CN108070559A - A kind of primary human breast cancer cell isolation and culture method - Google Patents
A kind of primary human breast cancer cell isolation and culture method Download PDFInfo
- Publication number
- CN108070559A CN108070559A CN201611032625.1A CN201611032625A CN108070559A CN 108070559 A CN108070559 A CN 108070559A CN 201611032625 A CN201611032625 A CN 201611032625A CN 108070559 A CN108070559 A CN 108070559A
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- CN
- China
- Prior art keywords
- breast cancer
- human breast
- cancer cell
- primary human
- culture method
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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
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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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention provides a kind of primary human breast cancer cell isolation and culture method, including step：(a) dual anti-precooling PBS cleaning tissue specimen is contained；(b) tissue, mixed enzyme solution digestion are shredded；(c) it is thoroughly mixed culture medium and terminates digestion；(d) continuous screen filtration collects filtrate centrifugation；(e) cell precipitation is resuspended, is inoculated in blake bottle；(f) replacement mixing complete medium continues to cultivate after cultivating a couple of days.Short, the efficient method for obtaining human breast cancer cell is taken the present invention provides a kind of.
Description
Technical field
The invention belongs to cell biologies, and in particular to a kind of primary human breast cancer cell isolation and culture method.
Background technology
Breast cancer is a kind of malignant tumour for being usually happened at breast galandular epithelium tissue, is to seriously affect women's physical and mental health
Even one of most common malignant tumour of threat to life, the generation and transfer of tumour have with the microenvironment residing for tumour cell
Substantial connection.Tumor microenvironment is a complicated system, wherein tumour associated fibroblast cell (cancer-
Associated fibroblast, CAS) it is one of most important host cell of tumor microenvironment.The human breast carcinoma of original cuiture
CAF is directly separated from tissue, and significant change do not occur for biological characteristics and hereditary capacity, closest and reflect it
Tumor growth characteristic is research tumor microenvironment CAF biological properties itself, gene expression, In Vitro Chemotherapy drug sensitivity test
Quickly screen effective chemotherapeutics.The model and research breast cancer microenvironment CAF and breast cancer of curative effect of medication in vitro test are thin
The ideal model of correlation between born of the same parents.But contain significant quantities of fat tissue in human breast carcinoma so that separation and Purification of Human mammary gland
There are certain difficulty by cancer CAF.The present invention provides a kind of human breast carcinoma tissue fibroblast cell primary cultural method of comparatively perfect,
It lays a good foundation for research tumor of breast microenvironment CAF and its with breast cancer cell biological interaction, is also the generation of tumour
The research of development and complex treatment provides means.
The content of the invention
The purpose of the present invention is establish a kind of method for taking short, efficient acquisition human breast cancer cell.
Above-mentioned involved in order to solve the problems, such as, the present invention takes following method to obtain primary human breast cancer cell：
The present invention provides a kind of primary human breast cancer cell isolation and culture method, including step：(a) dual anti-precooling is contained
PBS cleaning tissue specimen；(b) tissue, mixed enzyme solution digestion are shredded；(c) it is thoroughly mixed culture medium and terminates digestion；(d) continuous screen
Net filtration collects filtrate centrifugation；(e) cell precipitation is resuspended, is inoculated in blake bottle；(f) mixing is replaced after cultivating a couple of days completely
Culture medium continues to cultivate.
Optional tissue specimen is fresh tissue specimen；
Optional mixed enzyme solution be the type i collagen enzyme of 0.05%-0.1%, 0.05%-0.1% II Collagenase Types and
The hyaluronidase of 0.1%-0.15%, digestion time 1.5h-2h；
Optional mixing complete medium is the H-DMEM containing 10%FBS and 1640 (2: 1)；
The method of optional continuous screen filtration is that postdigestive tissue fluid is successively by 80 mesh, 100 mesh, 200 mesh screens
Filtering；
Optionally the method for differential velocity adherent is after inoculation is resuspended in mixing complete medium, is replaced after culture 2-4h fresh complete
Culture medium.
The present invention provides a kind of method for mixing enzyme digestion and obtaining primary human breast cancer cell, not only operating procedure letter
Just, human breast cancer cell activity height that is short, and obtaining is taken.
Description of the drawings
Fig. 1 culture 72h cells pictures (100 ×)
Specific embodiment
In order to which the purpose of the present invention and advantage is made more clearly to show, now specific embodiment is expanded on further.Herein
The specific embodiment illustrated is explained only for the present invention, is not intended to limit the present invention.
