Romanian Society of Pharmaceutical Sciences
 

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CHARACTERISATION OF AN IN VITRO AGED CELL CULTURE MODEL

MARKO BUNC 1, SARA M HANNING 2, JOSE M PRIETO 3, JULIJANA KRISTL 1, MINE ORLU 3*

1.University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
2.University of Auckland, School of Pharmacy, Private Bag 92019, Auckland 1142, New Zealand
3.University College London, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom

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The present study aims to provide a detailed protocol to obtain an aged cell culture model, which is conveniently characterised in terms of morphology, metabolism, and key biochemical markers of senescence. Commercial Human Dermal Fibroblasts (HDF) (67-year-old female Asian donor) were cultured over a span of 35 passages to determine at what passages they express the phenotypes found in vivo in the geriatric population. The growth rate and density (counting, Trypan Blue) at confluence declined 4- and 3-fold, respectively. During 15 passages in vitro ageing the fibroblast metabolic capacity (XTT) was heavily reduced (by 75%), while conversely the content of cellular proteins (SRB) slightly increased (2 fold). Senescent cells (SA-β-gal positive) were continuously emerging from 0 to 20%. Freezing/thawing at this stage restored metabolic performance to original values which declined at a similar rate as before. Passages 5 - 10 of our selected aged HDFs were found to mimic in vivo tissue of older adults to the greatest extent.