CN106676073A - Culture method of breast cancer MCF-7 cell stem cell levitated spheres - Google Patents
Culture method of breast cancer MCF-7 cell stem cell levitated spheres Download PDFInfo
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- CN106676073A CN106676073A CN201710046574.6A CN201710046574A CN106676073A CN 106676073 A CN106676073 A CN 106676073A CN 201710046574 A CN201710046574 A CN 201710046574A CN 106676073 A CN106676073 A CN 106676073A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0695—Stem cells; Progenitor cells; Precursor cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The invention discloses a culture method of breast cancer MCF-7 cell stem cell levitated spheres. The culture method comprises the following steps: 1) collecting human breast cancer MCF-7 cells at a logarithmic growth phase, paving the collected cells on a 75 to 83 percent culture bottle, dissociating the cells with 0.25 percent by mass pancreatin till the cells become round, and discarding the pancreatin; 2) adding a DMEM/H-G complete culture solution to end a reaction, blowing and beating with a dropper to form a single-cell suspension, centrifuging at the speed of 1,000 revolutions per minute for 5 minutes, discarding supernatant, adjusting the cell concentration, and performing passage in different bottles in the ratio of 1 to 3; 3) re-suspending the MCF-7 cells subjected to passage in a DMEM/F-12 culture medium which does not contain serum, adjusting the cell concentration to 0.5*10<5>/mL, putting into a penicillin glass bottle, covering with a bottle plug, forming an oxygen-lack environment, culturing at the temperature of 37 DEG C in 5% CO2 without substituting the culture medium, mechanically blowing and beating into a single-cell suspension after a microcapsule is formed, and performing conventional passage. The breast cancer MCF-7 cell stem cell levitated spheres are cultured by an oxygen-free culture method, and spherical cell clusters remarkably growing in a suspending way appear on the second day of culture.
Description
Technical field
The present invention relates to a kind of cell culture processes, specially a kind of training of MCF-7 Breast Cancer Cell stem cell suspension ball
The method of supporting.
Background technology
There is sub-fraction in tumor cell group has the tumor stem cell of self-renewing, unlimited multiplication capacity, is tumour
The root of generation, Preventive and conventional therapy failure.Stem cell suspension ball, is commonly called as microballoon, and current cultural method is more, but
It is that to form the time more long.
The content of the invention
The technical problem to be solved in the present invention is to overcome existing stem cell suspension ball to form time defect more long, there is provided
A kind of cultural method of MCF-7 Breast Cancer Cell stem cell suspension ball.
In order to solve the above-mentioned technical problem, the invention provides following technical scheme：
A kind of cultural method of MCF-7 Breast Cancer Cell stem cell suspension ball, it is characterised in that comprise the following steps：
1）, collect exponential phase MCF-7 Human Breast Cancer Cells, 75%~83% blake bottle is paved with, with 0.25% mass fraction
Trypsin digestion cell abandons pancreatin to cell rounding；
2）, add use DMEM/H-G complete culture solution terminating reactions, with dropper blow and beat turn into single cell suspension, 1000 revs/min
Centrifugation, 5 minutes, abandons supernatant, cell concentration is adjusted, by 1:3 sub-bottles are passed on；
3）, the MCF-7 cells after passage are resuspended in the DMEM/F-12 culture mediums without serum, and add 20 ng/mL's
The B27 of EGF, the bFGF of 20 ng/mL, the BSA of 0.4% mass fraction and 2% mass fraction, adjust the concentration of cell for 0.5 ×
105/ mL, is placed in penicillin vial, and the vial needs to be sterilized by autoclaving, covers tightly bottle stopper, manufactures anoxic ring
Border, cultivates, not replacement medium under the conditions of 37 DEG C, 5%CO2, and after microballoon capsule is formed, machinery piping and druming is single cell suspension,
Routine passage.
Wherein, the vial is height 5cm, and diameter 2.4cm, bore is the vial of the 10mL of 1.9cm.
