CN101984049A - Method for separating mesenchymal stem cells from dispose tissues - Google Patents
Method for separating mesenchymal stem cells from dispose tissues Download PDFInfo
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Abstract
The invention relates to a method for separating mesenchymal stem cells from dispose tissues. The method comprises the following steps: putting dispose tissues to a biological bag for transportation or a sterilized reagent bottle; in aseptic conditions, removing red blood cells preliminarily; digesting dispose extract to obtain adipose-derived stem cells; removing red blood cells once again, and counting the adipose-derived stem cells; and carrying out activity detection to the adipose-derived stem cells, and cultivating the adipose-derived stem cells to obtain the mesenchymal stem cells. In the method, a large quantity of adipose-derived stem cells which are in good conditions and have good multi-difference capability can be obtained with a small quantity of dispose tissue. The method has the advantages of easiness and good reproducibility.
Description
Technical field
The present invention relates to the method for a kind of separating mesenchymal stem cell, especially the method for separating mesenchymal stem cell from fatty tissue.
Background technology
Since calendar year 2001 was found adipose-derived stem cell, because it has enormous amount, the relative characteristic of simple of acquisition mode was so have great advantage and wide prospect in clinical application.The research of fat stem cell is a focus of present stem-cell research and stem-cell therapy, in the middle of relevant clinical treatment and clinical trial are being carried out.The researchist has proved that not only it has the various mesoblastemas ability of (comprising adipocyte, scleroblast, chondrocyte and Skeletal Muscle Cell) that is divided into, and can also be induced the cell of other germinal layers such as becoming islet cells, neurocyte, liver cell through the comparatively complicated process of inducing.Along with deepening continuously of research, its using value clinically more and more is subjected to people's attention.
Constantly carry out along with the researchist is applied to clinical exploration to fat stem cell, various methods about its separation and vitro culture are in the news out.But for the isolating concrete grammar of fat stem cell and some details standard of a generally acknowledged strictness not.
As a kind of cellular product that will be applied to clinical cell therapy, should have following characteristics:
(1) tissue obtains simple.
(2) separation method is simple as far as possible, the interpolation allogenic material of trying one's best few, and can obtain the capacity cell.
(3) operating process controllability is strong, has very high repeatability.
(4) damage of pair cell itself reaches minimum.
Summary of the invention
Technical problem to be solved by this invention is, provides a kind of fatty tissue in a small amount that utilizes to obtain a large amount of in good condition, methods of separating mesenchymal stem cell from fatty tissue of keeping good multidirectional differentiation capability.
Technological line of the present invention is as follows:
(1) acquisition of fatty tissue, storage and transportation: the 20-500ml fatty tissue obtains/utilizes the fatty tissue waste 10-200g that other operations obtain by the hospital or the beauty salon of specialty by swelling method liposuction procedures; The fatty tissue of obtaining is put into biological shipping bags/sterilization reagent bottle (under the aseptic condition), can store 48 hours 4-20 ℃ (being preferably in 4 ℃) (be preferably within 4 hours and handle), maintains in the transportation under 4 ℃ of environment.
(2) tentatively remove red corpuscle: after layering is treated in the static placement of liposuction thing, carefully remove lower floor's liquid; Repeatedly limpid with the washing of D-Hanks liquid to elutant.
The fatty tissue that operation obtains shreds into about 1mm at first as far as possible
3The size tissue block is repeatedly limpid to elutant with the washing of D-Hanks liquid then.
(3) digestion liposuction thing: add a Collagen Type VI enzyme with the preparation of D-Hanks liquid to final concentration 0.01-2g/100ml, 37 ℃ of following 20-400 commentariess on classics/min concussions digested 15-120 minute.
(4) acquisition of fat stem cell: will digest after product centrifugal 5min under 4 ℃, centrifugal force 450g condition; The fat stem cell substratum is resuspended, removes impurity with 100 μ m aperture membrane filtrations and finally obtains fat stem cell.
(5) remove red corpuscle once more, utilize repetitive scrubbing, and the mode of erythrocyte splitting is removed red corpuscle once more.
