CN103667187B - A kind of isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank - Google Patents
A kind of isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank Download PDFInfo
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Abstract
The invention discloses a kind of isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank, comprise the following steps: (1) gathers human fat tissue; (2) obtain and separate human adipose-derived stem cell; (3) stem cell is cultivated; (4) detection, frozen; (5) build storehouse. The present invention adopts the epoxy glue protoenzyme that contains I type and VI Collagenase Type of D-Hanks balanced salt solution preparation to digest adipose tissue, makes the more thorough of tissue digestion, obviously reduces heteroproteose cell quantity and removes tissue block; With containing 10-15% hyclone, 103-105The MCDB-201 nutrient solution of U/mlLIF is cultivated the stem cell after separating, cell proliferation is fast, form good, can effectively suppress the differentiation of stem cell, ensure the characteristic of primary stem cell, can also promote the long term growth of human adipose-derived stem cell, and keep the features such as self and multi-lineage potential simultaneously; The stem cell obtaining is built to storehouse and preserve, ensured the more seed cell source of high-quality.
Description
Technical field
The present invention relates to the isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank thereof, belong to doctorLearn and biology techniques field.
Background technology
Stem-cell research is one of the most noticeable focus in medical science and field of biology in recent years. Stem cell isOne class has the cell of self and differentiation potential. By studying their differentiation potential, amplification ability, pointCell function after change and the problem such as safe ready of whether drawing materials, select most suitable stem cell source to carry out groupKnit cytothesis reparation. Human adipose-derived stem cell (adipose-derivedstemcells, ADSCs) is nearIn Nian Laicong human fatty tissue, separate a kind of stem cell with multi-lineage potential obtaining. Fat is found in researchFat stem cell can be divided into Gegenbaur's cell, cartilage cell, cardiac muscle cell, adipocyte, god under given conditionsThrough cell etc., there is lower immunogenicity and immunoloregulation function simultaneously, transplant allogeneADSCs will can not cause strong immune response and the rejection in later stage, is the homology heteroplastic transplantation of ADSCsAdvantage is provided. In addition, ADSCs wide material sources, can by liposuction or adipectomy fromIn any human body, obtain, safe no pain, and cultivate and stablize in vitro, amplification rate is fast, is difficult for old and feeble. CauseThis, ADSCs becomes the new focus of current research, no matter all have aspect regenerative medicine or gene therapy heavilyLarge application potential.
The liposuction procedures that people are undertaken by beauty treatment, hyperadiposity or other reasons or adipectomy, can not onlyEnough discarded adipose tissue after operation is separated and obtained required fat stem cell by extraction, reach treatment variousThe object of disease, can also reach by liposuction or lipectomy the effect of fat-reducing.
That Chinese patent ZL201010120944.4 discloses is a kind of " acquisition methods of people's adipose-derived adult stem cell andThe construction method of this stem cell bank ", facilitate people effectively to store and use fat stem cell resource, this patentMiddle employing NTx enzyme digests adipose tissue, and digestion product strainer filtering is removed and do not digested tissue block,Centrifugal rear with DMEM complete culture solution re-suspended cell, be finally inoculated in Tissue Culture Flask and cultivate, after 4-5 daysChange fresh medium, had digestive transfer culture after cell covers with; Chinese patent application CN201210018269.3 forExisting cell isolation method has been done further improvement, adopts the epoxy glue protoenzyme of I type, II type familial combined hyperlipidemia to enterRow digestion, although the method increases digestion effect, can not remove tissue block and a large amount of assorted completelyCell; In addition cell is seeded to while cultivation in blake bottle, the nutrient solution of use is preserved less stable, and cell increasesGrow lowlyer, have certain differentiation, cause the purity of cell in frozen stem cell bank still influenced.
For overcoming the deficiency of above-mentioned technology, we further improve and have optimized fatty dry in practical operationThe isolated culture method of cell, makes the more thorough of tissue digestion, obviously reduces heteroproteose cell quantity and removes tissuePiece, the culture medium of employing has that cell proliferation is fast, form good, effectively suppresses the differentiation of stem cell, ensures primaryThe characteristic of stem cell, can also promote the long term growth of human adipose-derived stem cell simultaneously, and keeps self and manyTo features such as differentiation potentials, thereby ensure the more seed cell source of high-quality.
Summary of the invention
The object of the invention is to overcome the deficiency of above separation and extraction technology, and a kind of people's fat providing is dry thinBorn of the same parents' isolated culture method, its have easy and simple to handle, have a extensive future, can be clinical and research and provide a large amount ofThe feature of stem cell resource.
