Background & Aims The neuroprotective effect of the spheroid reservoir bioartificial liver (SRBAL) was evaluated in a porcine model of drug-overdose acute liver failure (ALF). Methods Healthy pigs were randomized into three groups (standard therapy (ST) alone, ST + No-cell device, ST + SRBAL device) before placement of an implantable intracranial pressure (ICP) monitor and a tunneled central venous catheter. One week later, pigs received bolus infusion of the hepatotoxin D-galactosamine and were followed for up to 90 hours. Results At 48 hours, all animals had developed encephalopathy and biochemical changes confirming ALF; extracorporeal treatment was initiated and pigs were observed up to 90 hours after drug infusion. Pigs treated with the SRBAL, loaded with porcine hepatocyte spheroids, had improved survival (83%, n=6) compared to ST alone (0%, n=6, p=0.003) and No-cell device therapy (17%, n=6, p=0.02). Ammonia detoxification, peak levels of serum ammonia and peak ICP, and pig survival were influenced by hepatocyte cell dose, membrane pore size and duration of SRBAL treatment. Hepatocyte spheroids remained highly functional with no decline in mean oxygen consumption from initiation to completion of treatment. Conclusions The SRBAL improved survival in an allogeneic model of drug-overdose ALF. Survival correlated with ammonia detoxification and ICP lowering indicating that hepatocyte spheroids prevented the cerebral manifestations of ALF (brain swelling, herniation, death). Further investigation of SRBAL therapy in a clinical setting is warranted.

Cell therapies, which include bioartificial liver support and hepatocyte transplantation, have emerged as potential treatments for a variety of liver diseases. Acute liver failure (ALF), acute-on-chronic liver failure, and inherited metabolic liver diseases are examples of liver diseases that have been successfully treated with cell therapies at centers around the world. Cell therapies also have the potential for wide application in other liver diseases, including non-inherited liver diseases and liver cancer, and in improving the success of liver transplantation. Here we briefly summarize current concepts of cell therapy for liver diseases.

In utero cell transplantation (IUCT) can lead to postnatal engraftment of human cells in the xenogeneic recipient. Most reports of IUCTs have involved hematopoietic stem cells. It is unknown if human hepatocytes used for IUCT in fetal pigs will lead to engraftment of these same cells in the postnatal environment. In this study, fetal pigs received direct liver injections of 1×107 human hepatocytes in utero and were delivered by cesarean-section at term. Piglets received a second direct liver injection of 5×107 human hepatocytes 1 week postnatally. Serum was analyzed for human albumin at 2, 4, and 6 weeks post-engraftment. Piglet livers were harvested 6 weeks after transplantation and examined by immunohistochemistry, PCR and fluorescence in situ hybridization for human specific sequences. Piglets receiving IUCT with human hepatocytes that were postnatally engrafted with human hepatocytes showed significant levels of human albumin production in their serum at all post-engraftment time points. Human albumin gene expression, the presence of human hepatocytes and the presence of human beta-2 microglobulin were all confirmed 6 weeks post-engraftment. IUCT in fetal pigs using human hepatocytes early in gestation allowed for engraftment of human hepatocytes, which remained viable and functional for weeks after transplantation. IUCT followed by postnatal engraftment may provide a future means for large scale expansion of human hepatocytes in genetically-engineered pigs.

Hydrogel scaffolds provide a beneficial microenvironment in transected rat spinal cord. A combinatorial biomaterials‐based strategy provided a microenvironment that facilitated regeneration while reducing foreign body reaction to the three‐dimensional spinal cord construct. We used poly lactic‐co‐glycolic acid microspheres to provide sustained release of rapamycin from Schwann cell (SC)‐loaded, positively charged oligo‐polyethylene glycol fumarate scaffolds. The biological activity and dose‐release characteristics of rapamycin from microspheres alone and from microspheres embedded in the scaffold were determined in vitro. Three dose formulations of rapamycin were compared with controls in 53 rats. We observed a dose‐dependent reduction in the fibrotic reaction to the scaffold and improved functional recovery over 6 weeks. Recovery was replicated in a second cohort of 28 animals that included retransection injury. Immunohistochemical and stereological analysis demonstrated that blood vessel number, surface area, vessel diameter, basement membrane collagen, and microvessel phenotype within the regenerated tissue was dependent on the presence of SCs and rapamycin. TRITC‐dextran injection demonstrated enhanced perfusion into scaffold channels. Rapamycin also increased the number of descending regenerated axons, as assessed by Fast Blue retrograde axonal tracing. These results demonstrate that normalization of the neovasculature was associated with enhanced axonal regeneration and improved function after spinal cord transection.

Several studies in the past have formed 3-dimensional (3D) spheroids of primary hepatocytes in suspension culture. Unfortunately, primary hepatocytes in a suspension environment tend to lose their differentiated function over time, generally due to damage from fluid shear stress and eventual spheroid settling. We have therefore created a novel suspension culture system, by seeding H35 rat hepatoma cells, a hepatocyte-derived cell line, in a 24-well tissue culture polystyrene (TCPS) plate placed atop an orbital shaker to create 3D spheroids. To provide stability to the formed spheroids, we used a long-chain polymer, bovine serum albumin (BSA), dissolved in the cell culture medium and/or coated on TCPS surfaces placed in suspension configurations. Our results demonstrate that BSA coating of culture surfaces resulted in uniform and well-defined spheroids with little spheroid settling or "flattening" of cell colonies in either static or suspension configurations. In BSA-coated suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium. In static uncoated cultures, the normalized rat albumin production levels were enhanced by addition of BSA within culture medium. Thus, both addition of BSA to culture medium and application of BSA as a surface coating appear to be meaningful avenues for tailoring spheroid morphology and function. This 24-well plate suspension culture system may be a valuable tool for high throughput investigations of liver cell behavior in a stable, uniform, 3D spheroid state.