Bladder Tumor Organoid Model: A Method for Orthotopic Transplantation of Bladder Organoids into Recipient Murine Bladder

Samenvatting

Source: Kim, Y. et al. Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids. J. Vis. Exp. (2020)

In this video, we describe the orthotopic transplantation of cultured bladder tumor organoids into the murine bladder to study the role of the organ environment on developing tumors.

Protocol

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

1. Subculture Bladder Tumor Organoids

NOTE: Passage of bladder tumor organoids when they reach 100–150 µm in diameter is recommended.

1. Add 500 µL of collagenase/dispase to the organoid medium in a 24-well plate with tumor organoids. Pipette up and down the basement membrane matrix and the medium. Incubate for 20 min at 37 °C and harvest the cells into a 15 mL tube.
   NOTE: Examine the organoids isolated from the basement membrane matrix under a microscope. If the organoids are not detached from the basement membrane matrix, increase the incubation time or pipette more times.
2. Add 5 mL of prewarmed DMEM, centrifuge the tube at 400 x g for 3 min at 4 °C, and aspirate the supernatant.
3. Resuspend the pellet using 1 mL of prewarmed 0.25% trypsin-EDTA and 10 µM Y-27632. Incubate for 5 min in a 37 °C water bath. Vigorously pipette the cells up and down and neutralize the trypsin using 5 mL of DMEM with 10% FBS.
4. Centrifuge the tube at 400 x g for 3 min at 4 °C and aspirate the supernatant.
5. Resuspend the pellet using 1 mL of prewarmed organoid medium and count the number of single tumor cells.

2. Orthotopic Transplantation of Bladder Organoid (Figure 1A)

1. Prepare the bladder tumor organoids for orthotopic transplantation
   1. Before transplantation, culture the bladder tumor organoids for 5–7 days, as described above.
   2. Add 500 µL of collagenase/dispase to the organoid medium in a 24-well plate with the tumor organoids. Pipette up and down the basement membrane matrix and medium. Incubate for 20 min at 37 °C and collect the cells into a 15 mL tube.
   3. Add 5 mL of prewarmed DMEM, centrifuge the tube at 400 x g for 3 min at 4 °C, and aspirate the supernatant.
   4. Resuspend the pellet with 1 mL of DMEM and transfer the solution into a 90 mm Petri dish.
   5. Under a microscope, pick up the 10–100 tumor organoids by using a P200 micropipette and collect them into a microtube on ice.
   6. Centrifuge the tube at 400 x g for 3 min at 4 °C and carefully discard the supernatant.
   7. Maintain the cell pellet on ice until the mice are ready for surgery.
2. Submucosal bladder wall transplantation
   1. Prepare an 8- to 10-week old male nude mouse (CAnN.Cg-Foxn1nu/Crl) at least 1 week before the experiment to allow it to acclimate to a new environment. Inject enrofloxacin (5 mg/kg) subcutaneously 24 h before surgery.
   2. Clean the bench surface with soap and water. Autoclave the surgical instruments prior to the surgical procedure and perform surgery using sterile instruments.
   3. Keep the 29 G insulin syringe, pipette tips, and basement membrane matrix on ice. Administer ketoprofen (5 mg/kg) subcutaneously before administration of anesthesia.
   4. Anesthetize the mouse with 4% isoflurane in an induction chamber. Once general anesthesia is achieved, lay the mouse in a supine position and maintain anesthesia by mask inhalation of 2% vaporized isoflurane.
      NOTE: If the anesthetization time is over 30 min, apply eye ointment to both eyes using a cotton swab to avoid corneal drying.
   5. Apply povidone-iodine with sterile gauze and wipe it down with 70% ethanol. Repeat 3x with a new gauze or a cotton swab each time.
   6. Cover the anus and the surgical field using disposable, sterile surgical drapes.
   7. Using a dissecting microscope for magnification, make a small transverse incision (smaller than 1.5 cm) in the skin and muscular wall of the lower midline abdomen with sterile surgical scissors. Expose the bladder from the abdominal cavity and support it with saline-soaked cotton swabs.
      NOTE: If the bladder is full of urine, gently press the bladder to decompress it slightly.
   8. Resuspend the organoid pellets in 80 µL of organoid medium containing 50% high-concentration basement membrane matrix (Table 1 and Table of Materials).
   9. Inject the organoid suspension into the anterior aspect of the bladder dome using the 29 G insulin syringe under a dissecting microscope.
   10. Close the inner layer of the abdominal wall with antibacterial absorbable suture and then close the outer layer with 4-0 nylon suture. Disinfect the surgical site with povidone-iodine and 70% ethanol.
   11. Allow the mouse to recover under an infrared irradiator 10–15 min. Monitor the mouse until it regains consciousness and motility.
   12. One day after surgery, check the general condition of the mouse and anastomotic leakage. Administer ketoprofen (5 mg/kg) once daily for 3 days postoperatively and treat enrofloxacin (5 mg/kg) once daily for 10 days postoperatively.
   13. When the incision site has healed (10–14 days after surgery), remove the sutures. Monitor the growth of the mouse bladder tumor for 2–3 weeks after the tumor organoid injection.
   14. If bladder tumor growth is observed, euthanize the mouse using carbon dioxide inhalation, and harvest the entire bladder tumor. Wash it using cold DPBS (Figure 1B).
   15. To analyze the bladder tumor histology, stain the paraffin-embedded section of the tissue using hematoxylin and eosin (H&E) staining (Figure 1B).

