Figure 3.

Evaluation of the preconditioning effect on fetal- and perinatal hAFS viability and on secretome yield. (A) Apoptosis analysis by flow cytometry evaluation of PI and Annexin V expression on f-hAFS and p-hAFS after 24 h preconditioning in either 20% O₂ normoxic condition compared to the 1% O₂ hypoxic condition, and in complete control (Ctrl) medium versus serum-free (SF) medium (Ctrl f-hAFS_normo; Ctrl f-hAFS_hypo; SF f-hAFS_normo; SF f-hAFS_hypo; Ctrl p-hAFS_normo; Ctrl p-hAFS_hypo; SF p-hAFS_normo; SF p-hAFS_hypo, respectively). (B) Pie charts of live, early apoptotic and late apoptotic f-hAFS (n = 4 experiments, upper panel) and corresponding p-hAFS (n = 6 experiments, lower panel); late apoptotic Ctrl p-hAFS_hypo vs SF p-hAFS_hypo * p = 0.0392; no statistically significant differences were detected among all other comparisons. (C) Protein concentration of hAFS secretome yield obtained from serum-free culture conditions by BCA assay. Left panel refers to the entire secretome formulation, namely the in vitro hAFS-conditioned medium (hAFS-CM). Right panel refers to hAFS-EV formulations (hAFS-EVs); n = 8 experiments for f-hAFS-EVs and n = 11 experiments for p-hAS-EVs. All values are expressed as mean ± s.e.m of independent experiments.