(6) fat stem cell counting, the active detection and cultivation: get 100 μ l isolated cells and carry out cell counting with cell counting count board; Take out 100 μ l cells simultaneously and expect that by tongue blue painted method carries out viable count.According to the result of two countings according to 3*10
4/ cm
2Density be inoculated in the T-75 culturing bottle 37 ℃, CO
2Cultivate under concentration 5%, humidity 100% condition.
The invention has the beneficial effects as follows: can utilize in a small amount fatty tissue to obtain a large amount of in good condition, fat stem cells of keeping good multidirectional differentiation capability, and working method is simple, repeatable strong.
Description of drawings
Fig. 1 is the fat stem cell that adopts method of the present invention to obtain.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail:
Of the present invention from fatty tissue the method for separating mesenchymal stem cell, comprise the steps:
(1) acquisition of fatty tissue: fatty tissue is the hospital of specialty or the waste that the beauty salon obtains by swelling method liposuction procedures, and under aseptic condition, 4-20 ℃ of storage was less than 48 hours;
(2) tentatively remove red corpuscle: after layering is treated in the static placement of liposuction thing, carefully remove lower floor's liquid, repeatedly limpid with the washing of D-Hanks liquid to elutant;
(3) digestion liposuction thing: add a Collagen Type VI enzyme with the preparation of D-Hanks liquid to 0.01-2g/100ml, 37 ℃ of following 20-400 commentariess on classics/min concussions digested 15-120 minute;
(4) acquisition of fat stem cell: will digest after product centrifugal 5min under 4 ℃, centrifugal force 450g condition, the fat stem cell substratum is resuspended, removes impurity with 100 μ m aperture membrane filtrations and finally obtains fat stem cell;
(5) remove red corpuscle once more, utilize the mode of repetitive scrubbing and erythrocyte splitting to remove red corpuscle once more;
(6) fat stem cell counting, the active detection and cultivation: get 100 μ l isolated cells and carry out cell counting with cell counting count board; Take out 100 μ l cells simultaneously and expect that by tongue blue painted method carries out viable count, according to the result of two countings according to 3*10
4/ cm
2Density be inoculated in the T-75 culturing bottle 37 ℃, CO
2Cultivate under concentration 5%, humidity 100% condition.
In the step (1), the fatty tissue of obtaining is put into biological shipping bags or the storage of sterilization reagent bottle.
In the step (1), under aseptic condition, 4 ℃ of storages maintained in the transportation under 4 ℃ of environment less than 4 hours.
A kind of from fatty tissue the method for separating mesenchymal stem cell, comprise the steps:
(1) acquisition of fatty tissue: the fatty tissue waste of fatty tissue for obtaining in the operation, under aseptic condition, 4-20 ℃ of storage was less than 48 hours;
(2) tentatively remove red corpuscle: the fatty tissue that operation obtains shreds into about 1mm at first as far as possible
3The size tissue block is repeatedly limpid to elutant with the washing of D-Hanks liquid then;
(3) digestion liposuction thing: add a Collagen Type VI enzyme with the preparation of D-Hanks liquid to 0.01-2g/100ml, 37 ℃ of following 20-400 commentariess on classics/min concussions digested 15-120 minute;
(4) acquisition of fat stem cell: will digest after product centrifugal 5min under 4 ℃, centrifugal force 450g condition, the fat stem cell substratum is resuspended, removes impurity with 100 μ m aperture membrane filtrations and finally obtains fat stem cell;
(5) remove red corpuscle once more, utilize the mode of repetitive scrubbing and erythrocyte splitting to remove red corpuscle once more;
(6) fat stem cell counting, the active detection and cultivation: get 100 μ l isolated cells and carry out cell counting with cell counting count board; Take out 100 μ l cells simultaneously and expect that by tongue blue painted method carries out viable count, according to the result of two countings according to 3*10
4/ cm
2Density be inoculated in the T-75 culturing bottle 37 ℃, CO
2Cultivate under concentration 5%, 100% humidity condition.
In the step (1), the fatty tissue of obtaining is put into biological shipping bags or the storage of sterilization reagent bottle.
In the step (1), under aseptic condition, 4 ℃ of storages maintained in the transportation under 4 ℃ of environment less than 4 hours.
