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 (IC ₅₀ ) were experimentally determined, and margins of safety (MOS) were calculated by dividing IC ₅₀ by clinical maximum plasma concentration ( C _max ). 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.

The efficient delivery of small interfering RNAs (siRNAs) to immune and respiratory cells represents a key methodological challenge in developing inhaled RNA interference (RNAi) approaches. A central question is whether siRNA functionality is preserved following aerosolization, as the mechanical stress of nebulization may compromise siRNA integrity and silencing activity. Here, we report a proof-of-concept study using THP-1-derived macrophage-like cells as a tractable in vitro model to characterize jet nebulization for siRNA delivery. Three siRNA candidates targeting interleukin-1 beta (IL-1β) were computationally designed and validated for potent silencing activity and low cytotoxicity. Using a commercially available, off-the-shelf jet nebulizer combined with Lipofectamine RNAiMAX, we demonstrate that siRNA-lipoplexes retain their gene-silencing activity after aerosolization, achieving robust IL-1β knockdown. The delivery efficiency was influenced by siRNA-lipoplex complexation, highlighting the importance of formulation parameters. These findings establish a practical and accessible in vitro platform for evaluating nebulized siRNA functionality, providing a foundation for future studies in more complex and physiologically relevant airway models.