Isolation and Culture of Tumor Cells

Negative cells, which contain tumour cells, were plated in 12-well plates at a concentration of 0.5–1 × 10⁶ cells/mL of CellGro SCGM (Cell Genix, Freiburg, Germany, cat# 20802-0500), supplemented with 20% foetal bovine serum (FBS) (Euroclone, cat# ECS0180D) and 0.1% gentamicin (Gibco, Life Technologies Limited, Paisley, UK, cat# 15750-037), and cultured in 25 cm² tissue flasks (Corning Stone, Staffordshire, UK) at 37 °C and 5% CO₂. Viable tumour cells attached to the flask within 12–24 h. At the first medium change, the cells were put into a fresh flask. The cells at 75% to 100% confluence were detached with 0.25% trypsin and 0.02% EDTA (Euroclone) in a calcium/magnesium-free balanced solution. The culture medium was changed twice a week and cellular homogeneity was evaluated microscopically every 24–48 h. Tumour cells derived from early passages (3–5) cultures were cryopreserved in 90% FBS and 10% DMSO (Alchimia, Ponte San Nicolò, Italy, cat# CRN 001-00) and stored in liquid nitrogen for further experiments. To confirm the neoplastic origin of the cultured cells, the cells underwent morphological and immunocytochemical analysis, as previously described [9 (link),26 (link)]. We were able to isolate and grow tumour cells from all 10 patients enrolled in this study.