Bruce Jessica, Kaiko Gerard E, Keely Simon
Viruses, Infections/Immunity, Vaccines and Asthma (VIVA) Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia.
School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia.
Methods Mol Biol. 2019;2029:49-62. doi: 10.1007/978-1-4939-9631-5_5.
The gastrointestinal epithelium is a highly regenerative organ, where each cell is replaced in a cycle of 4-6 days, depending on the mammalian species. This highly proliferative state is driven by gastrointestinal stem and progenitor cells, located at the base of crypts. These cells give rise to at least six types of differentiated epithelial cells, each with a distinct function in maintaining homeostasis between the intestinal interface and the luminal environment. The isolation and culture of these cells from mammalian gastrointestinal tissue is a novel technique, which allows for the generation and maintenance of an in vitro culture system for adult epithelial cells. There are two predominant methods for isolation and propagation of gastrointestinal epithelial cells, the first is the organoid system developed in 2009, and the second is a later version known as the L-WRN spheroid system. In this chapter, we describe the method to isolate and culture human gastrointestinal stem and progenitor cells and culture them as three-dimensional spheroids using L-WRN cell conditioned media.
胃肠道上皮是一个高度再生的器官，根据哺乳动物的种类不同，每个细胞在4 - 6天的周期内被替换。这种高度增殖状态由位于隐窝底部的胃肠道干细胞和祖细胞驱动。这些细胞产生至少六种类型的分化上皮细胞，每种细胞在维持肠道界面和管腔环境之间的稳态中具有独特功能。从哺乳动物胃肠道组织中分离和培养这些细胞是一项新技术，它能够生成并维持成人上皮细胞的体外培养系统。胃肠道上皮细胞分离和增殖主要有两种方法，第一种是2009年开发的类器官系统，第二种是后来的版本，即L-WRN球体系统。在本章中，我们描述了使用L-WRN细胞条件培养基分离和培养人胃肠道干细胞和祖细胞并将其培养为三维球体的方法。
