Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes
Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes
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DOI：
10.1007/s10646-012-0965-5
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发表时间：
2012-11-01
期刊：
影响因子：
2.7
通讯作者：
Jha, Awadhesh N.
中科院分区：
文献类型：
--
作者：
Baron, Matthew G.;Purcell, Wendy M.;Jha, Awadhesh N.
鱼类原代细胞和细胞系的使用为化学毒性评估和生态毒理学环境样品评价提供了一种体外替代方法。然而，它们的用途并非没有限制，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（球状体）技术现已确立用于体外哺乳动物毒性研究，但迄今尚未考虑将其用于水生物种模型的环境应用。在这项研究中，我们报告了一个可重复的六孔板，回转介导的方法虹鳟鱼（虹鳟鱼）肝细胞球体培养的发展，并比较其功能和生化状态与二维（2D）单层肝细胞。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，将原发性肝球体形成分为两个阶段，未成熟（1-5天）和成熟（>= 6天）。成熟的球状体保留的形态特征（光滑的外表面，紧密的细胞-细胞接触），如通过光学和扫描电子显微镜所示的哺乳动物球状体先前所述。与传统的2D单层培养相比，成熟球体中葡萄糖产生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）漏出显著减少，且显著低于2D培养（P < 0.01）。因此，建议成熟的球状体可以保持高度的功能，生物化学和形态学状态随着时间的推移，在文化是上级传统的2D模型，并可以提供现实的器官型反应在体外。需要6-8天才能成熟的鳟鱼球状体适合用于急性毒理学试验，由于可以将单个球状体培养一个多月，这项工作有可能导致体外生物累积替代品，并对慢性接触进行高通量筛选。这是在未来的鱼类生态毒理学研究中开发替代体外工具的重要一步。
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 similar to 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.