Name: combining human organoids and organ-on-a-chip technology to model intestinal region-specific functionality
Uploaded: 2022-05-05T15:00:00
Description: Watch this Scientific Journal Video about Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality at JoVE.com

We thank Professor Mark Donowitz for providing the intestinal biopsy-derived organoids and Brett Clair for designing the scientific illustrations of the chip, portable and culture module. All the rest of the scientific illustrations were generated using the BioRender.

NOTE: All cell cultures should be handled using a proper aseptic technique.
The human intestinal organoids employed in this study were obtained from Johns Hopkins University and all methods were carried out in accordance with approved guidelines and regulations. All experimental protocols were approved by the Johns Hopkins University Institutional Review Board (IRB #NA 00038329).
1. Preparation of the cell culture reagents
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	<li>Prepare the hu...

<ol>
	<li>Fritz, 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=Expression+of+clinically+relevant+drug-metabolizing+enzymes+along+the+human+intestine+and+their+correlation+to+drug+transporters+and+nuclear+receptors:+An+intra-subject+analysis.">Expression of clinically relevant drug-metabolizing enzymes along the human intestine and their correlation to drug transporters and nuclear receptors: An intra-subject analysis.</a> Basic and Clinical Pharmacology and Toxicology. 124 (3), 245-255 (2019).</li><li>Okumura, R., Takeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+intestinal+homeostasis+by+mucosal+barriers.">Maintenance of intestinal homeostasis by mucosal barriers.</a> Inflammation and Regeneration. 38 (1), 1-8 (2018).</li><li>Delgado, M. E., Grabinger, T., Brunner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+at+the+intestinal+epithelial+front+line.">Cell death at the intestinal epithelial front line.</a> FEBS Journal. 283 (14), 2701-2719 (2016).</li><li>Mestas, J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+Differences+between+mouse+and+human+immunology.">Of mice and not men: Differences between mouse and human immunology.</a> The Journal of Immunology. 172 (5), 2731-2738 (2004).</li><li>Sun, H., Chow, E. C. Y., Liu, S., Du, Y., Pang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caco-2+cell+monolayer:+Usefulness+and+limitations.">The Caco-2 cell monolayer: Usefulness and limitations.</a> Expert Opinion on Drug Metabolism and Toxicology. 4 (4), 395-411 (2008).</li><li>Yu, 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=The+contributions+of+human+mini-intestines+to+the+study+of+intestinal+physiology+and+pathophysiology.">The contributions of human mini-intestines to the study of intestinal physiology and pathophysiology.</a> Annual Review of Physiology. 79, 291-312 (2017).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Sato, T., 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett&#8217;s+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett&#8217;s epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Spence, J. R., 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=Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro.">Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.</a> Nature. 470 (7332), 105-110 (2011).</li><li>Durel, J. F., Nerurkar, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+vertebrate+gut+morphogenesis.">Mechanobiology of vertebrate gut morphogenesis.</a> Current Opinion in Genetics and Development. 63, 45-52 (2020).</li><li>Gayer, C. P., Basson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanical+forces+on+intestinal+physiology+and+pathology.">The effects of mechanical forces on intestinal physiology and pathology.</a> Cellular Signalling. 21 (8), 1237-1244 (2009).