CN107858329A

CN107858329A - The method of fractionation of fatty mescenchymal stem cell and the test solution used from fat

Info

Publication number: CN107858329A
Application number: CN201711354906.3A
Authority: CN
Inventors: 王芳; 梅俊
Original assignee: British Liberal Life Science & Technology Co Ltd
Current assignee: British Liberal Life Science & Technology Co Ltd
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2018-03-30
Anticipated expiration: 2037-12-15
Also published as: CN107858329B

Abstract

The present invention relates to the method for fractionation of fatty mescenchymal stem cell from fat and the test solution used.On the one hand, the method for fractionation of fatty mescenchymal stem cell includes step from fat：Liposuction is obtained into the fat-like centrifugation doped with inflation fluid；The superiors' grease is removed, pipettes suspension lipochondrion and bottom cell precipitation respectively；Lipochondrion adds digestion enzyme mixation, vibration digestion, terminates digestion, centrifugation, cell precipitation is resuspended；Bottom cell precipitation is resuspended, and adds erythrocyte cracked liquid to crack, and centrifuges, and cell precipitation is resuspended；Merge gained cell suspension, filtering, be separated as primary fat mesenchymal stem cell.The method for further relating to separate and cultivating fat mesenchymal stem cell, obtained fat mesenchymal stem cell preparation, for the digestion enzyme mixation of fractionation of fatty mescenchymal stem cell, and for cultivating the culture medium of fat mesenchymal stem cell.Excellent technique effect as used in the description is presented in each side of the present invention.

Description

The method of fractionation of fatty mescenchymal stem cell and the test solution used from fat

Technical field

The invention belongs to biological technical field, is related to a kind of method of the fractionation of fatty mescenchymal stem cell from fat, special It is not to be related to a kind of isolated culture method of autologous fat mescenchymal stem cell.The invention further relates to above-mentioned autologous fat mesenchyma Culture medium used and related test solution in stem cell culture.The fat mesenchymal stem cell is separately cultured using the inventive method When, excellent technique effect of the present invention can be presented.These obtained autologous fat mescenchymal stem cells can be used for autologous Using.

Background technology

Mescenchymal stem cell (MCS) is the focus cell that adult stem has multi-lineage potential, is a kind of most of What is found in adult organ has the cell colony of bone formation ability and hematopoiesis function, can be carried from marrow, bleeding of the umbilicus, fat Take, under specific condition of culture, it is thin that mescenchymal stem cell can be divided into adipocyte, Gegenbaur's cell, cartilage cell and flesh Born of the same parents etc..

Mescenchymal stem cell is the main seed cell of current organizational project.After experienced the research of more scholars for many years, though Right organizational project has been obtained for remarkable progress, however, but due to lacking preferable screening technique at present, mescenchymal stem cell is not easy Acquisition, amplification in vitro difficulty etc. limit, and are restricted its application fed back in clinical rank.Therefore, one kind is found to be easy to obtain , immunogenicity is low, the seed cell of the clinical treatment rank that is easy to amplification in vitro is that current organizational project is in the urgent need to address One of the problem of.

People from adipose tissue isolate it is a kind of there is the stem cell of multi-lineage potential, and cultivate in vitro it is special Adipocyte, Gegenbaur's cell, cartilage cell and myocyte etc. are successfully divided into system, this stem cell broken up is recognized 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 is dry Cell, which has, breaks up more germinal layer totipotency features, including mesoderm, the ability of entoderm and ectoderm noble cells, can divide The treatment method for secreting the wound healing of many potential favourable growth factors and cytokine based on cell is quick Development.Fat-derived stem cells are the multi-functional mescenchymal stem cells of a group, can be divided into other cell lines in adipose tissue Non-fat (interstitial) fragment in substantial amounts of fat-derived cell be present, and be easily isolated acquisition.

As the auxiliary treating method of various tissue defects, the recommended application of fat-derived stem cells.In wound healing mistake Cheng Zhong, revascularization are that the formation of an important step new vessels is to support that granulation tissue is formed and keratinocyte survival is expanded Increase the necessary condition of healing wounds.Result, it is believed that fat-derived stem cells are to promote to create by quickly promoting revascularization The healing in face.In addition, fat-derived stem cells also show by cell differentiation and secretion promote collage synthesis and corium into The long factor of fibrocyte migration promotes the healing of the surface of a wound

Fat-derived stem cells and extracellular matrix support ingredient combination have been applied to the skin soft tissue of animal by people The surface of a wound, fat-derived stem cells are found to promote the vascularization ability of support.There is result of study to show, fat-derived cell is to wound The facilitation of face healing promotes the formation of epithelialization and granulation tissue relevant with fat-derived stem cells, and fat-derived dry It is more work(that cell, which has potential epidermal cell differentiation capability result of study also to prompt the amphi position differentiation capability of fat-derived stem cells, Can a reaction of the fat stem cell to the microenvironment of the surface of a wound.Remodeling of the fat-derived stem cells to local vascular includes directly dividing Vascular endothelial cell and secretion angiogenesis factor are turned to, rear both are then beneficial to the regeneration of blood vessel.It is i other words fat-derived dry Cell promotes the healing of the surface of a wound by cell differentiation and revascularization.In addition, people are by fat-derived stem cells and blood platelet source Property growth factor, i.e., a kind of well-known factor for participating in normal wound combines the treatment for the surface of a wound.Research As a result showing the use in conjunction of the two has synergy, it is possible to promotes being cured for the surface of a wound by improving the level of growth factor Close.

Theoretically, cell therapy has a extensive future, because versatile stem cell or progenitor cells pass through to surface of a wound missing tissues Regeneration and many advantage factors are persistently provided and promote the healing of the surface of a wound.Nevertheless, on fat-derived stem cells to wound The research of the mechanism of face healing is still up for further exploring.Remodeling of the fat-derived stem cells to local vascular includes directly dividing Vascular endothelial cell and secretion angiogenic growth factor are turned to, rear both are then beneficial to the regeneration of blood vessel.

The application prospect of fat mesenchymal stem cell is verified that it is used by the application on each disease animal model Come therapeutic intervention diabetes, hepar damnification reparation, the reconstruction of wing flesh, diabetes B sun rash, myocardial infarction, cerebral infarction, cognitive function barrier Hinder, apoplexy, interverbebral disc reparation, glioblastoma treatment, Cranial defect, post-traumatic vascular new life, rheumatic arthritis, lip it is stunned Split, heart failure, colitis, the urinary incontinence etc..There is researcher that fat mesenchymal stem cell is changed into cardiac muscle cell, both compiled again The ripe stem cell of journey, and can improve cardiopathic treatment.Also someone answers fat mesenchymal stem cell and Fibrin Glue It is expelled to after conjunction on the left ventricular wall of Following Myocardial Infarction In Rats, ADSCs has very big in clinical tissue engineering treatment miocardial infarction Prospect.Fat mesenchymal stem cell is put in network as seed cell, has successfully been repaired the head injury of dog, has been Clinical treatment head injury.Also research is injected into their heart using the fat mesenchymal stem cell of heart patient's stomach fat After dirty, reduce the damage of heart while also increase blood flow, stem cell injection passes through 6 months after entering heart, Bing Renxin Dirty ability of the receiving with oxygen blood improves, and the blood that heart left ventricle sends out also increases 3.5 times, and SPELT imagings prove to receive The ability of patient's heart pump of fat mesenchymal stem cell injection adds 5.7%, and nuclear magnetic scanning displays that the mean myocardial of patient Scar area drops to 15.4% by original 31.6%, and this achievement in research is issued in American Heart Association's science in 2010 In annual meeting.Fat mesenchymal stem cell can adjust growth hormone and cell factor in surrounding tissue in process of tissue reparation Deng preventing and treating Apoptosis, there is experiment to prove that fat mesenchymal stem cell can secrete various skin growth factors such as fibrocyte and give birth to Long factor (Basic fibroblastgrowth factor, bFGF) etc. promotes fibroblast to breed, and with anti-light old Change, anti-oxidant, crease-resistant, anti-ultraviolet radiation etc. act on.There is research with fat mesenchymal stem cell to treat patient's complexity anus repeatly Have been introduced into II phase clinical trials.The adipose tissue-derived mesenchymal stem cell transplantation of also employment enter a mastatrophy, it is not of uniform size or Need in the patient's chest enlarged the bosom, left and right udder size balance and symmetry, breast X-ray check, the breast being repaired is naturally soft complete Completely without calcification, no tumour phenomenon, without obvious injection scar.Nearest clinical studies show, intramuscular injection fat mesenchymal are done Cell also has the effect of certain to diabetes and Arteriosclerosis obliterans.At 6 months of injection fat mesenchymal stem cell Afterwards, from clinically, the tranquillization pain of patient is alleviated, and the distance of painless walking is obviously prolonged, and does not find to appoint What complication.There is experiment to show that fat mesenchymal stem cell induction feeds back diabetic, Follow-up After 23 into islet-like cells Individual month, it is found that the exogenous insulin dosage of patient declines, the average weight of patient adds and patient is without uncomfortable or secondary anti- Should.

