Method for culturing colorectal cancer cell lines through organoid adaptation and colorectal cancer cell lines

A colorectal cancer, domestication and culture technology, applied in the field of biomedicine, can solve the problems such as the inability to truly simulate the tumorigenesis mechanism, the difficulty in removing fibroblast contamination, and the difficulty in establishing a tumor cell line, so as to improve the success rate of line establishment and shorten the The effect of line establishment time and strong genetic stability

Active Publication Date: 2022-02-08
北京科途医学科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The method of transferring tumor driver genes is to transfer tumor driver genes such as telomerase gene, human papillomavirus gene and SV40 large T antigen gene into cells, so that the cells proliferate and form immortalized cell lines. This method introduces exogenous Driver genes cannot truly simulate the tumorigenesis mechanism
The spontaneous immortalization method is to cultivate the primary tumor cells, and after the tumor cells grow, the fast-growing fibroblasts are removed by subculture. This method has defects such as complicated operation procedures, long culture time, and difficulty in removing fibroblast contamination. , and the probability of spontaneous immortalization of tumor cells during culture is extremely low, making it difficult to establish tumor cell lines

Method used

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  • Method for culturing colorectal cancer cell lines through organoid adaptation and colorectal cancer cell lines
  • Method for culturing colorectal cancer cell lines through organoid adaptation and colorectal cancer cell lines
  • Method for culturing colorectal cancer cell lines through organoid adaptation and colorectal cancer cell lines

Examples

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Embodiment 1

[0051] This example is used to illustrate the method for culturing colorectal cancer organoid clusters using colorectal cancer isolated tissues.

[0052] 1. Acquisition of colorectal cancer tissue in vitro

[0053] The discarded colorectal cancer isolated tissues after surgical treatment or needle biopsy of colorectal cancer patients were placed in H + GPSA medium containing 1 vol% Hibernate A, 1 vol% GlutaMax, 1 vol% PenStrep and 1 vol% Amphotericin B , transported to the operating room at low temperature within 48 hours.

[0054] 2. Enzymatic hydrolysis of colorectal cancer tissue in vitro

[0055] The isolated colorectal cancer tissue was taken out and placed in a sterile environment, washed 3 times with freshly prepared H + GPSA medium containing 1 vol% HibernateA, 1 vol% GlutaMax, 1 vol% PenStrep and 1 vol% Amphotericin B, Remove impurities such as blood clots and necrotic tissue in the tissue.

[0056] Use sterile instruments to cut the cleaned colorectal cancer tissu...

Embodiment 2

[0061] This example is used to illustrate the method of using colorectal cancer organoids to domesticate and culture colorectal cancer cell lines.

[0062] (1) Take the colorectal cancer organoid mass cultured in Example 1 and centrifuge it at 300 g for 5 min. The obtained cell pellet is digested with Accutase. After the cell mass in the cell pellet is dispersed into a single cell suspension, the Centrifuge for 5 minutes under the same conditions to obtain the colorectal cancer single cell pellet in the colorectal cancer organoid;

[0063] (2) Resuspend the single cell pellet of colorectal cancer in the acclimatization medium mixed with 5 volume % Matrigel matrigel to obtain a cell concentration of 3 × 10 4 Individual / mL colorectal cancer single cell suspension;

[0064] (3) Inoculate the colorectal cancer single cell suspension obtained above into a 24-well plate at an inoculum volume of 300 μL / well, and place it at 37°C, 5% CO 2 Culture in an incubator for 0.5-2 hours, the...

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Abstract

The present disclosure relates to a method for culturing colorectal cancer cell lines through organoid domestication, using organoid carriers and domestication medium to perform multiple domestication and culture of colorectal cancer single cells in colorectal cancer organoids, during the domestication culture process Gradually reducing the amount of organoid carrier used, the colorectal cancer single cells in colorectal cancer organoids gradually adapted to the two-dimensional culture environment, and were eventually domesticated into immortalized colorectal cancer cell lines. This disclosure creatively proposes a brand-new method for culturing colorectal cancer cell lines through organoid domestication, which can significantly shorten the establishment time of colorectal cancer cell lines and increase the success rate of colorectal cancer cell line establishment; in addition , the method has simple culture conditions and is easy to implement, and can establish human-derived colorectal cancer cell lines from primary cells in vitro with low cost and high efficiency.

Description

technical field [0001] The present disclosure relates to the technical field of biomedicine, and in particular, relates to a method for culturing colorectal cancer cell lines through organoid adaptation and the colorectal cancer cell lines cultured by the method. Background technique [0002] Tumor cell lines are good models for studying the biological characteristics of tumor cells, the mechanism of carcinogenesis, and tumor treatment options in vitro. Each tumor cell line cultured from the tumor cells of the primary patient at least partially retains the individual pathogenic information of the primary patient. Therefore, the more tumor cell lines are established, the more conducive to the research of related tumors. Compared with other biological models used in tumor research, although tumor cell lines have lost many pathological features and genetic details related to tumor tissue, their advantages lie in simple culture, easy to achieve and control culture conditions, lo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09C12R1/91
CPCC12N5/0693C12N5/0679C12N2500/14C12N2501/11C12N2500/30C12N2500/38C12N2501/345C12N2501/415C12N2501/15C12N2501/405C12N2513/00C12N2533/90
Inventor 史媛媛李程肖金平孙志坚
Owner 北京科途医学科技有限公司
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