CN102433299A - Method for separating, culturing and purifying mouse adipose-derived stem cells - Google Patents
Method for separating, culturing and purifying mouse adipose-derived stem cells Download PDFInfo
- Publication number
- CN102433299A CN102433299A CN2011103661286A CN201110366128A CN102433299A CN 102433299 A CN102433299 A CN 102433299A CN 2011103661286 A CN2011103661286 A CN 2011103661286A CN 201110366128 A CN201110366128 A CN 201110366128A CN 102433299 A CN102433299 A CN 102433299A
- Authority
- CN
- China
- Prior art keywords
- cell
- tissue
- fatty tissue
- separation
- digestive system
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for separating, culturing and purifying mouse adipose-derived stem cells, which comprises: a cell separation stage, a cell culture stage and a cell purification stage. Particularly in a separation process, nutrition is supplied continuously, times for three times of liquid exchange are changed, and the cell purity is improved by using a differential digestion process. The method can separate high-activity and high-purity adipose-derived stem cells from the adipose tissue of a mouse, and lays a functional for subsequent further researches on adipose-derived stem cells.
Description
[technical field]
The present invention relates to the separation method of stem cell animal, relate in particular to the method for the separation of a kind of mouse fat stem cell, cultivation and purifying.
[background technology]
Fat stem cell has the potential of multidirectional differentiation; External orientable be divided into fat, skeletonization, cartilage, liver, myocardial cell etc.; Aspect Ren Yuan; Than bone marrow stem cell, its have draw materials easily, the amount of obtaining is big, reduce donor when drawing materials painful advantage, therefore on regenerative medicine, have great importance.Mouse is as an important model animal; Aspect fundamental research, have a wide range of applications; But the vitro culture of mouse fat stem cell but is a difficult problem always, relates to all respects such as used Digestive system, nutrient solution, and separation method of being taked and cultural method.For the method for available data, numerous and complicated mixed and disorderly, find that after actually operating effect is not an ideal very, its weak point is: operability is not strong, and the improved place of needs is arranged.The present invention is directed to some problems that occur in the practice process for this reason; On relevant link, some improvement have been done; Through repeatedly attempting, formed one more perfect relatively, the digestion separation system of workable mouse fat stem cell with separate the cultural method of cell afterwards.
[summary of the invention]
The technical problem that the present invention will solve provides the method for the separation of a kind of mouse fat stem cell, cultivation and purifying.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of separation of mouse fat stem cell, cultivation and purification process may further comprise the steps:
One, the separation of cell
(1) draws materials: get the male mouse of ICR in 4 ages in week, put to death, to its whole body sterilization;
(2) cut the fatty tissue at inguinal region place under the aseptic condition, put into the DPBS liquid that contains penicillium mould and Streptomycin sulphate and clean blood stains;
(3) blood vessel in the removal fatty tissue, washing changes in the new petridish once more; Fully shred, add Digestive system (10%FBS, 0.09% collagenase I; All the other compositions are DMEM/F12); Pipettor with being connected with the 1ml suction nozzle is is fully blown and beaten, until tissue can be very unobstructed the discrepancy suction nozzle, fatty tissue is changed in the centrifuge tube; Record adds the amount of Digestive system and the volume of final suspension, calculating the amount of fatty tissue, and make the volume of final Digestive system be fatty tissue 2-3 doubly;
(4) 37 ℃ of digestion 1h regularly shake, and tissue block is fully contacted with Digestive system;
(5) digested after, blow and beat Digestive system repeatedly, dispersion tissue's piece is processed cell suspension;
(6) low-speed centrifugal 5min blows and beats 1min at least, recentrifuge repeatedly with the fatty tissue on upper strata;
(7) discard the Digestive system in upper strata mature fat cell and middle level, use the cell of the resuspended bottom of basic medium again;
(8) cell suspension is filtered with nylon leaching net, gained filtrating is filtered once more, removes the majority of mature adipocyte, collects and filtrates, and filters the back at every turn and notes washing filter screen with an amount of basic medium again, and cell is residual on the minimizing filter screen;
