The study of age-related changes in the physiology, biochemistry, and molecular biology of isolated skin cell populations in culture has expanded and provide the basis for the understanding of the fundamental aspects of skin aging. In the field of molecular and cellular biology of aging and skin aging the terminology and theory about ‘‘cellular aging,’’ ‘‘cell senescence,’’ or ‘‘replicative senescence’’ is most commonly derived from the study of normal diploid cells (e.g. dermal fibroblast) in vitro through serial subcultivation. This process of cellular senescence, or replicative senescence in vitro is generally known as the Hayflick phenomenon, and the limited division potential of cells (dermal fibroblast) is called the Hayflick limit. After a limited number of serial passaging, fibroblast enter into the period of slowing-down of cell proliferation rate, followed the cessation of cell division known as “replicative senescence’’. After fibroblasts reaches replicative senescence, some cells can still stay alive and be metabolically active at a minimal level for sometime and generally resist undergoing apoptosis. For fibroblasts the range of cumulative population doublings (CPD, i.e. the total number of cell divisions) for the cell strains originating from embryonic tissues is between 50 and 70, whereas for those originating from adult biopsies it is generally less than 50 CPD. Additionally, gaseous composition, especially oxygen levels, and the quality of the nutritional serum and growth factors of the culture medium as well as the type of skin biopsy (sun-exposed vs sun-protected skin biopsy), can significantly affect the proliferative lifespan of fibroblasts in vitro. The in vitro system of fibroblast cellular aging provide the model for studying structural and functional aspects of skin aging.
There is a progressive and accumulative occurrence of a wide variety of phenotype changes during the whole serial passaging of fibroblasts before cessation of cell replication occurs. The emerging senescent phenotype of serially passaged fibroblasts can be categorized into the structural, physiological, and biochemical and molecular phenotypes. There are more than 200 such structural, physiological, biochemical, and molecular characteristics that have been studied during cellular aging of fibroblast that appear progressively in cell cultures.
The structural changes occur progressively during in vitro fibroblast senescence include: the increase in cell size, changed cellular morphology (from thin, long, and spindle-like to flattened and irregular cell shape), rod-like polymerization of the cytoskeletal actin filaments and disorganized microtubules, increased membrane rigidity, increased multinucleation, increased number of vacuoles and dense lysosomal autophagous bodies. In addition to the gross structural alterations, there are several ultrastructural changes by electron microscopic studies.
The physiological changes occur progressively during fibroblast in vitro serial subcultivation include: altered calcium flux, pH, viscosity, and membrane potential, reduced activity of ionic pumps, reduced mobility, reduced respiration and energy production, reduced response to growth factors and other mitogens, increased sensitivity to drugs, irradiation, and other stresses.
A large amount of data documented a plethora of changes of fibroblast during cellular senescence at the biochemical and molecular level which form the mechanistic bases of structural and physiological alterations. The biochemical and molecular changes of fibroblast occur progressively during fibroblast cellular aging include: cell cycle pause at the G1 phase near the S phase boundary (cessation of cell division), increased mRNA and protein levels of cell cycle inhibitors, increased mRNA and protein levels of inhibitors of proteases (decreased damaged protein degradation), decreased amount and activities of numerous house-keeping enzymes, decreased activities of macromolecular turnover pathways, reduced levels of DNA methylation, reduced length of telomeres, increased nuclear and mitochondria DNA damage, increased protein damage, increased macromolecular cross-linking, increased accumulation of reactive oxygen species (ROS).
The correlation between cellular aging in vitro and in vivo is often based on the evidence gathered from studies of fibroblast derived from aging skin biopsies and from premature aging syndromes on cellular proliferative capacity in vitro. These studies indicate that the genetic and intrinsic Hayflick limit of fibroblast in vitro subcultivation is a true reflection of what is going on during aging and skin aging.