Bulk Cryopreservation of Cancer Spheroids for High-Throughput Applications

Three-dimensional cancer spheroids provide physiologically relevant models that closely mimic in vivo tumor architecture, making them valuable tools for drug screening and tumor biology research. Despite their advantages, integration into high-throughput workflows is often hindered by labor-intensive and time-sensitive culture processes. In this study, we investigated the feasibility of bulk cryopreservation of cancer spheroid lines using Corning’s 12K Flask format, followed by assessment of post-thaw viability and performance in downstream assays. Our findings demonstrate that bulk freezing effectively preserves spheroid morphology and cellular viability across multiple cancer cell types. Post-thaw spheroids retained structural integrity and metabolic activity, supporting their use in screening applications. This scalable approach enhances experimental reproducibility and workflow flexibility, enabling broader adoption of 3D models in preclinical research. These results support the incorporation of cryopreserved spheroids into high-throughput platforms, advancing the utility of 3D culture systems in cancer drug discovery. Citation Format: Hilary Sherman, Rock Pulak. Bulk cancer spheroid preservation and post-thaw viability for high-throughput screening applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 664.