assessing epithelial barrier integrity of organoid-derived 2d monolayers using paracellular permeability assay

פיתוח תרביות חד-שכבתיות דו-ממדיות מאורגנואידים של מעי בקר

The culture of intestinal epithelial stem cells in three-dimensional (3D) cultures, known as intestinal organoids, marks a significant advancement in in vitro technology for investigating intestinal functions, nutrition, and interactions with pathogens1,2. These organoids mimic the complex structure of the in vivo intestinal epithelium by self-replicating and organizing into 3D formations that encompass various intestinal cell lineages3. This feature highlights their considerable potential to propel the understanding of intestinal biology forward.
The rising interest in applying intestinal organoid technology to farm animals necessitates the refinement of culture and maintenance techniques4,5. This technology&#39;s relevance is underscored by its potential impact on studying the gut health of farm animals, which plays a critical role in their productivity and, consequently, the economics of the food-animal industry by influencing animal welfare and operational costs6,7. Specifically, employing intestinal organoid cultures to explore the gut function of cattle is of paramount importance, given their role as reservoirs for zoonotic enteric pathogens, such as Salmonella spp. and Escherichia coli (E. coli) O157:H78. These pathogens are localized in particular segments of the gut, making it essential to differentiate intestinal organoid culture methods by gut segment to enhance precision in studies9.
A significant obstacle in the study of intestinal organoids is the restricted access to the epithelial cell&#39;s apical surface10. When cultured within an extracellular matrix (ECM), the cells naturally orient themselves so the basal surface faces outward, and the apical surface is directed inward10. To address this challenge, methods are presented that involve dissociating 3D organoids into single cells and seeding them into semi-permeable cell culture inserts. This setup establishes an interface between the apical surface and a basolateral compartment. This protocol demonstrates that cells derived from bovine intestinal organoids can form a coherent 2D monolayer, as evidenced by transepithelial electrical resistance (TEER) measurements and paracellular permeability assays. Additionally, the development of cellular polarity with brush borders and tight junctions in the organoid-derived 2D monolayer cells is confirmed through immunofluorescence and electron microscopy, reflecting the in vivo gut epithelium&#39;s properties.
In this study, the ileum represents the small intestinal tract, and the rectum signifies the large intestinal tract. These selections are based on relevant enteric pathogens such as Salmonella spp., which can translocate the ileum11, and E. coli O157:H7 known to primarily colonize the rectum9 in cattle. The selection of these specific intestinal segments highlights the necessity of tailoring intestinal organoid culture methods to the gut region for precision in research. These methods detail the procedure for effectively culturing an organoid-derived 2D monolayer interface from these intestinal segments, providing a robust model for exploring cattle gut health, pathogen infections, and interactions between the gut microbiome and host.

מחבר זרקור: פיתוח ואפיון מודלים חד-שכבתיים דו-ממדיים של מעיים מאורגנואידים של בקר לחקר אינטראקציה עם פתוגנים

התקדמות בטכנולוגיית אורגנואיד בקר באמצעות ממשקים חד-שכבתיים של מעי קטן וגדול

Our research goal is to establish robust techniques to generate staple intestinal monolayer interfaces derived from adult bovine small and large intestinal organoids. We employed a multimodal analytical approach to confirm the functional epithelial barrier formation within the established monolayers. Unlike three-dimensional models, 2D monolayers offer an exposed, accessible luminal surface crucial for studying host pathogen interactions.These monolayers reflecting the in vivo gut with multiple cell lineages now provide tailored protocols for investigating pathogens in specific gut sections such as small and large intestinal niches. Our research identified optimized culture conditions to create and sustain 2D monolayers from cells of bovine mall and large intestinal organoids. We confirmed the formation of functional epithelial barrier within these monolayers using transepithelial electrical registrants and paracellular permeability assays, as well as immunocytochemistry staining techniques.Our protocol addressed the research gap by focusing on the lack of intestinal organoid derived monolayer models of bovines. These models have been well studied in mice and humans, however, bovines, which are major reservoirs for intestinal pathogens with serious public health implications haven't been adequately explored. Our research establishes and characterizes a 2D bovine monolayer culture system.This innovative tool allows for the exploration of normal and disease states of bovine intestinal physiology, with potential applications extending into biomedical and translational research of public health significance.

הערכת שלמות מחסום אפיתל של חד-שכבות דו-ממדיות שמקורן באורגנואידים באמצעות בדיקת חדירות פארא-תאית

כדי להתחיל, שלפו את הצלחת המכילה תוספות תרבית תאים עם חד-שכבות דו-ממדיות שמקורן באורגנואידים מהאינקובטור. הסר את מדיית התרבית החד-שכבתית הן מהחדר האפי והן מהחדר הבזולטרלי של תוספות תרבית התאים להערכה. שטפו בעדינות את תאי האפיק והבסיס פעמיים עם 200 ו -500 מיקרוליטר של PBS שחומם מראש.הסר את תמיסת השטיפה מתא האפיקלי והחל 200 מיקרוליטר של 0.5 מיליגרם למיליליטר ארבעה קילודלטון FITC-דקסטרן נותב ב- PBS על התא האפי של תוספת תרבית התא. דוגרים על הצלחת באינקובטור לח בטמפרטורה של 37 מעלות צלזיוס ופחמן דו חמצני של 5% למשך 20 דקות. לאחר מכן דגמו 50 מיקרוליטר מהחדר הבזולטרלי של צלחת 24 הקידוחים והעבירו אותה לצלחת 96 בארות התואמת לקורא מיקרו-צלחות.החלף 50 מיקרוליטר של PBS טרי בתא basolateral של הבאר שנדגמה. באמצעות קורא מיקרו-לוחות מכויל מראש, מדוד מיד את עוצמת הפלואורסצנטיות באורך גל עירור של 495 ננומטר ובאורך גל פליטה של 535 ננומטר.

assessing-epithelial-barrier-integrity-organoid-derived-2d-monolayers

Research

JoVE Journal

Medicine

Assessing Epithelial Barrier Integrity of Organoid-Derived 2D Monolayers Using Paracellular Permeability Assay

201 Views.  Washington State University. כדי להתחיל, שלפו את הצלחת המכילה תוספות תרבית תאים עם חד-שכבות דו-ממדיות שמקורן באורגנואידים מהאינקובטור. הסר את מדיית התרבית החד-שכבתית הן מהחדר האפי והן מהחדר הבזולטרלי של תוספות תרבית התאים להערכה. שטפו בעדינות את תאי האפיק והבסיס פעמיים עם 200 ו -500 מיקרוליטר של PBS שחומם מ?...

Video: הערכת שלמות מחסום אפיתל של חד-שכבות דו-ממדיות שמקורן באורגנואידים באמצעות בדיקת חדירות פארא-תאית

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...