Patterns of amino acid metabolism by proliferating human mesenchymal stem cells.

The nutritional requirements of stem cells have not been determined; in particular, the amino acid metabolism of stem cells is largely unknown. In this study, we investigated the amino acid metabolism of human mesenchymal stem cells (hMSCs), with focus on two questions: Which amino acids are consumed and/or secreted by hMSCs and at what rates? To answer these questions, hMSCs were cultured on tissue culture plastic and in a bioreactor, and their amino acid profile was analyzed. The results showed that the kinetics of hMSCs growth and amino acid metabolism were significantly higher for hMSCs in tissue culture plastic than in the bioreactor. Despite differences in culture conditions, 8 essential and 6 nonessential amino acids were consumed by hMSCs in both tissue culture plastic and bioreactor cultures. Glutamine was the most consumed amino acid with significantly higher rates than for any other amino acid. The metabolism of nonessential amino acids by hMSCs deviated significantly from that of other cell lines. The secretion of alanine, glycine, glutamate, and ornithine by hMSCs showed that there is a strong overflow metabolism that can be due to the high concentrations of amino acids provided in the medium. In addition, the data showed that there is a metabolic pattern for proliferating hMSCs, which can contribute to the design of medium without animal serum for stem cells. Further, this study shows how to implement amino acid rates and metabolic principles in three-dimensional stem cell biology.

Quantifying in vitro growth and metabolism kinetics of human mesenchymal stem cells using a mathematical model.

Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100 cells/cm2 than when seeding at 1000 cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17-32h, specific glucose consumption in the order of 1.25 x 10(-1) to 3.77 x 10(-1) pmol/cell/h, specific lactate production in the order of 2.48 x 10(-1) to 7.67 x 10(-1)pmol/cell/h, specific glutamine production in the order of 7.04 x 10(-3) to 2.27 pmol/cell/h, and specific glutamate production in the order of 4.87 x 10(-1) to 23.4 pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitro.

Growth, metabolism, and growth inhibitors of mesenchymal stem cells.

Most therapeutic applications of bone marrow stromal cells (MSCs), or mesenchymal stem cells, require expansion of these cells. This study aimed to obtain more information about human MSCs regarding their expansion characteristics: growth, metabolism, and growth inhibitors. In addition, the same expansion factors were examined for (model species) goat and rat MSCs to evaluate differences between MSCs of mammalian species. MSC proliferation, nutrient consumption, and metabolite production were determined for five donors per species. In addition, the growth inhibitory concentrations of lactate and ammonia (NH3) were established. Results showed that goat MSCs grew significantly faster than human and rat MSCs and that goat cells metabolized glucose more efficiently into energy (Ylac/glc=0.8) than human (Ylac/glc=2.0) and rat MSCs (Ylac/glc=1.9). In addition, human (qGlc= -9.2pmol cell(-1) day(-1) and rat MSCs (qGlc= -5.9pmol cell(-1) day(-1)) consumed more glucose than goat MSCs (qGlc= -2.6pmol cell(-1) day(-1)). Glutamine was shown not to be important as energy source for human, goat, and rat MSCs. Regarding growth inhibition by metabolites, rat MSCs were more sensitive to lactate and NH3 (growth inhibiting at 16mM lactate and at 1.9mM NH3) than goat (lactate: 28.4mM, NH3: 2.9mM) and human MSCs (lactate: 35.4mM, NH3: 2.4mM). Human MSCs did not lose their differentiation potential when their growth was inhibited by lactate or NH3.

Reversible aggregation of lysozyme in a biodegradable amphiphilic multiblock copolymer.

Lysozyme-loaded poly(ethylene glycol terephthalate)-poly(butylene terephthalate) (PEGT/PBT) films were prepared using a water-in-oil emulsification solvent evaporation method. Infrared spectroscopic analysis of the dried films indicated the presence of non-covalent lysozyme aggregates in the polymer matrix. The use of methanol to enhance the drying rate of the films increased the relative amount of aggregates. Surprisingly, quantitative in-vitro release of fully active, non-aggregated lysozyme was observed, indicating that lysozyme forms reversible aggregates during encapsulation in PEGT/PBT films.