Unraveling the Extracellular Metabolism of Immortalized Hippocampal Neurons Under Normal Growth Conditions

Abstract

Metabolomic profiling of cell lines has shown many potential applications and advantages compared to animal models and human subjects, and an accurate cellular metabolite analysis is critical to understanding both the intracellular and extracellular environments in cell culture. This study provides a fast protocol to investigate in vitro metabolites of immortalized hippocampal neurons HN9.10e with minimal perturbation of the cell system using a targeted approach. HN9.10e neurons represent a reliable model of one of the most vulnerable regions of the central nervous system. Here, the assessment of their extracellular metabolic profile was performed by studying the cell culture medium before and after cell growth under standard conditions. The targeted analysis was performed by a direct, easy, high-throughput reversed-phase liquid chromatography with diode array detector (RP-HPLC-DAD) method and by headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS) for the study of volatile organic compounds (VOCs). The analysis of six different batches of cells has allowed to investigate the metabolic reproducibility of neuronal cells and to describe the metabolic “starting” conditions that are mandatory for a well-grounded interpretation of the results of any following cellular treatment. An accurate study of the metabolic profile of the HN9.10e cell line has never been performed before, and it could represent a quality parameter before any other targeting assay or further exploration.

Liquid chromatography coupled to high-resolution mass spectrometry metabolomics: A useful tool for investigating tumor secretome based on a three-dimensional co-culture model

“…The analysis of ERM provides a picture of the metabolites resulting from the exchange carried out between the cells and the culture medium. This approach has the following advantages: ensuring little (or non-existent) handling of cells, which avoids the production of artifacts, allowing the monitoring of metabolic activity in response to experimental disturbances without cell disruption, enabling the monitoring of metabolic changes over time within the same culture and avoid carrying out long and multiple extraction procedures, which also enable the production of technical artifacts that can lead to concealment or unwanted manipulation of biological results [ 24 ]. However, the disadvantages of working with the secretome include matrix effects related to the salty media composition, which is rich in sugars, lipids, proteins, and water and might interfere in the analysis.…”