Summary.-The establishment in culture and characterization of 4 human neuroblastoma (NB) cell lines and 1 human ganglioneuroblastoma cell line are described. Each cell line fulfilled at least 2 of 4 criteria for malignant or transformed cells: viz., subcultured more than 70 times, high saturation density with absence of contact inhibition, population-doubling time within a range of 10-40 h, and tumour formation in nu/nu mice. Each cell line also fulfilled at least 2 of 3 criteria for neuroblastoma cells: viz., humoral and cell-mediated immune reactivity toward NB-associated cell-surface antigen, intracellular storage and extra-cellular secretion of catecholamines, and characteristic neuroblast and ganglion-cell morphology.REPORTS of well-characterized cell lines from human neuroblastomas and related tumours (Tumilowicz et al., 1970;Biedler et al., 1973;Schlesinger et al., 1976;Gerson et al., 1977;Seeger et al., 1977), remain infrequent, despite the productiveness of this type of resource in other tumour systems. We have established 4 new neuroblastoma lines and one line from a ganglioneuroblastoma. This paper describes studies of their metabolism, population-doubling time, immunology, tumorigenicity and morphology.
MATERIALS AND METHODS
PatientsAll patients who provided tissue for culture attended the Royal Hospital for Sick Children in Glasgow. The diagnosis was confirmed histologically in each case. Details of the patients are given in Table I.
Cell suspensionsCell suspensions were obtained by dicing fresh surgically excised tumour tissue with scalpel blades in Eagle's minimal essential medium containing penicillin (100 iu/ml) and streptomycin (100 ,tg/ml) under sterile conditions in a laminar-flow cabinet. Fiveml samples of the cell suspension were incubated in 25cm2 or 75cm2 flasks in growth mediumll (GM) at 37°C for 48-72 h to permit attachment of cells to the base of the flask. Cultures were examined microscopically every 3 days and the growth medium changed with the same frequency. Subculture was performed at confluence by adding 0-25% trypsin-saline solution for 10-15 min at 37°C and then dislodging the loosened cells by shaking the bottle and by agitating the medium with a Pasteur pipette. The resulting cell suspension was then washed once in fresh GM and the cell suspension divided between 2 fresh culture flasks.Samples of exponentially growing lines ITo whom reprint requests should be sent.1 I To 150 ml Eagle's MEM add: 2 ml L-glutamine (29-2 mg/ml); 2 ml M Hepes buffer; 5000 iu penicillin; 5000 ,tg streptomycin; 15,000 iu mycostatin; 1 ml MEM nonessential amino acids; 25 ml foetal calf serum.