CN104357382A - Method for obtaining human adipose-derived stem cells and construction method for multilevel allogeneic adipose-derived stem cell bank - Google Patents

Abstract

The invention discloses a method for obtaining human adipose-derived stem cells and a construction method for a multilevel allogeneic adipose-derived stem cell bank, relates to an adipose-derived stem cell obtaining method and an adipose-derived stem cell bank construction method, and aims to solve the problems of great damage to cell activity caused by a fat collection method and a conventional human adipose-derived stem cell obtaining method and high aging rate of stem cells used by multiple persons after subculture of many times in a conventional adipose-derived stem cell bank. The method for obtaining the stem cells comprises the following steps of 1, screening donors; 2, collecting fats; 3, transporting the fats; 4, separating the human adipose-derived stem cells. The construction method for the stem cell bank comprises the following steps of 1, screening the donors; 2, collecting the fats; 3, transporting the fats; 4, separating the human adipose-derived stem cells; 5, culturing, subculturing and amplifying the human adipose-derived stem cells; 6, constructing a main cell bank; 7, establishing working cell banks. According to the methods, the cell activity is less damaged, and multiple levels of stem cell banks for multiple persons are established for the establishment of the adipose-derived stem cell bank.

Description

The acquisition methods of human adipose-derived stem cell and the construction process in multistage Conspecific variant fat stem cell used storehouse

Technical field

The present invention relates to a kind of acquisition methods of fat stem cell and the construction process in fat stem cell storehouse.

Background technology

Fat stem cell (Adipose-Derived Stem Cells, ADSCs or be called Adipose-DerivedMesenchymal Stromal cells, ADMSC) be the stem cell that the class obtained from fatty tissue has multi-lineage potential, it can be divided into scleroblast, chondroblast and adipocyte etc. under specific inductive condition.Zuk2001 first passage is separated human fat tissue and obtains the stem cell that this type of has cell therapy potential, thus has started the beginning of fat stem cell investigation and application.Find through the scientific researches of more than 10 years and practice, fat stem cell be a group be similar to mesenchymal stem cells MSCs have regeneration differentiation capability cell, it does not express the hematopoietic cell surface antigens such as CD34, CD45, and therefore can not break up under physiological status becomes hemocyte and vascular endothelial cell; Fat stem cell human leucocyte HLA-DR expresses feminine gender, therefore would not produce allosome rejection in migration process, from but an excellent cell kind of carrying out heteroplastic transplantation; Fat stem cell is same with bone marrow stem cell expresses CD105, CD73, CD90 etc., be adapted at that there is in Process of in vitro the feature such as adherent growth and formation colony, therefore can realize cultured and amplified in vitro, thus for various diseases treatment established the basis of a large amount of production and qualification.

Vitro culture also finds, compared with the mescenchymal stem cell of derived from bone marrow, fat stem cell has more stability and not easily Aging, is high quality seed cell rare in organizational project; Fat acquisition process is simple in addition, and simple liposuction procedures just can obtain significant quantities of fat cell, and operation on bone marrow is then painful large, is not easily accepted by normal health crowd; Calendar year 2001 and the Hattori etc. such as in fatty tissue, stem cell population is many, Zuk research in 2004 shows, the fatty tissue mesostroma vascular lamina cell content of extraction is approximately 4 × 10 ⁸l ^-1, and in these stromal vascular confluent monolayer cells, the ratio of human adipose-derived stem cell studies greatly fatty stem cell (4-20) × 10 in the fatty tissue of extraction about about 1-5% ⁶l ^-1, and in medullary cell mescenchymal stem cell ratio 30-50 year crowd in be only 25 ~ 400,000/, therefore same volume vs, in fat, stem cell is at least 1000 times in marrow.Fat stem cell has more lower immunogenicity than bone marrow stem cell; can not exotic be used as by body and repel when transplanting; just allotransplantation can be carried out without distribution type; therefore in the application of market; if after can carrying out unitized management, large-scale production, make unified standard preparation, then not only fat stem cell can individuation be applied; future can also carry out allosome, mass-producing disease treatment, substantially increases the effect in fat stem cell damage and disease reparation.End existing 102 adipose-derived stem cells of employing on clinicaltrials.gov on May 4th, 2014 to report as the clinical trial for the treatment of means, involved clinical disease comprises shaping, fistula, in heart failure, chronic lung disease, I/type II diabetes, keratopathy, autism, the multiple stubborn disease such as osteoarthritis, following progressively going deep into along with clinical study, the accumulation of ADSCs security efficacy data is more perfect, the fistula caused after first ADSCs pharmacological agent clone disease in the world gets the Green Light in Korea S and enters clinical treatment for 2012, the progressively listing of ADSCs medicine, no matter be that employing is autologous or the application and development of allosome ADSCs in the treatment and organ reparation of disease will have larger prospect.Another huge autologous application market of ADSCs is exactly beautifying and anti-aging industry, the pure fat that weight reducing liposuction obtains carries out moulding filling, often can not maintain for a long time, once fill fat absorbed after, just face the problem of again filling, if mixing ADSCs together while fat is filled, ADSCs is divided into fatty ability then can make filling effect be able to long sustaining, improve the effect of beauty treatment plasticity, become the developing direction of following beauty industry personalized customization.

Because the adipose-derived of fat stem cell culture of isolated technology adopted at present mainly contains 2 kinds: the fatty tissue block that (1) surgical site infections obtains, the discarded liquid fat obtained after (2) beauty treatment liposuction procedures.

Although the fat lump in surgical site infections source has complete weave construction and cytoactive, few and operatingly belong to health non-health owing to carrying out operating crowd more, not a major customer colony carrying out ADSCs mass-producing cell bank and set up.

Beauty treatment liposuction technique is divided into hand gun to aspirate roughly and mechanical suction pump aspirates two kinds of modes.Although the syringe liposuction method not adding extraneous power-assisted is little in the surging force of liposuction process to cell, easily causes the fatigue of operator, be therefore only applicable to the process that liposuction amount is few.And the liposuction technique adopting traditional beauty industry to carry out, focus on be fat-reducing and moulding, do not focus on lipolytic activity and the stem cell performance of sucking-off, the vacuum tightness that traditional vacuum sliding chute is taked is tall and big in 750mmHg, be damaged close to the adipocyte of 50%, Stem Cell Activity is poor, is unfavorable for the foundation needs of efficient stem cell bank.What water power liposuction process adopted is that fatty tissue breaks up by soft hydraulic pressure, and then carry out liposuction by vacuum tightness at the condition of negative pressure of about 300mmHg, this is very favourable to the downstream stem cell mask work in future undoubtedly.

Carry out fat stem cell preparation and set up for the autologous fat stem cell storehouse that future clinical is treated being suitable for healthy population and having planned the crowd that carrying out improves looks cultivates one's moral character, for vast the diseases of middle-and old-aged person patient, gathering acquisition autologous fat stem cell can make Stem Cell Activity reduce due to healthy factor, amplification rate is slow, differentiation capability is weak, and be not suitable for self-treating, therefore healthy supplying is adopted, carry out mass-producing amplification, set up Conspecific variant fat stem cell used storehouse and be following developing direction for the disease treatment of non-health crowd and injury repairing, because the immunogenicity of ADSCs is low, heteroplastic transplantation can be carried out, thus open a fan gate of allosome treatment.

Summary of the invention

The present invention destroys large to cytoactive when being acquisition methods and the collection fat that will solve existing human adipose-derived stem cell, in existing fat stem cell storehouse, stem cell is repeatedly gone down to posterity and uses the problem easily occurring stem cell catabiosis to many people, provides the construction process in the acquisition methods of human adipose-derived stem cell and multistage Conspecific variant fat stem cell used storehouse.

