CN107828725A - A kind of fat stem cell separation and the method for in vitro culture - Google Patents
A kind of fat stem cell separation and the method for in vitro culture Download PDFInfo
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Abstract
A kind of fat stem cell separation and the method for in vitro culture, it is characterised in that comprise the following steps：The acquisition of adipose tissue, the separation of fat stem cell, the in vitro culture of fat stem cell.The present invention is advantageous in that：The method of a kind of separation of fat stem cell and in vitro culture in the present invention compared with prior art, has advantages below：1st, the risk when injury and later phase clinical use to cell is reduced；2nd, operating method is simple and easy, repeatable strong；3rd, growth rate is fast, and cultivation cycle is shorter.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of method of fat stem cell separation and in vitro culture.
Background technology
Fat stem cell (adipose-derived stem cells, ADSCs) receives very after being found from 2001
Big attention.Show by research, fat stem cell have to fat, cartilage, skeletonization, into multi-lineage potentials such as fleshes.2012
In January in year, the autologous fat stem cell medicine Cuepistem of South Korea FDA approvals is successfully listed, for treating complexity clone disease
Concurrent anal fistula.October in the same year, U.S. FDA have approved Cytori Therapeutics company's fat stem cells and heart failure suffered from
The clinical test of person's validity.Fat stem cell not only there are all kinds of stem cells to have the advantage that, also with convenient material drawing, wound
It is small, source is sufficient, can once obtain a large amount of fresh stem cells, autologous use does not have the advantages that immunological rejection, and
Avoid the source trouble such as rare of the ethics puzzlement and stem cell of embryonic stem cell.Therefore fat stem cell has non-
Often wide potential applicability in clinical practice.
After the separation method of fat stem cell is more using Mechanical Method processing at present, divided using the method for enzymic digestion
From, it is in the culture in later stage to add dual anti-and serum more, although having certain benefit to culture, also increase injury to cell and
Risk when later phase clinical uses.
The content of the invention
It is an object of the invention to provide a kind of injury to cell is small, and the small fat of risk during later phase clinical use is done
Cell separates and the method for in vitro culture.
A kind of fat stem cell separation and the method for in vitro culture, it is characterised in that comprise the following steps：
(1) acquisition of adipose tissue：The fat that adipose tissue is aseptically obtained for the beauty parlor of specialty by liposuction
Fat discarded object, with 2-8 DEG C of preservation after extraction, storage time was no more than 48 hours；
(2) separation of fat stem cell：1500rpm is carried out to adipose tissue to centrifuge 5 minutes, discards upper strata grease and lower floor
Courageous and upright cellular layer, middle fat deposit is taken, using PBS fat 3-5 times, until cleaning fluid is limpid；Last time cleaning gained
Mixture carries out 1500rpm and centrifuged 5 minutes, aspirating adipose, records fatty volume；Obtain fatty according to 1:1 is carried out using digestive ferment
Digestion, 37 DEG C of constant-temperature table 150-280 rotating speeds digest 20-60 minutes；After digestion terminates, 2000rpm is centrifuged 5 minutes, is discarded
Layer oil layer and postdigestive fat deposit, leaving layer stem cell precipitation and a small amount of digestive juice, add isometric FBS and carry out eventually
Only；Mixture carries out 1500rpm centrifugations 5 minutes after termination, collects fat stem cell, is resuspended using PBS, 1500rpm cleanings 2
Time, remove the dual anti-and FBS used early stage；Gained cell count；
(3) in vitro culture of fat stem cell：It is resuspended using primary culture medium, 100um filterings, according to 3-5*10e5 density
It is inoculated in T75 blake bottles, 37 DEG C, 5% CO2gas incubator is incubated；24-48 hours after inoculation, observe cell attachment
State, primary culture medium full dose change liquid, remove not adherent suspension cell；Changing for primary culture medium was carried out every 2-3 days
Liquid, reach 80-90% to cell fusion degree；The fat stem cell for reaching 80-90% to degrees of fusion passes on, and discards former culture
Liquid, PBS 3 times, cell dissociation buffer carry out digestion process, collect gained fat stem cell, carry out cell count, use passage
Culture medium is inoculated in T75 blake bottles according to 3-5*10e5,37 DEG C, and 5% CO2gas incubator is incubated；Repeat above-mentioned step
It is rapid to arrive P3 to passage, preserved.
As a kind of preferable scheme, adipose tissue is preferred with belly, buttocks, thigh described in step (1).
It is further preferred that storage time described in step (1) is less than 4 hours.
