Aseptic Capillary Vitrification of Human Spermatozoa

Most of the proposed vitrification methods to obtain ultrarapid cooling rates are performed in open systems by plunging into liquid nitrogen, such as cryoloop, which would bring potential contamination risks as open systems expose the samples directly to LN2. It is important to explore an aseptic cooling method to cryopreserve sperm cells. Therefore, our group carried out the aseptic vitrification technology and focused on micro-drop methods and capillary-in-straw vitrification. The method of aseptic micro-drop technology by directly dropping human spermatozoa into a clean cooling agent (sterile liquid air) had been described “Cryopreservation and Freeze-Drying Protocols”. This part aimed to shortly describe the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries. The method was carried out in double straws packaged by a closed system. One filled sperm suspension straw inside another straw could protect against the cell suspension from bonding directly with the liquid nitrogen because it is wholly sealed, making it completely aseptic. In 2021, we improved the aseptic capillary-in-straw method using the innovative manipulation microtool with a 600 μm diameter pipette, which can be the alternative capillary as reported by Isachenko et al. Spermatozoa that are vitrified by this method are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment.