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 physiological, luminal, and vascular flow and peristalsis-like motions. In addition, we describe procedures for the assessment of critical intestinal functions, such as drug metabolism in the duodenum chip and barrier function in the colon chip.
To recapitulate epithelial-endothelial cellular interactions, which are essential for the maintenance of gut homeostasis and therefore the accurate modeling of intestinal tissue on chip, it is critical to establish functional and intact cell monolayers at both sides of PDMS membrane. The first step toward successful chip seeding is chemical activation of the PDMS surface that allows for development of a stable linkage between the PDMS surface and ECM proteins. Improper activation may hamper the attachment of cells and result in the formation of incomplete cellular monolayers. The activation solution must be prepared fresh as it is light sensitive and prone to degradation. During UV activation and subsequent ECM coating, it is important to ensure an even distribution of reagents within each channel. The specific composition and concentration of solutions used to coat the top and bottom channels have been optimized to suit the needs of the epithelial and endothelial cells, respectively. If changes to cellular composition of the intestine chip are required, the ECM conditions might need to be re-optimized to achieve optimal results.
Following ECM coating, it is important to seed HIMECs at high cell seeding density (8 x 106 cells/mL) to obtain a complete and homogenous monolayer on the bottom side of the PDMS membrane. If full cell confluency is not being achieved, confluency, it is advised to further increase the density of the endothelial cell suspension during the seeding or to delay the seeding of organoid fragments in the top channel of the chip until HIMECs become fully confluent. Use of fragmented organoids, rather than suspension of single cells obtained through enzymatic digestion of organoids, was previously shown to improve the success and reproducibility of the intestinal monolayer formation within the intestine chip14. Therefore, it is strongly advised to closely monitor the organoid digestion process to ensure the optimal time of incubation with the enzyme resulting in organoid fragments composed of around 10-30 cells (40-100 &#181;m in size). Inadequate dissociation can result in reformation of the cystic organoid structure in the microfluidic chips instead of the desired monolayer. In addition, it&#39;s critical to avoid excessive organoid dissociation into single cells that could lead to diminished cell survival, recovery, and failure to form confluent monolayer within the chip.
Incorporation of fluid flow (shear stress) and cyclic strain into the intestine chip culture, supported using the culture module, has been shown to enhance the formation of the functional intestinal barrier and to promote the spontaneous development of the 3D architecture of the epithelial tissue13. However, when performing microfluidic experiments with the use of Organs-on-Chips, it is critical to pre-warm and degas the cell culture media prior to use to avoid formation of microbubbles in the channels&#160;which could lead to cellular stress or even detachment&#160;from the membrane.
Besides the specific applications shown here, the intestine chip model can be used to address a variety of scientific questions, from basic science to discovery and testing of novel drugs or drug delivery technologies. It can facilitate studies of human intestinal development, stem cell maturation, and epithelial cell function; particularly in the context of evaluating the role of mechanics in the growth and homeostasis of the intestinal tissue. Recent discoveries revealed that dynamic equilibrium of the healthy intestinal epithelium relies on its ability to precisely coordinate various forces that define crypt-villi architecture, compartmentalize cell types, direct cell migration, and regulate cell identity, proliferation, and death in space and time32. The intestine chip provides an opportunity to better elucidate the interplay between mechanical forces (e.g., tension or shear stress), cell fate, and function to answer some of many fascinating questions that remain&#160;in the emerging field of intestinal mechanobiology. Moreover, it may allow for studies of sensing and intestinal transport processes, thanks to the presence of hormone-secreting enteroendocrine cells and correct localization of various nutrient transporters15,16. The intestine chip model can also be modified to incorporate immune cells as well as commensal or pathogenic microorganisms for studies on gut inflammatory disorders, host-pathogen, and host-microbiome interactions, as well as to precisely predict off-target toxicities of immuno-oncology products, as previously shown17,20,33,34,35.
In addition, the use of clinical biopsy specimens from individuals with specific genotypic and phenotypic characteristics can enable analysis of patient-specific disease mechanisms as well as response to therapies, thereby helping to advance personalized medicine in the future.
The major limitation of this method is that fragments from a large number of organoids (~60-80) are required to form a confluent intestinal epithelium on each chip. This is because organoid fragments require high-density seeding to ensure that an adequate number of intestinal stem cells are present to support the proliferative expansion of the monolayer and the establishment of a 3D tissue architecture. A fully differentiated intestinal epithelium on a chip possesses all the major intestinal epithelial cell types (absorptive enterocytes, Paneth cells, goblet cells and enteroendocrine cells) and transcriptional profile closely resembling the in vivo counterpart. However, the current model still lacks other important components of the living intestine, including intestinal fibroblasts, resident immune cells (e.g., macrophages, intraepithelial lymphocytes, and dendritic cells), and the enteric nervous system.
While these are potential drawbacks, previous studies have shown that the power of the Organ-on-a-Chip technology approach lies in its ability to emulate the structural and functional complexity of human organs by progressively integrating various components of in vivo tissues and their microenvironment one by one36,37. This synthetic biology approach to in vitro tissue engineering provides a way to study the contribution of individual cellular and molecular components to organ-level physiological and pathophysiological responses at varying levels of system complexity. Moreover, it allows for the biochemical, genetic, and microscopic analysis of the interacting tissues to be performed individually and in real-time, gaining insight into how intercellular signals and tissue-tissue interactions contribute to specific organ-level behaviors. Another notable characteristic of intestine chip technology is versatility. Precise control over microenvironmental elements enables fine tuning of media flow rates and strain parameters to reproduce natural forces experienced by cells in the human body, such as the shear stress sensed by the endothelial cells exposed to blood flow and the cyclic peristaltic motions of the intestinal tissue. Furthermore, synergistic engineering of organoids and organs-on-chips enables creation of vascularized Organs-on-Chips representing various regions of intestinal tissues that could be fluidically linked with each other to study physiological interactions between the small and large intestines. Thus, we believe that the intestine chip represents a superior model of the intestinal epithelium and might help to implement the 3Rs (Reduction, Refinement, and Replacement) principle in basic science as well as in the preclinical and regulatory setup.

