CN108315297A - A method of it detached from adipose tissue, purify fat stem cell - Google Patents
A method of it detached from adipose tissue, purify fat stem cell 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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Abstract
The present invention provides a kind of method for being detached from adipose tissue, purifying fat stem cell, and the method includes fat stem cell preparation, the secondary culture of fat mesenchymal stem cell, cells to harvest.+ 1% L GlutaMAX of 5ng/ml EGF+5ng/ml FGF+5ng/ml IFN γ+5ng/ml TNF α are added in the medium, the proliferation that can effectively facilitate MSC is added in cultivating system simultaneously in these factors, to effectively shorten incubation time.The present invention can not only effectively reduce cost but also can be quickly obtained good fat stem cell, reduce the injury to cell, shorten cultivation cycle, reduce pollution, and can improve cell purity.
Description
Technical field
The invention belongs to biotechnologies, specifically provide and a kind of to be detached from adipose tissue, purify fat stem cell
Method.
Background technology
Fat stem cell（Adipose-derived stem cells, ADSCs）It is isolated from adipose tissue
A kind of stem cell with multi-lineage potential.Fat stem cell can be divided into polyembryony confluent monolayer cells, including mesoderm, entoderm
And ectoderm cell, and can secrete many to the advantageous cell factor of tissue.Fat stem cell is multi-functional of a group
Mesenchymal stem cells can be divided into other cell lines, and there are a large amount of fat in non-fat (interstitial) fragment of adipose tissue
Fat stem cell, and it is easily isolated acquisition.Fat stem cell can promote the regeneration of cell with the repair function of recovery organization cell,
Restore young face；Can also physical function be made to be fully improved simultaneously, the diseases such as inferior health, early ageing can be effectively improved, by
It is interior and outer be really effective against aging.
A large amount of research confirms that ADSCs has other types of mesenchymal stem cell such as mesenchymal stem cells unexistent
Some advantages：Adipose tissue-derived is extensive, convenient material drawing, and stem cell amount to obtain is big, can be easily separated purifying, and adipose tissue is damaged for area
Hinder small, differentiation capability is strong.Thus, fat stem cell is a kind of good organizational project and regenerative medicine seed cell.
Fat stem cell clinically starts to apply and significant effect, is promoted at present in medical and beauty treatment, immunity
Etc. application it is relatively broad, and in angiocardiopathy, diabetes, neurogenic disease, disease of digestive tract, genital system diseases
Multinomial clinical research is carried out in terms of etc. human life and healthy major disease is endangered.
In conclusion fat stem cell application advantage, there are advantage, practical application, potential development etc. have it is non-
Normal advantage outstanding, so being referred to as " star stem cell ".Currently, the product preparation process of fat stem cell class is complicated, raw
Object activity is limited by various external conditions, saves inconvenience.
The immediate technical solution of present：Chinese patent CN104560868A discloses a kind of fat stem cell
Primary separation and culture method has been directed to collagenase type I and the mixing liquid of trypsase digestion adipose tissue；With containing
There is the DMEM of EGF and FBS to cultivate fat stem cell.
Technological deficiency at present：In art methods, Mechanical Method removal blood vessel, single enzyme digestion, use are still mostly used
Lysate splitting erythrocyte, and etc., the cell mass of the higher ADSCs of purity is obtained as far as possible；Blueness-chain is added in culture
The antibiotic such as mycin also cause different degrees of injury and in use exist centainly to strive although can prevent from polluting to cell
View.Fat stem cell is separately cultured that process is complicated for operation, time-consuming, production cost is higher.In addition to this, due to the use of compared with
More chemical reagent, vigor is relatively low after isolation, growth rate is slower by ADSCs.Using the digestive ferment of single varieties to fatty group
It knits and is digested, digestion, which is not thorough, leads to fat stem cell low separation efficiency.Generally speaking, the separation of ADSCs and cultural method
All have greatly improved space.
