Chronotoxici-Plate Containing Droplet-Engineered Rhythmic Liver Organoids for Drug Toxicity Evaluation.

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.

Bead-jet printing enabled sparse mesenchymal stem cell patterning augments skeletal muscle and hair follicle regeneration.

Transplantation of mesenchymal stem cells (MSCs) holds promise to repair severe traumatic injuries. However, current transplantation practices limit the potential of this technique, either by losing the viable MSCs or reducing the performance of resident MSCs. Herein, we design a "bead-jet" printer, specialized for high-throughput intra-operative formulation and printing of MSCs-laden Matrigel beads. We show that high-density encapsulation of MSCs in Matrigel beads is able to augment MSC function, increasing MSC proliferation, migration, and extracellular vesicle production, compared with low-density bead or high-density bulk encapsulation of the equivalent number of MSCs. We find that the high-density MSCs-laden beads in sparse patterns demonstrate significantly improved therapeutic performance, by regenerating skeletal muscles approaching native-like cell density with reduced fibrosis, and regenerating skin with hair follicle growth and increased dermis thickness. MSC proliferation within 1-week post-transplantation and differentiation at 3 - 4 weeks post-transplantation are suggested to contribute therapy augmentation. We expect this "bead-jet" printing system to strengthen the potential of MSC transplantation.