Calibasi-Kocal Gizem, Sever Tolga, Canda Aras Emre, Kadioglu Leman Evren, Ates Halil, Basbinar Yasemin, Ellidokuz Ender
Department of Translational Oncology, Institute of Oncology, Dokuz Eylul University, Izmir, Türkiye.
Department of Oncology, Institute of Health Sciences, Dokuz Eylul University, Izmir, Turkey.
Sci Rep. 2025 Apr 18;15(1):13452. doi: 10.1038/s41598-025-97650-8.
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 and CD133 stem cell populations. Collagenase produced the highest organoid counts, while Hyaluronidase supported the largest organoid expansion, with both enzymes generating larger organoid surface areas and a greater number of organoids compared to TrypLE and T/E. These results highlight Collagenase and Hyaluronidase as optimal choices for PDO generation, providing a framework for optimizing dissociation protocols. This study underscores the critical influence of enzymatic dissociation methods on the establishment and reliability of colorectal cancer patient-derived organoids, providing a foundation for optimizing PDO protocols and advancing their translational application in precision oncology.
癌症类器官是三维体外模型,能紧密复制原发肿瘤的基因、表型和异质性特征,使其成为癌症研究中有价值的工具。然而,缺乏标准化方案限制了它们的更广泛应用。本研究通过比较四种酶解法:胰蛋白酶样酶(TrypLE)、胰蛋白酶 - 乙二胺四乙酸(T/E)、胶原酶和透明质酸酶,评估酶解在从结直肠癌组织生成患者来源类器官(PDO)中的作用。使用这些酶处理结直肠癌组织,并通过台盼蓝排斥试验和流式细胞术7 - 氨基放线菌素D(7 - AAD)染色评估细胞活力、解离效率和分离质量。通过流式细胞术对以富含亮氨酸重复序列的G蛋白偶联受体5(LGR5)和CD133标记的癌症干细胞进行定量,同时使用共聚焦显微镜在11天内监测类器官的生成和生长。胰蛋白酶样酶和T/E在细胞活力保存方面表现出色,但解离效率有限,每毫克组织产生的细胞计数较低。相比之下,胶原酶和透明质酸酶表现出更好的组织解离效果,产生更高的总细胞计数以及LGR5和CD133干细胞群体的最高比例。胶原酶产生的类器官数量最多,而透明质酸酶支持最大程度的类器官扩增,与胰蛋白酶样酶和T/E相比,这两种酶产生的类器官表面积更大且数量更多。这些结果突出了胶原酶和透明质酸酶是生成PDO的最佳选择,为优化解离方案提供了框架。本研究强调了酶解方法对结直肠癌患者来源类器官的建立和可靠性的关键影响,为优化PDO方案并推进其在精准肿瘤学中的转化应用奠定了基础。