Prevention of malignant change in mammalian cells during prolonged culture in vitro

Mixed cultures of epithelial cells and fibroblasts, derived from primary cultures of the skin of embryo rats, grown always in rubber-stoppered T-60 flasks, first yielded a transplantable tumor from the 52nd passage, at the end of 13 months of frequently repeated subculture. A group of subcultures, derived from the 22nd passage, grown under the same medium, with the addition of 1% oxyhemoglobin, failed to yield a tumor in 23 months of repeated subculture. A return of these cultures to the regular medium with oxyhemoglobin, yielded a tumor in 4 months, after 12 more passages. Cultures of transformed cells that had regularly yielded a transplantable tumor, for 6 years, up to the 305th passage, continued to yield transplantable tumors when 1% oxyhemoglobin was added to the medium. The cells remained highly atypical, microscopically, and there was no indication of reversal of the malignancy. Although oxyhemoglobin in the medium of cell cultures seems to have had the ability to keep malignancy in abeyance, it did not reverse the established malignant transformation of the cells.