7. Rapidly plunge the straws into 2 L capacity dewar filled with
LN 2 .
8. Place a cryo-canister with cryo-goblets, and insert the straws
into cryo-goblets, and place the cryo-canister into a cryo-sleeve
to protect the straws accidentally falling from the cryo-goblets.
9. Place the cryo-canister enclosed with cryo-sleeve into a LN 2
tank for long-term storage.
3.6 Thawing
1. Remove the cryo-cane containing the OT samples from the
large LN 2 dewar, and quickly transfer into a 2 L capacity dewar
filled with LN 2 .
2. Remove a single straw by holding from cotton tip end followed
by holding at room temperature until ice totally melts.
3. Wipe the straw with 70% ethanol and kimwipes, and cut the
sealed end first, point the cut end to a Petri dish containing
3–4 mL TL-HEPES solution, expel the contents including the
OT pieces, and hold for 10 min at room temperature to allow
removal of DMSO.
4. Transfer the OT into a fresh TL-HEPES solution by using fine
forceps, and hold for another 10 min, and finally transfer into
another fresh TL-HEPES solution.
5. At this stage, the OT can either be grafted orthotopically, a
desired site heterotopically, or fixed in 10% neutral buffered
formalin for histological or analysis.
3.7 Ovarian Tissue
Transplantation
1. Set up standard surgical area in a laminar flow clean bench
having a stereo dissecting microscope with fiber optic light
source and a heating pad and all the necessary autoclaved
surgical equipment before the operation.
2. After weighing anaesthetize the recipient females by IP injection of ketamine and xylazine cocktail (rats, 80 mg/kg ketamine +9 mg/kg xylazine; mice, 90 mg/kg ketamine +10 mg/
kg xylazine). Inject (SC) postoperative pain reliever, banamine
(rats, 0.05 mg/kg; mice, 0.1 mg/kg), once the vital signs such
as the toe pinch reflex disappear. Apply ophthalmic ointment to
both eyes to protect corneas from drying. Place the animal into
the heated cage until the anesthesia has taken effect.
Ovarian Ɵssue pieces
IdenƟficaƟon
Heath seal
Air bubble
Fig. 2 Pre-labeled 0.5 cc French straw containing ovarian tissue pieces in freezing solution
476
Yuksel Agca and Cansu Agca
LN 2 .
8. Place a cryo-canister with cryo-goblets, and insert the straws
into cryo-goblets, and place the cryo-canister into a cryo-sleeve
to protect the straws accidentally falling from the cryo-goblets.
9. Place the cryo-canister enclosed with cryo-sleeve into a LN 2
tank for long-term storage.
3.6 Thawing
1. Remove the cryo-cane containing the OT samples from the
large LN 2 dewar, and quickly transfer into a 2 L capacity dewar
filled with LN 2 .
2. Remove a single straw by holding from cotton tip end followed
by holding at room temperature until ice totally melts.
3. Wipe the straw with 70% ethanol and kimwipes, and cut the
sealed end first, point the cut end to a Petri dish containing
3–4 mL TL-HEPES solution, expel the contents including the
OT pieces, and hold for 10 min at room temperature to allow
removal of DMSO.
4. Transfer the OT into a fresh TL-HEPES solution by using fine
forceps, and hold for another 10 min, and finally transfer into
another fresh TL-HEPES solution.
5. At this stage, the OT can either be grafted orthotopically, a
desired site heterotopically, or fixed in 10% neutral buffered
formalin for histological or analysis.
3.7 Ovarian Tissue
Transplantation
1. Set up standard surgical area in a laminar flow clean bench
having a stereo dissecting microscope with fiber optic light
source and a heating pad and all the necessary autoclaved
surgical equipment before the operation.
2. After weighing anaesthetize the recipient females by IP injection of ketamine and xylazine cocktail (rats, 80 mg/kg ketamine +9 mg/kg xylazine; mice, 90 mg/kg ketamine +10 mg/
kg xylazine). Inject (SC) postoperative pain reliever, banamine
(rats, 0.05 mg/kg; mice, 0.1 mg/kg), once the vital signs such
as the toe pinch reflex disappear. Apply ophthalmic ointment to
both eyes to protect corneas from drying. Place the animal into
the heated cage until the anesthesia has taken effect.
Ovarian Ɵssue pieces
IdenƟficaƟon
Heath seal
Air bubble
Fig. 2 Pre-labeled 0.5 cc French straw containing ovarian tissue pieces in freezing solution
476
Yuksel Agca and Cansu Agca