Protocol for single-cell dissociation of head and neck cancer organoids.

Here, we present a protocol for dissociating head and neck squamous cell carcinoma patient-derived organoids (PDOs) into viable single-cell suspensions, suitable for use in downstream applications such as drug screening and single-cell RNA sequencing. We describe steps for maximizing single-cell yield through incubation with 0.05% trypsin and gentle mechanical dissociation. We avoid higher trypsin concentrations and extended incubation times, as these conditions promote cell aggregation and significantly reduce cell viability. Additionally, we detail procedures for the assessment of single-cell yield by automated cell counter, 24-h live-cell imaging, and flow cytometry.