</li><li>Xu, Y., 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=Mechanical+stimulation+activates+Piezo1+to+promote+mucin2+expression+in+goblet+cells.">Mechanical stimulation activates Piezo1 to promote mucin2 expression in goblet cells.</a> Journal of Gastroenterology and Hepatology (Australia). 36 (11), 3127-3139 (2021).</li><li>Navabi, N., McGuckin, M. A., Lind&#233;n, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+cell+lines+form+polarized+epithelia+with+an+adherent+mucus+layer+when+cultured+in+semi-wet+interfaces+with+mechanical+stimulation.">Gastrointestinal cell lines form polarized epithelia with an adherent mucus layer when cultured in semi-wet interfaces with mechanical stimulation.</a> PLoS One. 8 (7), 68761 (2013).</li><li>Kasendra, M., 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=Development+of+a+primary+human+Small+Intestine-on-a-Chip+using+biopsy-derived+organoids.">Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids.</a> Scientific Reports. 8 (1), 1-14 (2018).</li><li>Kasendra, M., 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=Duodenum+intestine-chip+for+preclinical+drug+assessment+in+a+human+relevant+model.">Duodenum intestine-chip for preclinical drug assessment in a human relevant model.</a> eLife. 9, 50135 (2020).</li><li>Apostolou, 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=A+novel+microphysiological+colon+platform+to+decipher+mechanisms+driving+human+intestinal+permeability.">A novel microphysiological colon platform to decipher mechanisms driving human intestinal permeability.</a> Cellular and Molecular Gastroenterology and Hepatology. 12 (5), 1719-1741 (2021).</li><li>Jalili-Firoozinezhad, S., 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=Author+correction:+A+complex+human+gut+microbiome+cultured+in+an+anaerobic+intestine-on-a-chip.">Author correction: A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip.</a> Nature Biomedical Engineering. 3 (7), 583 (2019).</li><li>Yin, J., 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=Fluid+shear+stress+enhances+differentiation+of+jejunal+human+enteroids+in+Intestine-Chip.">Fluid shear stress enhances differentiation of jejunal human enteroids in Intestine-Chip.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 320 (3), 258-271 (2021).</li><li>Sontheimer-Phelps, 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=Human+colon-on-a-chip+enables+continuous+in+vitro+analysis+of+colon+mucus+layer+accumulation+and+physiology.">Human colon-on-a-chip enables continuous in vitro analysis of colon mucus layer accumulation and physiology.</a> Cellular and Molecular Gastroenterology and Hepatology. 9 (3), 507-526 (2020).</li><li>Tovaglieri, 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=Species-specific+enhancement+of+enterohemorrhagic+E.+coli+pathogenesis+mediated+by+microbiome+metabolites.">Species-specific enhancement of enterohemorrhagic E. coli pathogenesis mediated by microbiome metabolites.</a> Microbiome. 7 (1), 43 (2019).</li><li>Huh, 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=Microfabrication+of+human+organs-on-chips.">Microfabrication of human organs-on-chips.</a> Nature Protocols. 8 (11), 2135-2157 (2013).</li><li>Fujii, M., Matano, M., Nanki, K., Sato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genetic+engineering+of+human+intestinal+organoids+using+electroporation.">Efficient genetic engineering of human intestinal organoids using electroporation.</a> Nature Protocols. 10 (10), 1474-1485 (2015).</li><li>Kim, H. J., Huh, D., Hamilton, G., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gut-on-a-chip+inhabited+by+microbial+flora+that+experiences+intestinal+peristalsis-like+motions+and+flow.">Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow.</a> Lab on a Chip. 12 (12), 2165-2174 (2012).</li><li>Sarna, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+motility:+From+bench+side+to+bedside.">Colonic motility: From bench side to bedside.</a> Morgan &amp; Claypool Life Sciences. , (2010).