The filtrating that (9) will obtain at last is centrifugal, inhales and to abandon the buoyant adipocyte, with basic medium repetitive scrubbing 3 times;
Two, cell cultures
(10) last centrifugal back is resuspended with perfect medium, inoculates, place the incubator normal condition (37 ℃, 5%CO
2, 100% humidity) cultivate;
(11) fatty tissue of every 0.4-0.6ml separates the cell inoculation obtain in a hole of six orifice plates;
(12) change liquid first: add DPBS behind the former pickup kind 2h, repetitive scrubbing 5-6 time, most of heteroproteose cell of flush away and tissue block add fresh perfect medium and continue to cultivate;
(13) change liquid for the second time: after changing liquid 24h first it is carried out the washing second time, further flush away is attached cell and tissue block not, adds fresh perfect medium and continues to cultivate;
(14) change liquid for the third time: change first behind the liquid and wash for the third time behind the 72h, clean basically until the cell face, add fresh perfect medium and continue to cultivate; Per 3 days half of later stage amount is changed liquid once, grows to 90% and completely promptly goes down to posterity;
Three, purifying
(15) after cell covers with, add trysinization 2min, will digest the cell transfer and the termination of getting off rapidly, centrifugal resuspended back is inserted and is continued in the new ware to cultivate, and cell residual at the bottom of the preceding ware then discards; Utilize this method repeatedly to go down to posterity, reduce the ratio of heteroproteose cell gradually, thereby reach the purpose of purifying.
As preferably, the concentration of the said penicillium mould of step (2) is 400IU/ml; The concentration of said Streptomycin sulphate is 400 μ g/ml.
As preferably, cut out the resuspended and inoculation of the last centrifugal back of fatty tissue to step (10) from step (2) with perfect medium, whole process time is controlled in the 4h.
As preferably, in the step (8), using the aperture earlier is the nylon leaching net filtration cell suspension of 250 μ m, and again gained filtrating being used the aperture is that the nylon leaching net of 80 μ m filters.
As preferably, the moity of perfect medium is: foetal calf serum 20%, penicillium mould 100IU/ml; Streptomycin sulphate 100 μ g/ml, L-glutaminate 2mmol/L, Prostatropin 10ng/ml; Vitamins C 50 μ g/ml, DMEM/F12 supplies 100%.
As preferably, the moity of basic medium is: foetal calf serum 20%, and penicillium mould 100IU/ml, Streptomycin sulphate 100 μ g/ml, L-glutaminate 2mmol/L, DMEM/F12 supplies 100%.
Compare prior art, the present invention has mainly made improvement aspect following three:
One, interrupts the supply of nutrition in whole sepn process
What in more existing separable programmings, have has promptly interrupted nutrition supply in digestive process or in the washing process; Because whole separable programming required time is very long, this way possibly be unfavorable for keeping of later stage cytoactive, for this reason; We provide nutrition to cell in whole separable programming; The basic ingredient that contains cell growth in the Digestive system, cell washing are also with the substratum washing but not the DPBS that reports in the relevant document farthest keeps cell activity.
Two, the liquid program is changed in washing:
Exist a large amount of hemocytes and indigested tissue, the especially latter in the former foster fat stem cell of being commissioned to train and swim in fluid surface and be difficult to remove, hemocyte is if exist cracking subsequently always, and the objectionable impurities of generation can influence the growth activity of cell; It is too early to change liquid, and cell is not adherent fully as yet, not only influences the pick-up rate of fat stem cell, also can cause serious physical abuse to it simultaneously; It is slow excessively to change liquid, and the disadvantageous effect after the hemocyte cracking will embody, and simultaneously some indigested tissue block also can be adherent and be climbed out of cell, thereby influences the purity of cell, therefore changes liquid first and washs and change the accurate assurance of liquid time and seem most important.Yet change liquid first and change the problem that liquid is all considered physical abuse for the second time; Can not overwass; So also can residual a part of heteroproteose cell and tissue after twice washing; Can treat abundant adherent then it washing for the third time of cell, not have tangible impurity to add fresh perfect medium again until the cell face and continue to cultivate.
