Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Summary

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Abstract

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Introduction

Circadian clock
All organisms, from bacteria to mammals, have a complex and dynamic relationship with their environment. Within this relationship, adaptation to environmental changes is critical for the survival of organisms. Most organisms possess circadian rhythms that enable them to adapt and optimize their functions to diurnal cycles of approximately 24 h. The circadian clock is a hierarchical network of central and peripheral clocks that work in cooperation to maintain physiological homeostasis and keep organisms synchronized with daily changes^1,2. In mammals, the central or master ....

Protocol

All experiments using human tissues for the generation of HIEs were approved by an IRB at CCHMC (IRB#2014-0427). See the Table of Materials for details related to all materials used in this protocol.

NOTE: To illustrate the procedure outlined in this protocol, we utilized Bmal1-luc human intestinal enteroids (HIEs). These enteroids underwent stable lentiviral transduction²² with the pABpuro-BluF reporter plasmid, which contains the Bmal1 promot.......

Discussion

Bioluminescence assay offers several advantages for the investigation of circadian rhythms, which requires data collection from long-term time course experiments. First, it enables researchers to monitor the gene expression or protein of interest as cells move and proliferate. Without making unnecessary adjustments or disrupting the cells' functions, interested cellular events or gene expression can be recorded using bioluminescence readout, which gives reliable real-time data. Importantly, the bioluminescence assay .......

Materials

Name	Company	Catalog Number	Comments
35 x 10 Falcon tissue culture dishes	Fisher Scientific	08-772A
A 83-01	Sigma Aldrich	SML0788
Advanced DMEM/F12	Life Technologies	12634-028
B-27 Supplement (50x)	Gibco	17504-044
BD Micro-Fine IV Insulin Syringes	Fisher Scientific	14-829-1Bb	Mfrn: BD 329424
CHIR99021	Cayman Chemical	13122	GSK-3 inhibitor
Dexamethasone	Sigma Aldrich	D4902-500MG
D-Luciferin (potassium salt)	Cayman Chemical	14681
Gastrin I Human	Sigma Aldrich	G9020
GlutaMAX	Gibco	35050061
Growth Factor reduced (GFR) Matrigel	Corning	CB-40230C
HEPES	Gibco	15630080
IntestiCult Organoid Growth Medium (Human)	Stemcell Technologies	06010	Consist of IntestiCult OGM Human Basal Medium, 50 mL and Organoid Supplement, 50 mL. Mix both as 1:1 ratio to use as intestinal organoid growth medium
Kronos Dio Luminometer Machine	ATTO Corporation	AB-2550
N-2 Supplement (100x)	Gibco	17502-048
N-Acetyl-L-cysteine	Sigma Aldrich	A9165
pABpuro-BluF reporter plasmid	Addgene	46824
PBS without Calcium and Magnesium	Corning	21-040-CV
Penicillin-Streptomycin	Gibco	15140122
Recombinant murine EGF	PeproTech	315-09
Y-27632	R&D Systems	1254/10	ROCK inhibitor