</li><li>Basson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradigms+for+mechanical+signal+transduction+in+the+intestinal+epithelium+-+category:+Molecular,+cell,+and+developmental+biology.">Paradigms for mechanical signal transduction in the intestinal epithelium - category: Molecular, cell, and developmental biology.</a> Digestion. 68 (4), 217-225 (2003).</li><li>Wang, F., 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=Interferon-gamma+and+tumor+necrosis+factor-alpha+synergize+to+induce+intestinal+epithelial+barrier+dysfunction+by+up-regulating+myosin+light+chain+kinase+expression.">Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression.</a> The American Journal of Pathology. 166 (2), 409-419 (2005).</li><li>Nava, P., 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=Interferon-&#947;+regulates+intestinal+epithelial+homeostasis+through+converging+&#946;-Catenin+signaling+pathways.">Interferon-&#947; regulates intestinal epithelial homeostasis through converging &#946;-Catenin signaling pathways.</a> Immunity. 32 (3), 392-402 (2010).</li><li>Madara, J. L., Stafford, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-&#947;+directly+affects+barrier+function+of+cultured+intestinal+epithelial+monolayers.">Interferon-&#947; directly affects barrier function of cultured intestinal epithelial monolayers.</a> Journal of Clinical Investigation. 83 (2), 724-727 (1989).</li><li>Bruewer, M., 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=Proinflammatory+cytokines+disrupt+epithelial+barrier+function+by+apoptosis-independent+mechanisms.">Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms.</a> The Journal of Immunology. 171 (11), 6164-6172 (2003).</li><li>Jones, S. 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=Adhesion+molecules+in+inflammatory+bowel+disease.">Adhesion molecules in inflammatory bowel disease.</a> Gut. 36 (5), 724-730 (1995).</li><li>Uhlar, C. M., Whitehead, 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=Serum+amyloid+A,+the+major+vertebrate+acute-phase+reactant.">Serum amyloid A, the major vertebrate acute-phase reactant.</a> European Journal of Biochemistry. 265 (2), 501-523 (1999).</li><li>P&#233;rez-Gonz&#225;lez, C., Ceada, G., Matej&#269;i&#263;, M., Trepat, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digesting+the+mechanobiology+of+the+intestinal+epithelium.">Digesting the mechanobiology of the intestinal epithelium.</a> Current Opinion in Genetics &amp; Development. 72, 82-90 (2022).</li><li>In, J., 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=Enterohemorrhagic+escherichia+coli+reduces+mucus+and+intermicrovillar+bridges+in+human+stem+cell-derived+colonoids.">Enterohemorrhagic escherichia coli reduces mucus and intermicrovillar bridges in human stem cell-derived colonoids.</a> Cellular and Molecular Gastroenterology and Hepatology. 2 (1), 48-62 (2016).</li><li>Grassart, 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=Bioengineered+human+organ-on-chip+reveals+intestinal+microenvironment+and+mechanical+forces+impacting+shigella+infection.">Bioengineered human organ-on-chip reveals intestinal microenvironment and mechanical forces impacting shigella infection.</a> Cell Host and Microbe. 26 (3), 435-444 (2019).</li><li>Kerns, S. J., 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+immunocompetent+organ-on-chip+platforms+allow+safety+profiling+of+tumor-targeted+t-cell+bispecific+antibodies.">Human immunocompetent organ-on-chip platforms allow safety profiling of tumor-targeted t-cell bispecific antibodies.</a> eLife. 10, 67106 (2021).</li><li>Bhatia, S. N., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+organs-on-chips.">Microfluidic organs-on-chips.</a> Nature Biotechnology. 32 (8), 760-772 (2014).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+engineering+human+pathophysiology+with+organs-on-chips.">Reverse engineering human pathophysiology with organs-on-chips.</a> Cell. 164 (6), 1105-1109 (2016).</li></ol>