In the isolated culture method of human adipose-derived stem cell provided by the invention, it adopts D-Hanks balanced salt solutionThe epoxy glue protoenzyme of preparation digests adipose tissue, and described epoxy glue protoenzyme is by NTx enzyme familial combined hyperlipidemia glueProtoenzyme composition.
1% mother liquor compound method of described epoxy glue protoenzyme is: in 100mlD-Hanks balanced salt solution, add0.7g NTx enzyme, 0.3g type Ⅳ collagenase.
Using the final concentration while mixing collagenase digesting adipose tissue is 0.1%-0.2% (m/v). Preferably final concentrationBe 0.1% (m/v).
The fat stem cell that obtains of digestion will be containing will after cultivating 1-2 days in the MCDB-201 nutrient solution of hycloneOriginal nutrient solution is inhaled and is abandoned, and changes fresh medium, continues again to change fresh medium after cultivation 24h.
Also contain LIF described containing in the nutrient solution of hyclone.
Specifically, described nutrient solution is to contain 10-15% hyclone, 103-105The MCDB-201 of U/mlLIFNutrient solution.
Preferably, nutrient solution is to contain 12% hyclone, 103The MCDB-201 nutrient solution of U/mlLIF.
For achieving the above object, the present invention has adopted following technical scheme:
An isolated culture method for human adipose-derived stem cell, comprises the following steps:
(1) gather human fat tissue;
(2) obtain and separate human adipose-derived stem cell: get the adipose tissue of collection, in rejecting fat, naked eyes are visibleBlood vessel and pars fibrosa, shred into 1-2mm3Size tissue block, the D-Hanks that is 7.2-7.4 by pH valueBalanced salt solution washing is repeatedly limpid to eluate, removes residual blood, adds with adipose tissue isopyknic0.2-0.4%(m/v) epoxy glue protoenzyme, under 37 ° of C temperature conditions, evenly concussion digests 40-80 minute, thenBe placed in centrifuge with the centrifugal 4-10 minute of rotating speed of 1600-1800 rev/min, remove external fat, willThe D-Hanks balanced salt solution that bottom cell is 7.2-7.4 by pH value cleans repeatedly, the liquid washing downFilter through 100 eye mesh screens, remove digestion tissue, by filtrate with 1200 revs/min of centrifugal 5-10 minute,Abandoning supernatant, obtains stem cell;
(3) stem cell cultivate: by obtain stem cell according to 2-3 × 104/cm2Density be inoculated in blake bottle,Add 10mL to contain the nutrient solution of hyclone, be placed in 37 DEG C, CO2In the incubator of concentration 5%, cultivate,After 1-2 days, original fluid is inhaled and is abandoned, change the fresh nutrient solution containing hyclone, discard non-adherent cell,Within every 24 hours later, change nutrient solution once, treat that Growth of Cells reaches 80% fusion, adds 0.25% in blake bottlePancreas enzyme-EDTA digests, and goes down to posterity by 1:3, obtains the human adipose-derived stem cell going down to posterity after cultivating;
(4) detect, frozen: detect and frozenly carry out simultaneously, in cell cryopreservation process, carrying out immunophenotypeDetection, cell count and vitality test, biological efficacy detection, the detection of Cell Differentiation ability etc. are to determine that it isThe no criterion of acceptability that reaches; Frozen step is: in the people's adipose-derived adult stem cell obtaining, add hyclone, thanExample stem cell number/hyclone is 1-2 × 106Cell/0.9mL, fully mixes, stand-by; At cryovial and coldFreeze on box and encode; Above-mentioned people's adipose-derived adult stem cell suspension and cryopreserving liquid are joined freezing according to 1:1 ratioIn pipe, mix, capping, concussion, fully mixes, and is moved into rapidly in-80 ° of C refrigerators; After 24 hours, pressBe put into and on corresponding freezing frame, move into the medium-term and long-term preservation of liquid nitrogen according to numbering, result to be detected out after, will detect notQualified cell is together discarded together with cryopreservation tube by corresponding freeze-stored cell according to its numbering.
In described step (1), human fat tissue is the discarded object obtaining in human body liposuction procedures, gathers to peopleBody belly, thigh or subcutaneous fat.
Another object of the present invention is to provide the construction method in above-mentioned human adipose-derived stem cell storehouse. Being convenient to people effectively stores upDeposit and use stem cell resource.
