CN109266605A - A kind of method of eye pouch fat stem cell separation and in vitro culture - Google Patents
A kind of method of eye pouch fat stem cell separation and in vitro culture Download PDFInfo
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- 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
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- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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Abstract
A kind of method of eye pouch fat stem cell separation and in vitro culture, which comprises the following steps: the acquisition of adipose tissue, the separation of fat stem cell, fat stem cell in vitro culture.The invention has the beneficial effects that: the method for the separation of one of present invention fat stem cell and in vitro culture is compared with prior art, have the advantage that 1, cell is cultivated using the culture medium of serum-free, no heterologous serum composition is added, and reduces risk when using the injury of cell and later phase clinical；2, cell state is good, and vigor is good, and cellular morphology, proliferative capacity, surface marker expression and differentiation capability are good.
Description
Technical field
The present invention relates to stem cell separation and Vitro Culture Techniques field more particularly to a kind of separation of eye pouch fat stem cell
And the method for in vitro culture.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that adult stem has multi-lineage potential
Hot spot cell, be the cell colony with bone formation ability and hematopoiesis function found in a kind of most of adult organs, can
With from marrow, bleeding of the umbilicus, fat in extract, under specific condition of culture, mescenchymal stem cell can be divided into fat cell, at
Osteocyte, cartilage cell and myocyte etc..Mescenchymal stem cell is the main seed cell of current organizational project.It experienced for many years
After the research of more scholars, although organizational project has been obtained for remarkable progress, however, due to lacking ideal screening technique at present,
The limitations such as mescenchymal stem cell is not easy to obtain, amplification in vitro difficulty, make its clinical rank feeds back using being restricted.Cause
This, find it is a kind of be easily obtained, immunogenicity is low, the seed cell of the clinical treatment rank that is easy to amplification in vitro is to organize at present
Engineering one of problem in the urgent need to address.
People isolate a kind of stem cell with multi-lineage potential from adipose tissue, and cultivate in vitro it is special
Fat cell, osteoblast, cartilage cell and myocyte etc. are successfully divided into system, this stem cell broken up is recognized
It can be adipose-derived mesenchymal cell, i.e. fat stem cell or fat mesenchymal stem cell (adipose-derived stem
Cells, ADSCs), it is a kind of stem cell with multi-lineage potential isolated from adipose tissue in recent years.Fat
Stem cell has differentiation polyembryony layer totipotency feature, including mesoderm, the ability of entoderm and ectoderm noble cells, and energy
Many potential advantageous cell factors are secreted, the treatment method of the wound healing based on cell is fast-developing.Rouge
Fat derived stem cells are the multi-functional mescenchymal stem cells of a group, can be divided into other cell lines in the non-fat of adipose tissue
There is a large amount of fat-derived cell in (interstitial) fragment, and is easily isolated acquisition.
Fat stem cell receives very big attention after being found from 2001.Show that fat stem cell has after study
Oriented fat, cartilage, skeletonization, at multi-lineage potentials such as fleshes.In January, 2012, the autologous fat stem cell medicine of South Korea FDA approval
Object Cuepistem is successfully listed, for treating the concurrent anal fistula of complexity clone disease.October in the same year, U.S. FDA have approved
Clinical test of the Cytori Therapeutics company's fat stem cell to heart failure patients' validity.Fat stem cell is not
Only there are all kinds of stem cells to have the advantage that, also convenient, wound is small, source is sufficient with drawing materials, and can once obtain a large amount of new
Fresh stem cell, self use do not have the advantages that immunological rejection, and avoid the ethics puzzlement of embryonic stem cell with
The rare equal trouble in the source of stem cell.Therefore fat stem cell has boundless potential applicability in clinical practice.
After the separation method of fat stem cell mostly uses Mechanical Method to handle at present, divided using the method for enzymic digestion
From complex for operation step, low separation efficiency in the culture in later period to add dual anti-and serum more, although having to culture certain
Benefit also increases risk when injury and later phase clinical to cell use.
Summary of the invention
The present invention provides the method for a kind of separation of eye pouch fat stem cell and in vitro culture, it is therefore intended that overcomes current fat
The deficiency of stem cell separation and extraction technology, and a kind of isolated culture method of the eye pouch fat stem cell provided, have operation
Feature easy, separative efficiency is high, cell proliferation rate is fast, has a extensive future, can provide for organizational project and regenerative medicine
A large amount of seed cells.
