Automation of 3D Liver Spheroid generation and Acetaminophen dose– response on the MO:BOT enhances assay robustness and precision

Toxicity is a major cause of drug failure and post-marketing withdrawals, making sensitive and reproducible toxicity readouts essential for modern drug development. FDA Modernization Act 3.0 and related strategic roadmaps explicitly encourage the use of human-relevant in vitro models as New Approach Methodologies (NAMs) as an alternative to animal models. Yet, current preclinical drug discovery and toxicity testing with human in vitro models still depend on manual three-dimensional (3D) spheroid workflows that are challenging to standardize and scale. Although 3D human models better capture tissue-like architecture, cell-cell interactions, and organ-specific functions, 3D culture protocol standardization is precluded by user-dependent protocols, variable spheroid size and morphology, and fragmented steps for seeding, maintenance, and compound dosing. This lack of standardization weakens assay robustness, complicates cross-study comparison, and slows broader adoption in decision-making pipelines. In responses to these needs, we present the MO:BOT, a modular automation platform that unifies 3D organoid and spheroid seeding, medium exchange, image-based quality control, and compound dosing, within a single enclosed system. In this article, we present one use case application focused on an automated workflow to generate HepG2 liver spheroids and screening for acetaminophen (APAP) drug-response. In direct comparison with a manual workflow, the MO:BOT reduced the well-to-well variability in liver spheroid size, stabilized spheroid area over time, and yielded spheroids with higher viability. Automated medium exchange preserved spheroid architecture, demonstrating that appropriately tuned pipetting routines can maintain the integrity of delicate 3D structures. A MO:BOT automated APAP dose–response workflow produced a clear, sigmoidal hepatotoxicity profile with an EC ₅₀ in the clinically relevant range and concordant changes in viability, LDH release, and ALT activity are closely matching the responses. Together, these findings show that the fully automated 3D liver spheroid workflow implemented on the MO:BOT improves culture uniformity and assay readout precision, providing a standardized, scalable foundation for deploying human 3D human models as robust, decision-enabling tools in modern preclinical testing.