CN107557331A - A kind of method for separating and cultivating human adipose-derived stem cell - Google Patents
A kind of method for separating and cultivating human adipose-derived stem cell Download PDFInfo
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Abstract
The present invention relates to a kind of method for separating and cultivating human adipose-derived stem cell, including：(1) liposuction aspirates are digested using the mixture slaking enzyme solutions being made up of Collagenase I, clostridiopetidase A II and ACCUTASE, digestion neutralizes digestive ferment, centrifugation, filtering, then further removes heteroproteose cell with lymphocyte separation medium after terminating；(2) serum free medium culture fat stem cell is used.One aspect of the present invention can the quickly and efficiently dissociated cell from adipose tissue, not only increase the yield of stem cell, shorten a point cellifugal time, be also maximally maintained the activity of cell；Another aspect medium component is clear and definite, without any external source serum composition, can significantly improve the adherent ability and multiplication capacity of fat stem cell, be advantageous to fat stem cell and expand and keep in vitro its dryness, have a good application prospect.
Description
Technical field
The invention belongs to cell separation and culture field, more particularly to a kind of side for separating and cultivating human adipose-derived stem cell
Method.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is a branch for stem cell, is a kind of tool
Have the cell of self-replacation and Multidirectional Differentiation ability, be widely present in Various Tissues, as marrow, Cord blood and umbilical cord tissue,
Placenta tissue and adipose tissue etc..Mescenchymal stem cell has three outstanding features：1. the mescenchymal stem cell of external culture
It is adherent growth；2. high expression CD73, CD90 and the CD105 of mescenchymal stem cell, does not express CD31, CD34, CD45, HLA-
The marks such as DR, CD14, CD19 and CD11b；3. under suitable stimulus, it is thin that mescenchymal stem cell can be divided into skeletonization
The cell of the Various Tissues such as born of the same parents, adipocyte and nerve cell.
Fat mesenchymal stem cell, be called fat stem cell (Adipose-Derived Stem Cells, ADSCs) be from
A kind of isolated stem cell with multi-lineage potential of adipose tissue.Research has shown that fat stem cell is specifically being cultivated
Under the conditions of can break up polytype cell such as lipoblast, cardiac muscle cell and nerve cell.Fat stem cell has very
Low immunogenicity, therefore the fat stem cell for transplanting allosome will not cause strong immunological rejection, be allograft fat
Fat stem cell provides advantage.Fat stem cell wide material sources, it can be obtained using the method for liposuction or excision fat
Adipose tissue, the problem of being not present ethically using fat stem cell.Fat stem cell has very strong amplification ability in vitro, can
Substantial amounts of fat stem cell is obtained in the method by vitro culture.Fat stem cell is in the beauty such as chest enlarge, smoothing wrinkle and shaping etc.
Industry has to be widely applied very much, and also plays increasing effect in medical field fat stem cell.
Fat stem cell is preparing and problems with occurs in incubation：1. during fractionation of fatty stem cell
Some heteroproteose cells can be mixed into, the adherent of fat stem cell and growth can be influenceed.2. during fractionation of fatty stem cell, if
Enzymolysis time is oversize, and fat stem cell can be caused to damage.3. most scientific researches or medical institutions still use animal blood serum culture
Fat stem cell, but the Serology Quality difference of different batches is very big, its composition and function can not be consistent；And animal blood
Contain low-level cytostatic material, and potential virus and mycoplasma contamination clearly.4. fat stem cell is being trained
A certain degree of differentiation be present during supporting.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of method for separating and cultivating human adipose-derived stem cell, this method
On the one hand can the quickly and efficiently dissociated cell from adipose tissue, not only increase the yield of stem cell, shorten separation
The time of cell, the activity of cell also it has been maximally maintained；Another aspect medium component is clear and definite, without any external source blood
Clear composition, the adherent ability and multiplication capacity of fat stem cell can be significantly improved, is advantageous to fat stem cell and expands in vitro
With its dryness of holding, have a good application prospect.
