Peripheral blood was collected in EDTA with informed consent from otherwise healthy haemochromatosis patients (ethical approval: Use of venesected blood from patients with haemochromatosis to isolate and culture immune cells, REC reference 04/Q2708/41) as well as patients with autoimmune hepatitis and alcoholic liver disease (ethical approval via the University of Birmingham Human Biomaterials Resource Centre (NRES Committee North West – Haydock; Ref 15/NW/0079)). In the case of matched blood and liver samples, venous blood was obtained from patients undergoing liver transplantation before the induction of anaesthesia. Subsequently explanted diseased liver was obtained from the same patients to allow the isolation of matched blood and liver-derived cells (ethical approval: Cellular trafficking and immune response in the human liver, REC reference 06/Q2708/11). Venous blood was also obtained from consenting healthy donors (protocol approved by the NRES Committee West Midlands ethical board; REC reference 14/WM/1254) in heparin. Umbilical cord blood units were obtained from the Anthony Nolan Cell Therapy Centre Nottingham (ANCTC) under generic tissue bank ethics held by ANCTC and extended to the researchers under a material transfer agreement (MTA).
Peripheral blood mononuclear cells were then isolated from whole blood by layering on top of Lympholyte-H Cell Separation Media (Cedarlane, Burlington, Canada) before centrifugation at 800g for 25 minutes at room temperature allowing separation based on gradient density. PBMCs were aspirated from the cloudy partition layer and washed twice with sterile phosphate buffered saline (PBS) before counting using an improved
Neubauer haemocytometer with dead cells excluded using Trypan-blue (Sigma-Aldrich, Gillingham , UK) and further experimental procedure.
2.1.2 Lymphocyte isolation from human liver
Diseased liver tissue was taken with informed consent from patients undergoing liver transplant at Queen Elizabeth Hospital, Birmingham UK. Chronically inflamed livers from the following patient groups were studied: alcoholic liver cirrhosis (ALD), non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), auto-immune hepatitis (AIH), chronic hepatitis C virus infection (HCV) and chronic hepatitis B virus infection (HBV). Additionally, livers explanted for non-inflammatory chronic conditions including polycystic liver disease and α-1 antitrypsin deficiency were also studied in some cases, where a comparison between inflamed and non-inflamed liver tissue was required. Non-diseased liver tissue was obtained from surplus tissue from cadaveric registered organ donors or from uninvolved liver tissue removed at the time of resection for colorectal hepatic metastases. All samples were collected with informed patient consent and local research ethics committee approval (local research ethics committee approval 04/Q2708/41 and REC 2003/242).
Explanted livers were cut into slices by a qualified pathologist at Queen Elizabeth Hospital and then transported to the Centre for Liver Research for further processing. Under sterile conditions, liver slices weighing approximately 50 – 150 g were diced into 3 - 5 mm cubes using scalpels and then washed at least 5 times in PBS to remove any blood associated lymphocytes and erythrocytes. Once washed, tissue was then transferred to Stomacher 400 Circulator bags (Seward, Worthing, UK) in approximately 150 ml chilled RPMI-1640 lymphocyte medium (Sigma-Aldrich) and then stomached for 5 – 7 minutes,
depending on severity of fibrosis, at 230 rpm in a Stomacher 400 machine (Seward, Worthing). Mechanical homogenisation in this manner prevented loss of surface markers by enzymatic cleavage as well as being quicker and cheaper than enzymatic digestion. In cases where very small amounts of tissue were obtained (typically less than 20 g), mechanical digestion was performed using GentleMACS dissociation C tubes (Miltenyi Biotech, Bergisch Gladbach, Germany) in a GentleMACS dissociator, running the mouse spleen standard protocol.
Following homogenisation, liver tissue was filtered through a fine (63 micron) nytal mesh (John Stanniar and Co, Manchester, UK) and washed with twice the initial volume of chilled PBS. Large cellular debris was then removed by centrifugation at 55 g for 5 minutes. Supernatants were then re-centrifuged at 2,000 rpm at 4oC , the supernatant discarded and the cell pellet washed at 4oC with PBS a further 3 – 5 times depending on the extent of steatosis present in the liver tissue. Finally, washed cell pellets were re- suspended in PBS, layered on Lympholyte-H cell separation medium and centrifuged at 800 g for 25 minutes at room temperature. Liver derived lymphocytes (LDL) were aspirated from the partition band and washed twice with cold PBS before counting using an improved Neubauer haemocytometer with dead cells excluded using Trypan-blue (Sigma-Aldrich).
2.1.3 HSEC isolation and culture
Primary human hepatic sinusoidal endothelium cells (HSEC) were isolated from explanted liver disease tissue or from normal donor liver surplus to transplant requirements. Slices of liver tissue (20 – 50 g) were diced into 5 mm cubes using scalpels, washed with cold PBS as before and then incubated with collagenase type 1A (Sigma, St. Louis, USA) at 37oC
for 20 minutes in a shaker incubator. Following digestion the tissue was layered on top of a 33%/ 77% Percoll: 1M NaCl solution gradient and centrifuged at 500 g for 30 minutes. The interface layer was aspirated and cells washed in cold PBS three times before cholangiocytes were labelled using a cholangiocyte specific mAb for HEA125 (Progen, Heidelberg, Germany) at 4oC for 20 minutes. Following incubation, cholangiocytes were removed from the cell suspension by incubation with anti-mouse IgG1-coated Dynabeads (Invitrogen, Carlsbad, USA) and subsequent magnetic separation. 25 μL pre-washed and re-suspended Dynabeads were added to 1 mL of labelled cells in a polypropylene FACS tube and incubated for 20 minutes at 4oC before inserting the tube into a magnet for 2 minutes to allow labelled cells to adhere to the tube walls. With the tube still in the magnet, the supernatant containing non-labelled cells was then removed.
HSEC were then isolated from the resultant cell suspension by labelling using Dynabead- conjugated anti-CD31 antibody (10 μg/mL; Invitrogen) and subsequent magnetic separation. The protocol was identical to that for cholangiocyte removal except after magnetic incubation the supernatant was discarded and the adherent cells washed three times with PBS with an additional incubation in the magnet at each step, discarding non- adherent cells each time to ensure cell purity.
Isolated CD31+ HSEC were cultured in complete endothelial media (Gibco, Invitrogen, United Kingdom) supplemented with penicillin, streptomycin (100 µg/ml) and glutamine (2 mM, Gibco, CA, USA), heat-inactivated human AB serum (10% v/v, HD Supplies, Buckingham, UK), hepatocyte growth factor (10 ng/ml Peprotech, Rocky Hill, NJ, USA) and vascular endothelial growth factor (10 ng/ml Peprotech), referred to hereafter as HSEC media. Endothelial cells were grown in 25 cm2 tissue culture flasks coated with rat
tail collagen with regular medium exchanges until they became a confluent monolayer, then expanded in 75 cm2 tissue culture flasks coated with rat tail collagen.