Mouse intestinal epithelium pit cell line and constructing and culturing method thereof

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

Active Publication Date: 2019-05-14
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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  • Mouse intestinal epithelium pit cell line and constructing and culturing method thereof
  • Mouse intestinal epithelium pit cell line and constructing and culturing method thereof
  • Mouse intestinal epithelium pit cell line and constructing and culturing method thereof

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 factors:

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

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

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

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

[0049] Separation buffer I: 2 mM EDTA, prepared 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 was used to euthanize CD1 mice born for 2 weeks; the middle and lower jejunum and the middle and upper ileum were diss...

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Abstract

The invention belongs to the technical field of molecules and cellular biology, and particularly relates to a constructing method of a mouse intestinal epithelium pit cell line. The method comprises the steps that a single cell suspension of pit epithelium cells is prepared through a special separation method, cellular immortality is conducted after cell adherence. The form of intestinal epithelium primary cells is well maintained by the cell line, and high proliferation capability is achieved. Characteristic organ structures can be formed in three-dimensional culturing conditions, it is indicated by specific staining that cells which form the structure comprise multiple intestinal epithelium differentiated progeny. Pit cells of an established line can only grow in primary culturing conditions still, and the pit cells can be applied to the establishing of a mouse intestinal epithelium in vitro multi-dimensional study model which is used for studying intestinal physiologic functions andpathological mechanisms including infection, carcinogenesis and the like, and tools and platforms are provided for treatment targets and medicine screening and evaluating of intestinal diseases including intestinal cancer.

Description

Technical field [0001] The invention belongs to the technical field of molecular and cell biology, and specifically relates to a mouse intestinal epithelial crypt cell line, and a method for construction and culture thereof. Background technique [0002] Intestinal epithelial cells are one of the fastest renewal cell types in the body, and crypt cells provide a continuous supply of cells for this renewal. The cells leaving the crypt 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, these progeny cells play different functions together to 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. It is also the simplest research model for studying mammalian tissue renewal, pluripotent stem cells, binary lineage determination and programmed...

Claims

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Application Information

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Patent Type & Authority Applications(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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