Name: transplantation into the mouse ovarian fat pad
Uploaded: 2016-09-07T14:00:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Transplantation Into the Mouse Ovarian Fat Pad at JoVE.com

We would like to thank Dr. Andrew K. Godwin, University of Kansas Medical Center, for providing the human cells HIO118. These studies were supported by the grants from the New York Stem Cell Program (NYSTEM; T028095) and National Institutes of Health/National Institute of Child Health and Human Development (P50HD076210) to AFN, and by the grants from NYSTEM (C028125, C024174 and T028095), National Institutes of Health/National Cancer Institute (CA182413) and Ovarian Cancer Research Fund (327516) to AYN.

All the in vivo work described is approved by the Cornell University Institutional Animal Care and Use Committee.
1. Sample Preparations for Ovarian Fat Pad Assays
<ol>
	<li>Isolation and Culture of Primary Tubal Epithelium (TE) Cells
	<ol>
		<li>Euthanize donor 6- to 8-week-old virgin &#946;-actin-Enhanced Green Fluorescent Protein (EFGP) or &#946;-actin-Discosoma Red Fluorescent Protein (DsRed) mice by CO2 administration followed by cervical dislocation. Verify successful euthanasia by a toe pinch.</li>
		<li>Place an individual mouse on an absorbent drape, ventral side up, and then wipe the abdomen with 70% ethanol. Open the skin by making a lateral incision at the body midline and with fingertips pull the skin above and below the incision toward the head and tail of the mouse.</li>
		<li>Hold the peritoneum using blunt forceps and make a cut with fine scissors to open the body cavity. Gently push the coil of the intestine aside and locate the reproductive organs.</li>
		<li>With fine forceps pick up one uterine horn and cut 0.5 cm above the point where the uterine horns separate. While holding the uterine horn, dissect away connective tissue attached to the uterine horn, uterine tube, ovary and ovarian fat pad. Place dissected reproductive tract in a dish with 6 ml sterile Phosphate Buffered Saline (PBS). Proceed with the second reproductive tract.</li>
		<li>Transfer the dish to a Biosafety cabinet and wash the tissues 3 times in 6 ml sterile PBS. Continue the work under a dissection microscope located in the Biosafety cabinet. Grab the uterine horn with fine tweezers and disconnect the uterine horn from the uterine tube by severing at the utero-tubal junction. Cut out the ovarian bursa surrounding the ovary by using a 25 G needle. 
		Note: After the ovarian bursa has been removed, the dissection is complete and the individual uterine tube remains in the PBS solution.</li>
		<li>Transfer a single uterine tube in a 50 &#181;l drop of PBS to a 3.5 cm dish and mince into 0.1 mm pieces using 28 gauge needles. Transfer to 200 &#181;l digestion buffer 1 (Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) F12 (Ham&#39;s) medium supplemented with 300 units ml-1 Collagenase and 100 units ml-1 Hyaluronidase) and incubate for 1 hr at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>After the incubation, add 1 ml 0.25% Trypsin-Ethylenediaminetetraacetic acid (EDTA) and suspend the solution with the help of a 1 ml pipette tip (aka blue tip) 20 times for 3 minutes. Add 5 ml + 4 &#176;C DMEM/F12 (Ham&#39;s) medium containing 2% fetal bovine serum.</li>
		<li>Collect tissues by centrifugation (600 rcf, 5 min, room temperature (RT)), and add 1 ml digestion buffer 2 (DMEM F12 (Ham&#39;s) medium supplemented with 7 mg ml-1 Dispase II and 10 &#181;g ml-1 Deoxyribonuclease I (DNase I)). Suspend tissue pellet 20 times with help of a 1 ml pipette tip.</li>
		<li>Add 5 ml serum free complete mouse tubal epithelium growth medium (M-TE-GM, Table 1).</li>
		<li>Collect cells by centrifugation (600 rcf, 5 min, RT). Add 1 ml M-TE-GM, suspend and count cells by hemocytometer. Seed 1 x 105 cells in 1 ml per well in a 24-well low attachment plate and incubate in M-TE-GM at 37&#176;C in a 5% CO2 incubator for 4 days.</li>
		<li>Change the medium every day.</li>
		<li>Prepare single cell suspensions of cultured primary suspension TE cells from &#946;-actin-EGFP or &#946;-actin-DsRed mice.
		<ol>
			<li>Collect TE suspension cultures from 24-well low attachment plates. Centrifuge (600 rcf, 5 min, RT), and suspend cell pellets with 1 ml 0.25% Trypsin-EDTA. Incubate for 10 min at 37&#176;C in a 5% CO2 incubator.</li>
			<li>Stop trypsin activity by adding 5 ml DMEM/F12 (Ham&#39;s) medium containing 5% fetal bovine serum. Collect cell pellets by centrifugation (600 rcf, 5 min, RT).</li>
		</ol>
		</li>
		<li>Wash cell pellets twice with 4 ml of cold PBS (4&#176;C), add 1 ml PBS per cell pellet, and suspend. Count cells by hemocytometer and transfer to 1.7 ml centrifugation tubes. Working on ice, add 1 x 105 cells to 10 &#181;l PBS, then add 10 &#181;l basement membrane matrix. Suspend and transport on ice to the animal room. 
		Note: Perform all dissection and tissue culture work in a Biosafety cabinet under sterile conditions.</li>
	</ol>
	</li>
	<li>Preparation of Immortalized Human Cell Line
	<ol>
		<li>Separately grow lentivirus-EGFP and -mCherry labeled human immortalized ovarian surface epithelium cell lineHIO118 until 80 - 90% confluency on 10 cm dishes at 37&#176;C in a 5% CO2 incubator.</li>
		<li>Wash dishes twice with 10 ml PBS, add 1 ml 0.25% Trypsin-EDTA per dish and incubate for 10 min at 37&#176;C in a 5% CO2 incubator. Stop the reaction by adding 10 ml DMEM/F12 (Ham&#39;s) medium containing 5% fetal bovine serum. Collect cell pellets by centrifugation (600 rcf, 5 min, RT).</li>
