention, the present invention proposes a kind of method obtaining chorion mescenchymal stem cell.
According to embodiments of the invention, methods described includes：Using mixing enzyme preparation, chorion is carried out digestion process, obtain digestion and produce
Thing；And separate described chorion mescenchymal stem cell from described digestion product, wherein, described mixing enzyme preparation includes：Pancreas
Enzyme；Clostridiopetidase A IV；And clostridiopetidase A II.
Using digestion process method, can be by the protein degradation between cell in fine hair membrane tissue, thus isolating list
Individual cell.Inventor finds, single enzyme is difficult to by fine hair membrane digestion completely, and obtained chorion mescenchymal stem cell amount is relatively
Few, thus need repeatedly to be digested.But, repeatedly digestion easily causes the damage of cell it is difficult to obtain a large amount of and activity relatively
High chorion mescenchymal stem cell.And then, inventor finds, the effect being digested using mixing enzyme preparation is preferable, and no
Need to repeatedly digest, to reduce the damage to cell.Inventor includes pancreatin, clostridiopetidase A IV and collagen it was unexpectedly observed that adopting
The mixing enzyme preparation of enzyme II is digested, and is obtained in that the chorion mescenchymal stem cells a large amount of, purity is higher and activity is higher.
However, the effect on driving birds is not good using other kind of fermentoid.
According to embodiments of the invention, the method for described acquisition chorion mescenchymal stem cell can also have following adding
Technical characteristic：
According to embodiments of the invention, described mixing enzyme preparation includes：The pancreatin of 0.05 mass %～0.25 mass %；1
The clostridiopetidase A IV of～2 μ g/mL；And 0.1 mass % clostridiopetidase A II.Inventor it was unexpectedly observed that pancreatin, clostridiopetidase A IV and
The concentration of clostridiopetidase A II significantly affects digestive efficiency, and then affects yield, purity and the activity of chorion mescenchymal stem cell.?
Under above-mentioned optimum melting concn, the yield of chorion mescenchymal stem cell, purity and activity are higher.Concrete reality according to the present invention
Apply example, in mixing enzyme preparation pancreas enzyme concentration can for 0.08 mass %, 0.10 mass %, 0.13 mass %, 0.18 mass % or
0.21 mass %.When the excessive concentration of pancreatin, clostridiopetidase A IV and clostridiopetidase A II, chorion mescenchymal stem cell will be caused damage
Wound is so as to activity decrease, if pancreatin, clostridiopetidase A IV and clostridiopetidase A II concentration is too low it is impossible to abundant digest, thus make to obtain
The yield of chorion mescenchymal stem cell and purity are relatively low.Thus, acquisition chorion mesenchyma according to embodiments of the present invention is done
The method of cell be obtained in that in a large number, purity is higher and active preferable chorion mescenchymal stem cell.
According to embodiments of the invention, described mixing enzyme preparation and chorial amount ratio are 0.5～2:1, preferably 1:1.
Inventor finds, the addition of mixing enzyme preparation significantly affects digestion process effect, and then affects chorion mescenchymal stem cell
Yield and activity.Inventor is it was unexpectedly observed that working as mixing enzyme preparation with chorial amount ratio is 0.5～2:1, preferably 1:1
When, it is obtained in that the chorion mescenchymal stem cells a large amount of, purity is higher and activity is higher.If addition is excessive, easily cause
Cellular damage, activity decrease.
In practical situations both, those skilled in the art can select digestion process condition as needed, such as reaction temperature,
Time, considered critical is not made to this application, as long as chorion mescenchymal stem cell can be obtained.According to the present invention one
A little embodiments, described digestion process is concussion 30～60min under 35～37 DEG C of temperature, the rotating speed of 100～200rpm.According to
The preferred embodiments of the present invention, described digestion process is concussion 30min under 37 DEG C of temperature, the rotating speed of 150rpm.Inventor
It was unexpectedly observed that being obtained in that the chorion mescenchymal stem cells a large amount of, purity is higher and activity is higher with this understanding.Digestion
Treatment temperature significantly affects enzymatic activity, and the enzyme activity that all will result in too high or too low for temperature is too low, leads to digestive efficiency to reduce, gained
The cell concentration arriving is less.Digestion process overlong time, can cause cellular damage so as to activity reduces, the time is too short, and enzymic digestion is not
Fully, obtained cell concentration is less.If concussion overlong time, the structure of cell will be destroyed, cause to damage.
