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Caco-2 cell monolayer membrane forming culture method

A culture method and cell monolayer technology, applied in the field of Caco-2 cell monolayer film formation and culture, can solve the problems of long experimental period, difficult state guarantee, vacuoles, etc. for Caco-2 cell monolayer film formation, so as to save research. Cost, increasing seeding density, and the effect of improving cell status

Active Publication Date: 2020-08-07
WUHAN PROCELL LIFE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In view of this, the present invention proposes a Caco-2 cell monolayer membrane formation culture method to solve the problem that the Caco-2 cell monolayer membrane formation experiment period is long, the state is difficult to guarantee, and vacuoles appear during the experiment, which leads to the failure of the experiment. question

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  • Caco-2 cell monolayer membrane forming culture method
  • Caco-2 cell monolayer membrane forming culture method
  • Caco-2 cell monolayer membrane forming culture method

Examples

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Embodiment 1

[0033] The present embodiment provides a kind of Caco-2 cell monolayer membrane formation culture method, specifically as follows:

[0034] Caco-2 cells were cultured in MEM containing 20% ​​(v / v) fetal bovine serum, 1% (v / v) penicillin / streptomycin at 37°C, 5% CO 2 Constant temperature static culture, when the cell confluency reaches 60% (1-2d), remove the medium and add trypsin to digest for 3 minutes, then add fresh medium, blow the cells gently and fully to obtain a single cell suspension, according to the volume ratio Divide into two equally for the next subculture, so that the subculture is continued twice.

[0035] After the cells were cultured to the logarithmic growth phase, the cell morphology was observed under a microscope and photographed. Remove the medium and add trypsin to digest for 3 minutes, then add complete medium, and blow the cells gently and fully. Count with a cell counting plate and inoculate on a 6-well culture plate with an inoculum size of 1×10 ...

Embodiment 2

[0037] The present embodiment provides a kind of Caco-2 cell monolayer membrane formation culture method, specifically as follows:

[0038] Caco-2 cells were cultured in MEM containing 20% ​​(v / v) fetal bovine serum, 1% (v / v) penicillin / streptomycin double antibody at 37°C, 5% CO 2 Constant temperature static culture, when the cell confluency reaches 70% (1-2d), remove the medium and add trypsin to digest for 5 minutes, then add fresh medium, blow the cells gently and fully to obtain a single cell suspension, according to the volume ratio Evenly divide into two for the next subculture, so that the subculture is continued for 3 times.

[0039] After the cells were cultured to the logarithmic growth phase, the cell morphology was observed under a microscope and photographed. Remove the medium and add trypsin for 5 minutes, then add complete medium, and gently blow the cells fully. Count with a cell counting plate and inoculate on a 6-well culture plate with an inoculum size of...

Embodiment 3

[0041] The present embodiment provides a kind of Caco-2 cell monolayer membrane formation culture method, specifically as follows:

[0042] Caco-2 cells were cultured in MEM containing 20% ​​(v / v) fetal bovine serum, 1% (v / v) penicillin / streptomycin at 37°C, 5% CO 2 Constant temperature static culture, when the cell confluency reaches 60% (1-2d), remove the medium and add trypsin to digest for 3 minutes, then add fresh medium, blow the cells gently and fully to obtain a single cell suspension, according to the volume ratio Divide into two equally for the next subculture, so that the subculture is continued twice.

[0043] After the cells were cultured to the logarithmic growth phase, the cell morphology was observed under a microscope and photographed. Remove the medium and add trypsin to digest for 3 minutes, then add complete medium, and blow the cells gently and fully. Count with a cell counting plate, inoculate on a 12-well culture plate, and the inoculum size is 5×10 5...

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Abstract

The invention provides a Caco-2 cell monolayer membrane forming culture method. The method comprises the following steps: culturing Caco-2 cells by using an MEM culture medium containing 20% of fetalcalf serum and 1% of penicillin / streptomycin, when the cell convergence degree reaches 60-70%, performing pancreatin digestion, carrying out continuous subculture for 2-3 times, and after the cells are cultured to the logarithmic phase, carrying out excessive inoculation cell culture and cell adherent expansion, cleaning non-adherent cells with PBS to obtain Caco-2 monolayer membrane cells. A Caco-2 monolayer membrane model established by the method is good in state, and is arranged in a paving stone inlay shape, the cells are tightly connected, cavitation is basically not formed, the Caco-2 cell model is reliable and stable, the repeatability is good, and an experimental foundation is laid for subsequent drug absorption and transfer research.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for forming and culturing a Caco-2 cell monolayer membrane. Background technique [0002] Cells are the basic structural and functional units of organisms, and are also the research objects of cytological experiments. Almost all known diseases are closely related to cells, and cytological experiments are one of the most direct ways to study cells in the field of life sciences. Cells in good condition are one of the most fundamental and important experimental materials for cell experiments, and cell culture technology is the decisive technology to ensure the good state of cells. It is known that there are more than a thousand kinds of cells from different sources, and they are still increasing. Different batches or passages of different types of cells or even the same type of cells have their own most suitable culture schemes. Even for the same cell line, researchers have exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/84C12N2500/02C12N2523/00Y02A50/30
Inventor 冷毅斌吴娟李弘泽严睿韬寇小娟
Owner WUHAN PROCELL LIFE SCI & TECH CO LTD
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