There is scholar's research to find that adipose tissue participates in wound repair, after adipocyte is stimulated by wound, gene expression The inflammation such as generation irritability TNF secretion-a, IL-1, IL-6, intercellular adhesionmolecule1, chemotactic factor (CF), acute reflection albumen, acceptor The gene expression of disease matrix can all increase, and these cytokine regulatories are all that we understand the inherent therapy apparatus that adipocyte participates in repairing System, this regulation system are the precision controls for being worth us to think deeply.The scientific achievement of acquisition is the theoretical base of abundant wound repair Plinth.Clinical practice injures the depth skin lesion wound of subepidermal tissue fat deposit, and fatty adoptive therapy can reach the acceleration surface of a wound The ideal treatment of reparation, foreign study scholar also apply adipocyte, collagen and elastin laminin application organizational project Technology prepares the artificial skin containing adipocyte, and clinical practice is found caused by can reducing the tissue repair of full thickness dermal Wound Contraction, this phenomenon prompt the factor and cell with repair in our adipose tissues be present.

Fat mesenchymal stem cell extracellular matrix sticks strong rapid charater, and the people of in-vitro screening culture is adipose-derived Mescenchymal stem cell be able to the special sexual development of method at home and abroad.And stem cell biology performance, can accomplish promote function, Avoid potentially harmful gene and divergaence time and the different differentiation directions of control etc. are controlled to stem cell, controlled in basic scientific research, clinic When treatment, stem-cell therapy research direction, skin regeneration injury repair, the high structure aspect for intending organization engineering skin etc. have history The directive significance that phase is not replaced.

Obtain adipose-derived mescenchymal stem cell can with the adoptive therapy of applying clinical relevant disease cell therapy, with And beauty and shaping injection filling.

Fat mesenchymal stem cell (ADSCs) is as the mescenchymal stem cell from fat, and convenient material drawing, it extracts effect Rate is higher than mesenchymal stem cells MSCs 40 times, and growth rate is faster under condition of in vitro culture；, can with Multidirectional Differentiation ability With to adipocyte, Gegenbaur's cell, cartilage cell, cardiac muscle cell, or even neural cellular differentiation.Clinical I phases and II phase are real at present Testing all proves, applications of the ADSCs in a variety of organ damage reparations such as heart, rectum, mammary gland is safe and effective, Er Qieyi Playing the role of research confirms that ADSCs has promotion skin regeneration technique.

ADSCs shows its inherent advantages in many aspects.ADSCs belongs to monocyte family, is adult stem cell In one kind.It is grown from treated adipose tissue, during culture in Tissue Culture Dish with adherent, adherent fashion, and is had There is the ability of various differentiation, be not only divided into adipocyte, myocyte, cartilage cell, nerve cell, blood vessel can also be divided into Endothelial cell, osteocyte etc., therefore they are also widely used in organizational project.For example, ADSCs can pass through self-replacation Adipocyte is divided into, is played a role in regeneration；ADSCs can be divided into vascular endothelial cell or peripheral cell； ADSCs can secrete angiogenesis factor under anoxic or other conditions；When ADSCs and adipocyte are transplanted together, It can be divided into endothelial cell, prevent the generation of fibrosis and adiponecrosis, improve fat survival；According to nearest many Specific result of study shows that ADSCs can express cytokine profiles VEGF, IGF, TGF-β 1, bFGF, EGF etc., also pass through table Up to angiogenesis factors such as VEGF, IGF-1, to prevent the generation of apoptosis, the survival rate of adipocyte is maintained.

Prior art discloses many cultural methods about fat mesenchymal stem cell.For example, CN106479970A (201611050980.1) a kind of method of large-scale culture human adipose mesenchymal stem cells is disclosed.Big rule of the present invention The method of mould culture human adipose mesenchymal stem cells adds fat mesenchymal stem cell culture medium into glass blake bottle, in humidity 95%th, balance is incubated 30 minutes in 35 DEG C in 5%CO2 incubator；The temperature of adjustment fat mesenchymal stem cell culture medium arrives 35 DEG C, pH7.05, add the microcarrier and fat mesenchymal stem cell of pretreatment, 5RPM~20RPM stirrings 2h；It is warming up to 37 DEG C 96h is cultivated under the conditions of 15RPM-40RPM, 5%CO2, during which adds fat mesenchymal stem cell culture medium.With existing method phase It is more more than obtaining cell after the method propagation of, it is believed that the invention large-scale culture human adipose mesenchymal stem cells, cell viability and Multiplication capacity is strong, and can keep the form of fat mesenchymal stem cell and good stem cell properties very well.

CN104762260A (201510197879.8) is related to a kind of preparation method of fat mesenchymal stem cell, its feature It is, comprises the following steps：Step 1：Fat is obtained, pretreatment, enzymolysis, centrifugation, collects precipitation, it is dry thin to obtain fat mesenchymal Born of the same parents；Step 2：The fat mesenchymal stem cell is cultivated, when the fat mesenchymal stem cell grows to 80% fusion, abandons training Nutrient solution, add PBS, then add EDTA~trypsin solution digestion, terminate digestion, centrifugation, Secondary Culture.It is believed that the invention have with Lower advantage：1st, the stem cell purity that culture obtains is high；2nd, secreted out of in recovery fat mesenchymal stem cell incubation all kinds of The factor, the propagation of epidermal cell can be remarkably promoted, show that the new life of epidermal cell can be accelerated and substitute, reach significant anti-ageing Old effect.

It is dry thin that CN104974984A (application number 201510173453.9) discloses a kind of mesenchyma of adipose tissue-derived The amplification cultivation method of born of the same parents, comprises the following steps：By the mescenchymal stem cell fat stem cell of the adipose tissue-derived of separation Culture medium carries out resuspension processing, obtains the re-suspension liquid containing mescenchymal stem cell；The re-suspension liquid is seeded to stem cell incubator Middle carry out amplification cultivation；Amplification cultivation changes the fat stem cell culture medium after 24 hours, change the fat within every two days afterwards Fat stem cell media is once, to be grown when being fused to 80~90%, with carrying out Secondary Culture after pancreatin digestion process；Wherein, The fat stem cell culture medium contains 0.1~10v/v% hyclones, 1~10v/v% gentamicins and 80~98.9v/ V% DMEM in high glucose culture mediums；The stem cell incubator is the stem cell incubator that processing is coated with fibronectin splicing variants.It is believed that The amplification cultivation method of the mescenchymal stem cell of the invention adipose tissue-derived is using fibronectin splicing variants coating fat mesenchymal The solid support that stem cell growth depends on, screened and trained using the Synergistic of low concentration hyclone and high concentration gentamicin Support so that the mescenchymal stem cell of adipose tissue-derived can expand 400-500 times in two time-of-weeks, and purity is high, can orient It is induced to differentiate into adipose tissue.

