13C-Isotope-Assisted Assessment of Metabolic Quenching During Sample Collection from Suspension Cell Cultures.

Abstract

Metabolomics and fluxomics are core approaches to directly profile and interrogate cellular metabolism in response to various genetic or environmental perturbations. In order to accurately measure the abundance and isotope enrichment of intracellular metabolites, cell culture samples must be rapidly harvested and cold quenched to preserve the in vivo metabolic state of the cells at the time of sample collection. When dealing with suspension cultures, this process is complicated by the need to separate the liquid culture media from cellular biomass prior to metabolite extraction. Here, we examine the efficacy of several commonly used metabolic quenching methods, using the model cyanobacterium Synechocystis sp. PCC 6803 as an example. Multiple 13C-labeled compounds, including 13C-bicarbonate, 13C-glucose, and 13C-glutamine, were used as tracers during the sample collection and the cold-quenching process to assess the extent of metabolic turnover after cells were harvested from culture flasks. We show that the combination of rapid filtration followed by 100% cold (-80 °C) methanol quenching exhibits the highest quenching efficiency, while mixing cell samples with a partially frozen 30% methanol slurry (-24 °C) followed by centrifugation is slightly less effective at quenching metabolism but enables less laborious sample processing. By contrast, rapidly mixing the cells with a saline ice slurry (∼0 °C) is less effective, as indicated by high isotope-labeling rates after sample harvest, while mixing the cells with 60% cold methanol (-65 °C) prior to centrifugation causes significant metabolite loss. This study demonstrates a rigorous, quantitative, and broadly applicable method for assessing the metabolic quenching efficacy of protocols used for sample collection in metabolomics and fluxomics studies.