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><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&#039;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&#039;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&amp;euml; CM-1&amp;trade;&amp;nbsp;Culture Module</td><td>Emulate&amp;nbsp;Inc.</td><td>-</td><td>Culture module</td></tr><tr><td>Orb-HM1&amp;trade;&amp;nbsp;Hub Module</td><td>Emulate&amp;nbsp;Inc.</td><td>-</td><td>5% CO2, vacuum stretch, and power supply</td></tr><tr><td>Chip-S1&amp;trade; Stretchable Chip</td><td>Emulate&amp;nbsp;Inc.</td><td>-</td><td>Organ-Chip</td></tr><tr><td>Pod&amp;trade; Portable Modules</td><td>Emulate&amp;nbsp;Inc.</td><td>-</td><td>Portable module</td></tr><tr><td>UV Light Box</td><td>Emulate&amp;nbsp;Inc.</td><td>-</td><td>-</td></tr><tr><td>Chip Cradle</td><td>Emulate&amp;nbsp;Inc.</td><td>-</td><td>1 per square culture dish</td></tr><tr><td>Steriflip&amp;reg;-HV Filters</td><td>EMD Millipore</td><td>SE1M003M00</td><td>0.45&amp;nbsp;&amp;mu;m&amp;nbsp;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&amp;nbsp;vacuum&amp;nbsp;aspirator</td><td>Corning</td><td>4930</td><td>&amp;nbsp;-</td></tr><tr><td>Aspirating&amp;nbsp;pipettes</td><td>Corning / Falcon</td><td>357558</td><td>2 mL,&amp;nbsp;polystyrene, individually&amp;nbsp;wrapped</td></tr><tr><td>Aspirating tips</td><td>&amp;nbsp;-</td><td></td><td>Sterile (autoclaved)</td></tr><tr><td>Serological&amp;nbsp;pipettes</td><td></td><td>&amp;nbsp;-</td><td>2 mL, 5 mL,&amp;nbsp;10 mL,&amp;nbsp;and&amp;nbsp;25 mL&amp;nbsp;low endotoxin, sterile</td></tr><tr><td>Pipette</td><td></td><td></td><td>P20, P200,&amp;nbsp;P1000&amp;nbsp;and standard multichannel</td></tr><tr><td>Pipette&amp;nbsp;tips&amp;nbsp;</td><td></td><td></td><td>P20, P200,&amp;nbsp;and&amp;nbsp;P1000.</td></tr><tr><td>Conical tubes&amp;nbsp;(Protein&amp;nbsp;LoBind&amp;reg; Tubes)</td><td>Eppendorf</td><td>0030122216;&amp;nbsp;0030122240</td><td>15 mL, 50 mL tubes</td></tr><tr><td>Eppendorf&amp;nbsp;Tubes&amp;reg;&amp;nbsp;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&amp;nbsp;imaging</td></tr><tr><td>Water bath (or beads)</td><td>-</td><td>-</td><td>Set to 37&amp;deg;C</td></tr><tr><td>Vacuum set-up</td><td>&amp;nbsp;-</td><td>-</td><td>Minimum&amp;nbsp;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&amp;nbsp;(ER-1)</td><td>Emulate Inc.</td><td>-</td><td>Chip coating solution</td></tr><tr><td>Emulate Reagent-2&amp;nbsp;(ER-2)</td><td>Emulate Inc.</td><td>-</td><td>Chip coating solution</td></tr><tr><td>Dulbecco&amp;rsquo;s PBS&amp;nbsp;(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&amp;nbsp;Express</td><td>ThermoFisher&amp;nbsp;Scientific</td><td>12604013</td><td>Organoids&amp;nbsp;dissociation&amp;nbsp;and endothelium cells&amp;nbsp;detachment&amp;nbsp;solution</td></tr><tr><td>Advanced DMEM/F12</td><td>ThermoFisher&amp;nbsp;Scientific</td><td>12634028</td><td>Medium</td></tr><tr><td>IntestiCult&amp;trade; 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&amp;trade;&amp;nbsp;</td><td>InvivoGen</td><td>ANT-PM-1</td><td>antimicrobial agent</td></tr><tr><td>Attachment Factor&amp;trade;</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&amp;gamma;</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&amp;nbsp;Scientific</td><td>33-1500</td><td>tight junctions marker</td></tr><tr><td>anti-Claudin 4</td><td>ThermoFisher&amp;nbsp;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&amp;nbsp;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&amp;trade; IV VILO&amp;trade; Master Mix</td><td>Invitrogen</td><td>11756050</td><td>reverse transcriptase kit</td></tr><tr><td>TaqMan&amp;trade; Fast Advanced Master Mix</td><td>Applied Biosystems</td><td>4444557</td><td>qPCR reagent</td></tr><tr><td>QuantStudio&amp;trade; 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&amp;nbsp; family 3 subfamily A member 4</td></tr><tr><td>Pierce&amp;trade; 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 demonstrate the fold CYP3A4 induction responses in the duodenum chip from three different organoid donors. The chips were exposed to 20 &#181;M RIF or 100 nM VD3 for 48 h and were used to assess the mRNA levels (A) and/or the fold catalytic activity (B) of the CYP450 enzyme. Increased levels of testosterone metabolite, 6&#946;-hydroxytestosterone (6&#946;-OH-T), consistent with the elevated CYP3A4 mRNA gene expression was observed in the duodenum chip exposed to RIF and VD3 indicating appropriate induction responses across all three donors tested. The means &#177; SEM of n = 3 biologically independent chips are shown.
Figure 4&#160;depicts the representative results for the modeling of the leaky gut syndrome in the colon chip, including (A,C) changes in epithelial cell morphology and tight junction integrity, (B) decrease in the barrier function, (D) induction of apoptosis and (E) increased&#160;cytokine secretion in response to the IFN&#947; treatment.