The present invention takes following technical scheme:
The present invention is the method for a kind of separation of eye pouch fat stem cell and in vitro culture, is included the following steps:
1) acquisition of eye pouch adipose tissue: eye pouch adipose tissue aseptically passes through the elderly's eye pouch for hospital and performs the operation
The fatty waste removed is placed in sterile saline and is saved with 2-8 DEG C, and storage time is no more than 24 hours；
2) separation of eye pouch fat stem cell: by eye pouch adipose tissue with cleaning solution rinse 3-5 times, cut with aseptic operation and
Tweezers remove the trace of blood in fatty sample, and fatty sample is cut into 1mm3The tissue block of size is put into 50mL centrifuge tube, body
Product is 0.5-3mL；The ratio of addition digestive ferment, digestive ferment and adipose tissue is 5:1-1.5:1,37 DEG C of constant-temperature tables concussion digestion
40-60 minutes, after digestion, digestive juice moved into 50mL centrifuge tube, 1500rpm centrifugation 10 through 70 μm of strainer filterings, by filtrate
Minute, remove upper layer yellow grease and middle layer digestive juice, collect to precipitate and simultaneously normal saline flushing is added 1-2 times, 1500rpm from
The heart 6 minutes, collect precipitating, as eye pouch fat stem cell；
3) 2ml free serum culture base weight the in vitro culture of eye pouch fat stem cell: is added in eye pouch fat stem cell precipitating
It is outstanding, according to 10000-20000/cm2Density is inoculated in 6 orifice plates or culture dish, and in 37 DEG C, 5% carbon dioxide incubator is incubated
It educates, the serum free medium that inoculation more renews after 3-5 days, carried out a serum free medium every 2-4 days later changes liquid, until
Cell confluency reaches 70%-80%；Eye pouch fat stem cell is passed on, propagating method is to discard former culture medium, with clear
Washing lotion is cleaned cell 1-2 times, and recombination pancreatin is added, digests 1-2 minutes under the conditions of 37 DEG C, and it is anti-that culture medium termination digestion is added
It answers, cell suspension is transferred in 50ml centrifuge tube, then clean 6 orifice plates or culture dish with cleaning solution, cleaning solution is also shifted
Into 50ml centrifuge tube, 1500rpm is centrifuged 6 minutes, is collected cell precipitation, is 10000-20000/cm with cell density2Inoculation
To culture bottle, in 37 DEG C, 5% carbon dioxide incubator is incubated for；It repeats the above steps to passage to P2-P4 generation, uses cell
Calculating instrument carries out Trypan Blue counting, after 1500rpm is centrifuged 6 minutes again, is frozen carefully with cell density 1-5*10e6/mL
Born of the same parents.
Further, cleaning solution described in step (2) and (3) is mixed with dual anti-according to volume ratio 500:5 by physiological saline
It is made.
Further, digestive ferment described in step (2) and (3) by 0.1% Type I collagen enzyme, 0.05%IV Collagenase Type,
1.5-3:1.5-3:1 is mixed 0.25% pancreatin/EDTA by volume.
Further, serum free medium described in step (3) by Lonza serum free medium, PALL serum substitute,
20mmol/L L-Glutamine solution is mixed according to volume ratio 500:10:5.
The invention has the beneficial effects that: the method for one of present invention eye pouch fat stem cell separation and in vitro culture
With the prior art, having the advantage that 1, cell is cultivated using the culture medium of serum-free, no heterologous serum composition is added,
Reduce risk when using the injury of cell and later phase clinical；2, cell state is good, and vigor is good, cellular morphology, proliferation energy
Power, surface marker expression and differentiation capability are good.
Detailed description of the invention
Fig. 1 is 40 times of inverted microscope of primary eye pouch fat stem cell in vitro culture the 6th day that the embodiment of the present invention 2 separates
Figure；
Fig. 2 is the primary eye pouch fat stem cell in vitro culture inverted microscope 40 on the 10th that the embodiment of the present invention 2 separates
Scheme again；
Flow cytometer detection figure of the P2 that Fig. 3 separates for the embodiment of the present invention 2 and in vitro culture obtains for eye pouch fat stem cell；
Fig. 4 is that the isolated eye pouch fat stem cell in vitro culture of the embodiment of the present invention 3 is passed on to P1 for inverted microscope 40
Scheme again；
Fig. 5 is that the isolated eye pouch fat stem cell in vitro culture of the embodiment of the present invention 3 is passed on to P2 for inverted microscope 40
Scheme again.
