Holgersson Vanessa, Joyce Shelby, Brookman-Amissah Marianne, Lammel Tobias
Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg 405 30, Sweden.
Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg 405 30, Sweden.
Ecotoxicol Environ Saf. 2024 Dec;288:117327. doi: 10.1016/j.ecoenv.2024.117327. Epub 2024 Nov 16.
In vitro models based on permanent fish liver cell lines have proven to be versatile tools for examining chemical biotransformation and toxicity. However, their in vivo relevance remains uncertain due to their potentially de-differentiated phenotype. Here, we investigate whether a 3D cell culture environment can restore hepatocyte-like properties of the Rainbow trout liver cell line RTL-W1. Utilizing ultralow attachment (ULA) microwell plates, we achieved controlled sizing and extended culture (3 weeks) of spheroidal aggregate cultures (spheroids). RTL-W1 cells within the spheroids remained viable and metabolically active, as confirmed by the CellTiter-Glo 3D assay. Transmission electron microscopy revealed that spheroids exhibit tissue-like arrangements, such as interdigitations, cell-cell junctions, and endo- or exocytic activity at the cell-cell interface. They also displayed ultrastructural characteristics typical of metabolically active cells/hepatocytes, including abundant endoplasmic reticulum (ER), Golgi apparatus, and mitochondria. RT-qPCR analysis showed upregulation of genes involved in xenobiotic and endogenous (lipid) metabolism in 3D cultures over time. Notably, for several genes, especially cyp1a, expression levels were significantly higher in spheroids than in monolayers cultured for the same duration. This was corroborated at the enzyme level by increased Cyp1a-dependent catalytic activity (EROD). Interestingly, increased Cyp1a expression did not lead to heightened susceptibility to benzo[a]pyrene toxicity, which requires bioactivation. However, RTL-W1 3D and 2D cell cultures exhibited differential susceptibility to toxicity from other model chemicals, such as the surfactant SDS and the metal copper (Cu). These findings support the hypothesis that RTL-W1 cells can re-differentiate to a hepatocyte-like phenotype when cultured in a 3D configuration and may exhibit distinct biological responses upon exposure to xenobiotics. Overall, this study advances our understanding of the potential of cell line-derived 3D in vitro models for research and providing more physiologically relevant data for regulatory contexts.
基于永久性鱼类肝细胞系的体外模型已被证明是用于研究化学物质生物转化和毒性的多功能工具。然而，由于其潜在的去分化表型，它们在体内的相关性仍不确定。在此，我们研究三维细胞培养环境是否能够恢复虹鳟鱼肝细胞系RTL-W1的类肝细胞特性。利用超低吸附（ULA）微孔板，我们实现了对球形聚集体培养物（球体）的大小控制和延长培养（3周）。球体中的RTL-W1细胞保持存活且代谢活跃，这通过CellTiter-Glo 3D检测得以证实。透射电子显微镜显示，球体呈现出组织样排列，如指状交叉、细胞间连接以及细胞-细胞界面处的内吞或外吞活性。它们还表现出代谢活跃细胞/肝细胞典型的超微结构特征，包括丰富的内质网（ER）、高尔基体和线粒体。RT-qPCR分析表明，随着时间推移，三维培养中参与外源性和内源性（脂质）代谢的基因上调。值得注意的是，对于几个基因，尤其是cyp1a，球体中的表达水平显著高于相同培养时间的单层细胞。在酶水平上，Cyp1a依赖性催化活性（EROD）增加证实了这一点。有趣的是，Cyp1a表达增加并未导致对需要生物活化的苯并[a]芘毒性的易感性增加。然而，RTL-W1三维和二维细胞培养物对其他模型化学品（如表面活性剂SDS和金属铜（Cu））的毒性表现出不同的易感性。这些发现支持了这样的假设，即RTL-W1细胞在三维结构中培养时可以重新分化为类肝细胞表型，并且在接触外源性物质时可能表现出不同的生物学反应。总体而言，这项研究增进了我们对细胞系来源的三维体外模型在研究中的潜力的理解，并为监管环境提供了更具生理相关性的数据。
