Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids.

Cancer organoids are three-dimensional in vitro models that closely replicate the genetic, phenotypic, and heterogeneity characteristics of original tumors, making them valuable tools in cancer research. However, the lack of standardized protocols limits their broader application. This study evaluates the role of enzymatic isolation in generating patient-derived organoids (PDOs) from colorectal cancer tissues by comparing four enzymatic methods: TrypLE, Trypsin-EDTA (T/E), Collagenase, and Hyaluronidase. Colorectal cancer tissues were processed using these enzymes, and cell viability, dissociation efficiency, and isolation quality were assessed via Trypan Blue exclusion assay and 7-AAD staining with flow cytometry. Cancer stem cells marked by LGR5 and CD133 were quantified via flow cytometry, while organoid generation and growth were monitored over 11 days using confocal microscopy. TrypLE and T/E demonstrated superior preservation of cell viability but limited dissociation efficiency, yielding lower cell count per milligram of tissue. In contrast, Collagenase and Hyaluronidase demonstrated superior tissue dissociation, yielding higher total cell counts and the highest proportions of LGR5