Embodiment 1
The product that obtains a expansion liposuction from hospital is total to 180ml, uses biological shipping bags to be transported to the laboratory within after the liposuction 4 ℃, 2 hours, handles under sterile state:
1. clean to scavenging solution colourlessly with the D-Hanks liquid of 37 ℃ of incubations repeatedly, guarantee that red corpuscle cleans up.
2. add a Collagen Type VI enzyme with the preparation of D-Hanks liquid to final concentration 0.1g/100ml, 37 ℃ down 100 commentariess on classics/min concussion digested 1 hour
3. will digest after product centrifugal 5min under 4 ℃, 450g condition; 10ml fat stem cell substratum is resuspended, removes impurity with 100 μ m aperture membrane filtrations and finally obtains fat stem cell.
4. fat stem cell suspension and erythrocyte cracked liquid were mixed with 1: 1 and hatched 2 minutes, centrifugal 5min under 4 ℃, centrifugal force 450g condition utilizes the resuspended fat stem cell of fat stem cell substratum.
5. get 100 μ l isolated cells and carry out cell counting with cell counting count board; Take out 100 μ l cells simultaneously and expect that by tongue blue painted method carries out viable count: the result shows and obtains about 1.2*10 altogether
7Individual cell, the motility rate of cell is more than 95%.
With cell according to 3*10
4/ cm
2Density be inoculated in the T-75 culturing bottle 37 ℃, CO
2Cultivate under concentration 5%, humidity 100% condition.Cultivate microscopically observation after 2 days, cell state is good, as Fig. 1.
7. separate obtaining fat stem cell, can be divided into adipocyte, scleroblast, sarcoplast, chondroblast, have multidirectional differentiation potential through inducing differentiation detection confirmation.
Embodiment 2
The fatty tissue waste 180g of fatty tissue for obtaining in the operation uses the sterilization reagent bottle, under aseptic condition, is transported to the laboratory within 4 ℃, 2 hours, handles under sterile state:
1. shred into about 1mm at first as far as possible
3The size tissue block is cleaned to scavenging solution colourlessly repeatedly with the D-Hanks liquid of 37 ℃ of incubations, guarantee that red corpuscle cleans up.
2. add a Collagen Type VI enzyme with the preparation of D-Hanks liquid to final concentration 0.2g/100ml, 37 ℃ down 100 commentariess on classics/min concussion digested 1 hour
3. will digest after product centrifugal 5min under 4 ℃, 450g condition; 10ml fat stem cell substratum is resuspended, removes impurity with 100 μ m aperture membrane filtrations and finally obtains fat stem cell.
4. fat stem cell suspension and erythrocyte cracked liquid were mixed with 1: 1 and hatched 2 minutes, centrifugal 5min under 4 ℃, centrifugal force 450g condition utilizes the resuspended fat stem cell of fat stem cell substratum.
5. get 100 μ l isolated cells and carry out cell counting with cell counting count board; Take out 100 μ l cells simultaneously and expect that by tongue blue painted method carries out viable count: the result shows and obtains about 1.2*10 altogether
7Individual cell, the motility rate of cell is more than 95%.
With cell according to 3*10
4/ cm
2Density be inoculated in the T-75 culturing bottle 37 ℃, CO
2Cultivate under concentration 5%, humidity 100% condition.Cultivate microscopically observation after 2 days, cell state is good, as Fig. 1.
