Simple vitrification for small numbers of human spermatozoa

Conventional freezing procedures and containers are not appropriate for spermatozoa from the testis because of their low number and poor in-situ motility, and various types of container have been utilized to freeze small numbers of spermatozoa. This study tried to develop a vitrification method for small numbers of spermatozoa using the Cell Sleeper, which is a closed type of cell-cryopreservation container. The container with spermatozoa were cooled in liquid nitrogen vapour and then stored in a cryotank. Sperm motility parameters improved significantly (P < 0.05) by vitrification in oil-free droplets rather than in droplets covered with oil. After vitrification of five spermatozoa per container, all spermatozoa were recovered and the viable sperm rate was significantly higher when spermatozoa were vitrified in a 3.5-μl droplet rather than in 0.5 μl (72.0% versus 38.0%; P < 0.01). Recovery, motility and viability rates of vitrified–warmed spermatozoa were similar between the Cell Sleeper and the CryoTop groups. In conclusion, the Cell Sleeper is a highly effective tool for the cryopreservation of small numbers of spermatozoa and limited cells can be vitrified quickly and simply without significant loss.Conventional freezing procedures are not appropriate for spermatozoa from the testis because of their low number and poor in-situ motility. Techniques for the cryopreservation of small numbers of spermatozoa have attempted to use various types of containers, but these are the only currently available options and the lack of an easily implemented technology has remained a major bottleneck. In the current study, we successfully established a simple cryopreservation method for small numbers of human spermatozoa using the Cell Sleeper, which is a vial type of cell-cryopreservation container and is equipped with an inner tray. Cell Sleeper is commercially available and easy to prepare for use. On single-sperm cryopreservation, the best result was obtained when spermatozoa were cryopreserved in an oil-free 3.5-μl droplet and it was the most convenient volume for handling small numbers of spermatozoa using an intracytoplasmic sperm injection pipette equipped with a micromanipulator. Furthermore, we could recover spermatozoa efficiently and quickly without significant loss. It may be concluded that the Cell Sleeper is a useful container for the cryopreservation of small numbers of spermatozoa.