Establishment of Colorectal Tumor Organoids

CRC specimens were provided by the Peking University Third Hospital. Fresh tumor samples were stored in the antibiotic-containing DMEM medium with 10% fetal bovine serum (C04001-500, VivaCell) after surgically resected and transported to the laboratory at 4°C for immediate processing. The establishment and culture of colorectal tumor organoids was performed as previously described (Wang et al., 2022 (link)). Briefly, after being washed gently at least five times with pre-chilled 1X DPBS, tumor tissues were cut into small pieces using surgical scissors and digested with 2.5 mg/mL type II and type IV collagenase (17101015 and 17104019, Invitrogen) to obtain single-cell suspensions. After digestion, the suspension was passed through a 40 μm cell strainer to remove undigested parts, and then centrifuged at 800 ×g for 5 min. The pellet was resuspended with Matrigel (356231, Corning) and dispensed into a 24-well cell culture dish. After 30 min of solidification of Matrigel, conditioned medium was then added. Conditioned medium was prepared according to the previously reported protocol (Miyoshi and Stappenbeck, 2013 (link)). At early passages, 10 μmol/L ROCK inhibitor Y-27632 (S6390, Selleck) was supplemented to the medium.