Construction method comprises the following steps:
(1) gather human fat tissue;
(2) obtain and separate human adipose-derived stem cell: get the adipose tissue of collection, in rejecting fat, naked eyes are visibleBlood vessel and pars fibrosa, shred into 1-2mm3Size tissue block, the DHanks that is 7.2-7.4 by pH valueBalanced salt solution washing is repeatedly limpid to eluate, removes residual blood, adds with adipose tissue isopyknic0.2-0.4%(m/v) epoxy glue protoenzyme, under 37 ° of C temperature conditions, evenly concussion digests 40-80 minute, thenBe placed in centrifuge with the centrifugal 4-10 minute of rotating speed of 1600-1800 rev/min, remove external fat, willThe D-Hanks balanced salt solution that bottom cell is 7.2-7.4 by pH value cleans repeatedly, the liquid washing downFilter through 100 eye mesh screens, remove digestion tissue, by filtrate with 1200 revs/min of centrifugal 5-10 minute,Abandoning supernatant, obtains stem cell;
(3) stem cell cultivate: by obtain stem cell according to 2-3 × 104/cm2Density be inoculated in blake bottle,Add 10mL to contain 10-15% hyclone, 103-105The MCDB-201 nutrient solution of U/mlLIF, is placed in37℃、CO2In the incubator of concentration 5%, cultivate, after 1-2 days, original fluid is inhaled and abandoned, change freshContaining the nutrient solution of hyclone, discard non-adherent cell, within later every 24 hours, change nutrient solution once, treat thinIntracellular growth reaches 80% fusion, adds 0.25% pancreas enzyme-EDTA and digest in blake bottle, go down to posterity by 1:3,Acquisition go down to posterity cultivate after human adipose-derived stem cell;
(4) detect, frozen: detect and frozenly carry out simultaneously, in cell cryopreservation process, carrying out immunophenotypeDetection, cell count and vitality test, biological efficacy detection, the detection of Cell Differentiation ability etc. are to determine that it isThe no criterion of acceptability that reaches; Frozen step is: in the people's adipose-derived adult stem cell obtaining, add hyclone, thanExample stem cell number/hyclone is 1-2 × 106Cell/0.9mL, fully mixes, stand-by; At cryovial and coldFreeze on box and encode; Above-mentioned people's adipose-derived adult stem cell suspension and cryopreserving liquid are joined freezing according to 1:1 ratioIn pipe, mix, capping, concussion, fully mixes, and is moved into rapidly in-80 ° of C refrigerators; After 24 hours, pressBe put into and on corresponding freezing frame, move into the medium-term and long-term preservation of liquid nitrogen according to numbering, result to be detected out after, will detect notQualified cell is together discarded together with cryopreservation tube by corresponding freeze-stored cell according to its numbering;
(5) build storehouse: by obtain every a human adipose-derived stem cell by donor name, ID card No. and storageDate preserves respectively, sets up the computer data of the detailed archives of every a stem cell, sets up human adipose-derived stem cellStorehouse.
Below, by test example, further set forth the beneficial effect of isolated culture method of the present invention:
Test example 1, the research of human adipose-derived stem cell extraction separation method
Adopt respectively following methods to digest the human fat tissue collecting, the composition of research digestive juice and denseThe impact of degree on cell separation, is then seeded to postdigestive cell in Tissue Culture Flask and cultivates, incubationIn cell state is carried out to record:
Method 1, according to the isolated culture method of embodiment 1-3, digestive juice mother liquor is to use 100mlDHanksIn balanced salt solution, add the method for 0.7g NTx enzyme, 0.3g type Ⅳ collagenase to obtain, select epoxy glueThe final concentration of protoenzyme is respectively 0.05%, 0.1%, 0.15%, 0.2%, 0.25%;
Method 2, according to the method for patent ZL201010120944.4 embodiment 1, the I that digestive juice is 0.2%Collagenase Type phosphate buffer, final concentration is 0.1%;
Method 3, according to the side of patent application CN201210018269.3 Instructions Page 2 preferred embodimentMethod, digestive juice mother liquor is with adding 0.5g NTx enzyme, 0.25g in 100mlD-Hanks balanced salt solutionThe method of II Collagenase Type, 0.25g type Ⅳ collagenase obtains, and epoxy glue protoenzyme final concentration is 0.15%-0.2%;
Method 4, according to the isolated culture method of embodiment 1-3, digestive juice mother liquor is to use 100mlD-HanksIn balanced salt solution, add the method for 0.6g NTx enzyme, 0.4g type Ⅳ collagenase to obtain, epoxy glue protoenzymeFinal concentration is 0.15%;
Method 5, according to the isolated culture method of embodiment 1-3, digestive juice mother liquor is to use 100mlD-HanksIn balanced salt solution, add the method for 0.5g NTx enzyme, 0.5g type Ⅳ collagenase to obtain, epoxy glue protoenzymeFinal concentration is 0.15%;
Method 6, according to the isolated culture method of embodiment 1-3, digestive juice mother liquor is to use 100mlD-HanksIn balanced salt solution, add the method for 0.8g NTx enzyme, 0.2g type Ⅳ collagenase to obtain, epoxy glue protoenzymeFinal concentration is 0.15%.
