Abstract
The circadian clock coordinates the daily rhythmicity of biological processes, and its dysregulation is associated with various human diseases. Despite the direct targeting of rhythmic genes by many prevalent and World Health Organization (WHO) essential drugs, traditional approaches can't satisfy the need of explore multi-timepoint drug administration strategies across a wide range of drugs. Here, droplet-engineered primary liver organoids (DPLOs) are generated with rhythmic characteristics in 4 days, and developed Chronotoxici-plate as an in vitro high-throughput automated rhythmic tool for chronotherapy assessment within 7 days. Cryptochrome 1 (Cry1) is identified as a rhythmic marker in DPLOs, providing insights for rapid assessment of organoid rhythmicity. Using oxaliplatin as a representative drug, time-dependent variations are demonstrated in toxicity on the Chronotoxici-plate, highlighting the importance of considering time-dependent effects. Additionally, the role of chronobiology is underscored in primary organoid modeling. This study may provide tools for both precision chronotherapy and chronotoxicity in drug development by optimizing administration timing.