The KB line of human epidermoid carcinoma cells grew with a minimum generation time of 15 hours when cultured in Eagle's medium containing human or calf serum. The maximum cell concentration was a function of the frequency of medium renewal, the time of incubation, and the amount of final cellular material present. Maximum cell density could be increased approximately twofold by the addition of various peptones to the complete medium, although these materials did not affect the minimum generation time. Fortification of the medium with glucose, glutamine, or vitamin mixture did not prolong the growth phase or affect the generation time.
When complete medium was incubated for 8 days at 37° C., it was found to be fully sufficient for subsequent growth of KB cells. When the medium was incubated in the presence of cells for as little as 16 hours, however, it was found to be insufficient for subsequent normal KB proliferation. The addition of certain peptones or an amino acid mixture partially reversed the deficiency.
In addition, beef extract, yeast extract, glycine, serine, asparagine, inositol, pyruvic acid, cholesterol, and coconut milk were tested but were unable to stimulate maximum cell production or growth rate with the KB line.
Renewal of the medium on stock cultures 24 hours prior to harvest and scraping of the cells with a rubber policeman yielded cells capable of growing at the maximum rate. Rapid serial subculture of the KB line resulted in selection of a culture which grew without a “lag” phase.
This work was supported in part by contract No. SA-43-ph-1933, Cancer Chemotherapy National Service Center, National Institutes of Health.