Division of Diabetes and Nutritional Sciences, Metal Metabolism Group, Faculty of Life Sciences and Medicine, King's College London, London, UK.
Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada.
Nat Protoc. 2016 Mar;11(3):490-8. doi: 10.1038/nprot.2016.029. Epub 2016 Feb 11.
This protocol describes how to reconstruct and culture the freshwater rainbow trout gill epithelium on flat permeable membrane supports within cell culture inserts. The protocol describes gill cell isolation, cultured gill epithelium formation, maintenance, monitoring and preparation for use in experimental procedures. To produce a heterogeneous gill epithelium, as seen in vivo, seeding of isolated gill cells twice over a 2-d period is required. As a consequence, this is termed the double-seeded insert technique. Approximately 5-12 d after cell isolation and seeding, preparations develop electrically tight gill epithelia that can withstand freshwater on the apical cell surface. The system can be used to study freshwater gill physiology, and it is a humane alternative for toxicity testing, bioaccumulation studies and environmental water quality monitoring.
本方案描述了如何在细胞培养插入物的平面可渗透膜支架上重建和培养淡水虹鳟鱼的鳃上皮。该方案描述了鳃细胞分离、培养的鳃上皮形成、维持、监测以及用于实验程序的准备。为了产生类似于体内所见的异质鳃上皮，需要在 2 天的时间内两次播种分离的鳃细胞。因此，这被称为双播种插入技术。大约在细胞分离和播种后 5-12 天，制备物会形成电封闭的鳃上皮，可以承受顶细胞表面的淡水。该系统可用于研究淡水鳃生理学，并且是毒性测试、生物累积研究和环境水质监测的一种更人道的替代方法。