A kind of separation of the present invention and the method for cultivating human adipose-derived stem cell, including：
(1) suction lipectomy is digested using the mixture slaking enzyme solutions being made up of Collagenase I, clostridiopetidase A II and ACCUTASE
Thing, digestion neutralize digestive ferment, centrifugation, filtering, then further remove heteroproteose cell with lymphocyte separation medium after terminating；
(2) serum free medium culture fat stem cell is used, wherein, serum free medium addition following components：
Rh-insulin：1~10 μ g/ml；
Recombinant human epidermal growth factor：10~50ng/ml；
Recombination human basic fibroblast growth factor：10~50ng/ml；
RhTGF-BETA-β：1~50ng/ml；
Recombinant human Patelet-Derived Growth Factor-BB：10~50ng/ml；
Hydrocortisone：0.5~10 μ g/ml；
Vitamin C：10~100 μ g/ml；
Reduced glutathione：1~5mM；
Recombinate human transferrin：0.5~10 μ g/ml；
Monoethanolamine：1~10 μ g/mL；
Glu：1~10mM；
Coacetylase：1~50 μ g/ml；
Recombinant human thrombin：1~10U/ml；
Gentamicin：1~100 μ g/ml；
Sodium selenite：1~100ng/ml；
Biotin：1~50 μ g/ml.
Mixture slaking enzyme solutions in the step (1) by 0.1%~0.4% (m/v) Collagenase I, 0.1%~
0.4% (m/v) clostridiopetidase A II and 1/4~1/2 × ACCUTASE (STEMCELL Technologies) compositions.
The volume ratio of mixture slaking enzyme solutions and liposuction aspirates in the step (1) is 1:1.
Digestion temperature in the step (1) is 37 DEG C, and digestion time is within half an hour.
Digestive ferment is neutralized using isometric culture medium containing 10%FBS in the step (1).
The filter screen filtration of 100 microns and 40 microns of diameter is specially used in filtering in the step (1) respectively.
Before the step (2) culture fat stem cell culture is coated with 1~10 μ g/mL recombinant human fibronectin polypeptide
Bottle.
The blake bottle is coated with overnight under conditions of 4 DEG C.
Serum free medium in the step (2) is DMEM-F12.
Cultivated in the step (2) and use TrypLE during passage after fat stem cellTMVitellophag.
Specifically, first, with the DMEM-F12 culture mediums of serum-free prepare the Collagenase I containing 0.3% (m/v) and
0.3% (m/v) solution of clostridiopetidase A II, then mixed liquor and ACCUTASE (STEMCELL two kinds of collagenase solutions
Technologies) with 1:1 volume mixture；The mixture slaking enzyme solutions finally given contain 0.15% Collagenase I,
0.15% clostridiopetidase A II and 1/2 × ACCUTASE.
The liposuction aspirates and mixture slaking enzyme solutions crossed with brine with 1:1 volume mixture, at 37 DEG C
Constant-temperature table in digest 15 minutes or so, the rotating speed of shaking table is 100rpm.After digestion terminates, add and isometric contain 10%
In FBS culture medium and digestive ferment.Then centrifuged ten minutes with 400g rotating speed, collect cell precipitation.
Cell is resuspended with physiological saline, allows cell suspension to be obtained successively by a diameter of 100 microns and 40 microns of screen pack
To the suspension of individual cells.In order to further remove heteroproteose cell, cell suspension is added to above lymphocyte separation medium, with 400g
Rotating speed centrifuge 30 minutes, directly collect the cellular layer between uppermost liquid level and separating liquid.
With physiology salt washing cell twice, supernatant is abandoned after centrifugation, cell is resuspended with fresh culture medium, cell is put into
Blake bottle culture.After culture 24 hours, liquid is changed, no adherent cell is washed away with physiological saline.In subsequent incubation
Liquid or passage are changed according to the actual growing state of cell.
Fat stem cell serum free medium provided by the invention contains a variety of growth factors and nutriment, and this can promote
Stem cell normally grows and is metabolized under serum-free culturing conditions：
Fibronectin is a kind of extracellular matrix protein, can between mediated cell, the adhesion of cell and extracellular matrix.With
Fibronectin coating blake bottle can promote cell preferably adherent.The present invention uses recombinant human fibronectin polypeptide coating blake bottle.
