Development of an optimized method for processing peripheral blood mononuclear cells for 1H-nuclear magnetic resonancebased metabolomic profiling

Abstract

ABSTRACT: Human peripheral blood mononuclear cells (PBMCs) are part of the innate and adaptive immune system, and form a critical interface between both systems. Studying the metabolic profile of PBMC could provide valuable information about the response to pathogens, toxins or cancer, the detection of drug toxicity, in drug discovery and cell replacement therapy. The primary purpose of this study was to develop an improved processing method for PBMCs metabolomic profiling with nuclear magnetic resonance (NMR) spectroscopy. To this end, an experimental design was applied to develop an alternative method to process PBMCs at low concentrations. The design included the isolation of PBMCs from the whole blood of four different volunteers, of whom 27 cell samples were processed by two different techniques for quenching and extraction of metabolites: a traditional one using organic solvents and an alternative one employing a high-intensity ultrasound probe, the latter with a variation that includes the use of deproteinizing filters. Finally, all the samples were characterized by 1H-NMR and the metabolomic profiles were compared by the method. As a result, two new methods for PBMCs processing, called Ultrasound Method (UM) and Ultrasound and Ultrafiltration Method (UUM), are described and compared to the Folch Method (FM), which is the standard protocol for extracting metabolites from cell samples. We found that UM and UUM were superior to FM in terms of sensitivity, processing time, spectrum quality, amount of identifiable, quantifiable metabolites and reproducibility.