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.

[1]  K. K. Frame,et al.  A model for density-dependent growth of anchorage-dependent mammalian cells. , 1988, Biotechnology and bioengineering.

[2]  B. Bode.  Recent molecular advances in mammalian glutamine transport. , 2001, The Journal of nutrition.

[3]  D. Prockop,et al.  The Wnt Signaling Inhibitor Dickkopf-1 Is Required for Reentry into the Cell Cycle of Human Adult Stem Cells from Bone Marrow* , 2003, Journal of Biological Chemistry.

[4]  J. Neu,et al.  Glutamine nutrition and metabolism: Where do we go from here? , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[5]  B. Larson,et al.  Human Multipotent Stromal Cells Undergo Sharp Transition from Division to Development in Culture , 2008, Stem cells.

[6]  H J Cruz,et al.  Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells. , 1999, Biotechnology and bioengineering.

[7]  Greg Lemon,et al.  Mathematical modelling of human mesenchymal stem cell proliferation and differentiation inside artificial porous scaffolds. , 2007, Journal of theoretical biology.

[8]  Feng Zhao,et al.  Effects of Oxygen Transport on 3‐D Human Mesenchymal Stem Cell Metabolic Activity in Perfusion and Static Cultures: Experiments and Mathematical Model , 2008, Biotechnology progress.

[9]  Michael W. Glacken,et al.  Catabolic Control of Mammalian Cell Culture , 1988, Bio/Technology.

[10]  S. Bruder,et al.  Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. , 2001, Trends in molecular medicine.

[11]  G. Moore,et al.  Spontaneous decomposition of glutamine in cell culture media. , 1962, Experimental cell research.

[12]  D J Prockop,et al.  Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[14]  I. Sekiya,et al.  Expansion of Human Adult Stem Cells from Bone Marrow Stroma: Conditions that Maximize the Yields of Early Progenitors and Evaluate Their Quality , 2002, Stem cells.

[15]  Udo Reichl,et al.  Segregated Mathematical Model for Growth of Anchorage‐Dependent MDCK Cells in Microcarrier Culture , 2008, Biotechnology progress.

[16]  S. Perez,et al.  Characterization of the Optimal Culture Conditions for Clinical Scale Production of Human Mesenchymal Stem Cells , 2006, Stem cells.

[17]  D J Prockop,et al.  Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. D. de Bruijn,et al.  Expansion of mesenchymal stem cells using a microcarrier‐based cultivation system: growth and metabolism , 2008, Journal of tissue engineering and regenerative medicine.

[19]  P. Rich.  The molecular machinery of Keilin's respiratory chain. , 2003, Biochemical Society transactions.

[20]  E. Pistikopoulos,et al.  Modeling Amino Acid Metabolism in Mammalian Cells‐Toward the Development of a Model Library , 2007, Biotechnology progress.

[21]  S. P. Asprey,et al.  Modelling of Mammalian Cells and Cell Culture Processes , 2004, Cytotechnology.

[22]  J. Masters,et al.  Changing medium and passaging cell lines , 2007, Nature Protocols.

[23]  Clemens A van Blitterswijk,et al.  A rapid and efficient method for expansion of human mesenchymal stem cells. , 2007, Tissue engineering.

[24]  D. Prockop.  Marrow Stromal Cells as Stem Cells for Nonhematopoietic Tissues , 1997, Science.

[25]  A. Braccini,et al.  The osteogenicity of implanted engineered bone constructs is related to the density of clonogenic bone marrow stromal cells , 2007, Journal of tissue engineering and regenerative medicine.

[26]  P. Genever,et al.  Glutamate signalling in non-neuronal tissues. , 2001, Trends in pharmacological sciences.