Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

The scope of our research group is fertility protection in young female cancer patient. It is important to improve existing protocols to increase the chances of a later pregnancy. That includes the vitrification of ovarian tissue and also the in-vitro growth of follicles.

With the in-vitro growth of ovarian follicles, the crucial point is the multi-step tissue culture approach prior to the follicular isolation procedure, as ovarian cortex tissue is very dense and limits follicular growth. Tissue vitrification is more complex than the slow freezing procedure, but it simplifies the whole process and has other advantages as well. We have shown that vitrification is a significant alternative to the common slow freezing protocol, which is supported by the previously reported five successful deliveries.

The stress of freezing and thawing, as well as the undersupply after reimplantation, together with the reperfusion damage, impacts follicular survival and leads to a significant decrease in the number of the follicles. Limiting the success of the therapy option, vitrification is thought to preserve the follicles with a similar or even greater success compared to slow-freezing approaches. We aim, therefore, to bring vitrification to a pre-clinical setting, that will provide all cryobank the possibility for tissue cryo-preservation, despite any personal and financial limitations.

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 one into the first well of the six well plate. Using the cell strainer, equilibrate the ovarian cortex tissue for five minutes in well one of the plate. Move the cell strainer with the cortex tissue to well two, containing five milliliters of vitrification solution two and equilibrate for five minutes.

Then place the strainer containing cortex tissue into well three of a six-well plate containing five milliliters of vitrification solution three for six minutes. Fill the prepared cryo vial with sterilized liquid nitrogen and place it into the cryo doer 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 one of a sterile six well plate, then transfer six milliliters of RS each to wells two and three. 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 cryopreserved 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.