CN102329773A - Method for separating adipose-derived mesenchymal stem cells from tumescent liquid - Google Patents
Method for separating adipose-derived mesenchymal stem cells from tumescent liquid Download PDFInfo
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- CN102329773A CN102329773A CN201110310461A CN201110310461A CN102329773A CN 102329773 A CN102329773 A CN 102329773A CN 201110310461 A CN201110310461 A CN 201110310461A CN 201110310461 A CN201110310461 A CN 201110310461A CN 102329773 A CN102329773 A CN 102329773A
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Abstract
The invention discloses a method for separating adipose-derived mesenchymal stem cells from tumescent liquid in the surgical liposuction surgery, and provides a new adipose-derived stem cell. The new adipose-derived stem cell does not have obvious difference with the adipose-tissue-derived mesenchymal stem cell in the aspects of cell morphology, immunophenotype and the like. Thus, a new way is provided for separating the adipose-derived stem cell.
Description
Technical field
The present invention relates to the method for separating out fat mescenchymal stem cell from inflation fluid, belong to biomedicine field.
Background technology
Mescenchymal stem cell [mesenchymal stem cells, MSC] is the important member of stem cell family, derives to grow early stage mesoderm and ectoderm.MSC finds in marrow at first, because of it has multidirectional differentiation potential, hematopoiesis support and promotes characteristics such as stem cell implantation, immunoregulation and self-replacation to receive people's attention day by day.Like mescenchymal stem cell in vivo or under the external specific inductive condition; Can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium; Still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as the ideal seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.
At present; The separation of fat stem cell mainly is the fatty tissue source from extracting; Existing patented claim all is from fatty tissue, to extract at present, for example: a kind of isolation cultivation method of fat stem cell (application number: CN201010568873.4, publication number: CN102002478A); The acquisition methods of people's adipose-derived adult stem cell and the construction process of this stem cell bank (application number: CN201010120944.4, publication number: CN102002475A); A kind of isolation cultivation method of fat mesenchymal stem cell and application thereof (application number: CN200410084407.3, publication number: CN1778905); The method of separating mesenchymal stem cell (application number: CN201010580537.1, publication number: CN101984049A) from fatty tissue.Also the someone reports the patent of separating out fat stem cell from inflation fluid as yet.
The expansion liposuction is the most extensive a kind of technology that is out at present and uses in section's liposuction procedures, mainly is perfusion excess hypotonic expansion liquid before operation, and this liquid has the effect of pain relieving, hemostasis, loose and cracking adipocyte.The inventor finds that from clinical application the inflation fluid that obtains in the liposuction procedures also contains a certain amount of fat stem cell.This is the source that provides new that obtains of stem cell, has increased new fat stem cell source approach.
Summary of the invention
The objective of the invention is to realize through following technical scheme: a kind of from inflation fluid the method for separating out fat mescenchymal stem cell; Be made up of following steps: the collection liposuction forward direction of (1) inflation fluid wants extraction location to pour into excessive hypotonic expansion liquid; Then fat that obtains in the liposuction procedures and the about 500ml of inflation fluid mixed solution are placed room temperature held 10min; After treating fat and inflation fluid separating fully; Red liquid with liquid-transfering gun absorption yellow fat lower part is the inflation fluid that contains fat stem cell., with its branch 50ml centrifuge tube of packing into.(2) processing of inflation fluid is put into whizzer with the centrifugal 5min of 1500rpm with the inflation fluid of collecting.Centrifugal absorption discards the liquid of centrifuge tube upper section, collecting precipitation.Every effective PBS liquid suspends, and adds the erythrocyte cracked liquid room temperature and leaves standstill 5 minutes, with the centrifugal 3min of 1200rpm, abandons supernatant.(3) washing of cell adding 5ml DMEM (not containing serum) dispels deposition, and cellular filter filters.Filter later liquid with the centrifugal 3min of 1200rpm, deposition is the fat mesenchymal stem cell that will obtain.Add DMEM (not containing serum) again and repeat above operated wash 3 times.
(4) cell cultures
To obtain cell inoculation to 75cm
2In the Tissue Culture Flask, inoculation adds the mescenchymal stem cell substratum.37 ℃, 5%CO
2Cultivate.
(5) cell is identified
To the cell of separation and Culture and adipose-derived stem cell carry out morphology, immunophenotype is identified and compare.
(6) inflation fluid source fat stem cell is carried out to fat, skeletonization and becomes cartilage three-dimensional differentiation potential to identify.
Further, the inflation fluid described in the step 1 adds the 20ml lignocaine and 1ml suprarenin prepares in 1000ml saline water.
Further, the add-on of the said PBS liquid of step 2 is 3 ml, and the volume that erythrocyte cracked liquid adds is 1-2 times of deposition and PBS liquid TV.
