Comparison of four methods for mass hepatocyte isolation from pig and human livers.

Using the pig liver, parameters for large scale hepatocyte isolation were studied in order to develop a technique suitable for human organs. These investigations led to a 5-step modification of the original 2-step method. Four groups were compared. A nonenzymatic EDTA perfusion technique has been shown to be inconvenient for mass cell isolation. The enzymatic 2-step perfusion, using 0.08% collagenase and 20-kg pigs, resulted in a mean hepatocyte viability of 61 +/- 1.9%, with a mean yield of 67 +/- 6.5% wet weight of the organ. The enzymatic 5-step method resulted in a mean hepatocyte viability of 74 +/- 1.7% with a mean yield of 80 +/- 1.8% wet weight. Five-step portal venous perfusion in combination with arterial perfusion resulted in 76 +/- 2.6% viability with a yield of 82 +/- 6.1%. The results were dependent on collagenase concentration and weight of the donors, improving with decreasing body weight. The 5-step method with combined arterial and portal vein perfusion developed for pig liver was used for mass human liver cell isolation with a minimum viability of 57% and a minimum yield of 58% wet weight.

Nonenzymatic versus enzymatic hepatocyte isolation from pig livers for larger scale investigations of liver cell perfusion systems.

A comparison of nonenzymatic and enzymatic hepatocyte isolation was performed on pig livers. The collagenase perfusion showed superior results: mean viability 72 +/- 10% versus a maximum viability of 21% using EDTA-perfusion. A five-step collagenase perfusion technique, developed for pig livers enables larger scale investigations, in order to develop methods for hepatocyte cultures in therapeutical liver cell perfusion systems.

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

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.

Comparison of pig hepatocyte isolation using intraoperative perfusion without warm ischemia and isolation of cells from abattoir organs after warm ischemia.

Enzymatic hepatocyte isolation using warm ischemic pig livers from an abattoir was compared with isolation using in situ perfused organs. Using organs from animals of 30 kg body mass (BM), the intraoperative perfusion showed superior results. The use of livers from abattoir pigs of 40-50 kg BM after warm ischemia resulted in a lower yield of hepatocytes and in high rates of injured cells. The mean yield in the intraoperative perfusion group was 68 +/- 11% (wet weight), the maximum yield in the abattoir organ group was 58%. The mean viability in the intraoperative perfusion group was 65 +/- 14% (trypan blue) compared with a maximum viability of 39% in the abattoir liver group. Additional purification by density gradient centrifugation improved the viability of the abattoir liver group to a mean of 95% (trypan blue). The use of pig livers from large abattoir animals required additional purification steps to improve viability since the cell yield is considerably lower than with intraoperative organ perfusion. In general, hepatocyte isolation from abattoir organs is not recommended.