Organoid Cultures for Assessing Intestinal Epithelial Differentiation and Function in Response to Type-2 Inflammation.

Abstract

During helminth infection of the gastrointestinal tract, a complex Type-2 inflammatory response involving immunological and mucosal components is mounted to clear the infection and reestablish a physiologically normal state. This response is characterized by the secretion of key interleukins, which impact epithelial lineage allocation and drive tuft and goblet cell hyperplasia to lead to eventual clearance of parasitic organisms. While there have been advances toward understanding Type-2 inflammatory responses in the intestine, detailed cellular and molecular mechanisms of epithelial responses to general inflammation and specific inflammatory cytokines remain to be explored. Intestinal organoids represent a physiologically relevant in vitro model to study how Type-2 inflammation impacts stem cell maintenance and differentiation and offer a new approach for investigators to test compounds that modulate mechanisms involved in worm clearance. The methods described in this chapter include: (1) intestinal crypt and single cell isolation; (2) organoid culture and cytokine treatment, as well as methods for downstream organoid analyses; (3) gene expression analysis by qRT-PCR; (4) protein analysis by western blot, immunohistochemistry, and florescence-activated cell sorting; and (5) organoid self-renewal by serial passaging.