Cellular senescence in vitro and organismal ageing Maier, A.B.

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Cellular senescence in vitro and organismal ageing

Maier, A.B.

Citation

Maier, A. B. (2008, December 11). Cellular senescence in vitro and organismal ageing. Retrieved from https://hdl.handle.net/1887/13335

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Colony formation and colony size do not reflect the onset of replicative senescence in human fibroblasts

Andrea B. Maier, Ilko L. Maier, Diana van Heemst, Rudi G.J. Westendorp

J. Gerontol. A Biol. Sci. Med. Sci. 2008; 63, 655-659

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Colony formation assay and replicative senescence

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Summary

Replicative senescence of human fibroblasts in vitro has been used as a model for in vivo ageing. The onset of replicative senescence varies between several months to years. A colony formation assay, critically dependent on the growth speed can be performed within weeks, and has been reported being an indicator for the onset of replicative senescence. Earlier we could not find a correlation between the growth speed in mass cultures and the onset of replicative senescence of human fibroblast strains. Therefore, we studied the colony formation assay in 23 fibroblast strains that varied widely in their replicative capacity. Neither the number nor the size of colonies were related to the onset of replicative senescence. The number of cells within the colonies was modestly correlated to the growth speed of the mass cultures. We conclude that the colony formation assay does not reflect the onset of replicative senescence in human fibroblasts.

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Introduction

The onset of replicative senescence in human fibroblasts in vitro has been used as a model for in vivo ageing (Hayflick, 1965; Martin et al., 1970; Smith et al., 1978;

Cristofalo et al., 1998; Smith et al., 2002; Tesco et al., 1998) and has also been studied in relation to the occurrence of various age associated diseases (Kill et al., 1994; Bridger and Kill, 2004; Gleason and Goldstein, 1978; Goldstein et al., 1978;

Pratsiniset al., 2002). Because the time to reach the onset of replicative senescence in culture may last from months to several years, surrogate indicators for the onset of replicative senescence have been developed, such as the colony formation assay (Smith et al., 1978). Exponentially replicating cells plated at low density and incubated for 14 days develop into colonies. The distribution of these colonies, especially the percentage of cells able to form colonies with 16 cells or more, has been reported to be significantly associated with the onset of replicative senescence (Smithet al., 1978). It is this colony formation assay that has been used in studies testing the relation between chronological age and the replicative capacity of human fibroblasts (Smith et al., 2002).

Colony formation is critically dependent on the attachment frequency which varies widely between 3-60% for human fibroblasts (Smith et al., 1978; Pomp et al., 1996). 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 (Maier et al., 2007).

To our knowledge, neither the attachment frequency nor the growth speed have 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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Material and Methods

Cell culture and replicative capacity by serial subcultivation

Subjects at the age of 90 years were recruited from the second cohort of the Leiden 85-plus Study, a population based follow-up study of the very old (der Weil, 2002).

Three millimeter full thickness skin biopsies were taken from the non sun exposed mid-upper medial arm of 68 subjects (Maier et al., 2007). All subjects gave informed consent and the medical ethical committee of the Leiden University Medical Centre approved the study.

Fibroblast mass cultures were established and grown under standardized conditions until the onset of replicative senescence as described elsewhere (Maier et al., 2007). In brief, fibroblasts were cultured with D-MEM:F12 (1:1) medium supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate, 10 mM HEPES, 2 mM glutamax I and antibiotics (100 Units per mL penicillin, 100 ȝg per mL streptomycin and 0.25-2.5 ȝg per mL amphotericin B) at 37°C and 5% CO2. All reagents were obtained from Gibco, Breda, the Netherlands. Medium was refreshed twice a week. At 95-100% confluency, cells were serially passaged in a 1:3.3 split ratio. If a strain was not subcultured for over 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 doublings (PDs) level, which corresponds to the sum of all previous population doublings, was calculated by tracking the increase in cell number in sequential passages. Cultures were monitored every two months for Mycoplasma contamination and all were found to be negative.

By 30 months after the start of fibroblast culturing, 57 out of 68 strains had reached the senescent state. 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 assays were performed.

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Colony formation assay

For each strain five hundred fibroblasts, derived from cultures during the early replicative phase at a confluence of 70-80%, were plated in triplo 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 two weeks, cells were fixed with 0.9% NaCl (Merck, Amsterdam, the Netherlands) and stained with methylene-blue (2.5 g per L; Sigma, Zwijndrecht, the Netherlands).

The total number of colonies formed at each plate was counted by using a stereomicroscope (Archiever, LW Scientific) at a 10x magnification. Colonies were defined as a cluster of cells containing 16 cells or more.

The colony size was determined by taking photos with a Zeiss reversal microscope (Axiovert 25, 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, Sliedrecht, the Netherlands). The number of cells within a colony was manually counted in case of small colonies of up to a number of 50 cells. In case of bigger colonies the number was estimated by counting the number of cells within a representative encircled part of the colony and subsequently related 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, 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.

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Statistical analyses

All statistical analyses were performed using SPSS 14.00 software (SPSS Inc.

Chicago, IL). The number of population doublings within single colonies was calculated as: ln(number of cells within colony) / ln(2), and the growth speed by dividing the number of population doublings 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.

Results

Table 1 shows the growth characteristics of all 23 established fibroblast mass cultures. The average growth speed of the mass cultures during the early replicative state was 0.29 PD per day (± 0.03), when the colony formation assays were performed (passage 8 through 18). The onset of replicative senescence varied widely from 51 to 108 PDs.

As shown in Figure 1, the number of colonies including 16 or more cells was neither related to the onset of replicative senescence, nor to the growth speed during the early replicative phase of the mass culture. Furthermore, the number of colonies formed was not significantly associated with other growth characteristics, like the duration of the initiation of the mass culture (r = -0.17, p = 0.44), the onset

Table 1. Growth characteristics of the 23 cell strains.

