Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout ( Salmo gairdneri )

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.