Vitrification and aseptic vitrification


During the freezing and thawing process, ice-crystal formation must be avoided by all means as this may result in cellular destruction. For this reason, the method of vitrification has been successfully used for freezing oocytes / embryos for a number of years now.

By “vitrification”, we understand the procedure of achieving a glass-like, amorphous state by the use of certain cryoprotectants at high levels of concentration and by ultra-rapid cooling (between 1000°C/min to 20.000°C/min during vitrification compared to 0.3°C/min while using the „Slow freezing“ method).

As the cells change directly into an amorphous state (vitrification), damages to the spindle apparatus and the chromosomes (due to ice-crystal formation) can be avoided.

Aseptic vitrification

In order to achieve high cooling rates during vitrification, the oocytes will be put on a specially designed carrier device in a small volume of vitrification solution and will be immersed in liquid nitrogen. These open hemi-straw plug devices are a strong point of criticism in vitrification, as it involves an increased risk of pathogen-related contamination and damage due to toxic substances by the direct exposure of the cells to liquid nitrogen.

In order to comply with the European Union’s directive on tissues and cells storage (2006), the cryopreservation of cells and tissues is now required to adhere to certain safety regulations. Since then, the use of a closed, aseptic system is mandatory during vitrification.

In order to comply with these high safety regulations, we were able to develop an aseptic vitrification method for oocytes. On the basis of legal regulations, the advantages of vitrification can now have a beneficial effect on cryopreservation. Sealed and thus aseptic straws present a problem relating to their heat-insulating barrier, which dramatically reduces the speed of cooling from >20.000°C/min to <2000°C/min. Solving this problem was crucial and represented a great challenge. At first, the cells were gradually exposed to increasing concentrations of cryoprotectants which induced an increase in intracellular concentrations of cryoprotectants hence reducing the likelihood of an osmotic shock.

As all cryoprotectants used are solutions having a potentially toxic effect on cells and the relatively high concentrations used for vitrification are often criticized, we assessed the relevant intracellular concentration of cryoprotectant for oocytes. Although the aseptic method involves exposing the cells to cryoprotectants for a prolonged period, we were able to prove that intracellular concentration of cryoprotectant is still low compared to “open” vitrification protocols and is well below those resulting from “slow freezing”.

Our data show that aseptic vitrification using VitriSafe represents a highly efficient and safe method of freezing egg cells. In spite of reduced cooling rates due to thermo-isolation during aseptic vitrification, acceptable results can be obtained and a negative impact on cell vitality and the development potential of the cells is not to be expected.

It is essential to maintain the high warming rates during the thawing process. The method we have developed complies with the European Union legislations and eliminates the risk of pathogen cross contamination and contact with toxic compounds during vitrification and storage in liquid nitrogen.

This vitrification technique using VitriSafe is clearly superior to the “slow-freezing” method.

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