The acquisition methods of the present inventor's fat stem cell, carries out according to the following steps:

One, the screening of donor:

Donor requires healthy, in donor blood, HCV, HBsAg, HBcAb, CMV, HIV, Syphilis, HLTV and EBV Viral diagnosis is negative, in blood, anerobe, aerophil, wide spectrum mycoplasma and fungal detection are feminine gender, get rid of have medical history, have familial inheritance medical history, have history of drug abuse, NIP, the patient that difficult without wound healing and compliance is poor, if donor is women, require that it is in non-pregnancy period and non-physiology phase state, donor to narcotic without anaphylaxis;

Two, fat acquisition:

Donor after screening step one carries out fat acquisition, fat acquisition employing suction strength is the water power liposuction technique of 200-350mmHg, after donor toponarcosis, the 1-2mm edge of a knife is cut at belly or thigh, inject 50-400mL inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction, the fat drawn directly is collected in the aseptic receptor with Screening Network, fatty tissue stops 5 minutes in the receiver, major part inflation fluid and the fat after screening are according to the different natural separation of its proportion, 50mL is adopted to be with cap Luer Lock syringe by the fatty tissue microballoon inhalation syringe of the 0.1-1mm diameter of acquisition,

Three, the transport of fat:

Fatty tissue step 2 collected adopts multi-functional fat acquisition, transport test kit transports, or the above-mentioned 50mL aseptic Luer Lock syringe having sucked fat is removed syringe needle, directly transports after being sealed by syringe with syringe cap;

Four, the separation of human adipose-derived stem cell:

In Biohazard Safety Equipment, fatty tissue is divided in 50mL centrifuge tube, in the centrifugal 10min of 1000-1500r/min, middle layer fatty tissue after centrifugal by pipette, extract shifts in new centrifuge tube, add equal-volume stroke-physiological saline solution or PBS cleaning, again in the centrifugal 10min of 1000-2000rpm, the upper-layer fat drawn after cleaning is placed in new centrifuge tube, add the type i collagen enzyme that mass percentage concentration is 0.03%-0.06%, lid is built in mixing, concussion digestion 15-30min in 37 DEG C of incubators, then PBS or physiological saline is added, and piping and druming tissue, cell suspension is filtered through 75 μm of aperture cellular filters, obtain single cell suspension, in the centrifugal 10min of 1000-2000rpm, obtain cell precipitation, add physiological saline or PBS relaunders cell once, centrifugal, precipitation is human adipose-derived stem cell.

The construction process in the multistage Conspecific variant fat stem cell used storehouse of the present invention, carries out according to the following steps:

One, the screening of donor:

Donor requires healthy, in donor blood, HCV, HBsAg, HBcAb, CMV, HIV, Syphilis, HLTV and EBV Viral diagnosis is negative, in blood, anerobe, aerophil, wide spectrum mycoplasma and fungal detection are feminine gender, get rid of have medical history, have familial inheritance medical history, have history of drug abuse, NIP, the patient that difficult without wound healing and compliance is poor, if donor is women, require that it is in non-pregnancy period and non-physiology phase state, donor to narcotic without anaphylaxis; The age of described donor is 18-40 year;

Two, fat acquisition:

Donor after screening step one carries out fat acquisition, fat acquisition employing suction strength is the water power liposuction technique of 200-350mmHg, after donor toponarcosis, the 1-2mm edge of a knife is cut at belly or thigh, inject 50-400mL inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction, the fat drawn directly is collected in the aseptic receptor with Screening Network, fatty tissue stops 5 minutes in the receiver, major part inflation fluid and the fat after screening are according to the different natural separation of proportion, 50mL is adopted to be with cap Luer Lock syringe by the fatty tissue microballoon inhalation syringe of the 0.1-1mm diameter of acquisition, adipocyte motility rate in syringe is greater than 80%, fat weight is 30-100 gram,

Three, the transport of fat:

Fatty tissue step 2 collected adopts multi-functional fat acquisition, transport test kit transports, or the above-mentioned 50mL aseptic Luer Lock syringe having sucked fat is removed syringe needle, directly transports after being sealed by syringe with syringe cap;

The specific operation process that the multi-functional fat acquisition of described employing, transport test kit carry out transporting is: with aseptic 50mL syringe, fat is transferred to the fat acquisition that DMEM conserving liquid is housed and transport in bottle, transport bottle flexible rubber ring is fixed in transport test kit, test kit both sides are placed in the position of ice bag and are put into ice bag, make transport test kit transport temperature remain on 4-12 DEG C, fat sample was transported to clean plant and carries out fat stem cell separation in 1 hour;

Described syringe cap carries out in the process of directly transporting after being sealed by syringe, and vertically put down by the cap of band cap syringe, inflation fluid residual in transportation is separated with adipocyte, and envrionment temperature residing for syringe is 4-12 DEG C;

Four, the separation of human adipose-derived stem cell:

In Biohazard Safety Equipment, fatty tissue is divided in 50mL centrifuge tube, in the centrifugal 10min of 1000-1500r/min, middle layer fatty tissue after centrifugal by pipette, extract shifts in new centrifuge tube, add equal-volume stroke-physiological saline solution or PBS cleaning, again in the centrifugal 10min of 1000-2000rpm, the upper-layer fat drawn after cleaning is placed in new centrifuge tube, add the type i collagen enzyme that mass percentage concentration is 0.03%-0.06%, lid is built in mixing, concussion digestion 15-30min in 37 DEG C of incubators, then PBS or physiological saline is added, and piping and druming tissue, cell suspension is filtered through 75 μm of aperture cellular filters, obtain single cell suspension, in the centrifugal 10min of 1000-2000rpm, obtain cell precipitation, add physiological saline or PBS relaunders cell once, centrifugal, precipitation is human adipose-derived stem cell,

Five, fat stem cell is cultivated, goes down to posterity, is increased:

Cell counting is carried out, with inoculum density 3 × 10 to the human adipose-derived stem cell that step 4 obtains ⁴individual/cm ²-8 × 10 ⁴individual/cm ²be inoculated in culturing bottle and carry out original cuiture, at 37 DEG C, the CO of 5% ₂in incubator, fresh culture is changed every 2-3 days, after cytogamy degree reaches 70%-80%, adopt 1 × TrypLE Select in 37 DEG C of digestion 3 minutes, add equal-volume PBS or normal saline dilution, repeatedly blow and beat, cell is cleaned up, then the cell after cleaning is moved in 50mL centrifuge tube, centrifugal 5min, centrifugal speed is 1000-2000rpm, abandon supernatant liquor, cell is resuspended in new serum free medium, according to the ratio that goes down to posterity of 1:3-6, go down to posterity in new culturing bottle, serum free medium is adopted to carry out amplification P1 culture, amplification procedure changed fresh serum-free media every 2-3 days, after cytogamy degree reaches 70%-80%, so repeat above-mentioned cell dissociation succeeding generations, complete P2 and P3 for going down to posterity and amplification cultivation, after P3 reaches 70%-80% for cytogamy degree, for setting up master cell bank, original cuiture used medium is serum free medium or MesenGro substratum,

Six, master cell bank is set up:

P3 step 5 obtained adopts PBS or physiological saline cleaning for human adipose-derived stem cell, 1 × TrypLESelect37 DEG C is adopted to digest 3 minutes, add PBS or physiological saline, and blow and beat, collect the cell suspension after dispersion in 50mL centrifuge tube, get part and carry out cell counting, Cell viability mensuration, the centrifugal 5min of all the other cell suspensions, centrifugal speed is 1000-2000rpm, supernatant liquor is stayed to carry out anerobe, aerophil, mycoplasma, fungi, syphilis and Viral diagnosis, abandon unnecessary supernatant liquor, by cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is divided in the cryopreservation tube of 40-70 root 2mL, often pipe 1mL, and cryopreservation tube sticks antifreeze bar-code label, carries out freezen protective;

Seven, working cardial cell storehouse is set up:

From liquid nitrogen container, get master cell bank cryopreservation tube 2-3 prop up, after recovery is melted rapidly in 37 DEG C of water-baths, cell is transferred in serum free medium or MesenGro substratum, according to 0.5 × 10 ⁴individual/cm ²-2 × 10 ⁴individual/cm ²inoculum density be inoculated in culturing bottle and carry out P4 culture, at 37 DEG C, the CO of 5% ₂in incubator, serum free medium or MesenGro substratum is adopted to cultivate, fresh culture is changed every 2-3 days, after cytogamy degree reaches 70%-80%, adopt 1 × TrypLE Select in 37 DEG C of digestion 3 minutes, add equivalent PBS or normal saline dilution collects postdigestive cell in 50mL centrifuge tube, centrifugal 5min, centrifugal speed is 1000-2000rpm, abandon supernatant liquor, cell is resuspended in new serum free medium, according to the ratio that goes down to posterity of 1:3-6, go down to posterity in new culturing bottle, serum free medium is adopted to carry out amplification P5 culture, amplification procedure changed fresh serum-free media every 2-3 days, after cytogamy degree reaches 70-80%, carry out working cardial cell storehouse and set up work,

The P5 cultivated adopts PBS or physiological saline to wash away substratum for cell, adopt 1 × TrypLE Select37 DEG C digestion 3 minutes, add PBS or physiological saline, and blow and beat with transfer pipet, collect the cell suspension after dispersion in 50mL centrifuge tube, get part and carry out cell counting, Cell viability mensuration, the centrifugal 5min of all the other cell suspensions, centrifugal speed is 1000-2000rpm, supernatant liquor is stayed to carry out anerobe, aerophil, mycoplasma, fungi, syphilis and Viral diagnosis, abandon unnecessary supernatant liquor, by cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is divided in 60-90 and props up in the cryopreservation tube of 2mL, and often pipe 1mL, cryopreservation tube sticks antifreeze bar-code label, carries out freezen protective, namely completes the structure in multistage Conspecific variant fat stem cell used storehouse.

