Primary liver cells were isolated by subjecting donor livers that cannot be transplanted as an organ to a modified three-step collagenase perfusion process.
Hepatocytes are isolated by a modified three-step collagenase perfusion. Livers are split into right and left lobe or not, placed in a steel bowl and perfused with buffer solutions through catheters placed into the portal or hepatic vein branches and fixed with sutures, and US Pharmacopoeia (USP)-grade platinum-cured silicone tubings. The perfusion system is run by three 3-channel calibrated peristaltic pumps. Four different GMP-grade buffers (buffers 1–4) used for perfusion custom made, based on HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]-buffered saline solution. Buffer 1 contained additional EGTA (0.5 mol/L), buffer 3 contained additional calcium chloride (0.6 g/L), and buffer 4 contained additional Pharmacopoeia European (Ph. Eur.)-grade human serum albumin. Buffer 1 was introduced into the liver with a pump rate of 100–400 ml/min. Buffer 2 was used to flush out residues of EGTA, which would inhibit enzymatic digestion. Buffer 3 was supplemented with Collagenase P or Liberase CI at concentrations of 100–1000 mg/l depending on the lot specific properties and used for enzymatic digestion of the tissue at 37°C for 25–45 min, depending on the individual terms of the organ. Digestion is stopped by ice cold buffer 4 when the liver tissue under the capsule is discernibly digested. The catheters are pulled out and the tissue is manually disrupted with sterile scissors and scalpels. The suspended hepatocytes are filtered through 750- and 500-μm filters into a 3-L bag containing 1000 ml of ice-cold buffer 4. After collection, the cells are distributed in 600-ml transfer packs and washed at 50 × g for 10 min. The supernatant is removed using a plasma extractor. The cell pellet is carefully resuspended in ice-cold buffer 4.