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.

Keywords: GC-MS; HPLC-DAD; metabolomics; primary cultured hippocampal neurons; reproducibility.

FIGURE 1

Representative absorbance chromatogram at 220 nm of the CFCM and CCM after 4 days of HN9.10e cell line culturing (V_inj = 5 μL). The chromatogram of the blank has been subtracted. 1 = L-histidine; 2 = L-threonine/L-glutamic acid; 3 = L-glutamine; 4 = oxalic acid; 5 = L-cysteine; 6 = glycolic acid; 7 = formic acid; 8 = NAD⁺; 9 = pyruvate; 10 = lactic acid; 11 = acetic acid; 12 = dopamine; 13 = citrate; 14 = L-methionine; 15 = fumaric acid; 16 = succinic acid; 17 = acetoacetic acid; 18 = L-tyrosine; 19 = L-leucine; 20 = propionic acid; 21 = L-phenylalanine; 22 = isobutyric acid; 23 = butyric acid; 24 = L-tryptophane.

FIGURE 2

Boxplots of RP-HPLC-DAD autoscaled and mean-centered data, showing the differences between metabolites determined by RP-HPLC-DAD in the CFCM and CCM (ns p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

FIGURE 3

Boxplots showing the differences between volatile metabolites determined by HS-GC-MS in the CCM before and after 4 days of HN9.10e cell line culturing (ns p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

FIGURE 4

Schematic representation of the HN9.10e cell line metabolism hypothesized on the base of the analysis of CCM. The key metabolites that significantly increase or decrease are shown and are represented inside the neuron. Their finding in the CCM supposes specific or aspecific transport outside the cells. BCATs, branched chain amino acids; BCKAs, branched chain α-keto acids; DNL, de novo lipogenesis; yellow box, decreased concentration; green box, increased concentration; red pathways, if excess of acetyl-CoA occurs; brown pathways, if deficit of NAD⁺ and acetyl-CoA occurs.