Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models

Transplantation allows the implantation of cells or tissue fragments into specific body sites in a recipient animal. It helps us study the role of the microenvironment in tumorigenesis and metastasis.

To begin, prep an anesthetized immunocompromised female mouse in the prone position. Make a surgical incision in the dorsomedial position to expose its reproductive tract. Locate the ovarian fat pads situated close to the ovary and oviduct. Using a sharp-tipped needle, make an incision puncturing halfway through the fat pad without compromising the ovary.

Next, inject a chilled suspension of experimental cells mixed with a suitable basement membrane matrix into the fat pad incision. Allow the matrix to solidify and secure the cells within. Place the ovarian fat pad back into the peritoneum and suture the incision. Allow the mouse to recover.

On successful transplantation, the fat pad provides a suitable microenvironment, facilitating the expansion of the injected cells. Check the mouse weekly to observe outgrowth of transplanted cells on the ovarian fat pad.

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.