An Open-Source Automated Pipeline for Quantitative Morphological Analysis of 3D-Bioprinted Cancer Cell Spheroids

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior, drug response, and cell-matrix interactions. Here, we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates, facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation, GelMA preparation, bioprinting, and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research, including studies of metastasis, drug resistance, cancer cell lipid metabolism, and TME-associated hypoxia.