CN103087982A - Kit and method capable of quickly separating adipose tissue-derived stem cells - Google Patents
Kit and method capable of quickly separating adipose tissue-derived stem cells Download PDFInfo
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- CN103087982A CN103087982A CN201110330948XA CN201110330948A CN103087982A CN 103087982 A CN103087982 A CN 103087982A CN 201110330948X A CN201110330948X A CN 201110330948XA CN 201110330948 A CN201110330948 A CN 201110330948A CN 103087982 A CN103087982 A CN 103087982A
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Abstract
The invention provides a kit and a method capable of quickly separating adipose tissue-derived stem cells. The kit comprises a washing liquid, a cell culture fluid A, a cell culture medium B and a cell digestive juice, wherein the washing fluid is normal saline (containing 0.9% sodium chloride); the cell culture medium A is a Knockout-DMEM liquid medium containing 100 ng/mL LIF and 100 ng/mL bFGF cytokines; the cell culture medium B is fetal bovine serum; and the cell digestive juice is a collagenase type I enzyme solution with a mass to volume percent concentration of 0.2%. Compared with a conventional separation culture method, the cells obtained by the kit have high purity and fast propagation. Generally, a primary cell culture can be completed in seven days; the primary cells are sub-cultured by using the culture solution, and cell density can reach 90% in 2-3 days. The kit and the method provide a rich source for further experimental study by using the adipose tissue-derived stem cells.
Description
Technical field
The invention belongs to field of tissue engineering technology, relate to a kind of test kit of separating out fat mescenchymal stem cell, the application of this test kit and a kind of method of separating out fat mescenchymal stem cell.
Background technology
Fat mesenchymal stem cell is the stem cell that a class has self, propagation and multi-lineage potential, has the body of being derived from, and draws materials extensively, is easy to collection, storage and transport, repels, avoids the plurality of advantages such as dispute of ethic without allosome.Flow cytometry analysis is found fat mesenchymal stem cell high expression level mesenchymal cell sign (CD44, CD105), integrin receptor (CD29, CD49b, CD49c, CD51), and not expressing hematopoiesis is sign (CD34, CD45) and endothelial cell marker CD31.Fat mesenchymal stem cell can be divided into osteocyte, chondrocyte, liver cell and adipocyte etc. in vivo and in vitro.Human adipose mesenchymal stem cells gos deep in the clinical application researchs such as organization engineering skin and cell therapy gradually at present, and has demonstrated wide application prospect.
At present, the separation and Culture of fat mesenchymal stem cell, amplification are not also had unified standard, have the shortcomings such as purity is low, primary incubation time is long.Various types of one-tenth fiber adult stem cell amplifications generally began to enter exponential growth at 2～3 days, be more or less the same, and primary cell the cell clone time difference occurs larger due to tissue-derived difference, so the primary cell generation time is to separate the key that obtains the purpose cell.
Summary of the invention
In order to solve purity, quantity and the primary incubation time problem of fat mesenchymal stem cell separation, the invention provides a kind of test kit of separating out fat mescenchymal stem cell.This test kit is purifying Mammals fat mesenchymal stem cell effectively, accelerates the primary cell amplification rate.Obtain the significant quantities of fat mescenchymal stem cell, be beneficial to carry out next step experimental study.
According to an aspect of the present invention, the test kit of separating out fat mescenchymal stem cell of the present invention comprises washings, cell culture fluid A, and cell culture fluid B and cell dissociation buffer, wherein:
Described cell washing liquid is physiological saline;
Described cell culture fluid A is for containing the Knockout-DMEM liquid nutrient medium (Gibco, 12660012) of 100ng/ml LIF (Sigma, L5158) and 100ng/ml bFGF (Sigma, F5392) cytokine;
Described cell culture fluid B is foetal calf serum;
Described cell dissociation buffer is collagenase I type enzyme liquid (Sigma, C-0130).
According to an aspect of the present invention, described fat mesenchymal stem cell is derived from Mammals.
According to an aspect of the present invention, described washings, nutrient solution A, nutrient solution B and cell dissociation buffer are aseptic.
According to an aspect of the present invention, described washings, nutrient solution A, nutrient solution B and cell dissociation buffer are independent packaging.
According to an aspect of the present invention, the cultured cells nutrient solution that is used for fat mesenchymal stem cell is the cell cultures mixed solution that cell culture fluid A and cell culture fluid B are prepared according to 4: 1 volume ratios.
According to an aspect of the present invention, described cell washing liquid contains 0.9% sodium-chlor, and it is 0.2% that described cell dissociation buffer can be the mass/volume percentage concentration.
Another object of the present invention is to provide a kind of method of separating out fat mescenchymal stem cell.The method is processed fatty tissue with test kit inner cell Digestive system, through the former culture of cell culture fluid A and B, and the final fat mesenchymal stem cell that obtains separation.
