Scalable Human Intestine Model with Accessible Lumen and Perfusable Branched Vasculature

Abstract

Two-dimensional cell culture and animal models inadequately represent human pharmacokinetics and diseases like inflammatory bowel disease and colorectal cancer. This means missed diagnostic and therapeutic opportunities, high drug attrition rates, and a portfolio of approved drugs that underdeliver the desired benefits to patient outcomes. This encourages the development of a more physiologically relevant intestine model. The objective of this work was to develop a 384-well plate organ-on-a-chip platform, IFlowPlateTM, that can accommodate up to 128 human intestine models with accessible lumens and perfusable branched vasculature in an ECM environment. Fibrin-Matrigel® was used a structurally supportive and biologically instructive substrate that enabled: (1) prolonged cell culture (at least 15 days) with routine refreshment of aprotinin-supplemented medium, (2) formation of a confluent Caco-2 monolayer with barrier function, and (3) de novo assembly of a vascular network with barrier function. A fluorescent dextran permeability assay was used for in situ real-time measurements of epithelial barrier function in a high-throughput manner. Mixed co-culture of endothelial cells and fibroblasts in fibrin-Matrigel® resulted in the formation of an interconnected network of patent vessels that retained an albumin surrogate tracer within the luminal space indicating endothelial barrier function. To improve the success rate of anastomoses between living vessels and fluidic channels, the modification of inherently hydrophobic PDMS and polystyrene culture surfaces with ECM protein was explored. To address the limitations of a cancer cell line-derived intestine model, the replacement of Caco-2 cells with biopsied-derived colon organoid cells was investigated. Different gel formulations were assessed for their ability to induce colon organoid fragments to form monolayers. Finally, the incorporation of multiscale intestinal topography and luminal flow was considered through a modified approach to plate fabrication, whereby moulded alginate is embedded in ECM and sacrificed to generate a scaffold. Work to make the moulded alginate more robust is presented.