1 To begin, obtain the tumor tissue2 from the patients, and process it with a chopper. 3 Tilt the Petri dish containing chopped tumor 4 upwards to 45 degrees, and carefully remove 2.5 milliliters 5 of the H-GPSA medium. 6 To wash the tumor pieces, add two milliliters 7 of RBC lysis buffer, and incubate in an orbital shaker 8 at a slow speed.
9 Then, discard the lysis buffer completely. 10 Next, add four milliliters of patient-derived 11 organoid medium to the tissue pieces, 12 and transfer the tissue chunks to an ultra low attachment 13 six well-plate. 14 Incubate the plate in an orbital shaker 15 for two to four weeks.
16 Every two days, change half of the spent medium 17 with a pre-warmed fresh patient-derived organoid medium. 18 To prevent tissue hypoxia, 19 transfer the patient-derived organoids 20 to a sterile glass Petri dish 21 and observe the tissue morphology under a microscope. 22 Then, using a scalpel, cut the adhesive tissue.
23 Patient-derived organoids obtained using the chopper method, 24 reached the desired rounded shape within one week 25 as compared to the manually chopped ones. 26 And the number of organoids formed 27 was significantly higher using this method.