The combination of organ-on-a-chip technology and intestinal organoids holds promise for accurate modeling of human intestinal physiology and pathophysiology. Here, we provide a simple and robust step-by-step protocol (outlined in Figure 1) for establishment of the intestine chip containing biopsy-derived small intestinal or colonic epithelium and intestinal microvascular endothelial cells co-cultured in a microfluidic device. This chip-based simulation of the human intestine incorporates ph...

The human intestine is a complex and multitasking organ capable of self-regeneration. It is divided into the small and large intestine. The primary function of the small intestine is to further digest food coming from the stomach, absorb all the nutrients, and pass the residue on to the large intestine, which recovers the water and electrolytes. The small intestine is further divided into multiple anatomically distinct regions: the duodenum, jejunum, and ileum, each of which is adapted to perform specific functions. For example, the duodenum helps&#160;break down of the chyme (stomach contents) to enable the proper absorption of nutrients involving proteins, carbohydrates, vitamins, and minerals in the jejunum. This proximal part of the small intestine is also the main site of intestinal drug absorption and metabolism, and it is characterized by the higher expression of drug-metabolizing enzymes (e.g., CYP3A4) compared to their expression in the ileum and colon1. In addition to its main role in digesting and absorbing of nutrients, the intestine is also an effective barrier against potentially harmful luminal contents, such as pathogenic microorganisms, microbial metabolites, dietary antigens, and toxins2,3. It is noteworthy that the human colon is inhabited by a large number of microorganisms, far exceeding that of total cells in the human body, which provide many benefits to nutrition, metabolism, and immunity. Therefore, the maintenance of the integrity of the mucosal barrier formed by intestinal epithelial cells is critical for allowing the symbiotic relationship between the gut microbiota and the host cells by physically separating them to avoid unnecessary immune cell activation2. In addition, programmed intestinal cell death plays an essential role as a self-protective mechanism preventing infected cells from persisting or proliferating&#8212;thereby disseminating potential pathogens3&#8212;while the continuous self-renewal of intestinal epithelium every four to seven days compensates for the cell loss ensuring barrier integrity and tissue homeostasis. Impairments of described intestinal functions, including nutrient absorption, barrier integrity, or imbalance in intestinal cell death and self-renewal, may result in the development of a range of gastrointestinal disorders, including&#160;malnutrition and inflammatory bowel disease (IBD)2,3.
Previously, animal models and transformed cancer-derived intestinal cell lines have been used to study the physiological and pathophysiological functions of human intestinal tissue. However, increasingly prominent concerns about the translatability of animal research to humans, caused by the presence of significant disparities between the two species, highlighted a need for human-relevant alternative methods4. The commonly used in vitro intestinal cell lines include T84, Caco-2, and HT29 cells. While they mimic certain aspects of the intestinal barrier function and membrane transport, they are characterized by an altered expression of drug metabolizing enzymes5, surface receptors, and transporters4. In addition, they lack intestinal segment specificity and fail to recapitulate the complexity of intestinal epithelium, with each model containing only one out of the five epithelial cell types present in the intestine6.
