Two-phase short-term culture method for cytogenetic investigations from human prostate carcinoma

An improved technique for primary short-term culture of prostate carcinoma cells in two phases, with and without serum, for subsequent cytogenetic analysis is reported and compared with four other methods. After mechanical disaggregation and a brief collagenase treatment of tumor specimens, cell clusters were seeded in RPMI 1640 and 15% fetal calf serum (FCS) without any other supplement in the first phase. The culture medium was changed to a serum-free medium supplemented with bovine pituitary extract (BPE) and epidermal growth factor (EGF) when the first outgrowth became apparent. During this second phase, fibroblast growth could be virtually abolished within 48 hr. The epithelial and prostatic origin of the cultured cells was confirmed by immunocytochemical methods in each culture. Metaphase analysis revealed chromosome aberrations in over 80% of cases (both clonal and nonclonal alterations) indicating the presence of neoplastic cells. Clonal numerical chromosome aberrations, found by conventional cytogenetic analysis, were used to provide the reliability of the culture system in interphase nuclei of corresponding uncultured tumor tissue by fluorescence in situ hybridization (FISH). The main points of the described method are: 1) combined mechanical/enzymatic disaggregation, 2) seeding of the disaggregated cell clumps rather than of single cells, 3) initialization of the cultures in RPMI 1640 medium with 18% FCS without any other supplements, and (4) stimulating of selective epithelial proliferation by changing the culture conditions through serum-free medium.