Figure 4A shows representative brightfield images of the control and IFN&#947;-treated (50 ng/mL, 48 h) epithelial monolayer of the colon chip. The chips were removed from the culture module and the epithelial morphology was assessed under the microscope daily. Images were acquired using a brightfield microscope and a 10x objective. Colon chips treated with IFN&#947; show a compromised cell morphology and loss of the columnar epithelium.
Figure 4B demonstrates a representative result of the apparent permeability assay performed on colon chips cultured under baseline conditions or upon stimulation with IFN&#947;. 3 kDa Dextran Cascade Blue tracer was added to the top channel reservoir to a final concentration of 100 &#181;g/mL. IFN&#947; at a final concentration of 50 ng/mL was dosed into the bottom channel&#160;on day 5 of the culture. Chips were perfused at 60 mL/h. Next, the media was collected daily (up to day 72 h post-exposure) from inlet and outlet reservoirs of both the top and bottom channels. 50 &#181;L of each sample was collected, processed as described in step 9.2.1 of the protocol, and examined for fluorescence at 375-420 nm using a plate reader. A significant increase of the epithelial paracellular permeability was observed following 48 h of IFN&#947; stimulation.
Figure 4C shows representative immunofluorescent images of the epithelial tight (Zonula Occludens 1 - ZO-1, Occludin, Claudin-4) and adherens (E-cadherin) junctions in control and IFN&#947;-treated (50 ng/mL, 48 h) colon chip. The chips were fixed with 4% Paraformaldehyde (PFA) for 15 min at room temperature and further processed as described in step 9.2.2 of the protocol. Images were acquired using a confocal microscope and 20x long-distance objective and processed using Fiji version 2.0 according to standard protocols. Treatment with IFN&#947; triggers displacement of ZO-1 and Claudin-4 and internalization of Occludin and E-cadherin, as shown by the increased cytoplasmic signal.
Figure 4D represents the time-course of Caspase 3 cleavage in colon chip in response to 50 ng/mL of IFN&#947; indicative of apoptosis activation. Epithelial cells were lysed on the chips and protein samples were purified according to standard methods. The intracellular content of the total and cleaved Caspase 3 was measured as described in step 9.2.3 of the protocol. IFN&#947; induces activation of apoptosis, as described previously29, in the colonic epithelial cells cultured on the chip, following 48 h of stimulation.
Figure 4E demonstrates the time-course of the cytokine&#39;s secretion from the colon chip upon stimulation with 50 ng/mL IFN&#947;. 200 &#181;L of effluent media were collected daily from the outlet reservoirs of both top and bottom channels. Effluent samples were stored at -80&#176;C and 24 h before the analysis were thawed overnight at 4&#176;C. The cytokine levels in the chip culture media were assessed using Meso Scale Discovery technology as described in step 9.2.4 of the protocol. IFN&#947; induced a polarized secretion of proinflammatory molecules in the colon chip, as shown by the basolateral secretion of Vascular Cell Adhesion Protein 1 (VCAM-1) and Interleukin 6 (IL-6) and the apical secretion of Intercellular Adhesion Molecule 1 (ICAM-1) and Serum Amyloid protein A (SAA). Serum levels of the soluble forms of VCAM-1, ICAM-1, IL-6, and SAA are used in clinical laboratories as an indicator of inflammation30,31.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63724/63724fig01v2.jpg" /> 
Figure 1: Establishment of the intestine chip15,16. (A) Schematic of the chip activation and coating. Briefly, the chips are placed in a chip&#160;cradle, perfused with ER1 solution and activated under UV light. Next, the chips are washed with ER2 and DPBS. Finally, the chips are coated with an ice-cold ECM solution, specific for each cell type, and incubated overnight at 37&#176;C. (B) Schematic depicting the process of introducing organoids in the chip. Organoids are transferred from the 24-well plate into a conical tube and incubated on ice in the presence of the BMM dissociation solution to recover organoids from the solubilized BMM. Organoid suspension is then centrifuged and evaluated for the presence of solubilized BMM residues. If a transparent layer of BMM is still visible above the cell pellet, the process should be repeated. If a well-defined pellet is observed with no visible gel residues, organoids are dissociated enzymatically and introduced in the top channel (blue) of the chip. The bottom channel (magenta) of the chip is seeded with tissue-specific microvascular endothelial cells. (C) Schematic showing the connection of the chips