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Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling

Abstract

Metabolite profiling of industrially important suspension-cultured mammalian cells is being increasingly used for rational improvement of bioprocesses. This requires the generation of global metabolite profiles that cover a broad range of metabolites and that are representative of the cells at the time of sampling. The protocol described here is a validated method for recovery of physiologically relevant amounts of key metabolites from suspension-cultured mammalian cells. The method is a two-step process consisting of initial quenching of the cells (to stop cellular metabolism and allow isolation of the cells) followed by extraction of the metabolites. The cells are quenched in 60% methanol supplemented with 0.85% (wt/vol) ammonium bicarbonate at −40 °C. Metabolites are then extracted from the quenched cells using two 100% methanol extractions followed by a single water extraction. Metabolite samples generated using this protocol are amenable to analysis by mass spectrometry–based techniques (e.g., gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry), NMR spectroscopy and enzymatic assays.

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Figure 1: Overview of protocol for metabolite extraction from suspension-cultured mammalian cells.
Figure 2: Effects of different quenching methods on yields of key intracellular metabolites.
Figure 3: Protocol for quenching of suspension-cultured mammalian cells.
Figure 4: Protocol for the extraction of metabolites from quenched cells.
Figure 5: Metabolite yields of key labile metabolites from quenched CHO cells compared with unquenched CHO cells.
Figure 6: Example of a raw GC-MS chromatogram of intracellular metabolites recovered from CHO cells in exponential growth phase using this protocol.

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Acknowledgements

We gratefully acknowledge the financial support of the UK Biotechnology and Biological Sciences Research Council, the UK Engineering and Physical Sciences Research Council and industrial members of the Bioprocessing Research Industry Club. We also thank A. Croxford, S. Anson and D. Knight for helpful discussions.

AUTHOR CONTRIBUTIONS

All authors discussed the steps of the protocol, its implications and applications. C.A.S. wrote the manuscript and R.H., G.M.S., R.G. and A.J.D. revised it.

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Correspondence to Alan J Dickson.

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Sellick, C., Hansen, R., Stephens, G. et al. Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling. Nat Protoc 6, 1241–1249 (2011). https://doi.org/10.1038/nprot.2011.366

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