Isolation of Liver Endothelial Cells

Tissue samples and blood samples from patients were obtained with written informed consent and with local ethics committee approval (LREC reference 06/Q2702/61, Birmingham, UK and 04/Q2708/41, Birmingham, UK). Liver endothelial cells were isolated from explanted livers or donor tissue surplus to surgical requirements using a collagenase digestion (collagenase type 1a, Sigma-Aldrich) as described previously60 (link). All tissue was collected from patients in the Liver Unit at Queen Elizabeth Hospital in Birmingham with informed consent and under local ethics committee approval. Briefly, digested tissue was placed over a 33% / 77% Percoll (Amersham Biosciences) density gradient. The endothelial cells were isolated by immunomagnetic selection using Abs against CD31 conjugated to Dynabeads (Life Technologies). The endothelial cells were then cultured in medium composed of human endothelial basal growth medium (Life technologies), 10% AB human serum (HD supplies), 10ng/ml vascular endothelial growth factor (VEGF), and 10ng/ml hepatocyte growth factor (HGF) (Peprotech). The cells were grown in collagen-coated culture flasks and were maintained at 37°C in a humidified incubator with 5% CO₂ until confluent.