The stem cell that above-mentioned digestion method is obtained is cultivated, and P0-2 is carried out to Continuous Observation for cell, knotReally as following table:
The impact that the different digestive juices of table 1 separate fat stem cell
Above-mentioned test can be found out, adopts digestive juice isolated adipose tissue of the present invention, obtains than additive methodStem cell growth speed is fast, and it is more thorough that adipose tissue digests, and obviously reduce heteroproteose cell quantity and remove tissue block,In its epoxy glue protoenzyme, the proportioning of each composition is appropriate, and in ratio method 4-6, the consumption of clostridiopetidase A is better; In addition, originallyThe final concentration of invention epoxy glue protoenzyme is that 0.1%-0.25% all can be effective, and considers large-scale production cost,Preferably final concentration is 1%.
Test example 2, vitro human fat stem cell culture studies
Adopt the cultural method of 1-3 of the present invention, select respectively following culture medium to cultivate: nutrient solution A, contain10%-15% hyclone, 103-105The MCDB-201 nutrient solution of U/mlLIF;
Nutrient solution B, containing the MCDB-201 nutrient solution of 10% hyclone;
Nutrient solution C, containing the low sugar DMEM nutrient solution of 10% hyclone; ;
Nutrient solution D, containing the DMEM in high glucose nutrient solution of 15% hyclone;
Result shows that cell is inoculated in nutrient solution A, and after inoculation, 4-7 days primary cells reach 80% fusion, haveTwo kinds of forms of endothelioid cells core fibroblast-like cells; 1st generation cell is cultivated 1-2 days, reaches 80% fusion, thinBorn of the same parents are fusiformis substantially, and only a few is endothelial cell sample form, and fusicellular percentage composition is greater than 97%; The 2ndCultivate 24-36 hour for cell, reach 80%-85% and merge, fusicellular percentage composition is greater than 99%; WhereinTaking hyclone content as 12%, LIF is as 103The effect of U/ml is best, and P2 is for rear fusicellular percentageContent is about 99.5%. Through qualification, this spindle cell is human adipose-derived stem cell.
Result shows that cell is inoculated in nutrient solution B, and after inoculation, 5-8 days primary cells reach 80% fusion, haveTwo kinds of forms of endothelioid cells core fibroblast-like cells; 1st generation cell is cultivated 2-3 days, and reach 75%-80% and merge,Cell is taking fusiformis as main, and minority is endothelial cell sample form, and fusicellular percentage composition is greater than 95%; The 2ndCultivate 1-2 days for cell, reach 80% fusion, fusicellular percentage composition is for being greater than 99%. Through qualification, shouldSpindle cell is human adipose-derived stem cell.
Result shows that cell is inoculated in nutrient solution C, and after inoculation, 10-12 days primary cells reach 70%-80% and meltClose, have two kinds of forms of endothelioid cells core fibroblast-like cells; 1st generation cell is cultivated 34 days, reaches 70%-80%Merge, cell is taking fusiformis as main, and minority is endothelial cell sample form, fusicellular percentage composition approximately 93%;2nd generation cell is cultivated 3-4 days, reaches 70%-80% and merges, and fusicellular percentage composition is greater than 97%. Through mirrorFixed, this spindle cell is human adipose-derived stem cell.
Result shows that cell is inoculated in nutrient solution D, and after inoculation, 7-10 days primary cells reach 80% fusion,There are two kinds of forms of endothelioid cells core fibroblast-like cells; 1st generation cell is cultivated 2-3 days, reaches 80% fusion,Cell is taking fusiformis as main, and minority is endothelial cell sample form, and fusicellular percentage composition is greater than 95%; The 2ndCultivate 1-2 days for cell, reach 80% fusion, fusicellular percentage composition is greater than 98%. Through qualification, this shuttleShape cell is human adipose-derived stem cell.
Experimental result shows, adopts nutrient solution provided by the invention, and compared with other nutrient solutions, incubation time is brightAobvious shortening, has that cell proliferation is fast, form good, effectively suppress the differentiation of stem cell, ensures primary stem cellThe features such as characteristic.
