Prevention of Hemolysis in Rapidly Frozen Erythrocytes by Using a Laser Pulse

This paper reports on the successful recovery of rapidly frozen unprotected erythrocytes by vitrification of the intracellular solution with a laser pulse prior to thawing. Erythrocytes that were frozen at 10,000 °C/min exhibited 100% hemolysis when thawed unless they were first irradiated by a 7 ns. laser pulse that selectively targeted the intracellular ice so that it was melted and resolidified into a glass phase. Up to 80% of the cells treated in this way remained intact after thawing. Wright's staining confirmed a healthy cell morphology and the retention of hemoglobin in the laser treated cells. While it is well known that small amounts of intracellular ice can be tolerated by cells, the findings of this study are the first to indicate that intracellular ice may be innocuous even when formed in substantial quantities provided that crystal growth and coalescence can somehow be avoided during warming.