7. separate obtaining fat stem cell, can be divided into adipocyte, scleroblast, sarcoplast, chondroblast, have multidirectional differentiation potential through inducing differentiation detection confirmation.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (6)
1. the method for a separating mesenchymal stem cell from fatty tissue is characterized in that, comprises the steps:
(1) acquisition of fatty tissue: fatty tissue is the hospital of specialty or the waste that the beauty salon obtains by swelling method liposuction procedures, and under aseptic condition, 4-20 ℃ of storage was less than 48 hours;
(2) tentatively remove red corpuscle: after layering is treated in the static placement of liposuction thing, carefully remove lower floor's liquid, repeatedly limpid with the washing of D-Hanks liquid to elutant;
(3) digestion liposuction thing: add a Collagen Type VI enzyme with the preparation of D-Hanks liquid to 0.01-2g/100ml, 37 ℃ of following 20-400 commentariess on classics/min concussions digested 15-120 minute;
(4) acquisition of fat stem cell: will digest after product centrifugal 5min under 4 ℃, centrifugal force 450g condition, the fat stem cell substratum is resuspended, removes impurity with 100 μ m aperture membrane filtrations and finally obtains fat stem cell;
(5) remove red corpuscle once more, utilize the mode of repetitive scrubbing and erythrocyte splitting to remove red corpuscle once more;
(6) fat stem cell counting, the active detection and cultivation: get 100 μ l isolated cells and carry out cell counting with cell counting count board; Take out 100 μ l cells simultaneously and expect that by tongue blue painted method carries out viable count, according to the result of two countings according to 3*10
4/ cm
2Density be inoculated in the T-75 culturing bottle 37 ℃, CO
2Cultivate under concentration 5%, humidity 100% condition.
2. according to claim 1 from fatty tissue the method for separating mesenchymal stem cell, it is characterized in that, in the step (1), the fatty tissue of obtaining is put into biological shipping bags or the sterilization reagent bottle stores.
3. according to claim 1 from fatty tissue the method for separating mesenchymal stem cell, it is characterized in that in the step (1), under aseptic condition, 4 ℃ of storages maintained in the transportation under 4 ℃ of environment less than 4 hours.
4. the method for a separating mesenchymal stem cell from fatty tissue is characterized in that, comprises the steps:
(1) acquisition of fatty tissue: the fatty tissue waste of fatty tissue for obtaining in the operation, under aseptic condition, 4-20 ℃ of storage was less than 48 hours;
(2) tentatively remove red corpuscle: the fatty tissue that operation obtains shreds into about 1mm at first as far as possible
3The size tissue block is repeatedly limpid to elutant with the washing of D-Hanks liquid then;
(3) digestion liposuction thing: add a Collagen Type VI enzyme with the preparation of D-Hanks liquid to 0.01-2g/100ml, 37 ℃ of following 20-400 commentariess on classics/min concussions digested 15-120 minute;
(4) acquisition of fat stem cell: will digest after product centrifugal 5min under 4 ℃, centrifugal force 450g condition, the fat stem cell substratum is resuspended, removes impurity with 100 μ m aperture membrane filtrations and finally obtains fat stem cell;
(5) remove red corpuscle once more, utilize the mode of repetitive scrubbing and erythrocyte splitting to remove red corpuscle once more;
(6) fat stem cell counting, the active detection and cultivation: get 100 μ l isolated cells and carry out cell counting with cell counting count board; Take out 100 μ l cells simultaneously and expect that by tongue blue painted method carries out viable count, according to the result of two countings according to 3*10
4/ cm
2Density be inoculated in the T-75 culturing bottle 37 ℃, CO
2Cultivate under concentration 5%, 100% humidity condition.
5. according to claim 4 from fatty tissue the method for separating mesenchymal stem cell, it is characterized in that, in the step (1), the fatty tissue of obtaining is put into biological shipping bags or the sterilization reagent bottle stores.
