Vitrification of erythrocytes, cryoprotective solutions and pure water by rapid solidification
Vitrification has been used successfully in the past to cryopreserve biologically active materials in the presence of high concentrations of cryoprotectants. Rapid cooling and rapid rewarming were investigated to reduce or eliminate the concentrations of cryoprotectant necessary for cryopreservation. Glycerol based cryoprotectants were unidirectionally quenched and rewarmed to determine the depth at which a glass could form upon quenching while also avoiding subsequent crystallization upon rewarming. It was determined that, at sufficient cooling rates, pure water could be vitrified in thicknesses of 700 microns by quenching on free standing diamond wafers, and that solutions of greater than 50% glycerol are required to vitrify thicknesses equivalent to that of a human kidney. This process has been adapted to cryopreserve erythrocytes resuspended in isotonic saline. The cell suspensions were either drawn into small diameter glass tubes (500 micron inner diameter), loaded between thin glass plates (130-170 micron plate thickness), or formed into thin discs by shearing a drop of the suspension on a diamond film. The tubes, plates and sheared droplets were then quenched by immersion into liquid nitrogen. Erythrocyte survival after rewarming was measured at up to 97% of the unfrozen controls. Additionally, erythrocyte intracellular 2,3-DPG, ATP, and K+ were measured for the quenched cells and compared to the unfrozen controls. 2,3-DPG levels dropped 17.9% ± 16.3%, ATP decreased 46.8% ± 13.4%, and 52.8% ± 3.4% of intracellular K+ remained after cryopreservation. The changes in intracellular indicators were similar to the changes observed in erythrocytes cryopreserved using the conventional glycerolized cryopreservation technique. Glass formation in erythrocyte suspensions upon cooling has been confirmed by differential scanning calorimetry (DS). Samples quenched in tubes, plates and on diamond films showed glass transition endotherms and crystallization exotherms, which were completely absent in the slowly cooled sample, indicating a partially, if not totally, glassy as quenched structure. This dissertation marks the first successful vitrification of liquid water in volumes large enough to contain biologically active materials as well as the first time erythrocytes have been successfully cryopreserved by vitrification through conductive heat transfer without the aid of cryoprotectants.
David J Schedgick,
"Vitrification of erythrocytes, cryoprotective solutions and pure water by rapid solidification"
(January 1, 2003).
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