High-oxygen gas-permeable culture improves viability and supports quantitative antifibrotic pharmacology in precision-cut liver slices.

Liver fibrosis remains a major unmet medical need, and quantitative evaluation of antifibrotic candidates requires ex vivo models that preserve tissue function while supporting higher-throughput pharmacological testing. Precision-cut liver slices (PCLSs) maintain native multicellular architecture but are limited by suboptimal oxygenation and insufficient throughput, particularly in fibrotic tissue. Therefore, we aimed to establish a high-oxygen culture system using commercially available gas-permeable plates to enable quantitative antifibrotic testing in rat PCLSs. Normal and choline-deficient high-fat diet-induced fibrotic PCLSs were cultured under 20% or 80% O2 in gas-permeable (G-Rex; NOK, InnoCell) or conventional nongas-permeable formats. High-oxygen gas-permeable conditions improved viability, assessed by cumulative lactate dehydrogenase release, and enhanced hepatic function, including albumin secretion and glycogen preservation. Under optimized conditions, transforming growth factor-β1 and platelet-derived growth factor-BB markedly induced Col1a1 and Acta2 expression and increased collagen type I production. Fibrotic PCLSs maintained activated stellate cell features and sustained collagen secretion for 96 h. This platform enabled quantitative pharmacological profiling: the activin receptor-like kinase 5 (ALK5) inhibitor SB-525334 consistently suppressed fibrogenic gene expression and collagen production, whereas the multikinase inhibitor nintedanib showed weaker inhibition. Miniaturization to gas-permeable 96-well plates preserved functional and pharmacological performance, increasing experimental yield per liver. Overall, this accessible, high-oxygen PCLS system overcomes oxygenation limitations and enables reliable, medium-throughput antifibrotic testing in a physiologically intact ex vivo fibrosis model.NEW & NOTEWORTHY Precision-cut liver slices preserve native hepatic architecture but are difficult to maintain and miniaturize for quantitative drug testing, particularly in fibrotic tissue. High-oxygen culture using readily available gas-permeable plates improves viability and hepatocyte function while sustaining fibrogenic readouts, enabling concentration-response antifibrotic testing in 96-well formats. This medium-throughput workflow increases data yield per liver and provides a practical, consistent platform for antifibrotic evaluation.