The use of arrested populations of human diploid fibroblasts for the study of senescence in vitro.

It has been shown that human diploid cells from various donor ages can be arrested in an essentially nonmitotic state by reducing the serum concentration of the incubation medium from 10 to 0.5 percent. Cells incubated at this serum level maintained the population distribution that was present when the cells reached confluency. The population, which has 90 percent of the cells in the G1 phase of the division cycle, was not static and exhibited a low level of mitotic activity with prolonged interdivision times. These cells also exhibited a greatly reduced (30 percent) protein content which occurred within the first 7 days of cultivation with 0.5 percent serum. Cells arrested by incubation with low serum medium and subsequently recovered to a proliferative state underwent an equivalent or greater number of population doublings with a concomitant extension of in vitro calendar time when compared to growth controls. This indicated that the number of mitotic events and not the length of time in culture was the primary determinant of in vitro lifespan. The ultimate passage level achieved by experimental cells as compared to controls was different in the three cell strains studied. A gradient of attained passage number according to donor age was established with cells from younger donors reaching a significantly greater number than those from older donors. The use of arrested cell populations for the study of cellular senescence offers a unique opportunity to have an in vitro system which may more closely approximate those in vivo tissues which normally do not exhibit a rapid rate of proliferation. By the use of this system numerous biochemical parameters can be investigated at various cell ages without the interference of proliferative processes.