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We recently published a study (1) in which we used colony size distribution (CSD) to estimate the in vitro proliferative potential of a large number of male and female donors aged 19–90þ years of age. In a commentary published in this issue of the Journal, Cristofalo and colleagues (2) have commented on some differences between the data and interpretation presented in our report and those of other authors. In this article, we respond to those comments in the order in which they were presented. First, Cristofalo and colleagues ‘‘suggest that CSD is not useful as a method for estimating total replicative life span’’ (2). This observation is based primarily on the failure of the authors to find a significant relationship between the estimated in vitro life span, which we calculated using CSD, and the proliferative potential obtained by technicians at the Coriell Institute for Medical Research, using standard subcultivation techniques (3). They suggested that the different results might have been due to a cohort effect in the cultures we examined. We consider this unlikely for the following reasons. The correlation between CSD and in vitro life span was observed in studies for numerous cell types over a period of several years. Furthermore, the linear relation [PD (population doublings) remaining 1⁄4 a 3 (% colonies with at least 16 cells) þ b] has very similar coefficients in all cell types tested (4–6). We first showed that the CSD could be used to predict the proliferative potential of normal human cells in culture (4). In these experiments, we used WI-38 (human fetal lung fibroblasts) cells grown and cloned in MCDB 102 with 10% FBS (fetal bovine serum); 10–20 WI-38 cells were seeded in each of several 60 mm tissue culture dishes and incubated at 378C, 5% CO2, and 98% 99% relative humidity (20% O2) undisturbed for 2 weeks. Three independent subcultivation series of WI-38 cells were cultured to the end of their in vitro life span and the CSD determined at 4 different PD for each subcultivation series. Colony size distribution was determined by counting the number of cells in each colony up to a maximum of 256 cells. All colonies with more than 256 cells were considered as a single group. The number of colonies of 1, 2–3, 4–7, 8–15, and so forth, cells were plotted. We found empirically that only the number of colonies with 16 or more cells was linearly related to the number of population doublings remaining (PDR) in the proliferative potential of the culture at the time the CSD was determined. Linear regression analysis revealed the relationship PDR 1⁄4 0.56 (% . 16) þ 0.6, r 1⁄4 0.98. We have found similar results for fibroblasts isolated from young and old human skin (5) and when we combined the data from WI-38, IMR90, Flow 2000 (human embryonic lung fibroblasts), and fibroblasts isolated from young and old donors (6). While we have no specific explanation for the inability of Cristofalo and colleagues to reproduce our results, several possibilities exist. In 1980 (6), we noted that since the in vitro life span of mass cultures can vary considerably depending on culture conditions, this exact relationship cannot be expected to apply to mass cultures maintained under culture conditions significantly different than those used in our studies. Since 30% to 50% of the cells fail to reattach at each subcultivation, the larger the number of subcultivations the lower the number of cumulative PD one would expect to obtain (7). Culture conditions that permit wide pH fluctuations and/or significant medium depletion during a subcultivation period might result in substantial cell death and also result in an erroneously low value for measured PD. Thus, the CSD loses it predictive value, as does prior life span determination by serial subculture, when cell culture conditions vary significantly from those originally used. If the culture conditions used at the Coriell Institute were uniformly better or worse than those used by us, the CSD would consistently underestimate or overestimate the actual in vitro life span, but the data actually appear to be random with respect to the estimated life span. Random variation could be caused by any of a number of cell culture conditions. For example, differences between various lots of serum or cell culture media used during the serial subculturing experiments could cause significant variation. Another variable is split ratio. Cells were cultured at a 1:2 or 1:3 split ratio in 15% FBS (3). The 1:2 or 1:3 split ratio could result in fewer apparent PD than the 1:4 ratio we used because of the failure of a substantial fraction of cells to attach at subcultivation. Also, the use of 15% FBS 779 COMMENTARY AND AUTHOR’S RESPONSE TO COMMENTARY

Author's Response to Commentary: Cell Senescence: An Evaluation of Replicative Senescence in Culture as a Model for Aging In Situ