The present invention can not only effectively reduce cost but also can be quickly obtained good fat stem cell, reduce the wound to cell
Evil shortens cultivation cycle, reduces pollution, and can improve cell purity.
Invention content
A kind of fractionation of fatty stem cell from adipose tissue of present invention offer simultaneously carries out secondary culture, final to obtain cell production
The method of product will solve the low separation efficiency being likely to occur in fat stem cell separation, incubation, period length, safety not
The problems such as sufficient.
The technical solution adopted by the present invention to solve the technical problems is：
Detection investigation, addition are taken in adipose tissue obtains, adipose tissue transports, prepared by adipose tissue and cell cultivation process
The methods of antibiotic ensures to obtain free of contamination fat stem cell to guarantee safety, and uses and changes in separation, incubation
Good digestion method, cultivating system and cultural method improve separative efficiency, shorten cultivation cycle and can obtain high-purity, characterize it is excellent
Good cell.
To achieve the above object, the present invention adopts the following technical scheme that：
A method of it detached from adipose tissue, purify fat stem cell, including prepared by fat stem cell, fat mesenchymal is dry
The secondary culture of cell, cell harvest.
The fat stem cell is prepared as：
（1）Adipose tissue is transferred to 50ml centrifuge tubes, often pipe 40ml, 800g, 8 minute；
（2）Adipose tissue after centrifugation is transferred in new centrifuge tube, isometric that 0.9% physiological saline, 800g 8 minutes is added；
（3）Supernatant is discarded, repeats step 2 washing adipose tissue twice；
（4）Add digestive juice into adipopexis in equal volume, 37 DEG C of shaking tables, 150rpm, 30-50min are put into after mixing well sealing；
（5）The adipose tissue digested is taken out, is centrifuged, 800g, 10 minutes；
（6）Upper-layer fat is discarded, the cell precipitation after centrifugation, centrifugation, 800g 10 minutes is resuspended with DMEM/F12 culture mediums；Weight
Multiple step 6 washs cell precipitation；
（7）The supernatant after centrifugation is discarded, cell precipitation and mixing is resuspended in DMEM/F12 culture mediums, first uses 40um strainer filterings thin
Born of the same parents；Filtered cell centrifugation, 800g 10 minutes abandon supernatant；
（8）Cell precipitation is resuspended in 1.0ml DMEM/F12 culture mediums, and the 0.3%NaCl solution of 4.0ml precoolings, room are added after mixing
Temperature is placed 10 minutes, splitting erythrocyte；
（9）Centrifugation, 400g 5 minutes discard supernatant；
（10）Cell precipitation, Trypan Blue cell count is resuspended with MSC serum free mediums, adjustment cell density is 2.0*10^6/ ml, each culture bottle first adds 14ml culture mediums, then adds 1.0ml cell suspensions, mixing；Cell changes liquid after 24 hours, and removal is not
Attached cell changes the liquid once every three days later；It cultivates to after the 5th day, cell density reaches 90%, and fat mesenchymal is dry thin
Born of the same parents' secondary culture.
Collagenase type I+0.1wt.% IV Collagenase Type+0.1wt.% pancreas the eggs containing 0.075 ~ 0.5wt.% in the digestive juice
White enzyme.
MSC serum free mediums be MSC serum free medium+5ng/ml EGF+5ng/ml FGF+5ng/ml IFN-γ+
5ng/ml TNF-α+1wt.% L-GlutaMAX。
The secondary culture of the fat mesenchymal stem cell is：
（1）The fat mesenchymal stem cell cell for preparing passage is taken out, culture medium is sopped up；
（2）Each culture bottle adds 0.25% pancreatin 1.0ml vitellophags, finds that 5.0ml ends are added in cell rounding immediately under microscope
Only liquid terminates digestion, will be transferred in 50ml centrifuge tubes after the soft pressure-vaccum of cell, physiological saline 20.0ml is used in combination to wash bottom culture bottle
Once, the cell of washing is transferred in centrifuge tube, 800g 6 minutes；
（3）Supernatant is abandoned in suction, cell precipitation, Trypan Blue cell count is resuspended with serum free medium, adjustment cell density is
4.0*10^5/ ml, each culture bottle first adds 23.0ml serum free mediums, then adds 2.0ml cell suspensions, and training is positioned over after mixing
Support static gas wave refrigerator in case.
