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Cells held in a liquid milieu undergo processes that cause progressive loss of viability. To prevent such degradation, cells need to be placed in conditions that essentially stop all chemical reactions for the duration of the time of storage. Because intracellular ice formation is lethal to most eukaryotic cells, stable storage of viable cells can be achieved only if intracellular vitrification without ice formation has occurred. This has been done by several methods, including equilibrium (slow) freezing, lyophilization (freeze-drying), and ice-free vitrification at subzero temperatures at moderate-to high cooling rates in the presence of cryoprotectants (‘conventional’ vitrification). In this paper, we discuss the mechanisms of vitrification, and specific aspects, advantages, and pitfalls of the different approaches. Particular emphasis is put on novel methods of cell preservation, such as cryoprotectant-free vitrification of sperm and high temperature vitrification by air/vacuum drying of progenitor and other nucleated cells.

Vitrification of laser treated human blastocysts using reduced concentrations of permeable cryoprotectants was carried out by submerging cut standard straws (CSS) into liquid nitrogen. The CSS were made by cutting a standard 0.25 ml straw at an angle of approximately 45 degrees . After laser assisted hatching, 6 day blastocysts (n=250) were loaded into droplets of approximately 0.75 microl in the CSS and were either plunged directly into liquid nitrogen or first encased in a standard 0.5 ml straw (aseptic technique) before being vitrified. Permeable cryoprotectants (ethylene glycol+Me(2)SO) at concentrations of 15% and 20% v:v were tested for their effect on post warming re-expansion and post transfer pregnancy rates. Our results indicate that the use of reduced concentrations of cryoprotectants and aseptic packaging of blastocysts did not have any statistically significant impact on the study outcomes.