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Method for domesticating and culturing colorectal cancer cell line through organoid and colorectal cancer cell line

A colorectal cancer, domestication and culture technology, applied in the field of biomedicine, can solve the problems of complex operation procedures, inability to truly simulate the tumorigenesis mechanism, and difficulty in removing fibroblast contamination.

Active Publication Date: 2021-04-06
北京科途医学科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 domesticating and culturing colorectal cancer cell line through organoid and colorectal cancer cell line
  • Method for domesticating and culturing colorectal cancer cell line through organoid and colorectal cancer cell line
  • Method for domesticating and culturing colorectal cancer cell line through organoid and colorectal cancer cell line

Examples

Experimental program
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Effect test

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 invention relates to a method for domesticating and culturing a colorectal cancer cell line through an organoid, which comprises the following steps of performing domestication culture on colorectal cancer single cells in colorectal cancer organoids for multiple times by using an organoid carrier and a domestication culture medium, and gradually reducing the use amount of the organoid carrier in the domestication culture process, such that the colorectal cancer single cells in colorectal cancer organs gradually adapt to a two-dimensional culture environment and are finally domesticated into an immortalized colorectal cancer cell line. The invention creatively provides a brand-new method for domesticating and culturing the colorectal cancer cell line through organoids, the method can remarkably shorten the line building time of the colorectal cancer cell line, and the line building success rate of the colorectal cancer cell line is increased; in addition, the method is simple in culturing condition and easy to implement, and the human-derived colorectal cancer cell line can be efficiently established from the primary cells in vitro at low cost.

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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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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