We would like to thank WSU Small Animal Internal Medicine service (Dr. Jillian Haines, Dr. Sarah Guess, Shelley Ensign LVT) and WSU VTH Clinical Studies Coordinator Valorie Wiss for their support in case recruitment and sample collection from citizen scientists (patient donors). This work was supported in part by the Office of The Director, National Institutes Of Health (K01OD030515 and R21OD031903 to Y.M.A.) and Japan Society for the Promotion of Science Overseas Challenge Program for Young Researchers (202280196 to I.N.). Figure 1A and Figure 3A were created with BioRender.com.

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

The authors do not have any conflict of interest to declare.

This study marks the pioneering demonstration of the compatibility of canine intestinal organoids with the development of a canine IBD Gut-on-a-Chip model. The integration of intestinal organoids and organoid-derived monolayer cultures into a microfluidic system (i.e., Gut-on-a-Chip system) has further evolved the technology, enabling the creation of in vitro intestinal models that closely mimic physiological dynamics and are more representative of biological conditions. In particular, since there are very few reports of Gut-on-a-Chip culture using IBD-derived organoids in humans, the current study using canine IBD-derived Gut-on-a-Chip may provide leading insights into the study of IBD in humans.
The successful development of canine intestinal epithelial 3D morphogenesis on a Gut-on-a-Chip requires careful attention to several critical steps. First, the hydrophobic surface of PDMS microfluidic channels may impede ECM adhesion and subsequent cell attachment, necessitating surface activation of PDMS prior to ECM coating and cell seeding (see protocol section 1). To achieve a stable monolayer culture, the removal of excess unattached cells is crucial following cell attachment (protocol steps 4.6-4.7). Additionally, dynamic stimulation, such as constant medium flow and peristaltic-like vacuum motion, is necessary for 3D morphogenesis of the intestinal epithelium (protocol step 5.2). Careful handling is essential to avoid air bubbles in the microchannel during any steps of the Gut-on-a-Chip culture.
If encountering poor cell seeding into the Gut-on-a-Chip, it could be due to a low cell number or poor cell attachment. To troubleshoot low cell numbers, it is important to inspect the health of prepared intestinal organoids by observing their growth in Matrigel. Cell viability can be assessed by Trypan blue staining after cell dissociation to ensure no more than 20% of cells are dead. If viable cell numbers are insufficient, optimizing organoid medium conditions can be attempted. Another possibility is incomplete organoid dissociation, resulting in an excess of cell clumps larger than 70 &#181;m that become trapped by the filter. To resolve this, one option is to extend the duration of pipetting during cell dissociation. Alternatively, the 15 mL conical tube can be gently agitated every minute while undergoing treatment with a trypsin-like protease. Poor cell attachment to the Gut-on-a-Chip may be due to improper ECM coating. During the coating process, it is advised to carefully check for the presence of air bubbles and prevent their formation by gently adding more coating solution as needed. Overcrowding of cells and a failure to wash away unattached cells can result in an insufficient initial monolayer. In such a case, a mild pulsing can be applied when pushing the syringe plunger. These troubleshooting steps can help identify and address issues during the Gut-on-a-Chip culture process.