According to a particular embodiment of the invention, after digestion process, by adding hyclone in digestion reaction system
(FBS), to terminate digestion reaction.
It should be noted that strictly not limiting for the method separating chorion mescenchymal stem cell from digestion product
Fixed, as long as being obtained in that chorion mescenchymal stem cell.According to embodiments of the invention, the suede higher in order to obtain purity
Trichilemma mescenchymal stem cell, described separation includes：Described digestion product is filtered, is collected filtrate；Described filtrate is carried out
Centrifugation, collects cell；And described cell is carried out, to obtain described chorion mescenchymal stem cell.Thereby, it is possible to
Effectively remove and be attached to digestive juice on cell, and avoid cell being caused damage, thus obtain purity higher and active relatively
Strong chorion mescenchymal stem cell.
According to embodiments of the invention, methods described includes：A size of 4cm × 4cm will be cut into using mixing enzyme preparation
Described chorion carry out digestion process, obtain digestion product；Described digestion product is diluted, and utilizes 100 eye mesh screens
Obtained dilution is filtered, is collected filtrate；And described filtrate is centrifuged 6min under the rotating speed of 1200rpm, abandon
Supernatant, obtains described chorion mescenchymal stem cell.
Inventor finds, chorion is cut to a size of 4cm × 4cm so that carrying out the cell area of digestion reaction relatively
Greatly, digestion reaction is promoted fully to occur.Then, digestion product is diluted, in order to subsequent filter.Then dilution is sieved,
To remove bulk tissue.Additionally, filtrate is centrifuged 6min under 1200rpm, can either sedimentation cell, be easy to collect, simultaneously relatively
Destroy structure and the activity of cell littlely.Thus, it is possible to obtain the chorion mesenchymas a large amount of, purity is higher and activity is higher do
Cell.
According to embodiments of the invention, methods described further includes that amplification is processed, and described amplification processes and includes：Will be described
Chorion mescenchymal stem cell is resuspended in culture medium, obtains mixed liquor；Described mixed liquor is inoculated in described culture medium, 37
DEG C, 5%CO2Under cultivate to clone occurs, long to passing on.Inventor finds, separates the chorion mescenchymal stem cell obtaining and is
Individual cells, are expanded to obtain substantial amounts of chorion mescenchymal stem cell.Under above-mentioned optimum amplification treatment conditions,
Its activity can also be made while amplification to improve further.
In another aspect of this invention, the present invention proposes a kind of kit.According to embodiments of the invention, described reagent
Box includes：Pancreatin；Clostridiopetidase A IV；And clostridiopetidase A II.According to a particular embodiment of the invention, described kit is used for obtaining suede
Trichilemma mescenchymal stem cell.
Using digestion process method, can be by the protein degradation between cell in fine hair membrane tissue, thus isolating list
Individual cell.Inventor finds, single enzyme is difficult to by fine hair membrane digestion completely, and obtained chorion mescenchymal stem cell amount is relatively
Few, thus need repeatedly to be digested.But, repeatedly digestion easily causes the damage of cell it is difficult to obtain a large amount of and activity relatively
High chorion mescenchymal stem cell.And then, inventor finds, the effect being digested using mixing enzyme preparation is preferable, and no
Need to repeatedly digest, to reduce the damage to cell.Inventor includes pancreatin, clostridiopetidase A IV and collagen it was unexpectedly observed that adopting
The mixing enzyme preparation of enzyme II is digested, and is obtained in that the chorion mescenchymal stem cells a large amount of, purity is higher and activity is higher.
