The overall goal of this protocol is to xenograft cryopreserved human ovarian tissue in combination with engineered endothelial cells that accelerate re-perfusion and provide paracrine stimulus that modulates follicular activation within the graft. This method provides a cell-based means of improving post-transplant viability of cryopreserved ovarian tissue and more broadly, establishes a robust model system for interrogating molecular regulation of human folliculogenesis. The main advantage of this technique is that it uses a single vehicle that of endothelial cells to both mitigate ischemia and provide a sustained source of secreted factors to engraft the tissue.
So this method provide global benefit for tissue viability. It can also provide insight into molecular regulators that have been implicated in follicular development. To make a fibrin clot, work quickly and add 360 microliters of HEPES buffer to 200 microliters of the fibrinogen stock solution.
Gently pipette to mix and keep the tube on ice. In the second microfuge tube, prepare thrombin working solution by adding 148.7 microliters of DMEM to 50 microliters of thrombin stock solution. Gently pipette to mix.
Then add 1.3 microliters of one mole per liter of calcium chloride. Gently pipette to mix and keep the sample on ice. Using the enzyme cell detachment medium Accutase, detach previously prepared engineered endothelial cells from the plate and transfer them to a third microfuge tube.
Then, spin down the cells. Next, using a hemocytometer and a covered glass, count the cells. To calculate the number of cells needed for the engraftment, convert a 20, 000 times area of ovarian tissue into millimeter squared by using a ruler to measure the dimensions.
The multiply the area by 20, 000 to determine the number of cells needed for the engraftment. Spin down the required cells. And use DMEM to re-suspend the pellet for a total volume of 16.8 microliters.
Keep the cell suspension on ice. Next, place a piece of ovarian tissue on a sterile gauze sponge for a few seconds to dry it. Then transfer the tissue onto a previously prepared plastic paraffin film in a 50 millimeter petri dish.
Add 39.2 microliters of the fibrinogen solution into the prepared cell suspension. Mix by gentle pipetting, and keep the suspension on ice. Then working quickly, add 14 microliters of the thrombin solution to the tube and gently pipette once to mix.
Pipette the mixture on top of the ovarian tissue in the shape of an elongated droplet. Incubate the petri dish with the clot at 37 degrees Celsius. Work quickly once you have added the thrombin solution to the fibrinogen cell suspension.
Synchronizing the future preparation in surgery is a challenge and it may be prudent to perform a practice run prior to expending precious human tissue. After anesthetizing the 10 to 14 week old female NSG mouse, and preparing it for surgery according to the text protocol, heat the animal and monitor the body temperature throughout the procedure. With a povidone iodine swab stick, clean the trimmed area and then use sterile alcohol to wipe it off.
Next, place a drop of sterile ocular lubricant vet ointment over each of the mouse's eyes to protect them from dehydration and damage to the cornea. After performing a bilateral oophorectomy according to the text protocol, inject buprenorphine subcutaneous. Then make a horizontal incision in the fascia above the gluteus maximus at a length that fits the clot dimensions.
Create a pocket within this virtual space by opening the scissors underneath the fascia. Pick up one piece of previously prepared encapsulated cortical tissue and place it in the pocket. Then use six O braided absorbable suture to suture the fascia before repeating the co-transplant on the contra-lateral side.
Close the dorsal wall with four O monofilament absorbable suture in simple interrupted stitches. Inject lidocaine subcutaneous at the incision site. Then use a sterile alcohol prep to clean the skin and carry out post-operative care according to the text protocol.
As seen here, two weeks after bilateral engraftment of E4-ORF1 treated human umbilical vein endothelial cells into NSG female mice, functionally perfused vessels formed from GFP labeled exogenous ECs or ExECs at the interface of host tissue and the graft. ExECs formed functional vessels when co-transplanted with ovarian tissue in cortical pieces co-transplanted with the mouse or human ExECs, increase the number of surviving follicles relative to control grafts. This increase in follicle development was associated with a decreased fibrotic area in ExEC assisted grafts.
Final fibrotic area values were calculated for each graft as the average of three sections analyzed. Importantly, grafts that were co-transplanted with human foreskin fibroblasts showed poor tissue viability and relatively few follicles. 14 weeks after engraftment, mice were stimulated with menotropins.
And the progression of follicle growth was monitored via MRI. Developing follicles were noted in both control and ExEC assisted grafts. However, more and larger sized follicles were noticed in the ExEC assisted grafts.
Compared to antral follicles derived from the same patient's tissue, the granulosa cell layer in the more advanced follicle displayed increased expression of the ovulatory markers CD44 and HABP1, as well as increased cell death and reduced proliferation. After watching this video, you will understand how to perform co-transplantation of cryopreserved ovarian tissue with endothelial cells in a fibrin clot. And once mastered, you should be able to complete this protocol in less than one hour.
Building on this approach, endothelial cells can be engineered to express any combination of secreted factors, thereby enabling interrogation of signaling pathways that may be relevant to fertility preservation, folliculogenesis, and/or tissue transplantation.
