The overall goal of this procedure is to consistently and rapidly produce tumors in the bladders of mice, which can then be used to test bladder cancer therapies. This is accomplished by first instilling cancer cells into mouse bladders using catheterization. Eight days later, the catheterization is repeated to instill an adenovirus expressing a luciferase reporter gene.
Then 24 hours later, viral gene expression is measured using a luciferase assay. Results are obtained that show the conditions under which optimal gene expression can be achieved. Visual demonstration of this technique is critical because the catheterization and installation steps are difficult to learn and because of the potential for damage to the bladder and or the causing leakage of the instilled material.
Two days before performing cell implantation played into T 25 flasks, one times 10 to the six MB 49 cells and high glucose DMEM supplemented with 10%FBS on the day of the procedure. Prepare tripsin for installation by using DMEM base medium to dilute sterile 0.25%tissue culture grade trypsin. Place the solution in a 37 degree Celsius water bath to equilibrate prewarm a heating pad and position it under the nose cones of the anesthesia system that will be used to deliver isof fluorine.
Place a clean absorbent pad over the heating pad after anesthetizing mice with 3%isof fluorine. Perform a toe pinch to verify level of sedation. Place them in a supine position on the heating pad and attach a nose cone with 2%isof fluorine.
Use a non-irritating lubricating gel to lubricate a new sterile 20 4G pediatric venous catheter. Then remove and discard the needle holding the hub of the catheter. Use the thumb and index finger of your opposite hand to spread the hind legs of the mouse and expose the urethral muus.
Gently insert the catheter into the urethra at a 45 degree angle, changing the angle to one parallel with the bench to insert it fully after full insertion. Lift the catheter gently keeping it parallel to the bench and visually confirmed that it is placed in the urethra and not the vagina, which is below it. Repeat with the remaining anesthetized mice.
Then reduce the isof fluorine concentration to 1%Attach a tip to a P 200 pipetter and remove urine from the bladder by applying suction to the external end of the catheter. Remove any remaining urine from the hub of the catheter, then discard the urine and leave the catheter in place Carefully pipette 80 microliters of the warm trips and solution into the hub of the catheter. Avoiding air bubbles.
Attach a one CC air-filled syringe to the catheter hub and slowly depress the plunger. 0.1 to 0.2 ccs to deliver the trypsin into the bladder. Leave the syringe and catheter assemblies in place for 15 minutes.
To prepare cells for implantation. Remove the medium from the previously plated MB 49 cells and add 500 microliters of 0.25%trypsin to the flask. When cells detach at five milliliters of complete DMEM to resuspend the cells remove a 50 microliter aliquot to count and centrifuge the remainder at 1000 RPM for five minutes while the cells are spinning.
Use a hemo cytometer to count the cells and calculate the cells per milliliter as well as the volume of medium needed to bring the cell pellet to four times 10 to the six cells per liter. Pour off the supernatant from the centrifuge cells and resus. Suspend the pellet in DMEM base medium.
Keep the cells at room temperature. After the 15 minute tripsin treatment, detach the airfield syringe from the catheter leaving the catheter in place. Use a P 200 pipetter to remove any remaining tripsin from the bladder by suction and discard immediately pipette 50 microliters of MB 49 cell suspension into the hub of the catheter.
Then attach the one CC air-filled syringe to the hub of the catheter and deliver the cells to the bladder by slowly depressing the plunger. 0.1 to 0.2 cc. Leaving the catheter syringe assembly in place.
Allow the cells to dwell on the bladder for 50 minutes. Then gently withdraw the catheter syringe assembly from the urethra. Reduce the isof fluorine to 0%and allow the mice to recover from anesthesia on the heating pad.
When a mouse has regained its writing reflex, return it to its cage. Caution, this protocol uses adenovirus, which is an infectious agent and should be handled under strict BSL two guidelines. All procedures performed with infectious agents were approved by the institutional biosafety Committee of the Medical University of South Carolina.
Eight days after implanting mice with MB 49 cells checked for hematuria by placing individual animals over a white piece of absorbent paper and gently pressing on the abdomen to blot drops of urine onto the paper. Urine that is pink to red indicates hematuria and is a sign that tumors have established thaw adenoviral stock on ice and use sterile room temperature PBS to dilute it to 10, to the nine pfu per 50 microliters after anesthetizing and catheterizing the mice and removing the urine as described earlier in this video, immediately pipette 50 microliters of virus suspension into the hub of the catheter, then attach an air-filled syringe and depress the plunger to deliver the virus to the bladder. Leaving the catheter syringe assembly place allow the virus to dwell on the bladder for 40 minutes.
Then gently withdraw the catheter syringe assembly from the urethra and discard into 10%bleach solution to inactivate any remaining virus. Allow the mouse to recover from anesthesia before returning it to its cage mark cages to indicate that mice have been instilled with adenovirus virus will be shed into the cage bedding for several days and all cages in bedding must be handled and disinfected appropriately. Hematuria is observed in nearly all mice within eight days after implantation of 200, 000 MB 49 cells as shown here.
Bladder weight more than doubles from 34.7 plus or minus 3.3 milligrams and non-tumor bearing mice to 87.5 plus or minus 19.2 milligrams and mice that have been implanted with MB 49 cells. In terms of gene delivery, imaging mice 24 hours after viral installation yields a stronger signal than after 48 hours. Adenoviral delivery is highly variable between animals, which should be taken into account when planning group size for statistical purposes.
If a small animal imaging system is not available, an alternative approach to measure expression of the luciferase transgene is to remove and homogenize the bladder for in vitro analysis as demonstrated here. Comparison between in vivo analysis using the IVUS 200 small animal system and in vitro analysis using the steady glow kit indicates excellent correlation between data sets. After watching this video, you should have a good understanding of how to catheterize a mouse bladder and instill tumor cells or therapeutic agents.
