CN101050452A - Method for separating stem cell of breast cancer - Google Patents
Method for separating stem cell of breast cancer Download PDFInfo
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- CN101050452A CN101050452A CNA2007100210781A CN200710021078A CN101050452A CN 101050452 A CN101050452 A CN 101050452A CN A2007100210781 A CNA2007100210781 A CN A2007100210781A CN 200710021078 A CN200710021078 A CN 200710021078A CN 101050452 A CN101050452 A CN 101050452A
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- stem cell
- breast cancer
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Abstract
This invention relates to a method for separating breast cancer stem cells. The method comprises: digesting breast cancer cells into single cell suspension, preparing Percoll discontinuous density gradient separating medium, mixing with single cell suspension, centrifuging to separate cell subsets, detecting by comparing the experimental tube and the control tube, adjusting the density of Percoll discontinuous density gradient separating medium until the content of the cell subsets related to the breast cancer stem cells reaches predetermined enrichment degree, and screening with a flow cytometry. The method utilizes Percoll screening or serum-free culture to enrich breast cancer stem cells, and then screens with a flow cytometry to ensure the screening efficiency. The ratio of the breast cancer stem cells after primary enrichment is higher than 25%, while that after low cytometry screening is higher than 99%.
Description
Technical field
The present invention relates to a kind of method for separating stem cell, especially a kind of method for separating stem cell of breast cancer belongs to the tumor research technical field.
Background technology
Al-Hajj in 2003 etc. utilize the difference of breast cancer cell surface antigen, use the antibody and the Flow Cytometry of specificity fluorescent mark, elect the breast cancer cell branch as independent colony, the cell that will sub-elect different phenotypes again is expelled to respectively in the immunodeficient mouse body, found that and has only surface antigen to be masked as ESA
+Lin
-CD44
+CD24
-/lowA small set of cell have continuity and become the knurl characteristic, and unique can self-replacation, start the generation of tumour, and its ratio is very little, only accounts for 2% of tumour cell, Here it is breast carcinoma stem cell.In addition, also available stem cell its functional attributes SP phenotype is come the sorting tumor stem cell.2004, Kondo etc. detected the SP cell in C6, MCF-7, B104 and four clones of Hela, and wherein the SP cell accounts for 2% among the MCF-7.But, similar with the stem cell in the healthy tissues, because breast carcinoma stem cell shared ratio in breast cancer tissue is low, only account for about 2%, directly come the sorting difficulty big, weak effect with flow cytometer, breast carcinoma stem cell purity is not high enough after the sorting, and generally speaking the efficient of sorting is not high.
In addition, 1987, discoveries such as Ponti can will make tumor stem cell survive by external dried serum-free culture mode, but not tumor stem cell is then dead gradually, thereby the content of tumor stem cell is improved.Though aforesaid method is simpler, the content of tumor stem cell is improved, this method is comparatively rough, makes rich gather limited in one's ability of tumor stem cell, can not reach the very high efficiency of separation and purity (as more than 95%).
Summary of the invention
The technical problem to be solved in the present invention is: at the shortcoming of above prior art existence, a kind of method for separating stem cell of breast cancer that can guarantee the high efficiency of separation and separation purity is proposed, for the various biological characteristicses of further studying breast carcinoma stem cell provide condition.
In order to solve above technical problem, one of method for separating stem cell of breast cancer of the present invention may further comprise the steps:
1) with breast cancer cell line or former generation breast cancer tissue be digested to single cell suspension;
2) press the predetermined density and number of plies configuration different densities Percoll parting liquid,, make Percoll discontinuous density gradient parting liquid by density height stack successively;
3) postdigestive single cell suspension is added to Percoll discontinuous density gradient parting liquid top, centrifugation;
4) stratified each cell subset in the Percoll parting liquid of the centrifugal back of sucking-off, PBS washes;
5) each subgroup in isolated each cell subset is respectively established experiment tube and control tube, experiment tube and control tube add Hoechst 33342 and the extremely predetermined final concentration of Hoechst 33342, Verapamil respectively, after water-bath and PBS wash, detect the highest subgroup of tumor stem cell ratio with flow cytometer;
6) reach predetermined Fu Judu as the highest subgroup of this tumor stem cell ratio, then use selected by flow cytometry apoptosis standby;
7) do not reach predetermined Fu Judu as the highest subgroup of this tumor stem cell ratio, then adjust the density of Percoll parting liquid, repeat above correlation step, reach the predetermined rich poly-Percoll parting liquid density of spending until filtering out tumor stem cell, standby with the tumor stem cell in the rich poly-subgroup of selected by flow cytometry apoptosis again.
