Abstract
Metabolomics is an emerging field of cell biology that aims at the comprehensive identification of metabolite levels in biological fluids or cells in a specific functional state. Currently, the major tools for determining metabolite concentrations are mass spectrometry coupled with chromatographic techniques and nuclear magnetic resonance, which are expensive, time consuming and destructive for the samples. Here, we report a time resolved approach to monitor metabolite dynamics in cell cultures, based on Surface Enhanced Raman Scattering (SERS). This method is label-free, easy to use and provides the opportunity to simultaneously study a broad range of molecules, without the need to process the biological samples. As proof of concept, NIH/3T3 cells were cultured in vitro, and the extracellular medium was collected at different time points to be analyzed with our engineered SERS substrates. By identifying individual peaks of the Raman spectra, we showed the simultaneous detection of several components of the conditioned medium, such as L-tyrosine, L-tryptophan, glycine, L-phenylalanine, L-histidine and fetal bovine serum proteins, as well as their intensity changes during time. Furthermore, analyzing the whole Raman data set with the Principal Component Analysis (PCA), we demonstrated that the Raman spectra collected at different days of culture and clustered by similarity, described a well-defined trajectory in the principal component plot. This approach was then utilized to determine indirectly the functional state of the macrophage cell line Raw 264.7, stimulated with the lipopolysaccharide (LPS) for 24 hours. The collected spectra at different time points, clustered by the PCA analysis, followed a well-defined trajectory, corresponding to the functional change of cells toward the activated pro-inflammatory state induced by the LPS. This study suggests that our engineered SERS surfaces can be used as a versatile tool both for the characterization of cell culture conditions and the functional state of cells over time.
Highlights
Metabolomics represents the global quantitative analysis of all metabolites in a biological system [1,2,3]
After 1, 2, 3 and 4 days in vitro (DIV), the conditioned medium was collected from each dish, centrifuged to avoid dead cells and cell debris, and transferred into a sterile Petri dish with Surface Enhanced Raman Scattering (SERS) Ag nanostructured substrates for Raman spectroscopy
In addition to the SERS characterization of cell culture conditions, we investigated whether our strategy could be applied for an indirect monitoring of the functional state of cells in culture over time
Summary
Metabolomics represents the global quantitative analysis of all metabolites in a biological system [1,2,3]. Different biological molecules, such as amino acids, sugars, proteins and salts, are required at a well-defined concentrations in order to allow specific metabolic processes, guaranteeing the optimal viability, as well as specific fate and functions of cells [7,8,9]. Wrong or poorly optimized feeding strategies may result in detrimental reduction of nutrients, accumulation of catabolites, lowering in cell growth rate, and functional changes of cells. These events, sometimes hardly identifiable, may result in misleading or inconsistent experiments. Robust tools able to properly monitor cell culture conditions over time are strongly needed, in order to optimize culture strategies and to describe possible functional changes of cells
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