While this Gut-on-a-Chip platform enables the creation of undulated 3D epithelial layers, we recognize the need for additional biological complexity to replicate the intestinal microenvironment fully. It is crucial to consider the interactions between epithelial and mesenchymal cells, the deposition of ECM for 3D regeneration, and the presence of crypt-villus characteristics that establish a suitable stem cell niche. Stromal cells, such as fibroblasts, play a vital role in the production of ECM proteins and the regulation of intestinal morphogenesis34,35,36. The inclusion of mesenchymal cells in this model has the potential to enhance both morphogenesis and the efficiency of cell attachment. Endothelial layers, which encompass capillary vasculature and lymphatic vessels, play a crucial role in governing molecular transport and the recruitment of immune cells37,38. The inclusion of patient-derived immune cells could be essential in modeling intestinal diseases as it allows for the demonstration of the interplay between innate and adaptive immunity as well as the establishment of tissue-specific immunity39. Following the completion of 3D morphogenesis on Gut-on-a-Chip, the organoid culture medium can be modified to an organoid differentiation medium. This can be a viable approach to induce additional cellular differentiation, depending on the experimental objectives.
Imaging the 3D microarchitecture in situ is challenging due to the long working distance required, which can be overcome with a long-distance objective. Additionally, the layer-by-layer microfabrication and bonding methods make it difficult to access upper layers for examination with SEM. For the current Gut-on-a-Chip design, one syringe pump per Gut-on-a-Chip microdevice is needed, occupying CO2 incubator space and preventing large-scale experiments. Innovations are needed to increase scalability for a user-friendly platform and high-throughput screening.
These current protocols allow for the spontaneous development of 3D epithelial layers in vitro, surpassing the limitations of traditional 3D organoids, 2D monolayers, and static microdevice culture systems. This dynamic in vitro intestinal microenvironment can be controlled by introducing co-culture of diverse cell types. Previous studies have explored methods for manipulating the Gut-on-a-Chip microenvironment, including co-culturing intestinal microbiome14,23 and peripheral mononuclear cells30. This reconstituted microenvironment has numerous potential applications, including drug testing, fundamental mechanistic studies, and disease modeling. The reconstructed microenvironment holds significant potential for a wide range of applications, such as drug testing23,40,41 and disease modeling12,13,14,30, as well as fundamental mechanistic investigations of intestinal morphogenesis42. A variety of assays can be performed by either collecting supernatants for assessment of metabolites43, by collecting cells for genomic examination2,32, or by visually examining the cells using live-cell dyes or fixation for subsequent immunofluorescence imaging23,44.
This study presents a reproducible protocol for developing 3D morphogenesis of canine intestinal epithelial layers in a Gut-on-a-Chip platform. The resulting 3D epithelial structure provides a more realistic representation of the intestinal microenvironment, which has immense potential for applications in various biomedical studies. By utilizing this intestinal architecture, we can conduct more translational research and potentially yield promising outcomes.

