To begin, prepare a coating solution containing growth factor reduced basement membrane matrix and organoid culture medium in a one to two ratio. Add 115 microliters of the coating solution to each well of a 48-well plate and place the plate at 37 degrees Celsius for 30 minutes in a humidified incubator. For the collagen method, add one milliliter of collagen gel to a 30 millimeter inner membrane insert, and let it solidify for 30 minutes at 37 degrees Celsius in a humidified incubator.
For harvesting the cellular mixture, wash the freshly resected melanoma tissue in a 10 milliliter Petri dish containing washing medium that is placed over ice. Using sterile tweezers, transfer the tumor into a second dish with washing medium for the next wash over ice. Throughout the three rinsing cycles using sterile scissors and a blade, remove excess connective tissue, fat, and residual blood.
Place the tumor in an empty Petri dish and mince it finely with sterile blades. Then transfer the mince tissue to a 50 milliliter conical tube containing 10 milliliters of the prepared digestion medium. Place the conical in a water bath at 37 degrees Celsius and vortex every five minutes.
After 25 minutes, add 30 milliliters of the ADMEM/F12 medium containing 10%FBS to stop the digestion. Next, filter the digested cell suspension through a 70 micrometer nylon cell strainer and rinse the strainer with supplemented ADMEM/F12. Using a pipette, retrieve any remaining media at the bottom of the filter.
Then pellet the cells at 300 x g for seven minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in the organoid culture medium. For culturing, seed 200, 000 cells in the pre-war matrigel-coated wells supplemented with 200 microliters of organoid culture medium.
Alternatively, seed 1 million cells mixed with one milliliter of collagen gel on the pre-solidified collagen gel membrane insert placed in a six-well plate and add the organoid culture medium to the well. For passage of 150 to 200 micrometer organoids, transfer them into a 15 milliliter conical and wash them with Dulbecco's PBS. Centrifuge the mixture at 300 x g for seven minutes at four degrees celsius.
After removing the supernatant, incubate the organoids with trypsin substitute and mix after every three minutes. To stop the digestion, add ADMEM/F12 with reduced serum medium containing 10%FBS. Centrifuge the mixture at 300 x g for seven minutes, and resuspend the palate in the organoid culture medium.
Finally, seed the single cell suspension into a new matrigel or collagen-coated well at the same initial seeding density for incubation. Microscopy images of the organoids in late in matrigel on day two and day seven indicated that the size of organoids gradually increased. Additionally, there was no statistically significant distinction in alpha-beta-T-cell proportion between the parental tumors and the respective organoids cultured in either matrigel or collagen.
