A kind of in vitro culture method of calf small intestinal epithelial cells

Abstract

The invention discloses an in-vitro culture method for small intestinal epithelial cells of a calf. A small intestine of the calf is taken, an intestinal canal is guided into an intestinal canal dilator so as to be dilated, manure in the small intestine is washed with a PBS fluid until a liquid flowing out is clear, then one end of the intestinal canal is sealed with a pair of haemostatic forceps, the other end of the intestinal canal is sealed after the intestinal canal is filled with a protease digestive fluid, the intestinal canal is put onto a shaker to be digested at the temperature of 37 DEG C for 1-1.5 h, a liquid is collected and washed twice in a centrifugal manner, and finally, a culture solution is added for inoculated culturing; cells grow against the wall of the intestinal canal after 12-24 h, 90% of cells are attached to the wall of the intestinal canal after 3-4 days, and the cells are the small intestinal epithelial cells. The protease digestive fluid is directly injected into the enteric cavity and is not contacted with the outer wall of the small intestine, accordingly, fibroblasts and muscle cells on the outer wall of the intestine cannot be digested, and the purity of the epithelial cells is guaranteed to the largest extent.

Description

technical field [0001] The invention relates to a biological culture technology, in particular to a method for culturing calf small intestinal epithelial cells in vitro. Background technique [0002] In mammals, the small intestine is the main place for food digestion and absorption, and the small intestinal mucosal epithelial cells are the functional cells of the intestine, participating in the digestion and absorption of intestinal chyme, immune barrier and stress response, etc. Endocrine and exocrine functions are closely related. Therefore, the isolation and culture of small intestinal epithelial cells in vitro is of great significance for studying the physiological functions of the small intestine, the mechanism of nutrient absorption, the effect of drugs, and the pathophysiological changes under the action of various pathogenic factors. Some developed countries have established a variety of small intestinal epithelial cell lines in the field of pharmaceutical developm...

AI Technical Summary

Problems solved by technology

The study found that the success rate of in vitro culture of small intestinal epithelial cells using adult cattle is extremely low, because there is a large amount of chyme and microbial flora in the intestinal tract of adult cattle, and bacteria may remain even after washing with a large amount of phosphate buffered saline (PBS) , resulting in primary culture contamination; and adult bovine intestinal epithelial cells are highly differentiated and can hardly adhere to the wall

(2) Low purity of epithelial cells

Although this method can separate and obtain a large number of epithelial cells, the disadvantages are also obvious, because when the protease digests the intestinal mucosal tissue, it will inevitably separate the smooth muscle cells and fibroblasts on the outer wall of the intestinal tube, resulting in both cultured cells. Epithelial cells have fibroblast-like cells, and epithelial cells are mixed with fibroblast-like cells

(3) The separation of fibroblasts and epithelial cells is cumbersome and affects cell viability

In order to separate fibroblasts and epithelial cells, the traditional method generally needs to add trypsin to digest and observe that fibroblast-like cells loosen and fall off, but stop digestion when the epithelial cells are not loose. Remove, and then subculture the remaining cells, and then use trypsinization to remove the remaining fibroblast-like cells after 80-90% of the cells adhere to the wall. Reduced function

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