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
<ol>
	<li>Prepare the hu...

<ol>
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Gauri Kulkarni, Athanasia Apostolou, Lorna Ewart, Carolina Lucchesi, and Magdalena Kasendra are current or former employees of Emulate Inc. and may hold equity. Emulate Inc. is the company that manufactures the organ chip devices and has published patents relevant to the work stated in this article.

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, ...

This protocol shows how to use biopsy-derived intestinal organoids and organ-on-a-chip technology to generate a physiologically relevant intestine chip which can then be used to predict pharmacokinetics and drug-drug interaction and to study intestinal epithelial barrier dysfunction. Uniting organoids in organ-on-a-chip technology allow us to reproduce the complexity of the intestinal epithelium. Additionally, it enables greater experimental control of the mechanical cues, flow distribution, and biochemical gradients.This method emulates the complex physiology of the human intestine, giving a greater understanding of the cellular and molecular basis of normal and pathological gut functions. This helps to better predict pharmacokinetics and pharmacodynamics and potential drug candidates to prevent inflammatory damage. Seeding is a critical step in the protocol.Excessive fragmentation or inaccurate seeding density of the intestinal organoid fragments may compromise the development and differentiation of the epithelial barrier. To begin, aspirate the medium from the static organoid culture. Recover media from enough wells to achieve a final seeding density of eight million cells per milliliter.Add 500 microliters of ice cold BMM dissociation solution to each well and attach the solubilized BMM off the well surface. Collect the suspension into a 15 milliliter low protein binding tube and place it on ice. Gently shake the tube every 15 minutes for one hour.Centrifuge at 300 times G for five minutes at four degrees Celsius to obtain an organoid pellet. If a transparent gel layer is observed above the pellet, aspirate solution from the tube. Resuspend the pellet and add an equal volume of BMM dissociation solution.Incubate on ice for 10 minutes and centrifuge. Repeat until no solubilized BMM residues are present. Then discard the supernatant and resuspend the pellet in two milliliters of organoid digestion solution.Incubate the tube at 37 degrees Celsius for one to three minutes and add eight milliliters of advanced DMEM F12 to stop the enzymatic reaction. Centrifuge and resuspend the pellet in complete organoid growth medium to achieve eight million cells per milliliter. Aliquot 360 microliters in sterile 1.5 milliliter low protein binding tubes.Next, remove the medium from the top channel of the coated chips and add 30 microliters of the cell suspension. Incubate the chips at 37 degrees Celsius overnight to adhere the organoid fragments to the membrane. Add three milliliters of pre-equilibrated complete organoid growth medium to the top inlet reservoir and three milliliters of pre-equilibrated endothelial cell growth medium in the bottom inlet reservoir.Add 300 microliters of the same media in the respective top and bottom outlet reservoirs. Transfer the tray carrying the portable modules into the culture module. On the culture module, repeatedly run the prime cycle until sufficient droplets form for successfully connecting the chips.Then slide the chip carrier into the portable module. Then start the two-hour long regulate cycle to pressurize the culture media in the portable module and chip after which the programmed conditions will resume. Next, change the stretch settings and start the culture module.After 24 hours, repeat the cycle with 10%stretch and the same frequency. To prepare dosing media or vehicle control, dilute the CYP inducer stock solutions or DMSO in complete organoid growth medium and endothelial cell growth medium. Pause the culture module and bring the trays to the biosafety cabinet.Replace the media in all the inlet and outlet reservoirs with two milliliters of dosing media with inducers or vehicle control. Return the portable modules back to the culture module and restart the flow at 30 microliters per hour. Replace the media with freshly prepared inducing solution every 24 hours and continue the culture for 48 to 72 hours.Then bring the trays to the biosafety cabinet and aspirate the dosing medium from all the reservoirs. Wash and replace the top inlet reservoir with warm advanced DMEM F12 medium and the bottom reservoir with endothelial cell growth medium. Replace the wash medium