Specific embodiment
The technical solution of the present invention is further described in detail With reference to embodiment.
Embodiment 1
The method of a kind of eye pouch fat stem cell separation proposed by the present invention and in vitro culture, includes the following steps:
1, preparation of reagents:
1) preparation of cleaning solution: physiological saline is mixed with dual anti-according to volume ratio 500:5；
2) digest the preparation of enzyme solution: 0.1% Type I collagen enzyme, 0.05%IV Collagenase Type, 0.25% pancreatin/EDTA press body
Product is mixed than 1.5:3:1；
3) preparation of serum free medium: Lonza serum free medium, PALL serum substitute, 20mmol/L L- paddy ammonia
Amide solution is mixed according to volume ratio 500:10:5.
2, the acquisition of eye pouch adipose tissue: eye pouch adipose tissue is that the fat that women and children remove from the operation of the elderly's eye pouch is discarded
Object is placed in sterile saline with 4 DEG C, saves 4 hours.
3, the separation of eye pouch fat stem cell: by eye pouch adipose tissue with cleaning solution rinse 3-5 times, cut with aseptic operation and
Tweezers remove the trace of blood in fatty sample, and fatty sample is cut into 1mm3The tissue block of size is respectively put into 50mL centrifuge tube
In, volume is about 0.5mL；2.5mL digestive ferment is added, 37 DEG C of constant-temperature table concussions digest 60 minutes, after digestion, digestive juice
Through 70 μm of strainer filterings, filtrate is moved into 50mL centrifuge tube, 1500rpm is centrifuged 10 minutes, removes upper layer yellow grease and centre
Layer digestive juice, collects and precipitates and normal saline flushing is added 1-2 times, and 1500rpm is centrifuged 6 minutes, collects precipitating, as eye pouch rouge
Fat stem cell；
4,2ml free serum culture base weight the in vitro culture of eye pouch fat stem cell: is added in eye pouch fat stem cell precipitating
It is outstanding, according to 10000-20000/cm2Density is inoculated in 6 orifice plates, and in 37 DEG C, 5% carbon dioxide incubator is incubated for, inoculation 5
The serum free medium more renewed after it, carried out a serum free medium every 4 days later changes liquid, until cell confluency reaches
To 80%；It is passed on, discards former culture medium, cleaned cell 2 times with cleaning solution, recombination pancreatin is added, disappears under the conditions of 37 DEG C
Change 1-2 minutes, culture medium is added and terminates digestion reaction, cell suspension is transferred in 50ml centrifuge tube, then clean 6 with cleaning solution
Cleaning solution is also transferred in 50ml centrifuge tube by orifice plate, and 1500rpm is centrifuged 6 minutes, is collected cell precipitation, is with cell density
10000-20000/cm22 T25 culture bottles are seeded to, in 37 DEG C, 5% carbon dioxide incubator is incubated for；Repeat above-mentioned step
It is rapid to arrive P2 generation to passage, Trypan Blue counting is carried out with cell counter, it is close with cell after 1500rpm is centrifuged 6 minutes again
Spend 4*10e6/ml freeze-stored cell.
Embodiment 2
The method of a kind of eye pouch fat stem cell separation proposed by the present invention and in vitro culture, includes the following steps:
1, preparation of reagents:
1) preparation of cleaning solution: physiological saline is mixed with dual anti-according to volume ratio 500:5；
2) digest the preparation of enzyme solution: 0.1% Type I collagen enzyme, 0.05%IV Collagenase Type, 0.25% pancreatin/EDTA press body
Product is mixed than 2:2:1；
3) preparation of serum free medium: Lonza serum free medium, PALL serum substitute, 20mmol/L L- paddy ammonia
Amide solution is mixed according to volume ratio 500:10:5.
2, the acquisition of eye pouch adipose tissue: eye pouch adipose tissue is that the fat that women and children remove from the operation of the elderly's eye pouch is discarded
Object is placed in sterile saline with 4 DEG C, saves 4 hours.
