title: Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.
abstract: The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.
authors.affiliation: Metanomics GmbH, Berlin, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Bordag
authors.affiliation: Biogen Idec Inc., Raleigh-Durham, North Carolina, United States of America.
authors.forename: Vijay
authors.initials: V
authors.lastname: Janakiraman
authors.affiliation: Metanomics GmbH, Berlin, Germany.
authors.forename: Jonny
authors.initials: J
authors.lastname: Nachtigall
authors.affiliation: Metanomics GmbH, Berlin, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: González Maldonado
authors.affiliation: Metanomics GmbH, Berlin, Germany.
authors.forename: Bianca
authors.initials: B
authors.lastname: Bethan
authors.affiliation: Metanomics Health GmbH, Berlin, Germany.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Laine
authors.affiliation: Metanomics GmbH, Berlin, Germany.
authors.forename: Elie
authors.initials: E
authors.lastname: Fux
mesh_terms: D000249:Adenosine Monophosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D016466:CHO Cells; D002244:Carbon / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods*; D002498:Centrifugation; D006224:Cricetinae; D003412:Cricetulus; D048168:Escherichia coli K12 / Q000302:isolation & purification*; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D018698:Glutamic Acid / Q000032:analysis; D008322:Mammals; D055442:Metabolome; D055432:Metabolomics / Q000379:methods; D012996:Solutions; D014618:Vacuum
chemical_list: D012996:Solutions; D018698:Glutamic Acid; D000249:Adenosine Monophosphate; D002244:Carbon; D000255:Adenosine Triphosphate