Intestinal epithelial morphogenesis has been largely studied through laboratory animal models, which are costly, time-consuming, and do not accurately represent human developmental processes1. Furthermore, conventional static 2D cell culture models lack the ability to mimic the complex spatial organization of a 3D epithelial architecture2. As a result, there is a need for a protocol to induce in vitro 3D morphogenesis using intestinal epithelial cells from human-relevant animal models to advance our understanding of gut epithelial architecture.
Companion dogs have developed intestinal anatomy and microbiome compositions that are remarkably similar to humans due to their shared environment and diet during domestication3. In addition to this similarity, both humans and dogs share various chronic morbidities that are thought to be attributed to intestinal health. Dogs, like humans, can spontaneously develop chronic conditions such as obesity, cognitive dysfunction, diabetes mellitus, inflammatory bowel disease (IBD), and colorectal adenocarcinoma4,5,6,7,8,9,10. Despite the development and use of human and murine epithelial cells in previous Gut-on-a-Chip studies2,11,12,13,14, canine intestinal epithelium has not been utilized until now. Our novel approach, utilizing canine intestinal organoid epithelium in a dynamic culture system with a 3D epithelial morphogenesis, has significant implications for both canine and human medicine.
Recent advancements in intestinal organoid culture have led to the establishment of canine intestinal organoid culture15. This culture system involves culturing intestinal stem cells under defined morphogen conditioning, resulting in a 3D model with self-renewing properties derived from adult stem cells16. However, conducting transport assays or host-microbiome cocultures poses difficulties with this 3D model because of the enclosed nature of the intestinal lumen17. To address this, researchers have generated a 2D monolayer derived from intestinal organoids, allowing for exposure of the luminal surface18,19. However, both 3D organoids and 2D monolayers are maintained under static conditions, which do not accurately reflect the in vivo biomechanics of the intestinal microenvironment. Combining patient-derived canine organoids technology with in vitro 3D morphogenesis presents an opportunity for translational research into chronic multifactorial diseases. This approach enables researchers to develop more effective treatments benefiting both humans and dogs and further advance translational research, aligning with the One Health Initiative, which is a collaborative approach that recognizes the interconnectedness of human, animal, and environmental health. It promotes interdisciplinary cooperation to address complex health challenges and achieve optimal health outcomes for all. By understanding the interdependencies between humans, animals, and ecosystems, the initiative aims to mitigate risks from emerging infectious diseases, environmental degradation, and other shared health concerns20,21,22.
This protocol outlines comprehensive methods for culturing canine intestinal epithelial cells obtained from patient organoids on a Gut-on-a-Chip microdevice with a polydimethylsiloxane (PDMS)-based porous membrane. Establishing the 3D epithelial morphogenesis by integrating canine intestinal organoids and this Gut-on-a-Chip technology enables us to study how the gut develops and maintains its cellular organization and stem cell niche. This platform offers a valuable opportunity to investigate the impact of microbiome communities on gut health and understand how these communities generate microbial metabolites that contribute to intestinal pathophysiology14,23. These advancements can now be extended to canine intestinal samples, providing researchers with opportunities to explore the intricate relationship between the gut microbiome and host physiology. This opens up avenues for gaining valuable insights into the underlying mechanisms of intestinal pathophysiology and understanding the potential role of microbial metabolites in both canine and human health, as well as various disease conditions. The protocol used for canine Gut-on-a-Chip is reproducible, making it a suitable experimental model for comparative medicine, as this approach enables investigation of host-microbiome interactions, pathogen infections, and probiotic-based therapeutic effects in both dogs and humans.