The present invention uses anaerobic cultivation culture MCF-7 Breast Cancer Cell stem cell suspension ball, and occurring for second day for culture is bright
The sphaerocyst group of aobvious floating life length, number of cells is no less than the visible individual cells and cell fragment being dispersed between 50, suspension ball,
Passage continues suspension growth if in serum free medium, such as gives cell attachment life after the normal culture medium containing hyclone
Long, cellular morphology is different, and major part is polygon.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for specification, with reality of the invention
Applying example is used to explain the present invention together, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the cell mass for compareing the lower MCF-7 cells accumulations of a culture；
Fig. 2 is the microballoon for compareing the two MCF-7 cells for cultivating 0h；
Fig. 3 is the microballoon for compareing the two MCF-7 cells for cultivating 24h；
Fig. 4 is the microballoon for compareing the two MCF-7 cells for cultivating 48h；
Fig. 5 is the microballoon for compareing the two MCF-7 cells for cultivating 72h；
Fig. 6 is to compare two microballoons for cultivating MCF-7 cells to carry out serum-containing media culture；
Fig. 7 is the microballoon of the MCF-7 cells that 0h is cultivated under cultural method of the invention；
Fig. 8 is the microballoon of the MCF-7 cells that 24h is cultivated under cultural method of the invention；
Fig. 9 is the microballoon of the MCF-7 cells that 48h is cultivated under cultural method of the invention；
Figure 10 is the microballoon of the MCF-7 cells that 72h is cultivated under cultural method of the invention；
Figure 11 is that the microballoon of the MCF-7 cells cultivated under cultural method of the invention carries out serum-containing media culture.
Specific embodiment
The preferred embodiments of the present invention are illustrated below, it will be appreciated that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment
MCF-7 Human Breast Cancer Cells system in the present invention, purchased from Chinese Academy of Medical Sciences Shanghai cellular resources storehouse
A kind of cultural method of MCF-7 Breast Cancer Cell stem cell suspension ball, it is characterised in that comprise the following steps：
1）, collect exponential phase MCF-7 Human Breast Cancer Cells, 75%~83% blake bottle is paved with, with 0.25% mass fraction
Trypsin digestion cell abandons pancreatin to cell rounding；
2）, add use DMEM/H-G complete culture solution terminating reactions, with dropper blow and beat turn into single cell suspension, 1000 revs/min
Centrifugation, 5 minutes, abandons supernatant, cell concentration is adjusted, by 1:3 sub-bottles are passed on；
3）, the MCF-7 cells after passage are resuspended in the DMEM/F-12 culture mediums without serum, and add 20 ng/mL's
The B27 of EGF, the bFGF of 20 ng/mL, the BSA of 0.4% mass fraction and 2% mass fraction, adjust the concentration of cell for 0.5 ×
105/ mL, is placed in penicillin vial, and the vial needs to be sterilized by autoclaving, covers tightly bottle stopper, manufactures anoxic ring
Border, cultivates, not replacement medium under the conditions of 37 DEG C, 5%CO2, and after microballoon capsule is formed, machinery piping and druming is single cell suspension,
Routine passage；
The MCF-7 cells after passage are cultivated using two kinds of conventional culture methods simultaneously carried out with cultural method of the invention
Contrast：
The control one, conventional culture methods of MCF-7 cells：Cultivated with the DMEM/H-G of the hyclone containing 10% mass fraction,
Cell is in 37 DEG C, the CO of 5% saturated humidity2Cultivated in incubator, this is normal MCF-7 groups of cells.
The control two, microballoon of commonsense method culture MCF-7 cells：Reference literature, is resuspended in after MCF-7 passages and is free of
The DMEM/F-12 culture mediums of serum, and 20 ng/mL EGF, 20 ng/mL bFGF, 0.4% BSA and 2% B27 are added, with thin
Born of the same parents' concentration 0.5 × 105/ mL is inoculated in 6 orifice plates（3 mL/ holes）, cultivated under the conditions of 37 DEG C, 5%CO2, it is new every 1d plus 1mL
Fresh culture medium, after microballoon capsule is formed, machinery piping and druming is single cell suspension, with cell concentration 0.5 × 105/ mL is passed on, about often
Zhou Chuandai 1 time, is made single cell suspension during cell detection, this group of cell is defined as MCF-7S groups of cells.
