The Long-Term Culture of Human Fibroblasts Reveals a Spectroscopic Signature of Senescence

Abstract

Aging is a complex process which leads to progressive loss of fitness/capability/ability, increasing susceptibility to disease and, ultimately, death. Regardless of the organism, there are some features common to aging, namely, the loss of proteostasis and cell senescence. Mammalian cell lines have been used as models to study the aging process, in particular, cell senescence. Thus, the aim of this study was to characterize the senescence-associated metabolic profile of a long-term culture of human fibroblasts using Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopy. We sub-cultivated fibroblasts from a newborn donor from passage 4 to passage 17 and the results showed deep changes in the spectroscopic profile of cells over time. Late passage cells were characterized by a decrease in the length of fatty acid chains, triglycerides and cholesterol and an increase in lipid unsaturation. We also found an increase in the content of intermolecular β-sheets, possibly indicating an increase in protein aggregation levels in cells of later passages. Metabolic profiling by NMR showed increased levels of extracellular lactate, phosphocholine and glycine in cells at later passages. This study suggests that spectroscopy approaches can be successfully used to study changes concomitant with cell senescence and validate the use of human fibroblasts as a model to monitor the aging process.

FTIR Spectroscopy and Blood-Derived Extracellular Vesicles Duo in Alzheimer’s Disease

“…The Amide I region was thus further analyzed in serum- and plasma-derived EVs spectra from Controls and AD cases. For AD cases, while an increase in intermolecular β-sheets was found, a decrease in antiparallel β-sheets was evident; supporting an increase in protein aggregation towards a decrease in oligomeric structures [ 53 ]. In agreement with our FTIR spectra observations for bdEVs, altered patterns in the Amide I region were also reported for human blood, blood peripheral mononuclear leukocytes or CSF, particularly an increase in peaks intensity assigned to β-sheets in AD cases [ 18, 23–25, 54 ].…”

Age-Related Fourier-Transform Infrared Spectroscopic Changes in Protein Conformation in an Aging Model of Human Dermal Fibroblasts

“…Another metabolomic study using nuclear magnetic resonance (NMR) spectroscopy additionally identified a decrease in phosphocholine, ADP and ATP levels [18]. Furthermore, a previous study in our group revealed that the aging process can be studied using human fibroblasts from a newborn subcultured from passage 3 until passage 17, and, using FTIR spectroscopy, there were detected changes in protein conformation, namely, an increased antiparallel β-sheet conformation and a decrease in proteins in intermolecular β-sheet conformation, as well as a decrease in fibril formation [22]. According to aging studies using fibroblasts from donors of different ages, as far as we are concerned, there are still few studies that are focused on metabolic approaches or changes in protein conformation rather than genetic alterations [17,23].…”

“…Cells were detached from the flask and centrifuged for 3 min at 1000 rpm at room temperature (RT). After that, the supernatant was discarded and the pellet was resuspended in complete cell culture medium and plated into new flasks [22]. At passage 12, cells were counted and cell viability was assessed using trypan blue, as previously described [24].…”

“…The supernatant was discarded, and the pellet was kept at −80 • C until FTIR analysis. The number of cells used for FTIR was based on a previous study using human fibroblasts and FTIR spectroscopy [22].…”

“…One can use fibroblasts that are collected from the same donor, usually a young donor, and age them in vitro until cells reach senescence, so that the sequential cell passages can be analyzed and compared. In this case, there is less heterogeneity of results, as the cells are from the same donor and there is less influence of external factors; however, this is a timeconsuming process, as the cells need to be cultured until they reach senescence [14,[18][19][20][21][22]. The other approach uses fibroblasts from donors of different ages.…”