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Cellular ageing in fibroblast cultures from elderly aged 90 years old

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. Background. Link between cellular ageing in vitro and chronological ageing in vivo ? Inconsistent results:

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Cellular ageing in fibroblast cultures from elderly aged 90 years old

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  1. 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

  2. Background • 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?

  3. Study design • 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

  4. Fibroblast growth kinetics in mass cultures • Phase I • initiation of the culture • Phase IIa • steady proliferation • Phase IIb • decrease in proliferation • Phase III • growth arrest PD III III IIb IIb IIa IIa I I days Hayflick 1961

  5. Growth kinetics (n=68) Culture: 183-510 days

  6. Results • 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

  7. 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

  8. Modeling growth kinetics (two examples) Transition 2a/2b S324: 47 PD (147 days) S182: 67 PD (229 days)

  9. 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

  10. Conclusions • 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)

  11. Acknowledgements • Andrea Maier • Corine de Koning-Treurniet • Joke Blom • Ton de Craen • Simon Mooijaart • Rudi Westendorp

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