		<li>Wash cell pellets twice with 10 ml of cold PBS (4&#176;C), add 1 ml PBS per cell pellet, and suspend. Count cells by hemocytometer and transfer to 1.7 ml centrifugation tubes.</li>
		<li>Working on ice, add 1.0 x 106 Lenti-mCherry labeled cells + 1.0 x 106 Lenti-EGFP labeled cells to 10 &#181;l PBS. Add 10 &#181;l basement membrane matrix. Suspend and transport on ice to the animal room.</li>
	</ol>
	</li>
	<li>Preparation of the Uterine Tube
	<ol>
		<li>Dissect individual uterine tubes from 6- to 8-week old virgin &#946;-actin-DsRed mice in PBS as described in steps 1.1.1-1.1.5. Transfer single uterine tubes into a 200 &#181;l drop of PBS under a dissection microscope. Gently uncoil uterine tubes with the help of tweezers and 28 gauge needles by removing the mesosalpinx, a portion of the broad ligament which supports the segments of the uterine tube. Place single uncoiled uterine tubes in a drop of 200 &#181;l ice-cold PBS until the recipient&#39;s ovarian fat pad is exposed and prepared to receive the transplant.</li>
	</ol>
	</li>
</ol>
2. 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 immunodeficiency (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 recipient mouse with a single intra-peritoneal 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 eyes to prevent dryness while the animal is under anesthesia.&#160;Inject the animal subcutaneously with the analgesic Ketoprofen at a dosage of 4 mg/ g body weight to treat for 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 an area 2 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. 
	Note: Critical step: Precise skin incision facilitates wound healing, the use of a scalpel is recommended in step 2.4.</li>
	<li>Using blunt fine forceps pull the ovarian fat pad carefully through the incision towards the midline, minimizing damage to 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 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 all the way 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 into the fat pad incision. For uterine tube transplantation, pick up the uterine tube with fine forceps and place in 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 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 2 stiches of surgical suture and the skin with two small wound clips or surgical suture.</li>
	<li>Repeat steps 2.4-2.8 to transplant a PBS-basement membrane matrix-mixture into the contra-lateral fat pad of the animal which will serve as control. 
	Critical step: After the surgery place the animal on a heating pad, because heat loss is rapid in anesthetized mice.</li>
	<li>Let 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 recumbence. 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 10 days after surgery when the wound has fully closed.</li>
</ol>
3. Histology and Image Acquisition
<ol>
	<li>Graft Collection and Preservation
	<ol>
		<li>Prepare 4% paraformaldehyde solution by mixing 26 ml ddH2O with 4 ml 10x PBS and 10 ml 16% paraformaldehyde solution.</li>
		<li>Dissect in one block, the engrafted ovarian fat pad, ovary, uterine tube, 0.5 cm uterus as described in steps 1.1.1-1.1.4. Immediately place in 2 ml 4% paraformaldehyde and fix for 2 hr, at RT. From the same animal, dissect the non-engrafted contralateral ovarian fat pad system transplanted with PBS-basement membrane matrix mixture as a control (see step 2.9). Fix and process in the same way.</li>
		<li>After fixation wash tissues three times with PBS for 5 min and incubate overnight in PBS at 4 &#176;C.</li>
		<li>For the whole-mount imaging of the ovarian fat pad proceed to step 3.2.1. 
		Note: After the image acquisition tissues can be frozen (3.3.1) or processed for paraffin embedding (3.3.2). Overnight incubation in 30% sucrose in PBS at 4 &#176;C improves quality of frozen tissues.</li>
	</ol>
	</li>
	<li>Image Acquisition
	<ol>
		<li>Place whole-mount ovarian fat pad systems in PBS filled dishes and image using an inverted microscope equipped with epi-fluorescence attachment and high-definition color camera, with 4, 10x objectives, and a stereomicroscope equipped with a fluorescence attachment, color camera and Plan Apo 1xWD70, ED Plan 1.5xWD45 objectives.</li>
	</ol>
	</li>
	<li>Histology
	<ol>
		<li>Following whole-mount image acquisition (3.2.1) place a 200-500 &#181;l drop of embedding medium for frozen tissue specimens on a 2 x 1 cm piece of laboratory film and using forceps, transfer the tissue to the drop. Pick up the film at one edge and transfer the tissue-medium drop to a 1.8 ml cryogenic vial. Close the vial and plunge into liquid nitrogen. Store frozen tissues at -80 &#176;C.</li>
		<li>Alternatively, proceed with the standard paraffin embedding. Use 20-40 volumes of tissue volume for each step.
		<ol>
			<li>Immerse tissues once in 65% ethanol for 30 min, shaking gently at RT.</li>
			<li>Immerse tissues once in 70% ethanol for 30 min, shaking gently at RT. At this stage samples can be stored for months at RT.</li>
			<li>Immerse tissues once in 90% ethanol for 30 min, shaking gently at RT.</li>
			<li>Immerse tissues once in 95% ethanol for 30 min, shaking gently at RT.</li>
			<li>Immerse tissues twice in absolute ethanol (200 proof) for 30 min, shaking gently at RT.</li>
			<li>Immerse tissues twice in chloroform for 30 min, shaking gently at RT.</li>
			<li>Immerse tissues once in paraffin for 30 min, shaking gently at 58 &#176;C.</li>
			<li>Immerse tissues once in paraffin for 12 hr, shaking gently at 58 &#176;C.</li>
			<li>Immerse tissues once in paraffin for 3 hr, at 58 &#176;C.</li>
