Ejaculated fresh human sperm were analyzed for concentration and motility before being washed in order to remove the seminal plasma. Then the sperm were diluted in Lyophilized solution (LyoS) 1:1 (v/v) in α-MEM Eagle medium containing 0.25M sucrose, 0.25M trehalose and 0.6% (w/v) human serum albumin (HSA).
A sample was taken for DNA integrity evaluation using the Halosperm G2 kit. Droplets of 10ul were then directly exposed to sterile liquid air produced by our bench top device. The frozen pellets were kept in closed 10ml glass vials (n=6). Part of the frozen drops were thawed for evaluation using Hepes medium warmed to 37°C.
The vials were opened, in sterile liquid air and placed in our lyophilization device. Prior to use the device was sterilized using an autoclave. Pressure was set to 10mTorr, shelf temperature was -35°C and condenser temperature was set to -110°C. We dried the sperm for 48 hours.
Dried droplets (20) were directly exposed to 0.2ml of α-MEM Eagle medium pre-warmed to 37°C or to 0.2ml of warm LyoS. Sperm cells were evaluated for concentration and at least 200 sperms were evaluated in each group for DNA integrity with the Halosperm G2 Kit.
Fresh sperm concentration was 10•106 cells/ml and motility was more than 50%, DNA integrity was 81.06±9.2%. Post thaw motility was 65-80% compared to the fresh (normalized) same specimen. Cells concentration of the group rehydrated with LyoS was 8.25•106 cells/ml and DNA integrity was 81.3%±3.5%. Cells concentration of the groups rehydrated with medium was 5.375•106 cells/ml and DNA integrity was 71.19%±7.7%.
Biological results of human sperm showed 81% DNA integrity after freeze-drying and rehydration.
A Novel Bench Top Device for Freeze Drying Sperm Cells
A. Arav and Y. Natan
Presentation at the 52nd annual meeting of the Society for Cryobiology, Ostrava, Czech Republic (2015)
Figure 1: Frozen-dried human sperm after 2 years storage