Albert D. Moscioni

A bioartificial liver to treat severe acute liver failure.

ObjectiveTo test the safety and efficacy of a bioartificial liver support system in patients with severe acute liver failure. Summary Background DataSummary Background Data authors developed a bioartificial liver using porcine hepatocytes. The system was tested in vitro and shown to have differentiated liver functions (cytochrome P450 activity, synthesis of liver-specific proteins, bilirubin synthesis, and conjugation). When tested in vivo in experimental animals with liver failure, it gave substantial metabolic and hemodynamic support. MethodsSeven patients with severe acute liver failure received a double lumen catheter in the saphenous vein; blood was removed, plasma was separated and perfused through a cartridge containing 4 to 6 X 109 porcine hepatocytes, and plasma and blood cells were reconstituted and reinfused. Each treatment lasted 6 to 7 hours. ResultsResults patients tolerated the procedure(s) well, with neurologic improvement, decreased intracranial pressure (23.0 ± 2.3 to 7.8 ± 1.7 mm Hg; p < 0.005) associated with an increase in cerebral perfusion pressure, decreased plasma ammonia (163.3 ± 21.3 to 112.2 ± 9.8 μMoles/L; p < 0.01), and increased encephalopathy index (0.60 ± 0.17 to 1.24 ± 0.22; p < 0.03). All patients survived, had a liver transplant, and were discharged from the hospital. ConclusionsConclusions bioartificial liver is safe and serves as an effective “bridge” to liver transplant in some patients.

Selective intraportal hepatocyte transplantation in analbuminemic and Gunn rats.

Although significant progress has been achieved in isolated hepatocyte transplantation, the optimal site of cell implantation has not yet been determined. We have developed a novel experimental method of intraportal hepatocyte transplantation that allows easy assessment of the morphology and function of transplanted hepatocytes. Donor hepatocytes were harvested from Sprague-Dawley rats by in situ EDTA/collagenase perfusion. Fifteen recipient Nagase analbuminemic rats (NAR) underwent cannulation of the gastroduodenal vein under ether anesthesia. Either the posterior or anterior liver lobes were selectively infused with cells by occluding the portal venous supply of the nontransplanted liver lobes. Normal donor hepatocytes (2±107) suspended in normal saline were infused over 1 min (4 ml). Recipients were treated with cyclosporine for the duration of the experiment. Plasma albumin levels were determined by ELISA, before and at various intervals after transplantation. In NAR rats transplanted with normal hepatocytes, there was a significant (P<0.003) and sustained (12 weeks) increase in plasma albumin levels. Control NAR rats transplanted with NAR hepatocytes (n=8) showed no significant changes in plasma albumin levels. Similarly, normal Wistar hepatocytes were infused in-traportally into the posterior lobes of Gunn rats (n=4), which lack the ability to conjugate bilirubin. Pre-and posttransplantation bile was collected following bile duct cannulation. Bile analysis by HPLC, demonstrated a significant (P=0.04) increase in the level of bilirubin conjugates following transplantation and a corresponding decrease in total serum bilirubin (P=0.04). Our experimental data demonstrate that direct selective intraportal infusion of hepatocytes is an effective technique of hepatocyte transplantation in the rat.

Repeated intraportal hepatocyte transplantation in analbuminemic rats

The optimal site for implantation of isolated hepatocytes has not been established. We have developed a novel technique which allows repeated infusion of hepatocytes into the portal system via an indwelling catheter. Seven Nagase Analbuminemic rats (NAR) underwent single intraportal infusion of 2 x 10(7) isolated normal albumin-producing rat hepatocytes. Another seven NAR rats underwent placement of indwelling catheters into the portal venous system via the gastroduodenal vein. Each of them received six batches of 5 x 10(6) normal albumin producing hepatocytes. Seven control NAR rats were infused repeatedly (intraportally) with saline only. Plasma albumin (ELISA) showed significant increase in experimental animals and was more pronounced (p < 0.05) in rats transplanted repeatedly than in those given a single dose of cells. Immunohistochemical staining of the liver sections confirmed the presence of transplanted albumin producing hepatocytes. Rats transplanted with a single large batch of isolated hepatocytes showed liver tissue damage, whereas those subjected to repeated cell infusions had normal liver histology. We have developed a novel intraportal transplantation method which allows successful engraftment of a large number of isolated hepatocytes.