CN106520686A (application number 201610887111.8) provides a kind of culture side of fat mesenchymal stem cell Method.Fat mesenchymal stem cell cultural method of the present invention includes following incubation step：The primary fat mesenchymal of acquisition is dry thin Born of the same parents, which are seeded in stem cell media, carries out culture processing, wherein, containing stem cell factor and in vain in the stem cell media The factor of interleukin 3, and the stem cell factor and the content ratio of the interleukin-13 factor are (5-20 μm of ol/L):(5-20ug/ml).This Invention cultural method can improve the amplification ability of fat mesenchymal stem cell, and obtain higher transfer ability.

CN101984049A (application number 201010580537.1) discloses one kind separating mesenchymal from adipose tissue and done The method of cell, it is characterised in that comprise the following steps：(1) acquisition of adipose tissue：Adipose tissue for specialty hospital or The discarded object that beauty parlor is obtained by swelling method liposuction procedures, aseptically, 4-20 DEG C of storage are less than 48 hours；(2) just Step removes red blood cell：Liposuction thing is static to be placed after being layered, and carefully removes lower floor's liquid, is washed repeatedly with D-Hanks liquid It is limpid to eluate；(3) liposuction thing is digested：Add with the Collagenase Type that D-Hanks liquid is prepared to 0.01-2g/ 100ml, 20-400 turns/min concussion digestion 15-120 minutes at 37 DEG C；(4) acquisition of fat stem cell：Product exists after digesting 4 DEG C, 5min is centrifuged under the conditions of centrifugal force 450g, fat stem cell culture medium is resuspended, and the removal of impurity is gone with 100 μm of aperture membrane filtrations It is final to obtain fat stem cell；(5) red blood cell is removed again, is removed again using the mode of washing and erythrocyte splitting repeatedly red Cell；(6) fat stem cell counting, Activity determination and culture：The cell that 100 μ l are separated is taken to carry out cytometer with cell counting count board Number；Take out the method that 100 μ l cells dyes by trypan blu e simultaneously and carry out viable count, according to two results counted according to 3*10⁴/cm²Density be inoculated in T-75 blake bottles, 37 DEG C, CO2 concentration 5%, cultivated under the conditions of humidity 100%.According to It is substantial amounts of in good condition, the good Multidirectional Differentiation ability of holding to believe that the inventive method can utilize a small amount of adipose tissues to obtain Fat stem cell, and operating method is simple and easy, repeatable strong.But in document method when being cracked to cell Mescenchymal stem cell is directed to simultaneously, this survival for stem cell is unfavorable.

The training of effective acquisition fat mesenchymal stem cell disclosed in CN106222134A (application number 201610605996.8) In the method for supporting, when being separated to fat, the fat granule part in intermediate layer after centrifugation is only drawn, and does not obtain centrifugation bottom Cell precipitation.

CN102329783A (application number 201110310461.5) discloses the inflation fluid in a kind of operation from surgical fat suction The method of middle fractionation of fatty mescenchymal stem cell, is comprised the steps of：(1) to wanted extracting part before the collection liposuction of inflation fluid Position irrigates excessive hypotonic expansion liquid, and then the fat obtained in liposuction procedures is placed in inflation fluid and places 10min at room temperature, is treated After fat is kept completely separate with inflation fluid, the lower sections of red liquid of yellow fat is drawn with liquid-transfering gun, it is as dry containing fat The inflation fluid of cell；(2) inflation fluid being collected into is distributed into 50ml centrifuge tubes by the processing of inflation fluid, be put into centrifuge with 1500rpm centrifuges 5min；The liquid for discarding centrifuge tube upper part is drawn in centrifugation, collects precipitation, is suspended, is added per effective PBS liquid Enter erythrocyte cracked liquid to be stored at room temperature 5 minutes, 3min is centrifuged with 1200rpm, abandons supernatant；(3) washing of cell adds 5ml DMEM (being free of serum) dispels precipitation, cellular filter filtering, filters later liquid and centrifuges 3min with 1200rpm, precipitation is For the fat mesenchymal stem cell to be obtained, the operated wash 3 times that DMEM (being free of serum) repeats the above is added；(4) cell Culture：Cell will be obtained to be seeded in 75cm2 Tissue Culture Flasks, be inoculated with, addition mescenchymal stem cell culture medium, 37 DEG C, 5% CO2 is cultivated.But the CN102329783A is separated just for the stem cell in inflation fluid part, rather than for doping The fat liquor for having inflation fluid carries out the separation of stem cell.

Although prior art discloses some all cultural methods described above about fat mesenchymal stem cell, but this Inventor has found that these methods seem to be not particularly suited for obtain significant quantities of fat mescenchymal stem cell from fat, such as these For method when for obtaining fat mesenchymal stem cell from fat, its cell yield is low and/or Cell viability is low.

Therefore, this area is still expected to have new method to prepare fat mesenchymal stem cell, and particularly expectation has applicable In the method that significant quantities of fat mescenchymal stem cell is obtained from fat.

The content of the invention

It is an object of the invention to provide a kind of new method to prepare fat mesenchymal stem cell, is especially to provide one kind Suitable for obtaining the method for significant quantities of fat mescenchymal stem cell from fat, and expect that this method can show cell receipts Characteristic and/or other excellent properties such as rate height and/or Cell viability height.The present invention has been had now surprisingly been found that using the present invention Method can obtain the technique effect of one or more aspects as described herein.The present invention is accomplished based on this discovery.

Therefore, the method that first aspect present invention provides separation and culture fat mesenchymal stem cell, it includes as follows Step：

(1) the fatty sample doped with inflation fluid will be obtained in centrifuge tube by liposuction, 500~1000rpm centrifugations 2~ 5min；

(2) the superiors' grease is removed, and pipettes the lipochondrion and bottom cell precipitation of suspension respectively, it is standby；

(31) into lipochondrion obtained by step (2) with volume ratio 1：0.8~1.2 adds digestion enzyme mixation, at 37 DEG C 20~40min of digestion is vibrated with 100rpm；After digestion terminates, add isometric complete medium termination and digest, 1000~ 2000rpm centrifuges 3~8min, abandons supernatant, and cell precipitation is resuspended in PBS；

(32) bottom cell precipitation obtained by step (2) is resuspended with PBS, addition equivalent erythrocyte cracked liquid standing 5~ 15min is cracked；After red blood cell fully cracks, 1000~2000rpm centrifuges 3~8min, abandons supernatant, and PBS is resuspended cell and sunk Form sediment；

(4) combining step (31) and (32) gained cell suspension, 100 μm of strainer filterings, are separated as primary fat Mescenchymal stem cell；

(5) the primary fat mesenchymal stem cell isolated is seeded to blake bottle and trained according to 30000 cells/cm^2 Support, add complete medium, 37 DEG C of 5%CO2 cultures；

(6) change liquid after cultivating 2 days, remove non-adherent cell, to be generated when growing to one week, harvest P1 fat subsitutes mesenchyma is dry thin Born of the same parents；

(7) a small amount of cell is taken to continue to be passaged to of future generation until in P5 generations, harvest and observe each according to step (5) and step (6) Generation fat mesenchymal stem cell, and carry out coherent detection.