Beneficial effect of the present invention:

1, cytoactive is destroyed little.Traditional fat acquisition mode is the vibrations assisted liposuction technology that vacuum tightness is greater than 750mmHg, large to the destruction of fatty tissue, fat stem cell in the fat gathered also is damaged, the motility rate of stem cell is generally lower than 40%, traditional liposuction is suitable for losing weight merely liposuction, requires then cannot meet to the re-using of fat.Therefore the fat stem cell quantity obtained in identical fatty volume is in the conventional way just few, and this technology adopts the water power liposuction technique of low vacuum, by vacuum degree control at 200-350mmHg, little to cytoclasis, overcomes the problems referred to above completely.

2, easy and simple to handle.The fat lump that general lipsuction and surgical operation are come is large, with more reticular tissue and vascular tissue, therefore stripping process will be carried out in early stage in operation, and present method is sieved through incipient cell, the tissue obtained exists to be dispersed in adipocyte substantially, does not need the stripping carrying out reticular tissue and blood vessel.

3, without Mechanical Crushing program.The fatty tissue that shatter value water power drawn in patent of the present invention is high has carried out organizing screening with screen cloth again, the distribution of particle sizes obtained is within the scope of 0.1-1mm, particularly be distributed within the scope of 0.1-0.5mm, be particularly suitable for next step fat stem cell separation test, this approach avoid the program adopting the machinery of larger fatty tissue traditional liposuction obtained to shred, namely simplify technique, additionally reduce histiocytic destructive process.

4, save time.Because cell mass is little, therefore the collagenase digesting process that the present invention adopts carries out can only adopting a kind of collagenase and concentration is very low, general enzymic digestion process adopts collagenase I, II, IV two or three to combine to digest, the concentration of lipase is also greater than 0.1%, digestion time is more than 1-2 hour, as long as the present invention is concentration 0.04-0.05% then, digestion time is also only at 5-20min, and the general 15min that adopts is just passable, therefore greatly provides cost savings and the time.

5, autologous stem cells storage is mainly carried out in the fat stem cell storehouse of forefathers.Autologous storage mainly uses to oneself, and the fat stem cell quantity of the amplification of needs is few, does not therefore relate to and sets up multilevel cell storehouse.Autologous storehouse is mainly personal from depositing.But there is the fat stem cell of a lot of situation oneself normally to obtain, as 1) there is liposuction psychological disorders as client, diabetes etc. cause wound-healing abilities poor, varix or phlebitis person, as skin has inflammation, allergy, local skin to have infection focus and more scar person, the main organs dysfunction persons such as cardiopulmonary, skin major relaxation and the very few person of subcutaneus adipose tissue, morbid obesity person or maternal obesity syndrome person cannot carry out liposuction procedures, these crowds are liposuction contraindication, obtain autologous fat stem cell just difficulty; 2) adopt the age of fat stem cell transplanting lower than the crowd of 18 years old for hope, because the needs of body development, be not suitable for carrying out liposuction procedures; 3) activity of fat stem cell, quantity and differentiation capability, nutrition adjustment and immunoregulation capability are all along with the change at age changes to some extent, therefore for the age more than 60 years old, when needing to carry out fat stem cell transplanting, the autologous fat stem cell difficulty secured good health; 4) pollution of day by day serious food problems, house ornamentation, virus infraction, Air haze, depletion of the ozone layer etc. cause radiation hazradial bundle day by day serious, these impacts healthy on people have influence on each organ, each tissue, each cell, and this also comprises fat stem cell.Therefore in the crowd seeming health, the gene of its fat stem cell and epigenetics quietly change, under this situation, adopts the fat stem cell reparation of oneself to transplant with regard to the fat stem cell supplied not as selection health of screening through strict age, genetic diseases, genovariation etc.

6, the liposuction versions building storehouse employing has multiple, what this patent adopted is the liposuction versions that can obtain the high fatty micelle of dispersity, the fat utilization rate obtained is high, even the fat stem cell storehouse setting up many person-portions does not need too many fat yet, the injury that causes donor is reduced to minimum.

7, adopt stem cell repeatedly to go down to posterity and to use to many people and there will be stem cell catabiosis, therefore in order to set up the stem cell bank that many person-portions allosome uses, multistage stem cell bank will be set up, this patent adopts two-stage cell bank method, the first step is master cell bank, props up P3 fat subsitutes stem cell form by 40-70.The second stage is working cardial cell storehouse, is made up of P5 fat subsitutes stem cell.After working cardial cell storehouse cell is finished, adopt 3 master cell bank cells from master cell bank, just can set up again the working cardial cell storehouse of a 60-90 part.Therefore from a health donors, the use of 800-2100 (40/3*60=800,70/3*90=2100) person-portion can just be given.

Accompanying drawing explanation

Fig. 1 is multistage fat stem cell storehouse Library development flow; Fig. 2 is frozen front fat mesenchymal stem cell aspect graph; Fig. 3 is that fat stem cell is divided into osteocyte; Fig. 4 is fat stem cell differentiating cartilage-forming cell; Fig. 5 is that fat stem cell is divided into adipocyte; Fig. 6 is the fat stem cell cell cycle after cultivating.

Embodiment

Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.

Embodiment one: the acquisition methods of present embodiment human adipose-derived stem cell, carry out according to the following steps:

One, the screening of donor:

Donor requires healthy, in donor blood, HCV, HBsAg, HBcAb, CMV, HIV, Syphilis, HLTV and EBV Viral diagnosis is negative, in blood, anerobe, aerophil, wide spectrum mycoplasma and fungal detection are feminine gender, get rid of have medical history, have familial inheritance medical history, have history of drug abuse, NIP, the patient that difficult without wound healing and compliance is poor, if donor is women, require that it is in non-pregnancy period and non-physiology phase state, donor to narcotic without anaphylaxis;

Two, fat acquisition:

Donor after screening step one carries out fat acquisition, fat acquisition employing suction strength is the water power liposuction technique of 200-350mmHg, after donor toponarcosis, the 1-2mm edge of a knife is cut at belly or thigh, inject 50-400mL inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction, the fat drawn directly is collected in the aseptic receptor with Screening Network, fatty tissue stops 5 minutes in the receiver, major part inflation fluid and the fat after screening are according to the different natural separation of its proportion, 50mL is adopted to be with cap Luer Lock syringe by the fatty tissue microballoon inhalation syringe of the 0.1-1mm diameter of acquisition,

Three, the transport of fat:

Fatty tissue step 2 collected adopts multi-functional fat acquisition, transport test kit transports, or the above-mentioned 50mL aseptic Luer Lock syringe having sucked fat is removed syringe needle, directly transports after being sealed by syringe with syringe cap;

Four, the separation of human adipose-derived stem cell:

In Biohazard Safety Equipment, fatty tissue is divided in 50mL centrifuge tube, in the centrifugal 10min of 1000-1500r/min, middle layer fatty tissue after centrifugal by pipette, extract shifts in new centrifuge tube, add equal-volume stroke-physiological saline solution or PBS cleaning, again in the centrifugal 10min of 1000-2000rpm, the upper-layer fat drawn after cleaning is placed in new centrifuge tube, add the type i collagen enzyme that mass percentage concentration is 0.03%-0.06%, lid is built in mixing, concussion digestion 15-30min in 37 DEG C of incubators, then PBS or physiological saline is added, and piping and druming tissue, cell suspension is filtered through 75 μm of aperture cellular filters, obtain single cell suspension, in the centrifugal 10min of 1000-2000rpm, obtain cell precipitation, add physiological saline or PBS relaunders cell once, centrifugal, precipitation is human adipose-derived stem cell.

Embodiment two: present embodiment and embodiment one unlike: the age of donor described in step one is 18-40 year.Other is identical with embodiment one.

Embodiment three: present embodiment and embodiment one or two unlike: in step 2, the suction strength of water power liposuction technique is 210-300mmHg.Other is identical with embodiment one or two.

