Fig. 1.

Schematic shows an overview of steps for the collection of normoxic or hypoxic CdM-BMSCs. After BMSCs reached greater than 80% confluency at Passage 3, the medium was aspirated, and BMSCs were rinsed 3 times with PBS. Then, α-MEM was added to culture flasks with BMSCs, and the culture flasks were put into an incubator under the normoxic or hypoxic conditions for 24 h. For the normoxic group, BMSCs were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and 20% O₂. The hypoxic group was cultured at 37 °C in 1% O₂, 5% CO₂, and 94% N₂ in a hypoxic incubator. The primary CdM was collected after incubation for 24 h, and the cell debris was removed using a syringe filter. The primary CdM was then sequentially concentrated to 15-fold by the centrifugation through Amicon Ultra Centrifugal Filter (5000 g for 2 h at 4 °C) according to the manufacturer’s protocol. Finally, the novel donor heart preservation solution was produced by diluting 300 ul α-MEM (Vehicle group), concentrated normoxic (N-CdM group), or hypoxic CdM (H-CdM group) from the filtrate tube of the top unit in 2700-ul Custodiol cardioplegic solution. BMSCs bone marrow mesenchymal stem cells, CdM conditioned medium, CdM-BMSCs conditioned medium-derived from bone marrow mesenchymal stem cells, N-CdM normoxic conditioned medium, H-CdM hypoxic-conditioned medium