The aim of this study was to establish the optimal conditions for the culture of equine hepatocytes in a monolayer configuration. The obtained results show that the rate of MTT metabolism correlated with the number of cultured cells and a linear increase of MTT reduction rate was observed in cases when the cell density varied between 1.25 × 10⁴ to 5 × 10⁴ viable cell/well of 96-well plate. Hepatocytes reached the optimal cell attachment rate and MTT reduction at a cell density of 5 × 10⁴ cells/well. The number of attached cells to a plastic culture dish was also related to incubation time. The greatest ability of hepatocytes to attach to the culture dish was observed after 10 h of incubation and it was found to be 84.1 ± 2.5% of seeded hepatocytes. It was also found that fetal bovine serum was more efficient than horse serum for the attachment of equine isolated hepatocytes in a monolayer culture. The highest rate of cell attachment (assessed microscopically and with MTT reduction test) was observed when cells were plated with the culture medium supplemented with FBS or HS at a concentration of 5%. However, medium supplementation with higher than 5% serum concentration (10% of FBS or HS) significantly decreased MTT reduction rate. The rate of MTT metabolism and cell attachment in hepatocytes cultured in WE supplemented with FBS or HS was also dependent on the plating time and were the highest after 10 h of seeding.
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