Tumor organoids have been widely used as an in vitro cancer model to study tumor biology. Our protocol provide a detailed procedure to isolate, culture, and genetically manipulate bladder tumor organoids serving as a critical tool to study various aspects in cancer biology. Using our protocol, we can genetically manipulate the bladder tumor organoids to express or knock out the gene of our interest.
Also, with our orthotopic transplantation method, we can create an intact tumor microenvironment tumor organoids. Demonstrating the procedure will be Yubin Kim, a graduate student from our laboratory. Begin by establishing bladder tumor organoids from the murine bladder tumor.
Add one milliliter of 10 millimolar HEPES in DMEM to the tumor fragments and mince the tissue with a sterilized razor blade. To dissociate the fragments into cell suspension, add nine milliliters of DMEM with HEPES, collagenase type one and two, and thermolysin to the tumor pieces, and incubate them for 1.5 to two hours at 37 degrees Celsius and 5%carbon dioxide on an orbital shaker. After the incubation, transfer the cell suspension to a 50 milliliter tube.
Centrifuge the tube at 400 times g for five minutes at four degrees Celsius and aspirate the supernatant. Resuspend the pellet in five milliliters of ACK lysing buffer and incubate the tube at room temperature for three to five minutes to lyse any red blood cells. Meanwhile, coat a well in a 24-well plate with 150 microliters of ice cold growth factor reduced basement membrane matrix and place the plate in an incubator for 30 minutes.
Add 20 milliliters of DMEM into the tube with the cells and repeat the centrifugation. Aspirate the supernatant and resuspend the pellet in one milliliter of 0.25%trypsin EDTA and 10 micromolar Y-27632 dihydrochloride. Incubate the tube for five minutes in a 37 degree Celsius water bath.
Then neutralize the trypsin by adding 10 milliliters of DMEM with 10%FBS. Filter the cell suspension through a 100 micrometer cell strainer on a new 50 milliliter tube to remove undigested debris. Centrifuge the tube at 400 times g for five minutes at four degrees Celsius and aspirate the supernatant.
Then resuspend the pellet with one milliliter of DMEM and count the cells with a hemocytometer. Transfer three to four times 10 to the four tumor cells to a 1.5 milliliter microtube on ice and centrifuge it at 400 times g for three minutes at four degrees Celsius. Carefully remove the supernatant and resuspend the cells in 500 microliters of prewarmed organoid medium and 10 micromolar Y-27632.
Transfer the cell suspension into the previously prepared membrane matrix coated well and place the 24-well plate back into the incubator. Replace the medium every two days with 500 microliters of prewarmed organoid medium. Split the tumor organoids 12 hours before the Lentivirus-mediated transduction.
On the next day, quickly thaw a virus containing aliquot in a 37 degree Celsius water bath. Add the thawed virus to 250 microliters of organoid medium with 10 micromolar Y-27632 and eight micrograms per milliliter of hexadimethrine bromide. Replace the organoid medium in the 24-well plate with 500 microliters of the virus containing medium and incubate the plate for 12 to 16 hours at 37 degrees Celsius and 5%carbon dioxide.
To prepare the bladder tumor organoids for orthotopic transplantation, add 500 microliters of collagenase dispase to the organoid medium in the 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 five milliliters of prewarmed DMEM.
Then centrifuge the tube at 400 times g for three minutes at four degrees Celsius and aspirate the supernatant. Resuspend the pellet with one 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. Mouse bladder tumor organoids were established and cultured for nine days.
If tumor cells do not form tumor organoids, they were potentially killed during the dissociation step and enzymatic digestion time may need to be adjusted. Bladder tumor organoids exhibited strong GFP signals with successful lentiviral infection. After concentration, a total of 250 microliters of virus containing media was enough to infect 30, 000 single tumor cells on the basement membrane matrix and maintain a 90 to 100%infection efficiency.
Bladder tumor allographs were harvested three weeks after orthotopic transplantation and the histology of the tumor was analyzed using H&E staining. Orthotopic transplants of tumor organoids can grow as bladder tumors for two to three weeks. Following this procedure, researchers can perform immunohistochemistry of resulting tumor organoids or carry out a quantitative RT-PCR for genes of interest.
These experiments can further characterize the tumor organoids. Our protocol allows us to manipulate the genes in cancer cells easily compared to the mouse model while maintaining in vivo characteristics of tumors in order to study the specific functions of various genes involved in tumorigenesis of bladder cancer.
