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Copyright © 2021 Talke F. zur Bruegge et al. This is an open access article distributed under the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. https://creativecommons.org/licenses/by/4.0/

Abstract

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.

Details

Title
Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation
Author
Talke F zur Bruegge 1   VIAFID ORCID Logo  ; Liese, Andrea 1 ; Donath, Sören 2 ; Kalies, Stefan 2 ; Kosanke, Maike 3 ; Dittrich-Breiholz, Oliver 3 ; Czech, Sandra 1 ; Bauer, Verena N 1 ; Bleich, André 1 ; Buettner, Manuela 1   VIAFID ORCID Logo 

 Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany 
 Institute of Quantum Optics, Leibniz University Hannover, Hannover, Germany; Lower Saxony Center for Biomedical Engineering, Implant Research and Development (NIFE), Hannover, Germany 
 Research Core Unit Genomics, Hannover Medical School, 30625 Hannover, Germany 
Editor
Yunhee kang
Publication year
2021
Publication date
2021
Publisher
Hindawi Limited
ISSN
1687966X
e-ISSN
16879678
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2576545993
Copyright
Copyright © 2021 Talke F. zur Bruegge et al. This is an open access article distributed under the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. https://creativecommons.org/licenses/by/4.0/