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 clock located in the suprachiasmatic nucleus (SCN) receives external cues, such as light, and transmits the information to the peripheral clocks via an advanced interplay of neural and humoral signaling pathways³. In addition to the central clock, peripheral tissues possess their own cell-autonomous circadian clock mechanism, maintained by a transcriptional-translational negative feedback loop (TTFL) regulating tissue-specific clock-controlled genes (CCGs)^4,5. This molecular machinery produces approximately 24 h rhythmicity in cellular and physiological events, such as gene expressions, signaling pathways, immune responses, and digestion. The circadian clock is present in almost all mammalian cells, and it has been demonstrated that up to 50% of the genes' expression patterns exhibit circadian rhythmicity⁶. Considering the abundance of CCGs, disruption of this clock mechanism may result in critical physiological problems. Hence, investigations into circadian rhythms are necessary to elucidate essential biological mechanisms and develop novel therapeutic strategies.

Luciferase reporter system
In circadian studies, real-time monitoring is critical for a better understanding of cellular behaviors and responses because it allows tracking of temporal changes in gene expression and/or protein levels, providing insights into the molecular mechanisms regulated by the circadian clock. Furthermore, real-time monitoring enables researchers to study the effects of environmental changes on molecular mechanisms^7,8. There are numerous techniques for real-time monitoring studies, including bioluminescence assay, which is widely used to track gene expression or protein levels over time. Bioluminescence assay is a method to detect biological processes using light production as a readout. In this assay, an oxidative enzyme that produces bioluminescence (e.g., luciferase) is either transiently or stably transfected into cells of interest, and the bioluminescence readout is measured in the presence of a substrate (e.g., luciferin) over time. For example, the luciferase enzyme produces bioluminescence by oxidizing the substrate luciferin in the presence of ATP⁹. Due to its short half-life, 3-4 h¹⁰, firefly luciferase is a powerful tool for circadian studies in terms of providing real-time dynamic monitoring with minimal background noise^11,12,13. For the insertion of DNA with a luciferase-tagged promoter or open reading frame (ORF), the lentiviral gene delivery system is a reliable method that provides high transduction efficacy, stable integration, and low immunogenicity. Stable transduction of a bioluminescent reporter provides robust expression in dividing and non-dividing cells, generating consistent data for circadian studies¹⁴.

Organoid as a model
Traditional immortalized two-dimensional cell lines have been instrumental in biological studies ranging from uncovering fundamental molecular mechanisms of circadian rhythms to drug screening. Despite the convenience of utilizing homogenized cell lines, they lack multicellular structures and intercellular interactions. In contrast, organoids are in vitro 3D multicellular "organ-like" structures that mimic organ structure in a dish by displaying similarity to in vivo tissue architecture and multicellularity, including stem, progenitor, and differentiated cell types^15,16. Possessing self-organization, multicellularity, and functionality features makes organoids a remarkable in vitro model representing the cellular and physiological processes occurring in real tissues¹⁷. Different types of organoids can be derived from pluripotent stem cells via directed differentiation or adult stem cells harvested from various organs, including the small intestine, brain, liver, lung, and kidney^18,19. Since organoid structures possess a real tissue-like architecture and function with multicellularity and dynamic cell-to-cell interaction, they are superior to homogenized cell lines for understanding the cellular events occurring in in vivo tissues. Organoids are also easily manipulated and can be grown under controlled conditions, making them useful for circadian studies²⁰.

The main purpose of this work is to introduce a real-time monitoring method utilizing a bioluminescence assay specifically tailored for studying circadian rhythms in multicellular 3D organoids. Real-time monitoring of cellular events using a bioluminescence assay technique has been widely performed for cell cultures lacking the multicellular complexity and intercellular communications that exist in real tissues. 3D organoids present unique opportunities to investigate the functions of circadian rhythms in multicellular systems in vitro. For example, one could investigate circadian rhythms in the organoids with altered cell compositions or organoids derived from patients' diseased tissues. This protocol enables the utilization of a bioluminescence assay to investigate different aspects of circadian rhythms in a more physiologically relevant in vitro model, organoids, which will help us better understand the roles of circadian rhythms in peripheral organs.

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 promoter fused to luciferase, showing the activity of the Bmal1 promoter²¹.

1. Lentiviral transduction

1. Carry out the lentiviral transduction²² under stem cell-enriched conditions for HIEs. Specifically, ensure the formation of spheroids in HIEs within 3-4 days after passage in the intestinal growth media, which creates stem cell-enriched conditions.
2. After the lentiviral transduction²², carry out antibiotic selection with 2 µg/mL puromycin, recover the puromycin-resistant HIEs, and passage them with relatively high density (i.e., 1 well into 2 wells) for 2-3x. If the growth rate of the HIEs is slow, add 10 µM ROCK inhibitors (Y-27632) and 10 µM GSK-3 inhibitor (CHIR-99021) to the intestinal organoid growth medium.

