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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Organoid basal medium</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&#8211;streptomycin</td>
			<td>Corning</td>
			<td>MT30009CI</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Organoids and organoid medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01&#160;</td>
			<td>PeproTech&#160;</td>
			<td>9094360</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</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 &#181;M</td>
		</tr>
		<tr>
			<td>HEK293 cells engineered to secrete Noggin&#160;</td>
			<td>Baylor College of Medicine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murine EGF</td>
			<td>PeproTech&#160;</td>
			<td>315-09-1MG</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>Murine Wnt-3a</td>
			<td>PeproTech&#160;</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&#160;</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>1x&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-1</td>
			<td>100 &#181;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&#160;</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067-25MG</td>
			<td>&#160;10 &#181;M</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>StemCellTechnologies</td>
			<td>72308</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>[Leu15 ]-Gastrin I human&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G9145-.5MG</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>Reagents</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&#38;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>Materials and Equipment</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>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LAS X imaging software&#160;</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.

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

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

Our research goal is to create a method for growing 3D structures using intestinal cells from animals similar to humans, specifically dogs. Intestinal cellular morphogenesis has been largely studied through laboratory animal models, which are costly, time consuming, and do not accurately represent human developmental processes. Furthermore, conventional static 2D cell culture models lack the ability to mimic the complex spatial organization of a 3D abSerial architecture.Achieving the attachment of individual cells derived from intestinal organoids onto the ECM-coated PDMS surface has posed a significant challenge. A pivotal step in addressing this challenge in both ensuring a uniform ECM coating through surface activation and allowing it to dry overnight. The integration of patient-derived canine organoid technology with in-vitro 3D Morphogenesis offers a promising avenue for conducting translational research into chronic multifactorial diseases, aligning with the One Health Initiative.The protocol for canine IBD, got on a chip, offers a replicable model for comparative medicine, enabling studies on intestinal morphogenesis, host-microbiome interactions, infections, drug and probiotic screenings, and has cross-species applicability.

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

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

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

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