In method described in any embodiment according to a first aspect of the present invention, wherein step (7), the coherent detection is Refer to detection cellular morphology and immunophenotypic characterization.In one embodiment, the immunophenotypic characterization refer to CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD34 are detected.

In method described in any embodiment according to a first aspect of the present invention, wherein step (1), the inflation fluid bag Contain：0.1mg/ml lidocaines and 0.02ug/ml adrenaline.

In method described in any embodiment according to a first aspect of the present invention, wherein step (1), centrifuged with 800rpm 3min。

In method described in any embodiment according to a first aspect of the present invention, wherein step (31), lipochondrion is with mixing Synthase is with volume ratio 1：1 ratio adds.

Method described in any embodiment according to a first aspect of the present invention, wherein being included in the digestive ferment mixed liquor： 0.05% clostridiopetidase A I types, 0.05% clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes.Use in the present invention The above-mentioned digestion enzyme mixation being made up of four kinds of enzymes, very high karyocyte can be obtained in step (4) and harvest quantity, had Nucleus harvest quantity is generally up to (7.5~15) × 10^7 (in terms of lipochondrion obtained by every 1ml steps (2)).It has been found that It is very important influence factor that the combination of digestive ferment harvests quantity for karyocyte, such as includes the digestion of above-mentioned four kinds of enzymes In enzyme mixation, if wherein lack any or two or three of enzyme, karyocyte harvest quantity can not be more than 3 × 10 ^6 (in terms of lipochondrion obtained by every 1ml steps (2)).In one embodiment, also added in the digestion enzyme mixation 0.002% isopropanol；Had now surprisingly been found that, it may be possible to due to lidocaine presence (because, it was found that, when swollen Step (4), which obtains karyocyte harvest quantity, when not adding lidocaine in swollen liquid can reach (15~20) × 10^7), mixed Karyocyte harvest quantity can be made to reach (55~65) × 10^7 (with every 1ml steps (2) institute when adding isopropanol in synthase liquid Lipochondrion meter), but the karyocyte that (lidocaine be present) when not adding isopropanol harvest quantity can not more than 15 × 10^7 (in terms of lipochondrion obtained by every 1ml steps (2)).

In the present invention, when representing cell quantity, " 10^7 " represents 10 7 powers；In the present invention, culture area is represented When, " cm^2 " represents square centimeter；Other situations for being related to " ^ " symbol, are respectively provided with similar meaning；The implication of this symbol is in ability Also it is known in domain.

In method described in any embodiment according to a first aspect of the present invention, wherein step (31), 37 DEG C with 100rpm vibration digestion 3min.

In method described in any embodiment according to a first aspect of the present invention, wherein step (31), after terminating digestion, with 1500rpm centrifuges 5min.

In method described in any embodiment according to a first aspect of the present invention, wherein step (32), it is red thin to add equivalent Cellular lysate liquid stands 10min and cracked.

In method described in any embodiment according to a first aspect of the present invention, wherein step (32), red blood cell fully splits Xie Hou, 1500rpm centrifuge 5min.

Method described in any embodiment according to a first aspect of the present invention, wherein the complete medium includes：Low sugar Type DMEM, 10%FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ Ml IGF.Use in the present invention comprising above-mentioned EGF, bFGF, TGF-β, tetra- kinds of cell factors of IGF complete medium when, In the step (6) cell of harvest by blake bottle it is every square centimeter in terms of cell quantity up to (90~180) × 10^6；But such as When fruit does not add any or two or three or four kinds of cell factors in this complete medium, then received in step (6) The cell obtained by blake bottle it is every square centimeter in terms of cell quantity be only capable of the scope that reaches (0.05~0.5) × 10^6, it is seen that simultaneously Four kinds of cell factors for adding above-mentioned quantity are necessary for high yield acquisition stem cell.In addition, the present inventor tries in supplement It also found in testing, when adding 0.025% sorbierite into the complete medium, between the fat that can further improve the present invention The harvest yield of mesenchymal stem cells, for example, particularly in the step (6) harvest cell by blake bottle it is every square centimeter in terms of cell number Amount is up to (500~650) × 10^6.Therefore, in one embodiment of the invention, also included in the complete medium 0.025% sorbierite, in view of the price of sorbierite is far below above-mentioned various cell factors, therefore add the technical meaning of sorbierite It is very significant.

Method described in any embodiment according to a first aspect of the present invention, wherein also including making obtained P1 generation to P5 generations Any generation or more generations fat mesenchymal stem cell carry out preparation preparation process.

Method described in any embodiment according to a first aspect of the present invention, wherein also including making obtained P1 generation to P5 generations Any generation or the fat mesenchymal stem cell in more generations the step of carrying out cell cryopreservation, the optional cell to being frozen answers Soviet Union, and the optional cell to recovery carry out the preparation of preparation.

Method described in any embodiment according to a first aspect of the present invention, the DMEM are low-sugar type DMEM.

Method described in any embodiment according to a first aspect of the present invention, the low-sugar type DMEM formulas composition are as follows： It is anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, sodium chloride 6400mg, anhydrous It is sodium dihydrogen phosphate 109mg, succinic acid 75mg, sodium succinate 100mg, L- R-gene 84mg, L- hydrochloric acid cystine 63mg, sweet Propylhomoserin 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L-Leu 105mg, LYS 146mg, L- Methionine 30mg, L-phenylalanine 66mg, Serine 42mg, L-threonine 95mg, L-Trp 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, core Flavine 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, water are fitted Amount adds to 1000mL.

Method described in any embodiment according to a first aspect of the present invention, the complete medium composition are：100mL's FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF, 0.025% sorbierite, anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, chlorination Sodium 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- R-gene 84mg, L- salt Sour cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L-Leu 105mg, L- salt Sour lysine 146mg, METHIONINE 30mg, L- phenylalanine 66mg, Serine 42mg, L-threonine 95mg, L- color ammonia Sour 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

Method described in any embodiment according to a first aspect of the present invention, the fat are obtained from any position of human body Fat.

Method described in any embodiment according to a first aspect of the present invention, the frozen stock solution composition that the cell cryopreservation uses For：1 part of DMSO：2 parts of human serum albumins：7 parts of complete mediums.

Further, second aspect of the present invention provides a kind of fat mesenchymal stem cell preparation, and it is by including such as What the method for lower step was prepared：

(8) P1 for making to obtain carries out the preparation of preparation for any generation to P5 generations or the fat mesenchymal stem cell in more generations； Or, so as to get any generation in P1 generation to P5 generations or the fat mesenchymal stem cell in more generations carry out cell cryopreservation, optional pair The cell frozen is recovered, and the optional cell to recovery carries out the preparation of preparation.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein step (1) In, the inflation fluid includes：0.1mg/ml lidocaines and 0.02ug/ml adrenaline.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein step (1) In, 3min is centrifuged with 800rpm.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein step (31) In, lipochondrion is with mixed enzyme with volume ratio 1：1 ratio adds.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein the digestion Included in enzyme mixation：0.05% clostridiopetidase A I types, 0.05% clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA Enzyme.In the present invention using the above-mentioned digestion enzyme mixation being made up of four kinds of enzymes, can be obtained in step (4) very high has Nucleus harvests quantity, and karyocyte harvest quantity is generally up to (7.5~15) × 10^7 (with fat obtained by every 1ml steps (2) Fat particle meter).It has been found that it is very important influence factor that the combination of digestive ferment harvests quantity for karyocyte, such as In digestion enzyme mixation comprising above-mentioned four kinds of enzymes, if wherein lack any or two or three of enzyme, karyocyte Harvesting quantity can not be more than 3 × 10^6 (in terms of lipochondrion obtained by every 1ml steps (2)).In one embodiment, it is described to disappear Change in enzyme mixation and also add 0.002% isopropanol；Have now surprisingly been found that, it may be possible to due to the presence of lidocaine (because it was found that, when not adding lidocaine in inflation fluid, step (4) obtains karyocyte harvest quantity and can reached (15~20) × 10^7), in mixed enzyme solution add isopropanol when can make karyocyte harvest quantity reach (55~65) × 10^7 (in terms of the lipochondrion obtained by every 1ml steps (2)), but the karyocyte that (lidocaine be present) when not adding isopropanol Harvesting quantity can not be more than 15 × 10^7 (in terms of lipochondrion obtained by every 1ml steps (2)).

