Label-free interferometry platform for drug response profiling of bioprinted tumor organoids at single-organoid resolution.

Organoids have become mainstay tools for drug discovery and personalized medicine. High-throughput imaging readouts for drug screening of tumor organoids are of particular interest as organoid-level quantification of responses provides insights into heterogeneity, which is relevant for predicting therapeutic efficacy and anticipating emergence of resistance. However, screening extracellular matrix (ECM)-embedded organoids remains technically challenging. Standard methods with manual cell seeding in thick ECM constructs impede imaging efficiency, whereas bulk endpoint assays are easy to implement but fail to resolve single-organoid-level drug response data. Here we present a protocol to bioprint cells within a temperature-sensitive ECM in thin, flat, square-shaped patterns in 96-well plates to establish three-dimensional (3D) cultures for efficient, high-throughput, time-resolved quantitative phase imaging at single-organoid resolution. Quantitative phase imaging of 3D-bioprinted organoids using high-speed live cell interferometry coupled with machine learning analyses enables label-free quantification of biomass, growth kinetics and drug response profiles of thousands of individual organoids per experiment. We demonstrate that the protocol can be leveraged to automatically generate plates containing thousands of organoids for high-throughput imaging and drug screening experiments, quantify growth and drug response heterogeneity, resolve rare phenotypes and identify predictive features of drug response profiles for fundamental studies and therapeutic decision-making. This protocol can be completed in 2 weeks or less and can be adapted to organoids derived from a variety of cell sources and to alternative screening paradigms. The protocol requires familiarity with coding and experience with 3D cell culture, optical assembly and software installation.