A kind of fat stem cell extracting methodTechnical field
The invention belongs to stem cell extractive technique fields, and in particular to a kind of fat stem cell extracting method.
Background technique
Fat stem cell (adipose-derivedstemcells, ADSCs) is that a kind of separate from adipose tissue mentionsTake out can adherent growth, mesoderma origin with plasticity, adhesion and multi-lineage potential adult stem cell.ADSCs can be divided into derived from mesoblastic various kinds of cell type, including Skeletal Muscle Cell, cardiac muscle cell, cartilage cell, fatCell, osteoblast etc..It can additionally be divided into derived from ectodermic nerve cell and derived from the pancreatic cell of entoderm.ADSCs there may also be the generation for helping vascular endothelial cell and hematopoietic cell.Since adipose tissue largely exists in human body, materialsBe easy, a small amount of tissue can obtain a large amount of stem cells, can stable proliferation and decline rate is low in vitro, be suitable for large-scale culture,Small on the influence of gathered person's health, in addition negative pressure of vacuum liposuction has been mature shaping technique, therefore ADSCs is shownGone out other stem cells cannot and application prospect.
The extracting mode of adipose-derived stem cell mostly uses tissue stationary culture and enzyme digestion at present, tissue adherent method andEnzyme digestion is easy to operate, short processing time, but fatty oil droplet is easy to contact with cell culture bottom of bottle in operation, causesCell is not easy adherent, and is attached to cell culture bottom of bottle containing a large amount of red blood cell after extracting, causes fat stem cell without footEnough spaces are adherent, are proliferated, and then cause fat stem cell adherent rate and survival rate low.
Summary of the invention
The purpose of the present invention is to provide above-mentioned deficiency in the prior art is directed to, it is dry thin that the present invention provides a kind of fatBorn of the same parents' extracting method effectively solves fat stem cell during the extraction process since the presence of fatty oil droplet and red blood cell leads to cellThe low problem low with survival rate of adherent rate.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of fat stem cell extracting method, comprising the following steps:
(1) fat obtains: the sterile acquisition rouge out of the age 18 ~ 45 years old, volunteer's body of unsystematic disease, infectious disease and venereal diseaseFat tissue；
(2) it washs: taking 20 mL of adipose tissue of sterile acquisition, adipose tissue is resuspended with the cleaning solution of 1 ~ 2 times of volume, stand 5 ~10 min stay upper-layer fat tissue after layering, the cleaning solution for adding 1 ~ 2 times of volume is resuspended, and stands 5 ~ 10 min, abandon subnatantBody, 400 ~ 500 rpm are centrifuged 8 ~ 10 min after so washing repeatedly 5 ~ 8 times, abandon lower liquid；
(3) it digests: the digestive ferment enzyme solution of 1 ~ 2 times of volume is added, be placed in 37 DEG C, digest 30 ~ 60 in 150 ~ 200 rpm shaking tablesMin, 400 ~ 500 rpm are centrifuged 8 ~ 10 min, abandon supernatant fluid；
(4) clean: the physiological saline that 2 ~ 3 times of volumes are added is resuspended, and 400 ~ 500 rpm are centrifuged 8 ~ 10 min, abandons supernatant fluid；
(5) splitting erythrocyte: being added 1 ~ 2 mL erythrocyte cracked liquid, be incubated for 4 ~ 5 min after mixing on ice, vibrates every 1 minOnce；
(6) it terminates cracking: 10 ~ 20 mL PBS, 300 ~ 400 g is added and are centrifuged 5 ~ 8 min, abandon supernatant；
(7) it cultivates: 15 mL mesenchymal stem cell serum-free complete mediums is added and are resuspended, is transferred to Tissue Culture Flask, is placed in 37DEG C, 5% CO2Cell incubator culture；
(8) multiple cropping: after 48 h of culture, taking cell culture medium supernatant, be placed in sterile centrifugation tube, 400 ~ 500 rpm centrifugation 8 ~ 10Min, the centrifuge tube after taking out centrifugation, abandons supernatant, after the resuspension of 15 mL mesenchymal stem cell serum-free complete mediums is added, turnsIt moves in new Tissue Culture Flask, is put into cell incubator and continues to cultivate；
(9) it passes on: replacing mesenchymal stem cell serum-free complete medium every 3 d, it is long to Tissue Culture Flask area to cell60% when, with 10 mL brine 2 times, be added 5 mL pancreatin digestive juices digest 1 ~ 3 min after, be added 5 mL mesenchymasStem cell serum-free complete medium terminates digestion, collects cell after digestion, is P0 for cell, by 10000/cm2CellDensity is passed on, and is expanded to P6 generation.
