Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs – “4-way”, “7-way”, and “10-way” – each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS “physiome-on-a-chip” approaches in drug discovery.

Abstract.Investigation of the pharmacokinetics (PK) of a compound is of significant importance during the early stages of drug development, and therefore several in vitro systems are routinely employed for this purpose. However, the need for more physiologically realistic in vitro models has recently fueled the emerging field of tissue-engineered 3D cultures, also referred to as organs-on-chips, or microphysiological systems (MPSs). We have developed a novel fluidic platform that interconnects multiple MPSs, allowing PK studies in multi-organ in vitro systems along with the collection of high-content quantitative data. This platform was employed here to integrate a gut and a liver MPS together in continuous communication, and investigate simultaneously different PK processes taking place after oral drug administration in humans (e.g., intestinal permeability, hepatic metabolism). Measurement of tissue-specific phenotypic metrics indicated that gut and liver MPSs can be fluidically coupled with circulating common medium without compromising their functionality. The PK of diclofenac and hydrocortisone was investigated under different experimental perturbations, and results illustrate the robustness of this integrated system for quantitative PK studies. Mechanistic model-based analysis of the obtained data allowed the derivation of the intrinsic parameters (e.g., permeability, metabolic clearance) associated with the PK processes taking place in each MPS. Although these processes were not substantially affected by the gut-liver interaction, our results indicate that inter-MPS communication can have a modulating effect (hepatic metabolism upregulation). We envision that our integrative approach, which combines multi-cellular tissue models, multi-MPS platforms, and quantitative mechanistic modeling, will have broad applicability in pre-clinical drug development.

An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host–pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

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Mice are not little people"-a refrain becoming louder as the gaps between animal models and human disease become more apparent. At the same time, three emerging approaches are headed toward integration: powerful systems biology analysis of cell-cell and intracellular signaling networks in patient-derived samples; 3D tissue engineered models of human organ systems, often made from stem cells; and micro-fluidic and meso-fluidic devices that enable living systems to be sustained, perturbed and analyzed for weeks in culture. Integration of these rapidly moving fields has the potential to revolutionize development of therapeutics for complex, chronic diseases, including those that have weak genetic bases and substantial contributions from gene-environment interactions. Technical challenges in modeling complex diseases with "organs on chips" approaches include the need for relatively large tissue masses and organ-organ cross talk to capture systemic effects, such that current microfluidic formats often fail to capture the required scale and complexity for interconnected systems. These constraints drive development of new strategies for designing in vitro models, including perfusing organ models, as well as "mesofluidic" pumping and circulation in platforms connecting several organ systems, to achieve the appropriate physiological relevance.

An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the Nlinked protein glycosylation (Pgl) pathway in Campylobacter jejuni pathogenicity. The gutimmune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin producing goblet cells, human enterocytes, and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host-pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylationdeficient strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealedthat 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome, is enabled by application of the gut-immune model and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

Intestinal homeostasis is tightly regulated by the orchestrated actions of a multitude of cell types, including enterocytes, goblet cells, and immune cells. Disruption of intestinal barrier function can increase susceptibility to pathogen invasion and destabilize commensal microbial-epithelial–immune interaction, manifesting in various intestinal and systemic pathologies. However, a quantitative understanding of how these cell types communicate and collectively contribute to tissue function in health and disease is lacking. Here, we utilized a human intestinal epithelial-dendritic cell model and multivariate analysis of secreted factors to investigate the cellular crosstalk in response to physiological and/or pathological cues (e.g., endotoxin, nonsteroidal anti-inflammation drug (NSAID)). Specifically, we demonstrated that treatment with diclofenac (DCF), an NSAID commonly used to treat inflammation associated with acute infection and other conditions, globally suppressed cytokine secretion when dosed in isolation. However, the disruption of barrier function induced by DCF allowed for luminal lipopolysaccharide (LPS) translocation and activation of resident immune cells that overrode the anti-inflammatory influence of DCF. DCF-facilitated inflammation in the presence of LPS was in part mediated by upregulation of macrophage migration inhibitory factor (MIF), an important regulator of innate immunity. However, while neutralization of MIF activity normalized inflammation, it did not lead to intestinal healing. Our data suggest that systems-wide suppression of inflammation alone is insufficient to achieve mucosal healing, especially in the presence of DCF, the target of which, the COX-prostaglandin pathway, is central to mucosal homeostasis. Indeed, DCF removal postinjury enabled partial recovery of intestinal epithelium functions, and this recovery phase was associated with upregulation of a subset of cytokines and chemokines, implicating their potential contribution to intestinal healing. The results highlight the utility of an intestinal model capturing immune function, coupled with multivariate analysis, in understanding molecular mechanisms governing response to microbial factors, supporting application in studying host–pathogen interactions.