6. according to claim 4 from fatty tissue the method for separating mesenchymal stem cell, it is characterized in that in the step (1), under aseptic condition, 4 ℃ of storages maintained in the transportation under 4 ℃ of environment less than 4 hours.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329773A (en) * | 2011-10-14 | 2012-01-25 | 成都清科生物科技有限公司 | Method for separating adipose-derived mesenchymal stem cells from tumescent liquid |
| CN102443566A (en) * | 2012-01-20 | 2012-05-09 | 天津和泽干细胞科技有限公司 | Acquisition method of adipose-derived stem cells |
| CN104164403A (en) * | 2014-07-25 | 2014-11-26 | 大连大学 | Method for extracting and culturing adipose-derived stem cells |
| 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 |
| CN105168251A (en) * | 2015-09-09 | 2015-12-23 | 广州赛莱拉干细胞科技股份有限公司 | Stem cell preparation as well as preparation method and application thereof |
| CN105754929A (en) * | 2014-12-16 | 2016-07-13 | 西比曼生物科技（上海）有限公司 | Reagent combination for efficiently quickly extracting karyocytes |
| CN106222134A (en) * | 2016-07-29 | 2016-12-14 | 中卫华医（北京）生物科技有限公司 | The cultural method of effective acquisition fat mesenchymal stem cell |
| CN106754687A (en) * | 2017-02-06 | 2017-05-31 | 贵州泛特尔细胞生物技术有限公司 | A kind of fat stem cell isolated culture method |
| CN107828726A (en) * | 2017-12-15 | 2018-03-23 | 北京焕生汇生物技术研究院 | A kind of method of the fractionation of fatty mescenchymal stem cell from fat |
| CN109837241A (en) * | 2019-03-20 | 2019-06-04 | 济南赛尔生物科技股份有限公司 | The separation of fat stem cell and cultural method |
| CN110079497A (en) * | 2019-03-20 | 2019-08-02 | 济南赛尔生物科技股份有限公司 | Method for separating and culturing adipose-derived stem cells |
| CN112063583A (en) * | 2020-11-16 | 2020-12-11 | 广州杜德生物科技有限公司 | Method for efficiently separating and extracting adipose-derived mesenchymal stem cells from adipose tissue |
| CN115125196A (en) * | 2022-08-24 | 2022-09-30 | 蚌埠医学院第一附属医院（蚌埠医学院附属肿瘤医院） | Adipogenic induction differentiation method for 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 |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329773A (en) * | 2011-10-14 | 2012-01-25 | 成都清科生物科技有限公司 | Method for separating adipose-derived mesenchymal stem cells from tumescent liquid |
| CN102443566A (en) * | 2012-01-20 | 2012-05-09 | 天津和泽干细胞科技有限公司 | Acquisition method of adipose-derived stem cells |
| CN104164403A (en) * | 2014-07-25 | 2014-11-26 | 大连大学 | Method for extracting and culturing adipose-derived stem cells |
| 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 |
| CN105754929A (en) * | 2014-12-16 | 2016-07-13 | 西比曼生物科技（上海）有限公司 | Reagent combination for efficiently quickly extracting karyocytes |
| CN105168251A (en) * | 2015-09-09 | 2015-12-23 | 广州赛莱拉干细胞科技股份有限公司 | Stem cell preparation as well as preparation method and application thereof |
| CN106222134A (en) * | 2016-07-29 | 2016-12-14 | 中卫华医（北京）生物科技有限公司 | The cultural method of effective acquisition fat mesenchymal stem cell |
| CN106754687A (en) * | 2017-02-06 | 2017-05-31 | 贵州泛特尔细胞生物技术有限公司 | A kind of fat stem cell isolated culture method |
| CN107828726A (en) * | 2017-12-15 | 2018-03-23 | 北京焕生汇生物技术研究院 | A kind of method of the fractionation of fatty mescenchymal stem cell from fat |
| CN109837241A (en) * | 2019-03-20 | 2019-06-04 | 济南赛尔生物科技股份有限公司 | The separation of fat stem cell and cultural method |
| CN110079497A (en) * | 2019-03-20 | 2019-08-02 | 济南赛尔生物科技股份有限公司 | Method for separating and culturing adipose-derived stem cells |
| CN109837241B (en) * | 2019-03-20 | 2022-05-17 | 济南赛尔生物科技股份有限公司 | Method for separating and culturing adipose-derived stem cells |
| CN112063583A (en) * | 2020-11-16 | 2020-12-11 | 广州杜德生物科技有限公司 | Method for efficiently separating and extracting adipose-derived mesenchymal stem cells from adipose tissue |
| CN112063583B (en) * | 2020-11-16 | 2021-03-23 | 广州杜德生物科技有限公司 | Method for efficiently separating and extracting adipose-derived mesenchymal stem cells from adipose tissue |
| CN115125196A (en) * | 2022-08-24 | 2022-09-30 | 蚌埠医学院第一附属医院（蚌埠医学院附属肿瘤医院） | Adipogenic induction differentiation method for adipose-derived stem cells |
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