Primary epithelial ovarian cancer (EOC) cells

Ascites samples were collected from patients with ovarian cancer at the Velindre Cancer Centre, Cardiff, UK and anonymously coded (EOC003 and EOC009). Ascites was stored at 4° C immediately after collection and processed within 24 h. Approximately 400 mL of ascites was centrifuged at 1000 rpm for 5 min to separate primary EOC cells from the fluid. The supernatant was stored at –70° C for subsequent use with autologous tumour cells. Red blood cell lysis buffer (Sigma Aldrich, UK) was added to the pellet according to the manufacturer`s instructions, where appropriate. Tumour cell pellets were frozen in 10% DMSO and 90% autologous supernatant (passage 0). A further 100 mL of ascites was used to generate primary EOC cultures, by separating into 20 mL aliquots and adding to 20 mL of complete (RPMI 1640) medium, supplemented with 10% (v/v) fetal calf serum (FCS), 200 µM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% (v/v) autologous ascitic fluid supernatant. Cells were maintained at 37° C and 5% CO₂. The resulting primary cultures were passaged when cells had reached confluence. All reagents were purchased from Gibco or Thermo Scientific (Paisley, UK).