Critical cooling and warming rates to avoid ice crystallization in small pieces of mammalian organs permeated with cryoprotective agents.

Measurements were made by differential scanning calorimetry on small pieces of rabbit kidney permeated with 2, 3-butanediol containing mainly the levo- and dextro-isomers. The critical cooling rates necessary to vitrify the pieces of organ, and the corresponding critical warming rates which are required to avoid crystallization in the vitrified samples, were determined. The dynamic method used for these determinations is described. The glass-forming tendency and the stability of the amorphous state were both greater in the kidney tissue samples than in the bulk cryoprotective solution. This result is discussed in the context of the lowering of the freezing point of water in emulsions and the promotion of supercooling in hydrogels and porous materials. In corresponding experiments with rat hearts impregnated with 1,2-propanediol, only the critical warming rate was reduced.

Reduction in toxicity for red blood cells in buffered solutions containing high concentrations of 2,3-butanediol by trehalose, sucrose, sorbitol, or mannitol.

Erythrocytes were stored at 4 degrees C in solutions of phosphate-buffered saline containing 2,3-butanediol and 4% (w/w) trehalose, sucrose, sorbitol, or mannitol. The 2,3-butanediol contained 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (2,3-butanediol 97% dl). The concentrations of 2,3-butanediol were 30 and 35% (w/w). A solution of 30% 2,3-butanediol showed relatively low toxicity. Hemolysis was only 2% after 5 h, but increased to 6% after 21 h and reached 60% after 46 h. Adding 4% (w/w) of one of the above compounds drastically decreased the toxicity. The two most efficient were the sugars trehalose and sucrose. With 30% 2,3-butanediol and 4% of any of the four compounds, hemolysis was about 0.6% after 2 days of storage. Furthermore, with trehalose or sucrose, hemolysis remained below 3% for 1 month. With sorbitol or mannitol, hemolysis slowly increased to 2% after 7 days and then increased rapidly. Even with 35% 2,3-butanediol, solutions containing trehalose or sucrose showed low toxicity. Hemolysis was also measured after redilution to buffered solution without 2,3-butanediol and without the additive, to mimic perfusion of organs with cryoprotectants and washing. Minima of hemolysis were observed after a few days of storage. The present solutions also have high glass-forming tendencies. They could be of great interest for organ vitrification.