Multiorgan MPS Models for the Studies of Immunometabolic Networks

Introduction: Microphysiological Systems (MPS) are engineered organotypic culture models (OCM) that mimic salient features of tissue and organ physiology by modeling the tissue’s native 3D environment under fluidic perfusion. MPS can be used to study a variety of biological processes, pathological states, and new therapies in the context of human biology. While MPS can be an alternative to animal experiments in limited circumstances, their inherent ability to scale biological complexity makes them a valuable research tool. The combination of scalable complexity paired with computational biology opens a new window that enables us to construct an interactome of different cell types under a variety of conditions, thus gaining a powerful tool to uncover causality with greater accuracy. We and others have used MPS and OCM in proof-of-principle studies to recreate facets of multiorgan interaction and the intricate interplay between different tissues and the immune system. We showed that the interaction between multiple tissues and immune cells leads to vastly more physiological, in vivo-like behavior than if cells are cultured individually. Despite this advancement, most MPS and other OCM models studying immunology rely on using circulating peripheral mononuclear cells (PBMCs) in the absence of native, tissue-resident immune environments. However, these environments are critical in mimicking both the homeostasis and immune responses of that tissue.

Materials and

Methods: We recently developed a new platform technology termed Multiorgan Tissue Interaction Vessel (MOTIVE). The perfusion vessels are 3D-printed from biocompatible Digital ABS material and can be designed to house any number of tissue constructs placed individually in their respective compartments. Compartments are composed of multiple stacked microporous membrane inserts, which allow us to create layers of interacting tissues and their tissue-resident immune environment. Compartments are arranged in an array of chambers that are actively perfused with circulating medium with or without PBMCs, thus mimicking interorgan crosstalk that includes tissue-resident immunity and the involvement of circulating immune cells. The MOTIVE platform can house up to three individual vessels with their corresponding peristaltic pumps. Flow speeds range from 400 µl/min to 4 ml/min to simulate systemic circulation. The flow direction and speed can be controlled remotely via Bluetooth to minimize handling time. We have utilized MOTIVE to reconstruct the gut-liver axis of mutliple individuals and used single-cell RNA sequencing data of the original tissue to predict the donors reponses to functional challanges.

Results, Conclusions, and Discussions: After obtaining the tissue, we first performed a deep single-cell RNA sequencing study to determine the exact cellular composition of each processed organ. We also performed TCR-sequencing of enriched T cell populations in each organ to determine the heterogeneity and clonal relationships among T cells. Further, we characterized the T cell landscape of the colonic intrapetihelial layer as well as the lamina propria, liver, and blood, and showed vast differences among the different T cell subsets found across tissues. The liver and colon largely consist of shared and unique tissue-resident memory populations, while T cells in circulation largely consist of naïve and central memory T cells. This again underpins the importance of including tissue-resident immune environments, specific to each organ, in immune-competent OCM models rather than relying on activated PBMCs. After transcriptomic characterization, we established gut organoid and primary hepatocyte cultures from the donors and isolated individual tissue-resident immune environments for future in vitro studies. To date, we have reconstructed two donors using the MOTIVE-2i platform and performed several inflammatory challenges aimed at establishing the physiological relevance of the model. The immunocompetent constructs were placed into the MOTIVE-2i platform and perfused with Williams E media supplemented with IL-2. We then introduced into the lumen of the gut compartment either LPS (TLR4 agonist), Poly:IC (TLR3 agonist) or 5-OP-RU, a bacteria-derived antigen specific to mucosa-associated invariant T cells. After 2 days of culture, we evaluated the ability of the different treatments to induce inflammation and tissue damage. While there was a slight decrease of TEER during treatment with LPS, we observed significant disruption of the gut barrier, with both T cell antigen stimulation with 5-OP-RU and Poly I:C. Interestingly, each treatment elicited different types of immune activation in each tissue which was donor-dependent. Antigen stimulation and conditioning led to the most pronounced induction of inflammatory cytokines and chemokines in the intestinal compartment universally in both donors while hepatic reponses were highly dependent on the individuals tissue-resident immune environment.
Our work represents an important steppingstone in our quest to develop patient-specific models of interorgan crosstalk, that incorporate the complexity of tissue immune environments.

Acknowledgements (Optional): This work is supported by the grant R35-GM146900.