Figure 1.

Schematic diagram of the sequential incubation steps, adapted from a previously reported technique for adding and removing the vitrification cocktail, VS55 [16]. For vessel segments, the process was facilitated by gravity perfusion of the solutions through the lumen of the artery segment [6]. Similarly, after vitrification and rewarming, the tissue samples were returned to physiological medium by eluting the cryoprotectants sequentially, using stepped changes. The cryoprotectant solution used at each step in the dilution was supplemented with 300mM mannitol as an osmotic buffer, to prevent excess cell swelling.