C-17: The first bench top device for producing clean liquid air (CLAir): A breakthrough in vitrification with open systems

Abstract

Vitrification using open systems has shown superior results over closed systems, particularly for oocytes cryopreservation. However, direct exposure of oocytes, embryos or other reproductive tissue to liquid nitrogen (LN) carries the risk of contamination with viruses, bacteria, fungi and spores typically detected in commercial standard LN. Here we report a new bench top system capable to produce clean and sterile liquid air, thus avoiding altogether the potential for contamination and disease transmission through standard LN. We describe here the first system for producing clean liquid air called CLAir (FertileSafe, Israel) composed of a sterilized metal container with an inner sink to increase the surface area in contact with filtered air. The outer surface of the metallic sink is cooled with LN poured into a special area of the device. After a few minutes the filtered air is liquefied within the sterile container and it is ready for use. The temperature of the liquefied air has been found consistently in the range of −190 °C to −196 °C. Cooling rate of above 20,000 C/min were measured using small drop of 0.1 μl VS placed on Cryoleaf (Origio, Denmark). Two different size systems have been tested, one able to produce 70ml of clean liquid air (CLAir, FertileSafe, Ness-Ziona, Israel) in 10 min and the other 500 ml of clean liquid air within 30 min (CLAir XL, FertileSafe, Israel). The clean liquid air is poured into a sterile Styrofoam cup into which the biological samples are plunged. After plunging the biological samples (oocytes , sperm, embryos or ovarian slices) in the clean liquefied air they are transferred to an empty straw or vial and then heat sealed and stored in standard LN tanks. Bio-burden test showed no colonies of bacteria, fungi or yeast, as opposed to commercial LN. In addition, bovine GV oocytes were vitrified using the Cryoleaf carrier and were plunged into clean liquid air (CLAir, FertileSafe, Israel) showing upon warming results of 91% survival (33/36). We also vitrified human oocytes and sperm using this method which will be presented in the meeting. In summary, the use of safe vitrification protocols is important to avoid contamination or cross-contamination when using open systems with commercial LN. CLAir is a breakthrough system allowing each laboratory the convenience and safety to produce clean and sterile liquid air for vitrification of oocytes, embryos or reproductive tissue.