Further, inoculum density is 5000/cm in the step 4
2, the add-on of mescenchymal stem cell substratum is 10 ml.
Further, the immunophenotype in the step 5 is identified and is meant CD73, CD90, and CD105 and CD45, CD34, CD14, CD11b, CD19, HLA-DR detects.
Further, the differentiation potential in the step 6 identifies it is that isolated cell is carried out to fat, skeletonization and becomes the test of cartilage three-dimensional differentiation potential.
The material and the reagent that adopt in the such scheme of the present invention are following:
Inflation fluid: lignocaine 20ML/L suprarenin 1ML/L;
Erythrocyte cracked liquid Solarbio, Beijing;
DMEM (not containing serum) HyClone, the U.S.;
The mescenchymal stem cell substratum stemcell U.S.;
Antibody BD, the U.S.;
Centrifuge tube Coring, the U.S.;
Transfer pipet Coring, the U.S.;
Cell filter screen BD, the U.S.;
Liquid-transfering gun BRAND, Germany;
Whizzer Xiang Lu, the Hunan;
Culturing bottle Coring, the U.S.;
Cell culture incubator Sanyo, Japan;
Flow cytometer, the U.S..
The invention a kind of new fat stem cell source, the mescenchymal stem cell of originating with fatty tissue at aspects such as cellular form, immunophenotypes does not have significant difference.Therefore, the present invention provides new approach for the separation of fat stem cell.
Description of drawings
Fig. 1 fatty tissue source fat stem cell.
Fig. 2 inflation fluid source fat stem cell.
Fig. 3 cell growth curve.
Fig. 4 cytodifferentiation potential evaluation--Osteoblast Differentiation experimental result.
Fig. 5 cytodifferentiation potential is identified--become fat Analytical Chemical Experiment result.
Fig. 6 cytodifferentiation potential is identified--become cartilage Analytical Chemical Experiment result.
Embodiment
Embodiment
1. inflation fluid obtains
The liposuction forward direction wants extraction location to pour into excessive hypotonic expansion liquid; Then fat that obtains in the liposuction procedures and the about 500ml of inflation fluid mixed solution are placed room temperature held 10min; After treating fat and inflation fluid separating fully; Liquid-transfering gun extracts lower floor's red liquid, is the inflation fluid that contains fat stem cell.
2. the processing of inflation fluid
The inflation fluid of collecting is put into whizzer with the centrifugal 5min of 1500rpm.Centrifugal absorption discards the liquid of centrifuge tube upper section, collecting precipitation.Every effective 3ml PBS liquid suspends, and the volume ratio of press 1:2 adds erythrocyte cracked liquid, and room temperature left standstill 5 minutes, with the centrifugal 3min of 1200rpm, abandon supernatant get precipitate subsequent use.
3. the separation of fat stem cell
The gained throw out is suspended with low sugar DMEM substratum, and cellular filter filters, then with filtering liq with the centrifugal 3min of 1200rpm, deposition is will obtain cell.Add DMEM (not containing serum) again and repeat above operated wash 3 times, remove erythrocyte cracked liquid, obtain cell and be the purpose cell.
4. the cultivation of fat stem cell
To obtain cell inoculation to 75cm
2In the Tissue Culture Flask, add 10ml mescenchymal stem cell substratum, 37 ℃, 5%CO
2Cultivate, 48h changes liquid to be continued to cultivate.
5. cell is identified
The separation and Culture attached cell is carried out immunophenotype CD73, CD90, CD105 and CD45, CD34, CD14; CD11b, CD19, the HLA-DR detected result shows, when cell reached for the 5th generation; Immunophenotype CD73, CD90, CD105 all are high expression level, and phenotype CD45; CD34, CD14, CD19, HLA-DR all present low the expression.And cell can be to becoming fat, skeletonization and becoming the differentiation of cartilage three-dimensional.This mescenchymal stem cell definition with international artery cell association regulation conforms to.Therefore, isolated cell really is a fat mesenchymal stem cell.
Compare with fatty tissue derived mesenchymal stem cell.
Immunophenotype detected result when table 1. cell reached for the 5th generation
。
Though separating the mescenchymal stem cell quantity of self-expanding liquid is starkly lower than and the equal-volume adipose tissue-derived stem cell; But growth kinetics of cells shows, but its rate of propagation is very fast, when growing to the 8th day; Density is equal to and adipose tissue-derived stem cell basically, and cellular form is no significant difference also.
Separate the inflation fluid that obtains in 5 person-times of suction lipectomy processes according to the identical operations method, all obtained fat stem cell, explain that this method is reliable and stable.
