CN109321517A - The efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method - Google Patents
The efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method Download PDFInfo
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- CN109321517A CN109321517A CN201811259326.0A CN201811259326A CN109321517A CN 109321517 A CN109321517 A CN 109321517A CN 201811259326 A CN201811259326 A CN 201811259326A CN 109321517 A CN109321517 A CN 109321517A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method characterized by comprising one, reagent；Two, two step enzyme digestions separate amnion mesenchymal stem cell；Three, amnion mesenchymal stem cell secondary culture.The present invention is compared with existing amnion mesenchymal stem cell separation method, amnion mesenchymal stem cell is separated using two step enzyme digestions, the first step removes amniotic epithelial cells, and digestion is twice, removes amniotic epithelial cells more abundant, the amnion mesenchymal stem cell purity of acquisition is higher, and it is easy to operate, without tissue and cell filtration, avoids amnion mesenchymal stem cell from entering in digestion mucus using DNase I and can not separate, the stem cell population of acquisition is more.The non-animal derived property ingredient of entire separation process, safety is good, applies convenient for subsequent research.
Description
Technical field
The present invention relates to a kind of separation of stem cell, amplification method, and in particular to a kind of human amnion mesenchymal stem cell
It efficiently separates, amplification method, belongs to field of biotechnology.
Background technique
Mescenchymal stem cell has self-replacation, Multidirectional Differentiation and immunoregulatory biological characteristics, has become research
Hot spot.Though the mescenchymal stem cell research of derived from bone marrow is more, it is sampled certainly will be by traumatic acquisition, and with individual
Growth, quality and quantity are declined.Currently, researchers at home and abroad have been able between obtaining in umbilical cord, bleeding of the umbilicus, dental pulp
Mesenchymal stem cells, but the cell quantity obtained is limited.Seek a kind of method for efficiently separating mesenchyma, and tissue-derived convenience,
It is not constrained by ethics, is inevitable development trend.Cell origin of the placenta tissue as regenerative medicine most worthy, and not
It is constrained by ethics.Amnion has richer mescenchymal stem cell than placenta, it has also become the object of each researcher's favor.
Structure is complicated for amnion tissue, including epithelium layer, basilar memebrane, compacted zone, fibrocyte layer and spongy layer, and sheep
Film mescenchymal stem cell is located at the innermost layer of amnion.The separation method of amnion mesenchymal stem cell is many kinds of at present, and or
Cell purity and Activity Results it is different.
This research removes amnioic epithelium stem cell with trypsase and EDTA first by two step enzyme digestions, then uses glue
Protoenzyme I and DNase I, digestion separation obtain amnion mesenchymal stem cell.
Currently, the method for separation amnion mesenchymal stem cell is mainly enzymatic isolation method.Structure is complicated for amnion tissue, simple enzyme
Solution can not separating amnion mesenchymal stem cell and amniotic epithelial cells, be difficult to obtain the mescenchymal stem cell of high-purity
System；Secondly also have after removing amniotic epithelial cells using cell scraper, then carry out the separation of amnion mesenchymal stem cell, this side
Method be easy to cause pollution, and separates amniotic epithelial cells and be also not thorough；There are also tissue separator is utilized, it is even to obtain amnion tissue
Slurry, then enzymic digestion is carried out, operation is relatively cumbersome, and higher cost.
Summary of the invention
The object of the present invention is to provide the efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method, to overcome
Disadvantages mentioned above present in the prior art and deficiency.
Technical problems to be solved needed for the present invention can be achieved through the following technical solutions:
The efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method characterized by comprising
One, reagent
Two, two step enzyme digestions separate amnion mesenchymal stem cell
Three, amnion mesenchymal stem cell secondary culture.