with one milliliter of probe substrate solution.Perfuse the chips at a high flow rate of 1, 000 microliters per hour for five minutes and aspirate both top and bottom outlet reservoirs. Return the chips to the culture module and incubate for one hour under a constant flow of 300 microliters per hour. After one hour of treatment, stop the flow and bring the trays to the biosafety cabinet.Collect 100 microliters of effluent from the top outlet reservoir and add it to a tube containing 200 microliters of stop solution. Place the tubes immediately on dry ice and store the samples at minus 80 degrees Celsius. Wash both the channels with 200 microliters of sterile DPBS.Then block the bottom channel outlet with a 200 microliter filter pipette tip. Perfuse 50 microliters of the dissociation solution through the bottom channel and incubate for two minutes at room temperature. Ensure complete detachment of the endothelial cells and remove the dissociation solution from the channel by pipetting.Repeat the wash. Next, block the top channel outlet and perfuse 75 microliters of protein lysis buffer. Leave the pipette tip inserted and incubate for five minutes at room temperature.Collect the cell lysates in a 1.5 milliliter tube by pipetting five to 10 times. Repeat perfusion and collection to obtain complete detachment of cells and store the lysates at minus 80 degrees Celsius until analysis. For RNA lysis, follow the same procedure while using 150 microliters of RNA lysis buffer.First, prepare an interferon gamma dosing solution by diluting the stock solutions in degassed endothelial cell growth medium. In the biosafety cabinet, remove the medium from the bottom channel inlet reservoirs and replace it with three milliliters of the dosing solution daily. Then place the trays into the culture module and perfuse the chips at a high flow rate of 1, 000 microliters per hour for five minutes.Switch the flow rate to 60 microliters per hour and continue the fluidic culture. Add 100 microliters of DPBS per well into a 96-well black walled plate. Using a multichannel pipette, add 50 microliters of the effluent from all the reservoirs to the respective wells.To prepare a standard curve, perform a three-fold dilution of the medium containing 100 micrograms per milliliter of three kilodalton dextran cascade blue in DPBS. Then perform serial delusions using a three-fold dilution of endothelial cell growth medium in DPBS. Distinct morphological features of the duodenum and colon chips were highlighted by the presence of the villi-like formations in the duodenum chip representing the architecture of the small intestine.In the duodenum chip exposed to the cytochrome P450 inducers rifampicin and vitamin D3, there was an elevated mRNA gene expression for the drug metabolizing enzyme cytochrome P450 3A4 and enhanced catalytic activity of the cytochrome P450 enzyme, indicating appropriate induction responses across all three organoid donors. Upon treating the epithelial monolayer of the colon chip with interferon gamma, compromised cell morphology and loss of the columnar epithelium were observed. Also, treatment with interferon gamma led to increased cytoplasmic signal of tight and adherent junction proteins compared to vehicle control, showing the displacement of tight junction markers ZO-1 and claudin 4 and internalization of occludin and E-cadherin.A significant increase in the epithelial paracellular permeability was observed following 48 hours of interferon gamma stimulation on colon chips. Similarly, interferon gamma induces activation of apoptosis indicated by increase in the intracellular content of the cleaved Caspase 3. Interferon gamma induced a polarized secretion of pro-inflammatory molecules in the colon chip as shown by the basolateral secretion of VCAM-1 and interleukin-6 and the apical secretion of ICAM-1 and serum amyloid protein A.For a successful intestine chip culture, one should utilize undifferentiated organoids and follow the fragmentation and seeding density instructions.Following successful establishment of the intestine chip, we can study intestinal absorption, transport, and metabolism, nutrient levels, and intestinal hormone secretion, host microbe pathogen interactions, drug safety and efficacy, patient-specific disease mechanisms, and responses to therapies.

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JoVE Journal

Bioengineering

13.0K 조회수.  Emulate Inc. Boston. 이 프로토콜은 생검 유래 장 오가노이드 및 장기 온 칩 기술을 사용하여 생리학적으로 관련된 장 칩을 생성한 다음 약동학 및 약물-약물 상호 작용을 예측하고 장 상피 장벽 기능 장애를 연구하는 데 사용할 수 있는 방법을 보여줍니다. organ-on-a-chip 기술에 유기체를 결합하면 장 상피의 복잡성을 재현 할 수 있습니다. 또한 기계적 단서, 유동 분포 및 생화학적 구배에 대한 더 ?...