Rh-insulin can improve the anabolism ability of cell, stimulate the growth of cell.
EGF is a kind of growth factor with multiple functions, there is strong rush splitting action to cell.
Basic fibroblast growth factor, transforming growth factor-β and PDGF-BB all have
Promote cell propagation and the growth factor of division, the combination of these three factors has been demonstrated that mescenchymal stem cell can be remarkably promoted
Propagation and strengthen stem cell differentiation capability.
Hydrocortisone is a kind of glucocorticoid, has and promotes gluconeogenesis and raising protein catabolism etc. to make
With.
Vitamin C is a kind of antioxidant, can protect cells from the threat of free radical, and vitamin C also participates in carefully
The metabolism of born of the same parents, it can significantly promote the propagation of various mescenchymal stem cells.
Reduced glutathione is three peptides containing sulfydryl (SH), has active oxidation reduction system in human body
The important physiologically active such as system, activation SH enzymes, detoxication.Reduced glutathione also participates in tricarboxylic acid cycle and glycometabolism, rises
To the effect of coenzyme.
Transferrins is the main siderophillin in cell, and transferrins can combine iron ion, reduce its toxicity and be
Cell metabolism provides ferro element.
Monoethanolamine participates in phospholipid metabolism, is the necessary nutriment of cell growth.
Glu is the significant energy source of cell growth, participates in the synthesis and metabolism of protein and lipid, moreover it is possible to
Improve the oxidation resistance of cell.Glu is not sufficiently stable, and Glu can be added before culture medium is prepared.
Coacetylase is the coenzyme of body acetylization reaction, and very important work is played in sugar, lipid and protein metabolism
With.
Fibrin ferment can promote mescenchymal stem cell Fibronectin Secretion, and so as to strengthen, mescenchymal stem cell is adherent and promotion is thin
Born of the same parents breed.
Gentamicin is a kind of antibiotic of wide spectrum.
Sodium selenite is the necessary trace element in cell growth, and oxidation resistant effect is played in cell metabolism.
Biotin is also known as biotin, is water soluble vitamin, falls within vitamin B complex.It is synthesis it is ascorbic must
Material is wanted, is fat and the indispensable material of protein eubolism.
Beneficial effect
(1) the mixture slaking liquid used in the present invention is a kind of digestive ferment combination by optimization, uses this mixing
Enzyme solutions only need just the cell inside adipose tissue can be all dissociateed for 15 minutes, hence it is evident that are consumed less than conventional enzyme solution
The time taken.This can not only improve the yield of stem cell, also be maximally maintained the activity of stem cell.
(2) the other stem cell culture of clinical grade avoids using animal blood serum first, because the serum of animal origin there are
The risk of pathogen contamination, and complicated component, very big difference is had between batch.The present invention is trained using serum free medium
Stem cell is supported, adds a variety of growth factors and nutrient in the medium, cell attachment can be effectively promoted and significantly carried
The multiplication capacity of high cell, there are good clinical value and potentiality.
(3) traditional cultural method uses trypsin digestion cell.Although the efficiency of trypsin digestion cell is very high, if
The grasp of digestion dynamics is bad to cause very big damage to cell.And pancreatin is usually to derive from pig or cattle tissue, pancreatin
Activity stopped with hyclone, animal component is so also the introduction of in cell cultivation process.TrypLETMIt is a kind of base
Because of engineering enzyme, the composition without animal origin.TrypLETMCan effectively and gentleer dissociation attached cell, this like cell
Easy damaged is not allowed in digestion, ensure that the vigor of cell well.And TrypLETMActivity do not need hyclone in
Only, need to only be diluted with physiological saline or culture medium.
