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.

<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, ...

combining-human-organoids-organ-on-chip-technology-to-model

Research

JoVE Journal

Bioengineering

13.3K Views.  Emulate Inc. Boston. Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

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

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

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

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., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

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., ...

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

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 complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

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

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

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

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

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

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

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

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

Regional Decellularized Lung Tissue Isolation to Study Cell-Cell and Cell-ECM Interactions

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of suitable donor lungs and both acute and chronic rejection after transplantation. Ascertaining novel bioengineering approaches for the replacement of diseased lungs is imperative for improving patient survival and avoiding complications associated with current transplantation methodologies. An alternative approach involves the use of decellularized whole lungs lacking cellular constituents that are typically the cause of acute and chronic rejection. Since the lung is such a complex organ, it is of interest to examine the extracellular matrix components of specific regions, including the vasculature, airways, and alveolar tissue. The purpose of this approach is to establish simple and reproducible methods by which researchers may dissect and isolate region-specific tissue from fully decellularized lungs. The current protocol has been devised for pig and human lungs, but may be applied to other species as well. For this protocol, four regions of the tissue were specified: airway, vasculature, alveoli, and bulk lung tissue. This procedure allows for the procurement of samples of tissue that more accurately represent the contents of the decellularized lung tissue as opposed to traditional bulk analysis methods.

Author Spotlight: Dissection and Isolation of Region-Specific Decellularized Lung Tissue  

Presented here is a protocol for the isolation of regional decellularized lung tissue. This protocol provides a powerful tool for studying complexities in the extracellular matrix and cell-matrix interactions.

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of ...

AI Program for Trypanosomiasis Detection and Classification

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of hotspot areas under active surveillance is a fundamental procedure for controlling disease transmission. Microscopic examination is a commonly used diagnostic method. It is, nevertheless, primarily reliant on skilled and experienced personnel. To address this issue, an artificial intelligence (AI) program was introduced that makes use of a hybrid deep learning technique of object identification and object classification neural network backbones on the in-house low-code AI platform (CiRA CORE). The program can identify and classify the protozoan trypanosome species, namely Trypanosoma cruzi, T. brucei, and T. evansi, from oil-immersion microscopic images. The AI program utilizes pattern recognition to observe and analyze multiple protozoa within a single blood sample and highlights the nucleus and kinetoplast of each parasite as specific characteristic features using an attention map.
To assess the AI program&#39;s performance, two unique modules are created that provide a variety of statistical measures such as accuracy, recall, specificity, precision, F1 score, misclassification rate, receiver operating characteristics (ROC) curves, and precision versus recall (PR) curves. The assessment findings show that the AI algorithm is effective at identifying and categorizing parasites. By delivering a speedy, automated, and accurate screening tool, this technology has the potential to transform disease surveillance and control. It could also assist local officials in making more informed decisions on disease transmission-blocking strategies.

Author Spotlight: AI-Driven Trypanosome Species Detection from Microscopic Images 

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of ...