Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity
Purpose: To establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity. Materials and Methods: Oral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto cult...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-05-01
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| Series: | Clinical and Translational Radiation Oncology |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405630822000209 |
| _version_ | 1818009646069186560 |
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| author | A.R. Thomsen C. Aldrian B. Luka S. Hornhardt M. Gomolka S. Moertl J. Hess H. Zitzelsberger T. Heider N. Schlueter S. Rau B. Monroy Ordonez H. Schäfer G. Rücker M. Henke Katharina Huber |
| author_facet | A.R. Thomsen C. Aldrian B. Luka S. Hornhardt M. Gomolka S. Moertl J. Hess H. Zitzelsberger T. Heider N. Schlueter S. Rau B. Monroy Ordonez H. Schäfer G. Rücker M. Henke Katharina Huber |
| author_sort | A.R. Thomsen |
| collection | DOAJ |
| description | Purpose: To establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity. Materials and Methods: Oral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto culture plates. Outgrowing cells were tested for cytokeratin 5/14 and Ki67, expanded, radiated at different doses, and seeded onto circumscribed areas before being allowed to spread centrifugally. In this newly developed spreading assay, cell-covered areas were measured by image analysis. For statistical analysis, a linear mixed regression model was used; additionally, results were correlated to the radiation dose applied. Colony forming efficiency (CFE) was used to validate the results. DNA damage repair was analysed by gammaH2AX and 53BP1 foci quantification using immunofluorescence microscopy 24 h and 96 h after irradiation. Results: Stable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm2 up to a median of 119.2 mm2 (range: 54.4–290). Radiated cells spread to only 100.7 mm2 (2 Gy; range: 55.3–266.7); 73.2 mm2 (4 Gy; 15–240.4); 47 mm2 (6 Gy; 2–111.9), and 22.7 mm2 (8 Gy; 0–80). Similarly, CFE decreased from 0.223 (0 Gy) to 0.0028 (8 Gy). Using an individual donor as a random factor, cell spread correlated with CFE, where radiation dose was the main driver (decrease by 0.50, adjusted for area). Upon irradiation with 6 Gy, radiation-induced DNA damage was increased after 24 h in all samples, and even after 96 h in 5 out of 7 samples, as detected by a higher number of gammaH2AX/53BP1 foci in irradiated cells (mean 3.7 for 24 h; mean 0.6 for 96 h). Conclusion: In vitro propagation of keratinocytes derived from a small biopsy is feasible. Radiation impairs cellular migration and proliferation, and the newly described spreading assay allows ranking for cellular radioresistance. The keratinocyte model also supports classical functional assays such as clonogenic survival and DNA double strand repair. The clinical relevance awaits upcoming investigations. |
| first_indexed | 2024-04-14T05:45:29Z |
| format | Article |
| id | doaj.art-21ed2d871e264cba8f498b0bdd61fcea |
| institution | Directory Open Access Journal |
| issn | 2405-6308 |
| language | English |
| last_indexed | 2024-04-14T05:45:29Z |
| publishDate | 2022-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Clinical and Translational Radiation Oncology |
| spelling | doaj.art-21ed2d871e264cba8f498b0bdd61fcea2022-12-22T02:09:18ZengElsevierClinical and Translational Radiation Oncology2405-63082022-05-01345156Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivityA.R. Thomsen0C. Aldrian1B. Luka2S. Hornhardt3M. Gomolka4S. Moertl5J. Hess6H. Zitzelsberger7T. Heider8N. Schlueter9S. Rau10B. Monroy Ordonez11H. Schäfer12G. Rücker13M. Henke14Katharina Huber15Department of Radiation Oncology, University Medical Center, University of Freiburg, Freiburg/Breisgau, Germany; German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (dkfz), Heidelberg, Germany; Corresponding author at: Department of Radiation Oncology, University Medical Center, University of Freiburg, Freiburg/Breisgau, Germany.Department of Radiation Oncology, University Medical Center, University of Freiburg, Freiburg/Breisgau, Germany; German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (dkfz), Heidelberg, GermanyDivision for Cariology, Department of Operative Dentistry and Periodontology, Center for Dental Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, GermanyFederal Office for Radiation Protection, Ingolstädter Landstr. 1, 85764 Oberschleißheim, GermanyFederal Office for Radiation Protection, Ingolstädter Landstr. 