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Culture medium and application thereof

A medium and basal medium technology, applied in tissue culture, cell culture active agents, microorganisms, etc., can solve the problems of high price and unfavorable promotion of intestinal epithelial stem cell culture system, and achieve the effect of improving the efficiency of in vitro culture

Active Publication Date: 2018-05-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing intestinal stem cell in vitro culture system needs to add growth factors such as EGF, Noggin and R-spondin (ENR) or ENR and Wnt-3a (ENRW), these growth factors are expensive, which is not conducive to the promotion of intestinal epithelial stem cell culture system, especially Limits the large-scale expansion of intestinal stem cells in vitro for the treatment of intestinal diseases

Method used

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  • Culture medium and application thereof
  • Culture medium and application thereof
  • Culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Culture of mouse small intestinal crypts and single stem cells based on 2ki system

[0040] According to the use of the 2ki stem cell medium provided by the present invention in culturing stem cells, the isolated mouse small intestinal lacuna and mouse Lgr5+ small intestinal adult stem cells prepared in this example were cultured. Using small intestinal lacuna and Lgr5+ small intestinal adult stem cells cultured in vitro as small intestinal organoids, the role of the small molecule 2ki in this culture process was explored. According to the method reported in the literature (Sato, T. et al. (2009). "Single Lgr5stem cells build crypt-villus structures invitro without a mesenchymal niche." Nature 459, 262-5), from Lgr5-EGFP-IRES-creERT2 transgenic mice (Can be purchased through The Jackson Laboratory organization, Stock Number: 008875, its feature description: the green fluorescent protein gene GFP is artificially inserted into the downstream of the promoter of t...

Embodiment 2

[0045] Example 2 Long-term culture of mouse small intestinal crypts based on 2ki system

[0046] Using small intestinal lacuna and Lgr5+ small intestinal adult stem cells cultured in vitro as small intestinal organoids, the role of the small molecule 2ki in this culture process was explored. Subsequently, we used this system for long-term culture to confirm that the culture system will not affect the functions and characteristics of small intestinal crypts and stem cells. The specific method is as follows:

[0047] (1) The small intestine was isolated from Lgr5-EGFP-IRES-creERT2 transgenic mice, cut longitudinally and washed three times with pre-cooled PBS buffer with a pH value of 7.4 to remove chyme, and then scraped carefully with a scalpel Remove intestinal villi on the lining of the small intestine. The remainder was cut into 5 mm length intestinal pieces and incubated with PBS buffer containing 10 mM EDTA on ice for 40 minutes. Replace the EDTA-containing PBS buffer s...

Embodiment 3

[0052] Example 3 In vitro culture of mouse colonic crypts based on 2ki system

[0053] According to the use of the 2ki stem cell culture medium provided by the present invention in culturing stem cells, the isolated mouse colon lacuna prepared in this example was cultured. Using the technique of culturing colonic lacunae into small intestinal organoids in vitro, the role of the small molecule 2ki in the culture process was discussed. According to the method reported in the literature (Sato, T. et al. (2011). "Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium." Gastroenterology 141:1762-1772), from Lgr5-EGFP - IRES-creERT2 transgenic mouse (can be purchased through The Jackson Laboratory, Stock Number: 008875, its characteristic description: the green fluorescent protein gene GFP is artificially inserted into the downstream of the promoter of the endogenous Lgr5 gene, so the transgenic mouse expresses The cells with...

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Abstract

The invention provides a culture medium. The culture medium is prepared from a basic culture medium, a serum replacement, double antibodies, glutamate / ester, N-acetylcysteine, CHIR-99021 and LDN-193189. The culture medium provided by the embodiment has the advantages that the culture medium is used for in-vitro culturing of intestinal crypt or stem cells, so that the amount of the obtained intestineal crypts is greater than the amount of intestinal crypts in the prior art, the ratio of stem cells in the obtained intestinal crypts is higher, and the amount of the obtained intestinal stem cellsis more; the culture medium is used for the in-vitro culturing of the intestinal crypts, so that the dryness of the stem cells in the intestinal crypts can be maintained for a long time, the cell proliferation in the intestinal crypts is more vigorous, and the cell differentiation in intestinal crypts is normal and ordered; the culture medium is favorable for the popularization of an intestinal epithelial stem cell culture system and an intestinal crypt organ culture system, is favorable for the in-vitro massive proliferation of the intestinal stem cells, and provides a favorable platform forthe treatment of intestinal diseases.

Description

technical field [0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to culture medium and its application, more specifically, the present invention relates to the medium, the use of CHIR-99021 and LDN-193189 in the preparation of medium, and the method for cultivating intestinal crypts And a method for obtaining intestinal stem cells. Background technique [0002] The intestinal epithelium is the main structure of the human body to perform digestion and absorption functions. It renews very quickly, and its renewal is mainly driven by the intestinal epithelial stem cells located in the crypt structure at the bottom. Therefore, the research on intestinal stem cells is closely related to human health. In recent years, the establishment of an in vitro culture system for intestinal epithelial stem cells has greatly promoted the research on intestinal epithelial stem cells, which facilitates the direct manipulation and ob...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/068C12N2501/01C12N2501/155C12N2501/405
Inventor 陈晔光李叶华刘媛
Owner TSINGHUA UNIV
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