Method for separating crypts from colonic mucosa tissues and inducing generation of colonic organoids

Abstract

The invention relates to the field of stem cell organoid research, and discloses a method for separating crypts from colonic mucosa tissues and inducing to generate colonic organoid. Compared with the prior art, the method has the following advantages and effects that the elastin is degraded through Dispase, the connection between the colon epithelium and the intrinsic layer is loosened on the premise that cells are intact, the three micromolecules of Thiazovivin (TZV), SB431542 and CHIR99021 are added into the OGM culture medium to improve the organoid induction success rate, and the method has the advantages that the method is simple and convenient to operate, and the cost is low. The human colon-like organs can be reliably obtained with relatively low cost and relatively high efficiency; meanwhile, the method has the advantages that the method does not depend on a high-skill epithelial layer stripping technology; more primary colonic progenitor cells are obtained; the induction success rate of the primary culture medium on the colon-like organs is improved; and popularization and application are facilitated.

Description

Technical field [0001] The present invention relates to the field of stem cells \ class organ research, and in particular, to the separation of the mucosa and induce a method of producing colon organs. Background technique [0002] ORGANOIDS refers to a tissue analog formed by using adult stem cells or multi-capable cells for in vitro 3D (3D) culture. Class organ 3D culture is an emerging in vitro study system for studying the growth, differentiation, and organ formation of organized stem cells. Although the type of organ is not a true body organ, it can simulate the real organs in the structure and function, and can maximize the structural structure and function in vivo, and can have a long-term stable passage. [0003] The colon is one of the most complex organs of human structure and function. The colonic epithelium has a strong self-renewal ability, and its update speed is the fastest in the organs of adult mammals. At present, three-dimensional culture of colorectic organs i...

AI Technical Summary

Problems solved by technology

However, traditional methods still have certain limitations

For example, the isolation steps of intestinal crypts reported by Sato T et al. are cumbersome and require careful mechanical separation of the epithelial layer, which is highly dependent on the skill level of the operator, which is not conducive to technology promotion; and the primary culture medium in the long-term culture conditions it creates The success rate of induction of colon organoids is not high (Sato T, Stange D E, Ferrante M, et al. Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium[J]. 5): 1762-1772.)

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