Kelly S P, Fletcher M, Pärt P, Wood C M
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada.
Methods Cell Sci. 2000;22(2-3):153-63. doi: 10.1023/a:1009816923262.
Techniques for the in vitro 'reconstruction' of freshwater rainbow trout branchial epithelia using the primary culture of gill cells on permeable polyethylene terephthalate cell culture filter supports are described. Representing models of the freshwater fish gill, epithelia grown by two separate techniques are composed of branchial pavement cells with or without the inclusion of mitochondria-rich (MR) cells. The generation of epithelia consisting of pavement cells only (via a method called single seeded inserts = SSI) involves an initial period of flask culture during which time MR cells, that appear unable to attach to the culture flask base, are excluded from the general cell populace. Alternately, the generation of a heterogeneous epithelia consisting of both pavement cells and MR cells (via a method called double seeded inserts = DSI) is facilitated by the direct seeding of cells into cell culture filter inserts. Critical to this second procedure is the repeat seeding of filter inserts over a two day period. Repeat seeding appears to allow MR cells to nest amongst the attached cell layer generated by the first day's seeding. The use of cell culture filter supports allows free access to both the apical and basolateral compartment of the epithelium and is ideal for experimental manipulation. Cells are grown under symmetrical conditions (apical media/basolateral media) and epithelium growth is measured as a function of transepithelial resistance (TER). When the epithelia exhibit a plateau in growth they can be subjected to asymmetrical conditions (freshwater apical/media basolateral) in order to assess gill cell function as in vivo.
本文描述了一种在体外“重建”淡水虹鳟鱼鳃上皮的技术，该技术利用鳃细胞在可渗透的聚对苯二甲酸乙二酯细胞培养滤膜支架上进行原代培养。作为淡水鱼鳃的模型，通过两种不同技术培养的上皮由鳃扁平细胞组成，其中一些包含或不包含富含线粒体（MR）的细胞。仅由扁平细胞组成的上皮（通过一种称为单接种插入物 = SSI的方法）的生成涉及一个初始的烧瓶培养阶段，在此期间，似乎无法附着在烧瓶底部的MR细胞被排除在一般细胞群体之外。另外，由扁平细胞和MR细胞组成的异质上皮（通过一种称为双接种插入物 = DSI的方法）的生成是通过将细胞直接接种到细胞培养滤膜插入物中来实现的。此第二步操作的关键是在两天内重复接种滤膜插入物。重复接种似乎允许MR细胞嵌套在第一天接种产生的附着细胞层中。使用细胞培养滤膜支架可使上皮的顶端和基底外侧隔室都能自由接触，非常适合进行实验操作。细胞在对称条件下（顶端培养基/基底外侧培养基）生长，上皮生长以跨上皮电阻（TER）来衡量。当上皮生长达到平稳期时，可使其处于不对称条件下（淡水顶端/培养基基底外侧），以便像在体内一样评估鳃细胞功能。
