• Home
  • Search Results
  • Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells.

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

Current protocols in immunology (2020-09-18)
Cayetano Pleguezuelos-Manzano, Jens Puschhof, Stieneke van den Brink, Veerle Geurts, Joep Beumer, Hans Clevers
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
N-Acetyl-L-cysteine, suitable for cell culture, BioReagent
Sigma-Aldrich
Nicotinamide, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
DAPT, ≥98% (HPLC), solid
Sigma-Aldrich
PD 0325901, ≥98% (HPLC)
Sigma-Aldrich
Phalloidin–Atto 647N, BioReagent, suitable for fluorescence, ≥80% (HPLC)
Sigma-Aldrich
SB 202190 monohydrochloride hydrate, ≥98% (HPLC)