to the culture module, which supports media flow and cyclic peristalsis-like stretch. The priming cycle enables the generation of&#160;the cell culture medium&#160;droplets over the chip ports and at the end of the portable module&#160;resistors. This ensures the creation of the liquid-liquid interface between the chip and the portable module, facilitating the chip sliding into the module and their secure connection. Lastly, the portable modules are placed in trays, and trays are inserted into the culture module. (D) Experimental timeline highlighting the major steps for the establishment of the biopsy-derived intestine chip platform, including duodenum and colon chip. Brightfield images illustrate the endothelial and organoid-derived cells at day 0, before and after seeding in the chip, as well as the formation of confluent and 3D epithelial tissue at day 8 of chip culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63724/63724fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63724/63724fig02.jpg" /> 
Figure 2: Collection of effluent media from the intestine chip. (A) Schematic depicting the collection of media effluent from the portable module. Media are collected from the outlet reservoirs of the top and bottom channel and stored at -80 &#176;C until further ELISA or LC-MS / LC-MS/MS analysis. (B) Schematic showing the collection of effluent samples using a multichannel pipette and their transfer into a 96-well black-walled plate for downstream assessment of epithelial apparent permeability (Papp). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63724/63724fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63724/63724fig03.jpg" /> 
Figure 3: CYP3A4 induction in the duodenum chip15. (A) Induction of CYP3A4 mRNA levels in duodenum chip by 48 h exposure to 20 &#181;M RIF and 100 nM VD3. Mean &#177; SEM, N = 3 chips/condition/donor, Two-way ANOVA, Tukey&#39;s post hoc test, ****p &#60; 0.0001 (compared across DMSO controls). (B) Induction of CYP3A4 activity in the duodenum chip exposed to prototypical inducers as assessed by LC-MS/MS quantification of probe substrate metabolite: 6&#946;-hydroxytestosterone. A significant increase in CYP3A4 activity was observed in the duodenum chip treated with 20 &#181;M RIF or 100 nM VD3 for 48 h. Mean &#177; SEM, N = 3 chips/condition/donor, Two-way ANOVA, Tukey&#39;s post hoc test, ****p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63724/63724fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63724/63724fig04.jpg" /> 
Figure 4: Disruption of the epithelial barrier in the colon chip using IFN&#947;16. (A) Brightfield images showing the morphology of the epithelial cells under baseline conditions and upon stimulation with 50 ng/mL IFN&#947; for 48 h. Scale bar: 100 &#181;m. (B) Apparent permeability of the epithelial barrier to 3 kDa Dextran over the course of a 72 h stimulation with 50 ng/mL of IFN&#947;. Mean &#177; 95% CI, N = 5-9 chips/condition, Two-way ANOVA, Tukey&#39;s post hoc test, ****p &#60; 0.0001. (C) Immunofluorescence images depicting the disruption of the epithelial tight and adherens junctions upon stimulation with 50 ng/mL of IFN&#947; for 48 h. Zonula Occludens 1 (ZO-1) (red) tight junctions, E-cadherin (red) adherens junctions, Occludin (red) tight junctions, Claudin-4 (red) tight junctions, 4&#8242;,6-diamidino-2-phenylindole (DAPI) (blue) nuclei. Scale bar: 50 &#181;m. (D) Induction of Caspase 3 cleavage in colon chip by the treatment with 50 ng/mL of IFN&#947;. Four different time points were assessed across 72 h of exposure. Mean &#177; 95% CI, N = 3-6 chips/ condition, Two-way ANOVA, Tukey&#39;s post hoc test, ****p &#60; 0.0001. (E) Increased secretion of cytokines: Vascular Cell Adhesion Protein 1 (VCAM-1) and Interleukin 6 (IL-6), in the bottom channel, and Intercellular Adhesion Molecule 1 (ICAM-1) and Serum Amyloid protein A (SAA) in the top channel effluent media, over the course of a 72 h stimulation of the colon chip with 50 ng/mL of IFN&#947;. Mean &#177; 95% CI, N = 3 chips/ condition, Two-way ANOVA, Tukey&#39;s post hoc test, *: p &#60; 0.05, **: p &#60; 0.01, ***: p &#60; 0.001, ****p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63724/63724fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.5K Views.  Emulate Inc. Boston. Dit protocol laat zien hoe biopsie-afgeleide intestinale organoïden en organ-on-a-chip-technologie kunnen worden gebruikt om een fysiologisch relevante darmchip te genereren die vervolgens kan worden gebruikt om farmacokinetiek en geneesmiddelinteractie te voorspellen en om intestinale epitheliale barrièredisfunctie te bestuderen. Door organoïden te verenigen in organ-on-a-chip-technologie kunnen we de complexiteit van het darmepitheel reproduceren. Bovendien maakt het een grotere experime...