<table><tbody><tr><td>&lt;strong&gt;Organoid basal medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-061</td><td>2 mM, glutamine substitute</td></tr><tr><td>1 M HEPES</td><td>VWR Life Science</td><td>J848-500ML</td><td>10 mM</td></tr><tr><td>100x penicillin&amp;ndash;streptomycin</td><td>Corning</td><td>MT30009CI</td><td>1x</td></tr><tr><td>&lt;strong&gt;Organoids and organoid medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A-83-01&amp;nbsp;</td><td>PeproTech&amp;nbsp;</td><td>9094360</td><td>500 nM</td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>Gibco</td><td>17504-044</td><td>1x</td></tr><tr><td>CHIR99021</td><td>Reprocell</td><td>04-0004-base</td><td>2.5 &amp;micro;M</td></tr><tr><td>HEK293 cells engineered to secrete Noggin&amp;nbsp;</td><td>Baylor College of Medicine</td><td></td><td></td></tr><tr><td>Murine EGF</td><td>PeproTech&amp;nbsp;</td><td>315-09-1MG</td><td>50 ng/mL</td></tr><tr><td>Murine Wnt-3a</td><td>PeproTech&amp;nbsp;</td><td>315-20-10UG</td><td>100 ng/mL</td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma</td><td>A9165-25G</td><td>1 mM</td></tr><tr><td>N2 MAX Media supplement&amp;nbsp;</td><td>Gibco</td><td>17502-048</td><td>1x&amp;nbsp;</td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636-100G</td><td>10 mM</td></tr><tr><td>Noggin Conditioned Medium</td><td>NA</td><td>NA</td><td>10% vol/vol&amp;nbsp;</td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-1</td><td>100 &amp;micro;g/ml</td></tr><tr><td>R-spondin1 (Rspo1) cells</td><td>Trevigen</td><td>3710-001-01</td><td>Rspo1 cells</td></tr><tr><td>R-Spondin-1 Conditioned Medium</td><td>NA</td><td>NA</td><td>20% vol/vol&amp;nbsp;</td></tr><tr><td>SB202190</td><td>Sigma-Aldrich</td><td>S7067-25MG</td><td>&amp;nbsp;10 &amp;micro;M</td></tr><tr><td>Y-27632</td><td>StemCellTechnologies</td><td>72308</td><td>10 &amp;micro;M</td></tr><tr><td>[Leu15 ]-Gastrin I human&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G9145-.5MG</td><td>10 nM</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4% Paraformaldehyde solution</td><td>Fisher Scientific</td><td>AAJ19943K2</td><td></td></tr><tr><td>Alexa Fluor 647 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A22287</td><td>x250 dilution</td></tr><tr><td>Anti-Rabbit IgG H&amp;amp;L labeled with Alexa Fluor 555</td><td>Abcam</td><td>ab150078</td><td>x1,000 dilution</td></tr><tr><td>Anti-ZO-1 polyclonal antibody</td><td>Thermo Fisher Scientific</td><td>61-7300</td><td>x50 dilution</td></tr><tr><td>Cell Recovery Solution</td><td>Corning</td><td>354253</td><td></td></tr><tr><td>Collagen I, Rat Tail 3 mg/mL</td><td>Gibco</td><td>A10483-01</td><td></td></tr><tr><td>Diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>62248</td><td>x1,000 dilution</td></tr><tr><td>EMS Glutaraldehyde Aqueous 50%</td><td>Electron Microscopy Sciences</td><td>16320</td><td></td></tr><tr><td>Matrigel Matrix</td><td>Corning</td><td>356255</td><td></td></tr><tr><td>Poly(ethyleneimine) solution</td><td>Sigma</td><td>408700-250ML</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12604-021</td><td></td></tr><tr><td>&lt;strong&gt;Materials and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24-well culture plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>87V Industrial Multimeter</td><td>Fluke Corporation</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5910R</td><td></td></tr><tr><td>CO2 incubator</td><td>Eppendorf</td><td>C170i</td><td></td></tr><tr><td>DMi8 fluorescence microscope</td><td>Leica microsystems</td><td>DMi8</td><td></td></tr><tr><td>Dry oven</td><td>Fisher Scientific</td><td>15-103-0519</td><td></td></tr><tr><td>FlexCell FX-5000 Tension system</td><td>Flexcell International Corporation</td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td>Leica microsystems</td><td>DMi1</td><td></td></tr><tr><td>SP8-X inverted confocal microscope</td><td>Leica microsystems</td><td>SP8-X</td><td></td></tr><tr><td>Syringe pump</td><td>Braintree Scientific</td><td>model no. BS-8000 120V</td><td></td></tr><tr><td>Syringe, 3 mL sterile</td><td>BD Biosciences</td><td>14-823-435</td><td></td></tr><tr><td>Syringes, 1 mL sterile</td><td>BD Biosciences</td><td>14-823-434</td><td></td></tr><tr><td>UV/ozone generator</td><td>Jelight Company</td><td>model no. 30</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LAS X imaging software&amp;nbsp;</td><td>Leica microsystems</td><td></td><td></td></tr></tbody></table>

