Irradiation and Processing of Patient-Derived Tumor Organoids for Radiosensitivity Assessment Using Live Imaging

To begin, obtain patient derived tumor organoid or PDTO and add three milliliters of fresh enriched DMEM F/12 culture medium to the PDTO every three to four days. Using the small animal research radiation platform irradiator, irradiate the PDTO containing domes in the open field mode. Immediately after irradiation wash each PDTO containing Nagrigel dome with two milliliters of enriched DMEM F/12 medium.

Then using a P 1000 resuspend the PDTO in one to three milliliters of a commercially obtained recombinant enzyme. Transfer the cell suspension into a 50 milliliter centrifuge tube and incubate for five minutes at 37 degrees Celsius. Centrifuge the tube to pellet the cells and aspirate the supernatant to leave approximately one milliliter of recombinant enzyme in the tube.

Then wash the cells with enriched DMEM F/12 medium and filter the cell suspension in a 40 micrometer mesh filter to eliminate cell clusters, stain a small sample of the filtered cells with 10%tripin blue in PBS and count them in an automated cell counter. Resuspend the cells to achieve a density of 800 cells in 50 microliters of enriched medium with 66%magrigel. Next plate, each dome in separate wells of an imaging compatible 48 well culture plate and incubate the plate to let the magrigel solidify.

Finally, add 0.5 to one milliliter of enriched DMEM F/12 medium to each well for live imaging.