Quantification of In Vitro Replicative Lifespan Elongation Activity of Hormones, Antioxidants, Plant Extract and Bacterial Exudate by Updated “Overlay Method”

Background/Objectives: Many products that claim to have anti-aging effects have been reported, but their relative potency is not clear. In this study, the in vitro replicative lifespan extension (RLE) activity of various groups of physiologically active substances was compared by using the updated “overlay method”. Methods: Human dermal and periodontal ligament fibroblasts (HDFa, HPLF) were inoculated into the inner 60 wells of 96-well microplate, surround by sterile water to prevent the water evaporation. At Day 1 and Day 8, the cells were overlayed with wide ranges of concentrations (0.01–100 µM) of samples without medium change. Viable cell number was measured by the MTT method at Day 15 and then corrected for the variation in cell growth due to the location of inoculated cells. The RLE value was calculated as the maximum cell proliferation rate relative to the control. Results: Cell density of HDFa and HPLFs at subculture decreased with the passage number, and their growth was stopped at 56 or 85 population doubling levels (PDLs), respectively. Hydrocortisone showed the highest RLE values among six hormones, followed by three plant extracts, sodium ascorbate and quercetin. On the other hand, other antioxidants, chlorogenic acid, phenylpropanoids, vanilloids, and bacterial products showed little or no RLE effects. However, for HPLF cells, hydrocortisone did not show RLE effects while oxytocin showed slight stimulation. Conclusions: When differences in proliferation due to cell seeding position were corrected, the biphasic dose response curve of most of the compounds significantly reduced. The present study suggests the significant role of hormones for the regulation of the long-term aging process. To confirm systemic or clinical anti-aging effects, further in vitro and in vivo experiments are needed.