Cryopreservation of Mouse Ovarian Tissue Following Prolonged Exposure to an Ischemic Environment

In cases in which ovarian tissue is to be cryopreserved for tissue or gene banking it is important to maintain its integrity and viability. This study examined how delays between the death of an animal and the collection/cryopreservation of its ovarian tissue influenced follicle viability. Mouse ovaries were placed in PBS+antibiotic (in vitro) or left within the body (in situ) at room temperature for 0, 3, 6, 12, or 24 h following the death of the donor. These ovaries were cryopreserved at 1°C/min on dry ice or in a −84°C freezer using a passive cooling device or by conventional slow cooling (0.3°C/min). The ovaries were grafted under the kidney capsule of ovariectomized recipient mice and collected 2 weeks later, and the size and number of follicles were determined. Cryopreserved ovarian tissue grafted immediately after the death of the donor contained numerous viable and healthy follicles independent of the cooling procedure (dry ice, 134 ± 32; −84°C, 165 ± 54; slow, 214 ± 55 follicles per half ovary). Tissues stored in vitro before cryopreservation retained viable follicles up to 12 h after death (dry ice, 30 ± 15; −84°C, 86 ± 45; slow, 93 ± 33), whereas tissue left in situ had significantly reduced follicle numbers within 3 h of death (dry ice, 36 ± 12; −84°C, 19 ± 6; slow, 28 ± 7). No significant difference was found between the cooling rates tested, indicating that a passive cooling container which cools at 1°C/min is a suitable alternative to conventional slow cooling. We conclude that ovarian tissues for cryobanking should be cryopreserved as soon as possible after collection or death of the animal to ensure maximal follicular survival.