surface activation and extracellular matrix coating of a pdms-based gut-on-a-chip device

在犬 IBD Gut-on-a-Chip 系统中建立 3D 形态发生

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.

作者聚焦：犬 IBD Gut-on-a-Chip 模型的开发和应用，用于 3D 肠道形态发生研究 

使用炎症性肠病患者肠道类器官在犬肠道芯片中的三维形态发生

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 Surface Activation and Extracellular Matrix Coating of a PDMS-Based Gut-on-a-Chip Device at JoVE.com

Surface Activation and Extracellular Matrix Coating of a PDMS-Based Gut-on-a-Chip Device  

基于PDMS的肠道芯片器件的表面活化和细胞外基质涂层

首先，将肠道芯片设备放入 60 摄氏度的干燥烤箱中 30 分钟以消除水分。然后使用紫外线臭氧发生器，将设备暴露在紫外线和臭氧处理下 60 分钟。使用活页夹固定上微通道的入口、旁路和出口管。种植下微通道的入口管，然后断开下下微通道的出口管。确保下微通道的旁路管保持打开状态。使用P100微量移液器，通过下微通道的出口孔加入100微升1%聚乙烯亚胺溶液。重新连接下微通道的出口管。固定下微通道的入口旁路和出口管，然后使用活页夹固定上微通道的入口管。现在断开上微通道的出口管，确保旁路管保持打开状态。接下来，通过上微通道的出口孔加入100微升1%聚乙烯亚胺溶液。重新连接上微通道的出口管，让设备在室温下孵育 10 分钟。用0.1%戊二醛重复该过程，然后用去离子水除去任何多余的表面活化溶液。将薯片在烤箱中以 60 摄氏度烘干过夜。第二天，从烤箱中取出薯片，让它在生物安全柜中冷却 10 分钟。使用活页夹固定上微通道的入口、旁路和出口管，包括下微通道的入口管。断开下微通道的出口管。通过下微通道的出口孔加入 20 微升细胞外基质或 ECM 混合物。然后重新连接下微通道的出口管。用粘结夹固定下微通道的入口、旁路和出口管，包括上微通道的入口管。断开上微通道的出口管后，通过上微通道的出口孔加入 20 微升 VCM 混合物。重新连接上微通道的出口管，然后从下通道的出口管上取下粘合夹，确保两个出口管都打开。将芯片放入 37 摄氏度 5% 二氧化碳培养箱中一小时。

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Research

JoVE Journal

Medicine

Surface Activation and Extracellular Matrix Coating of a PDMS-Based Gut-on-a-Chip Device

528 次观看.  Washington State University. 首先，将肠道芯片设备放入 60 摄氏度的干燥烤箱中 30 分钟以消除水分。然后使用紫外线臭氧发生器，将设备暴露在紫外线和臭氧处理下 60 分钟。使用活页夹固定上微通道的入口、旁路和出口管。种植下微通道的入口管，然后断开下下微通道的出口管。确保下微通道的旁路管保持打开状态。使用P100微量移液器，通过下微通道的出口孔加入100微升1%聚乙烯亚胺溶液。...

视频: Surface Activation and Extracellular Matrix Coating of a PDMS-Based Gut-on-a-Chip Device  

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.

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 ...