Journal: Experimental Biology and Medicine
Article Title: Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells
Figure Lengend Snippet: Schematic overview of the isolation of PHH and NPC from human liver resections. Liver cells were isolated by a two-step EGTA/collagenase P perfusion technique (1). The cell suspension was centrifuged initially at 50 × g , 5 min, 4℃ (2). PHH (pellet) were separated from the NPC (supernatant). PHH were purified by a Percoll density gradient centrifugation at 1250 × g , 20 min, 4℃ (A and B). The supernatant of PHH isolation (2) was centrifuged first at 300 × g , 5 min, 4℃ (3). KC were found in the supernatant of the first centrifugation step. Cells were treated with a second centrifugation at 650 × g , 7 min, 4℃. Both cell pellets were pooled and re-suspended in HBSS. Afterwards, the cell suspension was transferred to a two-layer (25% / 50%) Percoll density gradient and centrifuged at 1800 × g , 20 min, 4℃ (4). Dead cells on top of the 25% Percoll layer were discarded. NPC were located around the interphase of the 25% and the 50% Percoll layer. KC were separated from the NPC fraction by an adherence separation step (5). LEC and HSC were separated by MACS®. For this purpose, the remaining cells were centrifuged at 300 × g , 5 min, 4℃, and labelled with CD31-conjugated MicroBeads (6). CD31-negative HSC passed the MACS® separation column (7). The column was removed from the magnetic field and the CD31-positive LEC were dissolved from the column (8). (A color version of this figure is available in the online journal.)
Article Snippet: Percoll, Trypan Blue and HBSS were provided by Biochrom (Berlin, Germany).
Techniques: Isolation, Purification, Gradient Centrifugation, Centrifugation, Magnetic Cell Separation