Dependence of the adaptive capacity of liver mitochondria on preparation method

Abstract

When conducting studies on isolated hepatocytes, it is important to obtain cells that retain the functional properties that are characteristic of the whole organ. Increased blood viscosity during liver perfusion, decreased perfusion pressure in blood vessels, and hence hypoxia, are among the factors that may affect the functional state of isolated hepatocytes. The functional state of cells can be estimated by the adaptive capacity of mitochondria, by inducing maximal respiration rate by uncoupling respiration and oxidative phosphorylation due to the addition of FCCP. The research aimed to investigate the adaptive capacity of mitochondria of isolated hepatocytes using in situ and in vitro liver perfusion. Hepatocytes were isolated by the two-staged Seglen method by in situ and in vitro liver perfusion. Isolated hepatocytes, after 15-minute incubation in the medium without addition or with respective oxidative substrate – glutamine, pyruvate, succinate, monomethyl succinate, α-ketoglutarate, dimethyl-α-ketoglutarate (at a concentration of 2 mM) or glucose (10 mM) – were added into the respiratory chamber and FCCP was added in increasing concentrations. It was established that at in situ liver perfusion maximal rate of uncoupled respiration and the optimal concentration of FCCP was higher than at in vitro liver perfusion. Addition of exogenous substrates to a medium increased the respiration rate of hepatocytes. Upon in situ liver perfusion maximal uncoupled respiration rate increased at all causes except glucose, and at in vitro liver perfusion – only when dimethyl-α-ketoglutarate, succinate and monomethyl succinate were used. The optimal concentration of FCCP at in vitro liver perfusion increased due to the addition of glutamine, pyruvate and monomethyl succinate to the medium, and at in situ liver perfusion – only upon glucose oxidation. In both perfusion methods, the highest maximal rate of uncoupled respiration is with the use of monomethyl succinate and the optimal FCCP concentration – upon pyruvate oxidation. Therefore, in situ liver perfusion is better method to obtain stable and metabolically active hepatocytes in support respiratory processes at a high level then in vitro perfusion.