2-PHASE SHORT-TERM CULTURE METHOD FOR CYTOGENETIC INVESTIGATIONS FROMHUMAN PROSTATE CARCINOMA

An improved technique for primary short-term culture of prostate carci noma cells in two phases, with and without serum, for subsequent cytog enetic analysis is reported and compared with four other methods. Afte r mechanical disaggregation and a brief collagenase treatment of tumor specimens, cell clusters were seeded in RPM1 1640 and 15% fetal calf serum (FCS) without any other supplement in the first phase. The cultu re medium was changed to a serum-free medium supplemented with bovine pituitiary extract (BPE) and epidermal growth factor (EGF) when the fi rst outgrowth became apparent. During this second phase, fibroblast gr owth could be virtually abolished within 48 hr. The epithelial and pro static origin of the cultured cells was confirmed by immunocytochemica l methods in each culture. Metaphase analysis revealed chromosome aber rations in over 80% of cases (both clonal and nonclonal alterations) i ndicating the presence of neoplastic cells. Clonal numerical chromosom e aberrations, found by conventional cytogenetic analysis, were used t o provide the reliability of the culture system in interphase nuclei o f corresponding uncultured tumor tissue by fluorescence in situ hybrid ization (FISH). The main points of the described method are: 1) combin ed mechanical/enzymatic disaggregation, 2) seeding of the disaggregate d cell clumps rather than of single cells, 3) initialization of the cu ltures 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. (C) 1994 Wiley-Liss , Inc.