Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout (Salmo gairdneri) |
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Authors: | T Kocal B A Quinn I R Smith H W Ferguson M A Hayes |
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Institution: | (1) Fish Pathology Laboratory, Department of Pathology, Ontario Veterinary College, University of Guelph, N1G 2W1 Guelph, Ontario, Canada |
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Abstract: | Summary The influence of trout serum on the attachment and spreading of isolated trout hepatocytes maintained in primary culture at
different temperatures was evaluated. Hepatocytes were obtained from young rainbow trout (Salmo gairdneri) by collagenase dissociation and maintained in modified Leibowitz L15 medium at 10° or 27° C for 24 h in plastic dishes previously
coated with type I bovine collagen. In the absence of serum, fewer than 10% of hepatocytes attached and none of them spread
on the collagen substrate. Trout serum at concentrations as low as 1.25% in the medium resulted in a pronounced concentration-dependent
increase in hepatocyte attachment, as determined by direct counts by phase contrast microscopy, or by percentage of lactate
dehydrogenase activity attached to the dishes after washing away unattached cells. Attachment rates were greater at the lower
temperature (10° C). Trout serum also substantially increased the proportion of attached hepatocytes that spread as monolayers
on the collagen substrate, especially at 10° C. By comparison, fetal bovine serum had little influence on the attachment or
spreading of trout hepatocytes. These studies demonstrate a simple inexpensive method for preparing attached monolayer trout
hepatocyte cultures. This procedure may be useful in toxicologic or functional studies in which fish hepatocyte attachment
is an operational requirement. |
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Keywords: | hepatocytes rainbow trout primary culture attachment |
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