Nucleation and Crystal Growth in a Vitrification Solution Tested for Organ Cryopreservation by Vitrification

Nucleation and crystal growth are investigated for vitrification solution VS41A (dimethyl sulfoxide, formamide, and 1,2-propanediol) in an aqueous carrier solution giving, when added to these three cryoprotectants, a concentration of other solutes in the whole solution the same as that in Euro-Collins, with a 55% (w/v) cryoprotectant concentration. This concentration is assumed to achieve physical properties under 1 atmosphere similar to those of solution VS4 used under 1000 atmospheres. The thermal range and the kinetics of nucleation and crystal growth are investigated by DSC through different thermal treatments. It is found that the nucleation thermal range is below -90 degrees C and that of crystal growth is above -85 degrees C for a relatively long experimental time. The nucleation density is also studied through direct observations by cryomicroscopy and is related to the amount of crystallization calorimetrically recorded. The effect of storage below the glass transition shows the possibility of a slow increase in nucleation below the glass transition, as already observed by other authors for different aqueous solutions. Isothermal crystallization is analyzed within the Johnson-Mehl-Avrami model for temperatures above -75 degrees C. The corresponding samples have been cooled and warmed at the same rate of 40 degrees C/min and calculations give, at constant nuclei numbers, an activation energy of 9.3 +/- 0.3 kcal/mol and the Avrami exponent n = 2.2 +/- 0.05. This shows a two-dimensional crystal growth as observed by cryomicroscopy. The estimated critical warming rate relevant to the preservation of rabbit kidneys by vitrification is 270 degrees C/min with or without an increase in the nucleus density during storage. The present results support the possibility of using VS4 solution for vitrification of rabbit kidneys if pressure is not a limiting factor. Copyright 1993, 1999 Academic Press.