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

Innervation of Human Intestinal Organoids

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing.
Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into &#62;9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques ...

Developmental Biology

Enhanced Intestine-on-Chip Platform for Immunological Insight

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

Author Spotlight: Developing Immunocompetent Organ-on-Chip Models for Infectious Disease Research

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...

Immunology and Infection

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Medicine

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Establishment of 3D Morphogenesis in a Canine IBD Gut-on-a-Chip System

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Author Spotlight: Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Biology

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

A Gut-on-a-Chip Model to Study the Gut Microbiome-Nervous System Axis

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located in the gut. Though the interplay between the gut microbiome and the host has been extensively studied, how the gut microbiome interacts with the enteric nervous system remains largely unknown. To date, a physiologically representative in vitro model to study gut microbiome-nervous system interactions does not exist. 
To fill this gap, we further developed the human-microbial crosstalk (HuMiX) gut-on-chip model by introducing induced pluripotent stem cell-derived enteric neurons into the device. The resulting model, 'neuroHuMiX', allows for the co-culture of bacterial, epithelial, and neuronal cells across microfluidic channels, separated by semi-permeable membranes. Despite separation of the different cell types, the cells can communicate with each other through soluble factors, simultaneously providing an opportunity to study each cell type separately. This setup allows for first insights into how the gut microbiome affects the enteric neuronal cells. This is a critical first step in studying and understanding the human gut microbiome-nervous system axis.

neuroHuMiX is an advanced gut-on-a-chip model to study the interactions of bacterial, epithelial, and neuronal cells under proximal and representative co-culture conditions. This model allows unravelling of the molecular mechanisms underlying the communication between the gut microbiome and the nervous system.

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located ...

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells.
The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-&#945;, TGF-&#946;, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...

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

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