Above-mentioned evidence, the invention provides the isolated culture method of human adipose-derived stem cell, can be more effectiveSeparation, purifying, amplification fat stem cell, make the more thorough of adipose tissue digestion, obviously reduces heteroproteose cell quantityWith remove tissue block; The culture medium adopting in addition has that cell proliferation is fast, form good, effectively suppresses stem cellBreak up, ensure the characteristic of primary stem cell, can also promote the long term growth of human adipose-derived stem cell simultaneously, Yi JibaoHold the feature such as self and multi-lineage potential; And set up the stem cell bank of longer-term storage fat stem cell,For storage person is in the time suffering from the various diseases that need to adopt stem cell transplantation and correlation technique treatment, provide clinical suitableUse type fat stem cell, also provide a large amount of stem cell resources to clinical and research application, the inventive method behaviour simultaneouslyDo easy, have a extensive future.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further detailed explanation:
Embodiment 1:
An isolated culture method for human adipose-derived stem cell, comprises the steps:
(1) gather human fat tissue, this adipose tissue is discarded after people's stomach fat resection or lipsuctionAdipose tissue;
(2) obtain and separate human adipose-derived stem cell: get the adipose tissue of collection, in rejecting fat, naked eyes are visibleBlood vessel and pars fibrosa, shred into 1mm3Size tissue block, the D-Hanks that is 7.2-7.4 by pH valueBalanced salt solution washing is repeatedly limpid to eluate, removes residual blood, adds with adipose tissue isopyknic0.2%(m/v) (1% mother liquor compound method of this epoxy glue protoenzyme is epoxy glue protoenzyme: 100mlD-HanksIn balanced salt solution, add 0.7g NTx enzyme, 0.3g type Ⅳ collagenase), equal under 37 ° of C temperature conditionsEven concussion digestion 60 minutes, then be placed in centrifuge with the rotating speed of 1600 revs/min centrifugal 60 minutes, removeThe D-Hanks balanced salt solution that external fat is 7.2-7.4 by bottom cell by pH value cleans repeatedly, clearWash the liquid getting off and filter through 100 eye mesh screens, remove not digestion tissue, filtrate is centrifugal with 1200 revs/min8 minutes, abandoning supernatant, obtained stem cell;
(3) stem cell cultivate: by obtain stem cell according to 2 × 104/cm2Density be inoculated in blake bottle,Add 10mL to contain 12% hyclone, 103The MCDB-201 nutrient solution of U/mlLIF, is placed in 37 DEG C, CO2In the incubator of concentration 5%, cultivate, after 24 hours, original fluid is inhaled and abandoned, change fresh in tire ox bloodClear nutrient solution, discards non-adherent cell, within later every 24 hours, changes nutrient solution once, treats that Growth of Cells reachesTo 80% fusion, in blake bottle, add 0.25% pancreas enzyme-EDTA and digest, to go down to posterity by 1:3, acquisition is gone down to posterityHuman adipose-derived stem cell after cultivation;
(4) detect, frozen: detect and frozenly carry out simultaneously, in cell cryopreservation process, carrying out immunophenotypeDetection, cell count and vitality test, biological efficacy detection, the detection of Cell Differentiation ability etc. are to determine that it isThe no criterion of acceptability that reaches; Frozen step is: in the people's adipose-derived adult stem cell obtaining, add hyclone, thanExample stem cell number/hyclone is 1 × 106Cell/0.9mL, fully mixes, stand-by; At cryovial and freezingOn box, encode; Above-mentioned people's adipose-derived adult stem cell suspension and cryopreserving liquid are joined to cryovial according to 1:1 ratioInterior mixing, capping, concussion, fully mixes, and is moved into rapidly in-80 ° of C refrigerators; After 24 hours, according toNumbering is put into and on corresponding freezing frame, moves into the medium-term and long-term preservation of liquid nitrogen, result to be detected out after, detection is not conformed toThe cell of lattice is together discarded together with cryopreservation tube by corresponding freeze-stored cell according to its numbering.
