Organoid in droplet: Production of uniform pancreatic cancer organoids from single cells

Cancer organoids have improved our understanding of recapitulating the histology, genotypes, and drug response of patient tumors for personalized medicine. However, the existing cancer organoids are typically grown in animal-derived matrices (e.g., Matrigel), which suffer from poor reproducibility and low throughput due to uncontrollable origin of seed cells, undefined matrix, and manual manipulation. Here, we report a new strategy to massively generate uniform pancreatic cancer organoids (PCOs) in a droplet system from single cells. This system is composed of all-in-water fluids that allow to mildly encapsulate single tumor cell into isolated droplet, which subsequently proliferate and self-assemble into organoids, resembling the initial state of a tumor in the body. This high-throughput method can produce thousands of organoids in a single batch. The droplets can serve as templates for synthesizing defined microgels with proper stiffness similar to that of native tumors, facilitating functional expressions of PCOs. These organoids exhibit superior uniformity and controllability in terms of size and morphologies compared with organoids cultured in manually dropped Matrigel, due mainly to the controllable number of initiating cells and defined microgels. In addition, the established organoids maintain the key biomarkers of pancreatic tumor (e.g., KRT7, KRT19 and SOX9) and higher expression of genes associated with drug metabolism confirmed by RNA-seq and PCR analysis. Furthermore, they show distinguishing responses to four clinically used drugs in a reproducible manner in automatic pipetting workstation, indicating the feasibility of the proposed method in high-throughput drug testing. The established strategy has integrated the formation, 3D cultures, and analysis of PCOs derived from single cells in a whole system, which may provide a novel platform for advancing organoids research with standardized procedure in translational applications.