Collagenase P versus Liberase perfusion for large-scale isolation of primary liver cells from discarded human grafts

Aim: Human livers, discarded from transplantation due to excessive steatosis, fibrosis/cirrhosis or other reasons provide a suitable resource for primary human liver cells that can be used in artificial liver support systems. The therapeutic application of liver cells requires a high number and quality of cells. The outcome of the isolation procedure is strongly affected by the enzyme used for digestion of the organ. In this study we compared the effect of two different enzymes, crude collagenase and liberase, a highly purified collagenase, on the outcome of cell isolation process utilizing discarded grafts.

Methods: The organs (n=30) were rejected from the transplantation programm due to steatosis (n=15), fibrosis (n=2), cirrhosis (n=4) or other reasons (n=9). Human liver cell isolation was performed by an established five-step enzymatic perfusion technique using collagenase P (n=17) or liberase (n=13). There were no significant differences between the two investigated groups in age, body mass index, organ weight, preservation time, preservation solution or initial liver impairment.

Results: Cell isolation resulted in a significant (p ≤ 0.05) lower viability when using collagenase P (48.8±23.3%) than when using liberase (67.5±17.6%) perfusion. The mean cell yield obtained was comparable in both groups (collagenase P–64.7±13.7% and liberase–64.2±19.2%). Isolated liver cells were successfully cultured in hollow fiber based bioreactors for extracorporeal liver support in 11 cases of collagenase P perfusion (64.7%) and in 10 cases of liberase perfusion (76.9%).

Conclusion: Our study showed that the use of liberase resulted in a significantly higher quality of isolated liver cells than collagenase P. Thus we recommend the use of liberase for large-scale isolation of cells from discarded donor livers for the application in expracorporeal liver support systems.