Human corneal endothelial tolerance to glycerol, dimethylsulfoxide, 1,2-propanediol, and 2,3-butanediol

We exposed human corneas to various concentrations of four cryoprotectants by one of two methods: a gradual increase to the final concentration (ramp method) and a series of steps to the final concentration (step method). Endothelial damage was manifest as a decrease in the number of endothelial cells per unit area. The highest concentrations that did not cause a loss of endothelial cells by the ramp and step methods, respectively, were 4.3 and 2.0 M glycerol, 2.0 and 4.3 M dimethylsulfoxide, 2.0 and 3.0 M 1,2-propanediol, and 2.0 and 2.5 M 2,3-butanediol. The ramp method achieved higher final concentrations with the more slowly permeating glycerol, but required low toxicity. The step method achieved higher final concentrations with the more toxic cryoprotectants by limiting the exposure time, but required more rapid permeation. None of the four cryoprotectants was tolerated at concentrations sufficient for vitrification at practical cooling and warming rates.