To begin, cut the metal meshes into strips measuring 25 by 8 millimeters. Place the customized meshes in a sterilization container and autoclave for two hours. After autoclaving, place the sterilization container and 1.8 milliliter vials under the laminar flow bench.
Open the sterilization container and remove the metal grid. Fit the grid into the cap of the 1.8 milliliter vial and close the vial. After collecting human ovarian cortex tissue, remove the medulla and cut the tissue into desired shapes.
Add five milliliters of vitrification solution 1 into the first well of the six-well plate. Using the cell strainer, equilibrate the ovarian cortex tissue for five minutes in well 1 of the plate. Move the cell strainer with the cortex tissue to well 2 containing five milliliters of vitrification solution 2 and equilibrate for five minutes.
Then, place the strainer containing cortex tissue into well 3 of a six-well plate, containing five milliliters of vitrification solution 3 for six minutes. Fill the prepared cryo vial with sterilized liquid nitrogen and place it into the cryo dewar vessel filled with liquid nitrogen. Load the tissue samples onto the metal grid of the vitrification device within one minute.
Then insert the metal grid into the liquid nitrogen in the grid-based cryo vial. Add six milliliters of equilibration solution to well 1 of a sterile six-well plate, then transfer six milliliters of RS, each to wells 2 and 3. Incubate for one hour at room temperature.
Warm the rapid warming solution in a sterile sample beaker for one hour on a heating plate set at 37 degrees Celsius. Under the laminar flow bench, open the cryo-preserved vials containing vitrified ovarian cortical tissue. Rapidly transfer the mesh into the rapid warming solution.
Using sterile forceps transfer the tissue to the equilibration solution and incubate for three minutes on a rocking shaker. Then, rinse the tissue at room temperature for 10 minutes each with RS1 and RS2 on a rocking shaker. Dissolve calcein in 100 microliters of DMSO.
Transfer three microliters of calcein into the bottom of a 1.5 milliliter tube and store at minus 20 degrees Celsius. Add 0.007 grams of collagenase in a 1.5 milliliter tube, and store the tube at minus 20 degrees Celsius. Add 997 microliters of DPBS to the frozen three microliters of calcein and re-suspend to dissolve the calcein.
Then add cold collagenase powder to obtain 1000 microliters of working solution. Then add 500 microliters of working calcein solution and transfer two pieces of two millimeters of ovarian cortex fragments into the wells of a four well dish. Then incubate for 90 minutes at 37 degrees Celsius, protecting from light.
Finally, under fluorescence microscopy, determine the follicular viability.