Further, the efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method, comprising:
One, reagent
1. placenta saves liquid and cleaning solution
Injection penicillin and streptomycin sulphate are configured to the storing liquid of every milliliter of 10,000 units with injection physiological saline,
As 1% dual anti-storing liquid；
The dual anti-storing liquid of 5mL is added in the injection physiological saline of 500mL, adds 2mL Gentamicin Injection,
It is configured to placenta and saves liquid or cleaning solution；
2. trypsase storing liquid
The trypsase for weighing 0.5g is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.5% (m/
V) trypsase storing liquid；
3.EDTA storing liquid
0.2gEDTA disodium is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.2% (m/v)
EDTA storing liquid；
4. Collagenase I storing liquid
1g Collagenase I is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, the collagen of as 1% (m/v)
I storing liquid of enzyme；
I storing liquid of 5.DNase
0.2g DNase I is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.2% (m/v's)
I storing liquid of DNase；
6. culture medium and primary culture medium
Culture medium is the universal serum free medium of Lonza, adds serum replacement and L-Glutamine by operation instruction.
The 500 μ L of dual anti-storing liquid of the every 50mL addition 1% of primary culture medium, adds the Gentamicin Injection of 200 μ L；
Two, two step enzyme digestions separate amnion mesenchymal stem cell
1. placenta transports
Placenta (palpus multipara agrees to) after pregnant woman childbirth, is placed in the placenta Storage Box containing preservation liquid, 2~8
Degree Celsius it is shipped back laboratory in time.Sample reception room checks transport temperature, and whether placenta box is complete, avoids the occurrence of pollution, and
It is numbered；
2. amnion chorista separates
Placenta is placed in stainless steel pallet, is cleaned 1 time with cleaning solution；It will be around the sheep in the sub- face of placenta around umbilical cord with hand
Film is torn, and is put into culture dish, will be cut with the edge of bloodstain with operating scissors, and 75% medicinal alcohol is added and is rinsed,
It places into new culture dish, cleaning solution cleaning is added, and be cut into 1 × 1cm with tissue shear2The tissue block of size, then with cleaning
Liquid cleans one time, is transferred in 50mL centrifuge tube, and every pipe 10mL containing amnion tissue, 1300rpm/min are centrifuged 5min；
3. amniotic epithelial cells separate
After centrifugation, supernatant is abandoned, trypsase storing liquid 3mL, EDTA storing liquid 3mL is added in every pipe, supplements physiology salt
Water is to 30mL, and the concentration of trypsase and the concentration of EDTA are respectively 0.05% and 0.02% at this time.145rpm/min, 37 DEG C of vibrations
Swing digestion 15min；
After digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant.Repeat above-mentioned pancreas egg
The step of white enzyme and EDTA digest；
4. amnion mesenchymal stem cell separates
After above-mentioned digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant.By amnion tissue
It is put into culture dish, is cleaned twice with cleaning solution；
Amnion tissue is put into new 50mL centrifuge tube, every pipe shreds amnion tissue 1-3mm containing about tissue 10mL；
Collagenase I 2mL, I 2mL of DNAse is added, supplement physiological saline to 20mL, the concentration of Collagenase I and DNAse I are distinguished at this time
For 0.1% and 0.02%.145rpm/min, 37 DEG C of oscillations digest 60min；
After digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant and tissue.Every pipe is added
10mL primary culture medium, which is inoculated in the culture dish of 10cm, to be cultivated；
5. primary change liquid
Culture carried out partly changing liquid for the first time to the 3rd day, is partly changed according to the actual situation or changes liquid entirely within the 6th day, later every
Liquid was changed entirely every 3 days；
Three, amnion mesenchymal stem cell secondary culture
60% or more primary cell degrees of fusion of observation can pass P1 generation, and inoculum density is 8000~10000/cm2；After
Cell fusion degree can be passed on up to 80% or more.Passage is complete medium, without adding dual anti-or gentamicin.
Beneficial effects of the present invention:
The method that the present invention studies uses two step enzymic digestions compared with existing amnion mesenchymal stem cell separation method
Method separates amnion mesenchymal stem cell, and the first step removes amniotic epithelial cells, and digests twice, removes amniotic epithelial cells
More sufficiently, the amnion mesenchymal stem cell purity of acquisition is higher and easy to operate, is not necessarily to tissue and cell filtration, uses
DNase I avoids amnion mesenchymal stem cell from entering in digestion mucus and can not separate, and the stem cell population of acquisition is more.Entirely
The non-animal derived property ingredient of separation process, safety is good, applies convenient for subsequent research.
Detailed description of the invention
Fig. 1 is flow cytometer detection figure of the invention.