3, the separation of eye pouch fat stem cell: eye pouch adipose tissue cleaning solution is rinsed 3 times, is cut with aseptic operation and tweezer
The son removal trace of blood, and it is cut into 1mm3The tissue block of size is respectively put into 50mL centrifuge tube, and volume is about 1mL；Digestion is added
The ratio of enzyme, digestive ferment and adipose tissue is 1.5:1, and 37 DEG C of constant-temperature table concussions digest 60 minutes, and after digestion, observation disappears
Change as a result, adipose tissue digests clean noresidue.Digestive juice moves into 50mL centrifuge tube through 70 μm of strainer filterings, by filtrate,
1500rpm is centrifuged 10 minutes, removes upper layer yellow grease and middle layer digestive juice, is collected and is precipitated and normal saline flushing 2 is added
Secondary, 1500rpm is centrifuged 6 minutes, collects precipitating, as eye pouch fat stem cell.
4,2mL free serum culture base weight the in vitro culture of eye pouch fat stem cell: is added in eye pouch fat stem cell precipitating
It is outstanding, according to 10000-20000/cm2Density is inoculated in 6 orifice plates, and in 37 DEG C, 5% carbon dioxide incubator is incubated for.
5,1-5 days, daily to observe the adherent situation of cell: cell-free adherent.
6, it the 6th day, observes the adherent situation of cell: having cell adherent；The serum free medium more renewed.
8, the 8th day, the serum free medium more renewed.
9, the 10th day, cell confluency was up to 60%；Carry out serum free medium changes liquid.
10, the 12nd day, cell confluency was up to 80%, the serum free medium that more renews.
11, the 13rd day, passage: discarding old culture medium, clean cell 1-2 times with cleaning solution, recombination pancreatin is added, at 37 DEG C
Digestion 1 minute is added culture medium and terminates digestion, cell suspension is transferred in 50mL centrifuge tube, then clean 6 holes with cleaning solution
Cleaning solution is also transferred in 50mL centrifuge tube, 1500rpm by plate, is centrifuged 6 minutes.It discards supernatant, cell precipitation is seeded to 2
T25 culture bottle, inoculum density 10000-20000/cm2。
12, the 15th day, P1 passed P2 generation: carrying out had digestive transfer culture by above-mentioned steps, cell counter counts, count results cell
Quantity is 4.5*10e6, and Cell viability 94.06% repeats above-mentioned passage step, by cell inoculation into 3 T75 culture bottles.
13, the 17th day, P2 was collected for cell, cell counter counts, and result 7.84*10e6 carries out flow cytometer detection, with
Cell density 2.5*10e6/ml freeze-stored cell.
As a result as shown in Fig. 1-3 and table 1, Fig. 1 is that eye pouch fat stem cell in vitro culture the 6th day of the present embodiment separation is fallen
Set 40 times of microscope figures；Fig. 2 is 40 times of inverted microscope of eye pouch fat stem cell in vitro culture the 10th day of the present embodiment separation
Figure；Fig. 3 be separated in the present embodiment and the obtained P2 of in vitro culture for eye pouch fat stem cell flow cytometer detection figure；Table 1 is this
The expression rate for the eye pouch fat stem cell surface markers that separation and in vitro culture obtain in embodiment.As shown in Figure 1, this reality
Apply example separation eye pouch fat stem cell cultivate in vitro observed the adherent situation of cell within 6th；As shown in Figure 2, this reality
Apply example separation eye pouch fat stem cell cultivate in vitro observe cell confluency up to 60% within 10th；As Fig. 3 and table 1 can
Know, in the present embodiment separation and in vitro culture obtain eye pouch fat stem cell surface markers CD73, CD90, CD105,
CD29, CD44 expression rate are all 99% or more, and CD14, CD34, CD45, CD79a, HLA-DR expression rate are all 5% hereinafter, meet
The phenotypic characteristic of fat mesenchymal stem cell.
The expression for the eye pouch fat stem cell cell surface marker molecule that separation and in vitro culture obtain in 1 embodiment 2 of table
Rate
| Cell surface marker molecule | CD73 | CD90 | CD105 | CD29 | CD44 |
| Expression rate | 99.36% | 99.05% | 99.48% | 99.66% | 99.92% |
| Cell surface marker molecule | CD14 | CD34 | CD45 | CD79a | HLA-DR |
| Expression rate | 0.95% | 0.50% | 0.01% | 0.32% | 1.35% |
Embodiment 3
The method of a kind of eye pouch fat stem cell separation proposed by the present invention and in vitro culture, includes the following steps:
1, preparation of reagents:
1) preparation of cleaning solution: physiological saline is mixed with dual anti-according to volume ratio 500:5；
2) digest the preparation of enzyme solution: 0.1% Type I collagen enzyme, 0.05%IV Collagenase Type, 0.25% pancreatin/EDTA press body
Product is mixed than 3:1.5:1；
3) preparation of serum free medium: Lonza serum free medium, PALL serum substitute, 20mmol/L L- paddy ammonia
Amide solution is mixed according to volume ratio 500:10:5.