According to embodiments of the invention, described kit includes：The pancreatin of 0.05 mass %～0.25 mass %；1～2 μ
The clostridiopetidase A IV of g/mL；And 0.1 mass % clostridiopetidase A II.Inventor is it was unexpectedly observed that pancreatin, clostridiopetidase A IV and collagen
The concentration of enzyme II significantly affects digestive efficiency, and then affects yield, purity and the activity of chorion mescenchymal stem cell.Above-mentioned
Under optimal concentration, the yield of chorion mescenchymal stem cell, purity and activity are higher.According to a particular embodiment of the invention, pancreas
Enzyme concentration can be 0.08 mass %, 0.10 mass %, 0.13 mass %, 0.18 mass % or 0.21 mass %.When pancreatin, glue
Protoenzyme IV and the excessive concentration of clostridiopetidase A II, will cause to chorion mescenchymal stem cell to damage so as to activity decrease, if pancreas
Enzyme, clostridiopetidase A IV and clostridiopetidase A II concentration is too low it is impossible to abundant digest, thus the chorion mescenchymal stem cell making to obtain
Yield and purity are relatively low.Thus, kit according to embodiments of the present invention be obtained in that in a large number, purity higher and active preferably
Chorion mescenchymal stem cell.
The additional aspect of the present invention and advantage will be set forth in part in the description, and partly will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description
The above-mentioned and/or additional aspect of the present invention and advantage will become from reference to the description to embodiment for the accompanying drawings below
Substantially and easy to understand, wherein：
Fig. 1 shows microphotograph according to an embodiment of the invention, and wherein, (A) is to amplify 40 times, and (B) is to put
Big 100 times；
Fig. 2 shows cell phenotype identification and analysis figure according to an embodiment of the invention, and wherein, (A) does thin for interstitial
Born of the same parents indicate CDl05；(B) it is interstital stem cell mark CD90；(C) it is interstital stem cell mark CD73；(D) it is hematopoietic cell mark
CD19；(E) it is hematopoietic cell mark CD34；(F) it is hematopoietic cell mark CD45；(G) it is hematopoietic cell mark CD14；(H) it is
Human leucocyte antigen (HLA) HLA-DR；
Fig. 3 shows the microphotograph that multiplication factor according to an embodiment of the invention is 400 times, and wherein (A) is
Lipoblast, coloring agent is oil red O；(B) it is early stage Gegenbaur's cell, coloring agent is alizarin red；(C) it is late osteoblastic, dye
Toner is Von Kossa；(D) for the HE dyeing of chondroblast；(E) it is chondroblast, coloring agent is A Erxinlan；(F)
For chondroblast, coloring agent is toluidine blue；And
Fig. 4 shows cell viability analysis chart according to an embodiment of the invention.
Specific embodiment
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that it is following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carry out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, be can by city available from conventional products.
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that it is following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carry out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, be can by city available from conventional products.
Embodiment
In this embodiment, obtain chorion mescenchymal stem cell in following manner：
(1) chorion avillosum is cut to 4cm × 4cm size, physiology salt is washed three times, will kill in a large scale net as far as possible；
(2) again the 5g tissue block cleaned is transferred in 10cm plate, every plate adds the mixing enzyme preparation of 5ml, wherein
Containing 0.25% pancreatin, the clostridiopetidase A II of 2 μ g/mL clostridiopetidase As IV and 0.1 mass %, shake under 37 DEG C of temperature, the rotating speed of 150rpm
Swing 30min.Add hyclone to terminate pancreatin digestion, and add physiological saline in obtained digestion product, dilute 5 times,
Obtain dilution；
(3) dilution is passed through 100 mesh sieve net filtrations, collects filtrate, 1200rpm is centrifuged 6min, physiology salt is washed twice,
Abandon supernatant, obtain chorion mescenchymal stem cell；
(4) with 2ml primary culture medium resuspended chorion mescenchymal stem cell and inoculate, 37 DEG C, 5%CO2Lower culture until
Clone occurs, and long to passing on, period changes liquid once every 48h, to obtain the chorion mescenchymal stem cell expanding.
