Short-Term Fluctuations and Long-Term Trends in Anchorage-Independent Multiplication Among Cryopreserved Subpopulations of a Spontaneously Transformed Balb/3T3 Clone

A spontaneously transformed clone of Balb/3T3 cells which produced primitive pleomorphic sarcomas in nude mice was maintained in serial passage without cryopreservation. It was also cryopreserved at an early passage and thawed out at four successive intervals. The capacity of these 5 subpopulations to form colonies while suspended in agar (CFEag) was assayed repeatedly over a period of almost 3 years. The initial CFEag and the subsequent pattern of change were unique for each of the subpopulations. When later passages of the nonfrozen subpopulation were cryopreserved and then thawed, there was only a slight change in CFEag and colony size in agar. There were differences in multiplication rates of 3 of the subpopulations on plastic surfaces in the same rank order as their CFEag, but the differences were smaller. Short-term fluctuations in CFEag, however, were not paralleled by changes in multiplication rate on plastic. The nonfrozen subpopulation assumed the appearance of nontransformed fibroblasts when crowded for several days on plastic, and these “normalized” cells had a greatly reduced CFEag when assayed in agar. Neither the change in appearance nor the decrease in CFEag with prolonged incubation of crowded cells occurred with one of the cryopreserved subpopulations which had the same CFEag and growth rate on plastic as the nonfrozen subpopulation under optimal conditions. The evidence favors an epigenetic rather than a genetic origin of the variations in appearance and behavior of the subpopulations.