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.

This protocol reliably facilitates the spontaneous development of 3D intestinal morphogenesis in a Gut-on-a-Chip system. This approach utilizes canine intestinal epithelial cells obtained from intestinal organoids derived from dogs affected by inflammatory bowel disease (IBD) (Figure 1B). Occasional clustering of 3D morphogenesis of canine intestinal epithelial cells can be observed throughout the microchannel following 6-9 days of medium flow (Figure 3C). These morphological changes can be monitored using phase-contrast techniques.In this study, we utilized organoids derived from two dogs diagnosed with IBD. Notably, successful 3D morphogenesis was observed in two biological replicates, each of which was performed with two technical replicates. The findings from this study provide a foundation for future investigations involving intestinal organoids derived from other canine donors. These results demonstrate the potential applicability and repeatability of our experimental approach, which has previously been reported in human samples. These findings provide further confirmation that Gut-on-a-Chip technology is applicable to canine intestinal epithelial cells as previously reported with the studies utilizing human intestinal epithelial cells2.
This protocol showed that immunofluorescence staining can be used to evaluate the 3D structure of organoid-derived monolayers that have formed villus-like structures in microfluidic chips using conventional fluorescence microscopy (Figure 4 A,B). This protocol can be adapted to validate the differentiated and spatially organized cellular phenotypes through immunofluorescence staining. The visualization of a 3D morphogenesis within a Gut-on-a-Chip provides an excellent opportunity to investigate the host response during various pathological interactions14,23,31. When combined with epithelial cells derived from patient donors, as previously described in humans, this technology can be utilized to construct personalized models of intestinal diseases13. Through the integration of immunofluorescence imaging with targeted RNA visualization techniques like fluorescence in situ hybridization, it can be feasible to visually analyze the transcriptomes and proteomes of the host within a Gut-on-a-Chip system.
Preserving the integrity of the intestinal membrane is vital for maintaining intestinal homeostasis, and the Gut-on-a-Chip platform provides a valuable advantage by allowing for precise monitoring and quantification of this crucial function. The measurement of TEER using the Gut-on-a-Chip technology offers several benefits. For instance, previous studies have successfully evaluated TEER while co-culturing intestinal cells with non-pathogenic and probiotic bacteria32, as well as under leaky-gut conditions23. This allows researchers to study the impact of different conditions on the intestinal barrier function and to identify potential interventions to promote intestinal health.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65720/65720fig01.jpg" /> 
Figure 1: Establishment of patient-derived canine IBD Gut-on-a-Chip. (A) Integration of the patient-derived intestinal organoids and the Gut-on-a-Chip platform. Endoscopy biopsy can be performed to isolate intestinal crypt cells to develop donor-specific intestinal organoids. Epithelial cells can be dissociated into single cells from the organoids, then seeded into a PDMS-based Gut-on-a-Chip and cultured in a unique dynamic microenvironment. (B) Representative images of colonoids from an IBD dog. Scale bar = 100 &#181;m. (C) This schematic illustrates a Gut-on-a-Chip device consisting of a porous membrane placed between upper and lower microchannels. The upper microchannel is indicated by the blue area, while the lower microchannel is indicated by the red area. Vacuum chambers are present on each side of the microchannel, which deform the porous membrane to mimic peristaltic motion24. (D) A setup of canine Gut-on-a-Chip includes a PDMS-based Gut-on-a-Chip assembled with tubing that is placed on a cover slip2,24. The bypass tubing is critical to avoid pressure built-up within the microchannel during the handling (i.e., connecting to syringes). Binder clips are used to clamp the tubing. Volume-sensitive materials can be infused via the open holes of the upper or lower outlet. Organoid culture medium can be infused by connecting the syringes to the blunt end needles and the flow through the upper and lower inlet. Abbreviations: IBD = inflammatory bowel disease; PDMS = polydimethylsiloxane. <a href="https://www.jove.com/files/ftp_upload/65720/65720fig01large.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/65720/65720fig02.jpg" /> 
Figure 2: Formation of villus-like structures in canine IBD Gut-on-a-Chip. The dissociated epithelial cells were seeded in an ECM-coated Gut-on-a-Chip. Once the dissociated cells were attached to the PDMS membrane, apical flow was initiated for 3 days (D0-D3). When a confluent 2D monolayer is formed (D3), basolateral flow with frequent stretching is initiated (Stretching, AP, and BL Flow). After 2-3 days of dual flow and membrane stretching, the 2D monolayer begins to develop 3D morphogenesis, and villus-like structures are formed after 9 days of culture (3D morphogenesis, D9-D12). Abbreviations: ECM = extracellular matrix; PDMS = polydimethylsiloxane; AP = apical; BL = basolateral. <a href="https://www.jove.com/files/ftp_upload/65720/65720fig02large.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/65720/65720fig03.jpg" /> 
