Preparing Organoids from Tumors: A 3D In Vitro Tumor Model
Transcription
After mincing, place the tumor pieces into a 15-milliliter tube of digestion buffer for 1.5 to 2 hour digestion at 37 degrees Celsius. At the end of the digestion, sediment the digested tissue by centrifugation and discard the supernatant.
Flick the tube to loosen the cell pellet. Resuspend the cells in 1 milliliter of pre-warmed trypsin supplemented with 10 micromolar Y-27632 ROCK inhibitor for 5 minutes at 37 degrees Celsius.
At the end of the incubation, triturate the tissue slurry five times with the standard P1000 pipette tip. Return the tube to the water bath for an additional 5-minute incubation and trituration.
For matrix dome plating, wash the digested cells in 9 milliliters of cold medium, then collect the cells by centrifugation. Resuspend the cells in 1 milliliter of fresh medium for counting, and after counting, collect the cells by centrifugation once again. Dilute the tumor cells in the appropriate volume of matrix.
Carefully drop 200 microliters of matrix cell solution into each well of a 55 degree Celsius warmed 6-well plate. Allow the domes to solidify at room temperature for 2 minutes before placing the plate upside down in a 37 degrees Celsius incubator for 20 minutes.
When the domes have fully solidified, add 2 milliliters of prostate organoid medium supplemented with androgen R1881 and ROCK inhibitor to each well. Return the plate to the cell culture incubator.