(10) it identifies: carrying out the progress of surface marker expression using fat stem cell of the flow cytometer detection method to extractionDetection.
Cleaning solution is by 1 ~ 5 part of phosphatidyl choline, 1 ~ 5 part of NaTDC and 90 ~ 98 parts of physiological saline in above-mentioned steps (2)Composition.
Digestive ferment enzyme solution is by 0.1 ~ 0.2 part of collagenase type I powder, 99.8 ~ 99.9 parts of mesenchyma bases in above-mentioned steps (3)Basal culture medium composition.
The present invention has the advantages that
In 1 present invention, cell washing solution is mentioned by phosphatidyl choline, NaTDC and physiological saline composition in fat stem cellAdipose tissue is washed repeatedly before taking, can effectively remove fatty oil droplet；
In 2 present invention, digestive ferment is that collagenase type I powder is completely dissolved in mesenchyma basal medium, uses Millipore0.22 μm of sterilizing filter filtering.The effect of collagenase type I relatively mitigates, and the collagenous fibres in energy vitellophag interstitial are to dischargeCell, it is small to cellular damage, and the cell of separate out is present in mesenchyma basal medium, can utmostly keep cellActivity；
3 after cultivating 48 h, cell not adherent in Tissue Culture Flask supernatant is centrifuged, and abandons supernatant, is added new completeCulture medium gently blows and beats mixing, moves into new Tissue Culture Flask and continues to cultivate, and sees a large amount of cells after 24 h under the microscopeIt climbs out of.This step substantially increases the survival rate of cell, increases the growth rate of cell.
Detailed description of the invention
Fig. 1 fat stem cell P0 is for the cell state figure after multiple cropping when 3 d of culture.
Fig. 2 fat stem cell P0 be commissioned to train feeding 4 d when cell state figure.
Fig. 3 fat stem cell P1 be commissioned to train feeding 24 h when cell state figure.
Fig. 4 fat stem cell P3 be commissioned to train feeding 24 h when cell state figure.
Fig. 5 fat stem cell P6 be commissioned to train feeding 24 h when cell state figure.
Fig. 6 fat stem cell P3 is for flow cytometer detection result figure.A:CD90+；B:CD73+；C:CD105+；D:hMSC feminine gender is mixedClose object.
Specific embodiment
The present invention will be described in detail below with reference to specific embodiments: the present embodiment is based on the technical solution of the present inventionUnder tested, the detailed implementation method and specific operation process are given, but protection scope of the present invention be not limited to it is followingEmbodiment.
Agents useful for same is shown in Table 1 in specific embodiment.
