Colony size distributions as a measure of in vivo and in vitro aging.

Abstract

Recently it has been demonstrated that cell cultures from older human subjects, even during their first few in vitro passages, exhibit diminished replicative ability when compared to parallel cultures derived from young subjects (5). However, these studies have examined the replicative behavior of cell populations. Because human diploid cell cultures are heterogeneous in nature, it is also vital to examine the proliferative behavior of human cells as a function of aging at the level of the individual cell Recent studies on human fetal lung cells (WI-38) have indicated that the distribution of sizes of colonies derived from single cells may be an excellent indicator of the total proliferative capacity (in vitro life-span) of human diploid cell cultures (8).The studies described in this report compare the behavior of cloned human adult skin fibroblasts and fetal lung fibroblasts. In addition, the relationship between colony size distribution (CSD) and in vivo donor age is evaluated. MATERIALS AND METHODSCell cultures were established from 2-mm skin biopsies performed on nonhospitalized volunteer members of the Baltimore Longitudinal Study as described (5). Cell cultures from old (>65 years) and young (20-5 years) donors were examined after 5-15 population doublings (PDs) in vitro.The in vitro life-spans of these cell lines were determined at the Gerontology Research Center by described techniques (5). At low in vitro population doubling level (PDL), frozen stocks of these cultures were prepared by resuspending trypsinized cell suspensions in Eagle's minimal essential medium containing 10% fetal bovine serum and 5% dimethyl sulfoxide and cooled to the temperature of liquid nitrogen at a rate of '14/min.These frozen stocks of adult human skin fibroblasts were rapidly thawed in a 370 water bath, returned to tissue culture, and sent to the W. Alton Jones Cell Science Center where the colony size distributions were determined. Mass cultures were maintained in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate-buffered Eagle's basal medium with 10% fetal bovine serum (Rehatuin, Reheis Chemical Co., Kankakee, IL).All clonal growth experiments were performed in MCDB 102 which was prepared according to the procedures of Ham et al. (9). Each freshly prepared batch of MCDB 102 was tested for its ability to support clonal growth of human diploid fibroblasts at between the 20th and 30th PD. Only those batches that gave results essentially equivalent to those reported previously (9) were used. The lot of fetal bovine serum used was prescreened for its ability to promote clonal growth of WI-38 cells in Eagle's basal medium.Rapidly proliferating cultures were trypsinized prior to confluency. The cultures growing in 25-cm2 flasks were rinsed once with 5 ml of buffered medium without serum and washed with 5 ml of 0.25% crude trypsin in Eagle's basal medium. All except 0. The clonal plates were incubated undisturbed for 14 days at 370 in a 5% C02/95% air atmosphere at 98-100% relative humidity.A 2-week incubation period was ch...

Cytogerontology since 1881: A reappraisal of August Weismann and a review of modern progress

“…A similar difference was also reported in a comparison of cultures from young (21-36 years) and old (63-92 years) donors (Schneider and Mitsui 1976). A further correlation between in vitro and in vivo ageing was found by Smith et al (1978) in studies on age-related changes in colony size distributions among individual fibroblasts.…”

Characterization of Multipotential Mesenchymal Progenitor Cells Derived from Human Trabecular Bone

“…Passaged cultures proceeded through the same stages; however, the rate of growth in the log phase and the final number of cells after a fixed period in culture gradually diminished as a function of continued passaging. This decrease in the rate of growth, as well as the total number of cells as a function of increasing passage number, could be attributed to a small population of cells in each passage developing the broad, flattened morphology characteristic of cells entering G 0 , thus resulting in a smaller number of actively dividing cells remaining in the population [14,30,34]. Therefore, loss of doubling potential was accelerated in the DMEM-F12K cultures by the onset of cellular senescence.…”

“…Some cells appeared to begin dividing immediately upon migration from the bone fragment, and others failed to yield colonies at all or did so only after several days in culture. Previous studies have shown that fibroblasts, which are capable of quickly adhering and establishing rapidly growing colonies, undergo an increased number of total population doublings as compared with cells that slowly divide and form smaller colonies [30]. Our data support previous research into the aging characteristics of bone marrow stromal cells, where the confirmed presence of two types of adherent cells in primary culture, a rapidly dividing spindleshaped cell type and a much slower dividing broad and flattened cell type, was observed [14,31].…”

Clonal selection in cultured human fibroblasts: role of protein synthetic errors.

“…Indeed, it has also been shown that a large proportion of cells in a mass culture possess very limited proliferative capacity (32) and that the maximum MPD of clones which make up the mass culture are bimodally distributed (31, 33). Thus, one subpopulation of clones has low replicative potential whereas the other reaches the higher maximum MPD approximately equal to that of the mass culture.…”

“…2). Three clones (13,26,32) were assayed only at one stage. Of the remaining clones, two groups could be identified: group A, comprising seven clones (2,10,13,18,29,30,36), in which the error frequency remained relatively constant through subsequent passage although clones 10 and 29 were only assayed singly at two passage levels; group B, comprising two clones (8 and 14) in which an abrupt increase in error frequency at later passage was observed.…”