Cellular ageing in fibroblast cultures from elderly aged 90 years old

Diana van Heemst, Dept. Gerontology and Geriatrics, Leiden University Medical Center, Leiden, The Netherlands

• Link between cellular ageing in vitro and chronological ageing in vivo?

• Inconsistent results:-Inverse relationship: replicative lifespan -donors age (Martin 1970, Schneider 1976,Smith 1978, Goldstein 1978, Allsopp 1992)-No correlation (Cristofalo 1998)-High inter-individual variation

• Aim: Variability in growth kinetics in fibroblasts from elderly aged 90 years?

Background

• Leiden 85-plus Study– prospective population based study, inhabitants of Leiden, The Netherlands

- birth cohort 1912-1914, follow-up 5 years

- age of 90 years (n=68):

- good physical and mental health

- fibroblast cultures started from 3 mm skin biopsies

- standardised procedures

Study design

Fibroblast growth kinetics in mass cultures

I

IIa

IIbIII

Hayflick 1961

I

IIa

IIbIII

PD

days

• Phase I– initiation of the culture

• Phase IIa– steady proliferation

• Phase IIb– decrease in proliferation

• Phase III– growth arrest

Growth kinetics (n=68)

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Culture:

183-510

days

• no strain failed to proliferate• all easily cultured• all typical growth phases

–initiation–proliferation–senescence (n=10)

• reproducibility CV (sd): 11.17 (+/- 9.5) % (n=33)• huge variability in growth kinetics

Results

Modeling growth kinetics (n=10)

Phase 1, 2a, 2b: no differences in speed of growth (0.304 (+/- 0.028) PD/day in 2a and 0.076 (+/- 0.017) PD/day in 2b)

Transition 2a/2b: striking differences: 42-67 PD, 113-229 days

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Modeling growth kinetics (two examples)

Transition 2a/2b

S324: 47 PD (147 days) S182: 67 PD (229 days)

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Predicted Valuesdagen

popd2dagen

studienr: S182

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70,00Predicted Valuesdagen

popd2dagen

studienr: S324

Transition phase 2b/3 (n=10)

Phase 3: not subcultured 75 days without increase in cell density (77-156 days)

Transition 2b/3: striking differences: 53-80 PD, 294-470 days

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• High intra-biopsy reproducibility• Very high proliferative capacity left

– mixture of clones, cell clone with the highest proliferative capacity responsible for replicative capacity

• High variation in growth kinetics in fibroblasts from elderly in transitions 2a/2b and 2b/3• Future prospects: mechanism transition points (ß-galactosidase), relation with subject characteristics (health, remaining life span)

Conclusions

• Andrea Maier

• Corine de Koning-Treurniet

• Joke Blom

• Ton de Craen

• Simon Mooijaart

• Rudi Westendorp

Acknowledgements