2, the acquisition of eye pouch adipose tissue: eye pouch adipose tissue is that the fat that women and children remove from the operation of the elderly's eye pouch is discarded
Object is placed in sterile saline with 4 DEG C of preservation 4h.
3, the separation of eye pouch fat stem cell: eye pouch adipose tissue cleaning solution is rinsed 3 times, is cut with aseptic operation and tweezer
The son removal trace of blood, and it is cut into 1mm3The tissue block of size is respectively put into 50ml centrifuge tube, and volume is about 1mL；3mL is added to disappear
Change enzyme, 37 DEG C of constant-temperature table concussions digest 60 minutes, and after digestion, observation digests as a result, adipose tissue digestion is completely without residual
It stays；Digestive juice moves into 50ml centrifuge tube through 70 μm of strainer filterings, by filtrate, and 1500rpm is centrifuged 10 minutes, removes upper layer yellow oil
Rouge and middle layer digestive juice are collected and precipitate and be added normal saline flushing 2 times, and 1500rpm is centrifuged 6 minutes, collect precipitating, as
Eye pouch fat stem cell.
4,2mL free serum culture base weight the in vitro culture of eye pouch fat stem cell: is added in eye pouch fat stem cell precipitating
It is outstanding, according to 10000-20000/cm2Density is inoculated in 6 orifice plates, and in 37 DEG C, 5% carbon dioxide incubator is incubated for,.
5, the 5th day, the serum free medium more renewed observed the adherent situation of cell: having cell adherent；
6, it the 8th day, observes the adherent situation of cell: having cell adherent；The serum free medium more renewed；
7, the 11st day, cell confluency was up to 85%.P1 generation is passaged to from P0 band: being discarded old culture medium, is cleaned with cleaning solution
Cell 1-2 times is added recombination pancreatin, digests 2 minutes at 37 DEG C, and culture medium is added and terminates digestion, cell suspension is transferred to
In 50ml centrifuge tube, then with cleaning solution 6 orifice plates are cleaned, cleaning solution is also transferred in 50ml centrifuge tube, 1500rpm, is centrifuged 6 points
Clock.It discards supernatant, cell precipitation is seeded to 2 T25 culture bottles, inoculum density 10000-20000/cm2。
8, the 13rd day, observation cell confluency was passaged to P2 generation, inoculation 2 from P1 band up to 90%, according to above-mentioned passage step
In a T75 culture bottle.
9, the 15th day, cell is collected, carries out flow cytometer detection, the results are shown in Table 2, meets fat mesenchymal stem cell table
Type, with cell density 1-5*106/ml freeze-stored cell.
As a result as shown in Fig. 4-5 and table 2, Fig. 4 is that the eye pouch fat stem cell in vitro culture passage of the present embodiment separation is arrived
P1 schemes for 40 times of inverted microscope；Fig. 5 is that the eye pouch fat stem cell in vitro culture of the present embodiment separation is passed on to P2 generation inversion
40 times of microscope figures；Table 2 is the eye pouch fat stem cell surface markers that separation and in vitro culture obtain in the present embodiment
Expression rate.Such as Fig. 4 and Fig. 5 it is found that cellular morphology is clear after the eye pouch fat stem cell of the present embodiment separation is cultivated in vitro, it is in
Polygonal, fusiform, vortex shape growth, P2 for when contaminating cell it is few；As seen in Table 2, separation and external training in the present embodiment
Support obtained eye pouch fat stem cell surface markers CD73, CD90, CD105, CD29, CD44 expression rate all 99% with
On, CD14, CD34, CD45, CD79a, HLA-DR expression rate are all 5% hereinafter, the phenotype for meeting fat mesenchymal stem cell is special
Sign.
