Effective Quenching Processes for Physiologically Valid Metabolite Profiling of Suspension Cultured Mammalian Cells

Abstract

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.

Fast filtration for metabolome sampling of suspended animal cells

“…The average amount of residual amino acid in the filter was calculated to be 0.19 % ± 0.01 % of the total amino acid amount in the cell suspension used with a maximum for glycine of 0.22 % ± 0.04 %. These values are in accordance to those 8 presented by Sellick et al (2009) for direct quenching in methanol with an additional washing step.…”

“…Validation of fast filtration as means of metabolome sampling A microstructure heat exchanger method proposed by Wiendahl et al (2007) and the use of methanol/ammonium bicarbonate as proposed by Sellick et al (2009) have been used for quenching animal cell suspensions to 0 °C in the sub-second scale, but residual metabolic activity still occurs below 0 °C (Sauter 2003). Therefore it is key to authentic metabolome sampling of 7 animal cells to not only cool the cells down as fast as possible but also to keep overall sampling time to a minimum.…”

“…Unlike for bacteria and yeast (Bolten and Wittmann 2008;Faijes et al 2007;Schaub et al 2006;Villas-Bôas et al 2005), there are no standard protocols for sampling and extraction of suspended animal cells. A number of different rapid sampling methods are currently used (Sellick et al 2009;Wiendahl et al 2007;Dietmair et al 2010), but have not been compared to other methods in depth.…”

“…A standard quenching method for bacteria and yeast is cold methanol, though other protocols using a microstructure heat exchanger 3 or fast filtration are also applied (Wellerdiek et al 2009;Bolten and Wittmann 2008). Dietmair et al (2010) showed that using methanol, as proposed by Sellick et al (2009), causes increased cell permeabilization and leakage of intracellular components. Thus, alternative methods for the quenching of suspended animal cells are required.…”

“…A number of different extraction methods are currently employed for bacteria and yeast as well as for animal cells using boiling methanol, chloroform/methanol and acidic extractions with acidic acetonitrile/methanol or acetic acid. For suspended animal cells methanol is most commonly used as extraction agent (Ritter et al 2008;Bennett et al 2008;Sellick et al 2009;Dietmair et al 2010).…”