Recently, human intestinal organoid cultures established from fresh biopsies of the small intestine and colon7,8 or induced pluripotent stem cells (iPSC)9 were introduced as alternative experimental models with&#160;potential to complement, reduce and perhaps replace animal experimentation in the future. While iPSCs can be obtained in a non-invasive manner, establishing organoids from iPSCs requires the use of complex and lengthy protocols (with several experimental steps) and generates cultures resembling human fetal tissue. In contrast, biopsy-derived organoids are highly scalable, as they can leverage the inherent renewal capacity of intestinal tissue and can be indefinitely passaged and propagated in vitro. Importantly, biopsy-derived organoids maintain the disease and intestinal region-specific characteristics of the primary tissue from which they were developed and emulate the cellular diversity of the intestinal epithelium. Organoids can be used as patient-specific avatars in vitro to unravel the biology and pathogenesis of various gastrointestinal disorders and improve their therapeutic management. Although intestinal organoids have achieved an impressive degree of physiological functionality, they still fail to reproduce the complexity of the native organs due to their lack of critical stromal components&#8212;including blood vessels, connective tissue, peripheral nerves, and immune cells&#8212;as well as mechanical stimulation. Mechanical parameters, such as flow, shear stress, stretch, and pressure, are known to influence tissue morphogenesis and homeostasis in vivo and were previously shown to improve maturation of cells in vitro10,11,12,13. An additional important drawback of organoid systems is the inaccessibility of the lumen and, thus, to the apical side of the epithelium. This presents a challenge for investigating various mechanisms associated with the polarized expression of ion and drug transporters, host-microbiome interactions, and pharmaceutical toxicity testing. Lastly, organoid cultures suffer from considerable variability in size, morphology, and function, owing to the stochastic nature of the&#160;in vitro self-organization process and cell fate choices. Therefore, to realize the full potential of intestinal organoids in disease modeling, drug screening, and regenerative medicine, it is necessary to explore new strategies that reduce the variability in organoid development, improve the access to the luminal compartment, and incorporate missing cell-cell interactions.
Organ-on-a-Chip technology has introduced many techniques for the incorporation of mechanical forces and fluid flow to intestinal cell cultures in vitro. However, since most of the initial proof-of-concept studies have used cancer-derived cell lines that did not exhibit sufficient cellular diversity, the relevance of these systems has been questioned. Recently, we have synergistically combined intestinal organoids and organ-on-a-chip technology to incorporate the best features of each approach into one in vitro system14,15,16. The resulting intestine-chip recapitulates&#160;the multicellular&#160;architecture of intestinal epithelium, the presence of epithelial-endothelial tissue interface, and the mechanical forces of fluid flow and stretch, enabling emulation of organ-level functions in vitro. Additionally, the use of primary-tissue-derived organoids (which can be sampled from different regions of the human intestine) as a starting material increases this model&#8217;s versatility, as chips representing human duodenum, jejunum, ileum, and colon can be established following similar seeding and culture procedures. Importantly, Intestine-Chips enable real-time assessment of: the intestinal barrier integrity; activity of the brush border and drug-metabolism enzymes; production of mucins; secretion of cytokines; and interaction of intestinal cells with pathogenic and commensal microorganisms, as demonstrated in the previously published reports. Notably, when intestine chips were established using organoids generated from different individuals&#8217; tissue, these models captured the expected inter-donor variability in the functional responses to various drugs and treatments. Altogether,&#160;merging organoids with Organ-on-a-Chip technology opens the door to more advanced, personalized, in vivo-relevant&#160;models that could improve the physiological relevance and accuracy of the in vitro findings as well as their extrapolation to humans. Here, a detailed protocol is presented for&#160;establishing the intestine chip and its application in the studies of physiological functions of the two intestinal segments: duodenum and colon. Firstly, the methods for the assessing the activity