Clinical risk factors and pattern of initial fungal contamination in endoscopic biopsy-derived gastrointestinal cancer organoid culture

Background/Aims: Because gastrointestinal tract is not sterile, primary culture has contamination risk despite of massive washing with antimicrobial media. Mi-crobial contamination can play a key role in initial failure during biopsy-derived primary tumor culture. Methods: Tumor tissue was acquired from esophageal and gastric tumors using endoscopic biopsy. Three-dimensional cultures were performed, and separated spheroids were cultured in media for 7 to 10 days and then transferred to Matrigel (Corning Inc.). We investigated risk factors and patterns of initial fungal contamination. Results: Initial tumor contamination was observed in 23% (7/30) of esophageal cancer and 20% (3/15) of gastric cancer samples. Two cases of bacterial contamination occurred during the establishment of culture protocol. Moderate to thick whitish plaques (p < 0.001) and food retention in lumen (p < 0.001) were risk factors for initial fungal contamination. After exclusion of high risk patients for contamination, no fungal contamination occurred in primary organoid cultures. Fungal contamination was usually detected within 3 days after tumor preparation. How-ever, unusual fungal contamination (GC11 and EC29) was recognized after several passages. Growing spherical shapes resembled cancer organoids. Although they rapidly proliferated and multiple daughter spheroids appeared, the media were translucent. After several passages, yeasts and pseudohyphae were detected on the edges of the solid spherical structures and media. Conclusions: Moderate to thick whitish plaques and food retention are clinical risk factors for initial fungal contamination during biopsy-derived cancer organoid culture. Most initial fungal contamination was detected within 3 days, but it could be unusually recognized after several passages.