Change the liquid program through it being carried out above-mentioned washing, progressively reduce the ratio of heteroproteose cell, reduced physical abuse simultaneously, thereby better keep mouse fat stem cell growth activity fat stem cell in the suitable time; 3 washings are changed behind the liquid heteroproteose cell and are removed with organizing basically, and the long-term cultivation that is beneficial to the later stage covers with until cell and goes down to posterity.
Three, the purifying of cell: have various kinds of cell in the fatty tissue; Various cells such as epithelial cell, mature fat cell, fatty parent cell, hemocyte are arranged; Especially endotheliocyte is mainly derived from vessel wall, in separable programming, has the operation of rejecting blood vessel can reduce the introducing of heteroproteose cell.In any case but operation all can more or less have heteroproteose cell to get into culture system; For this reason; We are behind the biological characteristics of having understood fat stem cell and heteroproteose cell, and main trysinization temporal differences required when going down to posterity according to the two has been found out a suitable digestion time; Utilize the differential digestion method that heteroproteose cell and fat stem cell are separated, repeatedly go down to posterity and finally obtain highly purified fat stem cell.Facts have proved that the cell that goes out through this method separation and Culture can be good at keeping the correlation properties of fat stem cell, the fat stem cell purity that obtains simultaneously is very high.
The invention has the beneficial effects as follows:
Can from the fatty tissue of mouse, isolate high reactivity, highly purified fat stem cell, for the later stage lays a good foundation to the further research of fat stem cell.
[embodiment]
(1) separation of cell
1. draw materials: get the male mouse of ICR in 4 ages in week, 75% alcohol whole body soaking disinfection 5min is put to death in the cervical vertebra dislocation; Have report to show the age of donor to the differentiation potential important influence in fat stem cell later stage, we select the mouse in this age in week for this reason.Facts have proved that this mouse separates the Study on Differentiation that the fat stem cell that obtains can well be used for the later stage in stage.
2.75% alcohol disinfecting belly, the low level cross sections is opened the abdominal cavity, under aseptic condition, cuts the fatty tissue at inguinal region place rapidly, when getting fatty tissue, will note distinguishing and discard lymphoglandula, in order to avoid influence the purity of later stage cell.Putting into the DPBS that contains two anti-(400IU/ml penicillium mould, 400 μ g/ml Streptomycin sulphates) washes 3 times.
3. scalpel and tweezers are used, and remove the blood vessel in the fatty tissue.
The concrete operations step is following:
(1) will be soaked in first absorption a moment at the bottom of ware of fatty tissue among the DPBS, remove the liquid on surface, with make things convenient for that the later stage fatty tissue can be firm be attached to ware at the bottom of, be convenient to next step operation.
(2) fatty tissue that will fully remove DPBS is transferred in the new ware, and drawout exposes the lines of blood vessel, and it managed straight as far as possible.
(3) with the parallel placement of scalpel with blood vessel, do not contact blood vessel near blood vessel as far as possible, along the lines cutting-out of blood vessel, at the opposite side of scalpel avascular tissue is torn with tweezers.
(4) opposite side that again scalpel is parallel to blood vessel downcuts, and the bits of tissue that will contain blood vessel with tweezers is torn, the blood vessel in just can effectively removal tissue, and this method is especially effective for the microvascular removal in organizing.
4. the fatty tissue that will remove behind the blood vessel washs 3 times again; Change in the new ware, inhale and remove remaining DPBS, fatty tissue is fully shredded with sterile scissors; Add a spot of Digestive system; Pipettor with being connected with the 1ml suction nozzle is is fully blown and beaten, until tissue can be very unobstructed the discrepancy suction nozzle, again fatty tissue is changed in the centrifuge tube;
5. add a certain amount of Digestive system washing suction nozzle and ware again; Fully change in the centrifuge tube residual fatty tissue as far as possible; Record adds the amount of Digestive system and the volume of final suspension, calculating the amount of fatty tissue, and make the volume of final Digestive system be fatty tissue 2-3 doubly.