of the drug-metabolizing enzyme&#160;CYP3A4&#160;in the duodenum chip, as well as its induction by prototypical compounds such as rifampicin and Vitamin D3, are described. Secondly, the steps required to model &#8220;leaky gut&#8221; in the colon chip are outlined in the protocol, with the disruption of the epithelial barrier being performed using hallmark cytokines implicated in the pathogenesis of the IBD. Briefly, organoids derived from human biopsies are propagated in vitro, subjected to enzymatic digestion, and introduced in the top channel of the chip. In the presence of continuous perfusion with growth-factor-enriched media&#160;they develop into a confluent epithelial monolayer with 3D architecture and a readily accessible apical cell surface. The bottom, &#34;vascular&#34; chip compartment is seeded with microvascular endothelial cells isolated from the small or large intestine. The epithelium and endothelium are separated by a porous stretchable membrane, which facilitates the paracrine interactions between the two tissues and, when subjected to cyclic deformations, emulates peristalsis-like motions of the human gut. The co-culture is maintained under the dynamic flow conditions generated by luminal and vascular perfusion with appropriate cell culture media. Finally, we describe numerous types of assays and endpoint analyses&#160;that can be performed directly on-chip or from sampled cell culture effluents.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>small intestine Human Intestinal Microvascular Endothelial Cells</td>
			<td>AlphaBioRegen</td>
			<td>ALHE15</td>
			<td>0.5 cells M/ml ; cryopreserved</td>
		</tr>
		<tr>
			<td>colon Human Intestinal Microvascular Endothelial Cells</td>
			<td>AlphaBioRegen</td>
			<td>ALHE16</td>
			<td>0.5 cells M/ml ; cryopreserved</td>
		</tr>
		<tr>
			<td>Biopsy-derived Human Duodenal Organoids</td>
			<td>John Hopkin&#39;s University</td>
			<td>-</td>
			<td>The organoids were provided by Professor Mark Donowitz (Institutional Review Board Number: NA_00038329).</td>
		</tr>
		<tr>
			<td>Biopsy-derived Human Colonic Organoids</td>
			<td>John Hopkin&#39;s University</td>
			<td>-</td>
			<td>The organoids were provided by Professor Mark Donowitz (Institutional Review Board Number: NA_00038329).</td>
		</tr>
		<tr>
			<td>Zo&#235; CM-1&#8482;&#160;Culture Module</td>
			<td>Emulate&#160;Inc.</td>
			<td>-</td>
			<td>Culture module</td>
		</tr>
		<tr>
			<td>Orb-HM1&#8482;&#160;Hub Module</td>
			<td>Emulate&#160;Inc.</td>
			<td>-</td>
			<td>5% CO2, vacuum stretch, and power supply</td>
		</tr>
		<tr>
			<td>Chip-S1&#8482; Stretchable Chip</td>
			<td>Emulate&#160;Inc.</td>
			<td>-</td>
			<td>Organ-Chip</td>
		</tr>
		<tr>
			<td>Pod&#8482; Portable Modules</td>
			<td>Emulate&#160;Inc.</td>
			<td>-</td>
			<td>Portable module</td>
		</tr>
		<tr>
			<td>UV Light Box</td>
			<td>Emulate&#160;Inc.</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Chip Cradle</td>
			<td>Emulate&#160;Inc.</td>
			<td>-</td>
			<td>1 per square culture dish</td>
		</tr>
		<tr>
			<td>Steriflip&#174;-HV Filters</td>
			<td>EMD Millipore</td>
			<td>SE1M003M00</td>
			<td>0.45&#160;&#956;m&#160;PVDF filter</td>
		</tr>
		<tr>
			<td>Square Cell Culture Dish (120 x 120 mm)</td>
			<td>VWR</td>
			<td>82051-068</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Handheld&#160;vacuum&#160;aspirator</td>
			<td>Corning</td>
			<td>4930</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Aspirating&#160;pipettes</td>
			<td>Corning / Falcon</td>
			<td>357558</td>
			<td>2 mL,&#160;polystyrene, individually&#160;wrapped</td>
		</tr>
		<tr>
			<td>Aspirating tips</td>
			<td>&#160;-</td>
			<td></td>
			<td>Sterile (autoclaved)</td>
		</tr>
		<tr>
			<td>Serological&#160;pipettes</td>
			<td></td>
			<td>&#160;-</td>
			<td>2 mL, 5 mL,&#160;10 mL,&#160;and&#160;25 mL&#160;low endotoxin, sterile</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td></td>
			<td></td>
			<td>P20, P200,&#160;P1000&#160;and standard multichannel</td>
		</tr>
		<tr>
			<td>Pipette&#160;tips&#160;</td>
			<td></td>
			<td></td>
			<td>P20, P200,&#160;and&#160;P1000.</td>
		</tr>
		<tr>
			<td>Conical tubes&#160;(Protein&#160;LoBind&#174; Tubes)</td>
			<td>Eppendorf</td>