2. Preparation of organoids for bioluminescence assay

1. Day 1: Passage HIEs to 35 mm round dishes.
   1. Briefly, for expansion of HIEs, mix the HIEs with growth factor-reduced (GFR) 3D culture matrix and seed them onto 24-well plates with three droplets of 10 µL of 3D culture matrix per well. Add 350 µL of the HIE growth medium and change it every other day.
      NOTE: The density of HIE in the 3D culture matrix is critical for the growth of enteroids. Approximately, the number of spheroids should exceed more than 50 in each droplet. If the number is low, the growth rate of the HIE is negatively affected.
   2. When HIEs grow sufficiently (approximately 7 days after the post-passage), collect HIEs from four wells into 1.5 mL microcentrifuge tubes.
   3. Wash the HIEs with the cold PBS using a benchtop mini centrifuge (2,000 × g) for 40 s quick spin to remove debris and excessive 3D culture matrix by pipet.
   4. Add 0.5 mL of the cold PBS into the pellet and apply physical turbulence by syringing up to 10x with a 1 mL insulin syringe to dissociate the HIEs.
   5. Spin down the dissociated HIEs with a benchtop mini centrifuge as in step 2.1.3 and gently remove the supernatant by pipet. Then, place the microcentrifuge tube with the HIE pellet on ice to cool down for approximately 3 min.
   6. Add GFR 3D culture matrix to the microcentrifuge tube and mix it with the HIE pellet.
      NOTE: Calculate the amount of the 3D culture matrix based on the number of 35 mm round dishes that will be used for the experiment. We generally seed 30 µL of 3D culture matrix droplets per round dish.
   7. Seed the mixture of HIEs and the 3D culture matrix on the center of the 35 mm round dish and incubate the dish at 37 °C for 20 min to solidify the 3D culture matrix.
      NOTE: The density of seeding HIEs should be determined experimentally. Based on the intensity of bioluminescence, one can determine the number of HIEs during this process.
   8. Add 2 mL of prewarmed intestinal organoid growth medium to each dish and incubate the dishes in a CO₂ incubator until the next step.
2. Day 3: Initiation of differentiation
   1. To initiate differentiation of the HIEs, remove the growth medium and add 2 mL of prewarmed differentiation medium.
      NOTE: See Table 1 for the recipes of the HIE differentiation media²³.
3. Day 4: No additional process is required.
4. Day 5: Replace the medium of the enteroids with 2 mL of prewarmed differentiation medium.
5. Day 6: Clock synchronization and recording bioluminescence
   1. To synchronize the molecular clock of enteroids, add 2 µL of 100 µM dexamethasone (Dex) to make the final concentration 100 nM in each dish and then incubate the dishes in a CO₂ incubator (37 °C, 5% CO₂) for 1 h²⁴.
   2. During the Dex resetting, prepare the incubating luminometer. Check the CO₂ and temperature of the incubating luminometer and set to 5% and 37 °C, respectively.
      NOTE: It is suggested to wipe the inside of the incubating luminometer with 70% alcohol before and after every use of the machine to keep it sterile.
   3. After the clock synchronization, replenish the enteroids with 3 mL of prewarmed differentiation medium containing 200 µM D-luciferin.
      NOTE: As luciferin is light-sensitive, the luciferin-containing medium should be wrapped with foil and prewarmed in a 37 °C heat bath before applying it to the dish.
   4. Place the dishes in an 8-well dish table of the incubating luminometer and cover the sample dish table with its lid. Place two humidifier chambers with wet tissue or sponge on the top of the sample dish table. This is one-time process.
      NOTE: After the experiment is started, the lid of the incubating luminometer should be kept closed until the experiment is terminated. Make sure to wet the tissues/sponges enough.
   5. Close the lid of the machine and initiate the program to measure bioluminescence for the desired number of days and resolution.
      1. On the software, select the condition settings by clicking the Condition tab and adjust the settings on this panel: number of dishes, measurement time, background processing, and filter type.
         NOTE: For this demonstration, we selected all dishes from A to H; default measurement time, which is determined by the software based on the number of dishes; waiting time for background processing, and filter type F0. Since there are three filter options, three luminescent colors can be measured simultaneously.
      2. After entering all suitable settings based on the experiment, save the settings by clicking OK.
      3. To start running the experiment, click the Run tab | Start button (green triangle icon). Observe the data as raw, noise filtered, or detrended by making appropriate selections on the top of the screen of the software while running experiments. Click on Raw, Noise Filtered, or Detrended tabs to observe the selected data.
      4. Observe signals for up to 5 days.
         NOTE: After 5 days of monitoring in an incubating luminometer, enteroids may begin to die due to media depletion. To prevent any possible circadian disruption during the experiment, we do not perform media change for the duration of the experiment.
      5. At the end of the experiment, stop the run by clicking the Stop button (blue square icon), go to the File tab, and click Save as. Save the data in the Kronos format; export the data to a spreadsheet in the File section by clicking the File option and then selecting Export as excel format under the File for further analysis.

Results

Bioluminescence recording was conducted to assess the circadian rhythmicity of human intestinal enteroids (HIEs) under two distinct conditions: stem cell-enriched conditions using intestinal organoid growth medium (Figure 3) versus differentiation-inducing conditions, which was achieved by replacing the intestinal organoid growth medium with a differentiation medium. On the day of the experiment, we synchronized the circadian clocks by performing a 1 h treatment with 100 nM Dexamethasone. Su...

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