2.5. Human Fibroblast Proliferation by MTT Assay in Aging Conditioned Medium

The MTT assay allows the simple, accurate and reliable counting of metabolically active cells [36,37,38]. Since mitochondrial activity is directly related to the number of viable cells, this assay is usually performed to assess the proliferation capacity and cytotoxic potential of different compounds in different cell lines [38]. The MTT assay reflects the effects produced by a substance or treatment upon cell viability, which may be interpreted as toxic effects (cytotoxicity) if cell viability is compromised or stimulating effects (proliferation) if cell viability increases, comparing the treatments with the untreated control group.

To perform cell counting of live cells, cell viability was checked by staining with trypan blue solution. Live cells were counted in a Bürker chamber under the microscope.

To emulate the aging process, normal human dermal fibroblasts (NHDFs) were cultured overnight at a density of 8000 cells/well in a 96 well plate in growth medium. Twenty-four hours later, conditioned medium containing 500 μM hydrogen peroxide (H₂O₂) was added to the cells for 3 h, according to previously published work showing senescence induction in that range of doses [39,40]. After the incubation period, the medium was replaced by fresh medium containing the ingredient at different concentrations (0.01%, 0.005%, 0.001% and 0.0005%) for 96 h. Epidermal growth factor (EGF, 20 ng/mL) was included as a positive control and NHDF without H₂O₂ pre-treatment as a non-aging control (Control). The medium in all conditions contained only 0.5% FBS. After 96 h of incubation, the medium was removed, wells were washed with PBS and cell viability was then quantified using the MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT assay was performed following the ECVAM Guidelines as established in the ECVAM Database Service on Alternative Methods to Animal Experimentation (MTT assay protocol nr. 17) [41]. In brief, MTT solution 1:11 was added to each well. Plates were incubated in the refrigerated incubator at 37 °C for 3 h. The MTT reactive was removed, and DMSO 100% was added to each well to solubilize formazan crystals prior to absorbance measurements at 550 nm and 620 nm as a reference on a scanning multi-well spectrophotometer. A biological replicate with 8 technical replicates per condition was performed. The data were statistically analyzed using an ordinary one-way ANOVA test and Student’s t-test. Statistical significance was set at p < 0.05, with a 95% confidence interval. Absorbance values lower than those of control cells indicated a reduction in the rate of cell proliferation. Conversely, a higher absorbance rate indicated an increase in cell proliferation.