Background

The majority of embryos in the past have been frozen by the slow cooling method. An alternative method of cryopreservation became increasingly popular with embryologists in the last decade. This method is characterized by very rapid cooling rates (>20,000° C per min) and is usually referred to as "vitrification". The method offers simplicity of operation and higher embryonic survival and pregnancy rates as compared with the conventinal, slow cooling rates.

Vitrification can be defined as an extreme elevation in viscosity, i.e., solidification of solutions, without ice crystal formation, at low temperature. This phenomenon can be induced by either applying an extreme cooling rate or by using high concentrations of cryoprotectant solutions. To achieve the maximal rates of cooling and to reduce chilling injury and ice crystal formation without having to use high concentrations of cryoprotectant, embryos are exposed directly to LN2 in tiny droplets of cryoprotectant. Carriers that use this method are referred to as "open" systems. Open systems present the risk of contamination because most microorganisms can survive at low temperatures, including in LN2 (-196° C).

The first to demonstrate the possibility of cross contamination was Piasecka-Serafin. In that study, 94% percent of sterile samples became infected with Escherichia coli and Staphylococcus aureus within 2 h after placing them in a container holding contaminated LN2. More recently, Criado, in a study about a new open vitrification system, found that 45% of the Cryotops (vitrification open carriers) tested became contaminated when exposed to contaminated LN2.

Various systems (referred to as "closed" systems) have been developed to reduce the risk of contamination from exposure of the sample to the LN2. In such systems, the straw, after aspiration of the sample, is sealed before being plunged into LN2.

However, recent work indicates that no method can compete with the cooling and warming rates of a sample that is surrounded by a thin film of cryoprotectant and exposed directly to LN2. Without direct contact, the cooling rate is compromised. This is especially true for human oocytes, for which extremely high (>20,000, preferably >50,000° C/min ) cooling and warming rates seem to be indispensable (whereas, for many embryos, including mouse, bovine, ovine and caprine blastocysts, the slightly compromised cooling rate provided by a closed system is appropriate).

If there is direct contact between sample and LN2, the risk of contamination exists. The risk can be reduced with the filtration of LN or by exposing the LN to UV light. Filters are not 100% effective whereas UV irradiation has been proven to be 100% effective. NTERILIZER now provides an effective and easy to implement method to sterilize LN2 with UV radiation for the procedure of vitrification.