Protocol for culture, drug treatment, and dissociation of patient-derived 3D ovarian cancer microspheroids for flow cytometry

Single-cell technologies provide insights into tumor heterogeneity and treatment response. Here, we present a protocol to culture patient-derived ovarian tumor cells as 3D microspheroids. We describe steps for preparing multi-well dishes and isolating ovarian tumor cells for spheroid generation and culture. We then detail procedures for drug treatment, recovery, and dissociation of spheroids followed by single-cell analysis via flow cytometry. This scalable and cost-efficient protocol largely preserves the cellular composition of the original material. • Protocol to culture patient-derived 3D tumor microspheroids for single-cell analysis • Guidance on gentle recovery and dissociation of microspheroids into single cells • Steps for drug response testing with single-cell level readout using flow cytometry Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Single-cell technologies provide insights into tumor heterogeneity and treatment response. Here, we present a protocol to culture patient-derived ovarian tumor cells as 3D microspheroids. We describe steps for preparing multi-well dishes and isolating ovarian tumor cells for spheroid generation and culture. We then detail procedures for drug treatment, recovery, and dissociation of spheroids followed by single-cell analysis via flow cytometry. This scalable and cost-efficient protocol largely preserves the cellular composition of the original material.