Growth characteristic Mean (SD) Range

Initiation of the culture (day) 25 (3) 20-31

Growth speed

Early replicative phase (PD per day) 0.29 (0.03) 0.23-0.36 Late replicative phase (PD per day) 0.08 (0.02) 0.05-0.13

Onset of decline growth speed (PD) 56 (11) 38-81

Onset of replicative senescence (PD) 74 (13) 51-108

Replicative age of tested strains (passage) 14 (3) 8-18

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of the decline in growth speed (r = 0.1, p = 0.64), or the growth speed during the late replicative phase of the mass culture (r = 0.33, p = 0.12).

The colonies were composed of an average of 463 (± 293) cells, corresponding to an average growth speed within the colonies of 0.58 (± 0.06) PD per day and to 8.1 PDs during the incubation period of 14 days. As presented in Figure 2, the mean number of cells within the colonies was not significantly associated with the onset of replicative senescence of the mass cultures. There was however, a modest correlation between the number of cells within the colonies and the growth speed during the early replicative phase of the mass culture (r = 0.41, p = 0.05). No significant relation was found with the duration of the initiation of the culture (r = - 0.07,p = 0.75), the onset of the decline in growth speed (r = -0.1, p = 0.65) and the growth speed during the late replicative phase (r = -0.18, p = 0.42).

To test if the number of cells within the biggest colonies were predictive for the onset of replicative senescence of the mass culture, the number of cells within the biggest colony, and, the mean of the eight biggest colonies were related to the onset of replicative senescence. The mean number of cells within the biggest colony of the strains was 2102 (±1193; 0.77 [± 0.07] PD per day) and it was 1142 (± 664; 0.69 [± 0.08] PD per day) cells for the eight biggest colonies. There was no significant association with the onset of replicative senescence of the mass culture (r = -0.238, p = 0.274, and r = -0.206, p = 0.345 respectively).

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 PDs that had occurred before the colony formation assay was performed.

The remaining replicative capacity was calculated by subtraction of the number of PDs until the assay was performed from the PDs at the onset of replicative senescence. This correction did not improve the relation between the remaining PDs and the number of colonies formed (r = -0.120, p = 0.585) or mean number of cells within the colonies (r = 0.191, p = 0.384).

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Figure 1. Colony formation, expressed as percentage of 500 seeded cells, dependent on (A) the onset of replicative senescence and (B) the growth speed during the early replicative phase of fibroblast strains from 23 individuals. 01020304050 406080100120 onset of senescence (PD) colo

ny fo rm ati on (%

)

r = 0.28 p = 0.195 01020304050 0,20,250,30,350,4 growth speed early replicative phase (PD/day)

r = 0.249 p = 0.252

(A)(B)

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0

5001000

1500

2000

2500 0,20,250,30,350,4 growth speed early replicative phase (PD/day)

0

5001000

1500

2000

2500 406080100120 onset of senescence (PD)

nu mb er of cel lsw ith inco lon ies Figure 2. Number of cells within colonies (mean ± SD) dependent on (A) the onset of replicative senescence and (B) the growth speed the early replicative phase of fibroblast strains from 23 individuals. r = -0.16 p = 0.5r = 0.41 p = 0.05

(A)(B)

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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 = 0.402).

Discussion

In earlier studies the colony formation assay has been used as an indicator for the replicative potential of the parental mass culture (Smith et al., 1977, 1978, 2002).

This has led to the conclusion that there is no association between donor age and replicative capacity of human fibroblasts (Smith et al., 2002). Here, we show that the outcome of the colony formation assay is not associated with the onset of replicative senescence of human fibroblast strains with a wide variation in replicative capacity.

The formation of colonies out of a single cell is crucial dependent on the attachment of the cells after seeding and a remaining replicative capacity of the attached single cell of more than 4 PDs, 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 (Smith et al., 1978; Pomp et al., 1996), 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 at al.

(1978), but it was not correlated with the number of formed colonies. To our knowledge, no study has been conducted testing the relation between the attachment frequency of human fibroblast mass cultures and their cellular growth characteristics.

Fibroblast mass cultures consist out of a mixture of cell clones with varying growth potential (Bayreuther et al., 1988) and the number of clones within each

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mass culture is decreasing with a prolonged culture period (Holliday et al., 1977).

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 (Holliday et al., 1977). By contrast, the colony survival assay is performed by seeding cells of mass cultures during the early replicative phase consisting out of multiple fibroblast clones that vary widely in remaining and mitotic activity. However, Smith et al. reported a decreasing number of colonies with more than 16 cells after a prolonged in vitro history of an individual culture (1978), indicating that other factors than clonal dynamics may also contribute to the number of formed colonies.

We found no relation 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 fibroblasts strain during the same replicative phase. This 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 (Maier et al., 2007), therefore a relation 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 to 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 et al. (1978) who were able to show a correlation between the number of colonies and the replicative capacity, the mean number of PDs within a colony is far too low to be indicative for the maximal number of PDs of the mass

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culture with a high replicative capacity (8.1 PDs per 14 days within clones versus total 50-108 PDs).

Other researchers were able to replicate the relation between the replicative capacity and colony formation in fibroblast strains with a replicative capacity between 18 and 75 PDs (Smith et al., 1978, 2002). 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 out of 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 lowering 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 (Puck and Markus, 1956), 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 et cetera, or stress induced premature senescence (SIPS) (Brown and Attardi 2005; den Reijer et al., 2008). The relation between cellular stress resistance after treatment and the replicative capacity has not been studied in detail yet. However, colony formation and colony size seem to be dependent on factors, independently of the replicative capacity of human fibroblast strains.

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