Modelling the loss of metabolic capacities of cultured hepatocytes: application to measurement of Michaelis-Menten kinetic parameters in in vitro systems

Abstract

1. The loss of metabolic capacities during culture time constitutes a major limitation for the use of hepatocyte primary cultures in in vitro metabolism measurements. A new strategy is presented that permits one to calculate the Michaelis-Menten parameters V_max and K_m from extended experiments, by modelling V_max as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. V_max and K_m were used to calculate intrinsic clearance, and comparisons were made with methods already described in the literature. Intrinsic clearances given by our method were scaled to in vivo hepatic clearances that were close to those reported in the literature. 3. Our method could quantify the V_max decrease with culture time from estimates of time parameters, t_1/2 or t₅₀. In our system, this V_max decrease was in agreement with P450 cytochrome inactivation rates published for the rat liver. 4. In conclusion, we propose a convenient, simple and useful general method for both Michaelis-Menten parameter estimation and modelling of variations in the metabolic capacities observed in in vitro systems. Such an approach should improve the usefulness of hepatocytes in primary cultures for long-term metabolism experiments.