			<li>Place tissues in metal histology molds and fill molds with 58 &#176;C paraffin. Add a plastic histology cassette on top of the paraffin and cool down paraffin to +4 &#176;C. Extract paraffin-tissue block and store at RT. 
			Note: Paraffin temperature should not exceed 58 &#176;C for optimal preservation of tissue suitable for immunohistochemical staining.</li>
		</ol>
		</li>
		<li>Alternatively, Prepare for the Paraffin Embedding with Specimen Processing Gel.
		<ol>
			<li>Aspirate the PBS and transfer whole-mount tissues to 70% ethanol. Incubate at room temperature for 2 hr. In a water bath, preheat specimen processing gel to 60 &#176;C.</li>
			<li>Pipette 200 &#181;l of the specimen processing gel onto a laboratory film-lined Petri dish. Using fine dissection forceps transfer the whole-mount tissue system to the specimen processing gel drop.</li>
			<li>Allow the specimen processing gel plug to solidify at room temperature, or solidify the plug for 10 min at 4 &#176;C.</li>
			<li>Place the specimen processing gel embedded tissue in histology tissue cassettes sandwiched between biopsy foam pads. Embed in paraffin as in steps 3.3.2-3.3.2.10. 
			Note: Specimen processing gel embedding prevents loss and allows preservation of small fragile tissue samples. 
			Note: Tissues preserved by freezing or paraffin embedding are suitable for immunohistochemical staining14,15.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We have established an ovarian fat pad transplantation assay which allows for the accurate delivery and detection of epithelial cells and tissues. The ovarian fat pad assay transplantation procedure is less technically challenging than intrabursal injection10-12 and allows for more uniform and precise location of transplanted cells as compared to intraperitoneal injection9. It is also well suited for long-term transplantation of an entire organ, such as the uterine tube.
Importantly, the ovarian fat pad provides a familiar microenvironment for epithelia of the female reproductive system. It should be particularly suitable for studies of the molecular and cellular properties of human and mouse ovarian and tubal epithelium cells during regeneration and malignant transformation. In contrast to other organs, few studies have focused on identification and characterization of stem cells in the epithelia of the female reproductive tract18,19. Such studies are of particular importance because many cancers are believed to originate from adult stem cells20. Yet cellular origins of epithelial ovarian cancers remain highly debatable18. Epithelial ovarian cancers account for the majority of ovarian cancers and is in 5th place among all cancer related deaths of women in the US21. Thus development of an orthotopic assay with several advantages over the existing transplantation techniques9-12 should greatly facilitate studies in this area.
Given the superficial location of the ovarian fat pad, small animal imaging systems can be used to follow up on engraftments labeled with strong fluorescent or bioluminescent reporters. It also should be possible to establish minimally invasive high resolution live imaging assays, such as non-linear microscopy22. The ovarian fat pad can also be kept in organ cultures, thereby allowing three-dimensional culture of normal and neoplastic epithelia under conditions closely recapitulating the organization of cells and the extracellular matrix. Future studies should address these promising possibilities.
Several critical steps have to be considered. Providing a heating pad and keeping rodents warm during surgery improves greatly the survival rate. Sterility of instruments handling the fat pad ensures that the engrafted systems are kept sterile. In our experience, significant bleeding results in growth retardation of transplants. Importantly, the fat pad incision should be made precisely because tearing the fat pad or puncturing it through causes bleeding. Coagulants should be used to stop bleeding as necessary. If engraftments do not develop, a higher concentration of injected cells should be considered. The first successful outgrowths can be observed as early as one week after the transplantation and most engraftments should be visible one month after the procedure. For transplantation, needles with larger diameter (23-25 G) can be used to prevent shearing of large cells (over 10 &#181;m in diameter). However, such needles may be more traumatic to the fat pad and their use should be tested in advance. For our experiments we have used 6 to 8 week old donor and recipient mice. For both purposes younger or older mice can be used. However, the number of injected cells may need to be adjusted. The smaller size of ovarian fat pads in younger donors will also need to be considered.
As with any methods, there are some limitations of the mouse ovarian fat pad transplantation assay. Although syngeneic immuno-competent mice can be used for murine primary cells/tissues, our technique requires NSG or SCID mice for transplantation of human material. It is likely possible to humanize the fat pad to support the growth of human epithelial cells. However, we have not tested this approach yet. Use of younger females may to lead to lower breeding costs; however, mature virgin female mice (age 8 to 10 weeks) have a larger fat pad, thereby allowing transplantation of larger specimens. Finally, laboratory personnel should be trained in animal surgery to ensure timely and successful execution of the procedure.