6.37 ℃ digestion 1h, during every take out centrifuge tube at a distance from 5min, shake, make the abundant mixing of tissue block and Digestive system;
7. after having digested, blow and beat postdigestive suspension repeatedly with suction pipe, dispersion tissue's piece is processed cell suspension;
8.1200rpm centrifugal 5min blows and beats 1min at least with pipettor with the fatty tissue on upper strata repeatedly, recentrifuge, and this step can help improving the yield of fat stem cell.
9. discard the Digestive system in upper strata mature fat cell and middle level, use the cell of the resuspended bottom of basic medium again.Note when drawing the middle level Digestive system drawing from the surface of liquid, can help like this inhaling the adipocyte that swims in fluid surface, cell will fully be blown and beaten into individual cells when resuspended, otherwise the later stage can influence the yield of cell when filtering.
10. using the aperture earlier is the nylon leaching net filtration cell suspension of 250 μ m, removes not dispersive tissue block, collects filtrating.It is the nylon leaching net filtration of 80 μ m that filtrating is used the aperture again, removes the majority of mature adipocyte, collection filtrating, and attention with the basic medium washing filter screen of proper amount of fresh, reduces remaining in the cell on the filter screen again after each the filtration.
(1200rpm 5min), inhales and to abandon the buoyant adipocyte, with basic medium repetitive scrubbing 3 times 11. the filtrating that will obtain at last is centrifugal.It is ditto said that the method for upper strata suspension is abandoned in suction; The abundant wash clean of adipocyte is very important; Have report show residual adipocyte and fat stem cell altogether cultivation can cause the differentiation of fat stem cell, so the flush away adipocyte to keep for the dryness of fat stem cell be crucial.
(2) cell cultures
12. last centrifugal back is resuspended with perfect medium, inoculates, place the incubator normal condition (37 ℃, 5%CO
2, 100% humidity) cultivate.
13. contain a lot of hemocytes owing to separate in the cell obtain, and the hemocyte quantity that finally obtains when operating differs greatly, therefore confirms that through Cytometric method inoculum density is that ten minutes is inaccurate at every turn.The amount of the fat stem cell that contains in a certain amount of fatty tissue in the mouse in same strain, same age in week generally is metastable, and we come the inoculum density of final decision cell with the amount of fatty tissue for this reason.Through finding: it is proper in a hole of six orifice plates that the fatty tissue about every 0.4-0.6ml separates the cell inoculation that obtains, and can cover with in general 5-7 days.
14. change the light ware face of crossing of liquid first, can not acutely shake back and forth, light tiltedly culture plate is inhaled and is abandoned DPBS, and 5-6 time repeatedly, this moment is flush away part heteroproteose cell only, also has quite the heteroproteose cell of the amount of being not remove, but can not wash excessively, prevents that pair cell from causing damage, and changes liquid;
15. for the second time change liquid: change first behind the liquid 24h it is carried out washing second time and change liquid, thorough washing once more, method is the same; Further flush away is attached cell not, and changes liquid, and still have more heteroproteose cell to residue in the ware this moment; But cell is fully not adherent yet, therefore can not too much wash yet.
16. change liquid for the third time: change first to wash for the third time behind the 72h behind the liquid and change liquid, this moment, cell was fully adherent, but thorough washing is clean until the cell face.Per 3 days half of later stage amount is changed liquid once, grows to 90% and completely promptly goes down to posterity.
(3) purifying
For obtaining highly purified fat stem cell, the purification process of cell relates generally in sepn process with the later stage and goes down to posterity in the operation, and the purifying front in the sepn process is existing to be detailed, and at this later stage purification process in the process that goes down to posterity is described mainly.
