Effective quenching processes for physiologically valid metabolite profiling of suspension cultured Mammalian cells.
Global metabolite analysis approaches, coupled with sophisticated data analysis and modeling procedures (metabolomics), permit a dynamic read-out of how cellular proteins interact with cellular and environmental conditions to determine cell status. This type of approach has profound potential for understanding, and subsequently manipulating, the regulation of cell function. As part of our study to define the regulatory events that may be used to maximize production of commercially valuable recombinant proteins from cultured mammalian cells, we have optimized the quenching process to allow retention of physiologically relevant intracellular metabolite profiles in samples from recombinant Chinese hamster ovary (CHO) cells. In a comparison of a series of candidate quenching procedures, we have shown that quenching in 60% methanol supplemented with 0.85% ammonium bicarbonate (AMBIC) at -40 degrees C generates a profile of metabolites that is representative of a physiological status based upon examination of key labile cellular metabolites. This represents a key feature for any metabolomic study with suspension cultured mammalian cells and provides confidence in the validity of subsequent data analysis and modeling procedures.
Sellick
Christopher A
CA
Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M139PT, UK. christopher.sellick@manchester.ac.uk
Hansen
Rasmus
R
Maqsood
Arfa R
AR
Dunn
Warwick B
WB
Stephens
Gillian M
GM
Goodacre
Royston
R
Dickson
Alan J
AJ
D000227:Adenine Nucleotides / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000737:chemistry; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D008401:Gas Chromatography-Mass Spectrometry; D019298:Glucose-6-Phosphate / Q000032:analysis / Q000378:metabolism; D055432:Metabolomics / Q000379:methods*; D000432:Methanol / Q000737:chemistry; D009243:NAD / Q000032:analysis / Q000378:metabolism; D051381:Rats
D000227:Adenine Nucleotides; D001639:Bicarbonates; D009243:NAD; C027043:ammonium bicarbonate; D019298:Glucose-6-Phosphate; D000432:Methanol