Transplantation assays, which involve the introduction of cells and tissues into specific body sites in mice, represent an essential approach for studies of tissue regeneration and carcinogenesis. Orthotopic transplantations of cells, that is, their placement into a native milieu, are particularly important for characterization of adult stem cells. The existence of mammary stem cells was first suggested by the transplantation of epithelial mammary gland fragments into gland-free mammary fat pads of mice1. Engraftment assays to humanized mouse fat pad2 were used to recapitulate the regenerative potential and normal development of human primary breast epithelial cells. Moreover, serial and limiting dilution transplantations of phenotypically distinct cell types into the cleared mammary fat pad were crucial to test the self-renewal capacity and multilineage differentiation of mammary stem cells3-5. Transplantation assays in combination with genetic lineage tracing provided evidence of the cell of origin in mammary carcinogenesis5. Kidney capsule transplantations are commonly used for testing properties of putative prostate stem cells6. Orthotopic transplantation of normal and neoplastic mouse and human pancreatic organoids revealed genes and pathways altered in cancer progression7. The importance of the native environment has also been supported by the demonstration of diverse regeneration after engraftments of sweat glands into different locations, such as mammary fat pads, shoulder fat pads, and back skin8.
The peritoneal cavity and the ovarian bursa are usually used as places for orthotopic transplantation of epithelial cells of the female reproductive tract9-12. Both of these methods have a number of limitations. Intraperitoneal injection of cells leads to their implantations in different areas of peritoneal cavity, thereby complicating monitoring cellular growth. Furthermore, variations in the microenvironment, such as the extent of vascularization, innervation and immune cell representation, may be quite significant in different areas of the peritoneal cavity. The ovarian bursa has a very limited volume, allowing injection of no more than 10 &#181;l of liquid. This significantly limits the amount of cells that can be administered. Furthermore, injections into the ovarian bursa can be quite technically challenging and the procedure requires a significant amount of time.
To address these limitations, we have taken advantage of the ovarian fat pad properties. The mouse ovarian fat pad has a large size, is adjacent to the ovary and the uterine tube, and is easily accessible by surgery. It has been reported that equine trophoblast can be transplanted into the mouse ovarian fat pad13. However the details of this method were not described. It was also not reported if this method can be applied to adult cells and tissues. Here, we describe a reliable approach for the transplantation of mouse and human cells of the female reproductive tract and show that this method is also applicable to long-term organ transplantation.

<table><tbody><tr><td>25 gauge needle</td><td>BD Becton Dickinson</td><td>305122</td><td></td></tr><tr><td>28 gauge needle (hypodermic syringe with attached needle)</td><td>Kendall</td><td>30339</td><td>Part Number: 8881500014</td></tr><tr><td>basic fibroblast growth factor-2</td><td>Sigma-Aldrich</td><td>F9786</td><td></td></tr><tr><td>basement membrane matrix (Geltrex)</td><td>Invitrogen</td><td>A1413202</td><td></td></tr><tr><td>&amp;beta;-actin-DsRed mice</td><td>The Jackson Laboratory</td><td>6051</td><td>B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J</td></tr><tr><td>&amp;beta;-actin-EGFP mice</td><td>The Jackson Laboratory</td><td>3291</td><td>C57BL/6-Tg(CAG-eGFP)1Osb/J</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M7522</td><td></td></tr><tr><td>bovine albumin</td><td>Sigma-Aldrich</td><td>A3311</td><td></td></tr><tr><td>chloroform</td><td>VWR</td><td>EM-CX1058-1</td><td></td></tr><tr><td>collagenase/hyaluronidase</td><td>Stem Cell Technologies</td><td>7912</td><td></td></tr><tr><td>cryogenic vials</td><td>Thermo Scientific Nunc</td><td>377267</td><td></td></tr><tr><td>dispase II</td><td>Worthington</td><td>NPRO2</td><td></td></tr><tr><td>DMEM F12 (HAM&#039;s)</td><td>Corning</td><td>10-092-CV</td><td></td></tr><tr><td>DNaseI (Deoxyribonuclease I)</td><td>Stem Cell Technologies</td><td>7900</td><td></td></tr><tr><td>embedding medium for frozen tissue specimens (O. C. T.)</td><td>Sakura Finetek&amp;nbsp;</td><td>4583</td><td></td></tr><tr><td>epidermal growth factor</td><td>Sigma-Aldrich</td><td>E4127</td><td></td></tr><tr><td>ethanol 200 proof</td><td>Koptec</td><td>V1001</td><td>to prepare 70%&amp;nbsp;</td></tr><tr><td>fetal bovine serum</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>filter tip 0.1-10/20 &amp;micro;l XL&amp;nbsp;</td><td>USA Scientific</td><td>1120-3810</td><td></td></tr><tr><td>FOXJ1 antibody</td><td>eBioscience</td><td>E10109-1632</td><td>used at concentration 1:1,000 (no unmasking)</td></tr><tr><td>hydrocortisone</td><td>Sigma</td><td>H0135</td><td></td></tr><tr><td>Ketoprofen</td><td>Zoetis</td><td>4396H</td><td></td></tr><tr><td>laboratory film (Parafilm M)</td><td>American National Can</td><td>PM-999</td><td></td></tr><tr><td>L-Glutamine</td><td>Corning</td><td>25-005-Cl</td><td></td></tr><tr><td>insulin-transferin-sodium selenite</td><td>Sigma-Aldrich</td><td>I884</td><td></td></tr><tr><td>isoflurane, 250 ml</td><td>Santa Cruz Animal Health</td><td>sc-363629Rx</td><td></td></tr><tr><td>low attachment plate 24-well</td><td>Costar</td><td>3473</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>Corning</td><td>25-025-Cl</td><td></td></tr><tr><td>metal histology molds</td><td>Electron Microscopy Sciences</td><td>62527-22</td><td></td></tr><tr><td>microscope, inverted</td><td>Nikon</td><td>Eclipse TS 100</td><td></td></tr><tr><td>microscope, stereo</td><td>Nikon</td><td>SMZ800</td><td></td></tr><tr><td>Nod/SCID/gamma (NSG) mice</td><td>The Jackson Laboratory</td><td>05557</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>paraformaldehyde 16% solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>paraffin (Paraplast, X-TRA)</td><td>Fisher Thermo Scientific</td><td>23-021-401</td><td></td></tr><tr><td>PAX8 antibody</td><td>Proteintech</td><td>10336-1-AP</td><td>used at concentration 1:1,000 (no unmasking)</td></tr><tr><td>PBS (Phosphate-Buffered Saline)</td><td>Corning</td><td>&amp;nbsp;21-030-CV</td><td></td></tr><tr><td>penicillin streptomycin</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>severe combined immunodeficiency (SCID/NCr) mice, BALB/C background)</td><td>NCI-Frederick Animal Production Program</td><td>01S11</td><td></td></tr><tr><td>sodium pyruvate</td><td>Corning</td><td>25-000-Cl</td><td></td></tr><tr><td>specimen processing gel (HISTOGEL)</td><td>Richard-Allan Scientific</td><td>HG-4000-012</td><td></td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Trypsin-EDTA, 25%</td><td>Corning</td><td>25-053-Cl</td><td></td></tr></tbody></table>

