The overall goal of this ovarian fat pad transplantation assay is to facilitate studies of normal and transformed epithelia of the female reproductive tract in the most favorable native environment. This method can help to answer key questions in the field, such as differentiation, regenerative, and neoplastic potential of specific cell populations of the female reproductive tract. The main advantage of this technique is that the mouse ovarian fat pad allows transplantation of cells and large tissue fragments.
It's easily accessible for surgery and imaging. The peritoneal cavity and the ovarian bursa are frequently used for sites of orthotopic transplantation of epithelial cells of the female reproductive tract. These methods have a number of limitations.
After euthanizing beta-actin EGFP or beta-actin DS red mice according to the text protocol, place an individual mouse on an absorbent drape, ventral side up. Open the skin by making a lateral mid-line incision, and with the fingertips, pull the skin above and below the incision toward the head and tail of the mouse. Using blunt forceps, hold the peritoneum and use fine scissors to open the body cavity.
Then, gently push the coil of the intestine aside and locate the reproductive organs. With fine forceps, pick up one uterine horn and cut 0.5 centimeters above the point where the uterine horns separate. Then, while holding the uterine horn, dissect away connective tissue attached to the uterine horn, uterine tube, ovary and ovarian fat pad.
Place the dissected reproductive tract in a dish with six milliliters of sterile PBS and proceed with the second reproductive tract before culturing primary tubal epithelium, or TE, cells according to the text protocol. After dissecting individual uterine tubes from the uterine horns of six-to eight-week old virgin beta-actin DS red mice in PBS according to the text protocol, under a dissecting microscope, transfer single uterine tubes into a 200 microliter drop of PBS. Using tweezers and 28-gauge needles, gently uncoil uterine tubes by removing the mesosalpinx, a portion of the broad ligament which supports the segments of the uterine tube.
Place single, partially uncoiled uterine tubes in a 200 microliter drop of ice-cold PBS until the recipient's ovarian fat pad is exposed and prepared to receive the transplant. Move the animal to a bio-safety cabinet to carry out a fat pad transplantation. After anesthetizing a syngeneic mouse according to the text protocol, use number 40 clippers to shave an area twice the size of the surgical area.
Use povidone-iodine, followed by 70%ethanol, to perform three surgical scrubs. Next, move the animal under a dissecting microscope. Then, using a scalpel, make an incision in the dorsomedial position directly above the ovarian fat pad and expose the reproductive tract.
Afterwards, cover the area with a sterile drape. Next, with blunt fine forceps, pull the ovarian fat pad carefully through the incision towards the midline, minimizing damage to the nerves and major blood vessels. To prevent growth retardation of transplants, the fat pad incision should be made as precise as possible because tearing or puncturing the fat pad all the way through causes bleeding.
Coagulants should be used to stop bleeding as necessary. Use a 28-gauge beveled needle to make a 2-to 4-millimeter deep incision into the ovarian fat pad 3 to 4 millimeters above the ovary, ensuring that the needle only goes approximately halfway through the fat pad. For cell transplantations, fill a syringe with a 30-gauge needle with 10 to 20 microliters of the cell basement membrane matrix mixture and inject it into the fat pad incision.
For uterine tube transplantation, use fine forceps to pick up the uterine tube and place it in 10 to 20 microliters of basement membrane matrix kept on ice. Then, with a 0.1 to 10 to 20 microliter XL graduated filter tip, pick up the tissue and basement membrane matrix suspension and release it into the fat pad incision. Wait five minutes for the basement membrane matrix to solidify and place the reproductive tract back into the peritoneum.
With two stitches of surgical suture, close the muscles and use small wound clips or surgical sutures to close the skin. After the transplantation, allow the animal to recover according to the text protocol. To collect and preserve grafts, prepare a 4%paraformaldehyde, or PFA, solution by mixing 26 milliliters of doubly-distilled water with four milliliters of 10X PBS and 10 milliliters of 16%PFA solution.
After dissecting in one block the engrafted ovarian fat pad, ovary, uterine tube and uterus, immediately place the tissue in two milliliters of 4%PFA and fix at room temperature for two hours. From the same animal, similarly fix and process the non-engrafted contralateral ovarian fat pad control transplant. After fixation, wash the tissues with PBS three times for five minutes each and incubate in PBS at four degrees Celsius overnight.
Carry out whole-mount imaging and histology according to the text protocol. In this experiment, a uterine tube from a beta-actin DS red mouse was transplanted and engrafted into the ovarian fat pad as seen in this fluorescence image. Shown here are outgrowths of primary mouse tubal epithelial cells from beta-actin EGFP mice and beta-actin DS red mice eight days after transplantation into a syngeneic mouse.
This image shows engraftments of mixed HI0118 cells labeled with either Lenti-eGFP or Lenti-mCherry 10 days after transplantation into an NSG mouse. In these panels, the arrows point out a 43-day old graft from the transplantation of mixed HI0118 cells into a SCID mouse. In this fluorescent image, a complete uterine tube from a beta-actin DS red mouse is shown six days after transplantation into the ovarian fat pad.
A frozen section from the fluorescent fat pad is counterstained with DAPI in this panel. This uterine tube graft at 40 days post-transplantation into an NSG-recipient mouse displays complete preservation of uterine tube components and a lack of fibrosis and inflammation. Finally, sections were stained for the tubal epithelial markers Pax8 and FOXJ1 and counterstained with methyl green to demonstrate the preservation of ciliated and secretory cells, respectively.
Once mastered, this technique can be done in 20 to 30 minutes if done properly. While attempting this procedure, it is important to prepare the surgical plates, reagents and instruments in advance. Following this procedure, other methods, such as intravital imaging and injections of anti-cancer drugs, can help answer additional questions such as cancer formation and effects of anti-cancer therapy.
This technique paves the way for researchers in the fields of reproductive biology and ovarian cancer to explore normal stem-cell functions and cancer development in tissues of the female reproductive tract. After watching this video, you should have a good understanding of how to isolate cells and uterine tube tissue and then transplant them into the fat pad. Don't forget that working with animals in sterile conditions during survival surgery is extremely important for a successful outcome.
Providing a heating pad to keep rodents warm greatly improves survival rate.