			<td>0030122216;&#160;0030122240</td>
			<td>15 mL, 50 mL tubes</td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Tubes&#174;&#160;lo-bind</td>
			<td>Eppendorf</td>
			<td>022431081</td>
			<td>1.5 mL tubes</td>
		</tr>
		<tr>
			<td>96 wells black walled plate</td>
			<td>-</td>
			<td>-</td>
			<td>for epithelial permeability analysis</td>
		</tr>
		<tr>
			<td>Microscope (with camera)</td>
			<td>-</td>
			<td>-</td>
			<td>For bright-field&#160;imaging</td>
		</tr>
		<tr>
			<td>Water bath (or beads)</td>
			<td>-</td>
			<td>-</td>
			<td>Set to 37&#176;C</td>
		</tr>
		<tr>
			<td>Vacuum set-up</td>
			<td>&#160;-</td>
			<td>-</td>
			<td>Minimum&#160;pressure: -70 kPa</td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Biotium</td>
			<td>220033</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td>Cell culture flask</td>
		</tr>
		<tr>
			<td>Emulate Reagent-1&#160;(ER-1)</td>
			<td>Emulate Inc.</td>
			<td>-</td>
			<td>Chip coating solution</td>
		</tr>
		<tr>
			<td>Emulate Reagent-2&#160;(ER-2)</td>
			<td>Emulate Inc.</td>
			<td>-</td>
			<td>Chip coating solution</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS&#160;(DPBS)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>MT25055CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>93595</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>TryplE&#160;Express</td>
			<td>ThermoFisher&#160;Scientific</td>
			<td>12604013</td>
			<td>Organoids&#160;dissociation&#160;and endothelium cells&#160;detachment&#160;solution</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>ThermoFisher&#160;Scientific</td>
			<td>12634028</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>IntestiCult&#8482; Organoid Growth Medium (Human)</td>
			<td>Stem Cell technologies</td>
			<td>06010</td>
			<td>Organoid Growth Medium</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium MV 2</td>
			<td>Promocell</td>
			<td>C-22121</td>
			<td>Endothelial medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td>Serum</td>
		</tr>
		<tr>
			<td>Primocin&#8482;&#160;</td>
			<td>InvivoGen</td>
			<td>ANT-PM-1</td>
			<td>antimicrobial agent</td>
		</tr>
		<tr>
			<td>Attachment Factor&#8482;</td>
			<td>Cell Systems</td>
			<td>4Z0-210</td>
			<td>coating solution for flask</td>
		</tr>
		<tr>
			<td>Matrigel - Growth Factor Reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Solubilized basement membrane matrix</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td>ECM component</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td>ECM component</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stem Cell technologies</td>
			<td>72304</td>
			<td>organoid media supplement</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Reprocell</td>
			<td>04-0004-10</td>
			<td>organoid media supplement</td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td>Basement mebrane matrix dissociationsolution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9576</td>
			<td>30%, Sterile</td>
		</tr>
		<tr>
			<td>Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>MT25055CV</td>
			<td>Sterile, Water</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>solvent</td>
		</tr>
		<tr>
			<td>3KDa Dextran Cascade Blue</td>
			<td>Invitrogen</td>
			<td>D7132</td>
			<td>10 mg powder</td>
		</tr>
		<tr>
			<td>Rifampicin (RIF)</td>
			<td>Sigma</td>
			<td>Cat# R3501</td>
			<td>CYP inducer</td>
		</tr>
		<tr>
			<td>Testosterone hydrate</td>
			<td>Sigma</td>
			<td>T1500</td>
			<td>CYP substrate</td>
		</tr>
		<tr>
			<td>1,25-dihyroxy Vitamin D3 (VD3)</td>
			<td>Sigma</td>
			<td>Cat# D1530</td>
			<td>CYP inducer</td>
		</tr>
		<tr>
			<td>Acetonitrile with 0.1% (v/v) Formic acid</td>
			<td>Sigma</td>
			<td>159002</td>
			<td>LCMS stop solution</td>
		</tr>
		<tr>
			<td>IFN&#947;</td>
			<td>Peprotech</td>
			<td>300-02</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (PFA)</td>
			<td>EMS</td>
			<td>157-4</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum (NDS)</td>
			<td>Sigma</td>
			<td>566460</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Occludin</td>
			<td>ThermoFisher&#160;Scientific</td>
			<td>33-1500</td>
			<td>tight junctions marker</td>
		</tr>
		<tr>
			<td>anti-Claudin 4</td>
			<td>ThermoFisher&#160;Scientific</td>