transplantation into the mouse ovarian fat pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

Experimental cells and tissues can be accurately transplanted into the ovarian fat pad under a dissection microscope (Figure 1). The examples include primary mouse tubal epithelium cells derived from mice ubiquitously expressing EGFP (Figure 2A) or DsRed (Figure 2B) and human immortalized ovarian surface epithelium cells HIO11816,17cells labeled with mCherry and EGFP lentiviruses (Figure 2C-F). The approach is also applicable for long-term transplantation of organs, such as the mouse uterine tube (Figure 3). According to immunohistochemical staining, both ciliated (FOXJ1 positive14) and secretory (PAX8 positive15) cells are preserved in the tubal epithelium of transplanted tissues (Figure 3D and E).
<img alt="Figure 1" src="/files/ftp_upload/54444/54444fig1.jpg" /> 
Figure 1: Ovarian Fat Pad Transplantation. (A) Exposed area of transplantation (arrow). (B) An incision is made by a 28 G needle (arrow). (C) UT/basement membrane matrix mixture engraftment by pipette tip (arrow). (D) UT engrafted (arrow, bright red, UT of &#946;-actin-DsRed mice). FP, fat pad; Ov, ovary; UT, uterine tube. Scale bar = 2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54444/54444fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54444/54444fig2.jpg" /> 
Figure 2: Detection of Transplanted Mouse Primary Tubal Epithelium Cells and Human Immortalized Ovarian Surface Epithelium Cells. (A, B) Outgrowths of primary mouse tubal epithelium cells (arrows) of &#946;-actin-EGFP mice (A, green) and &#946;-actin-DsRed (B, red) 8 days after transplantation into a syngeneic mouse. (C, D) Engraftments of mixed HIO118 cells (arrows) labeled with either Lenti-EGFP (green) or Lenti-mCherry (red) 10 days after transplantation into NSG mice. (D) Enlarged area from (C, arrow). (E, F) Graft developed 43 days after transplantation of the same cells as in C, D to SCID mouse (arrows). A-D, F, fluorescence; E, bright field, FP, fat pad; Ov, ovary; UT, uterine tube. (A, B, D-F) Scale bar = 500 &#181;m; (C) Scale bar = 1,600 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54444/54444fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54444/54444fig3.jpg" /> 
Figure 3: Characterization of Uterine Tube Transplant. (A) Complete uterine tube (arrow) of &#946;-actin-DsRed mouse 6 days after transplantation into the ovarian fat pad (arrow, bright red). (B) Fluorescent frozen section of the fat pad with transplant shown in (A). Red, DsRed; Blue, 4&#39;,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining. (C) Uterine tube graft (arrow) 40 days after transplantation into NSG recipient mouse (arrow). Paraffin section. Note correct preservation of all principle uterine tube components, and lack of transplantation-associated fibrosis and inflammation. Hematoxylin and eosin staining. (D, E) Detection of tubal epithelium markers Paired-box gene 8 (PAX8) and Forkhead box J1 (FOXJ1; brown nuclear color, arrowheads) in cells of the graft (arrows) shown in (C). Immunoperoxidase system. Counterstaining with methyl green. FP, fat pad; Ov, ovary; UT, uterine tube. (A) Scale bar = 1,000 &#181;m; (B)&#160;Scale bar = 200 &#181;m; (C-E) Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54444/54444fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Component</td>
			<td>1x DMEM F12 (Ham&#8217;s) medium</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Sodium pruvate</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>10 ng ml-1</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor-2</td>
			<td>10 ng ml-1</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>500 ng ml-1</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>5 &#181;g ml-1</td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>5 &#181;g ml-1</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>5 ng ml-1</td>
		</tr>
		<tr>
			<td>Bovine albumin</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>100 units ml-1 / 100 &#181;g ml-1</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle (MEM) non-essential amino acids</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>10-4 M</td>
		</tr>
	</tbody>
</table>
Table 1: Mouse Tubal Epithelim Growth Medium (M-TE-GM). Components listed in the left column are dissolved in 1x DMEM F12 (Ham&#39;s) medium at the indicated concentrations, right column. The final medium composition is serum free.

