In-vitro monolayer culture and representing method for constructing mouse intestinal epithelium by using Transwell

Abstract

The invention discloses an in-vitro monolayer culture and representing method for constructing mouse intestinal epithelium by using Transwell. The method comprises the following steps of (1) respectively inoculating intestinal tract crypt structures containing intestinal tract stem cells and intestinal tract epithelial myofibroblasts to the upper surface and the lower surface of a Transwell nestedpolycarbonate membrane, nesting Transwell in a porous cell culture plate, adding an upper-layer cell and a lower-layer cell into an optimized culture medium, and carrying out co-culture to form a single-layer intestinal tract epithelium in vitro; and (2) regularly detecting the transepithelial resistance value of the single-layer intestinal epithelium every day in the culture process, representing that the intestinal in-vitro single-layer culture and the in-vivo intestinal epithelium have the same cell composition and structural characteristics by immunofluorescence and electron microscope results, and analyzing the permeability of the single-layer intestinal epithelium by taking isothiocyanate labeled glucan as a tracing reagent. The intestinal tract crypt and intestinal tract epithelialmyofibroblasts are co-cultured for the first time to form an in-vitro intestinal tract epithelial monolayer culture model with similar structural characteristics, cell composition and permeability toin-vivo intestinal tract epithelium.

Description

technical field [0001] The invention relates to a method for culturing and characterizing an in vitro monolayer of mouse intestinal epithelium by using Transwell. Background technique [0002] The small intestine is the largest nutrient absorption organ in the human body, and the nutrient perception, absorption, and transport at the intestinal level have received increasing attention. At present, the in vitro models used to evaluate the nutrient sensing, transport and absorption functions of the small intestine are mainly divided into two categories: one is based on intestinal tissue, mainly based on Uss perfusion system; the other is based on tumor cell lines. The basic model, mainly Caco-2 or Caco-2 and HT29 co-culture combined with transmembrane analysis technology (Transwell) can produce polarized epithelial layer for in vitro epithelial cell response research. However, the above-mentioned in vitro research models of intestinal function have certain defects. The model c...

AI Technical Summary

Problems solved by technology

However, the above-mentioned in vitro research models of intestinal function have certain defects. The model cells based on tumor cell lines are single and cannot truly reproduce the multicellular composition characteristics of the intestinal tract. The lack of cell-cell interaction may easily lead to over-evaluation of absorption evaluation; However, the model based on intestinal tissue has the problems of high technical requirements, large amount of animals used, relatively poor experimental repeatability and stability, and limited time for in vitro testing.

The discovery of intestinal stem cells and the establishment of in vitro 3D culture technology have provided new ideas for solving the above problems. The characteristics greatly limit the application of this model in the evaluation of nutrient absorption and transport

Images