4.3. Two Methods Used for Hypoxic Preconditioning of BM-MSCs

In the current study, two hypoxic preconditioning methods were used. The first, “physical” hypoxia was achieved by culturing cells for 6 h in a gas mixture composed of 2% O₂ (balanced with N₂) and 5% CO₂. The second, “pharmacological” hypoxia was obtained by culturing cells for 6 h with the PHDs inhibitor, Vadadustat (AKB-6548, Akebia, Cambridge, MA, USA) in a concentration of 40 μM. The incubation time for gene expression analysis was chosen based on preliminary RT-PCR time-course analysis of two HIF-1α-related genes (VEGF and GLUT1) to determine the time point at which their expression increased most following exposure to experimental factors. The 2% O₂ concentration was chosen to be lower than the physiological bone marrow oxygen partial pressure of 3–5% (reflecting hypoxia for BM-MSCs) but without causing anoxia. Vadadustat concentration was selected based on MTT assay [11] and Western blot analysis of HIF-1α level to achieve HIF-1α stabilization similar to that in 2% O₂ hypoxia with minimal effect on cell metabolic activity. Vadadustat was prepared as a 5 mM stock solution in DMSO (Merck, Darmstadt, Germany) according to the manufacturer’s instructions, meaning that no more than 0.8 % (v/v) DMSO was present in the culture medium (without causing a noticeable cytotoxic effect in the MTT assay [11]). After 6 h of incubation, cells were harvested, washed with PBS (Merck, Darmstadt, Germany) and frozen at −80 °C for further analysis. The incubation time was experimentally chosen as optimal to visualize transcriptomic changes associated with hypoxic preconditioning of MSCs.