Ultra-violet sterilization of liquid nitrogen prior to vitrification.

Sir, Regarding the biosafety of direct exposure of tissue/cells to liquid nitrogen (LN2) during the vitrification procedure when using ‘open carriers’, debated by Wang (2009) and Isachenko et al. (2009) in the July 2009 issue of this journal, we would like to disagree. We strongly dispute the proposed inefficacy of ultra-violet (UV) treatment of LN2 to guarantee the absence of contamination by viruses of biological material. UV disinfection is a well-accepted technology for the inactivation of bacterial and protozoan pathogens. Until recently, UV was also considered a viable technology for disinfection of viruses (Linden et al., 2007). At UV doses typically used in liquid disinfection (the UV dose is defined as the smallest amount of UV radiation able to guarantee the complete death of a given pathogen), UV is very effective against almost all known pathogenic viruses, with the single exception of adenoviruses (Gerba et al., 2002). However, according to US Environmental Protection Agency regulations (2006), the inactivation of adenoviruses can be achieved with a UV dose of 200 000 mWs/ cm. Hence, an adequate amount of UV radiation de-activates the growth of all kinds of micro-organisms, from viruses like Hepatitis (which require an 8000 UV dose) to fungi like Aspergillus niger (330 000 UV dose) (Srikanth, 1995). The use of UV radiation to sterilize water or other liquids is well known; however, this method is difficult to apply to LN2, which evaporates rapidly. Furthermore, with some liquids, the UV radiation may be absorbed before it can reach the micro-organism to be de-activated; fortunately however, LN2 is largely transparent to the radiations from the most common commercial UV sources. Recently, we demonstrated that direct UV sterilization is applicable to LN2; our study showed that decontamination of a small volume of LN2 from bacteria and fungi like Aspergillus [the most UV-resistant micro-organism ever observed in LN2 (Bielanski et al., 2003; Morris, 2005)] via UV irradiation is feasible and straightforward (Parmegiani et al., 2009). Our method for sterilizing LN2 is based on emitting the minimum dose of UV radiation necessary to kill micro-organisms that can survive at the boiling point of nitrogen (2195.82 8C) and which is irradiated in a temperature-controlled regimen, within a short time interval, before the LN2 completely evaporates. With our method, sterile LN2 can be easily obtained for any use and, in particular, for a safe vitrification procedure. Therefore, in the case of vitrification by ‘open carriers’, after cooling in UV-sterilized LN2, biological material could be enclosed in a sterile pre-cooled device for hermetical isolation at storage as suggested by Wang (2009) and/or stored in the vapour phase of LN2 (Cobo et al., 2007; Eum et al., 2009). Since some closed systems for vitrification also involve direct contact between the cells and LN2 during the warming procedure, UV sterilization of LN2 may be appropriate in these cases. In our opinion, the fact that LN2 can be quickly and safely sterilized could encourage the clinical application of human cell/tissue vitrification, both with ‘open carriers’ and with closed systems.