Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation
In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardizati...
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| Format: | Article |
| Language: | English |
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Wiley
2021-01-01
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| Series: | Stem Cells International |
| Online Access: | http://dx.doi.org/10.1155/2021/9041423 |
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| author | Talke F. zur Bruegge Andrea Liese Sören Donath Stefan Kalies Maike Kosanke Oliver Dittrich-Breiholz Sandra Czech Verena N. Bauer André Bleich Manuela Buettner |
| author_facet | Talke F. zur Bruegge Andrea Liese Sören Donath Stefan Kalies Maike Kosanke Oliver Dittrich-Breiholz Sandra Czech Verena N. Bauer André Bleich Manuela Buettner |
| author_sort | Talke F. zur Bruegge |
| collection | DOAJ |
| description | In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln, Zo-1, and Cldn4, the proliferation marker Ki67, and the proinflammatory cytokine Tnfα. Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development. |
| format | Article |
| id | doaj-art-c658cf2180534c15b46d8822f951e304 |
| institution | Kabale University |
| issn | 1687-966X 1687-9678 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Stem Cells International |
| spelling | doaj-art-c658cf2180534c15b46d8822f951e3042025-02-03T01:05:32ZengWileyStem Cells International1687-966X1687-96782021-01-01202110.1155/2021/90414239041423Intestinal Organoids in Colitis Research: Focusing on Variability and CryopreservationTalke F. zur Bruegge0Andrea Liese1Sören Donath2Stefan Kalies3Maike Kosanke4Oliver Dittrich-Breiholz5Sandra Czech6Verena N. Bauer7André Bleich8Manuela Buettner9Institute for Laboratory Animal Science, Hannover Medical School, Hannover, GermanyInstitute for Laboratory Animal Science, Hannover Medical School, Hannover, GermanyInstitute of Quantum Optics, Leibniz University Hannover, Hannover, GermanyInstitute of Quantum Optics, Leibniz University Hannover, Hannover, GermanyResearch Core Unit Genomics, Hannover Medical School, 30625 Hannover, GermanyResearch Core Unit Genomics, Hannover Medical School, 30625 Hannover, GermanyInstitute for Laboratory Animal Science, Hannover Medical School, Hannover, GermanyInstitute for Laboratory Animal Science, Hannover Medical School, Hannover, GermanyInstitute for Laboratory Animal Science, Hannover Medical School, Hannover, GermanyInstitute for Laboratory Animal Science, Hannover Medical School, Hannover, GermanyIn recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln, Zo-1, and Cldn4, the proliferation marker Ki67, and the proinflammatory cytokine Tnfα. Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development.http://dx.doi.org/10.1155/2021/9041423 |
| spellingShingle | Talke F. zur Bruegge Andrea Liese Sören Donath Stefan Kalies Maike Kosanke Oliver Dittrich-Breiholz Sandra Czech Verena N. Bauer André Bleich Manuela Buettner Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation Stem Cells International |
| title | Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation |
| title_full | Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation |
| title_fullStr | Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation |
| title_full_unstemmed | Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation |
| title_short | Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation |
| title_sort | intestinal organoids in colitis research focusing on variability and cryopreservation |
| url | http://dx.doi.org/10.1155/2021/9041423 |
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