On the malignant transformation of cells during prolonged culture under hypoxic conditions in vitro

Four independent series of mixed cultures of epithelial cells and fibroblasts, from the skin (mainly epidermis) of rat embryos, grown under a layer of fluid medium in stationary T-60 flasks, with or without free access of ambient air to the surface of the medium, eventually yielded malignant tumors, when the cultures were injected into rats of the same strain. These cultures were composed of both types of cell, and proved transplantable when injected subcutaneously or intraperitoneally, even into untreated rats of the same strain as the embryos. Exposure of the cells to a high concentration of readily available oxygen in the medium resulted in at least a delay, and probably the complete prevention, of the malignant transformation of the cells. Observations of the malignant transformation of cells grown for prolonged periods in vitro have become commonplace, but the cause of the phenomenon has remained obscure. Neither a virus nor any known physical or chemical carcinogenic agent has been proved to be the cause. The transformation of the cells was not reversed during 13 months of exposure of the cultures of malignant cells to a high concentration of oxygen in the medium. The experimental results of this investigation, as well as the theoretical considerations, suggest that intracellular hypoxia, due to the inadequate rate of diffusion of the oxygen in the ambient air through the stationary layer of liquid medium, acted at least as the initiating agent, with unknown (intrinsic or extrinsic) factors possibly acting as the promoting carcinogenic agent.