Practice finds that fat stem cell generally can digest down about 2min after adding pancreatin, and heteroproteose cell at this moment between Duan Ze be difficult to digest; Utilize this characteristic; We will digest the cell transfer and the termination of getting off rapidly behind trysinization 2min, centrifugal resuspended back is inserted and continued in the new ware to cultivate; Residual cell then discards at the bottom of the preceding ware, through this method fat stem cell and heteroproteose cell is separated.Certainly, this process also can't be avoided digesting a spot of heteroproteose cell, but the propagation of fat stem cells is rapid; Next utilizes this method through repeatedly going down to posterity; The ratio of heteroproteose cell is reduced gradually, thereby reach the purpose of purifying, in general 3 generations, can obtain quite highly purified cell.
It should be noted that being fetched into of from the 2nd step fatty tissue is last in the 12nd step centrifugally places incubator to cultivate preferably to be controlled in the 4h.
Related experiment below present embodiment has also carried out:
1, for the influence of the time pair cell adherent rate of changing liquid first; We have designed following experiment: support back 1h former being commissioned to train respectively, 2h, 4h, 24h transfer to the suspension on upper strata and continue to cultivate 48h in the new ware; The result finds that the supernatant liquid that changes liquid behind the 1h places a new ware to continue to cultivate; Still have 20% attached cell nearly, then the adherent cell of three can be ignored basically, explains that thus fat stem cell is adherent basically complete in 2h.
2, the cell that obtains with the used Digestive system digestion of present embodiment; At 2h, 24h it is changed liquid first respectively after the former pickup kind; 24h changes the liquid group first and only was maintained to for 17 generations and just stops propagation as a result, the cell serious aging, and it is in good condition during 17 generations to change the liquid group behind the 2h first; Still have higher multiplication capacity, and can be maintained until for 30 generations and go down to posterity still can continuing.
3, utilize the used purification process of present embodiment, with the third generation fat stem cell that obtains, the surface antigen CD44 expression rate that uses flow cytometer to detect the mesenchyme derived cell is 99.75%, CD105 is 68.77%; Multipotential cell surface antigen S ca-1 is 99.01%; Basically conform to existing report, we have also detected the expression rate of heteroproteose cell surface markers simultaneously, and wherein hemopoietic stem cell surface antigens c D45 is 0.25%; Surface endothelial cell antigens CD31 is 0.79%; Inoblast surface antigen HLA-DR is 0.24%, and the ratio of heteroproteose cell is very low, and cell purity is higher.
The fat stem cell of 4, turning out with present embodiment; Become the result of fat and osteogenic induction to show; It can be good at being divided into sophisticated adipocyte and scleroblast external; And control group does not break up, and explains that this culture system can keep its external undifferentiated state, and good vitro differentiation potential is also arranged simultaneously.
Claims (5)
1. the separation of a mouse fat stem cell, cultivation and purification process may further comprise the steps:
One, the separation of cell
(1) draws materials: get the male mouse of ICR in 4 ages in week, put to death, to its whole body sterilization;
(2) cut the fatty tissue at inguinal region place under the aseptic condition, put into the Du Shi phosphate buffered saline buffer (DPBS) that contains penicillium mould and Streptomycin sulphate and clean blood stains;
(3) blood vessel in the removal fatty tissue, washing changes in the new petridish once more, fully shreds, and adds Digestive system, and the moity of Digestive system is 10% foetal calf serum (FBS), 0.09% collagenase I, surplus is DMEM/F12; Pipettor with being connected with the 1ml suction nozzle is is fully blown and beaten, until tissue can be very unobstructed the discrepancy suction nozzle, fatty tissue is changed in the centrifuge tube; Record adds the amount of Digestive system and the volume of final suspension, calculating the amount of fatty tissue, and make the volume of final Digestive system be fatty tissue 2-3 doubly;
(4) 37 ℃ of digestion 1h regularly shake, and tissue block is fully contacted with Digestive system;
(5) digested after, blow and beat Digestive system repeatedly, dispersion tissue's piece is processed cell suspension;
(6) the centrifugal 5min of 1200rpm blows and beats 1min at least, recentrifuge repeatedly with the fatty tissue on upper strata;
(7) discard the Digestive system in upper strata mature fat cell and middle level, use the cell of the resuspended bottom of basic medium again;
(8) cell suspension being used the aperture earlier is the nylon leaching net filtration cell suspension of 250 μ m; Again gained filtrating being used the aperture is the nylon leaching net filtration of 80 μ m; Remove the majority of mature adipocyte; Collect and filtrate, filter the back at every turn and note washing filter screen with an amount of basic medium again, cell is residual on the minimizing filter screen;
The filtrating that (9) will obtain at last is centrifugal, inhales and to abandon the buoyant adipocyte, with basic medium repetitive scrubbing 3 times;
Two, cell cultures
(10) last centrifugal back is resuspended with perfect medium, and inoculation places the incubator normal condition to cultivate;
(11) fatty tissue about every 0.4-0.6ml separates the cell inoculation obtain in a hole of six orifice plates;
(12) change liquid first: add behind the former pickup kind 2h and discard substratum, with DPBS repetitive scrubbing 5-6 time, most of heteroproteose cell of flush away and tissue block add fresh perfect medium continuation cultivation;
(13) change liquid for the second time: after changing liquid 24h first it is carried out the washing second time, further flush away is attached cell and tissue block not, adds fresh perfect medium and continues to cultivate;
(14) change liquid for the third time: change first behind the liquid and wash for the third time behind the 72h, clean basically until the cell face, add fresh perfect medium and continue to cultivate; Per 3 days half of later stage amount is changed liquid once, grows to 90% and completely promptly goes down to posterity;
Three, purifying
(15) after cell covers with, add trysinization 2min, will digest the cell transfer and the termination of getting off rapidly, centrifugal resuspended back is inserted and is continued in the new ware to cultivate, and cell residual at the bottom of the preceding ware then discards; Utilize this method repeatedly to go down to posterity, reduce the ratio of heteroproteose cell gradually, thereby reach the purpose of purifying.
2. according to separation, cultivation and the purification process of the said mouse fat stem cell of claim 1, it is characterized in that the concentration of the said penicillium mould of step (2) is 400IU/ml; The concentration of said Streptomycin sulphate is 400 μ g/ml.
3. according to separation, cultivation and the purification process of the said mouse fat stem cell of claim 1; It is characterized in that; Cut out the resuspended and inoculation with perfect medium of the last centrifugal back of fatty tissue to step (10) from step (2), whole process time is controlled in the 4h.
4. according to separation, cultivation and the purification process of the said mouse fat stem cell of claim 1; It is characterized in that; In the said step (8), using the aperture earlier is the nylon leaching net filtration cell suspension of 250 μ m, and again gained filtrating being used the aperture is the nylon leaching net filtration of 80 μ m.Separation, cultivation and purification process according to the said mouse fat stem cell of claim 1 is characterized in that, the moity of said perfect medium is: foetal calf serum 20%; Penicillium mould 100IU/ml; Streptomycin sulphate 100 μ g/ml, L-glutaminate 2mmol/L, Prostatropin 10ng/ml; Vitamins C 50 μ g/ml, DMEM/F12 complements to 100%.
5. according to separation, cultivation and the purification process of the said mouse fat stem cell of claim 1, it is characterized in that the moity of said basic medium is: foetal calf serum 20%; Penicillium mould 100IU/ml; Streptomycin sulphate 100 μ g/ml, L-glutaminate 2mmol/L, DMEM/F12 complements to 100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110366128 CN102433299B (en) | 2011-11-17 | 2011-11-17 | Method for separating, culturing and purifying mouse adipose-derived stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110366128 CN102433299B (en) | 2011-11-17 | 2011-11-17 | Method for separating, culturing and purifying mouse adipose-derived stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102433299A true CN102433299A (en) | 2012-05-02 |
| CN102433299B CN102433299B (en) | 2013-01-23 |
Family
ID=45981645
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110366128 Expired - Fee Related CN102433299B (en) | 2011-11-17 | 2011-11-17 | Method for separating, culturing and purifying mouse adipose-derived stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102433299B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667349A (en) * | 2013-11-15 | 2014-03-26 | 安徽农业大学 | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) |
| CN104073466A (en) * | 2014-01-02 | 2014-10-01 | 安徽农业大学 | Method for verifying spontaneous transformation of mice adipose-derived stem cells |
| CN104818246A (en) * | 2015-04-23 | 2015-08-05 | 青岛尤尼科技有限公司 | Separation culture method of rabbit adipose-derived stem cells |
| CN105586311A (en) * | 2016-03-06 | 2016-05-18 | 李倩 | Culture medium for culturing human adipose-derived stem cells |
| CN105602896A (en) * | 2016-02-03 | 2016-05-25 | 广西大学 | Isolated culture and identification method of porcine adipose-derived stem cells |
| 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 |
| CN108949522A (en) * | 2018-07-19 | 2018-12-07 | 重庆医科大学附属第三医院(捷尔医院） | Shaping and beauty autologous fat competent cell filter device |
| CN109415675A (en) * | 2016-03-17 | 2019-03-01 | 辛诺瓦生命科学股份有限公司 | Use shock wave or the method for mechanical shock separation, dissociation and/or lytic cell |
| CN109439616A (en) * | 2018-11-14 | 2019-03-08 | 汪玉宝 | A kind of stem cell excretion body is promoting embryo's spilting of an egg and is increasing the method for hatching rate |
| CN109554339A (en) * | 2018-12-29 | 2019-04-02 | 中国医学科学院整形外科医院 | A method of efficiently separating fat mesenchymal stem cell from liposuction aspirates |
| CN111484973A (en) * | 2020-06-04 | 2020-08-04 | 广州同康生物科技有限公司 | Purification method of adipose-derived stem cells |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047804B (en) * | 2016-05-30 | 2019-11-12 | 浙江大学 | A kind of purification process of fat stem cell and stem cell are in osteogenic induction and at the application on chondrocyte induction |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778905A (en) * | 2004-11-22 | 2006-05-31 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| EP1261694B1 (en) * | 2000-02-26 | 2008-01-16 | Artecel, Inc. | Pleuripotent stem cells generated from adipose tissue-derived stromal cells and uses thereof |
| CN101314766A (en) * | 2007-05-31 | 2008-12-03 | 中国医学科学院基础医学研究所 | Method for isolated culture of human fat mesenchyma stem cell and special culture medium thereof |
-
2011
- 2011-11-17 CN CN 201110366128 patent/CN102433299B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1261694B1 (en) * | 2000-02-26 | 2008-01-16 | Artecel, Inc. | Pleuripotent stem cells generated from adipose tissue-derived stromal cells and uses thereof |
| CN1778905A (en) * | 2004-11-22 | 2006-05-31 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| CN101314766A (en) * | 2007-05-31 | 2008-12-03 | 中国医学科学院基础医学研究所 | Method for isolated culture of human fat mesenchyma stem cell and special culture medium thereof |
Non-Patent Citations (1)
| Title |
|---|
| BRUCE A. BUNNELL ET AL: "Adipose-derived stem cells:Isolation, expansion and differentiation", 《METHODS》 * |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667349B (en) * | 2013-11-15 | 2017-02-15 | 安徽农业大学 | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) |
| CN103667349A (en) * | 2013-11-15 | 2014-03-26 | 安徽农业大学 | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) |
| CN104073466A (en) * | 2014-01-02 | 2014-10-01 | 安徽农业大学 | Method for verifying spontaneous transformation of mice adipose-derived stem cells |
| 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 |
