The effectiveness of cryoprotectant-free vitrification was discovered by directly dropping small droplets of a human spermatozoa suspension into liquid nitrogen [7, 9, 22–25]. This effectively
protects spermatozoa from cryo-injuries associated with coolingwarming, because the rapid cooling of the cells by immersion
directly into liquid nitrogen reduces the chance of intracellular ice
formation [18, 19]. Vitrification of very small sample volumes of
spermatozoa in suspensions have been reported, ranging from 1 to
10 μL diluted 1:1 in 0.5 M sucrose medium [19] and from 20 to
30 μL by direct and quickly plunging/dropping into liquid
nitrogen [7].
However, cryopreservation of biological samples by direct
exposure to liquid nitrogen and their storage in standard liquid
nitrogen tanks would increase potential risks of contamination by
viruses, bacteria, fungi, and spores, which survive in liquid nitrogen
and pose a real threat [25–29]. It has been established that especially viruses have high cryostability [24, 30–32]. Bacteria also have
high cryostability [33], as demonstrated by many publications
regarding freezing-drying (cryoprotectant-free freezing with the
subsequent vacuumization) of lactic acid bacteria [34]. Mycoplasma
are cryostable pathogens, and high rates of Mycoplasma equigenitalium and Mycoplasma subdolum contamination have been reported
after cryoprotectant-free cryopreservation by direct plunging into
liquid nitrogen and storage at À196
C for 30 days [35]. Mycoplasma can also be effectively freeze-dried [36]. In fact, it is impossible to realize complete sterilization of commercially produced
liquid nitrogen contaminated with different pathogens.
To avoid the potential risks of contamination during cryopreservation, a benchtop device for the production of sterile liquid air
can be used [37]. The benchtop device produces clean liquid air
(CLAir, FertileSafe, Nes-Ziona, Israel) having a temperature
(À195.7
C) similar to the temperature of liquid nitrogen. Arav
and co-workers reported the use of this device for vitrification of
mice embryos and human oocytes [37]. The results showed that
such devices are safe and can be easily adopted in assisted reproduction laboratory to eliminate potential contamination of cells by
direct contact of these cells with the cooling liquid agent.
It has been reported that the vitrification of human pronuclear
oocytes through direct contact with a sterile cooling agent (liquid
air) is safe [38]. We have recently explored vitrification of human
spermatozoa in clean liquid air. The results implicated that
cryoprotectant-free vitrification by direct submerging of human
spermatozoa in clean liquid air (aseptic system) is a good alternative
for liquid nitrogen.
In this chapter, we describe the standardized aseptic technology
of cryoprotectant-free vitrification of human spermatozoa by direct
dropping into a clean cooling agent (sterile liquid air).
428
Mengying Wang et al.
protects spermatozoa from cryo-injuries associated with coolingwarming, because the rapid cooling of the cells by immersion
directly into liquid nitrogen reduces the chance of intracellular ice
formation [18, 19]. Vitrification of very small sample volumes of
spermatozoa in suspensions have been reported, ranging from 1 to
10 μL diluted 1:1 in 0.5 M sucrose medium [19] and from 20 to
30 μL by direct and quickly plunging/dropping into liquid
nitrogen [7].
However, cryopreservation of biological samples by direct
exposure to liquid nitrogen and their storage in standard liquid
nitrogen tanks would increase potential risks of contamination by
viruses, bacteria, fungi, and spores, which survive in liquid nitrogen
and pose a real threat [25–29]. It has been established that especially viruses have high cryostability [24, 30–32]. Bacteria also have
high cryostability [33], as demonstrated by many publications
regarding freezing-drying (cryoprotectant-free freezing with the
subsequent vacuumization) of lactic acid bacteria [34]. Mycoplasma
are cryostable pathogens, and high rates of Mycoplasma equigenitalium and Mycoplasma subdolum contamination have been reported
after cryoprotectant-free cryopreservation by direct plunging into
liquid nitrogen and storage at À196
C for 30 days [35]. Mycoplasma can also be effectively freeze-dried [36]. In fact, it is impossible to realize complete sterilization of commercially produced
liquid nitrogen contaminated with different pathogens.
To avoid the potential risks of contamination during cryopreservation, a benchtop device for the production of sterile liquid air
can be used [37]. The benchtop device produces clean liquid air
(CLAir, FertileSafe, Nes-Ziona, Israel) having a temperature
(À195.7
C) similar to the temperature of liquid nitrogen. Arav
and co-workers reported the use of this device for vitrification of
mice embryos and human oocytes [37]. The results showed that
such devices are safe and can be easily adopted in assisted reproduction laboratory to eliminate potential contamination of cells by
direct contact of these cells with the cooling liquid agent.
It has been reported that the vitrification of human pronuclear
oocytes through direct contact with a sterile cooling agent (liquid
air) is safe [38]. We have recently explored vitrification of human
spermatozoa in clean liquid air. The results implicated that
cryoprotectant-free vitrification by direct submerging of human
spermatozoa in clean liquid air (aseptic system) is a good alternative
for liquid nitrogen.
In this chapter, we describe the standardized aseptic technology
of cryoprotectant-free vitrification of human spermatozoa by direct
dropping into a clean cooling agent (sterile liquid air).
428
Mengying Wang et al.