Comparative example
Method according to embodiment obtains chorion mescenchymal stem cell, and difference is, in step (2), only uses 0.1 matter
The clostridiopetidase A II of amount % is digested.
Chorion mescenchymal stem cell after embodiment amplification is detected
1. cellular morphology：
Chorion mescenchymal stem cell is examined under a microscope, result is as shown in Figure 1.As can be seen that cell attachment, it is in
Fusiformis, lamellar growth.
2. cell phenotype identification：
Take the chorion mescenchymal stem cell reaching for 3 generations, become single cell suspension after digestion, by 4 × 105Individual often pipe is carried out point
Dress.1 × PBS washes once, and 300g is centrifuged 5min, abandons supernatant.100 μ l 1 × PBS re-suspended cells, often pipe be separately added into and carry fluorescence
Anti- CD90-FITC, CD73-PE, CD105-PE, CD34-PE, CD19-PE, CD45-PE, CD14-FITC and HLA- for mark
DR-PE (purchased from BD Pharmingen company of the U.S., by specification gives amount), if one manages as straight labeling antibody PE-isotype blank
Comparison, one manages as straight labeling antibody FITC-isotype blank, 4 DEG C of shaking tables, lucifuge reaction 40min.1 × PBS 3 times,
300g centrifugation 5min every time, 1% paraformaldehyde is fixed, 4 DEG C of placements, flow cytomery, and result as shown in Figure 2 and Table 1, can
To find out, cell phenotype is homogeneous, and cell expresses mesenchymal cell markers CD73, CD90, CD105, does not express hematopoietic cell markers
CD34, CD14, CD45, CD19, and do not express human leucocyte antigen (HLA) HLA-DR, comply fully with " international cell therapy association " with regard to
The standard that mescenchymal stem cell surface marker specifies.
Table 1 flow cytomery
| Cell phenotype | Detected value | Normal reference value |
| CD90 | 100% | >95% |
| CD73 | 98.33% | >95% |
| CD105 | 99.21% | >95% |
| CD34 | 0.10% | <2% |
| CD45 | 0% | <2% |
| CD14 | 0.33% | <2% |
| CD19 | 0.09% | <2% |
| HLA-DR | 0.% | <2% |
3. three is differentiation：
3.1 fat induction differentiation：
3.1.1 take and reach the chorion mescenchymal stem cell in 3 generations with 1.0 × 105/ hole is inoculated in 24 well culture plates, treats
When cell density reaches 100%, abandon the one-tenth fat induction A liquid that supernatant (gently being siphoned away with liquid-transfering gun) addition configures in advance
(adipogenic induction primary culture medium), changes induction B liquid (adipogenic induction maintenance culture medium) again into after 3 days.A liquid is changed again into, such as after 24h
This circulates 3-5 cycle, after the completion of change old B liquid with B liquid, with changing once, then place seven days within every three days, obtain induction carefully
Born of the same parents.
3.1.2 oil red O stain identification fat drips are formed
Inducing cell removes culture medium, and PBS washes once, adds 4% paraformaldehyde to fix 30min, distillation is washed 3 times, former
Liquid (0.5% oil red O) 6ml, plus distilled water 4ml, stand 5～10min, Filter paper filtering；Dyeing 20min, distillation washing 3 times, mirror
Lower observation is taken a picture, as shown in Figure 3.As can be seen that obvious fat drips after induction, oil red O stain is positive, and illustrates that cell has
There is the stronger ability to Adipocyte Differentiation.
3.2 Osteoinductive differentiation
3.2.1 take and reach the chorion mescenchymal stem cell in 3 generations with 5 × 104/ hole is inoculated in 24 well culture plates, normally
Abandon the Osteogenic Induction Medium that supernatant (gently being siphoned away with liquid-transfering gun) addition configures in advance after culture 24h, change liquid within every 3 days.
