Chapter 19
Aseptic Cryoprotectant-Free Vitrification of Human
Spermatozoa by Direct Dropping into a Cooling Agent
Mengying Wang, Evgenia Isachenko, Gohar Rahimi, Peter Mallmann,
and Vladimir Isachenko
Abstract
Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions.
Routinely applied cryopreservation techniques depend on permeating cryoprotectants and relatively slow
freezing rates. Cryoprotectant-free vitrification is an alternative and cost-effective method that is based on
rapid cooling of spermatozoa by direct plunging into a cooling agent to prevent lethal intracellular ice
crystallization and the detrimental effects of high salt concentrations. One of the problems with this
technique is that full sterilization of commercially produced liquid nitrogen, which could be contaminated
with different pathogens, is not possible. Here we use a benchtop device for the production of sterile liquid
air with the same temperature as liquid nitrogen (À195.7
C). This has been used to develop aseptic
technology for cryoprotectant-free vitrification of human spermatozoa.
Key words Human spermatozoa, Vitrification, Aseptic, Liquid nitrogen, Liquid air
1 Introduction
Cryopreservation of spermatozoa is an important technology in
reproductive medicine [1–4]. Since data on the cryopreservation
of human spermatozoa in the presence of cryoprotectants were first
published in the late 1950s [5], several cryopreservation methods
have been introduced, including conventional freezing and vitrification techniques [6–9].
Conventional cryopreservation methods presuppose the use of
cryoprotective agents (permeable cryoprotectants), which can be
toxic [10]. Negative impacts of conventional cryopreservation on
spermatozoa functions may also include intracellular ice crystal
formation, cellular dehydration, osmotic injury, cytoplasm damage,
functional destabilization, and mutagenesis [11–14]. This method
can lead to alterations in membrane permeability [15–17]. To avoid
these detrimental effects, cryoprotectant-free vitrification technology has been developed [17–21].
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_19, © Springer Science+Business Media, LLC, part of Springer Nature 2021
427
Aseptic Cryoprotectant-Free Vitrification of Human
Spermatozoa by Direct Dropping into a Cooling Agent
Mengying Wang, Evgenia Isachenko, Gohar Rahimi, Peter Mallmann,
and Vladimir Isachenko
Abstract
Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions.
Routinely applied cryopreservation techniques depend on permeating cryoprotectants and relatively slow
freezing rates. Cryoprotectant-free vitrification is an alternative and cost-effective method that is based on
rapid cooling of spermatozoa by direct plunging into a cooling agent to prevent lethal intracellular ice
crystallization and the detrimental effects of high salt concentrations. One of the problems with this
technique is that full sterilization of commercially produced liquid nitrogen, which could be contaminated
with different pathogens, is not possible. Here we use a benchtop device for the production of sterile liquid
air with the same temperature as liquid nitrogen (À195.7
C). This has been used to develop aseptic
technology for cryoprotectant-free vitrification of human spermatozoa.
Key words Human spermatozoa, Vitrification, Aseptic, Liquid nitrogen, Liquid air
1 Introduction
Cryopreservation of spermatozoa is an important technology in
reproductive medicine [1–4]. Since data on the cryopreservation
of human spermatozoa in the presence of cryoprotectants were first
published in the late 1950s [5], several cryopreservation methods
have been introduced, including conventional freezing and vitrification techniques [6–9].
Conventional cryopreservation methods presuppose the use of
cryoprotective agents (permeable cryoprotectants), which can be
toxic [10]. Negative impacts of conventional cryopreservation on
spermatozoa functions may also include intracellular ice crystal
formation, cellular dehydration, osmotic injury, cytoplasm damage,
functional destabilization, and mutagenesis [11–14]. This method
can lead to alterations in membrane permeability [15–17]. To avoid
these detrimental effects, cryoprotectant-free vitrification technology has been developed [17–21].
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_19, © Springer Science+Business Media, LLC, part of Springer Nature 2021
427