Human liver cell transplantation: Prolonged function in Athymic-Gunn and athymic-analbuminemic hybrid rats

Isolated cryopreserved human liver cells, attached to collagen-coated microcarriers, were injected intraperitoneally into mutant rat recipients genetically deficient in either bilirubin uridine diphosphoglucuronosyltransferase activity (Gunn rats) or albumin synthesis (Nagase analbuminemic rats). One group of the recipient Gunn and analbuminemic rats were made genetically immunodeficient by interbreeding with athymic rats with inherited T-cell deficiency. Injected microcarriers and cells formed aggregates on the surface of the pancreas. There was no morphologic evidence of rejection in athymic recipients, whereas immunocompetent recipients demonstrated rejection within 5 days of transplantation. Athymic-Gunn rat recipients demonstrated excretion of bilirubin glucuronides in bile for 30 days and reduction in their serum bilirubin levels. In recipient athymic-analbuminemic rats, plasma albumin levels increased from pretransplantation levels of 0.025-0.05 mg/ml to 3.9-4.8 mg/ml 8 days posttransplantation and remained nearly at that level throughout the 30 days of the study. A method of harvesting, attaching to microcarriers, cryopreserving, and in vivo testing of human hepatocytes with prolonged survival and function in athymic-Gunn and athymic-analbuminemic hybrid rats is reported. These rats are excellent animal models for testing xenograft function.

Automated liver cell processing facilitates large scale isolation and purification of porcine hepatocytes.

&NA; An automated method for large scale isolation and purification of porcine hepatocytes is described. Liver cells were harvested by a two‐step portal vein perfusion with ethylene‐diaminetetraacetate and collagenase. Hepatocyte purification was carried out using either a standard manual processing method (Procedure A) or an automated processing method using a filtration chamber and a programmable cell washer (Procedure B). Both methods produced high cell yields (Procedure A: 1.30 ± 0.55 × 1010 viable hepatocytes/liver; Procedure B: 1.38 ± 0.32 × 1010 viable hepatocytes/liver) and viability (Procedure A:89 ± 6.5%; Procedure B: 92 ± 3.9%). Hepatocyte purity was significantly greater after Procedure B than after Procedure A (93.1 ± 3.1% versus 83.1 ± 3%, p < 0.01). Isolated hepatocytes by either method were morphologically intact, as demonstrated by transmission electron microscopy showing integrity of plasma membranes and intracellular organelles. Cultured hepatocytes isolated by either method were functionally intact, although those isolated by Procedure A showed significantly lower activity of microsomal 7‐ethoxycoumarin‐O‐deethylase activity (p < 0.05) and mitochondrial succinate dehydrogenase activity (p < 0.01). In conclusion, use of the automated hepatocyte processing method resulted in efficient large scale preparation of porcine hepatocytes, with higher purity and greater retention of differentiated liver metabolic functions, and was found to be less time consuming and less labor intensive. ASAIO Journal 1995; 41:155‐161.

Long-term correction of albumin levels in the Nagase analbuminemic rat : repopulation of the liver by transplanted normal hepatocytes under a regeneration response

Numerous studies have reported successful transplantation of hepatocytes with demonstration of function. However, none have shown long-term correction of a liver-related metabolic defect. Male Nagase analbuminemic rats, immunosuppressed with cyclosporin-A, were transplanted with normal hepatocytes (2 x 10(7) cells/rat) isolated from allogeneic male Sprague-Dawley rat donors. Hepatocytes were selectively transplanted via the portal vein tributary into the posterior liver lobes of Nagase analbuminemic rats. Following 2 wk, to allow engraftment, selected transplanted rats (Group I) were reoperated and the portal venous branch supplying the anterior liver lobes was permanently ligated, resulting in their atrophy and induction of regeneration in the residual transplant-bearing lobes. Control rats consisted of: Group II-transplanted with normal hepatocytes without portal branch ligation; Group III-transplanted with analbuminemic hepatocytes with portal branch ligation; and Group IV-nontransplanted analbuminemic rats with portal branch ligation. The experimental period extended to 3 mo posttransplantation. All rats transplanted with normal hepatocytes demonstrated a significant elevation in serum albumin levels (ELISA). Group I rats had dramatic elevations in serum albumin to near normal levels (1.78 +/- 0.20 g/dl), and maintained these levels until the end of the experiment. Albumin levels in Group II rats reached 0.26 +/- 0.07 g/dl (p < 0.001), whereas Group III and IV rats showed no changes in serum albumin levels throughout the experiment. Immunohistology of liver tissue obtained from Group I rats, demonstrated large numbers (22.6 +/- 7.5%) of albumin-positive hepatocytes populating the recipient liver. This is the first report of near-total and sustained correction of a genetic defect in liver function in an experimental animal model following allogeneic hepatocyte transplantation.

Hepatocyte transplantation. A potential treatment for liver disease.

