AN INTEGRATED MICROPHYSIOLOGICAL SYSTEM FOR MULTI-EPITHELIAL BARRIERS: APPLICATION TO INTESTINAL AND EPIDERMAL BARRIERS

A modular organ-on-a-chip (OOC) system has been developed, incorporating multiple epithelial barriers and perfused immune cells, and enabling the monitoring of the structure, integrity, and response of the barriers to activation.Additionally, it facilitates the examination of the complex interaction between multi-epithelial barriers with immune cells.Fig. 1a schematically illustrates the system, while Fig. 1b provides details of the microfluidic chip connections and Fig. 1c shows the fabricated system.Triple intestinal barriers and intestinal and epidermal barriers were integrated and characterized.OOC technology enables precise cell positioning and in vivo-like polarization, mimicking tissue organization.While organoids provide multicellular structures, more advanced methods are required to induce cellular assemblies and recreate the extracellular environment [1].Epithelial cell polarity, regulated by tight junction (TJ) formation, is crucial for various barriers [2].Microfluidic-based cell culture seamlessly integrates diverse cell types and organs into a unified fluidic circuit.We previously developed a reconfigurable fluidic routing system for inter-chip communication [3].Here, we present an enhanced system that effectively regulates multiple fluidic parameters and enables flexible chip-to-chip interconnections, allowing for fluid redirection across different spatial and temporal dimensions without compromising the flow in other chips.To demonstrate the versatility of MOOCS, three intestinal cell monolayers were integrated into a single fluidic circuit (Fig. 2a), and the TransEpithelial Electrical Resistance (TEER) resulting from the different linkages was measured.Both the individual and the interconnected monolayers displayed stable and increasing TEER values (Fig. 2b).After 20 days, the chips were interconnected, and 100 L of DSS (5000 Da) was injected into one chip, leading to a decline in the total TEER.However, upon disconnecting the affected layer, the TEER returned to its initial level.This experimental setup allows for the execution of multiple experiments on the same cell culture by selectively eliminating the impacted cells.Then, three culture systems were tested: immune cells (THP-1), intestinal/immune cells (Caco-2/THP-1), and epidermal/immune cells (HaCaT/THP-1).Interactions with inflammation stimuli (LPS and DSS) were studied in interconnected/disconnected cellular systems (Fig. 3a).TEERs of disconnected layers increased over time (Fig. 3b).Caco-2 and HaCaT cultures were treated with DSS, showing significant cytotoxicity, especially in caco-2 cells (Fig. 3c).FITC-dextran (4 kDa) flux measurements revealed increased permeability when LPS was applied to Caco-2, HaCaT, or both layers (Fig. 3d).To investigate the response of the integrated cellular system, immune cells were activated by direct activation with LPS, activation through the caco-2 layer with LPS, activation through the HaCaT layer with DNCB, and activation through both layers with LPS and DNCB, respectively.IL-6 and IL-8 expression were measured in the basolateral compartment.Both LPS and DNCB induced significant immune cell responses, with direct treatment showing a higher inflammatory response compared to treatment through the caco-2/HaCaT layers.These findings highlight the successful integration of epithelial cell layers and their interaction with immune cells, providing a valuable model for studying drug screening.This system allows for flexible integration strategies of diverse cellular systems, enabling the investigation of cellular interactions when stimulated through various routes.