Fetal liver cells were obtained from 15 fetal livers at a mean yield of 1.8X109 cells. A marker expression analysis was performed immediately after cell isolation and before culturing. Flow cytometry analysis revealed a heterogeneous cell population characterized by expression of markers specific to hepatocytes, epithelial cells, hepatic progenitor cells, bipotential precursors of the hepatic and biliary lineages, hematopoietic progenitor cells and mesenchymal cells.
Fetal livers were obtained from tissue donations after selective, therapeutic abortions at a gestational age of 18 to 22 weeks.
Each fetal liver was perfused in situ for 10 minutes with 150-200 mL solution A [DPBS without calcium and magnesium containing ethylenediamine tetraacetic acid (EGTA, 2 mmol/L) and gentamicin (50 µg/mL)] to flush out the remaining blood and to loosen the desmosomal cell-cell junctions by chelation. Perfusion was continued for 10 minutes with solution B, which had a composition similar to that of solution A but without EGTA. Following perfusion with solution C [DPBS with calcium and magnesium] for 2 to 5 minutes, perfusion with solution D [DPBS with calcium, magnesium and collagenase] was performed for 7-10 minutes until macroscopic tissue disintegration was observed. The liver was excised and transferred into a 100-mm Petri dish. The capsule was ruptured with forceps and the mechanical disruption of the liver was completed by scraping tissue away from the biliary tree with a sterile, disposable cell scraper. The capsule material and the undigested tissue were removed by filtration of the cell suspension through a sterile 500-µm nylon mesh. The remaining undigested tissue on the filter was rinsed with 10-20 mL cold solution D. The cell pellet was washed 2 to 3 times and the cells were resuspended in a medium based on Williams’ E medium [DPBS with calcium, magnesium, glucose (15 mM), pyruvate (1 g/L), amphotericin B and penicillin/streptomycin] and were maintained at 150C in a 5% CO2 and 95% relative humidity environment until transplantation.