Table 1
Embodiment 1
The implementation time of the present embodiment: on October 18th, 2018；
Place: Hai Xi Cellular Bioengineering Inc., Fujian Province experimental center；
Volunteer: Foochow Victoria's shaping and beauty hospital volunteer；
A kind of fat stem cell extracting method, comprising the following steps:
(1) fat obtains: the sterile acquisition fat out of the age 18 years old, volunteer's body of unsystematic disease, infectious disease and venereal diseaseTissue；
(2) it washs: taking 20 mL of adipose tissue of sterile acquisition, adipose tissue is resuspended with the cleaning solution of 1 times of volume, stands 10Min stays upper-layer fat tissue after layering, the cleaning solution for adding 1 times of volume is resuspended, and stands 10 min, abandons lower liquid, so400 rpm are centrifuged 10 min after repeated washing 8 times, abandon lower liquid；
(3) it digests: the digestive ferment enzyme solution of 1 times of volume is added, is placed in 37 DEG C, 60 min, 400 rpm is digested in 200 rpm shaking tables10 min are centrifuged, supernatant fluid is abandoned；
(4) clean: the physiological saline that 2 times of volumes are added is resuspended, and 400 rpm are centrifuged 10 min, abandons supernatant fluid；
(5) splitting erythrocyte: being added 1mL erythrocyte cracked liquid, be incubated for 5 min after mixing on ice, primary every 1 min oscillation；
(6) it terminates cracking: 10 mL PBS, 400 g is added and are centrifuged 8 min, abandon supernatant；
(7) it cultivates: 15 mL mesenchymal stem cell serum-free complete mediums is added and are resuspended, is transferred to Tissue Culture Flask, is placed in 37DEG C, 5% CO2Cell incubator culture；
(8) multiple cropping: after 48 h of culture, taking cell culture medium supernatant, be placed in sterile centrifugation tube, and 400 rpm are centrifuged 10 min, takeCentrifuge tube after being centrifuged out abandons supernatant, after the resuspension of 15 mL mesenchymal stem cell serum-free complete mediums is added, is transferred to newTissue Culture Flask in, be put into cell incubator and continue to cultivate；
(9) it passes on: replacing mesenchymal stem cell serum-free complete medium every 3 d, it is long to Tissue Culture Flask area to cell60% when, with 10 mL brine 2 times, be added after 5 mL pancreatin digest 1 min, 5 mL are added, and that mesenchyma is added is dry thinBorn of the same parents' Serum-free complete medium terminates digestion, collects cell after digestion, is P0 for cell, by 10000/cm2Cell densityIt is passed on, and is expanded to P6 generation.
(10) it identifies: carrying out the progress of surface marker expression using fat stem cell of the flow cytometer detection method to extractionDetect Fig. 6.
Above-mentioned cleaning solution is by 1 part of phosphatidyl choline, 1 part of NaTDC and 98 parts of physiological saline compositions.
Above-mentioned digestive ferment enzyme solution is by 0.1 part of collagenase type I powder, 99.9 parts of mesenchyma basal medium compositions.
The fat stem cell P0 that the present embodiment extracts is shown in that Fig. 1, cell are most for cell state when cultivating 3 d after multiple croppingRounded, shuttle shape, tissue block is relatively more at this time；Fat stem cell P0 be commissioned to train feeding 4 d when cell state see Fig. 2, cellMost rounded, shuttle shape；Fat stem cell P1 be commissioned to train feeding 24 h when cell state see Fig. 3, cell rounded, shuttle shape mostly,Number of dead cells is reduced, and impurity is few；Fat stem cell P3 be commissioned to train feeding 24 h when cell state see Fig. 4, cell is classicalMescenchymal stem cell form, dead cell is almost nil, and impurity is few；Fat stem cell P6 be commissioned to train feeding 24 h when cell state seeFig. 5, cell both ends are longer, and profile is more unintelligible, and a small amount of impurity occur, illustrate that fat stem cell tends to differentiation.
Embodiment 2
The implementation time of the present embodiment: on November 20th, 2018；
Place: Hai Xi Cellular Bioengineering Inc., Fujian Province experimental center；
Volunteer: Foochow Victoria's shaping and beauty hospital volunteer；
A kind of fat stem cell extracting method, comprising the following steps:
(1) fat obtains: the sterile acquisition fat out of the age 45 years old, volunteer's body of unsystematic disease, infectious disease and venereal diseaseTissue；
(2) it washs: taking 20 mL of adipose tissue of sterile acquisition, adipose tissue is resuspended with the cleaning solution of 2 times of volumes, stands 8Min abandons lower liquid, and the cleaning solution for adding 2 times of volumes is resuspended, and stands 8 min, abandons lower liquid, so repeated washing 5 times500 rpm are centrifuged 8 min afterwards, abandon lower liquid；
(3) digest: the digestive ferment enzyme solution of 2 times of volumes be added, is placed in 37 DEG C, digests 30min in 200 rpm shaking tables, 500 rpm fromHeart 8min abandons supernatant fluid；
(4) clean: the physiological saline that 3 times of volumes are added is resuspended, and 500 rpm are centrifuged 8 min, abandons supernatant fluid；
(5) splitting erythrocyte: being added 2 mL erythrocyte cracked liquids, be incubated for 4min after mixing on ice, primary every 1 min oscillation；
(6) it terminates cracking: 20 mL PBS, 400 g is added and are centrifuged 5 min, abandon supernatant；
(7) it cultivates: 15 mL mesenchymal stem cell serum-free complete mediums is added and are resuspended, are transferred to Tissue Culture Flask, it is clear completeCulture medium is placed in 37 DEG C, 5% CO2Cell incubator culture；
(8) multiple cropping: after 48 h of culture, taking cell culture medium supernatant, be placed in sterile centrifugation tube, and 500 rpm are centrifuged 8min, take outCentrifuge tube after centrifugation abandons supernatant, after the resuspension of 15 mL mesenchymal stem cell serum-free complete mediums is added, is transferred to newIn Tissue Culture Flask, it is put into cell incubator and continues to cultivate.
(9) it passes on: replacing mesenchymal stem cell serum-free complete medium every 3 d, it is long to Tissue Culture Flask to cellArea 60% when, with 10 mL brine 2 times, be added 5 mL pancreatin digest 1.5 min after, be added 5 mL be added betweenMesenchymal stem cells Serum-free complete medium terminates digestion, collects cell after digestion, is P0 for cell, by 10000/cm2'sCell density is passed on, and is expanded to P6 generation.
(10) it identifies: carrying out the progress of surface marker expression using fat stem cell of the flow cytometer detection method to extractionDetection.
Above-mentioned cleaning solution is by 5 parts of phosphatidyl cholines, 5 parts of NaTDCs and 90 parts of physiological saline compositions.
Above-mentioned digestive ferment enzyme solution is by 0.2 part of collagenase type I powder, 99.8 parts of mesenchyma basal medium compositions.
Embodiment 3
The implementation time of the present embodiment: on December 11st, 2018；
Place: Hai Xi Cellular Bioengineering Inc., Fujian Province experimental center；
Volunteer: Foochow Victoria's shaping and beauty hospital volunteer；
A kind of fat stem cell extracting method, comprising the following steps:
(1) fat obtains: the sterile acquisition fat out of the age 30 years old, volunteer's body of unsystematic disease, infectious disease and venereal diseaseTissue；
(2) it washs: taking 20 mL of adipose tissue of sterile acquisition, adipose tissue is resuspended with the cleaning solution of 1.5 times of volumes, stands 5Min abandons lower liquid, and the cleaning solution for adding 1.5 times of volumes is resuspended, and stands 5 min, abandons lower liquid, so washes repeatedly 79 min are centrifuged all over rear 450 rpm, abandon lower liquid；
(3) it digests: the digestive ferment enzyme solution of 1.5 times of volumes is added, is placed in 37 DEG C, digests 45 min in 200 rpm shaking tables, 450Rpm is centrifuged 9 min, abandons supernatant fluid；
(4) clean: the physiological saline that 2.5 times of volumes are added is resuspended, and 450 rpm are centrifuged 9min, abandons supernatant fluid；
(5) splitting erythrocyte: being added 1.5 mL erythrocyte cracked liquids, be incubated for 5 min after mixing on ice, vibrates one every 1 minIt is secondary；
(6) it terminates cracking: 15 mL PBS, 350 g is added and are centrifuged 7 min, abandon supernatant；
(7) it cultivates: 15 mL mesenchymal stem cell serum-free complete mediums is added and are resuspended, is transferred to Tissue Culture Flask, is placed in 37DEG C, 5% CO2Cell incubator culture；
(8) multiple cropping: after 48 h of culture, taking cell culture medium supernatant, be placed in sterile centrifugation tube, and 450 rpm are centrifuged 9 min, takeCentrifuge tube after being centrifuged out abandons supernatant, after the resuspension of 15 mL mesenchymal stem cell serum-free complete mediums is added, is transferred to newTissue Culture Flask in, be put into cell incubator and continue to cultivate；
(9) it passes on: replacing mesenchymal stem cell serum-free complete medium every 3 d, it is long to Tissue Culture Flask area to cell60% when, with 10 mL brine 2 times, be added after 5 mL pancreatin digest 2 min, 5 mL are added, and that mesenchyma is added is dry thinBorn of the same parents' Serum-free complete medium terminates digestion, collects cell after digestion, is P0 for cell, by 10000/cm2Cell densityIt is passed on, and is expanded to P6 generation.