Embodiment 2:
An isolated culture method for human adipose-derived stem cell, comprises the steps:
(1) gather human fat tissue, this adipose tissue is discarded after people's thigh adipectomy or lipsuctionAdipose tissue;
(2) obtain and separate human adipose-derived stem cell: get the adipose tissue of collection, in rejecting fat, naked eyes are visibleBlood vessel and pars fibrosa, shred into 2mm3Size tissue block, the D-Hanks that is 7.2-7.4 by pH valueBalanced salt solution washing is repeatedly limpid to eluate, removes residual blood, adds with adipose tissue isopyknic0.3%(m/v) (1% mother liquor compound method of this epoxy glue protoenzyme is epoxy glue protoenzyme: 100mlD-HanksIn balanced salt solution, add 0.7g NTx enzyme, 0.3g type Ⅳ collagenase), equal under 37 ° of C temperature conditionsEven concussion digestion 80 minutes, then be placed in centrifuge with the rotating speed of 1800 revs/min centrifugal 4 minutes, removeThe D-Hanks balanced salt solution that external fat is 7.2-7.4 by bottom cell by pH value cleans repeatedly, clearWash the liquid getting off and filter through 100 eye mesh screens, remove not digestion tissue, filtrate is centrifugal with 1200 revs/min5 minutes, abandoning supernatant, obtained stem cell;
(3) stem cell cultivate: by obtain stem cell according to 3 × 104/cm2Density be inoculated in blake bottle,Add 10mL to contain 10% hyclone, 103The MCDB-201 nutrient solution of U/mlLIF, is placed in 37 DEG C, CO2In the incubator of concentration 5%, cultivate, after 2 days, original fluid is inhaled and abandoned, change the fresh hyclone that containsNutrient solution, discard non-adherent cell, later every 24 hours change nutrient solution once, treat that Growth of Cells reaches80% merges, and adds 0.25% pancreas enzyme-EDTA and digest in blake bottle, goes down to posterity by 1:3, obtains the training of going down to posterityHuman adipose-derived stem cell after supporting;
(4) detect, frozen: detect and frozenly carry out simultaneously, in cell cryopreservation process, carrying out immunophenotypeDetection, cell count and vitality test, biological efficacy detection, the detection of Cell Differentiation ability etc. are to determine that it isThe no criterion of acceptability that reaches; Frozen step is: in the people's adipose-derived adult stem cell obtaining, add hyclone, thanExample stem cell number/hyclone is 2 × 106Cell/0.9mL, fully mixes, stand-by; At cryovial and freezingOn box, encode; Above-mentioned people's adipose-derived adult stem cell suspension and cryopreserving liquid are joined to cryovial according to 1:1 ratioInterior mixing, capping, concussion, fully mixes, and is moved into rapidly in-80 ° of C refrigerators; After 24 hours, according toNumbering is put into and on corresponding freezing frame, moves into the medium-term and long-term preservation of liquid nitrogen, result to be detected out after, detection is not conformed toThe cell of lattice is together discarded together with cryopreservation tube by corresponding freeze-stored cell according to its numbering.
Embodiment 3:
An isolated culture method for human adipose-derived stem cell, comprises the following steps:
(1) gather human fat tissue, this adipose tissue is people's belly, thigh or Cutaneolipectomy or suctionThe postoperative discarded adipose tissue of fat;
(2) obtain and separate human adipose-derived stem cell: get the adipose tissue of collection, in rejecting fat, naked eyes are visibleBlood vessel and pars fibrosa, shred into 1mm3Size tissue block, the D-Hanks that is 7.2-7.4 by pH valueBalanced salt solution washing is repeatedly limpid to eluate, removes residual blood, adds with adipose tissue isopyknic0.4%(m/v) (1% mother liquor compound method of this epoxy glue protoenzyme is epoxy glue protoenzyme: 100mlD-HanksIn balanced salt solution, add 0.7g NTx enzyme, 0.3g type Ⅳ collagenase), equal under 37 ° of C temperature conditionsEven concussion digestion 40 minutes, then be placed in centrifuge with the rotating speed of 1600 revs/min centrifugal 10 minutes, removeThe D-Hanks balanced salt solution that external fat is 7.2-7.4 by bottom cell by pH value cleans repeatedly, clearWash the liquid getting off and filter through 100 eye mesh screens, remove not digestion tissue, filtrate is centrifugal with 1200 revs/min10 minutes, abandoning supernatant, obtained stem cell;
(3) stem cell cultivate: by obtain stem cell according to 2 × 104/cm2Density be inoculated in blake bottle,Add 10mL to contain 15% hyclone, 105The MCDB-201 nutrient solution of U/mlLIF, is placed in 37 DEG C, CO2In the incubator of concentration 5%, cultivate, after 24 hours, original fluid is inhaled and abandoned, change fresh in tire ox bloodClear nutrient solution, discards non-adherent cell, within later every 24 hours, changes nutrient solution once, treats that Growth of Cells reachesTo 80% fusion, in blake bottle, add 0.25% pancreas enzyme-EDTA and digest, to go down to posterity by 1:3, acquisition is gone down to posterityHuman adipose-derived stem cell after cultivation;
(4) detect, frozen: detect and frozenly carry out simultaneously, in cell cryopreservation process, carrying out immunophenotypeDetection, cell count and vitality test, biological efficacy detection, the detection of Cell Differentiation ability etc. are to determine that it isThe no criterion of acceptability that reaches; Frozen step is: in the people's adipose-derived adult stem cell obtaining, add hyclone, thanExample stem cell number/hyclone is 2 × 106Cell/0.9mL, fully mixes, stand-by; At cryovial and freezingOn box, encode; Above-mentioned people's adipose-derived adult stem cell suspension and cryopreserving liquid are joined to cryovial according to 1:1 ratioInterior mixing, capping, concussion, fully mixes, and is moved into rapidly in-80 ° of C refrigerators; After 24 hours, according toNumbering is put into and on corresponding freezing frame, moves into the medium-term and long-term preservation of liquid nitrogen, result to be detected out after, detection is not conformed toThe cell of lattice is together discarded together with cryopreservation tube by corresponding freeze-stored cell according to its numbering.