It is further preferred that the collocation method of digestive ferment is described in step (2), clostridiopetidase A I is added in 90mlPBS
72mg, clostridiopetidase A IV 18mg, after filtering, add 0.125%Trypsin-EDTA and mix, 100ml is made.
It is further preferred that the collocation method of primary culture medium is described in step (2), DMEM-F12 culture mediums 455ml
Middle addition HPL serum substitute 45ml, add 1000IU liquaemins.
It is further preferred that the collocation method of Secondary Culture base is described in step (3), DMEM-F12 culture mediums 455ml
HPL serum substitute 45ml are added, bFGF 10mg is added, adds 1000IU liquaemins.
It is further preferred that the method preserved described in step (3) is liquid nitrogen cryopreservation.
The present invention is advantageous in that：The method of a kind of separation of fat stem cell and in vitro culture in the present invention is with showing
There is technology to compare, there is advantages below：1st, the risk when injury and later phase clinical use to cell is reduced；2nd, operating method letter
It is single easy, repeatable strong；3rd, growth rate is fast, and cultivation cycle is shorter.
Brief description of the drawings
Fig. 1 is 40 times of figures of degrees of fusion inverted microscope of fat stem cell in vitro culture prepared by embodiments of the invention；
Fig. 2 is the flow cytometer detection figure for the stem cell that fat stem cell in vitro culture prepared by embodiments of the invention obtains.
Embodiment
A kind of fat stem cell separation and the method for in vitro culture, comprise the following steps：
(1) configuration of reagent：
The configuration of digestive ferment：Clostridiopetidase A I 72mg, clostridiopetidase A IV 18mg are added in 90mlPBS, after filtering, is added
0.125%Trypsin-EDTA is mixed, and 100ml is made；
The configuration of primary culture medium：HPL serum substitute 45ml are added in DMEM-F12 culture mediums 455ml, are added
1000IU liquaemins；
The configuration of Secondary Culture base：DMEM-F12 culture mediums 455ml adds HPL serum substitute 45ml, adds bFGF
10mg, add 1000IU liquaemins；
(2) acquisition of adipose tissue：Fatty waste 100ml, 4 DEG C of 4 small lucks of preservation are extracted under beauty parlor's aseptic condition
Transport to laboratory.
(3) separation of fat stem cell：1500rpm is carried out to adipose tissue to centrifuge 5 minutes, discards upper strata grease and lower floor
Courageous and upright cellular layer, middle fat deposit is taken, using PBS fat 3-5 times, until cleaning fluid is limpid；Last time cleaning gained
Mixture carries out 1500rpm and centrifuged 5 minutes, aspirating adipose, records fatty volume；Obtain fatty according to 1:1 is carried out using digestive ferment
Digestion, 37 DEG C of constant-temperature table 150-280 rotating speeds digest 20-60 minutes；After digestion terminates, 2000rpm is centrifuged 5 minutes, is discarded
Layer oil layer and postdigestive fat deposit, leaving layer stem cell precipitation and a small amount of digestive juice, add isometric FBS and carry out eventually
Only；Mixture carries out 1500rpm centrifugations 5 minutes after termination, collects fat stem cell 1.2*10e6, is resuspended using PBS,
1500rpm is cleaned 2 times, removes the dual anti-and FBS used early stage；Gained cell count；
(4) in vitro culture of fat stem cell：It is resuspended, 100um filterings, is connect according to 5*10e5 density using primary culture medium
Kind is in 2 T75 blake bottles, and 37 DEG C, 5% CO2gas incubator is incubated；36 hours after inoculation, cell attachment shape is observed
State, primary culture medium full dose change liquid, remove not adherent suspension cell；Liquid is changed every the primary culture medium of progress in 2-3 days,
Reach 90% to cell fusion degree, referring to Fig. 1；It is passaged in 2 T175 blake bottles, carried out changing liquid to cell every 2-3 days and melt
Right is 90%, according to 1:3 are passed on, and collect cell 3*10e altogether to P38Cell；Flow cytometer detection is carried out, as a result such as Fig. 2 institutes
Show, meet fat mesenchymal stem cell phenotype.
It should be appreciated that specific embodiment described above is only used for explaining invention, it is not intended to limit the present invention.By sending out
Among the obvious changes or variations that bright spirit is extended out is still in protection scope of the present invention.