The cell harvests：
1）Cell is observed, cell growth freezes or supply use to harvest is carried out when 80 ~ 90%；
2）Tissue Culture Flask is taken out, with 75% alcohol disinfecting bottom of bottle, is put into Biohazard Safety Equipment；
3）Old culture medium is abandoned with pipette suction；
4）10ml/ bottles of terminate liquid is added after cell obviously bounces back in every bottle of addition 0.25% pancreatin of 3ml, 37 DEG C of digestion 1min,
Terminate digestion, collect in all liq to 50ml centrifuge tubes, then every bottle plus 10ml physiological saline, imported after soft piping and druming 50ml from
In heart pipe；
5）1200rpm centrifuges 6min, abandons supernatant, and cell precipitation is suspended with 16ml physiological saline, and mixing takes 1ml to count and flow
Formula detects；
6）Add physiological saline to 40ml, 500 μ l supernatants is taken to do endotoxin detection, 1200rpm centrifuges 6min；
7）Supernatant is poured into clean centrifuge tube, does bacterium inspection and detection of mycoplasma；
8）The cell frozen：Centrifugation is suspended with 2.5ml FBS, is slow added into 2.5ml and is frozen mother liquor, is dispensed into after mixing
In cryopreservation tube, every pipe 1ml；
The cell of application：Centrifugation is resuspended with 0.9% normal saline solution, and cell quantity and volume are depending on service condition；
Cell suspension after resuspension is transferred to cell and feeds back bag or feed back in pipe；
9）Mark cell algebraically, density, bar code and operating time in outside tube wall；
10）The cell frozen is frozen by program temperature reduction box method, and the cell of application passes through cold chain transportation to application site.
The advantage of the invention is that：
1, digestive juice is the mixed enzyme solution of+0.1% trypsase of+0.1% IV Collagenase Types of 0.075% ~ 0.5% collagenase type I, can
To effectively improve enzymolysis efficiency, shorten preparation time.
2, with 0.3%NaCl solution splitting erythrocytes, the higher fat stem cell of purity can be obtained.
3,5ng/ml EGF+5ng/ml FGF+5ng/ml IFN-γ+5ng/ml TNF-α+1% is added in the medium
The proliferation that can effectively facilitate MSC is added in cultivating system simultaneously in L-GlutaMAX, these factors, is cultivated to effectively shorten
Time.
4. cell harvest when counted, the detection of flow cytometer detection, endotoxin, detection of mycoplasma, Bacteria Culture detection etc.,
There are the qualified just permission storage storage of every detection or supply client to use, from cell quantity, cell characterization and whether there is or not micro- lifes
Many-sided validity and safety that ensure that cell products such as object pollution.
The guarantee of the methods of detection investigation is taken to obtain in fat acquisition, fat preparation and cell cultivation process free of contamination
Fat stem cell is improved using the separation method and cultivating system of improvement in separation, incubation and is divided to guarantee safety
From efficiency, shortens cultivation cycle and the excellent cell of high-purity, characterization can be obtained.
Description of the drawings
Fig. 1 fat stem cells spread cell state figure of the bottle after 2 days：Cell is mostly rounded, and a few cell is in fusiformis,
Cell quantity is also less.
Fig. 2 fat stem cells spread cell state figure of the bottle after 5 days：Cell quantity showed increased, and classics are presented
Mescenchymal stem cell form.
Fig. 3 fat stem cells P0 passes the 3rd day cell state figure of P1：Cell growth uniformly, morphologic criteria, quantity compared with
It is more.
