Poster V8 - Colonization of intestinal organoids by bacteria in a microfluidic device

Introduction: Gut bacteria colonize the intestinal tract, and their quantity and composition change during disease. In some cases, it is necessary to prevent the colonization of specific bacterial species, such as E. coli O157, while in other cases, it may be desirable to promote the colonization of bacterial species, such as probiotics. Most of the gut bacteria are anaerobic, which presents a unique challenge when studying them in conjunction with the intestinal epithelium. Bacterial colonization promotes expression of epithelial mucins and glycotransferases and increases the secretion of antimicrobial peptides. Diseases and other factors such as inflammation influence the growth and quantify of bacteria as well as the colonization of bacteria, however, it is poorly understood. The objective of this study is to create an experimental system for studying the impact of bacterial colonization on the intestinal organoids and quantify how inflammation affects the bacterial colonization of organoids.

Materials and

Methods: The study used murine primary intestinal organoids and E. coli Nissle 1917 bacteria. Three methods were tested to investigate bacterial colonization of the organoids to occur over a period of 72 hours: a) culture of organoids in Matrigel extracellular matrix with the bacteria added to the culture media; b) culture organoid fragments with bacteria together in Matrigel, c) culture the organoids Matrigel with bacteria overlaid suspended in different concentrations of agar. After careful consideration, the first and the third approach with 5% agar was confirmed as providing the best conditions for bacterial colonization. This was verified using confocal microscopy before being translated to the microfluidic platform. A two-chamber microfluidic device was fabricated using standard soft lithography techniques as previously described (10.1096/fj.202001600RR). Organoid monolayers were established within these devices to study colonization, and the gene expression patterns were measured via qPCR. The cells were stimulated with an inflammatory cytokine (TNF-α), and the regulation of inflammatory markers was assessed along with the impact on colonization.

Results, Conclusions, and Discussions: Over a span of 72 hours, we observed successful colonization of the organoids, confirmed through confocal imaging which revealed bacterial penetration of the Matrigel layer and colonization within the organoids (Figure 1). Gene expression analysis via qPCR was conducted to assess the impact of long-term microbiota colonization on the regulation of key genes, including stem cell markers like LGR5, goblet cell marker MUC2, intestinal epithelial cell marker SI, enteroendocrine cell marker CHGA, and Paneth cell marker LYZ. Additionally, we quantified the regulation of cell proliferation markers Ki67 and SOX9 to determine whether prolonged microbiota colonization affects organoid growth or proliferation. The bacterial response to inflammation was measured using the expression for NF-κB. In this study, our aim was to establish an experimental platform for studying how colonization of organoids by gut bacteria. Current existing methods, such as microinjection or reversing the polarity of organoids, are intricate and challenging. We showed a couple of strategies for successful colonization of the organoids which was faithfully translated to the microfluidic organ on chip device. This allowed us to explore how gut bacteria interact with intestinal cells and influence gene expression patterns, affecting organoid proliferation, maturation, and development under normal conditions and inflammation. In future research, we intend to investigate the impact of hypoxia on colonization, using our microfluidic platform to develop the spatial oxygen distribution, especially in the context of colon cancer.

Acknowledgements (Optional): This research was supported in part by funds from the National Science Foundation [2240045] and the National Institutes of Health [U01CA265719].