P16-07 Automated Toxicology Screening in a Microfluidic, Assay-Ready Adult Stem Cell-Derived Colon Organoid Model for Evaluating Therapy-induced Gastrointestinal Toxicity

Gastrointestinal toxicity (GI) is a common side effect of various treatments, particularly chemotherapy. Since many drugs cause GI toxicity it is important to evaluate novel therapeutics in predictable human disease-models. In this study, we evaluated the use of our adult stem cell-derived (ASC) colon organoid on-a-chip model for automated GI toxicology screening of viability and barrier function. The assay-ready OrganoReady® Colon organoid microphysiological system (MIMETAS B.V.) demonstrated to replicate the histology of the human large intestine on a scalable platform. Our model integrates 64 microfluidic chips seeded with colon organoids. It operates under a pump-free, gravitydriven perfusion system, providing access to both apical and basolateral compartments within a microtiter plate format compatible with standard laboratory equipment. This setup combines physiological relevance with high-throughput capabilities, making it an ideal 3D model for pre-clinical drug development. Precultured organoids are fragmented and seeded in the Organoplate with an automated liquid handler (Biomek i5). Under perfusion, 3D tubules are formed against stabilized rat-tail Collagen-I, and barrier integrity is monitored via Trans-Epithelial Electric Resistance (TEER) measurement. We analyzed the cytoarchitecture of the colon tubules, demonstrating a differentiated and polarized phenotype, with functional stem cells using immunofluorescence staining of Ki67, Muc2, SI, and Integrin-b4. Additionally, the expression and function of key drug transporters such as PGP, and CYP3A4 were evaluated by performing live assays. This model system for human gut biology allows for multiple measurements from a single chip, including TEER, timepoint LDH and cytokine release, morphology assessment, and the choice of ATP measurement or immunofluorescence staining. Moreover, the model is proven to withstand shipment under controlled temperature environments, enabling end-users to perform large compound screening after a defined recovery period. Colon tubules were exposed to a selection of diarrhea-inducing compounds. After 72 hrs barrier integrity was determined by TEER measurement. In addition, cell viability was quantified by measuring LDH activity, and tubules were lysed for ATP measurement. Our model demonstrated strong potential in accurately recapitulating severe toxicity and distinguishing non-toxic compounds. We optimized this platform for large-scale screening of acute and chronic drug exposures, achieving a reproducible assay with a large assay window (Z’>0.5), making it well-suited for quantitative and mechanistic evaluation of experimental intestinal barrier modulators, or cytotoxic agents. This colon organoid-on-a-chip model presents a significant advancement in drug screening technology, enabling more accurate and personalized approaches to drug discovery and development, ultimately improving patient outcomes.