The human intestinal enteroids were obtained from the lab of Dr. Michael Helmrath at Cincinnati Children&#39;s Hospital Medical Center (CCHMC). This work was supported by R01 DK11005 (CIH) and the University of Cincinnati Cancer Center Pilot Funding. We are grateful for imaging support from the University of Cincinnati Live Microscopy Core (NIH S10OD030402).

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...

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

<ol>
	<li>Cox, K. H., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clock+genes+and+the+transcriptional+architecture+of+the+clock+mechanism.">Circadian clock genes and the transcriptional architecture of the clock mechanism.</a> J Mol Endocrinol. 63 (4), R93-R102 (2019).</li><li>Ayyar, V. S., Sukumaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms:+Influence+on+physiology,+pharmacology,+and+therapeutic+interventions.">Circadian rhythms: Influence on physiology, pharmacology, and therapeutic interventions.</a> J Pharmacokinet Pharmacodyn. 48 (3), 321-338 (2021).</li><li>Reppert, S., Weaver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+circadian+timing+in+mammals.">Coordination of circadian timing in mammals.</a> Nature. 418 (6901), 935-941 (2002).</li><li>Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+architecture+of+the+mammalian+circadian+clock.">Transcriptional architecture of the mammalian circadian clock.</a> Nat Rev Genet. 18 (3), 164-179 (2017).</li><li>Wolff, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+age-dependent+and+tissue-specific+circadian+transcriptome+in+male+mice.">Defining the age-dependent and tissue-specific circadian transcriptome in male mice.</a> Cell Rep. 42 (1), 111982 (2023).</li><li>Ruben, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+database+of+tissue-specific+rhythmically+expressed+human+genes+has+potential+applications+in+circadian+medicine.">A database of tissue-specific rhythmically expressed human genes has potential applications in circadian medicine.</a> Science Trans Med. 10 (458), 8806 (2018).</li><li>Huang, S., Lu, Q., Choi, M. H., Zhang, X., Kim, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+real-time+monitoring+of+circadian+oscillations+in+adult+mouse+brain+slices+to+study+communications+between+brain+regions.">Applying real-time monitoring of circadian oscillations in adult mouse brain slices to study communications between brain regions.</a> STAR Protoc. 2 (2), 100416 (2021).</li><li>Yagita, K., Yamanaka, I., Emoto, N., Kawakami, K., Shimada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+circadian+clock+oscillations+in+primary+cultures+of+mammalian+cells+using+Tol2+transposon-mediated+gene+transfer+strategy.">Real-time monitoring of circadian clock oscillations in primary cultures of mammalian cells using Tol2 transposon-mediated gene transfer strategy.</a> BMC Biotechnol. 10 (3), (2010).</li><li>Choy, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+noninvasive+fluorescent+and+bioluminescent+small+animal+optical+imaging.">Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging.</a> Biotechniques. 35 (5), 1022-1030 (2003).</li><li>Leclerc, G. M., Boockfor, F. R., Faught, W. J., Frawley, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+destabilized+firefly+luciferase+enzyme+for+measurement+of+gene+expression.">Development of a destabilized firefly luciferase enzyme for measurement of gene expression.</a> Biotechniques. 29 (3), 590-598 (2000).</li><li>Ramanathan, C., Khan, S. K., Kathale, N. D., Xu, H., Liu, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cell-autonomous+circadian+clock+rhythms+of+gene+expression+using+luciferase+bioluminescence+reporters.">Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.</a> J Vis Exp. (67), e4234 (2012).</li><li>Yoo, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc Natl Acad Sci U S A. 101 (15), 5339-5346 (2004).</li><li>Welsh, D. K., Yoo, S. H., Liu, A. C., Takahashi, J. S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+individual+fibroblasts+reveals+persistent,+independently+phased+circadian+rhythms+of+clock+gene+expression.">Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.</a> Curr Biol. 14 (24), 2289-2295 (2004).</li><li>Tiscornia, G., Singer, O., Verma, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat Protoc. 1 (1), 241-245 (2006).</li><li>Lee, S., Hong, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+as+model+systems+to+investigate+circadian+clock-related+diseases+and+treatments.">Organoids as model systems to investigate circadian clock-related diseases and treatments.</a> Front Genet. 13, 874288 (2022).</li><li>Zachos, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+enteroids/colonoids+and+intestinal+organoids+functionally+recapitulate+normal+intestinal+physiology+and+pathophysiology.">Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology.</a> J Biol Chem. 291 (8), 3759-3766 (2016).</li><li>Bartfeld, S., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+organoids+and+their+application+for+medical+research+and+patient+treatment.">Stem cell-derived organoids and their application for medical research and patient treatment.</a> J Mol Med. 95 (7), 729-738 (2017).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+development+and+disease+with+organoids.">Modeling development and disease with organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Rosselot, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+function+of+the+circadian+clock+in+intestinal+organoids.">Ontogeny and function of the circadian clock in intestinal organoids.</a> EMBO J. 41 (2), 106973 (2021).</li><li>Corr&#242;, C., Novellasdemut, L., Li, V. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+history+of+organoids.">A brief history of organoids.</a> Am J Physiol Cell Physiol. 319 (1), C151-C165 (2020).</li><li>Brown, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+period+length+of+fibroblast+circadian+gene+expression+varies+widely+among+human+individuals.">The period length of fibroblast circadian gene expression varies widely among human individuals.</a> PLos Biol. 3 (10), e338 (2005).</li><li>Van Lidth de Jeude, J. F., Vermeulen, J. L. M., Montenegro-Miranda, P. S., Vanden Brink, G. R., Heijmans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+lentiviral+transduction+and+downstream+analysis+of+intestinal+organoids.">A protocol for lentiviral transduction and downstream analysis of intestinal organoids.</a> J Vis Exp. (98), e52531 (2015).</li><li>Criss, Z. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drivers+of+transcriptional+variance+in+human+intestinal+epithelial+organoids.">Drivers of transcriptional variance in human intestinal epithelial organoids.</a> Physiol Genomics. 53 (11), 486-508 (2021).</li><li>Izumo, M., Sato, T. R., Straume, M., Johnson, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analyses+of+circadian+gene+expression+in+mammalian+cell+cultures.">Quantitative analyses of circadian gene expression in mammalian cell cultures.</a> PLoS Comput Biol. 2 (10), e136 (2006).</li><li>Hara-Miyauchi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+system+for+dynamic+imaging+of+cell+and+animal+behavior.">Bioluminescent system for dynamic imaging of cell and animal behavior.</a> Biochem Biophys Res Commun. 419 (2), 188-193 (2012).</li><li>Tung, J. K., Berglund, K., Gutekunst, C. A., Hochgeschwender, U., Gross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+in+live+cells+and+animals.">Bioluminescence imaging in live cells and animals.</a> Neurophotonics. 3 (2), 025001 (2016).</li><li>Xu, P., Elizalde, M., Masclee, A., Pierik, M., Jonkers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticosteroid+enhances+epithelial+barrier+function+in+intestinal+organoids+derived+from+patients+with+Chron's+disease.">Corticosteroid enhances epithelial barrier function in intestinal organoids derived from patients with Chron's disease.</a> J Mol Med (Berl). 99 (6), 805-815 (2021).</li><li>AlMusawi, S., Ahmed, M., Nateri, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+cell-cell+communication+and+signaling+in+the+colorectal+cancer+microenvironment.">Understanding cell-cell communication and signaling in the colorectal cancer microenvironment.</a> Clin Transll Med. 11 (2), 308 (2021).</li></ol>

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&#39; functions, interested cellular events or gene expression can be recorded using bioluminescence readout, which gives reliable real-time data. Importantly, the bioluminescence assay ...

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 changes1,2. 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&#39; 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 mechanisms7,8. 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 noise11,12,13. 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 &#34;organ-like&#34; structures that mimic organ structure in a dish by displaying similarity to in vivo tissue architecture and multicellularity, including stem, progenitor, and differentiated cell types15,16. 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 kidney18,19. 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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 x 10 Falcon tissue culture dishes</td>
			<td>Fisher Scientific</td>
			<td>08-772A</td>
			<td></td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>Sigma Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life Technologies</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro-Fine IV Insulin Syringes</td>
			<td>Fisher Scientific</td>
			<td>14-829-1Bb</td>
			<td>Mfrn: BD 329424</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman Chemical</td>
			<td>13122</td>
			<td>GSK-3 inhibitor</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D4902-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin (potassium salt)</td>
			<td>Cayman Chemical</td>
			<td>14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin I Human</td>
			<td>Sigma Aldrich</td>
			<td>G9020</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factor reduced (GFR) Matrigel</td>
			<td>Corning</td>
			<td>CB-40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult Organoid Growth Medium (Human)</td>
			<td>Stemcell Technologies</td>
			<td>06010</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Kronos Dio Luminometer Machine</td>
			<td>ATTO Corporation</td>
			<td>AB-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>pABpuro-BluF reporter plasmid</td>
			<td>Addgene</td>
			<td>46824</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine EGF</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>R&#38;D Systems</td>
			<td>1254/10</td>
			<td>ROCK inhibitor</td>
		</tr>
	</tbody>
</table>

monitoring circadian oscillations with a bioluminescence reporter in organoids

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.

Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Our current scope of research is to investigate the roles of circadian rhythms in intestinal 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 real-time monitoring method for circadian studies in three-dimensional organoids using a bioluminescence assay.Unlike performing bioluminescence assays in a conventional two-dimensional cell culture, this approach enables us to investigate circadian rhythms and their functions in a more physiologically relevant organoid model. Our findings show disrupted circadian rhythms in stem cell-enriched conditions while demonstrating robust circadian oscillations in differentiated human intestinal organoids, 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.

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...

Watch this Scientific Journal Video about Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems at JoVE.com

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.

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

monitoring-circadian-oscillations-with-bioluminescence-reporter

Research

JoVE Journal

Biology

629 visualizações.  University of Cincinnati College of Medicine. Nosso escopo atual de pesquisa é investigar os papéis dos ritmos circadianos na proliferação e diferenciação de células epiteliais intestinais. Estamos tentando determinar se o relógio circadiano regula a proliferação celular e a diferenciação das células-tronco intestinais. Este protocolo apresenta um método de monitoramento em tempo real para estudos circadianos em organoides tridimensionais usando um ensaio de bioluminescência.Ao contrário da realização de ensaios ...