The purpose of this report is to summarize data collected over the past several years in the field of hepatocyte transplantation. Herein, a novel method of intraperitoneal transplantation of hepatocytes attached to collagen-coated microcarriers (or a totally biodegradable collagen sponge) is described, resulting in correction of specific genetic liver defects in rats and improved survival in rats undergoing 90% partial hepatectomy. Transplanted cells/matrix form well-vascularized aggregates in the peritoneal cavity; transplanted hepatocytes can be identified by light microscopy using immunohistochemical methods. Prolongation of transplanted hepatocyte survival and function was demonstrated by pretreatment of cells with ultraviolet irradiation. The ability of transplanted cells to proliferate was examined using both biochemical and morphologic criteria. Finally, a method of harvesting, cryopreserving, andin vivo functional testing of human hepatocytes is described.

Carboxyfluorescein (CFSE) Labelling of Hepatocytes for Short-Term Localization following Intraportal Transplantation

Renewed interest in the transplantation of isolated hepatocytes into the liver as a potential therapy for liver disease has stimulated the development of methods for the identification of donor cells within the recipient organ. We describe a method for cellular tagging and in vivo identification of intraportally transplanted hepatocytes using an intracellular fluorescent dye, 5(6)-carboxyfluorescein diacetate, succinimidyl-ester (CFSE). Rat and porcine hepatocytes were isolated and labelled with CFSE. The optimal conditions for labelling consisted of a buffered saline suspension of hepatocytes (5 × 106 cells/mL) in 20.0 μM CFSE incubated for 15 min at 37°C. In vitro, labelled hepatocytes were cultured either on fibronectin-coated chamber slides or in culture flasks. Cultures were evaluated in situ by fluorescence photomicrography or by fluorescence-activated cell sorting (FACS) after cell detachment. Cell viability was assessed serially and cultured, labelled hepatocytes retained the dye for up to 3 wk (last day of study). CFSE did not effect hepatocyte viability and there was no evidence of intercellular diffusion of the dye. In vivo, syngeneic Lewis rats underwent selective portal vein infusion of freshly isolated, labelled hepatocytes (2.0 × 107 cells/2.0 mL saline/animal) into the posterior liver lobes. All recipients were sacrificed 48 h and 96 h later and their livers examined. Transplanted hepatocytes were identified by fluorescence microscopy in tissue sections and by FACS following collagenase digestion of the liver tissue. CFSE persisted in a population of viable, engrafted hepatocytes. FACS analysis demonstrated that 9 ± 3% of the hepatocytes in the posterior liver lobes were labelled 48 and 96 h after transplantation. At 96 h following transplantation, multiple engrafted hepatocytes could be observed by fluorescence microscopy around the central veins. CFSE labelling allows for both in vitro identification and in vivo localization of donor hepatocytes. Furthermore, it appears to be more stable and specific for labelling hepatocytes than other tested dyes (especially DiI).

Mass transfer in a hollow fiber device used as a bioartificial liver.

The transport of albumin, glucose and fluid in a hollow fiber bioartificial liver (BAL) was predicted by theory and measured experimentally. Results from the experiment were used in a three compartment transport model to estimate the transfiber fluid flux. Fluid convection driven by the transfiber pressure gradient transported solutes across the semi-permeable fibers of the BAL. Diffusion contributed significantly to the transfiber transport of both glucose and albumin. Modification of the BAL system hydraulic geometry significantly increased transfiber pressure gradient with corresponding increase in transfiber fluid flux. Transfiber solute transport was increased by osmotically driven transport which resulted from the revised flow geometry. Substantial delivery of hepatocyte synthesis products is possible with careful control of the physical parameters and operating conditions of the BAL.

A model for directed foreign gene delivery to rat liver cells in vivo

A novel technique for directed delivery of retroviral genes to rat liver cells in vivo is described. Vascular isolation of the liver was achieved in situ and perfusate containing retrovirus expressing the bacterial gene conferring resistance to Hygromycin-B was delivered selectively to the posterior liver lobes. After 15 min, normal blood flow to the liver was restored. The portal venous branch supplying the two anterior liver lobes was ligated either at the same time (Group I, n = 4) or 20 hr prior to perfusion (Group II, n = 4) to stimulate DNA synthesis in the posterior lobes. Controls (Group III, n = 4) were perfused with retrovirus without portal branch ligation. Hepatocyte transduction was assessed 7 days later by isolating the cells and assessing their viability in a selection medium. In Group I and II rats, 9.2 +/- 0.5 and 16.0 +/- 1.0%, respectively, of harvested hepatocytes, expressed the Hygromycin-B gene. In contrast, a significantly smaller number of hepatocytes (2.8 +/- 0.9%, P less than 0.003) expressed the gene in the absence of stimulation of DNA synthesis.