Investigation of exosomes in aged human fibroblasts cultured in serum-free medium

Introduction: Exosomes are small vesicles (30 to 100 nm) crucial for intercellular communication and influence various biological and pathological processes. This study examined exosome secretion in human skin fibroblasts in vitro. Materials and methods: Supernatants from young fibroblasts (passage 3) and aged fibroblasts (passage 12) cultured in DMEM medium, with or without 10% fetal bovine serum (FBS), were collected for analysis. After confirmation of exosome presence by scanning electron microscopy, the number of exosomes was measured using flow cytometry with magnetic beads coated with a specific antibody (anti-CD81). Additionally, the protein profile of these exosomes was examined using SDS-PAGE. Results: Electron microscopy revealed exosomes with diameters from 33 to 93 nm in fibroblast culture supernatants. Aged fibroblasts showed a significantly reduced abundance of exosomes in serum-starved conditions at 16 and 72 hours compared to controls (P<0.05). However, there was no significant difference in exosome abundance between young and aged fibroblasts in serum conditions. Young fibroblasts exhibited no significant differences in exosome levels across serum-starved and control groups at various time points. The concentration and mean fluorescent intensity (MFI) supported these findings. Electrophoresis showed exosome proteins ranging from 14 to 116 kDa, with no significant differences between age groups. Protein band density in serum-starved exosomes was lower than in controls, with only one exception noted. Conclusions: In aged fibroblasts, there were no significant changes in exosome quantity or protein patterns, but under serum-starved conditions, notable differences arose. The overall number of exosomes decreases under serum deprivation, though not significantly, while protein band density significantly decreases. Aged cells continue to secrete exosomes in serum deprivation, but their protein content diminishes. This finding improves our understanding of wound healing, cancer, and cell therapy.