Contamination of Stock Lines of Human Carcinoma Cells by Pleuropneumonia-like Organisms

Abstract

DURING January and February 1957 it was noticed that a line of human carcinoma cells (strain HeLa¹) maintained in this laboratory was not growing normally. The cells failed to form continuous sheets on the glass surfaces of the culture bottles, and after 10–14 days growth the cultures had macroscopic ‘moth-eaten’ areas at the edges in which the cells were rounded and degenerate. The tissue culture medium was 80 per cent Eagle's basal medium² and 20 per cent pooled human serum, and contained 80 units penicillin and 80 µgm. streptomycin per ml. When samples of tissue culture medium in which cells had been grown were seeded into nutrient broth, faint turbidity developed after two days incubation at 37°, but no micro-organisms were found in preparations stained by Gram's method. 5 per cent horse blood agar plates similarly seeded developed colonies, 0.5 mm. or less in diameter, after four days aerobic incubation at 37°. It was then suspected that the tissue cultures might be contaminated with pleuropneumonia-like organisms; when samples of used culture fluid were seeded on to solid medium suitable for the growth of pleuropneumonia-like organisms³, colonies with a morphology typical of these organisms grew after three days aerobic or anaerobic incubation at 37°. The colonial appearances were maintained during repeated subculture on this medium, in the absence of penicillin and thallium acetate. The organisms could not be isolated from fresh tissue-culture medium. Successive HeLa cell subcultures yielded profuse growths of pleuropneumonia-like organisms which seemed to multiply in association with the cells, since it has been shown that the organisms do not survive for more than 2 or 3 days in the tissue culture medium employed (Blyth, W., unpublished observations).