Cultivation results are contrasted：
MCF-7 cells adherent growth under conventional culture conditions is compareed, cellular morphology is polygon, different.It is conventional to pass
Dai Shi, part cell cells accumulation caused by blowing and beating inequality after pancreatin digestion is agglomerating, different from stem cell microballoon, as a result such as Fig. 1
It is shown；
Compare under two convenient stem cells condition of culture and the sphaerocyst group of obvious suspension growth, number of cells occur on the 3rd day
No less than 50, the visible individual cells and cell fragment being dispersed between suspension ball, passage continues to suspend if in serum free medium
Growth, such as gives cell attachment growth after the normal culture medium containing hyclone, and cellular morphology is different, and major part is polygon.
There is the sphaerocyst group of obvious suspension growth, number of cells in second day in cultural method cultivation of the invention
No less than 50, the visible individual cells and cell fragment being dispersed between suspension ball（Fig. 2）, pass on and continue if in serum free medium
Suspension growth, such as gives cell attachment growth after the normal culture medium containing hyclone, and cellular morphology is different, and major part is polygon
Shape.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although being described in detail to the present invention with reference to the foregoing embodiments, for a person skilled in the art, it still may be used
Modified with to the technical scheme described in foregoing embodiments, or equivalent is carried out to which part technical characteristic.
All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., should be included in of the invention
Within protection domain.
Claims (2)
1. a kind of cultural method of MCF-7 Breast Cancer Cell stem cell suspension ball, it is characterised in that comprise the following steps：
1）, collect exponential phase MCF-7 Human Breast Cancer Cells, 75%~83% blake bottle is paved with, with 0.25% mass fraction
Trypsin digestion cell abandons pancreatin to cell rounding；
2）, add use DMEM/H-G complete culture solution terminating reactions, with dropper blow and beat turn into single cell suspension, 1000 revs/min
Centrifugation, 5 minutes, abandons supernatant, cell concentration is adjusted, by 1:3 sub-bottles are passed on；
3）, the MCF-7 cells after passage are resuspended in the DMEM/F-12 culture mediums without serum, and add 20 ng/mL's
The B27 of EGF, the bFGF of 20 ng/mL, the BSA of 0.4% mass fraction and 2% mass fraction, adjust the concentration of cell for 0.5 ×
105/ mL, is placed in penicillin vial, and the vial needs to be sterilized by autoclaving, covers tightly bottle stopper, manufactures anoxic ring
Border, and cultivated under the conditions of 37 DEG C, 5%CO2, not replacement medium, after microballoon capsule is formed, machinery piping and druming is unicellular outstanding
Liquid, routine passage.
2. the cultural method of MCF-7 Breast Cancer Cell stem cell suspension ball as claimed in claim 1, it is characterised in that described
Vial is height 5cm, and diameter 2.4cm, bore is the vial of the 10mL of 1.9cm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111630158A (en) * | 2018-01-31 | 2020-09-04 | 韩国科学技术院 | Method for preparing cancer stem cell spheroids |
Citations (2)
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| CN102906249A (en) * | 2010-05-25 | 2013-01-30 | 独立行政法人国立癌症研究中心 | Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same |
| CN103289958A (en) * | 2013-07-09 | 2013-09-11 | 天津市人民医院 | Stem cell spheroid induction culture solution and stem cell spheroid culture method |
-
2017
- 2017-01-19 CN CN201710046574.6A patent/CN106676073A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906249A (en) * | 2010-05-25 | 2013-01-30 | 独立行政法人国立癌症研究中心 | Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same |
| CN103289958A (en) * | 2013-07-09 | 2013-09-11 | 天津市人民医院 | Stem cell spheroid induction culture solution and stem cell spheroid culture method |
Non-Patent Citations (5)
| Title |
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| 张斌等: "乳腺癌MCF-7细胞体外干细胞培养条件下雌激素受体表达与治疗敏感性初探", 《中国乳腺病杂志（电子版）》 * |
| 王鹏: "《生物实验室常用仪器的使用》", 31 August 2006, 中国环境科学出版社 * |
| 舒黛廉等: "《动物病原微生物检测技术》", 31 July 2014, 黄河水利出版社 * |
| 范原铭: "缺氧对人乳腺癌细胞微球体培养与生物学特性的影响及其机制研究", 《中国博士学位论文全文数据库（电子期刊）医药卫生科技辑》 * |
| 陆士新: "《干细胞与肿瘤》", 30 April 2009, 中国协和医科大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111630158A (en) * | 2018-01-31 | 2020-09-04 | 韩国科学技术院 | Method for preparing cancer stem cell spheroids |
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