The present invention selects fresh human breast carcinoma tissue, separates human breast cancer cell.Concrete operations are as follows：
1st, tissue specimen is rinsed 2-3 times with the PBS containing dual anti-precooling；
2nd, tissue specimen is fully shredded, adds in type i collagen enzyme, the II Collagen Type VIs of 0.05%-0.1% of 0.05%-0.1%
The mixed enzyme solution of the hyaluronidase of enzyme and 0.1%-0.15% digests 1.5h-2h in 37 DEG C of shaker water bath pots；
3rd, digestion is terminated with the H-DMEM containing 10%FBS and RPMI1640 (2: 1) mixing complete mediums；
4th, by postdigestive tissue successively through 80 mesh, 100 mesh, 200 mesh sieve net filtrations, collect filtrate, 1800rpm/min from
Heart 5min, abandons supernatant；
5th, cell precipitation is resuspended in sterile PBS cleaning 2 times, mixing complete medium；
6th, 37 DEG C, the CO of 5% (m/v)2, saturated humidity incubator in cultivate 2-3 days, discard not attached cell, add in
Fresh mix complete medium continues to cultivate, change the liquid once every three days；
The preferred embodiment of the invention is described in detail above, but the invention be not limited to it is above-mentioned
Embodiment, makes any modification within the spirit and principles of the invention, and equivalent substitution and improvement etc. should be included in this hair
In bright protection domain.
Claims (5)
1. a kind of primary human breast cancer cell isolation and culture method, including step：(a) dual anti-precooling PBS cleaning tissue mark is contained
This；(b) tissue, mixed enzyme solution digestion are shredded；(c) mix complete medium and terminate digestion；(d) continuous screen filtration collects filter
Liquid centrifuges；(e) cell precipitation is resuspended, is inoculated in blake bottle；(f) replacement mixing complete medium continues to train after cultivating a couple of days
It supports.
2. primary human breast cancer cell isolation and culture method according to claim 1, which is characterized in that the tissue
For flesh tissue.
3. primary human breast cancer cell isolation and culture method according to claim 1, which is characterized in that the step b
Middle mixed enzyme solution is the type i collagen enzyme of 0.05%-0.1%, the II Collagenase Types of 0.05%-0.1% and 0.1%-0.15%
Hyaluronidase, digestion time 1.5h-2h.
4. primary human breast cancer cell isolation and culture method according to claim 1, which is characterized in that the step c
Middle mixing complete medium is H-DMEM and RPMI1640 (2: 1) containing 10%FBS.
5. primary human breast cancer cell isolation and culture method according to claim 1, which is characterized in that the step d
The method of continuous screen filtration is that postdigestive tissue fluid by 80 mesh, 100 mesh, 200 mesh sieve net filtrations, collects filtrate successively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611032625.1A CN108070559A (en) | 2016-11-15 | 2016-11-15 | A kind of primary human breast cancer cell isolation and culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611032625.1A CN108070559A (en) | 2016-11-15 | 2016-11-15 | A kind of primary human breast cancer cell isolation and culture method |
Publications (1)
| Publication Number | Publication Date |
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| CN108070559A true CN108070559A (en) | 2018-05-25 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201611032625.1A Pending CN108070559A (en) | 2016-11-15 | 2016-11-15 | A kind of primary human breast cancer cell isolation and culture method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817665A (en) * | 2021-09-13 | 2021-12-21 | 哈尔滨中科赛恩斯生物技术有限公司 | Isolation culture and subculture method of mouse breast cancer cells |
Citations (3)
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| WO2006030473A1 (en) * | 2004-09-15 | 2006-03-23 | Apogenix Gmbh | Method for the purification and amplification of tumoral stem cells |
| CN102373186A (en) * | 2010-08-17 | 2012-03-14 | 刘东旭 | Multi-enzyme system used for separating patient tumor tissue-originated primary cells, application method thereof, purpose thereof, and relative kit thereof |
| CN104480062A (en) * | 2014-12-30 | 2015-04-01 | 广东海洋大学 | Separation and culture method for different cellular components of human mammary tissue |
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2016
- 2016-11-15 CN CN201611032625.1A patent/CN108070559A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006030473A1 (en) * | 2004-09-15 | 2006-03-23 | Apogenix Gmbh | Method for the purification and amplification of tumoral stem cells |
| CN102373186A (en) * | 2010-08-17 | 2012-03-14 | 刘东旭 | Multi-enzyme system used for separating patient tumor tissue-originated primary cells, application method thereof, purpose thereof, and relative kit thereof |
| CN104480062A (en) * | 2014-12-30 | 2015-04-01 | 广东海洋大学 | Separation and culture method for different cellular components of human mammary tissue |
Non-Patent Citations (2)
| Title |
|---|
| 崔军威等: "ALDHhiCD44+细胞在乳腺癌组织中的表达及其与相关细胞信号传导通路之关系研究", 《吉林医学》 * |
| 王宏等: "乌司他丁和泰索帝对人原代乳腺癌细胞增殖、侵袭及细胞因子IGF-1R、PDGFA和PAFR表达的影响", 《中国生物制品学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817665A (en) * | 2021-09-13 | 2021-12-21 | 哈尔滨中科赛恩斯生物技术有限公司 | Isolation culture and subculture method of mouse breast cancer cells |
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Application publication date: 20180525 |