Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Our current scope of research is to investigate the roles of circadian rhythms in intestine epithelial cell proliferation and differentiation. We are trying to determine whether the circadian clock regulates cell proliferation and differentiation from intestinal stem cells. This protocol presents a realtime monitoring method for circadian studies in three-dimensional organoid using a bioluminescence assay.

Unlike performing bioluminescence assay in a conventional two-dimensional cell culture, this approaches enables us to investigate circadian rhythms and their functions in a more physiologically relevant organoid model. Our findings show disruptive circadian rhythms in stem cell-enriched conditions while demonstrating robust circadian oscillations in differentiated human intestinal enteroids, suggesting a remodeling of circadian clock machinery from stem cells to differentiated cells. Usages of patient-derived organoids with circadian bioluminescent reporters will help us advance our understanding of circadian rhythms in peripheral organs and different disease states.

To begin, arrange all the materials required for packaging of human intestinal enteroids, or HIEs, onto the working platform. Mix the HIEs with growth factor-reduced 3D culture matrix. Seed the mixture onto a 24-well plate with three droplets of 10 microliters of 3D culture matrix per well and incubate for 20 minutes at 37 degrees Celsius.

Then, add 350 microliters of the HIE growth medium. After seven days, collect HIEs from four wells into 1.5 milliliter tubes. Centrifuge the HIEs at 2000 G for 40 seconds to remove the debris.

Using a pipette, remove the excessive 3D culture matrix from the tube. Add 0.5 milliliters of cold PBS into the tube. Using a one milliliter insulin syringe, apply physical turbulence by syringe up to 10 times to dissociate the HIEs.

Centrifuge the dissociated HIEs at 2000 G for 40 seconds and gently remove the supernatant. Then, place the tube on ice for approximately three minutes. Add growth factor-reduced 3D culture matrix to the tube and mix it with the pellet.

Seed the mixture of HIEs in the 3D culture matrix on the center of the 35 millimeter round dish. Incubate the dish at 37 degrees Celsius for 20 minutes to solidify the 3D culture matrix. Then, add two milliliters of pre-warmed intestinal organoid growth medium to the dish and incubate at 37 degrees Celsius with 5%carbon dioxide for 48 hours.

On day three, replace the growth medium with two milliliters of pre-warmed differentiation medium and incubate at 37 degrees Celsius with 5%carbon dioxide for 48 hours. On day five, replace the enteroids medium with two milliliters of pre-warmed differentiation medium, and continue incubation for 24 hours. Begin by removing the dish containing differentiated HIEs from the incubator.

To synchronize the molecular clock of enteroids, add two microliters of 100 micromolar dexamethasone to the dish and incubate at 37 degree Celsius with 5%carbon dioxide for one hour. During incubation, set the carbon dioxide percentage and temperature of the luminometer to 5%and 37 degrees Celsius respectively. After the clock synchronization, replenish the enteroids with three milliliters of pre-warmed differentiation medium containing 200 micromolar D-luciferin.

Place the dish in an eight-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 and close the lid of the machine. On the bioluminescence software, click on the condition tab to adjust the settings for the number of dishes, measurement time, background processing, and filter type.

Then, click on okay to save the settings. To start the experiment, click the run tab followed by the start button. Then, click on the raw, noise-filtered, or detrended tabs on the top of the screen to observe the signals for the selected data.

After recording, click on the stop button to stop the run. Go to the file tab, select save as, and save the data in the Kronos format. Finally, click on file and select export as Excel format to export the data to a spreadsheet.

Bioluminescence recording evaluated the circadian rhythmicity of HIEs under stem cell-enriched and differentiation-inducing conditions. The fast Fourier transform analysis revealed that, under differentiating conditions, Bmal1 luciferase bioluminescence exhibited robust circadian oscillations with a mean periodicity of 22.92 hours. In contrast, HIEs demonstrated disrupted circadian rhythms in stem cell-enriched conditions.