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.

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结肠炎研究中的肠道类器官：关注变异性和冷冻保存

近年来，干细胞衍生的类器官已成为细胞培养标准，广泛用于研究以前通过动物实验和使用常见肿瘤细胞系研究的各种科学问题。在他们最初的炒作之后，人们对他们的标准化问题提出了担忧。在这里，我们旨在提供一些对我们在标准化小鼠结肠上皮类器官方面的经验的见解，我们将其用作炎症性肠病研究的替代方法。考虑到良好的科学实践，我们研究了可能对使用这些类器官的实验的设计和结果提出挑战的各种因素。首先，为了分析抗生素/抗真菌剂的影响，我们使用 ZellShield® 进行了动力学实验并测量了紧密连接标记的基因表达水平Ocln、Zo-1和Cldn4、增殖标记Ki67和促炎细胞因子Tnfα。因为我们发现有和没有 ZellShield® 的培养没有差异，然后我们使用益生菌大肠杆菌进行了感染实验Nissle 1917 作为已经建立的模型设置，用于分析技术、实验间和生物学重复的影响。我们证明了实验间的差异对可重复性构成了最大的挑战，并解释了我们解决这些差异的策略。此外，我们使用新鲜分离和冷冻保存/解冻的类器官进行了感染实验，发现冷冻保存会影响早期传代期间的实验结果。以前冷冻保存的结肠样体表现出过早的外观和对细菌刺激的更高的促炎反应。因此，我们建议在用于实验之前分析冷冻保存的类器官的生长特性和可靠性，并在标准化条件下进行几个独立的实验。综合起来，