Real Time Analysis of Metabolic Profile in <em>Ex Vivo</em> Mouse Intestinal Crypt Organoid Cultures

Abstract

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors. Video LinkThe video component of this article can be found at

A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses

“…However, the assessment of organoids using XF demands a large amount of samples or crypts, being approx. > 200-300 crypts per experimental point 19 . Therefore, to compare it with O2-PLIM which provides analysis of single crypt regions at single cell / organoid resolution, the highly sensitive Agilent ® XF96 platform was selected as a comparative tool.…”

“…This was performed accordingly to published protocol 19 , with minor modifications. 9-10 organoids in 3 µl of Matrigel ® per well were seeded onto XF96 plate (Seahorse) and subsequently grown in ENRVC or EN (lacking R-spondin 1, sodium valproate and CHIR99021) media for 2 days prior to the analysis.…”

“…The standard approach is to analyze the extracellular flux, i.e. 'XF' 19,20 which informs an oxygen consumption rate (OCR) coupled with extracellular acidification (ECAR), but since this technology was initially targeted towards high-throughput analysis of 2D homogenous cell populations it cannot assess the complexity and heterogeneity of the multiple cell types present in organoid and 3D cultures. Disruptive methods can be also applied to the analysis of the mitochondrial function of stem cell niche, e.g.…”

An enduring role for quiescent stem cells

“…These findings have been made possible by important technical advances that allow for the analysis of key metabolic parameters, such as O 2 consumption, glycolytic rate, ATP production and respiratory capacity, in very small assay volumes, allowing for the study of rare populations. In the intestine, two recent papers have reported methods to evaluate the metabolic profile of both primary enteroids and isolated CBC ISCs (Bas and Augenlicht, 2014; Fan et al, 2015). In these studies, Lgr5 high colonic stem cells demonstrated decreased mitochondrial metabolism and increased glycolytic flux when compared with Lgr5 low daughter cells (Fan et al, 2015).…”

Evaluation of Compound Activity in Primary Human Intestinal Organoids Using Gene Expression and Histology

“…The influence of low‐abundance epithelial cell types, such as Paneth cells, can exert a profound effect on the health and function of neighboring cells (Sato et al., ; VanDussen et al., ). Functional changes such as ion transport, metabolic shifts, and intracellular transport in specific cell populations can be monitored in real time by altering culture conditions or introducing bioactive molecules into the culture (Bas & Augenlicht, ; Dekkers et al., ; Saxena et al., ).…”