[show abstract][hide abstract] ABSTRACT: Liver diseases still have a high mortality even though liver transplantation has become a standard treatment. Currently, hepatocyte transplantation has been proposed as another promising strategy. One limitation is the availability of human livers as a source of hepatocytes. Because of an unlimited supply, the use of porcine hepatocytes might address this problem. Regardless of the source, once isolated hepatocytes lose specific functionality due to the loss of the natural microenvironment. For this reason, we tested the ability of a self-assembling peptide nanofiber (SAPNF) to provide a provisional three-dimensional (3D) support to interact with cells to control their function in vivo. Isolated porcine hepatocytes were embedded in SAPNF, or collagen type I and transplanted by direct injection into the splenic pulp of SCID mice suffering from acute liver failure (ALF) by 90% hepatectomy. SAPNF porcine hepatocyte transplantation produced engraftment that was far superior to that obtained using collagen and prolonged the survival of mice with ALF, in contrast with controls. An ultrastructural evaluation using transmission electron microscopy indicated extensive cell-cell communication and preservation of hepatocyte architecture. The transplanted SAPNF hepatocytes showed higher expression of albumin and PAS and lower apoptotic events assessed by TUNEL staining. Hepatocytes culture in a truly 3D network allows in vivo maintaining of differentiated functions, and once transplanted between widely divergent species can function to correct acute liver failure in mice and prolong their survival.
[show abstract][hide abstract] ABSTRACT: We created a hepatic failure pig model that was suitable for the assessment of cell therapies, such as hepatocyte transplantation and bioartificial livers, using a laparoscopic surgical technique. In our model, all of three hepatic arteries were resected, 5, 7.5, or 10 ml of carbon tetrachloride (CCL4) was injected into the liver through the portal vein, and subsequently the portal vein was mechanically occluded for 30 min. After the portal occlusion was released, a liver biopsy was performed, and then the surgery was completed. Blood samples were regularly taken during the surgery in order to perform biochemical examinations. All of five pigs in which 5 ml of CCL4 was infused recovered spontaneously and survived; in contrast, all of five pigs that received 10 ml CCL4 died within 1.5 h after surgery. The pigs in which 7.5 ml CCL4 was administered developed liver failure and survived for 6.4 h on average (+/-1.4 SD). Induction of liver failure with the use of 7.5 ml CCL4 and 30-min hepatic ischemia fulfilled five of the six criteria that were proposed by Terblanche and Hickman: reversibility, reproducibility, death from liver failure, a therapeutic window, and a large-animal model. We believe that our model is the first report on creation of a reliable model for liver failure in pigs to assess the efficacy of liver-targeted cell therapies.
[show abstract][hide abstract] ABSTRACT: Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.