Two of method for separating stem cell of breast cancer of the present invention may further comprise the steps:
1) with breast cancer cell line or former generation breast cancer tissue be digested to single cell suspension;
2) in serum-free medium, add somatomedin, be prepared into serum-free medium;
3) postdigestive single cell suspension is placed serum-free medium, cultivate the scheduled time;
4) will be digested to single cell suspension through cultivating rich poly-tumor stem cell;
5) single cell suspension with step 4) is divided into experiment tube and control tube, adds CD in the experiment tube
44-FITC, CD
24-PE does not add in the control tube, and PBS washes the back with the resuspended fixed cell of PBS liquid that contains Paraformaldehyde 96, with the ratio of flow cytometer detection tumor stem cell after the serum-free culture richness is poly-;
6) reach predetermined Fu Judu as tumor stem cell content after the above serum-free culture, then use selected by flow cytometry apoptosis standby;
7) do not reach predetermined Fu Judu yet as tumor stem cell content after the above serum-free culture, then adjust the time of serum-free culture, repeat above correlation step, until reaching predetermined Fu Judu, standby with the rich poly-tumor stem cell of selected by flow cytometry apoptosis again.
In a word, the present invention has overcome the problem of the existence of existing separation method, at first by Percoll sorting or serum-free culture, make the tumor stem cell in the mammary cancer rich poly-, again by the flow cytometer refining-sorting, thereby guaranteed the efficient of sorting, through preliminary rich poly-back tumor stem cell relevant subgroup (SP cell or CD44
+CD24
-/lowCell) ratio can reach more than 25%, passes through the flow cytometer refining-sorting again, and the purity of tumor stem cell can reach more than 99%, thereby provides condition for the further various biological characteristicses of research breast carcinoma stem cell.
Description of drawings
The present invention is further illustrated below in conjunction with accompanying drawing.
Fig. 1 is the detection figure of the embodiment of the invention one, and the Percoll sorting is passed through in reflection, relevant subgroup (SP cell) enrichment of tumor stem cell among the MCF-7 MCF-7, and ratio has reached 25.23%.
Fig. 2 is the detection figure of the embodiment of the invention two, and reflection is by serum-free culture, the subgroup (CD44 that is correlated with of the tumor stem cell among the MCF-7 MDA-MB-231
+CD24
-/lowCell) rich poly-, ratio has reached 29.35%.
Embodiment
Embodiment one
Present embodiment is according to the relevant subgroup of the tumor stem cell among the following steps separating mammary cancerous cell line MCF-7:
1) breast cancer cell line MCF-7 is digested to single cell suspension with pancreas enzyme-EDTA.
2) six different densities Percoll parting liquids of configuration, density is respectively 1.035g/ml, 1.040g/ml, 1.050g/ml, 1.060g/ml, 1.070g/ml, 1.080g/ml, each density layer 2ml by density height stack successively, makes Percoll discontinuous density gradient parting liquid.
3) single cell suspension for preparing carefully is added to Percoll discontinuous density gradient parting liquid top, centrifugal 30 minutes of 2000 commentaries on classics/min.
4) stratified each cell subset in the Percoll parting liquid of the centrifugal back of careful sucking-off, PBS wash twice standby.
5) above-mentioned isolated each subgroup of each cell subset is respectively established experiment tube and control tube, every tube cell several 10
5Individual/ml, experiment tube add Hoechst 33342 to final concentration be 5 μ g/ml, control tube add again simultaneously Verapamil to final concentration be 50 μ g/ml, 37 ℃ of water-bath 90min, detect SP cell proportion in each subgroup with flow cytometer after PBS washes 2 times, find that the SP cell proportion is up to 25% (referring to Fig. 1) in the 4th layer (D subgroup).
6) the relevant subgroup (being the SP cell) of the tumor stem cell in the further sorting D of the flow cytometer subgroup is standby.
Embodiment two
Present embodiment is the relevant subgroup of the tumor stem cell among the separating mammary cancerous cell line MDA-MB-231 according to the following steps:
1) with breast cancer cell line or former generation breast cancer tissue be digested to the pancreas enzyme-EDTA single cell suspension.
2) in the DMEM/F12 serum-free medium, add somatomedins such as EGF, bFGF, Regular Insulin, be prepared into serum-free medium.
3) postdigestive single cell suspension is placed serum-free medium, cultivate 3 time-of-weeks.
4) with the rich poly-MDA-MB-231 of 3 week of serum-free culture back tumor stem cell, be digested to single cell suspension with pancreas enzyme-EDTA.
