Methods for Assessing Circadian Rhythms and Cell Cycle in Intestinal Enteroids

Abstract

Endogenous circadian clocks play a key role in regulating a vast array of biological processes from cell cycle to metabolism, and disruption of circadian rhythms exacerbates a range of human ailments including cardiovascular, metabolic, and gastrointestinal diseases. Determining the state of a patient's circadian rhythms and clock-controlled signaling pathways has important implications for precision and personalized medicine, from improving the diagnosis of circadian-related disorders to optimizing the timing of drug delivery. Patient-derived 3-dimensional enteroids or in vitro "mini gut" is an attractive model uncovering human- and patient-specific circadian target genes that may be critical for personalized medicine. Here, we introduce several procedures to assess circadian rhythms and cell cycle dynamics in enteroids through time course sample collection methods and assay techniques including immunofluorescence, live cell confocal microscopy, and bioluminescence. These methods can be applied to evaluate the state of circadian rhythms and circadian clock-gated cell division cycles using mouse and human intestinal enteroids.

Keywords: Bioluminescence assay; Cell cycle; Circadian rhythm; Immunofluorescence; Intestinal enteroids; Time course sampling.