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Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, the Netherlands.
Address correspondence to Andrea B. Maier, MD, Leiden University Medical Center, Department of Gerontology and Geriatrics, Postbox 9600, 2300 RC Leiden, the Netherlands. E-mail: a.b.maier{at}lumc.nl
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Key Words: Colony formation • Colony size • Growth speed • Replicative senescence • Fibroblasts
Colony formation is critically dependent on attachment frequency, which varies widely between 3% and 60% for human fibroblasts (3,12). It also depends on the ability of single cells to divide after attachment and on the growth speed during the incubation period. Earlier we have shown that the growth speed of exponentially replicating cultured fibroblasts in mass cultures and the onset of replicative senescence were not correlated (13).
To our knowledge, neither the attachment frequency nor the growth speed has been positively associated with the onset of replicative senescence of mass cultures. Therefore, we studied the correlation between the number of colonies formed, the mean number of cells within the colonies and the growth speed during the early replicative phase, and the onset of replicative senescence in 23 human fibroblast strains.
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MATERIALS AND METHODS |
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Fibroblast mass cultures were established and grown under standardized conditions until the onset of replicative senescence as described elsewhere (13). In brief, fibroblasts were cultured with Dulbecco's modified Eagle medium (DMEM)/F12 medium supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate, 10 mM HEPES, 2 mM glutamax and antibiotics (penicillin at 100 U/mL, streptomycin at 100 µg/mL, and amphotericin B at 0.25–2.5 µg/mL) at 37°C and 5% CO2. All reagents were obtained from Gibco (Breda, the Netherlands). Medium was refreshed twice a week. At 95%–100% confluence, cells were serially passaged in a 1:3.3 split ratio. If a strain was not subcultured for a period of 
35 days, the split ratio was changed to 1:1. Cultures reached the senescent state when cell density was stable or decreasing within a period of at least 45 days after the last subcultivation and after at least 75 days without subculturing. The cumulative population doubling (PD) level, which corresponds to the sum of all previous PD, was calculated by tracking the increase in cell number in sequential passages. Cultures were monitored every 2 months for Mycoplasma contamination, and all were found to be negative.
By 30 months after the start of fibroblast culturing, 57 of 68 strains had reached the senescent state. Colony formation assays were performed in 23 of these fibroblast strains (consisting of the 4 strains with the highest and the lowest replicative capacity and 19 randomly chosen strains).
Colony Formation Assay
For each strain, 500 fibroblasts derived from cultures during the early replicative phase at a confluence of 70%–80% were plated in triplicate into 94 mm petri dishes (Greiner, Alphen a/d Rijn, the Netherlands) containing 7.5 mL of culture medium with 10% FCS. Cells were grown for 14 days at 37°C and 5% CO2 to allow formation of colonies during which period medium was not changed. After 2 weeks, cells were fixed with 0.9% NaCl (Merck, Schiphol, the Netherlands) and stained with methylene blue (2.5 g/L, Sigma, Zwijndrecht, the Netherlands).
The total number of colonies formed on each plate was counted by using a stereomicroscope (LW Scientific, Lawrenceville, GA) at a 10x magnification. Colonies were defined as a cluster of cells containing 16 cells or more.
Colony size was determined by taking photos with a Zeiss reversal microscope (Axiovert 25; Sliedrecht, the Netherlands; 100x magnification) and Canon camera (Power Shot G6) from a minimum of 45 randomly chosen colonies, a subset of the eight biggest colonies and the biggest colony using the software program Axio Vision 4.5 (Zeiss). The number of cells within a colony was manually counted in the case of small colonies of up to a number of 50 cells. In the case of bigger colonies, the number was estimated by counting the number of cells within a representative encircled part of the colony and subsequently extrapolated to the surface of the colony.
Attachment Frequency
For analysis of attachment frequency, cells derived from cultures at a confluence of 70%–80% were seeded at a number of 15,000 cells per well on plastic well plates (Nunc, VWR, Amsterdam, the Netherlands). After the cells had fully attached, but before any subsequent growth (4 hours), cells were washed and attached cells were trypsinized and counted. The attachment frequency assays were repeated twice on separate days for each cell strain. Results from the two independent experiments were averaged.
Statistical Analyses
All statistical analyses were performed using SPSS 14.00 software (SPSS Inc., Chicago, IL). The number of PD within single colonies was calculated as: ln (number of cells within colony) / ln(2), and the growth speed was calculated by dividing the number of PD by the incubation period of 14 days. The associations between the outcome of the colony formation assay and the growth characteristics were tested using the Pearson correlation.