5) above-mentioned cell suspension is divided into experiment tube and control tube, every tube cell several 10
6Individual/ml, add CD in the experiment tube
44-FITC, CD
24-PE does not add in the control tube, and the 20min that keeps in Dark Place under the room temperature, PBS wash and use the resuspended fixed cell of PBS liquid 400ul that contains 1% Paraformaldehyde 96 after 2 times, and it (is CD44 that flow cytometer detects the relevant subgroup of tumor stem cell
+CD24
-/lowCell) content has reached nearly 30% (referring to Fig. 2).
6) the relevant subgroup (CD44 of the above-mentioned rich poly-tumor stem cell of the further sorting of flow cytometer
+CD24
-/low).
The method synthesis of above embodiment the advantage of existing separation method, practical, can guarantee tumor stem cell (SP cell or CD44 after the sorting
+CD24
-/lowCell) purity reaches more than 99%.
Claims (9)
1. method for separating stem cell of breast cancer may further comprise the steps:
1) with breast cancer cell line or former generation breast cancer tissue be digested to single cell suspension;
2) press the predetermined density and number of plies configuration different densities Percoll parting liquid,, make Percoll discontinuous density gradient parting liquid by density height stack successively;
3) postdigestive single cell suspension is added to Percoll discontinuous density gradient parting liquid top, centrifugation;
4) stratified each cell subset in the Percoll parting liquid of the centrifugal back of sucking-off, PBS washes;
5) each subgroup in isolated each cell subset is respectively established experiment tube and control tube, experiment tube and control tube add Hoechst 33342 and the extremely predetermined final concentration of Hoechst 33342, Verapamil respectively, after water-bath and PBS wash, detect the highest subgroup of tumor stem cell ratio with flow cytometer;
6) reach predetermined Fu Judu as the highest subgroup of this tumor stem cell ratio, then use selected by flow cytometry apoptosis standby;
7) do not reach predetermined Fu Judu as the highest subgroup of this tumor stem cell ratio, then adjust the density of Percoll parting liquid, repeat above correlation step, reach the predetermined rich poly-Percoll parting liquid density of spending until filtering out tumor stem cell, standby with the tumor stem cell in the rich poly-subgroup of selected by flow cytometry apoptosis again.
2. according to the described method for separating stem cell of breast cancer of claim 1, it is characterized in that: described digestion adopts pancreas enzyme-EDTA to realize.
3. according to claim 1 or 2 described method for separating stem cell of breast cancer, it is characterized in that: described step 2) four to eight different densities Percoll parting liquids of configuration, each contiguous parting liquid density difference is controlled at and is respectively 0.002g/ml-0.015g/ml.
4. according to claim 1 or 2 described method for separating stem cell of breast cancer, it is characterized in that: described step 2) six different densities Percoll parting liquids of configuration, density is respectively 1.035g/ml, 1.040g/ml, 1.050g/ml, 1.060g/ml, 1.070g/ml, 1.080g/ml, each density layer 2ml, by density height stack successively, make Percoll discontinuous density gradient parting liquid.
5. according to the described method for separating stem cell of breast cancer of claim 4, it is characterized in that: every tube cell several 10 in the described step 5)
5Individual/ml, it is 5 μ g/ml to final concentration that experiment tube adds Hoechst 33342, and it is 50 μ g/ml to final concentration that control tube adds Verapamil simultaneously again, and 37 ℃ of water-bath 90min detect SP cell proportion in each subgroup with flow cytometer after PBS washes 2 times.
6. method for separating stem cell of breast cancer may further comprise the steps:
1) with breast cancer cell line or former generation breast cancer tissue be digested to single cell suspension;
2) in serum-free medium, add somatomedin, be prepared into serum-free medium;
3) postdigestive single cell suspension is placed serum-free medium, cultivate the scheduled time;
4) will be digested to single cell suspension through cultivating rich poly-tumor stem cell;
5) single cell suspension with step 4) is divided into experiment tube and control tube, adds CD in the experiment tube
44-FITC, CD
24-PE does not add in the control tube, and PBS washes the back with the resuspended fixed cell of PBS liquid that contains Paraformaldehyde 96, with the ratio of flow cytometer detection tumor stem cell after the serum-free culture richness is poly-;
6) reach predetermined Fu Judu as tumor stem cell content after the above serum-free culture, then use selected by flow cytometry apoptosis standby;
7) do not reach predetermined Fu Judu yet as tumor stem cell content after the above serum-free culture, then adjust the time of serum-free culture, repeat above correlation step, until reaching predetermined Fu Judu, standby with the rich poly-tumor stem cell in selected by flow cytometry apoptosis place.
