Fetal liver cells (University of Pittsburgh)

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 15^0C in a 5% CO₂ and 95% relative humidity environment until transplantation.