Optimal conditions for studying the effects of platelet-rich plasma in vitro on the culture of dermal fibroblasts – T.I. Vlasova, E.P. Brodovskaya, K.S. Madonov, I.A. Khutorskaya

Objective. To study the effects of platelet-rich plasma (PRP) in vitro on the culture of dermal fibroblasts depending on the plasma concentration in the medium, method of activation, and cell density. Material and methods. To prepare PRP, we used one of the classic protocols for one-step centrifugation of blood from healthy donors (n=4). The study included series with different cell densities (hTERT-HDFa – d220 cell line): 2500/well, 5000/well and 10000/well – 96-well plate). Activated donor PRP was added to the experimental wells at the concentrations of 10.0%, 5.0%, 2.5%. We used freeze-thaw cycle as a control activator, with a series of experiments with additional activation using 10% CaCl2 (20 𝜇𝜇l/ml). We analyzed cell survival and metabolic activity with the MTT test. The evaluation of fibroblast migration activity was done in a scratch assay. The assessment of cell morphology and the determination of cell death mechanisms were conducted using fluorescent microscopy with preliminary staining of samples. StatTech v. 4.1.7 was used for statistical analysis. Results. The viability assessment of the hTERT-HDFa cells with different cell densities demonstrated the maximum metabolic activity of human skin fibroblasts at an initial planting density of 2500–5000 thousand cells per well (25 000–50 000/ml), and a decrease in cell growth intensity with increasing density. The optimal cell viability values were reached after 72 hours of observation. The effect of PRP activation with CaCl2 supplementing the freeze-thaw cycle before addition to the medium on fibroblast viability was negligible. In the scratch assay, the defect area closed faster with PRP used at the concentrations of 5–10%. Conclusion. To assess the biological effects of PRP on human skin fibroblast culture, we recommend to use a 5–10% concentration of the activated sample in the culture medium; the optimal planting density for this cell culture is 25 000–50 000 cells/ml. Key words: platelets, PRP therapy, human dermal fibroblasts, regeneration Conflicts of interest. The authors have no conflicts of interest to declare. Financing. The study was supported by the Russian Science Foundation grant No. 24-25-00278, https://rscf.ru/ project/24-25-00278/