A large scale isolation of pig hepatocytes for a bioartificial liver system

A bioartificial liver support using primary culture hepatocytes as a bioreactor requires a large scale isolation of hepatocytes for a clinical practice. In this study we developed a quick method to isolate 100g hepatocytes in single cell from a 20-25 kg pig. A pig under anesthesia was perfused from portal vein with heparinized saline followed by disperse- supplemented 0.05% collagenase. The liver was resected and re-perfused with 0.05% collagenase solution at 37°C for 10 minutes, resulting in isolation of a mixture of tissue fragments and some single cells. The tissue suspension was processed to a differential filtration using 4 layered metal filters with various mesh sizes ranging from 4.7 to 100 mesh. After larger tissue fragments were removed by smaller mesh-sized filters, most acinus- like tissue fragments retained on a 30-mesh filter. Thus, the 30-mesh filter was transferred to deep metal pan, received additional collagenase solution and incubated at 37°C under gentle agitation. This procedure allowed single cell to pass the 30-mesh filter gradually. The filter-passed cell suspension was further differentially filtrated with larger mesh-sized filters in the presence of collagenase solution. The filtrate after 100 mesh filter was collect, immediately cooled, and washed thrice by low speed centrifugation. Over 90% cells in the final cell preparation were in single cell and viable. By the combination of liver perfusion and differential filtration, about 100g hepatocytes in single cell were prepared from a 20-25 kg pig within 2 hours after the initiation of in vivo liver-perfusion.