Quantification of In Vitro Replicative Lifespan Elongation Activity of Pharmaceuticals, Natural Products and Radiation Using the “Overlay” Method

With an increasing number of comorbid diseases, the number of drug intake by elderly people and their chances to X-ray exposure inevitably increase. However, it is unclear how these treatments affect longevity. Primary human diploid fibroblasts with limited life span have been used as a model system for the study of cellular senescence. Since in vitro aging is a long process, the colony formation assay has often been used to monitor the aging progression. However, this method cannot accurately quantitate the anti-aging activity of the test samples due to the heterogeneous size distribution of the colonies. Therefore, we developed a new "overlay" method for quantitating the in vitro replicative lifespan elongation (RLE) activity of substances of interest. Cells were treated for 14 days with or without (as control) various concentrations of test samples in culture medium supplemented with fetal bovine serum (FBS), without medium changes, but overlayed with fresh medium to minimize the cell detachment and nutritional deprivation. From the dose-response curve, the elongation index of the survival time (EI) was calculated by the ratio of maximum viable cell number at the optimal concentration of the test sample to that of the control at Day 14. In general, six human fibroblasts prepared from dermal, oral and lung tissues required higher concentrations of FBS than cancer cells. Dermal fibroblasts were selected as the target cells, based on their higher hormetic responses. Quercetin, hydrocortisone, sodium ascorbate, vanillic acid and vanillin showed higher EI values than estradiol, curcumin, resveratrol, phellamurin, sodium butylate and X-ray irradiation, but did not reach the levels of plant extracts. The present method may be useful to quantitate the RLE activity of external and internal factors alone or in combination, in the presence of an appropriate positive control.