Embodiment four: one of present embodiment and embodiment one to three unlike: the adipocyte motility rate in step 2 syringe is greater than 80%, and fat weight is 30-100 gram.Other is identical with one of embodiment one to three.

Embodiment five: one of present embodiment and embodiment one to four unlike: adopt multi-functional fat acquisition described in step 3, the specific operation process that transport test kit carries out transporting is: with aseptic 50mL syringe, fat is transferred to the fat acquisition that DMEM conserving liquid is housed and transport in bottle, transport bottle flexible rubber ring is fixed in transport test kit, test kit both sides are placed in the position of ice bag and are put into ice bag, transport test kit transport temperature is made to remain on 4-12 DEG C, fat sample was transported to clean plant and carries out fat stem cell separation in 1 hour.Other is identical with one of embodiment one to four.

Described multi-functional fat acquisition, transport test kit are open in patent 201320810155.2.

Embodiment six: one of present embodiment and embodiment one to five unlike: described in step 3 with syringe cap will syringe seal after carry out in the process of directly transporting, the cap of band cap syringe is vertically put down, inflation fluid residual in transportation and adipocyte are separated, and envrionment temperature residing for syringe is 4-12 DEG C.Other is identical with one of embodiment one to five.

Embodiment seven: the construction process in the multistage Conspecific variant fat stem cell used storehouse of present embodiment, carry out according to the following steps:

One, the screening of donor:

Donor requires healthy, in donor blood, HCV, HBsAg, HBcAb, CMV, HIV, Syphilis, HLTV and EBV Viral diagnosis is negative, in blood, anerobe, aerophil, wide spectrum mycoplasma and fungal detection are feminine gender, get rid of have medical history, have familial inheritance medical history, have history of drug abuse, NIP, the patient that difficult without wound healing and compliance is poor, if donor is women, require that it is in non-pregnancy period and non-physiology phase state, donor to narcotic without anaphylaxis; The age of described donor is 18-40 year;

Two, fat acquisition:

Donor after screening step one carries out fat acquisition, fat acquisition employing suction strength is the water power liposuction technique of 200-350mmHg, after donor toponarcosis, the 1-2mm edge of a knife is cut at belly or thigh, inject 50-400mL inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction, the fat drawn directly is collected in the aseptic receptor with Screening Network, fatty tissue stops 5 minutes in the receiver, major part inflation fluid and the fat after screening are according to the different natural separation of proportion, 50mL is adopted to be with cap Luer Lock syringe by the fatty tissue microballoon inhalation syringe of the 0.1-1mm diameter of acquisition, adipocyte motility rate in syringe is greater than 80%, fat weight is 30-100 gram,

Three, the transport of fat:

Fatty tissue step 2 collected adopts multi-functional fat acquisition, transport test kit transports, or the above-mentioned 50mL aseptic Luer Lock syringe having sucked fat is removed syringe needle, directly transports after being sealed by syringe with syringe cap;

The specific operation process that the multi-functional fat acquisition of described employing, transport test kit carry out transporting is: with aseptic 50mL syringe, fat is transferred to the fat acquisition that DMEM conserving liquid is housed and transport in bottle, transport bottle flexible rubber ring is fixed in transport test kit, test kit both sides are placed in the position of ice bag and are put into ice bag, make transport test kit transport temperature remain on 4-12 DEG C, fat sample was transported to clean plant and carries out fat stem cell separation in 1 hour;

Described syringe cap carries out in the process of directly transporting after being sealed by syringe, and vertically put down by the cap of band cap syringe, inflation fluid residual in transportation is separated with adipocyte, and envrionment temperature residing for syringe is 4-12 DEG C;

Four, the separation of human adipose-derived stem cell:

In Biohazard Safety Equipment, fatty tissue is divided in 50mL centrifuge tube, in the centrifugal 10min of 1000-1500r/min, middle layer fatty tissue after centrifugal by pipette, extract shifts in new centrifuge tube, add equal-volume stroke-physiological saline solution or PBS cleaning, again in the centrifugal 10min of 1000-2000rpm, the upper-layer fat drawn after cleaning is placed in new centrifuge tube, add the type i collagen enzyme that mass percentage concentration is 0.03%-0.06%, lid is built in mixing, concussion digestion 15-30min in 37 DEG C of incubators, then PBS or physiological saline is added, and piping and druming tissue, cell suspension is filtered through 75 μm of aperture cellular filters, obtain single cell suspension, in the centrifugal 10min of 1000-2000rpm, obtain cell precipitation, add physiological saline or PBS relaunders cell once, centrifugal, precipitation is human adipose-derived stem cell,

Five, fat stem cell is cultivated, goes down to posterity, is increased:

Cell counting is carried out, with inoculum density 3 × 10 to the human adipose-derived stem cell that step 4 obtains ⁴individual/cm ²-8 × 10 ⁴individual/cm ²be inoculated in culturing bottle and carry out original cuiture, at 37 DEG C, the CO of 5% ₂in incubator, fresh culture is changed every 2-3 days, after cytogamy degree reaches 70%-80%, adopt 1 × TrypLE Select in 37 DEG C of digestion 3 minutes, add equal-volume PBS or normal saline dilution, repeatedly blow and beat, cell is cleaned up, then the cell after cleaning is moved in 50mL centrifuge tube, centrifugal 5min, centrifugal speed is 1000-2000rpm, abandon supernatant liquor, cell is resuspended in new serum free medium, according to the ratio that goes down to posterity of 1:3-6, go down to posterity in new culturing bottle, serum free medium is adopted to carry out amplification P1 culture, amplification procedure changed fresh serum-free media every 2-3 days, after cytogamy degree reaches 70%-80%, so repeat above-mentioned cell dissociation succeeding generations, complete P2 and P3 for going down to posterity and amplification cultivation, after P3 reaches 70%-80% for cytogamy degree, for setting up master cell bank, original cuiture used medium is serum free medium or MesenGro substratum,

Six, master cell bank is set up:

P3 step 5 obtained adopts PBS or physiological saline cleaning for human adipose-derived stem cell, 1 × TrypLESelect37 DEG C is adopted to digest 3 minutes, add PBS or physiological saline, and blow and beat, collect the cell suspension after dispersion in 50mL centrifuge tube, get part and carry out cell counting, Cell viability mensuration, the centrifugal 5min of all the other cell suspensions, centrifugal speed is 1000-2000rpm, supernatant liquor is stayed to carry out anerobe, aerophil, mycoplasma, fungi, syphilis and Viral diagnosis, abandon unnecessary supernatant liquor, by cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is divided in the cryopreservation tube of 40-70 root 2mL, often pipe 1mL, and cryopreservation tube sticks antifreeze bar-code label, carries out freezen protective;

Seven, working cardial cell storehouse is set up:

From liquid nitrogen container, get master cell bank cryopreservation tube 2-3 prop up, after recovery is melted rapidly in 37 DEG C of water-baths, cell is transferred in serum free medium or MesenGro substratum, according to 0.5 × 10 ⁴individual/cm ²-2 × 10 ⁴individual/cm ²inoculum density be inoculated in culturing bottle and carry out P4 culture, at 37 DEG C, the CO of 5% ₂in incubator, serum free medium or MesenGro substratum is adopted to cultivate, fresh culture is changed every 2-3 days, after cytogamy degree reaches 70%-80%, adopt 1 × TrypLE Select in 37 DEG C of digestion 3 minutes, add equivalent PBS or normal saline dilution collects postdigestive cell in 50mL centrifuge tube, centrifugal 5min, centrifugal speed is 1000-2000rpm, abandon supernatant liquor, cell is resuspended in new serum free medium, according to the ratio that goes down to posterity of 1:3-6, go down to posterity in new culturing bottle, serum free medium is adopted to carry out amplification P5 culture, amplification procedure changed fresh serum-free media every 2-3 days, after cytogamy degree reaches 70-80%, carry out working cardial cell storehouse and set up work,

The P5 cultivated adopts PBS or physiological saline to wash away substratum for cell, adopt 1 × TrypLE Select37 DEG C digestion 3 minutes, add PBS or physiological saline, and blow and beat with transfer pipet, collect the cell suspension after dispersion in 50mL centrifuge tube, get part and carry out cell counting, Cell viability mensuration, the centrifugal 5min of all the other cell suspensions, centrifugal speed is 1000-2000rpm, supernatant liquor is stayed to carry out anerobe, aerophil, mycoplasma, fungi, syphilis and Viral diagnosis, abandon unnecessary supernatant liquor, by cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is divided in 60-90 and props up in the cryopreservation tube of 2mL, and often pipe 1mL, cryopreservation tube sticks antifreeze bar-code label, carries out freezen protective, namely completes the structure in multistage Conspecific variant fat stem cell used storehouse.

