Abstract
Immunosurveillance of the gastrointestinal epithelium by mononuclear phagocytes (MNPs) is essential for maintaining gut health. However, studying the complex interplay between the human gastrointestinal epithelium and MNPs such as dendritic cells (DCs) is difficult, since traditional cell culture systems lack complexity, and animal models may not adequately represent human tissues. Microphysiological systems, or tissue chips, are an attractive alternative for these investigations, because they model functional features of specific tissues or organs using microscale culture platforms that recreate physiological tissue microenvironments. However, successful integration of multiple of tissue types on a tissue chip platform to reproduce physiological cell-cell interactions remains a challenge. We previously developed a tissue chip system, the gut organoid flow chip (GOFlowChip), for long term culture of 3-D pluripotent stem cell-derived human intestinal organoids. Here, we optimized the GOFlowChip platform to build a complex microphysiological immune-cell-epithelial cell co-culture model in order to study DC-epithelial interactions in human stomach. We first tested different tubing materials and chip configurations to optimize DC loading onto the GOFlowChip and demonstrated that DC culture on the GOFlowChip for up to 20 h did not impact DC activation status or viability. However, Transwell chemotaxis assays and live confocal imaging revealed that Matrigel, the extracellular matrix (ECM) material commonly used for organoid culture, prevented DC migration towards the organoids and the establishment of direct MNP-epithelial contacts. Therefore, we next evaluated DC chemotaxis through alternative ECM materials including Matrigel-collagen mixtures and synthetic hydrogels. A polysaccharide-based synthetic hydrogel, VitroGel®-ORGANOID-3 (V-ORG-3), enabled significantly increased DC chemotaxis through the matrix, supported organoid survival and growth, and did not significantly alter DC activation or viability. On the GOFlowChip, DCs that were flowed into the chip migrated rapidly through the V-ORG matrix and reached organoids embedded deep within the chip, with increased interactions between DCs and gastric organoids. The successful integration of DCs and V-ORG-3 embedded gastric organoids into the GOFlowChip platform now permits real-time imaging of MNP-epithelial interactions and other investigations of the complex interplay between gastrointestinal MNPs and epithelial cells in their response to pathogens, candidate drugs and mucosal vaccines.
Highlights
IntroductionDysregulation of the balance between the GI microbiome, immune system, and epithelium can disturb protective and tolerant responses to commensal and pathogenic bacteria (Kelsall and Rescigno, 2004)
Maintenance of gastrointestinal (GI) homeostasis is essential for proper gut health
The constraining factors of matrices are considered in this study, as we identify matrices optimal for mononuclear phagocytes (MNPs) and human gastric organoid (HGO) co-culture by comparing MNP chemotaxis through Matrigel, a gelatinous basement membrane mixture derived from mouse sarcoma cells that is used frequently for organoid culture, and chemotaxis through synthetic extracellular matrix (ECM)
Summary
Dysregulation of the balance between the GI microbiome, immune system, and epithelium can disturb protective and tolerant responses to commensal and pathogenic bacteria (Kelsall and Rescigno, 2004). Antigen sampling by mononuclear phagocytes (MNPs) including dendritic cells (DCs) and macrophages is essential to maintain this homeostasis (Mann and Li, 2014). MNPs sample their environment, maintaining tolerance to the complex milieu of commensal bacteria and food antigens, while inducing a robust response to pathogens. Sophisticated microphysiological systems (MPSs) that integrate gastrointestinal epithelial cell monolayers and immune components have been established by multiple groups (Kim and Ingber, 2013; Kim et al, 2016; Shin and Kim, 2018; Beaurivage et al, 2020). Few studies have developed MPS that utilize organoids in their 3-D configuration, and these systems have yet to introduce immune cell components (Lee et al, 2018; Sidar et al, 2019)
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