Fig. 2 is primary, the P1 of the present invention, the culture picture of each generation of P2, P3.Note: primary 15th day of upper left, upper right P1 the 3rd
It, lower-left P2 the 3rd day, bottom right P3 the 3rd day.
Appended drawing reference:
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this
Invention is not for limiting the scope of the invention.
Embodiment 1
The efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method,
One, reagent
1. placenta saves liquid and cleaning solution
Injection penicillin and streptomycin sulphate are configured to the storing liquid of every milliliter of 10,000 units with injection physiological saline,
As 1% dual anti-storing liquid.The dual anti-storing liquid of 5mL is added in the injection physiological saline of 500mL, adds 2mL injection
Gentamicin sulphate is configured to placenta and saves liquid or cleaning solution；
2. trypsase storing liquid
The trypsase for weighing 0.5g is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.5% (m/
V) trypsase storing liquid；
3.EDTA storing liquid
0.2gEDTA disodium is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.2% (m/v)
EDTA storing liquid；
4. Collagenase I storing liquid
1g Collagenase I is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, the collagen of as 1% (m/v)
I storing liquid of enzyme；
I storing liquid of 5.DNase
0.2g DNase I is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.2% (m/v)
I storing liquid of DNase；
6. culture medium and primary culture medium
Culture medium is the universal serum free medium of Lonza, adds serum replacement and L-Glutamine by operation instruction.
The 500 μ L of dual anti-storing liquid of the every 50mL addition 1% of primary culture medium, adds the Gentamicin Injection of 200 μ L；
Two, two step enzyme digestions separate amnion mesenchymal stem cell
1. placenta transports
Placenta (palpus multipara agrees to) after pregnant woman childbirth, is placed in the placenta Storage Box containing preservation liquid, 2~8
Degree Celsius it is shipped back laboratory in time.Sample reception room checks transport temperature, and whether placenta box is complete, avoids the occurrence of pollution, and
It is numbered.
2. amnion chorista separates
Placenta is placed in stainless steel pallet, is cleaned 1 time with cleaning solution；It will be around the sheep in the sub- face of placenta around umbilical cord with hand
Film is torn, and is put into culture dish, will be cut with the edge of bloodstain with operating scissors, and 75% medicinal alcohol is added and is rinsed,
It places into new culture dish, cleaning solution cleaning is added, and be cut into 1 × 1cm with tissue shear2The tissue block of size, then with cleaning
Liquid cleans one time, is transferred in 50mL centrifuge tube, and every pipe 10mL containing amnion tissue, 1300rpm/min are centrifuged 5min；
3. amniotic epithelial cells separate
After centrifugation, supernatant is abandoned, trypsase storing liquid 3mL, EDTA storing liquid 3mL is added in every pipe, supplements physiology salt
Water is to 30mL, and the concentration of trypsase and the concentration of EDTA are respectively 0.05% and 0.02% at this time.145rpm/min, 37 DEG C of vibrations
Swing digestion 15min.After digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant.It repeats above-mentioned
The step of trypsase and EDTA digest；
4. amnion mesenchymal stem cell separates
After above-mentioned digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant.By amnion tissue
It is put into culture dish, is cleaned twice with cleaning solution.Amnion tissue is put into new 50mL centrifuge tube, every pipe is containing about tissue
10mL, and shred amnion tissue 1-3mm.Addition Collagenase I 2mL, I 2mL of DNAse, supplement physiological saline to 20mL, at this time
The concentration of Collagenase I and DNAse I are respectively 0.1% and 0.02%.145rpm/min, 37 DEG C of oscillations digest 60min.Digestion knot
Shu Hou, is added appropriate physiological saline, and 1300rpm/min is centrifuged 5min；Abandon supernatant and tissue.10mL primary culture medium is added in every pipe
It is inoculated in the culture dish of 10cm and cultivates；
5. primary change liquid
Culture carried out partly changing liquid for the first time to the 3rd day, is partly changed according to the actual situation or changes liquid entirely within the 6th day, later every
Liquid was changed entirely every 3 days；
Three, amnion mesenchymal stem cell secondary culture
60% or more primary cell degrees of fusion of observation can pass P1 generation, and inoculum density is 8000~10000/cm2.After
Cell fusion degree can be passed on up to 80% or more.Passage is complete medium, without adding dual anti-or gentamicin.In P3 generation, takes thin
Born of the same parents' amount 1.0~1.2 × 106Cell/ml carries out cell surface marker detection.