Serum free medium described in step 5 is buy the products C TS StemPro MSCXeno Free from Life Technologies company.Described MesenGro buys from StemRD company.

The fat stem cell in present embodiment master cell bank and working cardial cell storehouse needs to carry out anaerobic bacterium bacterium, aerobic bacteria, fungi, mycoplasma, hepatitis B virus, hepatitis C virus, virus of AIDS, cytomegalovirus, Epstein-Barr virus inspection and treponema pallidum detection before frozen, guarantees that the sample in cell bank does not have external microbial contamination; Before cell cryopreservation and recovery after carry out cytoactive detection, guarantee frozen after cell damaged.

To the qualification of fat stem cell, employing be frozen front fat stem cell surface marker to be detected and cell differentiation detects (comprising adipogenic differentiate ability, cartilage differentiation ability, bone cell differentiation ability three kinds).For the stability inspection of fat stem cell cell cycle, tiny electrolytic cell, karyotyping, be check before the practical application of working cardial cell storehouse is provided.

Embodiment eight: present embodiment and embodiment seven unlike: Viral diagnosis described in step 6 is hepatitis B, the third liver, AIDS, EB, CMV and HTLV detect.Other is identical with embodiment seven.

Embodiment nine: present embodiment and embodiment seven unlike: in step 6, the concrete grammar of freezen protective is: first cryopreservation tube is placed in 4 DEG C of refrigerators, balance 30min, set up procedure frigorimeter simultaneously, after instrument being cooled to 4 DEG C, the cell cryopreservation tube balanced is put into and carries out the frozen process of program,-11 DEG C are cooled to from 4 DEG C successively with-0.7 DEG C/min speed, be cooled to-60 DEG C with-25 DEG C/min from-11 DEG C and stop 1 minute, with+10 DEG C/min speed from-60 DEG C of rewarmings to-25 DEG C, and then be cooled to-45 DEG C with-0.7 DEG C/min speed from-25 DEG C,-60 DEG C are cooled to from-45 DEG C with-5 DEG C/min speed,-95 DEG C are cooled to from-60 DEG C with-10 DEG C/min speed, and at the freezing procedure of this temperature equilibrium 10min, finally sample is transferred in-196 DEG C of liquid nitrogen containers, preserve for a long time in the liquid nitrogen gas phase of-150 ~-180 DEG C.Other is identical with embodiment seven.

Embodiment ten: present embodiment and embodiment seven unlike: in step 7, the concrete grammar of freezen protective is: first cryopreservation tube is placed in 4 DEG C of refrigerators, balance 30min, set up procedure frigorimeter simultaneously, after instrument being cooled to 4 DEG C, the cell cryopreservation tube balanced being put into and carries out the frozen process of program.-11 DEG C are cooled to from 4 DEG C respectively with-0.7 DEG C/min speed, be cooled to-60 DEG C with-25 DEG C/min from-11 DEG C and stop 1 minute, with+10 DEG C/min speed from-60 DEG C of rewarmings to-25 DEG C, and then be cooled to-45 DEG C with-0.7 DEG C/min speed from-25 DEG C,-60 DEG C are cooled to from-45 DEG C with-5 DEG C/min speed,-95 DEG C are cooled to and at the freezing procedure of this temperature equilibrium 10min from-60 DEG C with-10 DEG C/min speed, finally sample is transferred in-196 DEG C of liquid nitrogen containers, preserves for a long time in the liquid nitrogen gas phase of-150 ~-180 DEG C.Other is identical with embodiment seven.

Experiment 1:

One, fat acquisition and transport: fat acquisition is carried out to the donor after screening, the liposuction suction strength of setting water power fat suction equipment is 300mmHg, under hydrodynamic promotion, slow aspirating adipose being collected under the guiding of vacuum piping by the fat of absorption all has in the fatty collector of screen cloth up and down, wherein fatty fiber block is removed by upper screen, major part inflation fluid is by the Screening Network elimination below collector, for the fatty tissue microballoon of to be the aseptic fatty micelle diameters being collected in fatty collector middle level the be 0.1-0.5mm that fat stem cell extracts, this middle level fat micelle 100 grams is drawn in 4 50mL syringes, remove syringe needle, be transported to stem cell after being sealed by syringe with syringe cap and prepare workshop.

Two, adipocyte motility rate: get 2mL fatty tissue micelle, adds the physiological saline 4mL containing collagenase I, and in physiological saline, the concentration of collagenase I is 0.04% (w/v), at 37 DEG C of 5%CO ₂concussion digestion 12min in incubator, after cell repeats to break up after cancellationization, the sieved filter of cell through 75 μm of apertures, obtain single cell suspension, microscopic examination after dyeing with trypan blue 1:1, record total cell count and blue dead cell number, calculate according to adipocyte motility rate=100% × (adipocyte sum-dead cell number)/adipocyte sum formula, adipocyte motility rate is 82%.

Three, fat stem cell is separated: in Biohazard Safety Equipment, inflation fluid after layering in 4 50mL syringes of the splendid attire of reception fat micelle is removed, with isopyknic brine fat micelle twice, each 5 minutes, lipid layer is transferred in new centrifuge tube after 1200rpm is centrifugal, add the physiological saline containing type i collagen enzyme, make the type i collagen enzyme concn in end reaction liquid be 0.05%, at 37 DEG C of 5%CO ₂concussion digestion 15min in incubator, postdigestive cell suspension is centrifugal 10min at 1500 rpm, discard the adipocyte on upper strata and the physiological saline in middle level, the stromal vascular fraction (SVF) being rich in fat stem cell of collecting precipitation, with trypan blue, cell counting is carried out to lipid substrate vascular component, with inoculum density 6 × 10 ⁴/ cm ²be inoculated in Tissue Culture Flask and carry out original cuiture.

Four, master cell bank cell cultures: at 37 DEG C, the CO of 5% ₂in incubator, serum free medium CTS StemProMSC Xeno Free is adopted to cultivate, a fresh culture is changed every 2 days, until after cell grows to 80% degrees of fusion, add 1 × TrypLE Select37 DEG C and carry out digestion 3 minutes, add equivalent PBS or normal saline dilution collects postdigestive cell in 50mL centrifuge tube, the centrifugal 10min of 1500rpm.Abandon supernatant liquor, cell be resuspended in new serum free medium, adopt platform phenol indigo plant to carry out total cellular score record and viable count record, according to 1:3-1:6 ratio and inoculum density 0.5 × 10 ⁴individual/cm ²p0 is cultivated in new multilayer culture flask for passage, add serum free medium and carry out amplification P1 culture, amplification procedure changed fresh serum-free media every 2 days, until after cell grows to 70% degrees of fusion, so repeat above-mentioned cell dissociation succeeding generations, order completes amplification, the Secondary Culture of P2, P3 fat subsitutes stem cell, carries out master cell bank and set up work after P3 fat subsitutes stem cell fusion degree reaches 80%.

Five, master cell bank is set up: the P3 of cultivation adopts physiological saline to wash twice removal remaining medium for cell, add 1 × TrypLE Select37 DEG C of digestion 2 minutes, depart from after bottom culturing bottle until stem cell globulate, add normal saline dilution Digestive system and softly blow and beat cell suspension with transfer pipet, collecting the single cell after dispersion in 50ml centrifuge tube.Get after 10 microlitres of cells mixs according to ratio and the platform phenol of 1:1 blue staining fluid, recording stem cell motility rate is 98.5%.Cell suspension is adopted 1200rpm centrifugation 5min, stay cell conditioned medium liquid to carry out the items such as anerobe, aerophil, mycoplasma, fungi, hepatitis B, the third liver, syphilis, virus of AIDS, cytomegalovirus, HLTV, Epstein-Barr virus and intracellular toxin and detect.Abandon unnecessary supernatant liquor, by stem cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is sub-packed in 65 2mL cryopreservation tubes, and often pipe 1mL, cryopreservation tube sticks antifreeze bar-code label.

