RESOURCES

Abstract
Drug-induced liver injury (DILI) remains a major cause of acute liver failure, clinical trial attrition, and post-marketing drug withdrawal, yet predictive in vitro models are limited in accuracy, scalability, and human relevance. Here, we present a Liver-on-Micropillar (LoM) platform a fully animal-free, high-throughput, miniaturized human liver model designed for early-stage hepatotoxicity screening. The system combines a xeno-free medium with a xeno-free bioink to support co-culture of four human liver-relevant cell types: differentiated HepaRG, LX-2, HMEC-1, and differentiated THP-1 cells. Microlivers are bioprinted onto micropillar arrays compatible with standard 96-well plate formats. Functional characterization confirmed stable cell viability, albumin and urea production, as well as inducible CYP expression. To evaluate DILI predictivity, ten reference drugs were tested using assays to measure ATP content, XTT metabolic activity, and albumin secretion. Half-maximal inhibitory concentrations (IC50) were experimentally determined, and margins of safety (MOS) were calculated by dividing IC50 by clinical maximum plasma concentration (Cmax). The LoM platform correctly classified 90% of the tested compounds using a MOS threshold of 100. This scalable and reproducible model provides a human-relevant, regulatory-aligned alternative to animal testing and supports broader efforts to implement non-animal methodologies in drug safety evaluation.