Embodiment 4
The construction method in human adipose-derived stem cell storehouse, comprises the following steps: according to either party in embodiment 1-3The human adipose-derived stem cell that method obtains is built storehouse, and concrete grammar is: according to (1) in embodiment 1-3-(4)Step, obtains human adipose-derived stem cell; (5) build storehouse: by obtain every a human adipose-derived stem cell by donor surnameName, ID card No. and storage date preserve respectively, set up the computer data of the detailed archives of every a stem cellStorehouse, sets up human adipose-derived stem cell storehouse.
Claims (5)
1. an isolated culture method for human adipose-derived stem cell, is characterized in that, comprises the following steps:
(1) gather human fat tissue;
(2) obtain and separate human adipose-derived stem cell: getting the adipose tissue of collection, reject macroscopic blood vessel and fiber in fatPart, shreds into 1-2mm3Size tissue block, the D-Hanks balanced salt solution that is 7.2-7.4 by pH value washs repeatedly to washing outLiquid is limpid, removes residual blood, adds and the isopyknic 0.2-0.4% of adipose tissue (m/v) epoxy glue protoenzyme, at 37 DEG CEven concussion digestion 40-80 minute under temperature conditions, then be placed in centrifuge with the centrifugal 4-10 of rotating speed of 1600-1800 rev/minMinute, remove external fat, the D-Hanks balanced salt solution that is 7.2-7.4 by pH value by bottom cell cleans repeatedly, cleansThe liquid getting off filters through 100 eye mesh screens, removes not digestion tissue, and filtrate, with 1200 revs/min of centrifugal 5-10 minute, is abandonedRemove supernatant, obtain stem cell; Described epoxy glue protoenzyme is made up of NTx enzyme familial combined hyperlipidemia clostridiopetidase A, when digestion adipose tissueFinal concentration be 0.1%-0.2% (m/v), 1% (m/v) mother liquor compound method of described epoxy glue protoenzyme is: 100mLD-HanksIn balanced salt solution, add 0.7g NTx enzyme, 0.3g type Ⅳ collagenase;
(3) stem cell cultivate: by obtain stem cell according to 2-3 × 104/cm2Density be inoculated in blake bottle, add 10mLContaining the nutrient solution of hyclone, be placed in 37 DEG C, CO2In the incubator of concentration 5%, cultivate, after 1-2 days, original fluid is inhaledAbandon, change the fresh nutrient solution containing hyclone, discard non-adherent cell, within later every 24 hours, change nutrient solution once, treatGrowth of Cells reaches 80% fusion, adds 0.25% pancreas enzyme-EDTA and digest in blake bottle, goes down to posterity by 1:3, obtains the training of going down to posterityHuman adipose-derived stem cell after supporting; Described nutrient solution is to contain 10-15% hyclone, 103-105The MCDB-201 training of U/mlLIFNutrient solution;
(4) detect, frozen: detect and frozenly carry out simultaneously, in cell cryopreservation process, carrying out immunophenotype detection, cytometerNumber and vitality test, biological efficacy detect, Cell Differentiation ability detects to determine whether it reaches criterion of acceptability; Frozen stepFor: in the people's adipose-derived adult stem cell obtaining, add hyclone, ratio stem cell number/hyclone is 1-2 × 106Cell/ 0.9mL, fully mixes, stand-by; On cryovial and freeze box, encode; By above-mentioned people's adipose-derived adult stem cell suspension and frozenLiquid joins in cryovial and mixes according to 1:1 ratio, capping, and concussion, fully mixes, and is moved into rapidly in-80 DEG C of refrigerators;After 24 hours, be put into and on corresponding freezing frame, move into the medium-term and long-term preservation of liquid nitrogen according to numbering, result to be detected out after, will detectUnderproof cell is together discarded together with cryopreservation tube by corresponding freeze-stored cell according to its numbering.
2. the isolated culture method of human adipose-derived stem cell according to claim 1, is characterized in that: human fat tissue is peopleThe discarded object obtaining in body liposuction procedures or adipectomy, gathers from human abdomen, thigh or subcutaneous fat.
3. the isolated culture method of human adipose-derived stem cell according to claim 1, is characterized in that: described epoxy glue protoenzymeFinal concentration when digestion adipose tissue is 0.1% (m/v).
4. the isolated culture method of human adipose-derived stem cell as claimed in claim 1, is characterized in that: described nutrient solution is to contain12% hyclone, 103The MCDB-201 nutrient solution of U/mlLIF.