Fig. 4 fat stem cell flow cytometer detection result figures.From flow cytometer detection result, it can be seen that this programme detaches thin
Born of the same parents' phenotype meets the feature of fat stem cell（CD44/CD105 is the positive, and CD34/CD45 is feminine gender）.
Specific implementation mode
Embodiment 1
1, prepared by fat stem cell
（1）The qualified client of infectious disease detection leaves and takes fatty sample by liposuction procedures, and Cord blood transports to laboratory as early as possible.
（2）Adipose tissue is transferred to 50ml centrifuge tubes, often pipe 40ml, 800g, 8 minute.
（3）Adipose tissue after centrifugation is transferred in new centrifuge tube, 0.9% physiological saline of isometric addition, 800g, 8 points
Clock.
（4）Supernatant is discarded, repeats step 3 washing adipose tissue twice.
（5）Add digestive juice into adipopexis in equal volume（+ 0.1% pancreas egg of+0.1% IV Collagenase Types of 0.5% collagenase type I
White enzyme）, 37 DEG C of shaking tables, 150rpm, 30min are put into after mixing well sealing.
（6）The adipose tissue digested is taken out, is centrifuged, 800g, 10 minutes.
（7）Upper-layer fat is discarded, the cell precipitation after centrifugation is resuspended with DMEM/F12 basal mediums, centrifuges, 800g, 10
Minute.
（8）It repeats step 7 and washs cell precipitation.
（9）The supernatant after centrifugation is discarded, cell precipitation and mixing is resuspended in DMEM/F12, first uses 40um strainer filtering cells.
（10）Filtered cell centrifugation, 800g, 10 minutes.Abandon supernatant.
（11）Cell precipitation is resuspended in 1.0ml DMEM/F12 culture mediums, and the 0.3%NaCl that 4.0ml precoolings are added after mixing is molten
Liquid is placed at room temperature for 10 minutes, splitting erythrocyte.
（12）Centrifugation, 400g 5 minutes.Discard supernatant.
（13）With MSC serum free mediums（MSC serum-free basal medium+5ng/ml EGF+5ng/ml FGF+5ng/
ml IFN-γ+5ng/ml TNF-α+1% L-GlutaMAX）Cell precipitation is resuspended, platform expects that blue staining cell counts, adjusts cell
Density is 2.0*10^6/ml.Each T75 culture bottles first add 14ml culture mediums, then add 1.0ml cell suspensions, mixing.
（14）Cell changes liquid after 24 hours, removes non-attached cell.It changes the liquid once every three days later.
（15）Culture was to the 5th day or so, and cell density reaches 90% or so, fat mesenchymal stem cell passage training
It supports.
, fat mesenchymal stem cell secondary culture
（16）The fat mesenchymal stem cell cell for preparing passage is taken out, culture medium is sopped up.
（17）Each T75 culture bottles add 0.25% pancreatin 1.0ml vitellophags, find that cell rounding adds immediately under microscope
Enter 5.0ml terminate liquids and terminate digestion, will be transferred in 50ml centrifuge tubes after the soft pressure-vaccum of cell, physiological saline 20.0ml is used in combination to wash
Bottom culture bottle is primary, the cell of washing is transferred in centrifuge tube, 800g, 6 minutes.
（18）Supernatant is abandoned in suction, cell precipitation is resuspended with serum free medium, platform expects that blue staining cell counts, and adjustment cell is close
Degree is 4.0*10^5/ ml, each T175 culture bottles first add 23.0ml culture mediums, then add 2.0ml cell suspensions, are positioned over after mixing
Static gas wave refrigerator in incubator.
, cell harvest：
1）Cell is observed, cell growth freezes or supply client's use to harvest is carried out when 90%.
2）Tissue Culture Flask is taken out, with 75% alcohol disinfecting bottom of bottle, is put into Biohazard Safety Equipment.