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RESULTS |
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To eliminate the effect of different in vitro replicative histories between the strains, the onset of replicative senescence of the mass culture was also corrected for the PD that had occurred before the colony formation assay was performed. The remaining replicative capacity was calculated by subtraction of the number of PD until the assay was performed from the PD at the onset of replicative senescence. This correction did not improve the relationship between the remaining PD and the number of colonies formed (r = –0.120, p =.585) or the mean number of cells within the colonies (r = 0.191, p =.384).
To estimate the influence of the attachment frequency on the number of formed colonies, we used a randomly chosen subset of five fibroblast strains to determine the attachment frequency of these fibroblasts. The average attachment frequency of these strains was 67% (range 43%–79%) and was, in this small group of strains, not significantly associated with the number of formed colonies (r = 0.49, p =.402).
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DISCUSSION |
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The formation of colonies from single cells is crucially dependent on the attachment of the cells after seeding and a remaining replicative capacity of the attached single cell of >4 PD, which corresponds to the formation of a colony with 16 or more cells. The attachment frequency, which has been reported to vary between 3% and 60% for human fibroblasts (3,12), could dramatically influence the outcome of the colony formation. However, in the present study we found the attachment frequency to vary between 43% and 79%, which is on average comparable to that observed by Smith and colleagues (3), but it was not correlated with the number of formed colonies. To our knowledge, no study has been conducted testing the relationship between the attachment frequency of human fibroblast mass cultures and their cellular growth characteristics.
Fibroblast mass cultures consist of a mixture of cell clones with varying growth potential (16), and the number of clones within each mass culture decreases with a prolonged culture period (17). Therefore, the onset of replicative senescence of a mass culture may depend on the characteristics of the few remaining dominant cell clones with the highest remaining replicative capacity (17). In contrast, the colony survival assay is performed by seeding cells of mass cultures during the early replicative phase consisting of multiple fibroblast clones that vary widely in remaining and mitotic activity. However, Smith and colleagues (3) reported a decreasing number of colonies with more than 16 cells after a prolonged in vitro history of an individual culture, indicating that factors other than clonal dynamics may also contribute to the number of formed colonies.
We found no relationship between the mean number of cells within the colonies and the onset of replicative senescence. Single cells have slightly comparable growth characteristics compared to cells growing in a mass culture of the same fibroblast strain during the same replicative phase, which was shown in the present study by a moderate association between the growth speed within the colonies and the growth speed of the mass culture. Earlier, we have shown that the growth speed during the early replicative phase of a mass culture is not related to the onset of replicative senescence (13); therefore, a relationship between the number of cells within the colonies and the onset of replicative senescence was beforehand unlikely.
Two assumptions have to be fulfilled when the number of cells within a colony and the replicative capacity of a mass culture are linked: First, cells out of the few dominant clones that remain at the end of the replicative phase of the mass culture have to be studied; second, these cells, which will form the few dominant clones, have to reach their onset of replicative senescence within the incubation period of the colony formation assay. However, within an incubation period of 14 days, as proposed by Smith and colleagues (3), who were able to show a correlation between the number of colonies and the replicative capacity, the mean number of PD within a colony is far too low to be indicative of the maximal number of PD of the mass culture with a high replicative capacity (8.1 PD/14 days within clones vs a total of 50–108 PD).
Other researchers were able to replicate the relationship between the replicative capacity and colony formation in fibroblast strains with a replicative capacity between 18 and 75 PD (3,5). These studies differ with the present study in that the fibroblast strains being studied had a significantly lower replicative capacity. Dependent on the in vitro history of the strains with a lower remaining replicative capacity, some cells from the mass culture forming colonies with more than 16 cells could have reached the onset of replicative senescence within an incubation period of 14 days and, by that, lowered the average number of cells within all colonies. Other differences in clonal dynamics dependent on the replicative capacity have not been reported.
The colony formation assay, first published in 1956 by Puck and Marcus (18), has primarily been used to detect cells that have retained the capacity for production of a large number of progeny after treatment that induces cell reproductive death as a result of damage to chromosomes, apoptosis, or stress-induced premature senescence (19,20). The relationship between cellular stress resistance after treatment and replicative capacity has not been studied in detail yet. However, colony formation and colony size seem to be dependent on factors independent of the replicative capacity of human fibroblast strains.
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We thank Martijn den Reijer, Corine de Koning-Treurniet, and Johanna Blom for technical assistance.
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Received December 8, 2007
Accepted March 12, 2008
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