School of Biomedical and Biological Sciences, Plymouth University, Drake Circus, Plymouth, Devon, PL4 8AA, UK.
Ecotoxicology. 2012 Nov;21(8):2419-29. doi: 10.1007/s10646-012-0965-5. Epub 2012 Jun 26.
The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1-5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell-cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6-8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.
鱼类原代细胞和细胞系可作为体外替代方法，用于评估化学毒性和生态毒理学中的环境样品。然而，它们的使用并非没有局限性，例如培养时间短和功能丧失，特别是对于原代组织。虽然三维（球体）技术现已确立用于体外哺乳动物毒性研究，但迄今为止，它尚未被认为可用于水生模式物种的环境应用。在这项研究中，我们报告了一种可重复的六孔板旋转介导的虹鳟鱼（Oncorhynchus mykiss）肝细胞球体培养方法的开发，并将其功能和生化状态与二维（2D）单层肝细胞进行了比较。根据大小、形状以及功能和生化参数（蛋白质、葡萄糖、白蛋白和乳酸脱氢酶）的变化，将原代肝球体的形成分为两个阶段，即不成熟（1-5 天）和成熟（≥6 天）。成熟的球体保留了先前描述的哺乳动物球体的形态特征（光滑的外表面、紧密的细胞-细胞接触），这通过光镜和扫描电子显微镜得到了证实。与传统的 2D 单层培养相比，成熟球体的葡萄糖生成和白蛋白合成显著更高（P < 0.01），并且随着球体成熟而增加（P < 0.01）。在球体形成过程中，基础乳酸脱氢酶（LDH）渗漏显著降低，且显著低于 2D 培养物（P < 0.01）。因此，成熟的球体在培养过程中可以长时间保持高度的功能、生化和形态状态，优于传统的 2D 模型，并可在体外提供真实的器官型反应。成熟球体需要约 6-8 天才能达到成熟，因此适用于急性毒性测试，并且由于可以对单个球体进行超过一个月的培养，因此这项工作有可能开发出体外生物积累替代方法，并进行慢性暴露的高通量筛选。这是在未来鱼类生态毒理学研究中开发替代体外工具的重要一步。