3）Old culture medium is abandoned with pipette suction.
4）Terminate liquid 10ml/ is added after cell obviously bounces back in every bottle of addition 0.25% pancreatin of 3ml, 37 DEG C of digestion 1min
Bottle terminates digestion, collects in all liq to 50ml centrifuge tubes, then every bottle plus 10ml physiological saline, is imported after soft piping and druming
In 50ml centrifuge tubes.
5）1200rpm centrifuges 6min, abandons supernatant, and cell precipitation is suspended with 16ml physiological saline, and mixing takes 1ml to count
And flow cytometer detection.
6）Add physiological saline to 40ml, 500 μ l supernatants is taken to do endotoxin detection, 1200rpm centrifuges 6min.
7）Supernatant is poured into clean centrifuge tube, is bacterium inspection and detection of mycoplasma
8）The cell frozen：Centrifugation is suspended with 2.5ml FBS, is slow added into 2.5ml and is frozen mother liquor, is dispensed into after mixing
In cryopreservation tube, every pipe 1ml.
The cell of application：Centrifugation is resuspended with 0.9% normal saline solution, and cell quantity and volume are used according to client
Depending on situation.Cell suspension after resuspension is transferred to cell and feeds back bag or feed back in pipe.
9）Mark cell algebraically, density, bar code and operating time in outside tube wall.
10）The cell frozen is frozen by program temperature reduction box method, and the cell of application passes through cold chain transportation to application site.
Embodiment 2
1, prepared by fat stem cell
（19）The qualified client of infectious disease detection leaves and takes fatty sample by liposuction procedures, and Cord blood transports to laboratory as early as possible.
（20）Adipose tissue is transferred to 50ml centrifuge tubes, often pipe 40ml, 800g, 8 minute.
（21）Adipose tissue after centrifugation is transferred in new centrifuge tube, 0.9% physiological saline of isometric addition, 800g, and 8
Minute.
（22）Supernatant is discarded, repeats step 3 washing adipose tissue twice.
（23）Add digestive juice into adipopexis in equal volume（+ 0.1% pancreas of+0.1% IV Collagenase Types of 0.1% collagenase type I
Protease）, 37 DEG C of shaking tables, 150rpm, 40min are put into after mixing well sealing.
（24）The adipose tissue digested is taken out, is centrifuged, 800g, 10 minutes.
（25）Upper-layer fat is discarded, the cell precipitation after centrifugation is resuspended with DMEM/F12 basal mediums, centrifuges, 800g,
10 minutes.
（26）It repeats step 7 and washs cell precipitation.
（27）The supernatant after centrifugation is discarded, cell precipitation and mixing is resuspended in DMEM/F12, first uses 40um strainer filtering cells.
（28）Filtered cell centrifugation, 800g, 10 minutes.Abandon supernatant.
（29）Cell precipitation is resuspended in 1.0ml DMEM/F12 culture mediums, and the 0.3%NaCl that 4.0ml precoolings are added after mixing is molten
Liquid is placed at room temperature for 10 minutes, splitting erythrocyte.
（30）Centrifugation, 400g 5 minutes.Discard supernatant.
（31）With MSC serum free mediums（MSC serum-free basal medium+5ng/ml EGF+5ng/ml FGF+5ng/
ml IFN-γ+5ng/ml TNF-α+1% L-GlutaMAX）Cell precipitation is resuspended, platform expects that blue staining cell counts, adjusts cell
Density is 2.0*10^6/ml.Each T75 culture bottles first add 14ml culture mediums, then add 1.0ml cell suspensions, mixing.
（32）Cell changes liquid after 24 hours, removes non-attached cell.It changes the liquid once every three days later.
（33）Culture was to the 5th day or so, and cell density reaches 90% or so, fat mesenchymal stem cell passage training
It supports.
, fat mesenchymal stem cell secondary culture
（34）The fat mesenchymal stem cell cell for preparing passage is taken out, culture medium is sopped up.