(10) it identifies: carrying out the progress of surface marker expression using fat stem cell of the flow cytometer detection method to extractionDetection.
Above-mentioned cleaning solution is by 3 parts of phosphatidyl cholines, 3 parts of NaTDCs and 94 parts of physiological saline compositions.
Above-mentioned digestive ferment enzyme solution is by 0.15 part of collagenase type I powder, 99.85 parts of mesenchyma basal medium compositions.
Comparative example 1
(1) fat obtains: the sterile acquisition fat out of the age 18 years old, volunteer's body of unsystematic disease, infectious disease and venereal diseaseTissue；
(2) it washs: taking 20 mL of adipose tissue of sterile acquisition, adipose tissue is resuspended with the physiological saline of 1 times of volume, stands 10Min abandons lower liquid, and the cleaning solution for adding 2 times of volumes is resuspended, and stands 10 min, abandons lower liquid, so washes repeatedly 810 min are centrifuged all over rear 400 rpm, abandon lower liquid；
(3) it cultivates: 15 mL mesenchymal stem cell serum-free complete mediums is added and are resuspended, is transferred to Tissue Culture Flask, is placed in 37DEG C, 5% CO2Cell incubator culture；
(4) pass on: replacing mesenchymal stem cell serum-free complete medium every 3 d, when cell it is long to 60% when, it is raw with 10 mLIt is whole that 5 mL mesenchymal stem cell serum-free complete mediums are added after 5 mL pancreatin digestion, 2 min are added in reason salt water washing 2 timesOnly digest.Cell after cancellationization, by 10000/cm2Cell density passed on, and be expanded to P6 generation.
(5) it identifies: carrying out the progress of surface marker expression using fat stem cell of the flow cytometer detection method to extractionDetection.
Testing result:
The cultivated days of P0 in embodiment 1 ~ 3 and comparative example 1 are recorded respectively, and see Table 2 for details；P0 generation is counted, P3 generation and P6 are for cellQuantity and survival rate, see Table 3 for details.
Table 2
Table 3
Known by table 4, extracts fat stem cell according to the method for embodiment 1 ~ 3, the cultivated days in P0 generation are considerably less than in comparative exampleThe cultivated days in P0 generation illustrate that the present invention can be shortened the incubation time in P0 generation.
Known by table 5, according to the method for embodiment 1 ~ 3, the fat stem cell of extraction, cell quantity is more and survival rate is high, saysThe bright present invention can improve the recovery rate and survival rate of fat stem cell.
The P0 generation of embodiment 1 ~ 3 and comparative example 1, P3 generation and P6 are collected for cell, PBS is washed 2 times, adjusts cell concentration systemAt cell suspension, cell suspension is respectively added in label 1 ~ 6 pipe, makes the cell 1 × 10 of every pipe6A, pipe 1 plus 5 μ L mouse are anti-humanCD90-FITC, pipe 2 plus the anti-human CD105-PerCP-Cy 5.5 of 5 μ L mouse, pipe 3 plus the anti-human CD73-APC of 5 μ L mouse, pipe 4 are not addedAntibody, pipe 5 plus 10 μ L hMSC positive Isotype control mixtures and 10 μ LPE hMSC Negative isotype control mixtures, pipe 6 add10 μ L hMSC positive mixtures and 10 μ LPE hMSC feminine gender mixture room temperatures, which are protected from light, is incubated for 30 min, washes two repeatedly with PBSThree times, non-specific binding is reduced；300 μ LPBS are added and mix cell, flow cytometer detects CD90+、CD73+、CD105+With hMSC feminine gender mixture C D45+、CD34+、CD11b+、CD19+、HLA-DR+The expression of equal major surfaces marker, whereinCD90+、CD73+、CD105+Expression quantity answers >=95%, CD45+、CD34+、CD11b+、CD19+、HLA-DR+Expression quantity answers≤2%, in detailIt is shown in Table 4.1 fat stem cell P3 of embodiment is shown in Fig. 6 for flow cytometer detection result.
Table 4
Known by table 4, according to the method for embodiment 1 ~ 3, cell obtained, streaming phenotype is stable, and surface marker value is equalIt complies with standard, the fat stem cell for illustrating that the present invention extracts can satisfy clinical application demand.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent withModification, is all covered by the present invention.