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein step (31) In, digestion 3min is vibrated with 100rpm at 37 DEG C.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein step (31) In, after terminating digestion, 5min is centrifuged with 1500rpm.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein step (32) In, add equivalent erythrocyte cracked liquid standing 10min and cracked.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein step (32) In, after red blood cell fully cracks, 1500rpm centrifugations 5min.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, wherein described complete Culture medium includes：Low-sugar type DMEM, 10%FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF- β, 0.05 μ g/ml IGF.Use in the present invention comprising tetra- kinds of above-mentioned EGF, bFGF, TGF-β, IGF cell factors Complete medium when, in the step (6) cell of harvest by blake bottle it is every square centimeter in terms of cell quantity up to (90~180) ×10^6；But if when not adding any or two or three or four kinds of cell factors in this complete medium, then In the step (6) cell of harvest by blake bottle it is every square centimeter in terms of cell quantity be only capable of reaching (0.05~0.5) × 10^6's Scope, it is seen that while it is necessary that four kinds of cell factors for adding above-mentioned quantity obtain stem cell for high yield.In addition, this hair A person of good sense also found in complementary testing, when adding 0.025% sorbierite into the complete medium, can further improve this The harvest yield of the fat mesenchymal stem cell of invention, for example, particularly in the step (6) harvest cell with every square of blake bottle Centimetre meter cell quantity is up to (500~650) × 10^6.Therefore, in one embodiment of the invention, the culture completely 0.025% sorbierite is also included in base, in view of the price of sorbierite is far below above-mentioned various cell factors, therefore adds sorbierite Technical meaning be very significant.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, the DMEM are low Sugar-type DMEM.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, the low-sugar type DMEM formula compositions are as follows：Anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, chlorine Change sodium 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- R-gene 84mg, L- salt Sour cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L-Leu 105mg, L- salt Sour lysine 146mg, METHIONINE 30mg, L-phenylalanine 66mg, Serine 42mg, L-threonine 95mg, L- color ammonia Sour 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, the culture completely Base forms：100mL FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF, 0.025% sorbierite, anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous sulphur Sour magnesium 97.67mg, sodium chloride 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- hydrochloric acid Arginine 84mg, L- hydrochloric acid cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L- are bright Propylhomoserin 105mg, LYS 146mg, METHIONINE 30mg, L-phenylalanine 66mg, Serine 42mg, L- Soviet Union ammonia Sour 95mg, L-Trp 16mg, TYR 72mg, L- valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, leaf Sour 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, the fat be from The fat that any position of human body obtains.

Fat mesenchymal stem cell preparation described in any embodiment according to a second aspect of the present invention, the cell cryopreservation The frozen stock solution used forms：1 part of DMSO：2 parts of human serum albumins：7 parts of complete mediums.

Further, third aspect present invention provides a kind of digestive ferment for fractionation of fatty mescenchymal stem cell and mixed Liquid, including：0.05% clostridiopetidase A I types, 0.05% clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes.Appoint Choosing, wherein also adding 0.002% isopropanol.

Further, fourth aspect present invention provides a kind of culture medium for being used to cultivate fat mesenchymal stem cell, its For complete medium, including：Low-sugar type DMEM, 10%FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml BFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF.

Culture medium described in any embodiment according to a fourth aspect of the present invention, wherein, the low-sugar type DMEM formula groups Into as follows：Anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, sodium chloride 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- R-gene 84mg, L- hydrochloric acid Cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L-Leu 105mg, L- hydrochloric acid Lysine 146mg, METHIONINE 30mg, L- phenylalanine 66mg, Serine 42mg, L-threonine 95mg, L-Trp 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

Culture medium described in any embodiment according to a fourth aspect of the present invention, wherein, the complete medium composition is： 100mL FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF, 0.025% sorbierite, anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, Sodium chloride 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- R-gene 84mg, L- Hydrochloric acid cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L-Leu 105mg, L- Lysine hydrochloride 146mg, METHIONINE 30mg, L-phenylalanine 66mg, Serine 42mg, L-threonine 95mg, L- color Propylhomoserin 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

Further, fifth aspect present invention provides a kind of method of the fractionation of fatty mescenchymal stem cell from fat, It comprises the following steps：

(4) combining step (31) and (32) gained cell suspension, 100 μm of strainer filterings, are separated as primary fat Mescenchymal stem cell；Optional carries out carry out coherent detection to the fat mesenchymal stem cell.

Method described in any embodiment according to a fifth aspect of the present invention, its separated obtained fat mesenchymal are dry thin Born of the same parents can be used for further culture breeding or for clinical treatment.

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (4), the coherent detection is Refer to detection cellular morphology and immunophenotypic characterization.In one embodiment, the immunophenotypic characterization refer to CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD34 are detected.

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (1), the inflation fluid bag Contain：0.1mg/ml lidocaines and 0.02ug/ml adrenaline.

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (1), centrifuged with 800rpm 3min。

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (31), lipochondrion is with mixing Synthase is with volume ratio 1：1 ratio adds.

Method described in any embodiment according to a fifth aspect of the present invention, wherein being included in the digestive ferment mixed liquor： 0.05% clostridiopetidase A I types, 0.05% clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes.Use in the present invention The above-mentioned digestion enzyme mixation being made up of four kinds of enzymes, very high karyocyte can be obtained in step (4) and harvest quantity, had Nucleus harvest quantity is generally up to (7.5~15) × 10^7 (in terms of lipochondrion obtained by every 1ml steps (2)).It has been found that It is very important influence factor that the combination of digestive ferment harvests quantity for karyocyte, such as includes the digestion of above-mentioned four kinds of enzymes In enzyme mixation, if wherein lack any or two or three of enzyme, karyocyte harvest quantity can not be more than 3 × 10 ^6 (in terms of lipochondrion obtained by every 1ml steps (2)).In one embodiment, also added in the digestion enzyme mixation 0.002% isopropanol；Had now surprisingly been found that, it may be possible to due to lidocaine presence (because, it was found that, when swollen Step (4), which obtains karyocyte harvest quantity, when not adding lidocaine in swollen liquid can reach (15~20) × 10^7), mixed Karyocyte harvest quantity can be made to reach (55~65) × 10^7 (with every 1ml steps (2) institute when adding isopropanol in synthase liquid Lipochondrion meter), but the karyocyte that (lidocaine be present) when not adding isopropanol harvest quantity can not more than 15 × 10^7 (in terms of lipochondrion obtained by every 1ml steps (2)).

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (31), 37 DEG C with 100rpm vibration digestion 3min.

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (31), after terminating digestion, with 1500rpm centrifuges 5min.

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (32), it is red thin to add equivalent Cellular lysate liquid stands 10min and cracked.

In method described in any embodiment according to a fifth aspect of the present invention, wherein step (32), red blood cell fully splits Xie Hou, 1500rpm centrifuge 5min.

