Characterization and optimization of variability in a human colonic epithelium culture model
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Abstract
Animal models have historically been poor preclinical predictors of gastrointestinal (GI) directed therapeutic efficacy and drug-induced GI toxicity. Human stem and primary cell-derived culture systems are a major focus of efforts to create biologically relevant models that enhance preclinical predictive value of intestinal efficacy and toxicity. The inherent variability in stem cell-based cultures makes development of useful models a challenge; the stochastic nature of stem cell differentiation interferes with the ability to build and validate reproducible assays that query drug responses and pharmacokinetics. In this study, we aimed to characterize and reduce sources of variability in a complex stem cell-derived intestinal epithelium model, termed RepliGut® Planar, across cells from multiple human donors, cell lots, and passage numbers. Assessment criteria included barrier formation and integrity, gene expression, and cytokine responses. Gene expression and culture metric analyses revealed that controlling cell passage number reduces variability and maximizes physiological relevance of the model. In a case study where passage number was optimized, distinct cytokine responses were observed among four human donors, indicating that biological variability can be detected in cell cultures originating from diverse human sources. These findings highlight key considerations for designing assays that can be applied to additional primary cell-derived systems, as well as establish utility of the RepliGut® Planar platform for robust development of human-predictive drug-response assays.
Plain language summary
Animal models are frequently used as tools for studying gastrointestinal (GI) disease, but they inadequately replicate the complexities of the human gut, making them poor predictors of how humans respond to new drugs. Models using human stem cells are closer to human GI physiology, but their responses are not uniform owing to variability in the stem cells. We looked for the sources of this variability in the primary stem-cell derived RepliGut® Planar model. We found that limiting how long the cells were kept in culture reduced their variability and improved the physiological relevance of the model. These findings highlight key assay design considerations that also can be applied to other primary cell-derived systems. Reliable and physiologically relevant cell-based models can reduce animal testing, improve research accuracy, and ensure new treatments are more relevant and effective for patients.
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