Modeling Orthotopic Bladder Tumor in Mouse Model: A Procedure for Intravesical Administration of Cancer Cells into Murine Bladder

Intravesical instillation facilitates cancer cell delivery into the urinary bladder of a recipient animal. To begin, prep an anesthetized female mouse in the supine position on a heating pad to regulate its body temperature. Empty out the bladder of urine.

Next, assemble a catheter connected to a syringe containing poly-L-lysine or PLL solution. Lubricate the catheter tip to ensure smooth urethral insertion and gain access into the bladder. Following insertion, slowly inject the PLL solution into the bladder.

The slow delivery of PLL prevents vesicoureteral reflux - the backflow of urine from the bladder to the ureters and kidneys. Place a stopper on the catheter to prevent premature out-flow. The injected positively-charged PLL molecules interact and adhere to the negatively-charged glycosaminoglycan coating on the inner wall of the bladder.

Remove the catheter and vacate the bladder of unbound PLL solution and urine. Using another catheter-syringe assembly, slowly inject a chilled cancer cell suspension into the bladder to prevent vesicoureteral reflux and ensuing cell implantation in kidneys. Fix the stopper on the catheter.

The altered surface charge of the glycosaminoglycan layer enhances its electrostatic interaction with cancer cells, facilitating their adhesion. Remove the catheter and vacate the bladder of unadhered cancer cells and urine. Over time, the implanted cancer cells within the bladder proliferate to establish primary tumors.

Once a female mouse has been anesthetized, provide hydration by an intraperitoneal injection of Hartmann's solution at 0.1 milliliters per 10 grams of body weight. Repeat this every one or two hours during the anesthesia.

Next, apply sterile ophthalmic ointment to both eyes. Reapply as needed. Then, position the mouse supine on paper towels over a heat pad to maintain body temperature. Next, secure the hind legs with tape. Now, apply gentle pressure to the lower abdominal region and collect urine into a 1.5-milliliter tube.

Next, load a 1-milliliter syringe with sterile PLL and attach a 24-gauge IV catheter with the needle stylet removed. Apply lubricant to the tip of the catheter. Then, insert the catheter into the urethra and, using forceps, guide it to the bladder. Stop when resistance is felt. Then, slowly eject 50 microliters of PLL at a rate of 10 microliters every 20 seconds to avoid vesicoureteral reflux.

Now, leave the catheter in the bladder for 20 minutes with a stopper to prevent outflow. After 20 minutes, remove the catheter and vacate the bladder of any contents by gently pressing on the lower abdomen. Then, using a 1-milliliter syringe, flush any remaining contents out of the catheter.

Now, mix the MB49-PSA cells thoroughly by pipetting and load them into a 1-milliliter syringe. Attach the catheter and lubricate it as before. Insert the catheter into the bladder as before and commence with injecting the cells using a gradual ejection of 10 microliters per 20 seconds. Then, wait one hour.

After an hour, remove the catheter and vacate the bladder of any contents. Then, release the mouse. Position it on its ventral side and revive it with an injection of atipamezole. Now, monitor the mouse until it regains sternal recumbency and then return it to its home cage.