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

Protocol

1. Implantation of Cells

To implant Renca cells intrarenally, prepare Renca cells into a single-cell suspension at 2 x 10⁶ cells/mL in Hank's Balanced Salt Solution (HBSS).

1. 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 °C.
2. 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 cells/mL.
3. 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 cells into the kidney.

2. Surgical Area Preparation

1. 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.
2. 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.

3. Animal Preparation

1. 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.
2. When the animal is considered non-responsive, prepare the injection site (e.g., left flank) on Balb/c mice by plucking (with fingers) or shaving (using clippers) the fur.
3. Move the mice from the surgery prep area (e.g., 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.
4. 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.
5. Make a single incision (~1.0  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.
6. Hold the mouse with both hands, using the left hand to hold the head and the right hand to support the flank.
7. 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.
8. 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 5–10 s to reduce the backflow of cells. 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.
9. Remove the needle and close the incision with a thin layer of cyanoacrylate tissue adhesive.
10. 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.

Materials

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