Simple vitrification for small numbers of human spermatozoa

Yuji Endo, Yoshitaka Fujii, Kasumi Shintani, Momoyo Seo, Hiroaki Motoyama, Hiroaki Funahashi

Research output: Contribution to journalArticlepeer-review

50 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)301-307
Number of pages7
JournalReproductive BioMedicine Online
Issue number3
Publication statusPublished - Mar 2012


  • Cell sleeper
  • CryoTop
  • cryopreservation
  • simple vitrification method
  • single-sperm freezing

ASJC Scopus subject areas

  • Reproductive Medicine
  • Obstetrics and Gynaecology
  • Developmental Biology


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