Flow cytometer detection amnion mesenchymal stem cell surface marker
1) single cell suspension is taken, 1200rpm/min is centrifuged 5min；
2) cell precipitation is resuspended with PBS, adjusting cell concentration is 1.0~1.2 × 106cell/ml；
3) add antibody CD90, CD105, CD166, gently piping and druming mixes, and 4 DEG C are protected from light incubation 30min, while homotype pair is arranged
According to；
4) plus 1mlPBS is in flow cytometer detection pipe, 1200rpm, 5min, abandons supernatant；
5) add 100ulPBS, gently piping and druming mixes, upper machine testing.
As a result: amnion mesenchymal stem cell three kinds of surface markers CD90-95.13%, CD105-96.99%, CD166-
98.47%, positive rate is flow cytometer detection figure of the invention in 95% or more, Fig. 1, as shown in Figure 1.
The 15th day 80% or more cell fusion degree of originally culture, as shown in the upper left Fig. 2, P1 the 3rd day such as Fig. 2 upper right institute of generation
Show, the 3rd day generation of P2, the 3rd day generation of P3 was as shown in the bottom right Fig. 2 as shown in the lower-left Fig. 2.
The method of this research uses two step enzyme digestions point compared with existing amnion mesenchymal stem cell separation method
From amnion mesenchymal stem cell, the first step removes amniotic epithelial cells, and digests twice, makes more filling for amniotic epithelial cells removal
Point, the amnion mesenchymal stem cell purity of acquisition is higher and easy to operate, is not necessarily to tissue and cell filtration, uses DNase I
Amnion mesenchymal stem cell is avoided to enter in digestion mucus and can not separate, the stem cell population of acquisition is more.Entirely separated
The non-animal derived property ingredient of journey, safety is good, applies convenient for subsequent research.
A specific embodiment of the invention is illustrated above, but the present invention is not limited thereto, without departing from
Spirit of the invention, the present invention can also have various change.
Claims (6)
1. the efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method characterized by comprising
One, reagent
Two, two step enzyme digestions separate amnion mesenchymal stem cell
Three, amnion mesenchymal stem cell secondary culture.
2. the efficiently separating of a kind of human amnion mesenchymal stem cell according to claim 1, amplification method, feature exist
In: where one, reagent include:
1. placenta saves liquid and cleaning solution；
2. trypsase storing liquid；
3.EDTA storing liquid；
4. Collagenase I storing liquid；
I storing liquid of 5.DNase；
6. culture medium and primary culture medium.
3. the efficiently separating of a kind of human amnion mesenchymal stem cell according to claim 1, amplification method, feature exist
In: where two, two step enzyme digestions separate amnion mesenchymal stem cell
1. placenta transports；
2. amnion chorista separates；
3. amniotic epithelial cells separate；
4. amnion mesenchymal stem cell separates；
5. primary change liquid.
4. the efficiently separating of a kind of human amnion mesenchymal stem cell according to claim 1, amplification method, feature exist
In: where three, 60% or more primary cell degrees of fusion of observation can pass P1 generation, inoculum density is 8000~10000/cm2；
Later cell fusion degree can be passed on up to 80% or more；Passage is complete medium, without adding dual anti-or gentamicin.
5. the efficiently separating of a kind of human amnion mesenchymal stem cell according to claim 21, amplification method, feature exist
In: where one, reagent, comprising:
1. placenta saves liquid and cleaning solution
Injection penicillin and streptomycin sulphate are configured to the storing liquid of every milliliter of 10,000 units with injection physiological saline, as
1% dual anti-storing liquid；
The dual anti-storing liquid of 5mL is added in the injection physiological saline of 500mL, adds 2mL Gentamicin Injection, that is, matches
It is set to placenta and saves liquid or cleaning solution；
2. trypsase storing liquid
The trypsase for weighing 0.5g is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.5% (m/v's)
Trypsase storing liquid；
3.EDTA storing liquid
0.2gEDTA disodium is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.2% (m/v's)
EDTA storing liquid；
4. Collagenase I storing liquid
1g Collagenase I is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, the Collagenase I of as 1% (m/v)
Storing liquid；
I storing liquid of 5.DNase
0.2g DNase I is weighed, is dissolved in 100mL physiological saline, 0.22 μm of membrane filtration, as 0.2% (m/v's)
I storing liquid of DNase；
6. culture medium and primary culture medium
Culture medium is the universal serum free medium of Lonza, adds serum replacement and L-Glutamine by operation instruction；It is primary
The 500 μ L of dual anti-storing liquid of the every 50mL addition 1% of culture medium, adds the Gentamicin Injection of 200 μ L.
6. the efficiently separating of a kind of human amnion mesenchymal stem cell according to claim 3, amplification method, feature exist
In: where two, two step enzyme digestions separate amnion mesenchymal stem cell, comprising:
1. placenta transports
Placenta after pregnant woman childbirth is placed in containing in the placenta Storage Box for saving liquid, and 2~8 degrees Celsius are shipped back laboratory in time；
Sample reception room checks transport temperature, and whether placenta box is complete, avoids the occurrence of pollution, and it is numbered；
2. amnion chorista separates
Placenta is placed in stainless steel pallet, is cleaned 1 time with cleaning solution；It will be torn around the amnion in the sub- face of placenta around umbilical cord with hand
Under, it is put into culture dish, will be cut with the edge of bloodstain with operating scissors, 75% medicinal alcohol is added and is rinsed, then puts
Enter in new culture dish, cleaning solution cleaning is added, and be cut into 1 × 1cm with tissue shear2The tissue block of size, then it is clear with cleaning solution
It washes one time, is transferred in 50mL centrifuge tube, every pipe 10mL containing amnion tissue, 1300rpm/min are centrifuged 5min；
3. amniotic epithelial cells separate
After centrifugation, supernatant is abandoned, trypsase storing liquid 3mL, EDTA storing liquid 3mL is added in every pipe, and supplement physiological saline is extremely
30mL, the concentration of trypsase and the concentration of EDTA are respectively 0.05% and 0.02% at this time；145rpm/min, 37 DEG C of oscillations disappear
Change 15min；
After digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant；Repeat above-mentioned trypsase
The step of with EDTA digestion；
4. amnion mesenchymal stem cell separates
After above-mentioned digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant；Amnion tissue is put into
In culture dish, cleaned twice with cleaning solution；
Amnion tissue is put into new 50mL centrifuge tube, every pipe shreds amnion tissue 1-3mm containing about tissue 10mL；It is added
I 2mL of Collagenase I 2mL, DNAse supplements physiological saline to 20mL, and the concentration of Collagenase I and DNAse I are respectively 0.1% at this time
With 0.02%；145rpm/min, 37 DEG C of oscillations digest 60min；
After digestion, appropriate physiological saline is added, 1300rpm/min is centrifuged 5min；Abandon supernatant and tissue；10mL is added in every pipe
Primary culture medium is inoculated in the culture dish of 10cm and cultivates；
5. primary change liquid
Culture carried out partly changing liquid for the first time, and was partly changed according to the actual situation or change liquid entirely within the 6th day, later every 3 to the 3rd day
It changes liquid entirely.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257326A (en) * | 2019-06-11 | 2019-09-20 | 华夏源（上海）细胞基因工程股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell |
| CN110791477A (en) * | 2019-11-21 | 2020-02-14 | 陕西九州细胞基因工程有限公司 | Culture method of mesenchymal stem cells after cryopreservation and recovery of adipocytes |
| CN112574948A (en) * | 2020-08-18 | 2021-03-30 | 北京昱龙盛世生物科技有限公司 | Separation culture method of human amniotic mesenchymal stem cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257326A (en) * | 2019-06-11 | 2019-09-20 | 华夏源（上海）细胞基因工程股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell |
| CN110791477A (en) * | 2019-11-21 | 2020-02-14 | 陕西九州细胞基因工程有限公司 | Culture method of mesenchymal stem cells after cryopreservation and recovery of adipocytes |
| CN112574948A (en) * | 2020-08-18 | 2021-03-30 | 北京昱龙盛世生物科技有限公司 | Separation culture method of human amniotic mesenchymal stem cells |
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