3.2.2 osteogenic induction detection method
Alizarin red staining：Induction 14d chorion mescenchymal stem cell, obtains early stage Gegenbaur's cell, inhales and abandons induction broth,
PBS rinses, and 4% paraformaldehyde room temperature fixes 30min, and distilled water rinses, using 37 DEG C of incubation 30min of alizarin red working solution, double
Steam water to rinse.
Von kossa dyes：Induction 21d chorion mescenchymal stem cell, obtains late osteoblastic, reference cell Von
Kossas calcium staining kit is dyeed.
Respectively the early stage skeletonization after dyeing and late osteogenic are examined under a microscope, as shown in Figure 3.As can be seen that it is early
Phase, osteoblastic Alizarin red staining was positive, and illustrated that cell has the stronger ability to osteoblast differentiation.Late osteogenic
Cell Von kossa dyes as the positive, illustrates the osteoblastic calcium scoring of cell shape inducing differentiation it was demonstrated that cell has
The stronger ability to osteoblast differentiation.
3.3 chondrocyte induction differentiation
3.3.1 take and reach the chorion mescenchymal stem cell in 3 generations with 5 × 105/ pipe is inoculated in 15ml centrifuge tube, normally
Abandon supernatant (gently being siphoned away with liquid-transfering gun) the one-tenth chondrocyte induction culture medium that configures in advance of addition after culture 24h, before use plus
TGF-β 3, changes a not good liquor in every 3 days.
3.3.2 chondrocyte induction detection method
The micelle being formed was cultivated 4 weeks in above-mentioned system, changed liquid every 3 days, after induction finishes, carries out FFPE and cuts
Piece.Section is dyeed through 1% toluidine blue, A Erxinlan respectively, HE dyeing identification cartilage specificity proteoglycans.As shown in Figure 3.
As can be seen that HE dyeing, Toluidine blue staining, A Erxinlan stained positive, illustrate cell have very strong to cartilage cell
The ability of differentiation.
Vitality test
The vigor of the chorion mescenchymal stem cell obtained by the step (3) of comparing embodiment and comparative example, concrete steps
As follows：
Respectively the chorion mescenchymal stem cell obtained by the step (3) of embodiment and comparative example is washed 2 with physiology salt
Time, add the TrypLE Express without animal component to digest about 30s, under mirror, visible short fusiformis attached cell becomes round and suspends
After getting up, add physiological saline piping and druming cell and cell is transferred to centrifuge tube, 1200rpm is centrifuged 5min, discards supernatant, uses
The physiological saline of 2ml is resuspended, detects the vigor of cell by cell viability analyzer.
Chorion mescenchymal stem cell obtained by embodiment step (3) is 91.6%.Illustrate to utilize the method for the present invention
During obtaining chorion mescenchymal stem cell, digestion process mild condition, chorion mescenchymal stem cell is damaged less, make
The chorion mescenchymal stem cell activity obtaining is stronger.The vigor of the chorion mescenchymal stem cell obtained by comparative example is only
74.4% (as shown in Figure 4), and the yield of the chorion mescenchymal stem cell of embodiment step (3) is 3 times of comparative example.Explanation
The yield of chorion mescenchymal stem cell obtained by the method for the present invention and activity are all higher.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy describing with reference to this embodiment or example
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be in office
Combine in an appropriate manner in one or more embodiments or example.Additionally, in the case of not conflicting, the skill of this area
The feature of the different embodiments described in this specification or example and different embodiment or example can be tied by art personnel
Close and combine.
