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 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 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

13.3K 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., ...

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

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

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 ...

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

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

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

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).