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Published online 6 May 2009 | Nature 459, 19 (2009) | doi:10.1038/459019a
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Neuroscientists claim growing pains
Manufacturer denies that common lab feed can cause some neuron cultures to fail.
Leading neuroscientists are warning that difficulties with a staple laboratory product may be costing time and money.
The scientists say that variation between batches of a growth medium designed to sustain neurons in culture can, in their experience, cause experiments to fail or give low-quality results because of the poor survival and maturation of cells.
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As neurons in vivo depend on the presence and availability of creatine, mainly synthesized in astrocytes and taken up by neurons via a specific creatine transporter (Braissant et al. 2005), it is hard to understand why commercial cell culture media for neuronal cell cultures still do not contain creatine as an essential ingredient for neuronal development and differentiation (Andres et al. 2008), especially fully defined synthetic media. Fetal calf serum contains differing amounts of creatine. Thus some of the variation in performance of some of such media that still contain fetal calf serum may be due to differences in their creatine content. On the other hand, synthetic media for neuronal cell culture lack creatine all together. While some creatine my be carried over from cell isolation or by astrocyte contaminants, I would thus strongly advise the manufactures of synthetic media for neuronal cell cultures to specifically recommend the addition of freshly prepared creatine to their media at a final concentration of 5-10 mM (unfortunately, creatine is not very stable in solution for extended periods of time due to a non-enzymatic reaction converting creatine into creatinine). I am convinced that one will see the difference in cell viability and differentiation by adding creatine to these media (Andres et al. 2007), since creatine is known to exert a strong neuroprotective effect against a variety of cellular stressors (Brewer and Wallimann 2000). In addition, creatine is known to exert direct and indirect anti-oxidant activity thus acting as a cell protector and anti-aging substance (Guidi et al. 2008). Theo Wallimann, PhD, Prof. emeritus ETH Zurich. References Andres RH, Ducray AD, Schlattner U, Wallimann T, Widmer HR. Functions and effects of creatine in the central nervous system. Brain Res Bull. 2008 Jul 1;76(4):329-43. Epub 2008 Mar 24. Ducray AD, Schläppi JA, Qualls R, Andres RH, Seiler RW, Schlattner U, Wallimann T, Widmer HR. Creatine treatment promotes differentiation of GABA-ergic neuronal precursors in cultured fetal rat spinal cord. J Neurosci Res. 2007 Jul;85(9):1863-75. Braissant O, Henry H, Villard AM, Speer O, Wallimann T, Bachmann C. Creatine synthesis and transport during rat embryogenesis: spatiotemporal expression of AGAT, GAMT and CT1. BMC Dev Biol. 2005 May 26;5(1):9. G. Brewer and Wallimann T. Protective effect of the energy precursor creatine against toxicity of glutamate and beta-amyloid in rat hippocampal neurons. J. Neurochem. 74: 1968-1978 (2000) Guidi C, Potenza L, Sestili P, Martinelli C, Guescini M, Stocchi L, Zeppa S, Polidori E, Annibalini G, Stocchi V. Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA. Biochim Biophys Acta. 2008 Jan;1780(1):16-26.
As neurons in vivo depend on the presence and availability of creatine, mainly synthesized in astrocytes and taken up by neurons via a specific creatine transporter (Braissant et al. 2005), it is hard to understand why commercial cell culture media for neuronal cell cultures still do not contain creatine as an essential ingredient for neuronal development and differentiation (Andres et al. 2008), especially fully defined synthetic media. Fetal calf serum contains differing amounts of creatine. Thus some of the variation in performance of some of such media that still contain fetal calf serum may be due to differences in their creatine content. On the other hand, synthetic media for neuronal cell culture lack creatine all together. While some creatine my be carried over from cell isolation or by astrocyte contaminants, I would thus strongly advise the manufactures of synthetic media for neuronal cell cultures to specifically recommend the addition of freshly prepared creatine to their media at a final concentration of 5-10 mM (unfortunately, creatine is not very stable in solution for extended periods of time due to a non-enzymatic reaction converting creatine into creatinine). I am convinced that one will see the difference in cell viability and differentiation by adding creatine to these media (Andres et al. 2007), since creatine is known to exert a strong neuroprotective effect against a variety of cellular stressors (Brewer and Wallimann 2000). In addition, creatine is known to exert direct and indirect anti-oxidant activity thus acting as a cell protector and anti-aging substance (Guidi et al. 2008). Theo Wallimann, PhD, Prof. emeritus ETH Zurich. References: Andres RH, Ducray AD, Schlattner U, Wallimann T, Widmer HR. Functions and effects of creatine in the central nervous system. Brain Res Bull. 2008 Jul 1;76(4):329-43. Epub 2008 Mar 24. Ducray AD, Schläppi JA, Qualls R, Andres RH, Seiler RW, Schlattner U, Wallimann T, Widmer HR. Creatine treatment promotes differentiation of GABA-ergic neuronal precursors in cultured fetal rat spinal cord. J Neurosci Res. 2007 Jul;85(9):1863-75. Braissant O, Henry H, Villard AM, Speer O, Wallimann T, Bachmann C. Creatine synthesis and transport during rat embryogenesis: spatiotemporal expression of AGAT, GAMT and CT1. BMC Dev Biol. 2005 May 26;5(1):9. G. Brewer and Wallimann T. Protective effect of the energy precursor creatine against toxicity of glutamate and beta-amyloid in rat hippocampal neurons. J. Neurochem. 74: 1968-1978 (2000) Guidi C, Potenza L, Sestili P, Martinelli C, Guescini M, Stocchi L, Zeppa S, Polidori E, Annibalini G, Stocchi V. Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA. Biochim Biophys Acta. 2008 Jan;1780(1):16-26.