Metabolic Analysis of Fibroblast Conditioned Media and Comparison with Theoretical Modeling
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.
[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.
[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.
@article{"International Journal of Biological, Life and Agricultural Sciences:49509", author = "Priyanka Gupta and Paul Verma and Kerry Hourigan and Jayesh Bellare and Sameer Jadhav", title = "Metabolic Analysis of Fibroblast Conditioned Media and Comparison with Theoretical Modeling", 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.", volume = "6", number = "12", pages = "1055-5", }
