Protocol for high-throughput droplet-engineered TIL-retaining tumor organoids in triple-negative breast cancer.

Here, we present a protocol for generating high-throughput, droplet-engineered tumor organoids from triple-negative breast cancer (TNBC) tissues using the OrgFab® micro-sphere printing system. We describe tumor dissociation, bioink preparation, and automated organoid fabrication. We further detail rho-associated coiled-coil kinase (ROCK) pathway modulation to preserve tumor-infiltrating lymphocytes (TILs) and enhance T cell function, as well as flow cytometry-based immune characterization (Figure 1). This protocol enables scalable production of immune-competent tumor organoids for immunological analysis and drug-screening applications. For complete details on the use and execution of this protocol, please refer to Zhou et al.1.