2d monolayer formation and 3d morphogenesis establishment in a canine gut-on-a-chip system

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

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.

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.

Watch this Scientific Journal Video about 2D Monolayer Formation and 3D Morphogenesis Establishment in a Canine Gut-on-a-Chip System at JoVE.com

2D Monolayer Formation and 3D Morphogenesis Establishment in a Canine Gut-on-a-Chip System  

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503 조회수.  Washington State University. 시작하려면 10 마이크로몰 Y-27632 및 2.5 마이크로몰 CHIR-99021을 포함하는 오가노이드 배양 배지를 준비합니다. ECM을 gut-on-a-chip에 코팅한 후 상부 마이크로 채널의 바이패스 튜브를 열어 둔 상태에서 상부 마이크로 채널에서 출구 튜브를 분리합니다. 바인더 클립을 사용하여 하부 마이크로채널의 입구와 출구를 모두 고정합니다.P100 마이크로피펫을 사용하여 20마이크로리?...