Establishment and Applications of Intestinal Organoid Cultures

Description

Background: Two-dimensional in vitro cell culture and animal models have limitations to address questions related to human health and disease. Advances in three-dimensional organoid culture furnish robust technologies that can bridge the gap between 2D monolayer cell cultures on one side and animal models or human subjects on the other side. Organoids are in vitro miniaturized and less complex model systems of organs that recapitulate the complex organization and functionality of tissue features that resemble those in vivo. Importantly, in contrast to cultures of primary or immortalized cells, organoids are three-dimensional configurations that self-renew in vitro, allowing the capacity for expansion, differentiation, and damage repair. Organoid technology has gained enormous interest for studying intestinal stem cell activities, for modeling intestinal tissue development and disease, and for personalized medicine, drug screening and regenerative therapy in vitro. We have begun to establish an intestinal organoid culture system from mouse intestine using MatrigelÒ in organoid medium containing Epidermal Growth Factor/ R-spondin 1/Noggin which mimic the intestinal epithelium. In this context we are interested in establishing intestinal organoids that will be used to explore the expression of intestinal crypt stem cell proliferation and differentiation markers following exposure to opioids, environmental toxins, or specific microbes.

Materials and Methods: In order to establish successful organoid culture workflows in our laboratory, we first used commercially available intestinal organoids from mice under the recommended incubation and growth conditions (StemCell Technologies). Subsequently, small intestinal crypts were isolated from a single C57BL/6 five to six months old mouse by a tissue dissociation procedure. Approximately, 500 to 1500 crypts were embedded in 50 ml Matrigel domes and cultured in an organoid growth medium containing essential growth factors in 24 well cell culture plates at 37°C in a CO2 incubator, essentially following the already established workflow. For cryopreservation, mature organoids were resuspended in CryoStar™CS10 freezing medium. Mature organoids were also used for total RNA isolation and immunohistochemical staining for mRNA analysis and protein detection, respectively. The stem cell marker Lgr5, the proliferation marker Ki-67, and various indicators for cell differentiation (Paneth and goblet cells, enteroendocrine, and enterocytes were examined.

Result: We have successfully established a three-dimensional in vitro organoid culture system from isolated mouse small intestinal crypts. The isolated crypts produced organoids with a crypt-villus like configuration similar to the commercially available mouse organoids. Expression analyses indicated active stem cell renewal and differentiation. Future applications will be discussed.

Conclusion: We have an intestinal organoid generation workflow for in place that will allow us to the use organoids to study stem cell activities during exposure to drugs, toxic substances, and microbiota.

Period	15 Feb 2024
Event title	Oklahoma State University Center for Health Sciences Research Week 2024
Event type	Conference
Location	Tulsa, United States, OklahomaShow on map
Degree of Recognition	Regional