Nunclon sphera 96 well plates

LNCaP and PANC-1 cells were plated overnight (10,000 cells per well in Nunclon Sphera 96 well plates, 174925, ThermoScientific). To ensure spheroid formation, the plate was centrifuged at 1500 RPM for 10 mins at 20°C before overnight incubation. The spheroids were infected with different MOI of rSFV-GFP particles to quantify infectivity. Incucyte-based brightfield and fluorescence microscopy were performed over time to assess the morphology and infectivity of the spheroid respectively. The number of GFP-expressing cells in an image was used as a measure of rSFV-GFP infectivity. Confocal fluorescence microscopy was performed using the CD7 platform to quantify the depth at which rSFV-GFP particles can infect cells in the spheroid.

The neural crest cell-induction protocol was modified from previously described methods^{56 (link)}. hiPSCs were cultured in E8F defined medium. On days 0 and 1, the medium was changed to SBCHIR defined medium. On day 2, the medium was changed to DMHB defined medium. On day 10, hiPSC-derived neural crest cells were washed twice with PBS and incubated with Accutase (Stemcell Technologies) for 7 min and then cryopreserved. hiPSC-derived neural crest cells were thawed and cultured in NC defined medium with Y-27632 (10 µM). On day 11, the medium was changed to SNP defined medium. On day 13, hiPSC-derived sensory neuronal precursor cells were washed twice with PBS and incubated with Accumax solution (ICT) for 20 min and then cryopreserved. hiPSC-derived sensory neuronal precursors were thawed and seeded in Nunclon Sphera 96-well plates (Thermo Fisher) in sensory neurosphere-induction defined medium with Y-27632 (only on day 0). On day 3, the medium was changed to sensory neurosphere-maintenance defined medium, and sensory neurosphere-maintenance defined medium was carefully changed three times per week until day 14.

Spheroids were obtained from the A2780, A2780cis, A2780cisR, SKOV-3, and OVCAR-3 cell lines. Spheroids were cultured for up to three weeks, depending on the rate of spheroid formation (A2780 lines—7 days, SKOV-3—two weeks, OVCAR-3—3 weeks). The spheroid formation was followed by using light microscopy. Initially, 0.2 × 10⁶ cells per well suspended in the culture medium were seeded on a 24-well plate with an ultra-low attachment culture surface (Nunclon Sphera 24-well plates, Thermo Fisher Scientific, Waltham, MA, USA). The initial culture medium consisted of a 1–2-day culture medium that was appropriate for a given cell line, in a 1:1 ratio with a spheroid culture medium (DMEM: F12, 1: 1, supplemented with 2 g/L glucose, 5% FBS, penicillin, and streptomycin). During 1–3 weeks of culture (depending on the cell line), the culture medium was gradually changed 1:1 by gently discharging 1 mL of the old medium and supplementing it with 1 mL of the fresh medium for spheroids. The experiment was repeated three times using separate cell passages. For the experiments, the spheroid suspensions from 12 culture wells were pooled, the excess medium was removed, and 9 mL suspensions were distributed at 0.15 mL across the 96-well plate (Nunclon Sphera 96-well plates, Thermo Fisher Scientific, Waltham, MA, USA) and then treated with compounds, as described below.