To begin, obtain actively growing human gastric organoids with diameters between 200 and 700 micrometers. Thaw the extracellular matrix, or ECM, aliquot on ice for at least 45 minutes. And prewarm cell culture plates at 37 degrees Celsius in a 5%carbon dioxide incubator.
After removing the media from the wells of the culture plate, pipette ice cold PBS onto each ECM droplet. And scratch the gel with the P-1000 pipette tip. Collect the PBS with the ECM fragments containing organoids into a 15 milliliter conical tube.
Centrifuge the tube at 200G at four degrees Celsius for five minutes. After aspirating the supernatant, add 350 microliters of 0.25%Trypsin-EDTA into each tube and mix gently. Incubate the tubes in a water bath, tempered at 37 degrees Celsius for two to five minutes.
Add 600 microliters of ice cold DMEM, supplemented with penicillin and streptomycin to each tube, and vigorously pipette up and down 40 times. Centrifuge the tube as demonstrated, and re-suspend the cell palette in ice cold liquid ECM. For each sample, plate 40 microliters of the liquid ECM containing organoid fragments in a thin, horizontal line along the diameter of a 35 millimeter glass bottom dish.
After 15 to 30 minutes of incubation, carefully add two milliliters of organoid expansion media to the plate along the edge of the dish without disturbing the polymerized gel.