Watch this Scientific Journal Video about Transplantation Into the Mouse Ovarian Fat Pad at JoVE.com

Transplantation Into the Mouse Ovarian Fat Pad

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

transplantation-into-the-mouse-ovarian-fat-pad

Nanyang Technological University

Research

JoVE Journal

Medicine

11.3K Views.  Cornell University. We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of Müllerian origin.

Video: Transplantation Into the Mouse Ovarian Fat Pad

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Immunology and Infection

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Cancer Research

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Developmental Biology

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Biology

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Bioengineering

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining&#160;natural immunodeficiency up to day 17 postfertilization, making it ideal to study&#160;short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Here, we describe a protocol for developing a chick chorioallantoic membrane (CAM) xenografting model for human ovarian tissue and demonstrate the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle ...

Harvesting Peritoneal Fat Depots: A Technique to Study Ovarian Cancer Colonization of Peritoneal Adipose Tissues in Mouse Models

Source: Krishnan, V. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/52721">In Vivo</a><a href="https://www-jove-com.remotexs.ntu.edu.sg/t/52721">&#160;and&#160;Ex Vivo&#160;Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we use a euthanized female mouse model bearing ovarian cancer. We then describe a protocol to identify and extract fat depots or adipose tissues associated with the five primary peritoneal organs to study metastatic cancer cell colonization.

Source: Krishnan, V. et al. In Vivo&#160;and&#160;Ex Vivo&#160;Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Animal model studies

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

Source: Flesken-Nikitin, A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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 ...

Intact Ovary Harvest from Mouse: A Surgical Procedure to Isolate Complete Ovary from Murine Model

Source: Mason, J. B. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/56638">Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice.</a> J. Vis. Exp. (2018)
In this video, we describe a surgical procedure to harvest intact ovary from a mouse model.

Source: Mason, J. B. et al. Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in ...

Transplantation Into the Mouse Ovarian Fat Pad

Summary

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Chapters in this video

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

A Method for Islet Transplantation to the Omentum in Mouse

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Harvesting Peritoneal Fat Depots: A Technique to Study Ovarian Cancer Colonization of Peritoneal Adipose Tissues in Mouse Models

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

Intact Ovary Harvest from Mouse: A Surgical Procedure to Isolate Complete Ovary from Murine Model