Ovarian tissue-dissociated cells (human) (Massachusetts General Hospital)

Ovaries were surgically removed from six female subjects (ages 22-33 years) with gender identity disorder. The outer cortical layer was carefully removed, vitrified and cryopreserved. 1 mm-thick cortical fragments were cut into 10 X 10 mm pieces, incubated in an equilibration solution containing ethylene glycol (7.5%) and dimethylsulfoxide (DMSO, 7.5%) at 26 C for 25 min, and then incubated in a vitrification solution containing ethylene glycol (20%), DMSO (20%) and sucrose (0.5 M) at 26 C for 15 min prior to submersion in liquid nitrogen. For experimental analyses, cryopreserved ovarian tissue was thawed using the Cryotissue Thawing Kit and processed immediately for oogonial stem cell (OSC) isolation.