First cell cryopreservation tube is placed in 4 DEG C of refrigerators, and balance 30min, set up procedure frigorimeter, after instrument being cooled to 4 DEG C, putting into the cell cryopreservation tube balanced and carry out the frozen process of program simultaneously.-11 DEG C are cooled to from 4 DEG C respectively with-0.7 DEG C/min speed, be cooled to-60 DEG C with-25 DEG C/min from-11 DEG C and stop 1 minute, with+10 DEG C/min speed from-60 DEG C of rewarmings to-25 DEG C, and then be cooled to-45 DEG C with-0.7 DEG C/min speed from-25 DEG C,-60 DEG C are cooled to from-45 DEG C with-5 DEG C/min speed,-95 DEG C are cooled to and at the freezing procedure of this temperature equilibrium 10min from-60 DEG C with-10 DEG C/min speed, finally sample is transferred in-196 DEG C of liquid nitrogen exhibition temporary storage tank, preserve in the liquid nitrogen gas phase of-150 ~-180 DEG C, after the detection of cell every quality index is qualified, adopt liquid nitrogen transfer pipeline that this master cell bank cell is transferred to the medium-term and long-term preservation of final nitrogen storage tank.

Six, working cardial cell storehouse cell cultures: get master cell bank cryopreservation tube 3 from liquid nitrogen container, rocks rapid cellular recovery in 37 DEG C of water-baths, just will melt P3 completely immediately and be transferred to MesenGro substratum for cell, according to inoculum density 1 × 10 ⁴individual/cm2 is inoculated in culturing bottle and carries out P4 culture.At 37 DEG C, the CO of 5% ₂in incubator, serum free medium MesenGro is adopted to cultivate, fresh culture is changed every 2 days, until after cell grows to 80% degrees of fusion, 1 × TrypLE Select37 DEG C is adopted to carry out digestion 3 minutes, add equivalent PBS dilution and collect postdigestive cell in 50mL centrifuge tube, centrifugal 5min, centrifugal speed is 1500rpm, abandon supernatant liquor, cell is resuspended in new serum free medium, go down to posterity according to the ratio of 1:4, go down to posterity in new multilayer culture flask, serum free medium is adopted to carry out amplification P5 for cell cultures, amplification procedure changed fresh serum-free media every 2 days, after cytogamy degree reaches and grows to 70%-80%, carry out working cardial cell storehouse and set up work.

Seven, working cardial cell storehouse is set up: the P5 of cultivation for cell in culturing bottle, adopt physiological saline to wash twice and wash away substratum, adopt 1 × TrypLE Select37 DEG C digestion 2.5 minutes, add physiological saline and transfer pipet is is softly blown and beaten, collect the single cell after dispersion in 50ml centrifuge tube, carry out the blue staining cell counting of platform phenol, Cell viability measures, centrifugal 5min, centrifugal speed is 1400rpm, cell conditioned medium liquid is stayed to carry out anerobe, aerophil, mycoplasma, the items such as fungi and virus detect, abandon unnecessary supernatant liquor, by cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is divided in 75 2mL cryopreservation tubes, and often pipe 1mL, cryopreservation tube sticks antifreeze bar-code label.

First cell cryopreservation tube is placed in 4 DEG C of refrigerators, and balance 30min, set up procedure frigorimeter, after instrument being cooled to 4 DEG C, putting into the cell cryopreservation tube balanced and carry out the frozen process of program simultaneously.-11 DEG C are cooled to from 4 DEG C respectively with-0.7 DEG C/min speed, be cooled to-60 DEG C with-25 DEG C/min from-11 DEG C and stop 1 minute, with+10 DEG C/min speed from-60 DEG C of rewarmings to-25 DEG C, and then be cooled to-45 DEG C with-0.7 DEG C/min speed from-25 DEG C,-60 DEG C are cooled to from-45 DEG C with-5 DEG C/min speed,-95 DEG C are cooled to and at the freezing procedure of this temperature equilibrium 10min from-60 DEG C with-10 DEG C/min speed, finally sample is transferred in-196 DEG C of liquid nitrogen containers, preserves for a long time in the liquid nitrogen gas phase of-150 ~-180 DEG C.The detection carried out is needed: aseptic, the virus-free detection of frozen front and back, the active and rear Activity determination of recovery before cell cryopreservation, intracellular toxin inspection, cell surface marker inspection, cell differentiation in process.

Eight, in master cell bank/working cardial cell storehouse, fat stem cell sample allogenic material checks: before P3 is frozen for master cell bank, gets postdigestive eccentric cell supernatant liquor 3mL, carry out microorganism detection.Wherein the inspection of aerobic/anaerobic bacterium, fungi adopts direct culture method, the items such as virus, cytomegalovirus, HLTV, the Epstein-Barr virus such as hepatitis B virus, hepatitis C virus, acquired immune deficiency syndrome (AIDS) detect and adopt ELISA method, wherein the third liver and AIDS add survey PCR method, treponema pallidum takes Virus monitory (TRUST) and gelatin aggegation to detect (TPPA) two detection methods respectively, mycoplasma adopts plate method and PCR method, and intracellular toxin adopts limulus reagent test.Detected result is as table 1:

Table 1

Nine, in master cell bank/working cardial cell storehouse, fat stem cell surface marker checks

The fat stem cell in P5 generation is suspended in PBS, makes concentration about 2.5 × 10 ⁵the cell suspension of individual cell/mL, in 9 test tubes, adds above-mentioned cell suspension 400 μ L (about 1 × 10 respectively ⁵individual cell), add following antibody FITC-IgG1, FITC-CD45, FITC-CD14, HLA-DR, PE-IgG1, PE-CD34, FITC-CD79a, FITC-IgG1, FITC-CD90, PE-IgG1, PE-CD73, PE-CD105 respectively again, room temperature lucifuge hatches dyeing in 30 minutes, PBS washs the cell after 2 dyeing, and guarantee to carry out surface antigen detection by Beckman Coulter XL in 1 hour, detected result is as shown in table 2.Table 2 result shows, P5 expresses the equal >95% of positive indication for master cell bank fat stem cell surface antigen, and the equal <2% of negative indication, meets the feature request of mescenchymal stem cell.

Table 2P5 is for master cell bank fat stem cell immunophenotype detected result

Fat stem cell is to bone differentiation test: by P3 fat subsitutes stem cell with 3 × 10 ⁴individual cell/cm ²density be inoculated in 6 of 24 orifice plates and be added with in the hole of circular slide glass, adopt CTS StemPro MSC Xeno Free substratum, at 37 DEG C, the CO of 5% ₂when being cultured to fat stem cell to 60% full scale in incubator, change 4 holes into StemPro Osteoblast Differentiation substratum, fresh Osteoblast Differentiation substratum is changed once every 4 days, other two apertures does not change substratum, as a control group, remove substratum after being cultured to 15 days, rinse 2 times with PBS, 4%PFA fixes 30min and then rinses 3 times with PBS.0.1% sodium alizarinsulfonate-Trish-HCl (pH8.3) 37 DEG C, 10min, PBS use glycogelatin mounting fluid-tight sheet after rinsing 2 times.

Osteoblast Differentiation test shows after induction, have obvious tubercle to produce in cell, redden through Alizarin red staining posterior tubercle, illustrates that the present invention cultivates and the fat stem cell stored maintains differentiation capability (Fig. 3) to osteocyte.

Fat stem cell is tested to cartilage differentiation: by P3 fat subsitutes stem cell with 2 × 10 ⁵individual/mL is inoculated in 24 porocyte culture plates, adopts CTSStemPro MSC Xeno Free substratum, at 37 DEG C, the CO of 5% ₂fat stem cell degrees of fusion is cultured to when reaching 60% in incubator, 4 holes are changed into StemPro and become cartilage differentiation substratum, fresh one-tenth cartilage differentiation substratum is changed once every 4 days, other two apertures does not change substratum, as a control group, removes substratum after being cultured to 14 days, 2 times are rinsed with PBS, 4%PFA fixes 30min and then rinses 3 times with PBS, and adopt Alcian indigo plant to dye to it, PBS uses glycogelatin mounting fluid-tight sheet after rinsing 2 times.Cartilage differentiation test is become to show, cell after chondrocyte induction differentiation, forms spherical agglomerates gradually, with the dyeing of Alcian indigo plant, cell mass au bleu, illustrates that the present invention cultivates and the fat stem cell stored maintains differentiation capability (Fig. 4) to chondrocyte.

