Single-Cell Isolation and Patient-Derived Organoid Generation Using the Pala™ Single Cell Dispenser with Cancer Cell Lines Spiked into Blood as a Circulating Tumour Cell Model: A Platform for Precision Oncology and Drug Discovery

The isolation of cancer cells and rare circulating tumour cells (CTCs) and the initiation of patient-derived organoids (PDOs) represent two critical new approach methodologies (NAMs) for advancing precision oncology and drug discovery. However, current technologies encounter significant limitations, including high system pressures that compromise cell viability, sample loss, and reliance on marker-dependent enrichment strategies. Here, we performed a technical validation of the Pala Single Cell Sorter/Dispenser (Bio-Techne) using cancer cell lines spiked into healthy donor blood as a model for CTCs, alongside cells isolated from ovarian cancer (OC) patients and cervical cancer cell lines. The platform achieved up to 80% single-cell dispensing efficiency under gentle sorting conditions (<2 psi), successfully dispensing single cancer cells, cell clusters, and cancer cells spiked into blood (mimicking CTCs). Concurrently, three-dimensional organoid structures generated from dissociated OC samples and cervical cancer cell lines showed viable growth and cluster formation within one week. Compared to literature values for fluorescence-activated cell sorting (FACS), the Pala maintained higher post-sort viability (88% vs. 55-70%) and organoid initiation efficiency (68% vs. 42%). This work establishes the Pala as a flexible tool for patient cancer cell dispensing, CTC-mimic isolation, and PDO generation within drug discovery workflows. Clinical validation using authentic patient CTCs remains necessary prior to clinical implementation.