Method described in any embodiment according to a fifth aspect of the present invention, the fat are obtained from any position of human body Fat.

In the above-mentioned various operating procedures of the present invention, although its description specific steps are in some details or language is retouched The step of stating with described in the preparation example of following detailed description part is otherwise varied, however, those skilled in the art Approach described above step can be summarized according to the detailed disclosure of full text of the present invention completely.

Any embodiment of the either side of the present invention, can be combined with other embodiments, as long as they are not Contradiction occurs.In addition, in any embodiment of either side of the present invention, any technical characteristic goes for other realities The technical characteristic in scheme is applied, as long as they are not in contradiction.The invention will be further described below.

All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary When offering expressed implication and the inconsistent present invention, it is defined by the statement of the present invention.In addition, the various terms that use of the present invention and Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention The implication stated is defined.

Expansion liposuction is a kind of technology the most extensive applied in section's liposuction procedures that is out at present, and is mainly filled before surgery Excess hypotonic expansion liquid is noted, the liquid has the function that analgesic, hemostasis, loose and cracking adipocyte；The formula of inflation fluid is this Known to field, and generally comprise lidocaine and adrenaline.

In the present invention, term " fat mesenchymal stem cell " refers to the mescenchymal stem cell from fat.Therefore exist In the present invention, more particularly to the present invention linguistic context in, term " fat mesenchymal stem cell " can with " fat stem cell ", " stem cell ", " mescenchymal stem cell " used interchangeably, unless otherwise clearly indicating.

In the present invention, term " PBS " or " PBS " refer to phosphate buffer.Those skilled in the art are ripe Know the PBS used under situation of the present invention general formula and compound method and their general aspects such as pH value or pH Scope, and these PBS buffer solutions are typically the pre-mixing liquor (or prewired powder) that can be obtained by commercial sources, such as The PBS in field of the present invention is typically pH7.4 (± 0.1) commercialization buffer solution, such as the PBS of HyClone brands； PBS composition during the application of this area classics includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate radicals, In the present invention if not otherwise specified, PBS used using when composition be the composition.

The inventive method is separately cultured fat mesenchymal stem cell, and gained fat mesenchymal stem cell has very high pure Spend and there is very high yield.For example, in the embodiment of the present invention 1~3, included in the digestion enzyme mixation：0.05% Clostridiopetidase A I types, 0.05% clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes.Embodiment 1~3 using it is above-mentioned by The digestion enzyme mixation of four kinds of enzyme compositions, very high karyocyte can be obtained in step (4) and harvest quantity, karyocyte Quantity is harvested generally up to (7.5~15) × 10^7 (in terms of lipochondrion obtained by every 1ml steps (2)).Enter in reference implementation example 1 It has been found that it is very important influence factor that the combination of digestive ferment harvests quantity for karyocyte in the example of row supplement, Such as in the digestion enzyme mixation comprising above-mentioned four kinds of enzymes, if wherein lack any or two or three of enzyme, there is core Cell harvesting quantity can not be more than 3 × 10^6 (in terms of lipochondrion obtained by every 1ml steps (2)).Carried out in reference implementation example 1 Supplement in embodiment and have been found that when adding 0.002% isopropanol again into digestion enzyme mixation used, can be in step (4) karyocyte harvest quantity is made to reach (55~65) × 10^7 (in terms of lipochondrion obtained by every 1ml steps (2)) in, still (lidocaine being present) when not adding isopropanol, karyocyte harvest quantity can not be more than 15 × 10^7 (with every 1ml steps (2) Gained lipochondrion meter).In addition, it has been found that karyocyte harvest quantity in the supplement embodiment that reference implementation example 1 is carried out Raising may be relevant with the presence of lidocaine in inflation fluid, reason is, it was found that, when not adding benefit in inflation fluid Step (4), which obtains karyocyte harvest quantity, during cacaine can reach (15~20) × 10^7.These reference implementation examples 1~3 are entered The property of various fat mesenchymal stem cells obtained by capable supplement embodiment, in addition to above-mentioned harvest quantity is different, remaining property It is identical with embodiment 1~3, such as the phase of embodiment 1~3 that cellular morphology and streaming Phenotypic examination result refer to it Together.

In another example in the embodiment of the present invention 1~3, the composition of complete medium used is：Low-sugar type DMEM+10% The FBS+25mM HEPES+1 μ g/ml EGF+2 μ g/ml bFGF+0.02 μ g/ml μ of TGF-β+0.05 g/ml IGF, makes With comprising above-mentioned EGF, bFGF, TGF-β, tetra- kinds of cell factors of IGF complete medium when, the cell of harvest in the step (6) By blake bottle it is every square centimeter in terms of cell quantity up to (90~180) × 10^6；But the supplement examination that reference implementation example 1 is carried out Middle discovery is tested, if do not add any or two or three or four kinds of cell factors in this complete medium, In step (6) cell of harvest by blake bottle it is every square centimeter in terms of cell quantity be only capable of the model that reaches (0.05~0.5) × 10^6 Enclose, it is seen that while it is necessary that four kinds of cell factors for adding above-mentioned quantity obtain stem cell for high yield；It is in addition, of the invention People also found in the complementary testing that reference implementation example 1 is carried out, when adding 0.025% sorbierite into the complete medium, The harvest yield of the fat mesenchymal stem cell of the present invention can be further improved, such as is particularly harvested in step (6) thin Born of the same parents by blake bottle it is every square centimeter in terms of cell quantity up to (500~650) × 10^6.In view of the price of sorbierite is far below above-mentioned Various cell factors, therefore the technical meaning for adding sorbierite is very significant, particularly with significant economic implications.This The property for the various fat mesenchymal stem cells of supplement experiment gained that a little reference implementation examples 1~3 are carried out, except above-mentioned harvest quantity Different outer, remaining property is identical with embodiment 1~3, such as cellular morphology and streaming Phenotypic examination result refer to it Embodiment 1~3 it is identical.

The present invention is presented below as the excellent technique effect of one or more aspects：1. the optimized mixing enzymic digestion of the present invention Method can separate from lipochondrion obtains a large amount of original fat stem cells, is significantly higher than conventional method and (such as I types is used alone The situation of clostridiopetidase A), and cytoactive is high；2. present invention preserves the fat stem cell in inflation fluid, original is further increased The quantity of beginning fat stem cell；3. fatty sample size is few needed for the present invention, according to receptor's concrete condition, (cell should quantity, secondary Number etc.) collection capacity can be further reduced, at utmost reduce receptor's wound；4. the fat mesenchymal stem cell that the present invention optimizes is complete The FAQs such as full culture medium solves fat mesenchymal stem cell differentiation, paramophia, propagation is slow, phenotype is abnormal, wherein Four kinds of growth factors have been used in mixed way key effect, especially for cell proliferation rate and amount；5. the inventive method is trained Support P0, P1 the fat subsitutes stem cell obtained and be applied enough to clinical treatment, apply and can be completed in 10 days from collecting, and The autologous mesenchymal stem cell transplantations of Shi Jinhang can avoid missing the optimal treatment phase of patient.

Brief description of the drawings

Fig. 1：P0 to P5 generations cellular morphology figure.

Fig. 2：Streaming phenotypic map.

Embodiment

The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that on the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out general to the material and test method that are arrived used in experiment And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that But the present invention still makees description as detailed as possible herein.

