High yield hepatocyte isolation from pig livers for investigation of hybrid liver support systems: influence of collagenase concentration and body weight

Abstract

Cultured hepatocytes would be required in large quantities if a hybrid liver support system became available. We have therefore performed a study of larger scale enzymatic hepatocyte isolation on whole pig livers. A new method of open-loop and recirculating perfusion in five steps using both the portal venous and the hepatic arterial vascular systems has been developed. In a study of the yield and cell viability of hepatocytes afer isolation, the collagenase concentration of the perfusate was investigated over a range of 0.02 to 0.15%. The effect of animal weight on yield and cell viability was determined within the body weight range of 6 to 40 kg. At collagenase concentrations of less than 0.08%, yield and viability declined. As collagenase concentration increased, the yield continuously increased while the viability peaked at 0.1%. There was a decline of viability and yield with rising body weight. Optimal results were obtained with the lowest body weight (6 kg) which gave a yield of 95% (g wet weight) and a viability of 98% (trypan blue). This novel five-step collagenase perfusion technique with arterial perfusion appears to be reproducible and safe. Application of this method enables the large scale investigations to be undertaken towards the development of hybrid liver support.