Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells

TLDR

Human intestinal organoids derived from adult stem cells are miniature ex vivo models of the intestinal epithelium that enable studies of physiology and disease. The article presents protocols for establishing and maintaining small intestine and colon organoids from biopsies and outlines their downstream applications. The authors detail a set of protocols—including establishment, passage, differentiation, cryopreservation, staining, clonal culture, and support media production—that enable efficient handling of organoids, highlighting their advantages over immortalized cell lines. © 2020 The Authors.

Abstract

Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors. Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids Alternate Protocol: Differentiation of human intestinal organoids Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids Basic Protocol 4: Immunofluorescence staining of human intestinal organoids Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures Support Protocol 1: Production of Wnt3A conditioned medium Support Protocol 2: Production of Rspo1 conditioned medium Support Protocol 3: Extraction of RNA from intestinal organoid cultures.