title: Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction.
abstract: In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.
authors.affiliation: ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Hernández Bort
authors.affiliation: ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Vinoth
authors.initials: V
authors.lastname: Shanmukam
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Windwarder
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Laura
authors.initials: L
authors.lastname: Neumann
authors.affiliation: Biocrates Life Sciences, Innsbruck, Austria.
authors.forename: Ali
authors.initials: A
authors.lastname: Alchalabi
authors.affiliation: Biocrates Life Sciences, Innsbruck, Austria.
authors.forename: Guido
authors.initials: G
authors.lastname: Krebiehl
authors.affiliation: ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Gunda
authors.initials: G
authors.lastname: Koellensperger
authors.affiliation: ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hann
authors.affiliation: Biocrates Life Sciences, Innsbruck, Austria.
authors.forename: Denise
authors.initials: D
authors.lastname: Sonntag
authors.affiliation: ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Sandoz GmbH, Tirol, Austria.
authors.forename: Christine
authors.initials: C
authors.lastname: Heel
authors.affiliation: ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: nicole.borth@boku.ac.at.
authors.forename: Nicole
authors.initials: N
authors.lastname: Borth
mesh_terms: D000818:Animals; D001120:Arginine / Q000032:analysis; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D042541:Intracellular Space / Q000737:chemistry; D055432:Metabolomics / Q000295:instrumentation / Q000379:methods*; D013594:Syringes; D014364:Tryptophan / Q000032:analysis
chemical_list: D014364:Tryptophan; D001120:Arginine
keywords: CHO cells; Fast filtration; Metabolite extraction; Metabolomics; Quenching