（35）Each T75 culture bottles add 0.25% pancreatin 1.0ml vitellophags, find that cell rounding adds immediately under microscope
Enter 5.0ml terminate liquids and terminate digestion, will be transferred in 50ml centrifuge tubes after the soft pressure-vaccum of cell, physiological saline 20.0ml is used in combination to wash
Bottom culture bottle is primary, the cell of washing is transferred in centrifuge tube, 800g, 6 minutes.
（36）Supernatant is abandoned in suction, cell precipitation is resuspended with serum free medium, platform expects that blue staining cell counts, adjusts cell
Density is 4.0*10^5/ml, and each T175 culture bottles first add 23.0ml culture mediums, then add 2.0ml cell suspensions, are placed after mixing
The static gas wave refrigerator in incubator.
, cell harvest：
1）Cell is observed, cell growth freezes or supply client's use to harvest is carried out when 80%.
2）Tissue Culture Flask is taken out, with 75% alcohol disinfecting bottom of bottle, is put into Biohazard Safety Equipment.
3）Old culture medium is abandoned with pipette suction.
4）Terminate liquid 10ml/ is added after cell obviously bounces back in every bottle of addition 0.25% pancreatin of 3ml, 37 DEG C of digestion 1min
Bottle terminates digestion, collects in all liq to 50ml centrifuge tubes, then every bottle plus 10ml physiological saline, is imported after soft piping and druming
In 50ml centrifuge tubes.
5）1200rpm centrifuges 6min, abandons supernatant, and cell precipitation is suspended with 16ml physiological saline, and mixing takes 1ml to count
And flow cytometer detection.
6）Add physiological saline to 40ml, 500 μ l supernatants is taken to do endotoxin detection, 1200rpm centrifuges 6min.
7）Supernatant is poured into clean centrifuge tube, is bacterium inspection and detection of mycoplasma
8）The cell frozen：Centrifugation is suspended with 2.5ml FBS, is slow added into 2.5ml and is frozen mother liquor, is dispensed into after mixing
In cryopreservation tube, every pipe 1ml.
The cell of application：Centrifugation is resuspended with 0.9% normal saline solution, and cell quantity and volume are used according to client
Depending on situation.Cell suspension after resuspension is transferred to cell and feeds back bag or feed back in pipe.
9）Mark cell algebraically, density, bar code and operating time in outside tube wall.
10）The cell frozen is frozen by program temperature reduction box method, and the cell of application passes through cold chain transportation to application site.
Reference examples 1
Remaining condition is identical, using the digestion in 0.05%+0.1% trypsase alternate embodiment 1 of Type I collagen enzyme and implementation 2
Liquid, digestion time 40min；Culture medium is the DMEM in high glucose culture medium containing 10%FBS.
Embodiment 1, embodiment 2 and reference examples 1 have respectively been separately cultured three different samples, are carried out by following 2 aspects
The result detection and comparison of the present invention.
1. the enzymolysis efficiency of different digestive juices compares：
Human adipose-derived stem cell is digested using the digestive juice of different formulations, it is as a result as follows：Embodiment 1, embodiment 2 and control
Average living cells quantity difference of the example 1 per 100ml adipose tissue harvests is as follows：3.25*107It is a, 3.20*107It is a, 2.50*107
It is a.As shown in table 1, there are pole significant differences for living cells quantity obtained by the digestion of embodiment 1 and embodiment 2 with reference examples 1.Pass through
The digestion formula of liquid that these data can be seen that the present invention can significantly improve enzymolysis efficiency with respect to other, and cell yield is higher.
The explored enzymatic hydrolysis condition of invention is stable, efficient, and adipose tissue is thoroughly digested, and the cell in tissue is by as far as possible
It releases；When collagenase type I solubility is smaller（Embodiment 2）It needs that digestion time is appropriately extended, but digestion effect is not by shadow
It rings.