According to an aspect of the present invention, the method for described separating out fat mescenchymal stem cell can realize by following step:
Method of the present invention adopts collagenase I type enzyme effectively to process the lipoprotein colloid, utilizes cytokine (LIF, bFGF) combination to the promoter action of fat mesenchymal stem cell, thereby has guaranteed that primary cell separates quantity and reduces primary incubation time.
According to an aspect of the present invention, the method for described separating out fat mescenchymal stem cell can comprise:
1, fatty pre-treatment:
Aseptic collection fat is immersed in the cell culture fluid (A and B) that contains 1% pair anti-(mycillin), and the Glass Containers sealing is transported to the laboratory; In Biohazard Safety Equipment, approximately 1g is fatty for clip, with the blood stains in physiological saline cleaning fat.Shred fatty tissue with scissors and become rotten shape.
2, isolated cell:
To remain fatty tissue and shred into the meat gruel shape, transfer in centrifuge tube, add the 20ml cell dissociation buffer, 37 ℃ of digested overnight of mixing approximately 6 hours.Centrifugal, use the cell culture fluid re-suspended cell, the inoculation primary cell is put in 37 ℃, 5%CO2 incubator and is cultivated in the coated culturing bottle of 0.2% gelatin.72 as a child, and visible adherent one-tenth fibrous cell is Scroll-tupe growth (seeing Fig. 1).
Description of drawings
Fig. 1. separate the fat mesenchymal stem cell that obtains
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit the present invention with this.
Embodiment 1, for separating of the preparation of the test kit of fat mesenchymal stem cell
1, washings: get 9g NaCl and be settled to 1000ml with deionized water, autoclaving makes 0.9% sodium-chlor washings, is placed in 4 ℃ of refrigerators standby.
2, cell culture fluid: contain the Knockout-DMEM liquid nutrient medium of 100ng/ml LIF and 100ng/ml bFGF cytokine and aseptic foetal calf serum nutrient solution according to cell cultures mixed solution used in the volume ratio mixed preparing cell cultures of 4: 1.
3, cell dissociation buffer: preparation mass/volume percentage concentration is 0.2% collagenase I type enzyme solution.
Embodiment 2, for separating of the packing of the test kit of fat mesenchymal stem cell
The specification of test kit is 1 time/box, the amount of each component in every box: 1 bottle of washings (100ml/ bottle), cell training
Support liquid A1 bottle (80ml/ bottle), 1 bottle of cell culture fluid B (20ml/ bottle), 1 bottle of cell dissociation buffer (100ml/ bottle).Connect
Above-mentioned dosage carries out independent packaging to each component in test kit, obtains the test kit of separating out fat mescenchymal stem cell.
The separation of embodiment 3, fat mesenchymal stem cell and amplification cultivation
Aseptic collection fat is immersed in the cell culture fluid (A and B) that contains 1% pair anti-(mycillin), and the Glass Containers sealing is transported to the laboratory; The about fat of 1g of clip in Biohazard Safety Equipment cleans blood stains in fatty medium vessels with physiological saline.Fatty tissue is shredded into the meat gruel shape, transfer in centrifuge tube, add the 20ml cell dissociation buffer, 37 ℃ of digested overnight of mixing approximately 6 hours.Centrifugal, use the cell culture fluid re-suspended cell, the inoculation primary cell is put in 37 ℃, 5%CO2 incubator and is cultivated in the coated culturing bottle of 0.2% gelatin.
Claims (10)
1. the test kit of a separating out fat mescenchymal stem cell comprises:
Washings, it is the physiological saline that contains 0.9% sodium-chlor;
Nutrient solution A, it is the Knockout-DMEM liquid nutrient medium that contains 100ng/ml LIF and 100ng/ml bFGF cytokine;
Nutrient solution B, it is foetal calf serum;
Cell dissociation buffer, it is collagenase I type enzyme liquid.
2. test kit according to claim 1, it is characterized in that: described washings, nutrient solution A, nutrient solution B and cell dissociation buffer are aseptic.
3. test kit according to claim 1, it is characterized in that: described washings, nutrient solution A, nutrient solution B and cell dissociation buffer are independent packagings.
4. test kit according to claim 1, is characterized in that, described fat mesenchymal stem cell is derived from Mammals.
5. test kit according to claim 1, it is characterized in that: the technical indicator of described nutrient solution B is as follows: pH (7.0-7.5), total protein content (3-3.5/100ml), oxyphorase (≤20mg/100ml).
6. test kit claimed in claim 1, it is characterized in that: described nutrient solution A and nutrient solution B recently provide with the volume of 4: 1.
7. test kit according to claim 1, it is characterized in that: described physiological saline is to contain 0.9% sodium chloride solution, described cell dissociation buffer is the collagenase I type enzyme liquid of mass/volume percentage concentration 0.2%.
