Metabolic Analysis of Fibroblast Conditioned Media and Comparison with Theoretical Modeling
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 33122
Metabolic Analysis of Fibroblast Conditioned Media and Comparison with Theoretical Modeling

Authors: Priyanka Gupta, Paul Verma, Kerry Hourigan, Jayesh Bellare, Sameer Jadhav

Abstract:

Understanding the consumption and production of various metabolites of fibroblast conditioned media is needed for its proper and optimized use in expansion of pluripotent stem cells. For this purpose, we have used the HPLC method to analyse the consumption of glucose and the production of lactate over time by mouse embryonic fibroblasts. The experimental data have also been compared with mathematical model fits. 0.025 moles of lactate was produced after 72 hrs while the glucose concentration decreased from 0.017 moles to 0.011 moles. The mathematical model was able to predict the trends of glucose consumption and lactate production.

Keywords: Conditioned media, HPLC, metabolite analysis, mouse embryonic fibroblast.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1328210

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 2619

References:


[1] A. B. Choo, J. Padmanabhan, A. C. Chin, and S. K. Oh, "Expansion of pluripotent human embryonic stem cells on human feeders," Biotechnology and Bioengineering, vol. 88, pp. 321-31, Nov 5 2004.
[2] A. G. Smith, J. K. Heath, D. D. Donaldson, G. G. Wong, J. Moreau, M. Stahl, and D. Rogers, "Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides," Nature, vol. 336, pp. 688-90, Dec 15 1988.
[3] R. L. Williams, D. J. Hilton, S. Pease, T. A. Willson, C. L. Stewart, D. P. Gearing, E. F. Wagner, D. Metcalf, N. A. Nicola, and N. M. Gough, "Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells," Nature, vol. 336, pp. 684-7, Dec 15 1988.
[4] C. Xu, M. S. Inokuma, J. Denham, K. Golds, P. Kundu, J. D. Gold, and M. K. Carpenter, "Feeder-free growth of undifferentiated human embryonic stem cells," Nature biotechnology, vol. 19, pp. 971-4, Oct 2001.
[5] M. Amit, C. Shariki, V. Margulets, and J. Itskovitz-Eldor, "Feeder layerand serum-free culture of human embryonic stem cells," Biology of reproduction, vol. 70, pp. 837-45, Mar 2004.
[6] R. H. Xu, R. M. Peck, D. S. Li, X. Feng, T. Ludwig, and J. A. Thomson, "Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells," Nature methods, vol. 2, pp. 185-90, Mar 2005.
[7] A. B. Prowse, L. R. McQuade, K. J. Bryant, H. Marcal, and P. P. Gray, "Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media," Journal of proteome research, vol. 6, pp. 3796-807, Sep 2007.
[8] H. Eagle, S. Barban, M. Levy, and H. O. Schulze, "The utilization of carbohydrates by human cell cultures," The Journal of biological chemistry, vol. 233, pp. 551-8, Sep 1958.
[9] S. S. Ozturk, M. R. Riley, and B. O. Palsson, "Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production," Biotechnology and Bioengineering vol. 39, pp. 418-31, Feb 20 1992.
[10] T. Hassell, S. Gleave, and M. Butler, "Growth inhibition in animal cell culture. The effect of lactate and ammonia," Applied biochemistry and biotechnology, vol. 30, pp. 29-41, Jul 1991.
[11] S. D. Patel, E. T. Papoutsakis, J. N. Winter, and W. M. Miller, "The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures," Biotechnology progress, vol. 16, pp. 885-92, Sep-Oct 2000.
[12] A. Ouyang, R. Ng, and S. T. Yang, "Long-term culturing of undifferentiated embryonic stem cells in conditioned media and threedimensional fibrous matrices without extracellular matrix coating," Stem cells, vol. 25, pp. 447-54, Feb 2007.
[13] D. A. MacIntyre, D. Melguizo Sanchis, B. Jimenez, R. Moreno, M. Stojkovic, and A. Pineda-Lucena, "Characterisation of human embryonic stem cells conditioning media by 1H-nuclear magnetic resonance spectroscopy," PloS one, vol. 6, p. e16732, 2011.
[14] H. J. Cruz, J. L. Moreira, and M. J. Carrondo, "Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells," Biotechnology and Bioengineering, vol. 66, pp. 104-13, 1999.
[15] G. Higuera, D. Schop, F. Janssen, R. van Dijkhuizen-Radersma, T. van Boxtel, and C. A. van Blitterswijk, "Quantifying in vitro growth and metabolism kinetics of human mesenchymal stem cells using a mathematical model," Tissue engineering. Part A, vol. 15, pp. 2653-63, Sep 2009.
[16] L. Mohler, A. Bock, and U. Reichl, "Segregated mathematical model for growth of anchorage-dependent MDCK cells in microcarrier culture," Biotechnology progress, vol. 24, pp. 110-9, Jan-Feb 2008.
[17] A. Franchi, P. Silvestre, and J. Pouyssegur, ""Carrier activation" and glucose transport in Chinese hamster fibroblasts," Biochemical and biophysical research communications, vol. 85, pp. 1526-34, Dec 29 1978.