3D culture, passage, cryopreservation, recovery and identification method for in-vitro organoids based on small intestines of different segments of mice

Abstract

The invention discloses a 3D culture, passage, cryopreservation, recovery and identification method for in-vitro organoids based on small intestines of different segments of mice. The method comprisesthe following steps: (1) separating recesses of duodenum, jejunum and ileum segments of mice; (2) performing 3D culture on the recesses of the duodenum, jejunum and ileum segments of mice, and forming organoids; (3) performing passage on organoids of small intestines of mice; (4) performing cryopreservation on the organoids of small intestines of mice; (5) performing recovery on the organoids ofsmall intestines of mice; (6) preparing frozen sections of the organoids of small intestines of mice; and (7) performing immunofluorescence labeling and identification on the frozen sections of the organoids of small intestines of mice. Compared with the prior art, the method disclosed by the invention optimizes a recess extraction manner, an in-vitro culture system and a culture medium based on the recesses of the small intestines containing stem cells, and comprises the steps of complete culture, passage, cryopreservation, recovery and identification. The small intestine organoids obtained by the method are capable of performing repeated passage, and the passage organoids have character stability.

Description

technical field [0001] The invention relates to a method for in vitro organoid 3D culture, passage, cryopreservation, recovery and identification based on different sections of the small intestine of mice. Background technique [0002] The small intestine is the largest nutrient digestion and absorption organ in the human body. Due to its important role in metabolic disorders such as obesity and diabetes, there is growing interest in intestinal nutrient absorption, transport, perception, and hormone release. Currently, the in vitro models used for the study of nutrient sensing, transport, absorption and local hormone regulation in the small intestine are mainly divided into two categories: one is based on intestinal tissue, including ring method, everted intestinal sac, and Uss perfusion. Chamber and isolated intestinal perfusion, the above models can be used for the interaction of microorganisms, epithelial cells, immune cells, nerve cells and other cells in tissues under n...

AI Technical Summary

Problems solved by technology

[0003] However, the above-mentioned in vitro research models of intestinal function have certain defects: the models based on intestinal tissue, because the above-mentioned models need to obtain isolated tissues, and the number of experimental subjects will not be able to obtain sufficient quality and quantity of tissues, and in vitro The organization is time-effective, so there is only limited functional testing time, and the repeatability and stability of the experiment are poor

The cell-based model has a single cell composition and cannot truly reproduce the interaction between intestinal cells and cells, and between cells and the cell matrix

Images