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

Overview

In this video, we describe the intravesical procedure of delivering cancer cells into the bladder of a mouse model. This procedure helps generate an orthotopic bladder tumor model that can be used to develop therapeutic strategies.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Implanting the Tumor

NOTE: Each mouse is implanted with 1 x 10⁵ MB49-PSA cells in 50 µL of DMEM blank media in the bladder. Due to the dead space in the catheter, always prepare extra volume (at least 100 µL extra per mouse). An alternative approach would be to use an air-filled syringe.

1. Cells
   1. One day before implantation, when the cells are approximately 80% confluent, passage the MB49-PSA tumor cells in complete DMEM media (supplemented with 10% FBS, 2 mmol/L L-glutamine, 0.05 mg/mL Penicillin-Streptomycin, and 200 µg/mL Hygromycin B) at 37 °C and 5% CO₂ at a split ratio of 1:2.
   2. On the day of the procedure, harvest the cells by scraping them gently with a sterile cell scraper. Mix equal volumes of the cell suspension and a 0.4% trypan blue solution. Count the live cells using a hemocytometer. Do not implant if more than 20% of the cells are dead.
   3. Calculate the number of live cells required (2 x 10⁶ cells/mL) and transfer them to a 15 mL centrifuge tube. Centrifuge at 250 x g for 5 min at 4 °C and discard the supernatant. Resuspend the cell pellet in DMEM blank media. Repeat the wash three times to remove all traces of FBS before implantation.
   4. Keep the cell suspension on ice until the mice are ready for implantation.
2. Allow the 4- to 6-week-old female C57BL/6J mice to acclimatize for one week before the procedure. All animal work must be performed in a biological safety cabinet to maintain sterile conditions.
   1. Weigh each mouse and inject anesthesia (75 mg/kg ketamine and 1 mg/kg medetomidine) intraperitoneally at 0.1 mL per 10 g of body weight. Bodyweights should be between 17 and 22 g. Confirm anesthetization by observing no response after a toe pinch.
   2. For identification, mark the ear using an ear punch.
   3. Inject Hartmann's solution or compound sodium lactate intraperitoneally at 0.1 mL per 10 g of body weight every 1–2 h for hydration.
   4. Apply sterile ophthalmic ointment to both eyes using a sterile cotton bulb, since the blinking reflex is lost under anesthesia and the eyes dry out. Reapply when necessary.
   5. Lay the mice in the supine position on paper towels on top of a heat pack (activated by contact with air) to maintain body temperature and tape the hind legs down.
3. With a finger, apply gentle pressure to the lower abdominal region and collect urine from the bladder into a 1.5 mL microcentrifuge tube. This sample serves as the basal value of urinary PSA.
4. Withdraw sterile poly-L-lysine (PLL) into a 1 mL syringe and attach a 24-gauge IV catheter, with the needle stylet removed. Apply lubricant to the tip of the catheter and insert the catheter through the urethra into the bladder using forceps to guide the catheterization. Stop when resistance is felt.
   NOTE: Researchers should discuss with their veterinary staff about the need for the use of a lubricating gel with anesthetic for intravesical instillations and the use of post-procedure analgesics.
5. Instill 50 µL of PLL slowly, at a rate of 10 µL every 20 s, to avoid vesicoureteral reflux. Leave the catheter in the bladder for 20 min with a stopper to prevent out-flow.
6. After 20 min, remove the catheter and vacate the bladder of any contents by gently pressing on the lower abdominal region. Using an empty 1 mL syringe, flush any remaining contents out of the catheter.
7. Mix MB49-PSA cells thoroughly by pipetting and withdraw them into a 1 mL syringe. Attach the catheter and apply lubricant. Instill 50 µL of 1 x 10⁵ cells at a slow rate of 10 µL every 20 s. Replace the stopper on the catheter. Give control mice 50 µL of saline.
8. After 1 h, remove the catheters and vacate the bladder of any contents. Remove the tape on the hind legs and place the mice on their ventral sides. Revive the mice by subcutaneously injecting the reversal drug (1 mg/kg atipamezole) at 0.1 mL per 10 g of body weight.
9. Monitor the mice until motility is observed. Return the mice to their cages.

Materials

Name	Company	Catalog Number	Comments
MB49-PSA cells			Ref (Wu QH, 2004)
Dulbecco's Modified Eagle's Medium (DMEM) media	Biowest	L0102
Fetal Bovine Serum (FBS) South American	Biowest	S1810
Fetal Bovine Serum (FBS) South American, Premium	Biowest	S181B
Fetal Bovine Serum (FBS)	HyClone	SH30088.03
L-glutamine Biowest X0550	Biowest	X0550
Penicillin-Streptomycin	Biowest	L0022
Hygromycin B	Invitrogen	10687-010
C57BL/6J female mice	In Vivos		4-6 wk old
Anesthesia (75 mg/kg Ketamine and 1 mg/kg Medetomidine)	Local pharmacy
Reversal drug (1 mg/kg Atipamezole)	Local pharmacy
Ear punch	Electron Microscopy Sciences	72893-01
Hartmann's solution or Compound sodium lactate	B Braun
Ophthalmic ointment - Duratears sterile ocular lubricant ointment	Alcon
Heat pack - HotHands handwarmers	Heatmax Inc
Introcan Certo IV catheter	B Braun	4251300	24 G x 3/4″
Aquagel Lubricating jelly	Local pharmacy
Poly-L-lysine solution, 0.01%	Sigma	P4707