			<td>36-4800</td>
			<td>tight junctions marker</td>
		</tr>
		<tr>
			<td>anti-E-cadherin</td>
			<td>Abcam</td>
			<td>ab1416</td>
			<td>epithelial adherens junctions marker</td>
		</tr>
		<tr>
			<td>anti-VE-cadherin</td>
			<td>Abcam</td>
			<td>ab33168</td>
			<td>endothelial adherent junctions marker</td>
		</tr>
		<tr>
			<td>anti- Zonula Occludens 1 (ZO-1)</td>
			<td>Thermo Fischer</td>
			<td>339194</td>
			<td>tight junctions marker</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher&#160;Scientific</td>
			<td>62248</td>
			<td>nuclear stain</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink RNA Mini Kit</td>
			<td>Invitrogen</td>
			<td>12183020</td>
			<td>RNA lysis, isolation and purification kit</td>
		</tr>
		<tr>
			<td>SuperScript&#8482; IV VILO&#8482; Master Mix</td>
			<td>Invitrogen</td>
			<td>11756050</td>
			<td>reverse transcriptase kit</td>
		</tr>
		<tr>
			<td>TaqMan&#8482; Fast Advanced Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4444557</td>
			<td>qPCR reagent</td>
		</tr>
		<tr>
			<td>QuantStudio&#8482; 5 Real-Time PCR System</td>
			<td>Applied Biosystems</td>
			<td>A28573</td>
			<td>Real-time PCR cycler</td>
		</tr>
		<tr>
			<td>18S primer</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs99999901_s1</td>
			<td>Eukaryotic 18S rRNA</td>
		</tr>
		<tr>
			<td>CYP3A4 primer</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs00604506_m1</td>
			<td>Cytochrome&#160; family 3 subfamily A member 4</td>
		</tr>
		<tr>
			<td>Pierce&#8482; Coomassie Plus (Bradford) Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23236</td>
			<td>Protein quantification kit</td>
		</tr>
		<tr>
			<td>MSD Tris lysis buffer</td>
			<td>Meso Scale Diagnostics</td>
			<td>R60TX-3</td>
			<td>Protein lysis buffer</td>
		</tr>
		<tr>
			<td>Cleaved/Total Caspase-3 Whole Cell Lysate Kit</td>
			<td>Meso Scale Diagnostics</td>
			<td>K15140D</td>
			<td>Caspase 3 detection kit</td>
		</tr>
		<tr>
			<td>V-PLEX Vascular Injury Panel 2 Human Kit</td>
			<td>Meso Scale Diagnostics</td>
			<td>K15198D</td>
			<td></td>
		</tr>
		<tr>
			<td>V-PLEX Human Proinflammatory Panel II (4-Plex)</td>
			<td>Meso Scale Diagnostics</td>
			<td>K15053D</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 880</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>Zeiss LD plan-Neofluar 20x/0.40 Korr M27</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>20X long-distance objective lenses</td>
		</tr>
		<tr>
			<td>Zeiss AXIOvert.A1</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>Brightfield microscope</td>
		</tr>
		<tr>
			<td>Zeiss LD A-Plan 10X/0.25 Ph1</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>10X objective lenses</td>
		</tr>
	</tbody>
</table>

combining human organoids and organ-on-a-chip technology to model intestinal region-specific functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Figure 1D summarizes the timeline of the intestine chip culture and illustrates the intestinal endothelial cells and organoids before and upon seeding on the chip. Moreover, it demonstrates the distinct morphological differences between the duodenum and colon chips, highlighted by the presence of the villi-like formations in the duodenum chip and representative of the small intestinal architecture.
Figure 3A,B demonst...

Watch this Scientific Journal Video about Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality at JoVE.com

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Research

JoVE Journal

Bioengineering

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

人体软骨组织制作采用三维喷墨打印技术

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科研

生物工程

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

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Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

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Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

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The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

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The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

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The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...