Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

All procedures were approved and conducted under the guidelines of the Institutional Animal Care and Use Committee at POSTECH (IACUC number: POSTECH-2019-0055).

1. In Vitro Culture of Bladder Tumor Organoids

1. Establish bladder tumor organoids from the murine bladder tumor (Figure 1A).
   NOTE: The procedure for generating BBN-induced mouse bladder tumors is outlined in Shin et al.¹⁷.
   1. Provide 0.1% BBN-containing water in a dark bottle to mouse ad libitum for 6 months. Change BBN-containing water 2x a week.
      NOTE: A C57BL/6 male mouse with a body weight of approximately 25 g at 8–10 weeks of age was used. BBN-containing water can be administered to up to five mice in a single cage.
   2. After 6 months, euthanize the mouse using carbon dioxide inhalation and isolate the entire bladder tumor. Transfer it to a 90 mm Petri dish.
   3. Remove non-cancerous parts and necrotic regions using sterile surgical scissors and wash the bladder tumor fragments 2–3 times with cold 1x Dulbecco's phosphate-buffered saline (DPBS). Collect the fragments and transfer them to a new 90 mm Petri dish.
   4. Add 1 mL of Dulbecco's modified minimum essential medium (DMEM) with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).
   5. Mince the tumor tissue into pieces as small as possible (0.5–1 mm³) using a sterilized razor blade.
   6. Add 9 mL of DMEM with 10 mM HEPES, 250 µg/mL collagenase type I, 250 µg/mL collagenase type II, and 250 U/mL thermolysin. Incubate the minced tumor tissue for 1.5–2 h on an orbital shaker in an incubator (37 °C, 5% CO₂) to dissociate the fragments into the cell suspension. Transfer the cell suspension into a 50 mL tube.
      NOTE: If the size of tumor harvested from the mouse is greater than 1 cm³, treat it with 2x the amount of thermolysin or increase the incubation time.
   7. Centrifuge the tube at 400 x g for 5 min at 4 °C and aspirate the supernatant.
   8. Resuspend the pellet using 5 mL of ammonium-chloride-potassium (ACK) lysing buffer to lyse any red blood cells. Incubate the tube for 3­–5 min at room temperature (RT) until the complete lysis of red blood cells.
      NOTE: If the red blood cells are not observed, omit the lysing process.
   9. Add 20 mL of DMEM into the tube. Centrifuge the tube at 400 x g for 5 min at 4 °C and aspirate the supernatant.
   10. Resuspend the pellet with 1 mL of 0.25% Trypsin-EDTA and 10 µM Y-27632 dihydrochloride (Y-27632) to dissociate the pellet into single cells. Incubate the tube for 5 min in a 37 °C water bath.
       NOTE: Observe the tumor under a microscope to confirm complete dissociation into single cells. If chunks of cells persist, pipette the suspension further.
   11. Neutralize trypsin using 10 mL of DMEM with 10% fetal bovine serum (FBS). Filter the cell suspension through a 100 µm cell strainer on a new 50 mL tube to remove the undigested debris.
   12. Centrifuge the tube at 400 x g for 5 min at 4 °C and aspirate the supernatant.
   13. Coat a well in a 24 well plate using 150 µL of ice-cold growth factor reduced basement membrane matrix (Table of Materials) and place the 24 well plate in an incubator (37 °C, 5% CO₂) for 30 min to solidify the basement membrane matrix.
       NOTE: Thaw and maintain the basement membrane matrix at 4 °C to prevent solidification before use.
   14. Resuspend the pellet using 1 mL of DMEM and count the cells using a hemocytometer. Transfer 3–4 x 10⁴ tumor cells into a 1.5 mL microtube on ice.
   15. Centrifuge the microtube at 400 x g for 3 min at 4 °C and carefully discard the supernatant.
   16. Resuspend the cells with 500 µL of prewarmed organoid medium (Table 1) and 10 µM Y-27632 and transfer them into the coated well. Place the 24 well plate in an incubator (37 °C, 5% CO₂).
   17. Extra bladder tumor cells can be stocked with 1 mL of DMEM containing 10% FBS, 1% penicillin/streptomycin, and 10% dimethyl sulfoxide (DMSO) in 1.5 mL cryovials. Place them in a cryovial freezing container and transfer the container to a -80 °C freezer. After storage in the freezer overnight, transfer the cryovials into liquid nitrogen for long-term storage.
   18. Change the medium every 2 days using 500 µL of prewarmed organoid medium (Figure 1B).
2. 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 the 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.
   6. Repeat steps 1.1.14−1.1.18.

