Name: generating renal cell carcinoma model: a protocol to develop orthotopic rcc murine model by intrarenal implantation of cancer cells
Uploaded: 2023-04-30T13:53:27.000+00:00
Duration: 1 min 33 s
Description: Source: Murphy, K. A. et al. A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical ...

generating renal cell carcinoma model: a protocol to develop orthotopic rcc murine model by intrarenal implantation of cancer cells

Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells

To prepare the Renca tumor cells, first, attach the cell culture with 0.25% trypsin in HBSS after rinsing with PBS. Neutralize the reaction after 5 minutes with complete RPMI medium. Transfer the cells to a conical tube for centrifugation and resuspend the pellet in fresh HBSS.
After counting, adjust the volume to a 2 times 10 to the sixth cells per milliliter concentration in HBSS, and draw the cells into a 1-milliliter syringe equipped with an 18-gauge needle. Next, place a sterile surgical drape over a heating pad and confirm the appropriate level of sedation by toe pinch. Remove the hair from the left flank of the mouse and apply vet ointment to the eyes.
Then, swab the implantation site with 5% povidone-iodine antiseptic, and use a sterile scalpel to make a single 1 to 1.5-centimeter incision on the left flank, taking care not to penetrate the peritoneum. Using scissors, separate the dermis from the peritoneum, dabbing the incision site with sterile cotton gauze to remove any blood. Then, grasp the head with the left hand and support the flank with the right hand to locate the spleen through the peritoneum.
Using one finger from the right hand, palpate the mouse from underneath. The kidneys should become visible. Keeping a firm hold on the animal to provide tension across the peritoneum and kidney, have an assistant slowly deliver 0.1 milliliters of the Renca cells through the intact peritoneum into the center of the kidney. Hold the needle in place for 5 to 10 seconds to reduce the backflow of the cells. Then, close the incision with a thin layer of cyanoacrylate tissue adhesive.

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1. Implantation of Cells 
To implant Renca cells intrarenally, prepare Renca cells into a single-cell suspension at 2 x 106&#160;cells/mL in Hank&#39;s Balanced Salt Solution (HBSS).
<ol>
	<li>Remove adherent Renca cells from the tissue culture flask using 0.25% trypsin in HBSS. After ~5 min, add complete RPMI to the flask to neutralize the trypsin and transfer the cells to a centrifuge tube for washing. Centrifuge the cells at 300 relative centrifugal g force (rcf x g) for 5 min at 25 &#176;C.</li>
	<li>Decant the supernatant and resuspend the cells in HBSS. Count the cells using a hemocytometer and adjust the volume with HBSS to yield 2 x 106&#160;cells/mL.</li>
	<li>Draw the cells into a 1 mL syringe fitted with an 18-gauge needle to prevent shear stress on the cells. Replace the 18-gauge needle with a 28-gauge needle prior to the injection of the tumor cells. An injection of 0.1 mL delivers 2 x 105&#160;cells into the kidney.</li>
</ol>
2. Surgical Area Preparation
<ol>
	<li>Prepare a sterile surgical area in a biosafety cabinet by placing a sterile surgical drape over a heating pad. Gather all supplies, including forceps, scalpel, scissors, eye drops, 5% povidone-iodine antiseptic, and sterile gauze. Autoclave all instruments prior to use.</li>
	<li>Perform the surgery using an aseptic tip technique. Resterilize instruments between animals with a bead sterilizer. Wear sterile gloves, a lab coat, and a face mask.</li>
</ol>
3. Animal Preparation
<ol>
	<li>Anesthetize the mice with ketamine/xylazine (87.5 mg/kg and 12.5 mg/kg, respectively, i.p.) according to institutional guidelines. Determine anesthetic depth by performing a toe pinch.</li>
	<li>When the animal is considered non-responsive, prepare the injection site (e.g.,&#160;left flank) on Balb/c mice by plucking (with fingers) or shaving (using clippers) the fur.</li>
	<li>Move the mice from the surgery prep area (e.g.,&#160;the housing cage) to the surgical area and apply vet ointment to the eyes to prevent dryness. Liberally swab the prepared incision site with 5% povidone-iodine antiseptic.</li>
	<li>Inject bupivacaine hydrochloride (0.1 mL of a 2.5 mg/mL solution, s.c.) into the area where the incision will be made as a pre-operative analgesia.</li>
	<li>Make a single incision (~1.0 &#160;to 1.5 cm) on the left flank of the animal using a sterile scalpel or scissors carefully so as not to penetrate the peritoneum. Separate the dermis from the peritoneum with sterile scissors. Dab the incision site with sterile cotton gauze to remove any blood.</li>
	<li>Hold the mouse with both hands, using the left hand to hold the head and the right hand to support the flank.</li>
	<li>Identify the spleen through the peritoneum and, with one finger of the right hand, palpate the mouse from underneath; the kidney should now be visible through the peritoneum. Keep a firm hold of the animal to provide tension across the peritoneum and kidney.</li>
	<li>Have a second person inject the tumor cells (prepared in section 1.2) through the intact peritoneum into the center of the kidney using a 1-mL syringe fitted with a 28-gauge needle. Slowly deliver 0.1 mL (2 x 105) of cells. Once the cells are injected, hold the needle in place for 5&#8211;10 s to reduce the backflow of cells.&#160;It is important to note the need for extreme caution during this step, as the person injecting the cells will bring the needle into close proximity to the fingers of the person holding the mouse.</li>
	<li>Remove the needle and close the incision with a thin layer of cyanoacrylate tissue adhesive.</li>
	<li>Place the animal back into a clean cage with a sterile paper towel. Allow the animals to recover on a heating pad. Do not leave the animals unattended during the recovery period. Do not return the animals to standard housing conditions until they are upright and sternal. Follow institutional guidelines in all procedures using laboratory animals.</li>
</ol>