Brief description of the drawings
Fig. 1 is fat stem cell in the serum free medium of embodiment 1 and routine has the growth curve in serum free culture system；
Fig. 2 is aspect graph of the fat stem cell in P1 generations under the conditions of the serum free medium using embodiment 1.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
First, the mixture slaking enzyme solutions of digestion fat are prepared：
The Collagenase I containing 0.3% (m/v) and 0.3% (m/v) collagen are prepared with the DMEM-F12 culture mediums of serum-free
The solution of enzyme II, and it is degerming with the filter membrane that aperture is 0.2 μM.Again the mixed liquor and ACCUTASE of two kinds of clostridiopetidase As
Solution (STEMCELL Technologies) is with 1:1 volume mixture, the mixture slaking enzyme solutions finally given contain
0.15% Collagenase I, 0.15% clostridiopetidase A II and 1/2 × ACCUTASE solution.
2nd, with fibronectin coated cell blake bottle：
When the serum free medium provided using the present embodiment, each blake bottle will be fine even with recombined human in advance
Albumen is coated with.By taking T75 blake bottles as an example, coated process is described：5 milliliters, which are added, in T75 blake bottle uses physiology salt
Water-reducible concentration is 5.0 μ g/ml recombinant human fibronectin polypeptide solution, and 4 DEG C of coatings are overnight (about 12~16 hours).Adding
Before entering cell, Fibronectin solution is siphoned away.
3rd, separate and cultivate fat stem cell：
Liposuction aspirates are added in 250 milliliters of centrifuge tube, then add isometric physiological saline；Firmly rock
Centrifuge tube several times, then allows tube stand a few minutes, and adipose tissue can be floated over above physiological saline；Physiological saline is siphoned away, then
Fresh physiological saline is rejoined, washing blood of the fat several times inside fat with same operation repetition is washed off.
Liposuction aspirates and mixture slaking enzyme solutions are with 1:1 volume mixture, 15 points are digested in 37 DEG C of constant-temperature table
Clock or so, the rotating speed of shaking table is 100rpm.After digestion terminates, add in isometric culture medium containing 10%FBS and digest
Enzyme.Then centrifuged ten minutes with 400g rotating speed, collect cell precipitation.
Cell precipitation is resuspended with physiological saline, allows cell successively by a diameter of 100 microns and 40 microns of screen pack, to receive
Collect individual cells.
20 milliliters of lymphocyte separation medium Ficoll is added in a clean centrifuge tube of 50 milliliters, then cell
Suspension is added slowly to above separating liquid.Turn off the deceleration valve of centrifuge, centrifuged 30 minutes, collected above with 400g rotating speed
White cellular layer between liquid level and separating liquid.
Cell is resuspended with the physiological saline of 2 times of volumes, is centrifuged 10 minutes with 300g rotating speed, loses supernatant.Use physiology salt
Water cleans cell again, loses supernatant after centrifugation, and cell is resuspended with fresh culture medium, cell is put into blake bottle culture.Training
After supporting 24 hours, liquid is changed, no adherent cell is washed away with physiological saline.
Following nutriment is included in the free serum culture that the present embodiment is provided：10 μ g/ml rh-insulins,
20ng/ml recombinant human epidermal growth factors, 20ng/ml recombination human basic fibroblast growth factors, 10ng/ml recombined humans
Transforming growth factor-β, 20ng/ml recombinant human Patelet-Derived Growth Factor-BBs, 10ng/ml rhMGFs, 0.5
μ g/ml hydrocortisones, 50 μ g/ml vitamin Cs, 2mM reduced glutathiones, 5 μ g/ml restructuring human transferrin, 1 μ g/ml
Monoethanolamine, 2mM Glus, 50 μ g/ml coacetylases, 5U/ml recombinant human thrombins, 10 μ g/ml gentamicins, 10 μ g/ml
Biotin and 5ng/ml sodium selenites.Actual growing state according to cell in subsequent incubation changes liquid or biography
Generation.When the degrees of fusion of cell reaches 70~80%, TrypLE is usedTMVitellophag passes on, and the passage ratio of cell is 1:
3.4th, compare the serum free medium of the present embodiment and have the amplification ability of blood serum medium：
The fat stem cell separated with the present embodiment is respectively with two kinds of medium cultures：The serum free medium of the present embodiment
There is blood serum medium (DMEM-F12+10%FBS) with conventional.P2 for when, vitellophag, still use serum-free respectively
Culture medium and there is blood serum medium that suspension is made in cell to be added to inside six orifice plates.There are 2 milliliters inside the single hole of each six orifice plate
Culture medium, initiator cell quantity are all 6000；Two groups of cells have six orifice plates of 5 pieces of parallel inoculations.Six orifice plates are put back to culture
Case.One piece of six orifice plate is taken out inside incubator within every 2 days, digest cell and the cell count of the inside.To surplus when the 7th day
Under cell all change liquid, cell proliferation experiment can continue to the 10th day.
As shown in figure 1, the growth rate of two groups of cells shows obvious difference, p<0.01.Fat stem cell is in this reality
Growth rate of the growth rate apparently higher than in having blood serum medium in routine in the serum free medium of example offer is provided.
Claims (10)
1. a kind of method for separating and cultivating human adipose-derived stem cell, including：
(1) liposuction aspirates are digested using the mixture slaking enzyme solutions being made up of Collagenase I, clostridiopetidase A II and ACCUTASE, disappeared
Change neutralizes digestive ferment after terminating, centrifugation, filtering, then further remove heteroproteose cell with lymphocyte separation medium；
(2) serum free medium culture fat stem cell is used, wherein, serum free medium addition following components：
Rh-insulin：1~10 μ g/ml；
Recombinant human epidermal growth factor：10~50ng/ml；
Recombination human basic fibroblast growth factor：10~50ng/ml；
RhTGF-BETA-β：1~50ng/ml；
Recombinant human Patelet-Derived Growth Factor-BB：10~50ng/ml；
Hydrocortisone：0.5~10 μ g/ml；
Vitamin C：10~100 μ g/ml；
Reduced glutathione：1~5mM；
Recombinate human transferrin：0.5~10 μ g/ml；
Monoethanolamine：1~10 μ g/mL；
Glu：1~10mM；
Coacetylase：1~50 μ g/ml；
Recombinant human thrombin：1~10U/ml；
Gentamicin：1~100 μ g/ml；
Sodium selenite：1~100ng/ml；
Biotin：1~50 μ g/ml.
A kind of 2. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：The step
(1) the mixture slaking enzyme solutions in are by 0.1%~0.4%m/v Collagenase I, 0.1%~0.4%m/v clostridiopetidase A II and 1/
4~1/2 × ACCUTASE is formed.
A kind of 3. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：The step
(1) volume ratio of mixture slaking enzyme solutions and liposuction aspirates in is 1:1.
A kind of 4. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：The step
(1) the digestion temperature in is 37 DEG C, and digestion time is within half an hour.
A kind of 5. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：The step
(1) digestive ferment is neutralized using isometric culture medium containing 10%FBS in.
A kind of 6. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：The step
(1) filter screen filtration of 100 microns and 40 microns of diameter is specially used in the filtering in respectively.
A kind of 7. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：In the step
Suddenly it is coated with blake bottle with 1~10 μ g/mL recombinant human fibronectin polypeptide before (2) culture fat stem cell.
A kind of 8. method for separating and cultivating human adipose-derived stem cell according to claim 7, it is characterised in that：The culture
Bottle is coated with overnight under conditions of 4 DEG C.
A kind of 9. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：The step
(2) serum free medium in is DMEM-F12.
A kind of 10. method for separating and cultivating human adipose-derived stem cell according to claim 1, it is characterised in that：Described
After step (2) culture fat stem cell TrypLE is used during passageTMVitellophag.
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| CN110531057A (en) * | 2019-07-01 | 2019-12-03 | 启迪禾美生物科技(嘉兴)有限公司 | The method of high flux screening skin care item active compound |
| CN111420034A (en) * | 2019-01-08 | 2020-07-17 | 上海莱馥医疗科技有限公司 | Mixed stem cell preparation for treating psoriasis and preparation method thereof |
| CN115261312A (en) * | 2022-08-03 | 2022-11-01 | 湖北万海赛康生命科学有限公司 | Preparation method of mesenchymal stem cells |
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| CN115261312A (en) * | 2022-08-03 | 2022-11-01 | 湖北万海赛康生命科学有限公司 | Preparation method of mesenchymal stem cells |
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