ovarian fat pad transplantation assay: an in vivo technique to introduce cells into ovarian fat pad in mouse models

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

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.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Ovarian Fat Pad Transplantation
NOTE: Disinfect instruments between each animal surgery. Prepare and arrange all equipment in advance. Dorsal transplantations to the right ovarian fat pad are preferential as this avoids the spleen. However, recipients may have transplants in both ovarian fat pads.

<ol>
	<li>Use syngeneic mice or severe combined immunodeficient (SCID/NCr) mice as recipients of primary cells. For human cells, such as HIO118 cells, use Nod/SCID/gamma (NSG) mice or SCID mice.</li>
	<li>Anesthetize the recipient mouse with a single intraperitoneal injection of 2,2,2-Tribromoethanol at a dosage of 0.15 mL/10 g body weight. Place the animal on a heating pad in the animal hood and confirm proper anesthetization by loss of pedal reflex (toe pinch). Apply vet ointment on the eyes to prevent dryness while the animal is under anesthesia. Inject the animal subcutaneously with the analgesic Ketoprofen at a 4 mg/ g body weight dosage to treat post-surgical pain. 
	NOTE: As an alternative, use isoflurane for anesthesia. Place the animal in the induction chamber and adjust the oxygen flowmeter to 0.8-1.5 L/min and the isoflurane vaporizer to 2-3%. Remove the anesthetized animal from the chamber, let it breathe isoflurane from a mask and set the oxygen flowmeter to 0.4-0.8 L/min and the isoflurane vaporizer to 1.5%, then start the surgery.</li>
	<li>Shave the surgical area with #40 clippers; remove hair from a site two times the surgical area, prepare the shaved skin with three antiseptic scrubs of povidone-iodine, followed by 70% ethanol, and cover with a sterile drape.</li>
	<li>Move the animal under a dissection microscope located in a Biosafety cabinet. Expose the reproductive tract by an incision in the dorsomedial position directly above the ovarian fat pad. 
	CRITICAL STEP: Precise skin incision facilitates wound healing; the use of a scalpel is recommended in step 1.4.</li>
	<li>Using fine blunt forceps, pull the ovarian fat pad carefully through the incision towards the midline, minimizing damage to the nerves and major blood vessels. 
	CRITICAL STEP: If bleeding occurs, stop the surgery and consider transplanting to a different host animal.</li>
	<li>Under the control of the dissection microscope with the help of a 28 gauge beveled needle, make a 2-4 mm deep incision into the ovarian fat pad, 3-4 mm above the ovary. 
	CRITICAL STEP: Ensure that the incision only goes through half of the fat pad; puncturing through to the bottom side causes leakage. Be swift and proceed without delay after this step.</li>
	<li>For cell transplantations, fill a syringe (30-gauge needle) with 10-20 &#181;L of the cell-basement membrane matrix mixture and inject it into the fat pad incision. For uterine tube transplantation, pick up the uterine tube with fine forceps and place it in a 10-20 &#181;L basement membrane matrix kept on ice. Using a 0.1-10/20 &#181;L XL graduated filter tip (shortened by 3 mm), pick up the tissue and basement membrane matrix suspension and release it into the fat pad incision.</li>
	<li>Wait 5 minutes for the basement membrane matrix to solidify and place the reproductive tract back into the peritoneum. Close the muscles with two stitches of surgical suture and the skin with two small wound clips or surgical sutures.</li>
	<li>Repeat steps 1.4-1.8 to transplant a PBS-basement membrane matrix mixture into the animal&#39;s contra-lateral fat pad, which will serve as a control. 
	CRITICAL STEP: After the surgery, place the animal on a heating pad because heat loss is rapid in anesthetized mice.</li>
	<li>Let the animal recover on a heating pad in the native cage and observe the animal until fully awake from the anesthesia. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Place a handful of pre-wet food pellets inside the cage in addition to dry food on the cage after surgery. Apply post-surgical analgesics if needed for the next few days and examine the wound daily. Apply antibiotics according to the approved animal protocol if wound infection occurs. Remove wound clips ten days after surgery when the wound has fully closed.</li>
</ol>

<table><tbody><tr><td>28 gauge needle (hypodermic syringe with attached needle)&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Kendall</td><td>30339</td><td>Part Number: 8881500014</td></tr><tr><td>Basement membrane matrix&lt;br/&gt; (Geltrex)</td><td>Invitrogen</td><td>&amp;nbsp;A1413202</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Koptec&amp;nbsp;</td><td>V1001&amp;nbsp;</td><td>To prepare 70%&amp;nbsp;</td></tr><tr><td>Filter tip 0.1-10/20 &amp;micro;l XL&amp;nbsp;</td><td>USA Scientific&amp;nbsp;</td><td>1120-3810</td><td></td></tr><tr><td>Isoflurane, 250 ml&amp;nbsp;</td><td>Santa Cruz Animal Health&amp;nbsp;</td><td>sc-363629Rx</td><td></td></tr><tr><td>Ketoprofen</td><td>Zoetis</td><td>&amp;nbsp;4396H</td><td></td></tr><tr><td>Microscope, stereo</td><td>Nikon</td><td>&amp;nbsp;SMZ800</td><td></td></tr><tr><td>Nod/SCID/gamma (NSG) mice</td><td>The Jackson Laboratory</td><td>5557</td><td>NOD.Cg-Prkdc&lt;sup&gt;scid&lt;/sup&gt;Il2rg&lt;sup&gt;tm1Wjl&lt;/sup&gt;/SzJ</td></tr><tr><td>severe combined&lt;br/&gt; immunodeficiency (SCID/NCr)&lt;br/&gt; mice, BALB/C background)</td><td>NCI-Frederick Animal Production Program</td><td>01S11</td><td></td></tr></tbody></table>

Source: Flesken-Nikitin, A. et al. <a href="https://www.jove.com/t/54444">Transplantation Into the Mouse Ovarian Fat Pad.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an assay for transplantation of experimental cells and tissue fragments into the mouse ovarian fat pad. This method can be used to monitor cell regeneration and tumor transformation via non-invasive techniques.

Source: Flesken-Nikitin, A. et al. Transplantation Into the Mouse Ovarian Fat Pad. J. Vis. Exp. (2016)
In this video, we demonstrate an assay for ...

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.

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

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