Shelf life and safety evaluation of the dermal fibroblasts conditioned medium (DFCM) for future skin regeneration and rejuvenation

Abstract

Studies discovered that dermal fibroblasts conditioned medium (DFCM) as potential agent for skin regeneration and rejuvenation. However, the stability of these secreted proteins remains unknown. This study aimed to evaluate the stability of DFCM for skin regeneration and rejuvenation, focusing on storage conditions over time. Human dermal fibroblasts were cultured in serum-free fibroblast-specific medium (DFCM-FM) or keratinocyte- specific medium (DFCM-KM) for 72 hours, and the protein profile was analysed using liquid chromatography- mass spectrometry (LC-MS), Bicinchoninic Acid Protein Assay (BCA), and Enzyme-linked immunosorbent assay (ELISA). Then effects of DFCM on fibroblasts attachment, proliferation, migration, and cell characterisations were also assessed. DFCM-FM was formulated for skin care product and the physical and chemical stability evaluated. LC-MS identified 16 proteins in both DFCM, involved in skin remodelling and repair. Protein concentrations in DFCM-FM at 4 °C, -20 °C and -80 °C remained consistent up to 6 months. DFCM-KM stored at - 20 °C and -80 °C was stable for up to 6 and 12 months respectively. The collagen and fibronectin concentrations were more stable in DFCM-FM than in DFCM-KM, especially at lower storage temperatures. DFCM-FM enhanced fibroblasts attachment, proliferation, and migration over 6 months, while DFCM-KM was most effective at 1 month. Immunocytochemical staining revealed positive expression of collagen type I and Ki67 in both groups. Formulations of DFCM-FM (toner, serum, moisturizer, and mask) were stable under various temperature stress tests, maintaining appearance, pH, and viscosity. Microbiological and heavy metal contamination tests confirmed the formulations' safety. The study concludes that both DFCM exhibit promising storage stability under varying conditions, with DFCM-FM remaining stable for up to 6 months and DFCM-KM at -80 °C for up to 12 months. The formulations with DFCM-FM were confirmed to be safe and stable, highlighting their potential for clinical applications.