Fat stem cell is tested to Adipose Differentiation: by P5 fat subsitutes stem cell with 4 × 10 ⁴individual cell/cm ²density be inoculated in 6 holes of 24 orifice plates, add MesenGro substratum and carry out cell cultures, when cytogamy degree reaches 80%, change 4 culture hole into StemPro adipogenic differentiate substratum, fresh adipogenic differentiate substratum is changed once every 3 days, other two apertures does not change substratum, as a control group.After being cultured to 14 days, oil red O stain after Virahol aquation 5min.Adipogenic differentiate test shows, after induction, occurs obvious oil droplet in cell, and after oil red O stain, oil droplet becomes obviously red, illustrates that the present invention cultivates and the fat stem cell stored maintains to Adipocyte Differentiation ability (Fig. 5).

Osteoblast Differentiation test shows, after induction, cell stops growing and starts agglomerate gradually after 5 days.

Fat stem cell stability test: a master cell bank fat stem cell of recovering, adopts serum free medium, after cultivating P4 generation, and collecting cell number about 1 × 10 ⁶individual cell is in 50mL centrifuge tube, and the centrifugal 6min of 1300r/min, discards substratum.3mL PBS re-suspended cell, once, 1300r/min is centrifugal, and 6min removes PBS in washing.Add 1mL PBS re-suspended cell, cell suspension is moved in 1.5mL EP pipe, the centrifugal 6min of 1200r/min.Add the ethanol fixed cell of 70% of 1mL precooling, fix 2 hours for 4 DEG C.Centrifugally discard stationary liquid, 1mL PBS washes twice, with the dyeing of 0.5mL CellSignaling PI dye liquor, and 4 DEG C of lucifuge 30min.Flow cytomery: PI argon ion fluorescence excitation, excitation light wave wavelength is 488nm, and utilizing emitted light wave-wave is grown up in 630nm, produce red fluorescence, analyze PI fluorescence intensity, remove cell debris from SS/FS scattered light point diagram, the cell that delineation will be analyzed is A door, analyzing DNA content; By the Beckman flow cytometry software analysis cell cycle, result judges: on the histogram of PI fluorescence, and occurred a diploid peak before the G1/G0 phase, the S phase is complete, has no heteroploid DNA (Fig. 6).Therefore prove, in the fat stem cell storehouse adopting the present invention to set up, fat stem cell is stablized, and phenomena of apoptosis does not occur.

Master cell bank, working cardial cell storehouse stem cell recovery Cell viability detect: from liquid nitrogen container, get 1 pipe work cell bank cell, after in 37 DEG C of water-baths, recovery is melted rapidly, after fat stem cell suspension 10 μ L in pipe is mixed with trypan blue 10 μ L, the total cell count of cell and blue dead cell number is recorded immediately under microscope, calculate according to fat stem cell motility rate=100% × (adipocyte sum-dead cell number)/fat stem cell sum formula, fat stem cell motility rate is 91.6%.

Fat stem cell karyotyping: the Metaphase Chromosomes sample of P5 fat subsitutes stem cell prepares colchicine process, the methyl alcohol of employing 10 μ g/mL: glacial acetic acid=3:1 twice is fixing, Giemsa dye liquor dyeing after film-making; Analyze under mirror, go out report according to international human cytogenetics naming system, no abnormal many times of karyomit(e)s, karyomit(e) insert and deficient phenomena.

Claims (10)

1. the acquisition methods of human adipose-derived stem cell, is characterized in that the method is carried out according to the following steps:

One, the screening of donor:

Donor requires healthy, in donor blood, HCV, HBsAg, HBcAb, CMV, HIV, Syphilis, HLTV and EBV Viral diagnosis is negative, in blood, anerobe, aerophil, wide spectrum mycoplasma and fungal detection are feminine gender, get rid of have medical history, have familial inheritance medical history, have history of drug abuse, NIP, the patient that difficult without wound healing and compliance is poor, if donor is women, require that it is in non-pregnancy period and non-physiology phase state, donor to narcotic without anaphylaxis;

Two, fat acquisition:

Donor after screening step one carries out fat acquisition, fat acquisition employing suction strength is the water power liposuction technique of 200-350mmHg, after donor toponarcosis, the 1-2mm edge of a knife is cut at belly or thigh, inject 50-400mL inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction, the fat drawn directly is collected in the aseptic receptor with Screening Network, fatty tissue stops 5 minutes in the receiver, major part inflation fluid and the fat after screening are according to the different natural separation of its proportion, 50mL is adopted to be with cap Luer Lock syringe by the fatty tissue microballoon inhalation syringe of the 0.1-1mm diameter of acquisition,

Three, the transport of fat:

Fatty tissue step 2 collected adopts multi-functional fat acquisition, transport test kit transports, or the above-mentioned 50mL aseptic Luer Lock syringe having sucked fat is removed syringe needle, directly transports after being sealed by syringe with syringe cap;

Four, the separation of human adipose-derived stem cell:

In Biohazard Safety Equipment, fatty tissue is divided in 50mL centrifuge tube, in the centrifugal 10min of 1000-1500r/min, middle layer fatty tissue after centrifugal by pipette, extract shifts in new centrifuge tube, add equal-volume stroke-physiological saline solution or PBS cleaning, again in the centrifugal 10min of 1000-2000rpm, the upper-layer fat drawn after cleaning is placed in new centrifuge tube, add the type i collagen enzyme that mass percentage concentration is 0.03%-0.06%, lid is built in mixing, concussion digestion 15-30min in 37 DEG C of incubators, then PBS or physiological saline is added, and piping and druming tissue, cell suspension is filtered through 75 μm of aperture cellular filters, obtain single cell suspension, in the centrifugal 10min of 1000-2000rpm, obtain cell precipitation, add physiological saline or PBS relaunders cell once, centrifugal, precipitation is human adipose-derived stem cell.

2. the acquisition methods of human adipose-derived stem cell according to claim 1, is characterized in that the age of donor described in step one is 18-40 year.

3. the acquisition methods of human adipose-derived stem cell according to claim 1, is characterized in that the suction strength of water power liposuction technique in step 2 is 210-300mmHg.

4. the acquisition methods of human adipose-derived stem cell according to claim 1, it is characterized in that the adipocyte motility rate in step 2 syringe is greater than 80%, fat weight is 30-100 gram.

5. the acquisition methods of human adipose-derived stem cell according to claim 1, it is characterized in that the specific operation process adopting multi-functional fat acquisition described in step 3, transport test kit carries out transporting is: with aseptic 50mL syringe, fat is transferred to the fat acquisition that DMEM conserving liquid is housed and transport in bottle, transport bottle flexible rubber ring is fixed in transport test kit, test kit both sides are placed in the position of ice bag and are put into ice bag, make transport test kit transport temperature remain on 4-12 DEG C, fat sample was transported to clean plant and carries out fat stem cell separation in 1 hour.

6. the acquisition methods of human adipose-derived stem cell according to claim 1, it is characterized in that being undertaken in the process of directly transporting with syringe cap by after syringe sealing described in step 3, the cap of band cap syringe is vertically put down, inflation fluid residual in transportation and adipocyte are separated, and envrionment temperature residing for syringe is 4-12 DEG C.

7. the construction process in multistage Conspecific variant fat stem cell used storehouse, is characterized in that the method is carried out according to the following steps:

One, the screening of donor:

Donor requires healthy, in donor blood, HCV, HBsAg, HBcAb, CMV, HIV, Syphilis, HLTV and EBV Viral diagnosis is negative, in blood, anerobe, aerophil, wide spectrum mycoplasma and fungal detection are feminine gender, get rid of have medical history, have familial inheritance medical history, have history of drug abuse, NIP, the patient that difficult without wound healing and compliance is poor, if donor is women, require that it is in non-pregnancy period and non-physiology phase state, donor to narcotic without anaphylaxis; The age of described donor is 18-40 year;

Two, fat acquisition:

Donor after screening step one carries out fat acquisition, fat acquisition employing suction strength is the water power liposuction technique of 200-350mmHg, after donor toponarcosis, the 1-2mm edge of a knife is cut at belly or thigh, inject 50-400mL inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction, the fat drawn directly is collected in the aseptic receptor with Screening Network, fatty tissue stops 5 minutes in the receiver, major part inflation fluid and the fat after screening are according to the different natural separation of proportion, 50mL is adopted to be with cap Luer Lock syringe by the fatty tissue microballoon inhalation syringe of the 0.1-1mm diameter of acquisition, adipocyte motility rate in syringe is greater than 80%, fat weight is 30-100 gram,

Three, the transport of fat:

Fatty tissue step 2 collected adopts multi-functional fat acquisition, transport test kit transports, or the above-mentioned 50mL aseptic Luer Lock syringe having sucked fat is removed syringe needle, directly transports after being sealed by syringe with syringe cap;

