2969
Letters to the editor
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/
cm2. 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.
References
Bielanski A, Bergeron H, Lau PCK, Devenish J. Microbial contamination of
embryos and semen during long term banking in liquid nitrogen.
Cryobiology 2003;43:146 – 152.
Cobo A, Perez S, De los Santos MJ, Pellicer A, Remohı J. Comparison
between storage of vitrified oocytes by Cryotop method in liquid
nitrogen vs. vapour phase of liquid nitrogen tanks. Fertil Steril 2007;
88(Supp 1):S91.
Eum JH, Park JK, Lee WS, Cha KR, Yoon TK, Lee RD. Long-term liquid
nitrogen vapour storage of mouse embryos cryopreserved using
vitrification or slow cooling. Fertil Steril 2009;9:1928 –1932.
Gerba CP, Gramos DM, Nwachuku N. Comparative inactivation of
enteroviruses and adenovirus 2 by UV light. Appl Environ Microbiol
2002;68:5167 – 5169.
Isachenko V, Isachenko E, Weiss JM. Human ovarian tissue: vitrification
versus conventional freezing. Hum Reprod 2009;24:1767– 1768.
Linden KG, Thurston J, Schaefer R, Malley JP Jr. Enhanced UV inactivation
of Adenoviruses under polychromatic UV lamps. Appl Environ Microbiol
2007;73:7571 – 7574.
Morris GJ. The origin, ultrastructure, and microbiology of the sediment
accumulating in liquid nitrogen storage vessels. Cryobiology 2005;
50:231 – 238.
Parmegiani L, Accorsi A, Cognigni GE, Bernardi S, Troilo E, Filicori M.
Sterilization of liquid nitrogen with ultraviolet irradiation for safe
vitrification of human oocytes or embryos. Fertil Steril 2009. Epub
ahead of print.
Srikanth B. The basic benefits of ultraviolet technology. Water Conditioning
Purification, 1995; 26– 27.
US Environmental Protection Agency. Long term 2 enhanced surface
water treatment rule. Federal Register. Washington, DC: US
Environmental Protection Agency, 5 January 2006.
Wang Y. Reply: Human ovarian tissue: vitrification versus conventional
freezing. Hum Reprod 2009;24:1768 – 1769.
Lodovico Parmegiani1, Graciela Estela Cognigni and Marco Filicori
Reproductive Medicine Unit, GynePro Medical Centers, GynePro Medical, Via
T. Cremona, 8, 40137 Bologna, Italy
1
Correspondence address. Fax: 39-051-441-135; E-mail: l.parmegiani@
gynepro.it
doi:10.1093/humrep/dep329
Advanced Access publication on September 12, 2009