Mouse bladder tumor organoid medium
Advanced DMEM/F-12 (Basic medium)	10 mM HEPES (pH 7.4)
10 mM Nicotinamide	0.5x Serum-free supplement
2 mM L-alanyl-L-glutamine dipeptide	1% Penicillin/Streptomycin
1 mM N-acetyl-L-cysteine	50 ng/mL Murine epidermal growth factor
1 µM A 83-01

Table 1: Composition of bladder tumor organoid medium.

Resultaten

Figure 1: Orthotopic transplantation of bladder tumor organoids. (A) Schematic diagram of orthotopic transplantation of bladder tumor organoids to a nude mouse. (B) Representative images of bladders and...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
15 mL conical tube	 SPL	 50015
24-well plate	 Corning	 3526
29 G 1/2 insulin syringe	 SHINA	 B299473538
50 mL conical tube	 SPL	 50050
A8301	 Tocris	 2939	 Stock concentration: 25 mM
Absolute ethanol	 Daejung	 4023-2304
Absorbable suture	 Henry Schein	 039010
Advanced DMEM/F-12	 Thermo	 12634028
CAnN.Cg-Foxn1nu/Crl (nude mouse)	 Charles River	 194
Collagenase type I	 Thermo	 17100017	 Stock concentration: 20 mg/mL
Collagenase type II	 Thermo	 17100015	 Stock concentration: 20 mg/mL
Collagenase/dispase	 Sigma	 10269638001	 Stock concentration: 1 mg/mL
DMEM (Dulbecco's modified minimum essential media)	 Gibco	 11965-118
DMSO (Dimethyl sulfoxide)	 Sigma	 D8418
DPBS (Dulbecco's phosphate-buffered saline)	 Welgene	 LB 001-02
Matrigel growth factor reduced (GFR) Growth Factor Reduced (GFR)	 Corning	 354230	 Use for organoid culture in plate
Matrigel high concentration (HC)	 Corning	 354248	 Use for organoid transplantation
1.5 mL microtube	 Axygen	 MCT-150-C
Razor blade
Saline buffer	 JW Pharmaceutical
Trypsin-EDTA (0.25%)	 Gibco	 25200072
Ultracentrifugation tube	 Beckman Coulter	 331372
Y-27632 dihydrochloride	 Abmole	 M1817	 Stock concentration: 10 mM