| CN104818246A (en) * | 2015-04-23 | 2015-08-05 | 青岛尤尼科技有限公司 | Separation culture method of rabbit adipose-derived stem cells |
| CN104818246B (en) * | 2015-04-23 | 2017-11-28 | 青岛尤尼科技有限公司 | A kind of isolated culture method of rabbit fat stem cell |
| CN105602896A (en) * | 2016-02-03 | 2016-05-25 | 广西大学 | Isolated culture and identification method of porcine adipose-derived stem cells |
| CN105586311A (en) * | 2016-03-06 | 2016-05-18 | 李倩 | Culture medium for culturing human adipose-derived stem cells |
| CN105586311B (en) * | 2016-03-06 | 2019-02-22 | 集钧（上海）医疗科技发展有限公司 | It is a kind of for cultivating the culture medium of human adipose-derived stem cell |
| CN109415675B (en) * | 2016-03-17 | 2022-07-12 | 辛诺瓦生命科学股份有限公司 | Method for separating, dissociating and/or lysing cells using shock waves or mechanical shock |
| CN109415675A (en) * | 2016-03-17 | 2019-03-01 | 辛诺瓦生命科学股份有限公司 | Use shock wave or the method for mechanical shock separation, dissociation and/or lytic cell |
| US11866732B2 (en) | 2016-03-17 | 2024-01-09 | Synova Life Sciences, Inc. | Separation, dissociation and/or disaggregation of cells using shockwaves or mechanical impacts |
| CN108949522A (en) * | 2018-07-19 | 2018-12-07 | 重庆医科大学附属第三医院(捷尔医院） | Shaping and beauty autologous fat competent cell filter device |
| CN108949522B (en) * | 2018-07-19 | 2022-02-08 | 重庆医科大学附属第三医院(捷尔医院） | Autologous fat active cell filtering device for plastic cosmetology |
| CN109439616A (en) * | 2018-11-14 | 2019-03-08 | 汪玉宝 | A kind of stem cell excretion body is promoting embryo's spilting of an egg and is increasing the method for hatching rate |
| CN109554339A (en) * | 2018-12-29 | 2019-04-02 | 中国医学科学院整形外科医院 | A method of efficiently separating fat mesenchymal stem cell from liposuction aspirates |
| CN111484973A (en) * | 2020-06-04 | 2020-08-04 | 广州同康生物科技有限公司 | Purification method of adipose-derived stem cells |
| CN111484973B (en) * | 2020-06-04 | 2021-08-27 | 铜仁市泛特尔生物技术有限公司 | Purification method of adipose-derived stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102433299B (en) | 2013-01-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102433299B (en) | Method for separating, culturing and purifying mouse adipose-derived stem cells | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN105238751B (en) | Isolated culture method of umbilical cord tissue mesenchymal stem cells | |
| CN102127522A (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN106367389A (en) | Preparation method and application of human umbilical cord mesenchymal stem cell factors | |
| CN104762257B (en) | A kind of method preparing mescenchymal stem cell from umbilical cord | |
| CN105087474A (en) | Culture method of deciduous tooth pulp stem cells | |
| CN108795855A (en) | A kind of serum free medium of mescenchymal stem cell | |
| CN104762259A (en) | Culture medium for mesenchymal stem cells and large-scale culture method thereof | |
| CN108220229A (en) | A kind of preparation method for improving umbilical cord derived mesenchymal stem cell primary cell yield | |
| CN102851255A (en) | Efficient and fast method for separating mesenchymal stem cells | |
| CN101372682B (en) | Construction method of Epinephelus fuscoguttatus fin cell line | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN108753706A (en) | Application of the white flower loropetalum chinense in preparing umbilical cord mesenchymal stem cells culture supernatant | |
| CN105238738A (en) | Isolated culture method of piglet myocardial fibroblasts | |
| CN107354130B (en) | Human placenta chorion mesenchymal stem cell separation method | |
| CN102146359A (en) | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification | |
| CN105505865A (en) | Separation method for umbilical cord mesenchymal stem cells | |
| CN106222135A (en) | A kind of quick acquisition and the method for Purification of Human umbilical cord mesenchymal stem cells | |
| CN109628388A (en) | With digestive enzyme compositions from placenta blood vessel separating mesenchymal stem cell | |
| CN106497863B (en) | A kind of separation, purifying and the cultural method of cornea of rats endothelial cell | |
| CN101451121B (en) | Construction method of Epinephelus fuscoguttatus heart cell line | |
| CN1746297A (en) | Placenta derived mesenchymal stem cell and preparation thereof | |
| CN103305551B (en) | Method for screening donor cell line based on blastocyst gene expression level |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130123 Termination date: 20151117 |
|
| EXPY | Termination of patent right or utility model |