A mouse intestinal epithelial crypt cell line and its construction and culture method

A technology of crypt cells and construction methods, which is applied in the field of molecular and cell biology, can solve the problems of poor repeatability of research results, limited number of passages, and huge differences in characteristics, so as to achieve strong proliferation ability and avoid pollution

Active Publication Date: 2021-05-14
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most commercial or research primary cells have the problem of limited number of passages, and the characteristics of different generations of cells vary greatly, making the research results based on them poorly reproducible

Method used

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  • A mouse intestinal epithelial crypt cell line and its construction and culture method
  • A mouse intestinal epithelial crypt cell line and its construction and culture method
  • A mouse intestinal epithelial crypt cell line and its construction and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Isolation of intestinal crypt cells.

[0036] Reagent preparation:

[0037] Basic Medium:

[0038] Advanced DMEM / F12

[0039] L-glutamine L-glutamine (2mM)

[0040] Penn / Strep (100 units / ml)

[0041] Hepes (10 mM)

[0042] 1:100 N2 supplement

[0043] 1:50 B27 supplement

[0044] ENR medium: ready-to-use, on the basis of the basic medium, add 4 kinds of factors:

[0045] Epidermal growth factor EGF (final concentration 50ng / ml)

[0046] Noggin protein (final concentration 100 ng / ml or conditioned medium added at a volume ratio of 1:10)

[0047] R-spondin (conditional medium added at a volume ratio of 1:10)

[0048] Y27632 (ROCK inhibitor, final concentration 10µM)

[0049] Separation Buffer I: 2 mM EDTA in PBS, filtered and stored at 4°C.

[0050] Separation buffer II: 54.9 mM D-sorbitol, 43.4 mM sucrose, prepared in PBS, filtered and stored at 4°C.

[0051] 1) Euthanasia CD1 mice born 2 weeks old were killed; the middle and lower part of the jejunum and ...

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Abstract

The invention belongs to the technical field of molecular and cell biology, and specifically relates to a method for constructing a mouse small intestinal epithelial crypt cell line. The method uses a special separation method to prepare a single-cell suspension of crypt epithelial cells, and adopts it for immortality after adhering to the wall. change. This cell line well maintains the morphology of primary intestinal epithelial cells and has strong proliferation ability. It can form a characteristic organoid structure under three-dimensional culture conditions, and specific staining shows that the cells that make up this structure include a variety of intestinal epithelial differentiation progeny. After the establishment of the line, the crypt cells can still only grow under primary culture conditions, which can be applied to establish a multi-dimensional research model of mouse small intestinal epithelium in vitro, for the study of intestinal physiological functions and pathology including infection, cancer, etc. mechanism, and provide tools and platforms for therapeutic targets and drug screening and evaluation of intestinal diseases including intestinal cancer.

Description

technical field [0001] The invention belongs to the technical field of molecular and cell biology, and in particular relates to a mouse intestinal epithelial crypt cell line, its construction and culture method. Background technique [0002] Intestinal epithelial cells are one of the fastest-renewing cell types in the body, and crypt cells provide a steady supply of cells for this renewal. Cells leaving the crypts along the basement membrane gradually differentiate into different types of mature epithelial cells, such as Absorbing epithelial cells, goblet cells, Paneth cells, and M cells, etc., these progeny cells perform different functions and jointly maintain the physiological functions of the intestinal epithelial system. The intestinal epithelial system is an exquisite model for studying stem cell biology and cell lineage, and it is also the simplest research model for studying mammalian tissue renewal, pluripotent stem cells, dual lineage determination, and programmed ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N5/10
Inventor 王宁梁后杰何通川陈火明
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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