5. the construction method in people's adipose-derived adult stem cell storehouse, is characterized in that: concrete construction method is as follows:
(1) gather human fat tissue; Described human fat tissue is the discarded object obtaining in human body liposuction procedures or adipectomy,Gather from human abdomen, thigh or subcutaneous fat;
(2) obtain and separate human adipose-derived stem cell: getting the adipose tissue of collection, reject macroscopic blood vessel and fiber in fatPart, shreds into 1-2mm3Size tissue block, the D-Hanks balanced salt solution that is 7.2-7.4 by pH value washs repeatedly to washing outLiquid is limpid, removes residual blood, adds and the isopyknic 0.2-0.4% of adipose tissue (m/v) epoxy glue protoenzyme, at 37 DEG CEven concussion digestion 40-80 minute under temperature conditions, then be placed in centrifuge with the centrifugal 4-10 of rotating speed of 1600-1800 rev/minMinute, remove external fat, the D-Hanks balanced salt solution that is 7.2-7.4 by pH value by bottom cell cleans repeatedly, cleansThe liquid getting off filters through 100 eye mesh screens, removes not digestion tissue, and filtrate, with 1200 revs/min of centrifugal 5-10 minute, is abandonedRemove supernatant, obtain stem cell; Described epoxy glue protoenzyme is made up of NTx enzyme familial combined hyperlipidemia clostridiopetidase A, when digestion adipose tissueFinal concentration be 0.1%-0.2% (m/v), 1% (m/v) mother liquor compound method of described epoxy glue protoenzyme is: 100mLD-HanksIn balanced salt solution, add 0.7g NTx enzyme, 0.3g type Ⅳ collagenase;
(3) stem cell cultivate: by obtain stem cell according to 2-3 × 104/cm2Density be inoculated in blake bottle, add 10mLContaining the nutrient solution of hyclone, be placed in 37 DEG C, CO2In the incubator of concentration 5%, cultivate, after 1-2 days, original fluid is inhaledAbandon, change the fresh nutrient solution containing hyclone, discard non-adherent cell, within later every 24 hours, change nutrient solution once, treatGrowth of Cells reaches 80% fusion, adds 0.25% pancreas enzyme-EDTA and digest in blake bottle, goes down to posterity by 1:3, obtains the training of going down to posterityHuman adipose-derived stem cell after supporting; Described nutrient solution is to contain 10-15% hyclone, 103-105The MCDB-201 training of U/mlLIFNutrient solution;
(4) detect, frozen: detect and frozenly carry out simultaneously, in cell cryopreservation process, carrying out immunophenotype detection, cytometerNumber and vitality test, biological efficacy detect, Cell Differentiation ability detects to determine whether it reaches criterion of acceptability; Frozen stepFor: in the people's adipose-derived adult stem cell obtaining, add hyclone, ratio stem cell number/hyclone is 1-2 × 106Cell/ 0.9mL, fully mixes, stand-by; On cryovial and freeze box, encode; By above-mentioned people's adipose-derived adult stem cell suspension and frozenLiquid joins in cryovial and mixes according to 1:1 ratio, capping, and concussion, fully mixes, and is moved into rapidly in-80 DEG C of refrigerators;After 24 hours, be put into and on corresponding freezing frame, move into the medium-term and long-term preservation of liquid nitrogen according to numbering, result to be detected out after, will detectUnderproof cell is together discarded together with cryopreservation tube by corresponding freeze-stored cell according to its numbering;
(5) build storehouse: the every a human adipose-derived stem cell obtaining was protected respectively by donor name, ID card No. and storage dateDeposit, set up the computer data of the detailed archives of every a stem cell, set up human adipose-derived stem cell storehouse.
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| CN104357382A (en) * | 2014-10-08 | 2015-02-18 | 黑龙江天晴干细胞有限公司 | Method for obtaining human adipose-derived stem cells and construction method for multilevel allogeneic adipose-derived stem cell bank |
| CN104357387B (en) * | 2014-12-08 | 2017-02-22 | 深圳市赛欧细胞生物科技有限公司 | Method for separating human adipose-derived stem cells from human adipose tissues |
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| CN105907710A (en) * | 2016-05-05 | 2016-08-31 | 何红葖 | Isolated culture method of human adipose mesenchymal stem cells |
| CN106190967A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of autologous fat derived stem cell is for the preparation method of beautifying and antisenility |
| CN107475190B (en) * | 2017-10-13 | 2020-05-19 | 上海莱馥医疗科技有限公司 | Method for clinical-level efficient preparation and cryopreservation of fat SVF cells and application thereof |
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| CN107974430A (en) * | 2018-01-24 | 2018-05-01 | 北京臻溪谷医学研究中心（有限合伙） | A kind of construction method that obtains isolated culture method and stem cell bank of human adipose-derived stem cell |
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