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 border="0" cellpadding="0" cellspacing="0" width="1170">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">25 gauge needle</td>
			<td style="width:339px;">BD Becton Dickinson</td>
			<td style="width:155px;">305122</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">28 gauge needle (hypodermic syringe with attached needle)</td>
			<td style="width:339px;">Kendall</td>
			<td style="width:155px;">30339</td>
			<td>Part Number: 8881500014</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">basic fibroblast growth factor-2</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:155px;">F9786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">basement membrane matrix (Geltrex)</td>
			<td style="width:339px;">Invitrogen</td>
			<td style="width:155px;">A1413202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">&#946;-actin-DsRed mice</td>
			<td style="width:339px;">The Jackson Laboratory</td>
			<td style="width:155px;">6051</td>
			<td>B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">&#946;-actin-EGFP mice</td>
			<td style="width:339px;">The Jackson Laboratory</td>
			<td style="width:155px;">3291</td>
			<td>C57BL/6-Tg(CAG-eGFP)1Osb/J</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">&#946;-mercaptoethanol</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:155px;">M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">bovine albumin</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:155px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">chloroform</td>
			<td style="width:339px;">VWR</td>
			<td style="width:155px;">EM-CX1058-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">collagenase/hyaluronidase</td>
			<td style="width:339px;">Stem Cell Technologies</td>
			<td style="width:155px;">7912</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">cryogenic vials</td>
			<td style="width:339px;">Thermo Scientific Nunc</td>
			<td style="width:155px;">377267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">dispase II</td>
			<td style="width:339px;">Worthington</td>
			<td style="width:155px;">NPRO2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DMEM F12 (HAM&#39;s)</td>
			<td style="width:339px;">Corning</td>
			<td style="width:155px;">10-092-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNaseI (Deoxyribonuclease I)</td>
			<td style="width:339px;">Stem Cell Technologies</td>
			<td style="width:155px;">7900</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">embedding medium for frozen tissue specimens (O. C. T.)</td>
			<td style="width:339px;">Sakura Finetek&#160;</td>
			<td style="width:155px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">epidermal growth factor</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:155px;">E4127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">ethanol 200 proof</td>
			<td style="width:339px;">Koptec</td>
			<td style="width:155px;">V1001</td>
			<td>to prepare 70%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">fetal bovine serum</td>
			<td style="width:339px;">Sigma</td>
			<td style="width:155px;">F4135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">filter tip 0.1-10/20&#181;lXL&#160;</td>
			<td style="width:339px;">USA Scientific</td>
			<td style="width:155px;">1120-3810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FOXJ1 antibody</td>
			<td style="width:339px;">eBioscience</td>
			<td style="width:155px;">E10109-1632</td>
			<td>used at concentration 1:1000 (no unmasking)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">hydrocortisone</td>
			<td style="width:339px;">Sigma</td>
			<td style="width:155px;">H0135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">laboratory film (Parafilm M)</td>
			<td style="width:339px;">American National Can</td>
			<td style="width:155px;">PM-999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">L-Glutamine</td>
			<td style="width:339px;">Corning</td>
			<td style="width:155px;">25-005-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">insulin-transferin-sodium selenite</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:155px;">I884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">isoflurane, 250 ml</td>
			<td style="width:339px;">Santa Cruz Animal Health</td>
			<td style="width:155px;">sc-363629Rx</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">low attachment plate 24-well</td>
			<td style="width:339px;">Costar</td>
			<td style="width:155px;">3473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">MEM non-essential amino acids</td>
			<td style="width:339px;">Corning</td>
			<td style="width:155px;">25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">metal histology molds</td>
			<td style="width:339px;">Electron Microscopy Sciences</td>
			<td style="width:155px;">62527-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">microscope, inverted</td>
			<td style="width:339px;">Nikon</td>
			<td style="width:155px;">Eclipse TS 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">microscope, stereo</td>
			<td style="width:339px;">Nikon</td>
			<td style="width:155px;">SMZ800</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:331px;">Nod/SCID/gamma (NSG) mice</td>
			<td style="width:339px;">The Jackson Laboratory</td>
			<td style="width:155px;">05557</td>
			<td>NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">paraformaldehyde 16% solution</td>
			<td style="width:339px;">Electron Microscopy Sciences</td>
			<td style="width:155px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">paraffin (Paraplast, X-TRA)</td>
			<td style="width:339px;">Fisher Thermo Scientific</td>
			<td style="width:155px;">23-021-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">PAX8 antibody</td>
			<td style="width:339px;">Proteintech</td>
			<td style="width:155px;">10336-1-AP</td>
			<td>used at concentration 1:1000 (no unmasking)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">PBS (Phosphate-Buffered Saline)</td>
			<td style="width:339px;">Corning</td>
			<td style="width:155px;">&#160;21-030-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">penicillin streptomycin</td>
			<td style="width:339px;">Corning</td>
			<td style="width:155px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:331px;">severe combined immunodeficiency (SCID/NCr) mice, BALB/C background)</td>
			<td style="width:339px;">NCI-Frederick Animal Production Program</td>
			<td style="width:155px;">01S11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">sodium pyruvate</td>
			<td style="width:339px;">Corning</td>
			<td style="width:155px;">25-000-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">specimen processing gel (HISTOGEL)</td>
			<td style="width:339px;">Richard-Allan Scientific</td>
			<td style="width:155px;">HG-4000-012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">sucrose</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:155px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Trypsin-EDTA, 25%</td>
			<td style="width:339px;">Corning</td>
			<td style="width:155px;">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/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/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/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

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

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).