The portal vein was dissected and transected. The falciform and cardiac ligaments were transected and the liver mobilized to visualize the inferior vena cava. The vena cava was transected above and below the liver and any remaining attachments to the liver were dissected. The liver was removed with the capsule H 89 order intact. One side of the vena
cava was ligated and the other end was cannulated with a 22-gauge (22G) cannula in mice, 20G in rats and ferrets, and size 16 tubing in rabbits and pigs. The portal vein was cannulated with a 20G cannula in rats, ferrets, and rabbits, and with size 16 tubing in pigs. All cannulae were obtained from Terumo Medical Corp., Elkton, MD and the tubing was obtained from Masterflex, Cole-Palmer Instrument Co, Vernon Hills, IL. The cannulae www.selleckchem.com/products/VX-770.html in the portal veins were attached to a pump (Masterflex L/S peristaltic pump with Masterflex L/S easy load pump head and L/S 16G tubing, Cole-Palmer Instrument Co, Vernon Hills, IL) and distilled water was perfused through the portal vein at a rate of approximately 5 mL/minute (rat and ferret livers). Approximately 40 times the volume of the liver was perfused through this circuit. Subsequently, 1% Triton-X 100 with 0.1% Ammonium Hydroxide (Sigma-Aldrich) was
perfused through the livers to decellularize the organ. Approximately 50 times the volume of the liver was circulated through the vascular tree. Finally, a distilled water wash was circulated to wash out the decellularization detergent. DNA was isolated from a small piece from 6 different ferret
bioscaffolds and three different fresh ferret livers using the DNeasy Tissue kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. Similar masses of scaffold and control liver tissue were used. Hematoxylin and eosin (H&E), trichrome (Newcomer Supply, Middleton, WI), and Russel-Movat Pentachrome (American MasterTech Scientific Inc, Lodi, CA) staining were performed for ferret scaffold characterization after fixation, paraffin embedding and sectioning. Collagen, sGAG and elastin quantification (n = 3 samples) were performed as directed in the protocols associated with the Sircol, Blyscan and Fastin assay kits (Biocolor, Ltd., Newtownabbey, UK), respectively. MCE A Student’s t test were performed to compare the total amount of sGAG, O-sGAG, and elastin in fresh liver and bioscaffold samples. Ferret liver bioscaffold samples were prepared for scanning electron microscopy by lyophilizing the decellularized scaffold and cutting it into multiple sections. Ferret bioscaffold ECM components were analyzed by denaturing SDS-polyacrylamide gel electrophoresis and Western blot. Briefly, up to 70 μg of total protein extract (n = 3) were separated on a 4%-20% Tris-glycine gel (Invitrogen Corp., Carlsbad, CA) and blotted onto a Immobilon-P PVDF Membrane (Millipore Corp., Billerica, MA).