The expression for the eye pouch fat stem cell cell surface marker molecule that separation and in vitro culture obtain in 2 embodiment 3 of table
Rate
| Cell surface marker molecule | CD73 | CD90 | CD105 | CD29 | CD44 |
| Expression rate | 99.96% | 99.00% | 99.93% | 99.01% | 99.23% |
| Cell surface marker molecule | CD14 | CD34 | CD45 | CD79a | HLA-DR |
| Expression rate | 0.67 | 1.89 | 0.01 | 0.01 | 0.23 |
Specific embodiment described above is only used for explaining invention, is not intended to limit the present invention.By the spirit invented
The obvious changes or variations extended out are still in the protection scope of this invention.
Claims (4)
1. a kind of method of eye pouch fat stem cell separation and in vitro culture, which comprises the steps of:
(1) eye pouch adipose tissue is the fatty waste that hospital aseptically passes through that the operation of the elderly's eye pouch is removed, and is placed in
It is saved in sterile saline with 2-8 DEG C, storage time is no more than 24 hours；
(2) eye pouch adipose tissue cleaning solution is rinsed 3-5 times, is cut with aseptic operation and tweezers removes blood in fatty sample
Silk, and fatty sample is cut into 1mm3The tissue block of size is put into 50mL centrifuge tube, volume 0.5-3mL；Digestion is added
The ratio of enzyme, digestive ferment and adipose tissue is 5:1-1.5:1, and 37 DEG C of constant-temperature table concussions digest 40-60 minutes, and digestion terminates
Afterwards, filtrate is moved into 50ml centrifuge tube through 70 μm of strainer filterings by digestive juice, and 1500rpm is centrifuged 10 minutes, removes upper layer yellow oil
Rouge and middle layer digestive juice are collected and precipitate and normal saline flushing is added 1-2 times, and 1500rpm is centrifuged 6 minutes, collect precipitating, i.e.,
For eye pouch fat stem cell；
(3) 2ml serum free medium is added in eye pouch fat stem cell precipitating to be resuspended, according to 10000-20000/cm2Density inoculation
In 6 orifice plates or culture dish, in 37 DEG C, 5% carbon dioxide incubator is incubated for, the serum-free training that inoculation more renews after 3-5 days
Base is supported, carried out a serum free medium every 2-4 days later changes liquid, until cell confluency reaches 70%-80%；To eye pouch
Fat stem cell is passed on, and method is to discard former culture medium, cleans cell 1-2 times with cleaning solution, recombination pancreatin is added, 37
It is digested 1-2 minutes under the conditions of DEG C, culture medium is added and terminates digestion reaction, cell suspension is transferred in 50mL centrifuge tube, then is used
Cleaning solution cleans 6 orifice plates or culture dish, and cleaning solution is also transferred in 50ml centrifuge tube, and 1500rpm is centrifuged 6 minutes, collects
Cell precipitation is 10000-20000/cm with cell density2It is seeded to culture bottle, in 37 DEG C, 5% carbon dioxide incubator is carried out
It is incubated for；It repeats the above steps to passage to P2-P4 generation, Trypan Blue counting is carried out with cell counter, with cell density 1-
5*10e6/mL freeze-stored cell.
2. the method for a kind of eye pouch fat stem cell separation according to claim 1 and in vitro culture, which is characterized in that step
Suddenly cleaning solution described in (2) and (3) is mixed with dual anti-according to volume ratio 500:5 by physiological saline.
3. the method for a kind of eye pouch fat stem cell separation according to claim 1 and in vitro culture, which is characterized in that step
Suddenly digestive ferment described in (2) and (3) presses volume by 0.1% Type I collagen enzyme, 0.05%IV Collagenase Type, 0.25% pancreatin/EDTA
It is mixed than 1.5-3:1.5-3:1.
4. the method for a kind of eye pouch fat stem cell separation according to claim 1 and in vitro culture, which is characterized in that step
Suddenly serum free medium described in (3) is by Lonza serum free medium, PALL serum substitute, 20mmol/L L-Glutamine
Solution is mixed according to volume ratio 500:10:5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109692184A (en) * | 2019-01-31 | 2019-04-30 | 武汉汉密顿生物科技股份有限公司 | A kind of menses stem cell medicine and its preparation method and application |
| CN110669728A (en) * | 2019-11-07 | 2020-01-10 | 张伟 | Crocodile adipose-derived stem cell separation culture method and serum-free culture medium |
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