Although embodiments of the invention have been shown and described above it is to be understood that above-described embodiment is example
Property it is impossible to be interpreted as limitation of the present invention, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (8)
1. a kind of method obtaining chorion mescenchymal stem cell is it is characterised in that include：
Using mixing enzyme preparation, chorion is carried out digestion process, obtain digestion product；And
Separate described chorion mescenchymal stem cell from described digestion product,
Wherein, described mixing enzyme preparation includes：
Pancreatin；
Clostridiopetidase A IV；And
Clostridiopetidase A II,
Preferably, described mixing enzyme preparation includes：
The pancreatin of 0.05 mass %～0.25 mass %；
The clostridiopetidase A IV of 1～2 μ g/mL；And
The clostridiopetidase A II of 0.1 mass %.
2. method according to claim 1 it is characterised in that described digestion process be 35-37 DEG C temperature, 100～
30～60min is shaken under the rotating speed of 200rpm,
Preferably, described digestion process is concussion 30min under 37 DEG C of temperature, the rotating speed of 150rpm.
3. method according to claim 1 is it is characterised in that described separation includes：
Described digestion product is filtered, is collected filtrate；
Described filtrate is centrifuged, is collected cell；And
Described cell is carried out, to obtain described chorion mescenchymal stem cell.
4. method according to claim 1 is it is characterised in that include：
Carry out digestion process using described mixing enzyme preparation by being cut into a size of described chorion of 4cm × 4cm, digested
Product；
Described digestion product is diluted, and using 100 eye mesh screens, obtained dilution is filtered, collect filtrate；
And
Described filtrate is centrifuged 6min under the rotating speed of 1200rpm, abandons supernatant, obtain described chorion mescenchymal stem cell.
5. method according to claim 1 it is characterised in that further include amplification process,
Described amplification processes and includes：
Described chorion mescenchymal stem cell is resuspended in culture medium, obtains mixed liquor；
Described mixed liquor is inoculated in described culture medium, 37 DEG C, 5%CO2Under cultivate to clone occurs, long to passing on.
6. method according to claim 1 is it is characterised in that described mixing enzyme preparation and chorial amount ratio are 0.5
～2:1, preferably 1:1.
7. a kind of kit is it is characterised in that include：
Pancreatin；
Clostridiopetidase A IV；And
Clostridiopetidase A II,
Preferably, described kit includes：
The pancreatin of 0.05 mass %～0.25 mass %；
The clostridiopetidase A IV of 1～2 μ g/mL；And
The clostridiopetidase A II of 0.1 mass %.
8. kit according to claim 7 it is characterised in that described kit be used for obtaining chorion mesenchyma do thin
Born of the same parents.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109105364A (en) * | 2018-10-12 | 2019-01-01 | 希瑞干细胞科技有限公司 | A kind of chorion mesenchymal stem cell cryopreserving liquid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450609A (en) * | 2013-09-25 | 2015-03-25 | 深圳华大基因科技有限公司 | Method of separating and cultivating umbilical cord mesenchymal stem cells |
| CN104560870A (en) * | 2014-12-18 | 2015-04-29 | 江苏省北科生物科技有限公司 | Method for preparing decidua mesenchymal stem cell |
| CN104560869A (en) * | 2014-12-18 | 2015-04-29 | 江苏省北科生物科技有限公司 | Method for preparing chorionic mesenchymal stem cells |
-
2016
- 2016-11-08 CN CN201610983609.4A patent/CN106434544A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450609A (en) * | 2013-09-25 | 2015-03-25 | 深圳华大基因科技有限公司 | Method of separating and cultivating umbilical cord mesenchymal stem cells |
| CN104560870A (en) * | 2014-12-18 | 2015-04-29 | 江苏省北科生物科技有限公司 | Method for preparing decidua mesenchymal stem cell |
| CN104560869A (en) * | 2014-12-18 | 2015-04-29 | 江苏省北科生物科技有限公司 | Method for preparing chorionic mesenchymal stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 董敏: "高效分离人脐带间充质干细胞(HUCMSCs)方法的研究", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109105364A (en) * | 2018-10-12 | 2019-01-01 | 希瑞干细胞科技有限公司 | A kind of chorion mesenchymal stem cell cryopreserving liquid |
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