The specific operation process that the multi-functional fat acquisition of described employing, transport test kit carry out transporting is: with aseptic 50mL syringe, fat is transferred to the fat acquisition that DMEM conserving liquid is housed and transport in bottle, transport bottle flexible rubber ring is fixed in transport test kit, test kit both sides are placed in the position of ice bag and are put into ice bag, make transport test kit transport temperature remain on 4-12 DEG C, fat sample was transported to clean plant and carries out fat stem cell separation in 1 hour;

Described syringe cap carries out in the process of directly transporting after being sealed by syringe, and vertically put down by the cap of band cap syringe, inflation fluid residual in transportation is separated with adipocyte, and envrionment temperature residing for syringe is 4-12 DEG C;

Four, the separation of human adipose-derived stem cell:

In Biohazard Safety Equipment, fatty tissue is divided in 50mL centrifuge tube, in the centrifugal 10min of 1000-1500r/min, middle layer fatty tissue after centrifugal by pipette, extract shifts in new centrifuge tube, add equal-volume stroke-physiological saline solution or PBS cleaning, again in the centrifugal 10min of 1000-2000rpm, the upper-layer fat drawn after cleaning is placed in new centrifuge tube, add the type i collagen enzyme that mass percentage concentration is 0.03%-0.06%, lid is built in mixing, concussion digestion 15-30min in 37 DEG C of incubators, then PBS or physiological saline is added, and piping and druming tissue, cell suspension is filtered through 75 μm of aperture cellular filters, obtain single cell suspension, in the centrifugal 10min of 1000-2000rpm, obtain cell precipitation, add physiological saline or PBS relaunders cell once, centrifugal, precipitation is human adipose-derived stem cell,

Five, fat stem cell is cultivated, goes down to posterity, is increased:

Cell counting is carried out, with inoculum density 3 × 10 to the human adipose-derived stem cell that step 4 obtains ⁴individual/cm ²-8 × 10 ⁴individual/cm ²be inoculated in culturing bottle and carry out original cuiture, at 37 DEG C, the CO of 5% ₂in incubator, fresh culture is changed every 2-3 days, after cytogamy degree reaches 70%-80%, adopt 1 × TrypLESelect in 37 DEG C of digestion 3 minutes, add equal-volume PBS or normal saline dilution, repeatedly blow and beat, cell is cleaned up, then the cell after cleaning is moved in 50mL centrifuge tube, centrifugal 5min, centrifugal speed is 1000-2000rpm, abandon supernatant liquor, cell is resuspended in new serum free medium, according to the ratio that goes down to posterity of 1:3-6, go down to posterity in new culturing bottle, serum free medium is adopted to carry out amplification P1 culture, amplification procedure changed fresh serum-free media every 2-3 days, after cytogamy degree reaches 70%-80%, so repeat above-mentioned cell dissociation succeeding generations, complete P2 and P3 for going down to posterity and amplification cultivation, after P3 reaches 70%-80% for cytogamy degree, for setting up master cell bank, original cuiture used medium is serum free medium or MesenGro substratum,

Six, master cell bank is set up:

P3 step 5 obtained adopts PBS or physiological saline cleaning for human adipose-derived stem cell, 1 × TrypLESelect37 DEG C is adopted to digest 3 minutes, add PBS or physiological saline, and blow and beat, collect the cell suspension after dispersion in 50mL centrifuge tube, get part and carry out cell counting, Cell viability mensuration, the centrifugal 5min of all the other cell suspensions, centrifugal speed is 1000-2000rpm, supernatant liquor is stayed to carry out anerobe, aerophil, mycoplasma, fungi, syphilis and Viral diagnosis, abandon unnecessary supernatant liquor, by cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is divided in the cryopreservation tube of 40-70 root 2mL, often pipe 1mL, and cryopreservation tube sticks antifreeze bar-code label, carries out freezen protective;

Seven, working cardial cell storehouse is set up:

From liquid nitrogen container, get master cell bank cryopreservation tube 2-3 prop up, after recovery is melted rapidly in 37 DEG C of water-baths, cell is transferred in serum free medium or MesenGro substratum, according to 0.5 × 10 ⁴individual/cm ²-2 × 10 ⁴individual/cm ²inoculum density be inoculated in culturing bottle and carry out P4 culture, at 37 DEG C, the CO of 5% ₂in incubator, serum free medium or MesenGro substratum is adopted to cultivate, fresh culture is changed every 2-3 days, after cytogamy degree reaches 70%-80%, adopt 1 × TrypLE Select in 37 DEG C of digestion 3 minutes, add equivalent PBS or normal saline dilution collects postdigestive cell in 50mL centrifuge tube, centrifugal 5min, centrifugal speed is 1000-2000rpm, abandon supernatant liquor, cell is resuspended in new serum free medium, according to the ratio that goes down to posterity of 1:3-6, go down to posterity in new culturing bottle, serum free medium is adopted to carry out amplification P5 culture, amplification procedure changed fresh serum-free media every 2-3 days, after cytogamy degree reaches 70-80%, carry out working cardial cell storehouse and set up work,

The P5 cultivated adopts PBS or physiological saline to wash away substratum for cell, adopt 1 × TrypLE Select37 DEG C digestion 3 minutes, add PBS or physiological saline, and blow and beat with transfer pipet, collect the cell suspension after dispersion in 50mL centrifuge tube, get part and carry out cell counting, Cell viability mensuration, the centrifugal 5min of all the other cell suspensions, centrifugal speed is 1000-2000rpm, supernatant liquor is stayed to carry out anerobe, aerophil, mycoplasma, fungi, syphilis and Viral diagnosis, abandon unnecessary supernatant liquor, by cell according to cell ultimate density 2 × 10 ⁶individual/mL is resuspended in CryoStor CS10 frozen storing liquid, and is divided in 60-90 and props up in the cryopreservation tube of 2mL, and often pipe 1mL, cryopreservation tube sticks antifreeze bar-code label, carries out freezen protective, namely completes the structure in multistage Conspecific variant fat stem cell used storehouse.

8. the construction process in multistage Conspecific variant fat stem cell used storehouse according to claim 7, is characterized in that Viral diagnosis described in step 6 is hepatitis B, the third liver, AIDS, EB, CMV and HTLV detection.

9. the construction process in multistage Conspecific variant fat stem cell used storehouse according to claim 7, it is characterized in that the concrete grammar of freezen protective in step 6 is: first cryopreservation tube is placed in 4 DEG C of refrigerators, balance 30min, set up procedure frigorimeter simultaneously, after instrument being cooled to 4 DEG C, the cell cryopreservation tube balanced is put into, carry out the frozen process of program,-11 DEG C are cooled to from 4 DEG C successively with-0.7 DEG C/min speed, be cooled to-60 DEG C with-25 DEG C/min from-11 DEG C and stop 1 minute, with+10 DEG C/min speed from-60 DEG C of rewarmings to-25 DEG C, and then be cooled to-45 DEG C with-0.7 DEG C/min speed from-25 DEG C,-60 DEG C are cooled to from-45 DEG C with-5 DEG C/min speed,-95 DEG C are cooled to from-60 DEG C with-10 DEG C/min speed, and at the freezing procedure of this temperature equilibrium 10min, finally sample is transferred in-196 DEG C of liquid nitrogen containers, preserve for a long time in the liquid nitrogen gas phase of-150 ~-180 DEG C.

10. the construction process in multistage Conspecific variant fat stem cell used storehouse according to claim 7, it is characterized in that the concrete grammar of freezen protective in step 7 is: first cryopreservation tube is placed in 4 DEG C of refrigerators, balance 30min, set up procedure frigorimeter simultaneously, after instrument being cooled to 4 DEG C, the cell cryopreservation tube balanced being put into and carries out the frozen process of program.-11 DEG C are cooled to from 4 DEG C respectively with-0.7 DEG C/min speed, be cooled to-60 DEG C with-25 DEG C/min from-11 DEG C and stop 1 minute, with+10 DEG C/min speed from-60 DEG C of rewarmings to-25 DEG C, and then be cooled to-45 DEG C with-0.7 DEG C/min speed from-25 DEG C,-60 DEG C are cooled to from-45 DEG C with-5 DEG C/min speed,-95 DEG C are cooled to and at the freezing procedure of this temperature equilibrium 10min from-60 DEG C with-10 DEG C/min speed, finally sample is transferred in-196 DEG C of liquid nitrogen containers, preserves for a long time in the liquid nitrogen gas phase of-150 ~-180 DEG C.