Claims (7)
1. a kind of fat stem cell separation and the method for in vitro culture, it is characterised in that comprise the following steps：
(1) acquisition of adipose tissue：Adipose tissue gives up for the fat that the beauty parlor of specialty is aseptically obtained by liposuction
Gurry, with 2-8 DEG C of preservation after extraction, storage time was no more than 48 hours；
(2) separation of fat stem cell：1500rpm is carried out to adipose tissue to centrifuge 5 minutes, discards upper strata grease and lower floor is courageous and upright
Cellular layer, middle fat deposit is taken, using PBS fat 3-5 times, until cleaning fluid is limpid；Last time cleaning gained mixes
Thing carries out 1500rpm and centrifuged 5 minutes, aspirating adipose, records fatty volume；Obtain fatty according to 1:1 is digested using digestive ferment,
37 DEG C of constant-temperature table 150-280 rotating speeds digest 20-60 minutes；After digestion terminates, 2000rpm is centrifuged 5 minutes, discards upper strata grease
Layer and postdigestive fat deposit, leaving layer stem cell precipitation and a small amount of digestive juice, add isometric FBS and are terminated；Eventually
Mixture carries out 1500rpm centrifugations 5 minutes after only, collects fat stem cell, is resuspended using PBS, and 1500rpm is cleaned 2 times, removes
The dual anti-and FBS used early stage；Gained cell count；
(3) in vitro culture of fat stem cell：It is resuspended, 100um filterings, is inoculated with according to 3-5*10e5 density using primary culture medium
In T75 blake bottles, 37 DEG C, 5% CO2gas incubator is incubated；24-48 hours after inoculation, observe cell attachment shape
State, primary culture medium full dose change liquid, remove not adherent suspension cell；Liquid is changed every the primary culture medium of progress in 2-3 days,
Reach 80-90% to cell fusion degree；The fat stem cell for reaching 80-90% to degrees of fusion passes on, and discards original fluid,
PBS 3 times, cell dissociation buffer carry out digestion process, collect gained fat stem cell, carry out cell count, are trained using passage
Support base to be inoculated in T75 blake bottles according to 3-5*10e5,37 DEG C, 5% CO2gas incubator is incubated；Repeat the above steps
P3 is arrived to passage, is preserved.
2. a kind of fat stem cell separation according to claim 1 and the method for in vitro culture, it is characterised in that step
(1) adipose tissue described in is preferred with belly, buttocks, thigh.
3. a kind of fat stem cell separation according to claim 1 or 2 and the method for in vitro culture, it is characterised in that step
Suddenly storage time described in (1) is less than 4 hours.
4. a kind of fat stem cell separation according to claim 3 and the method for in vitro culture, it is characterised in that step
(2) collocation method of digestive ferment described in is that clostridiopetidase A I72mg, clostridiopetidase A IV 18mg are added in 90mlPBS, after filtering,
Add 0.125%Trypsin-EDTA to mix, 100ml is made.
5. a kind of fat stem cell separation according to claim 4 and the method for in vitro culture, it is characterised in that step
(2) collocation method of primary culture medium described in is that HPL serum substitute 45ml are added in DMEM-F12 culture mediums 455ml, then
Add 1000IU liquaemins.
6. a kind of fat stem cell separation according to claim 5 and the method for in vitro culture, it is characterised in that step
(3) collocation method of Secondary Culture base described in is that DMEM-F12 culture mediums 455ml adds HPL serum substitute 45ml, is added
BFGF 10mg, add 1000IU liquaemins.
7. a kind of fat stem cell separation according to claim 6 and the method for in vitro culture, it is characterised in that step
(3) method preserved described in is liquid nitrogen cryopreservation.
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Cited By (5)
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| CN109161521A (en) * | 2018-09-13 | 2019-01-08 | 佛山科学技术学院 | A kind of preparation method of human adipose-derived stem cell conditioned medium |
| CN109266605A (en) * | 2018-10-12 | 2019-01-25 | 希瑞干细胞科技有限公司 | A kind of method of eye pouch fat stem cell separation and in vitro culture |
| CN109554339A (en) * | 2018-12-29 | 2019-04-02 | 中国医学科学院整形外科医院 | A method of efficiently separating fat mesenchymal stem cell from liposuction aspirates |
| CN110079497A (en) * | 2019-03-20 | 2019-08-02 | 济南赛尔生物科技股份有限公司 | Method for separating and culturing adipose-derived stem cells |
| CN110885784A (en) * | 2018-09-11 | 2020-03-17 | 上海赛傲生物技术有限公司 | Clinical application-grade adipose-derived stem cells and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110885784A (en) * | 2018-09-11 | 2020-03-17 | 上海赛傲生物技术有限公司 | Clinical application-grade adipose-derived stem cells and preparation method thereof |
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| CN110079497A (en) * | 2019-03-20 | 2019-08-02 | 济南赛尔生物科技股份有限公司 | Method for separating and culturing adipose-derived stem cells |
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Application publication date: 20180323 |