<table><tbody><tr><td>Artificial tears ophthalmic ointment</td><td>Akorn</td><td>NDC 17478-162-35</td><td></td></tr><tr><td>Vetbond Tissue Adhesive&amp;nbsp;&amp;nbsp;</td><td>3M</td><td>CBGBIW011019</td><td></td></tr><tr><td>Betadine Solution (Povidoneiodine, 5%)&amp;nbsp; NDC&amp;nbsp;</td><td>Purdue Products, L.P.</td><td>67618-155-32</td><td></td></tr><tr><td>0.25% Marcaine (Bupivavaine HCl injection)</td><td>Hospira</td><td>0409-1587-50</td><td></td></tr><tr><td>Heating pad&amp;nbsp;</td><td>Sunbeam</td><td></td><td></td></tr><tr><td>Syringes BD</td><td></td><td>309659</td><td></td></tr><tr><td>Gauze&amp;nbsp;&amp;nbsp;</td><td>Venture</td><td>908291</td><td></td></tr><tr><td>Ketamine</td><td>Phoenix Pharmaceuticals NDC</td><td>57319-609-02</td><td></td></tr><tr><td>Xylazine&amp;nbsp;&amp;nbsp;</td><td>Akorn NDC</td><td>59399-111</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salts&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H2387</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Renca</td><td>ATCC</td><td>CRL-2947</td><td></td></tr></tbody></table>

Source: Murphy, K. A. et al. <a href="https://www.jove.com/video/55080" target="_blank">A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies.</a> J. Vis. Exp. (2017)
In this video, we develop an orthotopic mouse model of renal cell carcinoma. This model results in reproducible primary tumors in the injected kidney and metastatic tumors in the lung, allowing the evaluation of disease progression and treatment.

Source: Murphy, K. A. et al. A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical ...

The implantation of renal cell carcinoma, or RCC, cells from a donor mouse to an immunocompromised recipient mouse&#39;s kidney leads to the development of an orthotopic RCC model.
To develop an orthotopic RCC model, begin by placing an anesthetized immunodeficient mouse in the prone position on a dissection board. Now, use a scalpel to make a surgical cut on the left flank of the mouse, keeping the underlying peritoneum intact.
Palpate the mouse by applying gentle pressure from the bottom. This step helps visualize the kidney through the translucent peritoneum. Next, take a syringe containing RCC cell suspension in an appropriate medium. Using a needle, inject the cell suspension into the kidney without damaging the peritoneum.
Retract the needle and seal the incision site using a tissue adhesive. Allow the mouse to recover and monitor the tumor growth at desired intervals. Initially, the injected renal cancer cells get implanted into the kidney. These cells then proliferate to form tumors.
Subsequently, a few cells may detach from the primary tumor and disseminate within the peritoneal cavity. These migrating tumor cells can invade the lung tissue and metastasize to form secondary tumors.

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Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells

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Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells