Method of porcine intestinal crypt isolation and 3D type organ culturing

Abstract

The invention belongs to the technical field of animal tissue isolation and culturing, and particularly relates to a method of porcine intestinal crypt isolation and 3D type organ culturing. In the course of porcine intestinal crypt isolation, penicillin and streptomycin are added, so that in the later-stage culturing course, pollution caused by intestinal microorganisms is avoided; rotating speedand time for treating an intestinal segment of a horizontal rotary instrument and frequency and time of a vortex of a vortex instrument are optimized, so that the obtained crypt is complete in shape,and higher in survival rate and bud rate. According to the method disclosed by the invention, a formula of a culture solution for culturing the crypt and the 3D type organs is optimized, so that thecrypt can be well polarized to form the 3D type organs, and the growth and the polarization are stable; the isolating condition and the optimal culturing method of the porcine intestinal crypt are determined, and favorable foundation for subsequently utilizing the porcine intestinal 3D type organs to study enterovirus and bacterial infection is established.

Description

technical field [0001] The invention belongs to the technical field of animal histology. Specifically, it relates to a method for isolating porcine intestinal crypts and a method for culturing 3D organoids. Background technique [0002] The traditional two-dimensional (2D) cell lines used in the study of intestinal physiological and pathological processes are composed of a single type of cells, and each part of the intestinal tract has its own unique tissue structure and physiological functions. 2D cell lines cannot truly simulate the structure of the intestinal tract. Function and pathogenesis seriously restrict the study of intestinal physiological and pathological processes. In recent years, the development of intestinal 3D organoids has provided new and useful tools for the research on intestinal and intestinal diseases. Intestinal 3D organoid culture is to culture intestinal stem cells in vitro in the presence of Matrigel and various growth factors, so that they can g...

AI Technical Summary

Problems solved by technology

[0004] Pig intestinal diseases cause serious economic losses to the global pig industry every year. At present, people mainly use traditional 2D cell lines to carry out research on pig intestinal diseases, but because 2D cell lines cannot simulate the real situation in vivo, it affects their With the progress of research and the enhancement of people's awareness of animal welfare, the direct use of animal models for research is also limited. Therefore, it is urgent to develop a new type of in vitro model to simulate the real situation in vivo, and to carry out the study of pig intestinal tract. Physiological and pathological process research

The development of 3D organoids provides a useful way to solve this problem, but there are only 3 reports on the 3D organoid model of pig intestines, and the culture methods reported in the 3 literatures all have certain defects, among which Gonzalez In the isolation process of pig intestinal crypt isolation method reported by et al., the isolation effect needs to be observed continuously, and the time of incubation and shaking treatment should be extended according to the observation results, which is greatly affected by subjective factors, and the growth of organoids is not added during the culture process. Necessary nicotinamide, resulting in large differences in the morphology of subsequent cultured organoids (Gonzalez LM et al.Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.PLoS ONE.2013,8(6):e66465) The method introduced by Khalil et al. does not add antibiotics during the isolation process, which may lead to the failure of organoids to grow due to the contamination of intestinal microorganisms in the subsequent culture process. Leading to a large loss of intact crypts, because we found that the size of crypts in our study is much more than 100μm (Khalil HA et al. A Novel culture system for adult porcine intestinal crypts. Cell and TissueResearch. 2016, 365 (1): 123- 134); B. van der Hee was filtered with a 70 μm filter during the isolation process, which may lead to more loss of intact crypts, and the organoids isolated from B. van der Hee were severely cavitated during culture (B. van der Hee der Hee et al.Optimized procedures for generating an enhanced,nearphysiological 2D culture system from porcine intestinal organoids.Stem Cell Research.2018,28:165-171)

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