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

Beatrice Campanella,Riccardo Nieri,Massimo Onor,Edoardo Benedetti,Laura Colombaioni,Emilia Bramanti

doi:10.3389/fchem.2021.621548

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.

Highlights

Biomedical research, from drug development to biotechnological production, involves the employment of cell line cultures (León et al, 2013)
We propose the study of the extracellular metabolites of living immortalized hippocampal neuron (HN9.10e cell line) cell cultures in six replicated independent experiments by analyzing the cell-free culture medium (CFCM) and the cell culture medium (CCM) by a direct, easy, highthroughput reversed-phase liquid chromatography with diode array detector (RP-HPLC-DAD) method
This study aims to face with the metabolic reproducibility of neuronal cells and to describe the metabolic “starting” conditions for any experiment based on the cell culture model

Summary

Introduction

Biomedical research, from drug development to biotechnological production, involves the employment of cell line cultures (León et al, 2013). Cultured cell models in metabolomics are widespread employed in many areas of medical research as a valuable alternative to the use of animals in toxicology testing with benefits in term of a greater control of external variables and no ethical problems. Standard procedures for cultured mammalian cell metabolomics are researched worldwide (Hayton et al, 2017; Ivanisevic and Want, 2019; Villaret-Cazadamont et al, 2020), and many authors claim in their concluding comments that standardized procedures are still needed (Hayton et al, 2017; Hirsch and Schildknecht, 2019). Mammalian cells suffer from high variability, which has to be controlled and investigated in order to get reliable and reproducible results once the cell cultures undergo specific treatments (Wright Muelas et al, 2018). The metabolic performance of the cell lines can be a prime cause of irreproducibility of experiment outcome

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