8. the force method of a separating out fat mescenchymal stem cell comprises:
(1) blood stains in utilizing in washings flushing fat, scratch exterior skin with scalper at a fatty end (from 1cm place, top) circumference, with this end of clip fixed fat, with two tweezers fatty exterior skin of firmly tearing, separate again two radicular arterieses, at last venous endothelial is rejected;
(2) will remain fatty tissue and shred into the meat gruel shape, transfer in centrifuge tube, subsequently with cell dissociation buffer digestion (37 ℃ digested 6 hours);
(3) postdigestive liquid in the sucking-off centrifuge tube.Remove its hetero-organization and impurity.
(4) liquid of sucking-off is through whizzer centrifugal (2500rpm, 5 minutes).After centrifugal, pour out substratum, use the cell culture fluid re-suspended cell.
(5) primary cell of inoculation is in the coated culturing bottle of 0.2% gelatin, puts in 37 ℃, 5%CO2 incubator and cultivates.
Wherein washings is physiological saline;
Nutrient solution A and the nutrient solution B of cell cultures mixed solution for recently preparing according to 9: 1 volumes, wherein nutrient solution A is the Knockout-DMEM liquid nutrient medium, nutrient solution B is foetal calf serum;
And cell dissociation buffer collagenase I type enzyme liquid.
(6) every day observation of cell adherent growth situation.The most adherent rear full doses of cell are changed liquid, remove not attached cell and impurity, and amount was changed cell culture fluid in later every 3～4 days half.
9. method according to claim 8, wherein in step (5), adopt every 106 cells to be inoculated in the 25m2 culturing bottle.
10. method according to claim 8, wherein in step (6), the most adherent rear full doses of 24 hour cells are changed liquid, remove not attached cell and impurity.
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Cited By (8)
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| CN104928238A (en) * | 2014-03-21 | 2015-09-23 | 青岛中天干细胞工程有限公司 | Adipose mesenchymal stem cell preparation kit |
| CN105624106A (en) * | 2016-03-31 | 2016-06-01 | 崔长友 | Kit for culturing mesenchymal stem cells |
| CN105647861A (en) * | 2016-03-31 | 2016-06-08 | 赵慧慧 | Kit for culturing stem cells |
| CN105713872A (en) * | 2016-03-31 | 2016-06-29 | 赵顺英 | MSC (mesenchymal stem cell) culture kit |
| 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 |
| CN105779385A (en) * | 2016-03-31 | 2016-07-20 | 谷超 | Mesenchymal stem cell culture kit |
| CN106047702A (en) * | 2016-08-15 | 2016-10-26 | 北京昱龙盛世生物科技有限公司 | Kit for culture of adipose-derived stem cells and culture method |
| CN108300689A (en) * | 2018-01-16 | 2018-07-20 | 佛山科学技术学院 | A method of separation and primary culture placental decidua vera mescenchymal stem cell |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928238A (en) * | 2014-03-21 | 2015-09-23 | 青岛中天干细胞工程有限公司 | Adipose mesenchymal stem cell preparation kit |
| 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 |
| CN105624106A (en) * | 2016-03-31 | 2016-06-01 | 崔长友 | Kit for culturing mesenchymal stem cells |
| CN105647861A (en) * | 2016-03-31 | 2016-06-08 | 赵慧慧 | Kit for culturing stem cells |
| CN105713872A (en) * | 2016-03-31 | 2016-06-29 | 赵顺英 | MSC (mesenchymal stem cell) culture kit |
| CN105779385A (en) * | 2016-03-31 | 2016-07-20 | 谷超 | Mesenchymal stem cell culture kit |
| CN106047702A (en) * | 2016-08-15 | 2016-10-26 | 北京昱龙盛世生物科技有限公司 | Kit for culture of adipose-derived stem cells and culture method |
| CN106047702B (en) * | 2016-08-15 | 2018-07-20 | 北京昱龙盛世生物科技有限公司 | A kind of kit and cultural method for cultivating fat stem cell |
| CN108300689A (en) * | 2018-01-16 | 2018-07-20 | 佛山科学技术学院 | A method of separation and primary culture placental decidua vera mescenchymal stem cell |
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Addressee: Beijing QMLC Stem Cell Technology Co., Ltd. Document name: Notification of Publication and of Entering the Substantive Examination Stage of the Application for Invention |
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Address after: 100084, C building, Tsinghua Science and technology building, No. 1 Zhongguancun East Road, Beijing, Haidian District, 301 Applicant after: Beijing QMLC Stem Cell Technology Co., Ltd. Address before: 100097 Beijing city Haidian District minzhuang Road No. 3, Tsinghua Science Park building 204 Yuquan Huigu 7 Applicant before: Beijing QMLC Stem Cell Technology Co., Ltd. |
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Application publication date: 20130508 |