Establishment of a quantitative quality control framework for standardized murine intestinal organoids in toxicological modeling

Abstract The lack of standardized quality control (QC) protocols for intestinal organoids remains a major hurdle to their reliable application in toxicological assessments. In this study, we established a framework for standardizing and functionally validating murine intestinal organoids to ensure batch-to-batch consistency and analytical sensitivity. First, we verified the epithelial purity and cellular composition of the organoids using flow cytometric analysis of lineage markers. The proliferation and baseline cell death were established as essential QC benchmarks. Building on this standardized baseline, we confirmed that organoids cultured in basal medium exhibit a balanced distribution of specialized cell lineages, which can be further directed toward specific lineages under maturation conditions. To objectively evaluate physiological relevance, quantitative reference ranges were established for each lineage to maintain normal cellular homeostasis. Furthermore, we validated the functional characteristics of the model by assessing epithelial barrier integrity, active transport, and physiological responsiveness. Notably, we found that compromised barrier function significantly obscured cytotoxic responses to drug treatment, thereby limiting the analytical resolution of the assay. These results demonstrate that implementing standardized QC thresholds from initial growth and purity through functional validation is essential to ensure the reliability of organoid-based drug toxicity profiling.