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The formulation of the culture medium for a Chinese hamster ovary (CHO) cell line has been investigated in terms of the simultaneous replacement of glucose and glutamine, the most commonly employed carbon and nitrogen sources, pursuing the objective of achieving a more efficient use of these compounds, simultaneously avoiding the accumulation of lactate and ammonium in the medium. The key factor in this process is the selection of compounds that are slowly metabolized. Among the different compounds studied, galactose and glutamate provide the best results, allowing support of cell growth with an optimal balance between nutrient uptake and cell requirements and the generation of minimal quantities of lactate and ammonium. The attained results also highlight the capacity of the cells to redistribute their metabolism as a response to the changes in medium composition.

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.

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Background: Whether aging modifies mesenchymal stem cell (MSC) properties is unknown. Aim: To compare the differentiation capacity of human CD105 + MSCs obtained from young and elderly donors. Methods and results: Cells were obtained from young (n = 10, 24 T 6.4 years) and elderly (n = 9, 77 T 8.4 years) donors. Cell senescence was assessed by telomere length assays and lipofuscin accumulation. Cell pluripotentiality was analysed by adipogenic and osteogenic induction media, and myocyte phenotype was attempted with 5-azacytidine (5-AZ). Immunofluorescence, Western blot, transmission electron microscopy and fluo-4 confocal imaging were used to analyse the sarcomere, gap junctions and Ca 2+ dynamics. Cells obtained from young and elderly donors showed no significant differences in relative telomere length (40.1 T 6.4% and 40.3 T 3.6%, p = 0.9) and lipofuscin accumulation. Adipogenic and osteogenic potential of CD105 + MSCs was demonstrated. 5-AZ induced increased expression of sarcomeric proteins without complete sarcomere organization. Treated cells also showed increased presence of connexin-43 both in young and old donor-derived cells. Intercellular communications were verified by the observation of gap junctions and passage of Ca 2+ between neighbouring cells. Spontaneous Ca 2+ raises did not significantly increase after 5-AZ treatment in both age groups. Conclusion: Age does not influence the adipogenic and myogenic differentiation potential of human CD105 + MSCs.