2. the attached cell number and cultivation cycle of different culture media culture compare：
Human adipose-derived stem cell is cultivated using the culture medium of different formulations, P0 is counted for cell, it is as a result as follows：It is real
Applying the average cell number that example 1, embodiment 2 and reference examples 1 are cultivated is respectively：2.50*106It is a, 2.45*106It is a, 1.58*106It is a.
Using the medium culture fat stem cell of different formulations, 2.50*10 is finally harvested7A cell, when counting required culture
Between, the overall average incubation time of embodiment 1, embodiment 2 and reference examples 1 is respectively：14 days, 14 days, 20 days.As shown in table 1, real
The P0 for applying example 1 and embodiment 2 and reference examples 1 withholds and obtains cell number and cultivated days there are pole significant differences.
Table 1
The result shows that the present invention can improve separation effect in separation, incubation using the cultivating system and cultural method of improvement
Rate shortens cultivation cycle and can obtain the excellent cell of high-purity, characterization.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (6)
1. a kind of method for being detached from adipose tissue, purifying fat stem cell, it is characterised in that：It is dry that the method includes fat
Cell preparation, the secondary culture of fat mesenchymal stem cell, cell harvest.
2. a kind of method for being detached from adipose tissue, purifying fat stem cell according to claim 1, feature exist
In：The fat stem cell is prepared as：
（1）Adipose tissue is transferred to 50ml centrifuge tubes, often pipe 40ml, 800g, 8 minute；
（2）Adipose tissue after centrifugation is transferred in new centrifuge tube, isometric that 0.9% physiological saline, 800g 8 minutes is added；
（3）Supernatant is discarded, repeats step 2 washing adipose tissue twice；
（4）Add digestive juice into adipopexis in equal volume, 37 DEG C of shaking tables, 150rpm, 30-50min are put into after mixing well sealing；
（5）The adipose tissue digested is taken out, is centrifuged, 800g, 10 minutes；
（6）Upper-layer fat is discarded, the cell precipitation after centrifugation, centrifugation, 800g 10 minutes is resuspended with DMEM/F12 culture mediums；Weight
Multiple step 6 washs cell precipitation；
（7）The supernatant after centrifugation is discarded, cell precipitation and mixing is resuspended in DMEM/F12 culture mediums, first uses 40um strainer filterings thin
Born of the same parents；Filtered cell centrifugation, 800g 10 minutes abandon supernatant；
（8）Cell precipitation is resuspended in 1.0ml DMEM/F12 culture mediums, and the 0.3%NaCl solution of 4.0ml precoolings, room are added after mixing
Temperature is placed 10 minutes, splitting erythrocyte；
（9）Centrifugation, 400g 5 minutes discard supernatant；
（10）Cell precipitation, Trypan Blue cell count is resuspended with MSC serum free mediums, adjustment cell density is 2.0*10^6/ ml, each culture bottle first adds 14ml culture mediums, then adds 1.0ml cell suspensions, mixing；Cell changes liquid after 24 hours, and removal is not
Attached cell changes the liquid once every three days later；It cultivates to after the 5th day, cell density reaches 90%, and fat mesenchymal is dry thin
Born of the same parents' secondary culture.
3. a kind of method for being detached from adipose tissue, purifying fat stem cell according to claim 2, feature exist
In：Collagenase type I+0.1wt.% IV Collagenase Type+0.1wt.% the trypsase containing 0.075 ~ 0.5wt.% in the digestive juice.
