New Approaches to Characterization of Aging Human Fibroblasts at Individual Cell Level

Abstract

Considering the heterogeneity of cell populations and other critcal problems in mass cultured senescent human fibroblasts, we proposed several new approaches for studying true cellular aging, as follows. 1) To establish a correlation among aging indexes at the individual cell level, we demonstrated a relationship among 3H-thymidine incorporation activity, nuclear size, cell volume, and DNA contents at individual cell level. 2) To fractionate homogenous cell populations and examine the relationship between their life spans and aging indexes, we separated human cells into relatively homogenous populations by the sedimentation velocity method, and found that life span of fractionated cells was almost identical among various fractions, irrespective of their great differences in cell volume and 3H-thymidine incorporation indexes. This suggests that some aging indexes, such as cell volumes and 3H-thymidine incorporation activity, are reversible cell properties, and are not specific properties at the individual cell level. 3) To find age-specific cell properties in fractionated senescent cell populations, we performed a quantitative analysis with an image analyzer on electromicroscopic pictures of fractionated small and large cell populations from young and senescent cultures. We discovered that the differences between young and senescent cultures in nuclear size, mitochonria size and number of lysosomes are due solely to the presence of large cells in senescent cultures, and that an increase in the constricted endoplasmic reticulum is a common phenotype of fractionated senescent cell populations. This suggests that there is some loss of function in endoplasmic reticulum or changes in the membrane system. 4) To examine age-specific changes in membrane system at the individual cell level, we found that the amount of absorption of concanavalin A-bound red blood cells to fibroblast surfaces increases linearly with in vitro passage. Further examination of individual cells indicated that this passage-related change in surface membrane is not dependent on cell cycle phase, surface area, or metabolic age, but certainly is dependent upon division age.