Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids
Abstract 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 stud...
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Nature Portfolio
2025-04-01
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| Online Access: | https://doi.org/10.1038/s41598-025-97650-8 |
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| author | Gizem Calibasi-Kocal Tolga Sever Aras Emre Canda Leman Evren Kadioglu Halil Ates Yasemin Basbinar Ender Ellidokuz |
| author_facet | Gizem Calibasi-Kocal Tolga Sever Aras Emre Canda Leman Evren Kadioglu Halil Ates Yasemin Basbinar Ender Ellidokuz |
| author_sort | Gizem Calibasi-Kocal |
| collection | DOAJ |
| description | Abstract 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 LGR5positive and CD133positive 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. |
| format | Article |
| id | doaj-art-237a5ce534584ab0aeff06609d0a85aa |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-237a5ce534584ab0aeff06609d0a85aa2025-08-20T02:17:50ZengNature PortfolioScientific Reports2045-23222025-04-0115111110.1038/s41598-025-97650-8Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoidsGizem Calibasi-Kocal0Tolga Sever1Aras Emre Canda2Leman Evren Kadioglu3Halil Ates4Yasemin Basbinar5Ender Ellidokuz6Department of Translational Oncology, Institute of Oncology, Dokuz Eylul UniversityDepartment of Oncology, Institute of Health Sciences, Dokuz Eylul UniversityPrivate Clinic for Colorectal Cancer Surgery and Peritoneal CarcinomatosisDepartment of Pathology, Acibadem Izmir Kent HospitalInstitute of Oncology, Dokuz Eylul UniversityDepartment of Translational Oncology, Institute of Oncology, Dokuz Eylul UniversityDepartment of Internal Diseases, Gastroenterology, Faculty of Medicine, Dokuz Eylul UniversityAbstract 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 LGR5positive and CD133positive 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.https://doi.org/10.1038/s41598-025-97650-8Organoid cultureEnzymatic cell isolationColorectal cancer |
| spellingShingle | Gizem Calibasi-Kocal Tolga Sever Aras Emre Canda Leman Evren Kadioglu Halil Ates Yasemin Basbinar Ender Ellidokuz Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids Scientific Reports Organoid culture Enzymatic cell isolation Colorectal cancer |
| title | Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids |
| title_full | Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids |
| title_fullStr | Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids |
| title_full_unstemmed | Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids |
| title_short | Impact of enzymatic isolation on the propagation efficiency of patient-derived colorectal cancer organoids |
| title_sort | impact of enzymatic isolation on the propagation efficiency of patient derived colorectal cancer organoids |
| topic | Organoid culture Enzymatic cell isolation Colorectal cancer |
| url | https://doi.org/10.1038/s41598-025-97650-8 |
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