4. a kind of method for being detached from adipose tissue, purifying fat stem cell according to claim 2, feature exist
In：MSC serum free mediums are MSC serum free medium+5ng/ml EGF+5ng/ml FGF+5ng/ml IFN-γ+5ng/
ml TNF-α+1wt.% L-GlutaMAX。
5. a kind of method for being detached from adipose tissue, purifying fat stem cell according to claim 1, feature exist
In：The secondary culture of the fat mesenchymal stem cell is：
（1）The fat mesenchymal stem cell cell for preparing passage is taken out, culture medium is sopped up；
（2）Each culture bottle adds 0.25% pancreatin 1.0ml vitellophags, finds that 5.0ml ends are added in cell rounding immediately under microscope
Only liquid terminates digestion, will be transferred in 50ml centrifuge tubes after the soft pressure-vaccum of cell, physiological saline 20.0ml is used in combination to wash bottom culture bottle
Once, the cell of washing is transferred in centrifuge tube, 800g 6 minutes；
（3）Supernatant is abandoned in suction, cell precipitation, Trypan Blue cell count is resuspended with serum free medium, adjustment cell density is
4.0*10^5/ ml, each culture bottle first adds 23.0ml serum free mediums, then adds 2.0ml cell suspensions, and training is positioned over after mixing
Support static gas wave refrigerator in case.
6. a kind of method for being detached from adipose tissue, purifying fat stem cell according to claim 1, feature exist
In：The cell harvests：
1）Cell is observed, cell growth freezes or supply use to harvest is carried out when 80 ~ 90%；
2）Tissue Culture Flask is taken out, with 75% alcohol disinfecting bottom of bottle, is put into Biohazard Safety Equipment；
3）Old culture medium is abandoned with pipette suction；
4）10ml/ bottles of terminate liquid is added after cell obviously bounces back in every bottle of addition 0.25% pancreatin of 3ml, 37 DEG C of digestion 1min,
Terminate digestion, collect in all liq to 50ml centrifuge tubes, then every bottle plus 10ml physiological saline, imported after soft piping and druming 50ml from
In heart pipe；
5）1200rpm centrifuges 6min, abandons supernatant, and cell precipitation is suspended with 16ml physiological saline, and mixing takes 1ml to count and flow
Formula detects；
6）Add physiological saline to 40ml, 500 μ l supernatants is taken to do endotoxin detection, 1200rpm centrifuges 6min；
7）Supernatant is poured into clean centrifuge tube, does bacterium inspection and detection of mycoplasma；
8）The cell frozen：Centrifugation is suspended with 2.5ml FBS, is slow added into 2.5ml and is frozen mother liquor, is dispensed into after mixing
In cryopreservation tube, every pipe 1ml；
The cell of application：Centrifugation is resuspended with 0.9% normal saline solution, and cell quantity and volume are depending on service condition；
Cell suspension after resuspension is transferred to cell and feeds back bag or feed back in pipe；
9）Mark cell algebraically, density, bar code and operating time in outside tube wall；
10）The cell frozen is frozen by program temperature reduction box method, and the cell of application passes through cold chain transportation to application site.
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| CN109797136A (en) * | 2019-03-14 | 2019-05-24 | 湖南南华爱世普林生物技术有限公司 | A kind of isolated culture method of human adipose mesenchymal stem cells |
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| CN109260227A (en) * | 2018-08-29 | 2019-01-25 | 瑞太医药科技（杭州）有限公司 | It is a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease |
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| CN109797136A (en) * | 2019-03-14 | 2019-05-24 | 湖南南华爱世普林生物技术有限公司 | A kind of isolated culture method of human adipose mesenchymal stem cells |
| CN111484973A (en) * | 2020-06-04 | 2020-08-04 | 广州同康生物科技有限公司 | Purification method of adipose-derived stem cells |
| CN111484973B (en) * | 2020-06-04 | 2021-08-27 | 铜仁市泛特尔生物技术有限公司 | Purification method of adipose-derived stem cells |
| CN112481191A (en) * | 2020-12-14 | 2021-03-12 | 北京博奥体质宝健康科技有限公司 | Method for extracting CD90 high-expression target cells from adipose tissues |
| CN114672463A (en) * | 2022-03-15 | 2022-06-28 | 中国科学院苏州生物医学工程技术研究所 | Stem cell line capable of carrying out multi-modal tracing, preparation method and application thereof |
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