Kämpfer Angela A M, Shah Ume-Kulsoom, Chu Shui L, Busch Mathias, Büttner Veronika, He Ruiwen, Rothen-Rutishauser Barbara, Schins Roel P F, Jenkins Gareth J
IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany.
School of Medicine, Faculty of Medicine, Health and Life Science, Swansea, Wales UK.
In Vitro Model. 2022 Jun 13;2(3-4):89-97. doi: 10.1007/s44164-022-00025-w. eCollection 2023 Aug.
The development and improvement of advanced intestinal in vitro models has received increasing attention in recent years. While the availability of relevant in vitro models is pivotal to advance the replacement and reduction of animal use in research, their robustness is a crucial determinant for intra- and interlaboratory reproducibility. We have developed a standard protocol to build a triple culture model combining two types of human intestinal epithelial cells (Caco-2, HT29-MTX-E12) and macrophages (THP-1), which was tested for transferability and reproducibility between three laboratories. The epithelial tissue barrier development and triple culture stability were investigated as well as the models' responses to the non-steroidal anti-inflammatory drug diclofenac in terms of barrier integrity, cytotoxicity, and cytokine release. The results of two partner laboratories were compared to previously established benchmark results and quality criteria. For the epithelial co-cultures, the results were overall highly comparable between the laboratories. The addition of THP-1 cells resulted in increased variability and reduced reproducibility. While good correlation was achieved in several endpoints, others showed substantial response differences between the laboratories. Some variations may be addressed with training or demonstrations, whereas others might be related to fundamental differences in the cell lines introduced during routine cell culture and maintenance. Our results underline the importance of interlaboratory transfer studies using standardised experimental procedures, including defined quality criteria and benchmarks, as well as of training when newly establishing complex in vitro models in laboratories.
The online version contains supplementary material available at 10.1007/s44164-022-00025-w.
近年来，先进的肠道体外模型的开发和改进受到了越来越多的关注。虽然相关体外模型的可用性对于推进研究中动物使用的替代和减少至关重要，但其稳健性是实验室内和实验室间可重复性的关键决定因素。我们制定了一个标准方案来构建一个三重培养模型，该模型结合了两种类型的人肠道上皮细胞（Caco-2、HT29-MTX-E12）和巨噬细胞（THP-1），并在三个实验室之间测试了其可转移性和可重复性。研究了上皮组织屏障的发育和三重培养的稳定性，以及该模型对非甾体抗炎药双氯芬酸在屏障完整性、细胞毒性和细胞因子释放方面的反应。将两个合作实验室的结果与先前建立的基准结果和质量标准进行了比较。对于上皮共培养，各实验室之间的结果总体上具有高度可比性。添加THP-1细胞导致变异性增加和可重复性降低。虽然在几个终点上取得了良好的相关性，但其他终点在各实验室之间显示出显著的反应差异。一些差异可以通过培训或示范来解决，而其他差异可能与常规细胞培养和维护过程中引入的细胞系的根本差异有关。我们的结果强调了使用标准化实验程序（包括明确的质量标准和基准）进行实验室间转移研究的重要性，以及在实验室新建立复杂体外模型时进行培训的重要性。
在线版本包含可在10.1007/s44164-022-00025-w获取的补充材料。
