Bladder Tumor Organoid Model: A Method for Orthotopic Transplantation of Bladder Organoids into Recipient Murine Bladder
筆記録
To prepare the bladder tumor organoids for orthotopic transplantation, add 500 microliters of collagenase/dispase to the organoid medium in a 24-well plate. Pipette the basement membrane matrix and the medium up and down. Then, incubate the plate for 20 minutes at 37 degrees Celsius.
After the incubation, collect the cells into a 15-milliliter tube and add 5 milliliters of pre-warmed DMEM. Then, centrifuge the tube at 400 x g for 3 minutes at 4 degrees Celsius and aspirate the supernatant. Resuspend the pellet with 1 milliliter of DMEM and transfer the solution to a 90-millimeter Petri dish.
Under a microscope, pick up 10 to 100 tumor organoids with a P200 pipette and collect them into a microtube on ice. Centrifuge the microtube and carefully remove the supernatant. Then, maintain the pellet on ice until the mice are ready for surgery. Prior to submucosal bladder wall transplantation, keep the syringes, pipette tips, and basement membrane matrix on ice until ready to use.
After the mouse has been anesthetized, lay it in a supine position and maintain anesthesia by mask inhalation of 2% vaporized isoflurane. Apply povidone-iodine with a sterile gauze and wipe it down with 70% ethanol. Make a small transverse incision 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 a saline-soaked cotton swab.
Resuspend the organoid pellets in 80 microliters of organoid medium containing 50% high concentration basement membrane matrix and inject the suspension into the anterior aspect of the bladder dome using the insulin syringe. Close the incision with a 4-0 nylon suture and disinfect the surgical site with povidone-iodine and 70% ethanol. Allow the mouse to recover under an infrared irradiator for 10 to 15 minutes. Then, monitor the mouse again until it regains consciousness.