7. according to the described method for separating stem cell of breast cancer of claim 6, it is characterized in that: described digestion adopts pancreas enzyme-EDTA to realize.
8. according to claim 6 or 7 described method for separating stem cell of breast cancer, it is characterized in that: described step 2) in the DMEM/F12 serum-free medium, add EGF, bFGF, insulin-like growth factor, be prepared into serum-free medium.
9. described according to Claim 8 method for separating stem cell of breast cancer is characterized in that: every tube cell several 10 in the described step 5)
6Individual/ml, add CD in the experiment tube
44-FITC, CD
24Behind-the PE, the 20min that keeps in Dark Place under the room temperature, PBS wash after 2 times with the resuspended fixed cell of PBS liquid 400ul that contains 1% Paraformaldehyde 96, again with the relevant subgroup of flow cytometer detection sorting tumor stem cell.
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| CNA2007100210781A CN101050452A (en) | 2007-03-27 | 2007-03-27 | Method for separating stem cell of breast cancer |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993852A (en) * | 2009-08-13 | 2011-03-30 | 中国医学科学院基础医学研究所 | Culture medium and culture method of breast stem cells and breast stem cell-rich mixture |
| CN102178960A (en) * | 2011-04-02 | 2011-09-14 | 中国人民解放军第三军医大学第一附属医院 | Application of Tap73 gene to preparation of cancer stem cell chemosensitization medicine |
| CN102260647A (en) * | 2010-05-26 | 2011-11-30 | 卢英 | Method for isolating and purifying breast cancer stem cells |
| CN102676456A (en) * | 2012-05-10 | 2012-09-19 | 上海市胸科医院 | Method for separating lung cancer cell from hydrothorax |
| CN102770530A (en) * | 2009-11-05 | 2012-11-07 | 斯隆-凯特林癌症研究院 | Catenae: serosal cancer stem cells |
| CN103243075A (en) * | 2013-05-23 | 2013-08-14 | 成都美进生物科技有限公司 | Separation method of breast cancer stem cells |
| CN103266083A (en) * | 2013-05-23 | 2013-08-28 | 成都美进生物科技有限公司 | Culture method of breast cancer stem cell |
| CN110129271A (en) * | 2019-05-22 | 2019-08-16 | 中国人民解放军陆军军医大学第一附属医院 | A kind of method for separating of glioma stem cells |
| CN110396501A (en) * | 2019-08-01 | 2019-11-01 | 江苏省人民医院（南京医科大学第一附属医院） | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro |
-
2007
- 2007-03-27 CN CNA2007100210781A patent/CN101050452A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993852A (en) * | 2009-08-13 | 2011-03-30 | 中国医学科学院基础医学研究所 | Culture medium and culture method of breast stem cells and breast stem cell-rich mixture |
| CN101993852B (en) * | 2009-08-13 | 2013-12-25 | 中国医学科学院基础医学研究所 | Culture medium and culture method of breast stem cells and breast stem cell-rich mixture |
| CN102770530A (en) * | 2009-11-05 | 2012-11-07 | 斯隆-凯特林癌症研究院 | Catenae: serosal cancer stem cells |
| CN102260647A (en) * | 2010-05-26 | 2011-11-30 | 卢英 | Method for isolating and purifying breast cancer stem cells |
| CN102178960A (en) * | 2011-04-02 | 2011-09-14 | 中国人民解放军第三军医大学第一附属医院 | Application of Tap73 gene to preparation of cancer stem cell chemosensitization medicine |
| CN102178960B (en) * | 2011-04-02 | 2012-12-05 | 中国人民解放军第三军医大学第一附属医院 | Application of Tap73 gene in preparation of cancer stem cell chemosensitization medicine |
| CN102676456A (en) * | 2012-05-10 | 2012-09-19 | 上海市胸科医院 | Method for separating lung cancer cell from hydrothorax |
| CN103243075A (en) * | 2013-05-23 | 2013-08-14 | 成都美进生物科技有限公司 | Separation method of breast cancer stem cells |
| CN103266083A (en) * | 2013-05-23 | 2013-08-28 | 成都美进生物科技有限公司 | Culture method of breast cancer stem cell |
| CN110129271A (en) * | 2019-05-22 | 2019-08-16 | 中国人民解放军陆军军医大学第一附属医院 | A kind of method for separating of glioma stem cells |
| CN110396501A (en) * | 2019-08-01 | 2019-11-01 | 江苏省人民医院（南京医科大学第一附属医院） | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro |
| CN110396501B (en) * | 2019-08-01 | 2020-10-30 | 江苏省人民医院（南京医科大学第一附属医院） | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro |
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