Raman and infrared spectroscopy reveal that proliferating and quiescent human fibroblast cells age by biochemically similar but not identical processes

Abstract

Dermal fibroblast cells can adopt different cell states such as proliferation, quiescence, apoptosis or senescence, in order to ensure tissue homeostasis. Proliferating (dividing) cells pass through the phases of the cell cycle, while quiescent and senescent cells exist in a non-proliferating cell cycle-arrested state. However, the reversible quiescence state is in contrast to the irreversible senescence state. Long-term quiescent cells transit into senescence indicating that cells age also when not passing through the cell cycle. Here, by label-free in vitro vibrational spectroscopy, we studied the biomolecular composition of quiescent dermal fibroblast cells and compared them with those of proliferating and senescent cells. Spectra were examined by multivariate statistical analysis using a PLS-LDA classification model, revealing differences in the biomolecular composition between the cell states mainly associated with protein alterations (variations in the side chain residues of amino acids and protein secondary structure), but also within nucleic acids and lipids. We observed spectral changes in quiescent compared to proliferating cells, which increased with quiescence cultivation time. Raman and infrared spectroscopy, which yield complementary biochemical information, clearly distinguished contact-inhibited from serum-starved quiescent cells. Furthermore, the spectra displayed spectral differences between quiescent cells and proliferating cells, which had recovered from quiescence. This became more distinct with increasing quiescence cultivation time. When comparing proliferating, (in particular long-term) quiescent and senescent cells, we found that Raman as well as infrared spectroscopy can separate these three cellular states from each other due to differences in their biomolecular composition. Our spectroscopic analysis shows that proliferating and quiescent fibroblast cells age by similar but biochemically not identical processes. Despite their aging induced changes, over long time periods quiescent cells can return into the cell cycle. Finally however, the cell cycle arrest becomes irreversible indicating senescence.

Discrimination of Dental Pulp Stem Cell Regenerative Heterogeneity by Single-Cell Raman Spectroscopy

“…[16][17][18][21][22][23] However, concurrent with its successful utilisation in the present study to discriminate between high proliferative/multi-potent and low proliferative/uni-potent DPSCs, Raman Spectroscopy has recently been shown to distinguish between proliferative/non-proliferative, senescent/non-senescent, young/aged and viable/non-viable cells, within other MSC and fibroblast populations. 15,[29][30][31][32][33] The increased spectral intensities obtained for high proliferative/multi-potent DPSC subpopulation, A3 (18PDs), compared to more senescent A3 (60PDs) and to low proliferative/unipotent DPSCs, A1 and B1 (8PDs and 7PDs, respectively), are consistent with recent studies reporting alterations in the biomolecular compositions of senescent fibroblast populations, versus non-senescent counterparts; relating to band intensities and/or band positions. [31][32] Specifically, such studies have associated reductions in DNA (788, 1580 cm -1 ) and protein (1658 cm -1 ) peaks, in addition to elevated lipid (1732, 2850 and 2930 cm -1 ) peak contents during cellular senescence, with protein and lipid modifications predominating overall.…”

A guide to assessing cellular senescence in vitro and in vivo

“…Newer techniques able to analyse and detect other biochemical changes, such as the formation and breakage of chemical bonds, are also gaining attention in the field. For instance, vibrational spectroscopy techniques, such as Raman and infrared (IR) spectroscopy [136], have been applied to effectively differentiate senescent cells from their nonsenescent counterparts [137,138]. By scanning through a wide range of wavelengths and analysing the scattered light profiles, the biochemical composition of a cell can be mapped, which can be used to differentiate cell types and cellular states.…”

Raman and Atr-Ftir Spectroscopy Towards Classification of Wet Blue Bovine Leather Using Ratiometric and Chemometric Analysis

“…It summarises correct and incorrect spectra classification. It is useful for two-class classification and in measuring recall, precision and accuracy [18,51,52]. The confusion matrix for Raman and IR data obtained is presented in Tables 4 and 5.…”

“…Sample preparation is relatively simple as compared to other analytical techniques, such as high-performance liquid chromatography (HPLC) and colorimetric methods [18][19][20][21][22]. Finding the variations in the initial leather processing steps reduces the costs of down-stream processing.…”