In tests below, if not otherwise specified, the low-sugar type DMEM formulas composition is as follows：Anhydrous calcium chloride 265mg, nitre Sour iron 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, sodium chloride 6400mg, AMSP 109mg, fourth two Sour 75mg, sodium succinate 100mg, L- R-gene 84mg, L- hydrochloric acid cystine 63mg, glycine 30mg, L- hydrochloric acid group ammonia Sour 42mg, ILE 105mg, L-Leu 105mg, LYS 146mg, METHIONINE 30mg, L- phenylpropyl alcohol Propylhomoserin 66mg, Serine 42mg, L-threonine 95mg, L-Trp 16mg, TYR 72mg, Valine 94mg, D- Calcium pantothenate 4mg, choline tartrate 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

In tests below, if not otherwise specified, the complete medium composition is：100mL FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF, anhydrous calcium chloride 265mg, nitric acid Iron 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, sodium chloride 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- R-gene 84mg, L- hydrochloric acid cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L-Leu 105mg, LYS 146mg, METHIONINE 30mg, L- phenylpropyl alcohol ammonia Sour 66mg, Serine 42mg, L-threonine 95mg, L-Trp 16mg, TYR 72mg, Valine 94mg, D- are general Sour calcium 4mg, choline tartrate 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, Pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

In tests below, if not otherwise specified, included in the digestion enzyme mixation：0.05% clostridiopetidase A I types, 0.05% Clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes.In tests below, if not otherwise specified, red blood cell used splits Solution liquid is purchased from Solarbio.In tests below, if not otherwise specified, fatty sample used is obtained by expanding liposuction versions 's.

Embodiment 1：Separation and culture fat mesenchymal stem cell

(1) the fatty sample doped with inflation fluid will be obtained in centrifuge tube by liposuction, 800rpm centrifugations 3min；

(31) into lipochondrion obtained by step (2) with volume ratio 1: 1 add mixed enzyme (0.05% clostridiopetidase A I types+ Neutral proteinase+0.03%DNA the enzymes of 0.05% clostridiopetidase A II types+0.03%), digestion 30min is vibrated with 100rpm at 37 DEG C；Disappear After change terminates, add isometric complete medium and terminate digestion, 1500rpm centrifugation 5min abandon supernatant, and PBS is resuspended cell and sunk Form sediment；

(32) bottom cell precipitation obtained by step (2) is resuspended with PBS, adds equivalent erythrocyte cracked liquid and stand 10min Cracked；After red blood cell fully cracks, 1500rpm centrifugation 5min, supernatant is abandoned, cell precipitation is resuspended in PBS；

[sample to Other nucleated cells differential count (with sysmex blood counting instruments) and detect Cell viability and (blue dye is expected by platform Color), and sample for microorganism detection；Acquisition karyocyte quantity is 7.5 × 10^7-1.5 × 10^8 (with every 1ml steps (2) Gained lipochondrion meter), and Cell viability more than 90%]

(5) the primary fat mesenchymal stem cell isolated is seeded into T225 blake bottles according to 30000 cells/cm^2 to enter Row culture, add complete medium, 37 DEG C of 5%CO2 cultures；[the composition of complete medium：Low-sugar type DMEM+10%FBS+ The 25mM HEPES+1 μ g/ml EGF+2 μ g/ml bFGF+0.02 μ g/ml μ of TGF-β+0.05 g/ml IGF]

(6) change liquid after cultivating 2 days, remove non-adherent cell, (cell confluency is up to 90%) to be generated when growing to one week with On, harvest P1 fat subsitutes mescenchymal stem cell (by blake bottle it is every square centimeter in terms of cell quantity up to (90~180) × 10^6)；

(7) a small amount of cell is taken to continue to be passaged to of future generation until P5 generations (often pass a generation, cell according to step (5) and step (6) Up to 13~26 times of growths), harvest and observe each generation fat mesenchymal stem cell, and carry out coherent detection.

[testing result is shown：Cellular morphology activity, multiplication capacity, streaming phenotype etc. are stable, disclosure satisfy that clinical answer With；Per generation Cell viability more than 95%, streaming phenotype：CD73, CD90, CD105 positive rate more than 99%, CD19, CD11b, CD31, CD45, HLADR, CD34 are hardly expressed]

The present embodiment P0 to P5 generations cellular morphology as shown in figure 1, the present embodiment experiment gained streaming phenotype see Fig. 2, this In invention other embodiments and supplement embodiment, the cellular morphology figure essentially identical with the present embodiment and streaming phenotype are respectively provided with Figure.

Embodiment 2：Separation and culture fat mesenchymal stem cell

(1) the fatty sample doped with inflation fluid will be obtained in centrifuge tube by liposuction, 500rpm centrifugations 5min；

(31) into lipochondrion obtained by step (2) with volume ratio 1：1.2 addition mixed enzymes (0.05% clostridiopetidase A I types+ Neutral proteinase+0.03%DNA the enzymes of 0.05% clostridiopetidase A II types+0.03%), digestion 20min is vibrated with 100rpm at 37 DEG C； After digestion terminates, add isometric complete medium and terminate digestion, 2000rpm centrifugation 3min, abandon supernatant, cell is resuspended in PBS Precipitation；

(32) bottom cell precipitation obtained by step (2) is resuspended with PBS, adds equivalent erythrocyte cracked liquid standing 5min and enter Row cracking；After red blood cell fully cracks, 2000rpm centrifugation 3min, supernatant is abandoned, cell precipitation is resuspended in PBS；

Embodiment 3：Separation and culture fat mesenchymal stem cell

(1) the fatty sample doped with inflation fluid will be obtained in centrifuge tube by liposuction, 1000rpm centrifugations 2min；

(31) into lipochondrion obtained by step (2) with volume ratio 1：0.8 addition mixed enzyme (0.05% clostridiopetidase A I types+ Neutral proteinase+0.03%DNA the enzymes of 0.05% clostridiopetidase A II types+0.03%), digestion 40min is vibrated with 100rpm at 37 DEG C； After digestion terminates, add isometric complete medium and terminate digestion, 1000 centrifugation 8min abandon supernatant, and PBS is resuspended cell and sunk Form sediment；

(32) bottom cell precipitation obtained by step (2) is resuspended with PBS, adds equivalent erythrocyte cracked liquid and stand 15min Cracked；After red blood cell fully cracks, 1000rpm centrifugation 8min, supernatant is abandoned, cell precipitation is resuspended in PBS；

Fat mesenchymal stem cell (ADSCs) produced by the present invention can be applied to various parts for example applied to face with autologous Portion, exemplary application process are：After all detections of gained fat mesenchymal stem cell preparation meet standard, it can incite somebody to action ADSCs preparations directly carry out facial injection.It is, for example, possible to use 1ml injector for medical purpose, is used 30GA (external diameter 0.3mm or so) Syringe needle, each position suggests that dosage is 0.5-1ml, the filling available for positions such as facial wrinkles, temple, tear ditches.

In the above embodiment of the present invention 1, since primary i.e. P0 generations, contain in each fat subsitutes mescenchymal stem cell of gained There are a certain amount of fat drips, such as the eye fat mesenchymal stem cell for primary adherent culture, as shown in figure 1, being filled between fat Matter stem cell is in spindle shape, swirl shape growth, possesses typical mescenchymal stem cell form.Larger circle is eye fat in figure The fat drips adhered on fat mescenchymal stem cell, can be by passing on removal repeatedly.

Embodiment described above is only the preferred embodiment to absolutely prove the application and being lifted, the protection model of the application Enclose not limited to this.The equivalent substitute or conversion that those skilled in the art are made on the basis of the application, in the application Protection domain within.The protection domain of the application is defined by claims.

