Ovis aries (Whole ovary)
The authors of the paper from which this protocol is adopted noted that it is not perfect. Ki67 expression was reduced following cryopreservation, suggesting that cellular proliferation is reduced. Cryopreservation also caused arterial endothelial disruption. It is possible that there may be other negative effects that were not observed by the authors, however the results as a whole were called “encouraging for whole ovary cryopreservation, demonstrating maintained cell viability”.
• Leibovitz L-15 media
• Dimethyl sulfoxide (DMSO; Fisher Scientific, Loughborough, UK)
• Sucrose (Fisher Scientific)
• Heat inactivated fetal calf serum (FCS)
• 2.5F (0.75 mm outer diameter) i.v. cannula (Sims Portex Limited, Kent, UK)
• 2/0 mersilk suture (Ethicon, Edinburgh, UK)
• Perfusion tray
• Syringe pump
• 15 ml cryogenic vials
• Controlled-rate freezer
1. Cannulate the origin of the ovarian artery of one ovary from each tract using a 2.5F (0.75 mm outer diameter) i.v. cannula and tie it securely in place using 2/0 mersilk suture.
2. Dissect the ovary and cannulated pedicle free of the ovarian tract. Ligate subsidiary vessles using 2/0 mersilk suture.
3. Place the dissected ovary in a chilled perfusion tray and immerse in pre-chilled cryoprotective solution (Leibovitz L-15 media supplemented with 1.5 mol / l DMSO , 0.1 mol / l sucrose, and 10% heat inactivated FCS).
4. Perfuse pre-chilled cryoprotective solution through the ovary via the cannula for 60 min at a rate of 0.5 ml / min using a syringe pump.
5. Transfer each ovary with its cannulated pedicle to a 15 ml cryogenic vial and cover it with pre-chilled cryoprotective solution.
6. Cool the tissue to -9°C at -1°C / min using a planar freezer.
7. Remove the cryogenic vial from the freezer and hold it over liquid nitrogen until the first signs of ice formation are observed.
8. Place the tissue back into the freezer and cool to -40°C at a rate of -0.2°C / min, then to -140°C at a rate of -10°C / min.
9. Store the cryogenic vials in liquid nitrogen.
1. Remove the vials from liquid nitrogen and let sit at room temperature for 2 min.
2. Place the tubes in a 37°C water bath with gentle agitation 30 – 40 min.
3. Transfer each ovary to a perfusion tray and immerse in warm (37°C) thawing media (Leibovitz L-15 media supplemented with 1 mol / l DMSO, 0.1 mol / l sucrose and 10% FCS).
4. Via the pre-existing cannula, perfuse each ovary with warm thawing media for 10 min at 1 ml / min.
Onions VJ, Mitchell MR, Campbell BK, Webb R. Ovarian tissue viability following whole ovine ovary cryopreservation: assessing the effects of sphingosine-1-phosphate inclusion. Hum Reprod. 2008 Mar;23(3):606-18.
Added by: Carlton Hoyt on July 25, 2012