1, 85764 Oberschleißheim, GermanyFederal Office for Radiation Protection, Ingolstädter Landstr. 1, 85764 Oberschleißheim, GermanyResearch Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, Germany; Clinical Cooperation Group “Personalized Radiotherapy in Head and Neck Cancer”, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, Germany; Department of Radiation Oncology, University Hospital, LMU Munich, Munich, GermanyResearch Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, GermanyDepartment of Radiation Oncology, University Hospital, LMU Munich, Munich, GermanyDivision for Cariology, Department of Operative Dentistry and Periodontology, Center for Dental Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, GermanyDivision for Cariology, Department of Operative Dentistry and Periodontology, Center for Dental Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, GermanyDepartment of Radiation Oncology, University Medical Center, University of Freiburg, Freiburg/Breisgau, Germany; German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (dkfz), Heidelberg, GermanyDepartment of Radiation Oncology, University Medical Center, University of Freiburg, Freiburg/Breisgau, Germany; German Cancer Consortium (DKTK) Partner Site Freiburg, German Cancer Research Center (dkfz), Heidelberg, GermanyInstitute for Medical Biometry and Statistics, Medical Center – University of Freiburg, GermanyDepartment of Radiation Oncology, University Medical Center, University of Freiburg, Freiburg/Breisgau, GermanyFederal Office for Radiation Protection, Ingolstädter Landstr. 1, 85764 Oberschleißheim, GermanyPurpose: To establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity. Materials and Methods: Oral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto culture plates. Outgrowing cells were tested for cytokeratin 5/14 and Ki67, expanded, radiated at different doses, and seeded onto circumscribed areas before being allowed to spread centrifugally. In this newly developed spreading assay, cell-covered areas were measured by image analysis. For statistical analysis, a linear mixed regression model was used; additionally, results were correlated to the radiation dose applied. Colony forming efficiency (CFE) was used to validate the results. DNA damage repair was analysed by gammaH2AX and 53BP1 foci quantification using immunofluorescence microscopy 24 h and 96 h after irradiation. Results: Stable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm2 up to a median of 119.2 mm2 (range: 54.4–290). Radiated cells spread to only 100.7 mm2 (2 Gy; range: 55.3–266.7); 73.2 mm2 (4 Gy; 15–240.4); 47 mm2 (6 Gy; 2–111.9), and 22.7 mm2 (8 Gy; 0–80). Similarly, CFE decreased from 0.223 (0 Gy) to 0.0028 (8 Gy). Using an individual donor as a random factor, cell spread correlated with CFE, where radiation dose was the main driver (decrease by 0.50, adjusted for area). Upon irradiation with 6 Gy, radiation-induced DNA damage was increased after 24 h in all samples, and even after 96 h in 5 out of 7 samples, as detected by a higher number of gammaH2AX/53BP1 foci in irradiated cells (mean 3.7 for 24 h; mean 0.6 for 96 h). Conclusion: In vitro propagation of keratinocytes derived from a small biopsy is feasible. Radiation impairs cellular migration and proliferation, and the newly described spreading assay allows ranking for cellular radioresistance. The keratinocyte model also supports classical functional assays such as clonogenic survival and DNA double strand repair. The clinical relevance awaits upcoming investigations.http://www.sciencedirect.com/science/article/pii/S2405630822000209 |
| spellingShingle | A.R. Thomsen C. Aldrian B. Luka S. Hornhardt M. Gomolka S. Moertl J. Hess H. Zitzelsberger T. Heider N. Schlueter S. Rau B. Monroy Ordonez H. Schäfer G. Rücker M. Henke Katharina Huber Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity Clinical and Translational Radiation Oncology |
| title | Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity |
| title_full | Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity |
| title_fullStr | Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity |
| title_full_unstemmed | Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity |
| title_short | Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity |
| title_sort | biopsy derived oral keratinocytes a model to potentially test for oral mucosa radiation sensitivity |
| url | http://www.sciencedirect.com/science/article/pii/S2405630822000209 |
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