Claims

1. the method for fractionation of fatty mescenchymal stem cell from fat, it comprises the following steps：

(31) into lipochondrion obtained by step (2) with volume ratio 1：0.8~1.2 add digestion enzyme mixation, 37 DEG C with 20~40min of 100rpm vibration digestion；After digestion terminates, add isometric complete medium termination and digest, 1000~ 2000rpm centrifuges 3~8min, abandons supernatant, and cell precipitation is resuspended in PBS；

(32) bottom cell precipitation obtained by step (2) is resuspended with PBS, adds 5~15min of equivalent erythrocyte cracked liquid standing and enter Row cracking；After red blood cell fully cracks, 1000~2000rpm centrifuges 3~8min, abandons supernatant, and cell precipitation is resuspended in PBS；

(4) combining step (31) and (32) gained cell suspension, 100 μm of strainer filterings, are separated as filling between primary fat Matter stem cell；Optional carries out carry out coherent detection to the fat mesenchymal stem cell.

2. method according to claim 1, it is characterised in that：

In step (1), 3min is centrifuged with 800rpm；

In step (31), lipochondrion is with mixed enzyme with volume ratio 1：1 ratio adds；

In step (31), digestion 3min is vibrated with 100rpm at 37 DEG C；

In step (31), after terminating digestion, 5min is centrifuged with 1500rpm；

In step (32), add equivalent erythrocyte cracked liquid standing 10min and cracked；And/or

In step (32), after red blood cell fully cracks, 1500rpm centrifugations 5min.

3. method according to claim 1, it is characterised in that：

The inflation fluid includes：0.1mg/ml lidocaines and 0.02ug/ml adrenaline；

Included in the digestion enzyme mixation：0.05% clostridiopetidase A I types, 0.05% clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes and optional isopropanol；

The fat is the fat obtained from any position of human body；

In step (4), the coherent detection refers to detect cellular morphology and immunophenotypic characterization；For example, the immunophenotype Identification refers to detect CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD34；And/or

Its separated obtained fat mesenchymal stem cell can be used for further culture breeding or for clinical treatment.

4. separation and the method for culture fat mesenchymal stem cell, it comprises the following steps：

(4) combining step (31) and (32) gained cell suspension, 100 μm of strainer filterings, are separated as filling between primary fat Matter stem cell；

(5) the primary fat mesenchymal stem cell isolated is seeded to blake bottle and cultivated according to 30000 cells/cm^2, Add complete medium, 37 DEG C of 5%CO2 cultures；

(6) change liquid after cultivating 2 days, remove non-adherent cell, it is to be generated when growing to one week, harvest P1 fat subsitutes mescenchymal stem cells；

(7) a small amount of cell is taken to continue to be passaged to of future generation until in P5 generations, harvest and observe each generation according to step (5) and step (6) Fat mesenchymal stem cell, and carry out coherent detection.

5. method according to claim 4, it is characterised in that：

In step (1), 3min is centrifuged with 800rpm；

In step (7), the coherent detection refers to detect cellular morphology and immunophenotypic characterization；For example, the immunophenotype Identification refers to detect CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD34；

In step (31), digestion 3min is vibrated with 100rpm at 37 DEG C；

In step (31), after terminating digestion, 5min is centrifuged with 1500rpm；

In step (32), add equivalent erythrocyte cracked liquid standing 10min and cracked；

In step (32), after red blood cell fully cracks, 1500rpm centrifugations 5min；

The P1 for wherein also including making to obtain carries out the system of preparation for any generation to P5 generations or the fat mesenchymal stem cell in more generations Standby step；And/or

Wherein also include making obtained any generation in P1 generation to P5 generations or the fat mesenchymal stem cell progress cell cryopreservation in more generations The step of, the optional cell to being frozen is recovered, and the optional cell to recovery carries out the preparation of preparation.

6. method according to claim 4, it is characterised in that：

In step (1), the inflation fluid includes：0.1mg/ml lidocaines and 0.02ug/ml adrenaline；

Included in the digestion enzyme mixation：0.05% clostridiopetidase A I types, 0.05% clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes；

The complete medium includes：Low-sugar type DMEM, 10%FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml BFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF；

The DMEM is low-sugar type DMEM；

The low-sugar type DMEM formulas composition is as follows：Anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous sulphur Sour magnesium 97.67mg, sodium chloride 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- hydrochloric acid Arginine 84mg, L- hydrochloric acid cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L- are bright Propylhomoserin 105mg, LYS 146mg, METHIONINE 30mg, L-phenylalanine 66mg, Serine 42mg, L- Soviet Union ammonia Sour 95mg, L-Trp 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, leaf Sour 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL；And/or

The complete medium forms：100mL FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF, 0.025% sorbierite, anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous magnesium sulfate 97.67mg, sodium chloride 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- R-gene 84mg, L- hydrochloric acid cystine 63mg, the different bright ammonia of glycine 30mg, L- histidine monohydrochloride 42mg, L- Sour 105mg, L-Leu 105mg, LYS 146mg, METHIONINE 30mg, L-phenylalanine 66mg, L- silk ammonia Sour 42mg, L-threonine 95mg, L-Trp 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, tartaric acid Choline 7.2mg, folic acid 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, Glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL.

7. a kind of fat mesenchymal stem cell preparation, it is prepared by a method comprising the following steps to obtain：

(7) a small amount of cell is taken to continue to be passaged to of future generation until in P5 generations, harvest and observe each generation according to step (5) and step (6) Fat mesenchymal stem cell, and carry out coherent detection；

(8) P1 for making to obtain carries out the preparation of preparation for any generation to P5 generations or the fat mesenchymal stem cell in more generations；Or In any generation in P1 generation to P5 generations or the fat mesenchymal stem cell in more generations for making to obtain, carry out cell cryopreservation, optional to being frozen Cell recovered, and the optional cell to recovery carries out the preparation of preparation.

8. fat mesenchymal stem cell preparation according to claim 7, it is characterised in that：

In step (1), 3min is centrifuged with 800rpm；

In step (31), digestion 3min is vibrated with 100rpm at 37 DEG C；

In step (31), after terminating digestion, 5min is centrifuged with 1500rpm；

The DMEM is low-sugar type DMEM；

The low-sugar type DMEM formulas composition is as follows：Anhydrous calcium chloride 265mg, ferric nitrate 0.1mg, potassium chloride 400mg, anhydrous sulphur Sour magnesium 97.67mg, sodium chloride 6400mg, AMSP 109mg, succinic acid 75mg, sodium succinate 100mg, L- hydrochloric acid Arginine 84mg, L- hydrochloric acid cystine 63mg, glycine 30mg, L- histidine monohydrochloride 42mg, ILE 105mg, L- are bright Propylhomoserin 105mg, LYS 146mg, METHIONINE 30mg, L-phenylalanine 66mg, Serine 42mg, L- Soviet Union ammonia Sour 95mg, L-Trp 16mg, TYR 72mg, Valine 94mg, D-VB5 calcium 4mg, choline tartrate 7.2mg, leaf Sour 4mg, inositol 7.2mg, niacinamide 4mg, riboflavin 0.4mg, thiamine hydrochloride 4mg, pyridoxine hydrochloride 4mg, glucose 1000mg, Sodium Pyruvate 110mg, phenol red 9.3mg, appropriate amount of water add to 1000mL；

9. it is used for the digestion enzyme mixation of fractionation of fatty mescenchymal stem cell, including：0.05% clostridiopetidase A I types, 0.05% Clostridiopetidase A II types, 0.03% neutral proteinase, 0.03%DNA enzymes.

10. the culture medium for cultivating fat mesenchymal stem cell, it is complete medium, including：Low-sugar type DMEM, 10%FBS, 25mM HEPES, 1 μ g/ml EGF, 2 μ g/ml bFGF, 0.02 μ g/ml TGF-β, 0.05 μ g/ml IGF.