Claims (9)
1. the method for a separating out fat mescenchymal stem cell from inflation fluid, form by following steps:
(1) the collection liposuction forward direction of inflation fluid wants extraction location to pour into excessive hypotonic expansion liquid; Then fat that obtains in the liposuction procedures and inflation fluid are placed room temperature held 10min; After treating fat and inflation fluid separating fully; Red liquid with liquid-transfering gun absorption yellow fat lower part is the inflation fluid that contains fat stem cell.
2. the processing of (2) inflation fluid is put into whizzer with the centrifugal 5min of 1500rpm with the inflation fluid branch of the collecting 50ml centrifuge tube of packing into.
3. centrifugal absorption discards the liquid of centrifuge tube upper section, collecting precipitation, and every effective PBS liquid suspends, and adds the erythrocyte cracked liquid room temperature and leaves standstill 5 minutes, with the centrifugal 3min of 1200rpm, abandons supernatant; (3) washing of cell adding 5ml DMEM (not containing serum) dispels deposition; Cellular filter filters; Filter later liquid with the centrifugal 3min of 1200rpm, deposition is the fat mesenchymal stem cell that will obtain, and adds DMEM (not containing serum) again and repeats above operated wash 3 times;
(4) cell cultures
To obtain cell inoculation to 75cm
2In the Tissue Culture Flask, inoculation adds the mescenchymal stem cell substratum, and 37 ℃, 5%CO
2Cultivate;
(5) cell is identified
To the cell of separation and Culture and adipose-derived stem cell carry out morphology, immunophenotype is identified and compare.
4. (6) are carried out to fat, skeletonization and become cartilage three-dimensional differentiation potential to identify inflation fluid source fat stem cell.
5. the method for claim 1, wherein the described inflation fluid of step 1 adds the 20ml lignocaine and 1ml suprarenin prepares in 1000ml saline water.
6. the method for claim 1, wherein the add-on of the said PBS liquid of step 2 is 3 ml, the volume that erythrocyte cracked liquid adds is 1-2 times of deposition and PBS liquid TV.
7. like each described method of claim 1-3, wherein the described inoculum density of step 4 is 5000/cm
2, the add-on of mescenchymal stem cell substratum is 10 ml.
8. like each described method of claim 1-4, wherein the described immunophenotype of step 5 is identified and is meant CD73, CD90, and CD105 and CD45, CD34, CD14, CD11b, CD19, HLA-DR detects.
9. like each described method of claim 1-5, wherein the said differentiation potential of step 6 identifies it is that isolated cell is carried out to fat, skeletonization and becomes the cartilage three-dimensional to induce differentiation, and differentiation effect is observed in dyeing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965337A (en) * | 2012-12-04 | 2013-03-13 | 东南大学 | Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction |
| CN103074298A (en) * | 2013-01-04 | 2013-05-01 | 广州爱菲科生物科技有限公司 | Human fat mesenchymal stem cell bank and construction method thereof |
| CN108004207A (en) * | 2017-12-15 | 2018-05-08 | 英科博雅生命科技有限公司 | The method that significant quantities of fat mescenchymal stem cell is obtained from fat |
| CN115322963A (en) * | 2021-05-10 | 2022-11-11 | 东莞宣冠干细胞再生医学有限公司 | Method for separating and preparing primary clinical-grade adipose-derived stem cells |
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| CN1778905A (en) * | 2004-11-22 | 2006-05-31 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| CN101984049A (en) * | 2010-12-09 | 2011-03-09 | 协和干细胞基因工程有限公司 | Method for separating mesenchymal stem cells from dispose tissues |
| CN102002475A (en) * | 2010-03-10 | 2011-04-06 | 和泽生物科技有限公司 | Method for obtaining fat adult stem cells of human and method for establishing stem cell library |
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2011
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| CN1778905A (en) * | 2004-11-22 | 2006-05-31 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| CN102002475A (en) * | 2010-03-10 | 2011-04-06 | 和泽生物科技有限公司 | Method for obtaining fat adult stem cells of human and method for establishing stem cell library |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965337A (en) * | 2012-12-04 | 2013-03-13 | 东南大学 | Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction |
| CN103074298A (en) * | 2013-01-04 | 2013-05-01 | 广州爱菲科生物科技有限公司 | Human fat mesenchymal stem cell bank and construction method thereof |
| CN108004207A (en) * | 2017-12-15 | 2018-05-08 | 英科博雅生命科技有限公司 | The method that significant quantities of fat mescenchymal stem cell is obtained from fat |
| CN108004207B (en) * | 2017-12-15 | 2021-07-13 | 英科博雅生命科技有限公司 | Method for obtaining large amount of adipose mesenchymal stem cells from fat |
| CN115322963A (en) * | 2021-05-10 | 2022-11-11 | 东莞宣冠干细胞再生医学有限公司 | Method for separating and preparing primary clinical-grade adipose-derived stem cells |
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Application publication date: 20120125 |