2. Genetic Manipulation of Bladder Tumor Organoids Using Lentivirus-mediated Transduction (Figure 2A)

1. Produce the GFP-expressing lentiviral particles.
   1. On day 0, plate HEK 293T cells at a density of 5–6 x 10⁶ cells per 10 cm cell culture plate in cell line culture medium (i.e., DMEM with 10% FBS and 1% penicillin/streptomycin).
   2. On day 1, prepare the DNA transfection solution including transfer plasmid containing GFP (8 µg), lentiviral packaging plasmid (10 µg of pCMVR 8.74 and 3 µg of pMD2.G), and 1 mL of reduced serum medium (Table of Materials).
   3. Add 3 µL of transfection reagent (Table of Materials) per 1 µg of total plasmid according to the manufacturer's instructions and mix gently by pipetting. Incubate for 20 min at RT and add 9 mL of DMEM with 10% FBS.
   4. Aspirate the culture medium in the cell culture plate with HEK 293T cells. Carefully transfer 10 mL of the DNA transfection solution onto the HEK 293T cells and incubate in a cell culture incubator at 37 °C.
   5. On day 3, observe the cells under a fluorescence microscope (excitation at 488 nm and emission at 512 nm) to determine the transfection efficiency. Almost 90%–100% of the cells in the entire cell population should express GFP.
   6. Collect the supernatant (containing the virus) and filtrate the supernatant with a 0.45 µm polyethersulfone (PES) filter.
      NOTE: Use a low protein-binding filter such as a PES filter.
   7. To concentrate the virus, centrifuge the virus supernatant at 98,768 x g in an ultracentrifuge for 2 h at 4 °C in a swinging bucket rotor (Table of Materials) and carefully discard the supernatant.
   8. Resuspend the pellet in 2.5 mL of cold organoid medium.
   9. For long-term storage, aliquot 250 µL of lentiviral medium into cryogenic vials and snap-freeze them using liquid nitrogen. Store the frozen viral stocks in a -80 °C freezer.
2. Perform lentivirus-mediated transduction of the bladder tumor organoids.
   1. On day 2, split the tumor organoids as described above (step 1.2) 12 h before the lentivirus-mediated transduction.
   2. On day 3, quickly thaw an aliquot (step 2.1.9) containing virus in a 37 °C water bath and add the 250 µL of organoid medium with 10 µM Y-27632 and 8 µg/mL hexadimethrine bromide.
   3. Replace the organoid medium in the 24 well plate with tumor organoids by 500 µL of virus-containing medium and incubate for 12–16 h in an incubator (37 °C, 5% CO₂).
   4. On day 4, change the medium with 500 µL of fresh organoid medium.
      NOTE: After 12–16 h of incubation, the medium should be changed, because the medium containing lentivirus and hexadimethrine bromide is cytotoxic.
   5. On day 6, monitor the GFP signal from the tumor organoids 3 days after transduction under a fluorescence microscope (Figure 2B).
   6. On day 10, passage and stock the organoids 7 days after transduction as described in step 1.2, to maintain the genetically modified tumor organoid lines.

3. Orthotopic Transplantation of Bladder Organoid (Figure 3A)

1. Prepare the bladder tumor organoids for orthotopic transplantation.
   1. Before transplantation, culture the bladder tumor organoids for 5–7 days, as described above (step 1.2).
   2. Add 500 µL of collagenase/dispase to 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
   NOTE: This procedure is modified from the protocol published by Fu et al¹⁸.
   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 by 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 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 a 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 (step 3.1.7) in 80 µL of organoid medium containing 50% high-concentration basement membrane matrix (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 post-operatively and treat enrofloxacin (5 mg/kg) once daily for 10 days post-operatively.
   13. When 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 3B)¹⁶.
   15. To analyze the bladder tumor histology, stain the paraffin-embedded section of the tissue using hematoxylin and eosin (H and E) staining (Figure 3B)¹⁶.