Effect of selective nutrients in medium on human skin fibroblasts growth and metabolism

Abstract

A thorough understanding of cell metabolism and physiology is necessary for medium optimization, where cells can improve their yield and increase their efficiency of medium utilization or minimize the formation of toxic by-products. The objectives of the present study are to investigate the effect of culture conditions on the growth of human skin fibroblasts, and to characterize human skin fibroblasts growth and metabolism. Growth profiles of human skin fibroblasts by using various donor skin biopsies, seeding densities (SD), medium volume to cell growth area ratio (VAR), interval between medium changes (IMC), and way medium changes (WMC) were studied. Experiments were also conducted to determine the consumption or production of glucose, glutamine, amino acid, lactate and ammonia by fibroblasts. Human skin fibroblasts were cultured and used after three passages. Cell proliferation was measured using trypan blue exclusion test and 3-(4,5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Glucose, lactate and glutamine were measured using YSI biochemistry analyzer; amino acids were measured by gas chromatography; and ammonia was determined by enzymatic assay. The results show no significant difference on growth of human skin fibroblasts isolated from different donor skin biopsy. Fibroblasts with higher SD (1×104cell/cm2 and 2×104cell/cm2) have shorter lag phase and population doubling time, and higher saturation density than the lower SD (1×103cell/cm2 and 2×103cell/cm2). Results also shown that fibroblasts cells could grow in VAR between 0.1-1.0ml/cm2. Higher cell proliferation was obtained by fully changing the medium at IMC two days. Conditioned medium tested by WCM did not show any proliferative effect on fibroblasts. Percentage of nutrients consumption was 12.6% for glucose and 14.3% for glutamine; and percentage of metabolite production was 305.7% for lactate and 55.8% for ammonia. The overall apparent yield of lactate from glucose, Y’Lac,Glc (mmol mmol-1) and overall apparent yield of ammonia from glutamine, Y’Amm,Gln (mmol mmol-1), was calculated to be 2.3 and 0.96 respectively.