Figure 3: Canine intestinal organoid seeding and 3D morphogenesis in a Gut-on-a-Chip. (A) The experimental steps for the surface activation of a porous membrane in a PDMS-based Gut-on-a-Chip. The utilization of UV/Ozone treatment, PEI, and GA treatment in conjunction facilitates the cross-linking of amines present in ECM solutions. This process leads to the stable immobilization of ECM proteins onto the porous membrane. (B) The phase contrast images demonstrate the morphologies of cells immediately after seeding (left) and 3-5 h after seeding (right). The porous membrane after 3 h of seeding displays thinner and darker areas where single cells have attached, highlighting the attachment process. (C) The phase contrast images depict the 3D morphogenesis of intestinal monolayers within a Gut-on-a-Chip system. These monolayers were derived from dogs affected with IBD and these organoid cells were cultured for a period of 12 days under dynamic conditions, which involved fluid flow and stretching motions. Scale bars = 50 &#181;m (B,C). Abbreviations: IBD = inflammatory bowel disease; ECM = extracellular matrix; PDMS = polydimethylsiloxane; PEI = polyethylenimine; GA = glutaraldehyde. <a href="https://www.jove.com/files/ftp_upload/65720/65720fig03large.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/65720/65720fig04.jpg" /> 
Figure 4: Evaluation of the 3D morphological development in patient-derived canine Gut-on-a-Chip. (A) Immunofluorescence imaging of a canine IBD Gut-on-a-Chip, showcasing a top-down view of a fully developed 3D epithelium after 12 days of culture, which is evaluated by a fluorescence microscope. The tight junction protein (ZO-1) is visualized in yellow; the brush-border membrane (F-actin) appears in red; and the nuclei are stained with DAPI and appear blue. (B) Immunofluorescence imaging of a canine IBD Gut-on-a-Chip using a confocal microscope with a long-distance lens. As shown in the schematic, a fluorescent image of a cross section of a villus-like structure of a fully developed 3D epithelium after 12 days of culture is shown. In addition, Z-stacking shows a side view of the 3D epithelium, which reveals the formation of villus-like structures. The brush-border membrane (F-actin) appears in red, and the nuclei are stained with DAPI and appear blue. (C) Intestinal barrier function was evaluated and measured by TEER in patient-derived canine Gut-on-a-Chips. Stable TEER values were reached on Day 5 of culture on Gut-on-a-Chip. Error bars express the SEM of the measurements. TEER value was measured among two biological replicates with one technical replicate. Scale bars = 50 &#181;m (A), 25 &#181;m (B). Abbreviations: IBD = inflammatory bowel disease; DAPI = 4&#39;,6-diamidino-2-phenylindole; TEER = transepithelial electrical resistance. <a href="https://www.jove.com/files/ftp_upload/65720/65720fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Characterization of anti-ZO-1 polyclonal antibody in canine colonoid-derived monolayers and on Gut-on-a-Chip devices. (A) Immunofluorescence staining of ZO-1 in yellow with F-actin in red and their overlay image. Scale bar = 25 &#181;m. (B) Immunofluorescence visualization of ZO-1 in yellow in &#39;Leaky Gut Chips&#39; was conducted under various conditions, including stimulation with probiotic bacteria (LGG + Cytokines or VSL#3 + Cytokines) and germ-free controls without probiotic stimulation (Cytokines). Scale bar = 50 &#181;m. This figure is reproduced from Min et al.23. <a href="https://www.jove.com/files/ftp_upload/65720/FigureS1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Table S1: A summary of tissue donors&#39; information. A summary table of the donors&#39; age, sex, breed, histopathological evaluation, and canine IBD activity index (CIBDAI) score. The CIBDAI is a numeric scoring system used to infer clinical severity in canine IBD33. <a href="https://www.jove.com/files/ftp_upload/65720/65720_SupplementTable.JoVE.Nagao (2).xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies at JoVE.com

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.

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 ...

three-dimensional-morphogenesis-canine-gut-on-chip-using-intestinal

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JoVE Journal

Medicine

3.0K Views.  Washington State University. Ons onderzoeksdoel is om een methode te creëren voor het kweken van 3D-structuren met behulp van darmcellen van dieren die lijken op mensen, met name honden. Intestinale cellologische morfogenese is grotendeels bestudeerd door middel van proefdiermodellen, die kostbaar en tijdrovend zijn en de menselijke ontwikkelingsprocessen niet nauwkeurig weergeven. Bovendien missen conventionele statische 2D-celkweekmodellen het vermogen om de complexe ruimtelijke organisatie van een 3D abSeriële archi...

Video: Auteur in de schijnwerpers: ontwikkeling en toepassing van een IBD darm-op-een-chip-model voor 3D-darmmorfogenesestudies 

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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.

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, ...

Bioengineering

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...

Immunology and Infection

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 ...

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine. Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely. Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.

Experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures are described. Furthermore, the laboratory techniques to ensure consistent growth and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures are discussed.

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they ...

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 ...

Biology

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. ...

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

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