Jacek Rozga

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.

Clinical experience with a porcine hepatocyte-based liver support system.

The only clinically proven effective treatment of fulminant hepatic failure (FHF) is orthotopic liver transplant (OLT). However, many patients die before an organ becomes available. Thus, there is a need for development of an extracorporeal liver support system to “bridge” these patients either to OLT or spontaneous recovery. We developed a bioartificial liver (BAL) based on plasma perfusion through a circuit of a hollow-fiber cartridge seeded with matrix-anchored porcine hepatocytes to treat patients with severe acute liver failure. Two groups of patients were studied. Group 1 (n = 12): patients with FHF. All patients were successfully “bridged” to OLT. “Bridge” time to OLT was 21-96 hr (mean: 39.3 hr). All patients were discharged neurologically intact. Reversal of decerebration was noted in all 11 deep stage 4 coma patients. There was reduction in intracranial pressure (ICP mmHg, 18.2 ± 2.2 to 8.5 ± 1.2; p<0.004) and increase in cerebral perfusion pressure (CPP mmHg, 71.1 ± 4.0 to 84.7 ± 2.6; p<0.006). Laboratory values pre- and post- BAL treatment: glucose (mg/dl) 122 ± 11 to 183 ± 21, p<0.002; ammonia (μmol/l) 155.6 ± 13.2 to 121.6 ± 9.5, p<0.02; total bilirubin (mg/dl) 21.6 ± 2.8 to 18.2 ± 2.2, p<0.001; PT (sec) 23.2 ± 1.7 to 21.9 ± 1.0, p<0.3. Group II (n=8): patients with chronic liver failure experiencing acute exacerbation. Two patients survived and later underwent OLT. Six patients (not OLT candidates) died 1-14 days after last BAL treatment. Laboratory values pre- and post-treatment: ammonia (μmol/l) 201 ± 47 to 143 ± 25, p<0.06; total bilirubin (mg/dl) 22.8 ± 5.2 to 19.5 ± 4.4, p<0.01; PT (sec) 22.5 ± 2.0 to 21.8 ± 1.1, p<0.6. Conclusion: our clinical experience with the BAL suggests that it may serve as “bridge” to OLT in patients with FHF primarily by reversing intracranial hypertension, but it is not a substitute for OLT in patients with end-stage liver disease who are non-transplant candidates.

Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cells.

Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood‐forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow‐derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co‐cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell–cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcirption factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3‐dioxygenase, are improved in hepatocyte/BMSC co‐cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro.

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.

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.

Review: Artificial liver support systems

Despite recent advances in medical therapy, patients with fulminant hepatic failure (FHF) have a mortality rate approaching 90%. Many patients die because of failure to arrest the progression of cerebral edema. Liver transplantation has improved survival to 65% to 75%. However, there is a shortage of donors and approximately one half of the patients with FHF will die while awaiting liver transplantation. There is thus a need to develop an extracorporeal liver assist system to help keep these patients alive and neurologically intact until either an organ becomes available for transplantation or the native liver recovers from injury. Such a system could also be used during the period of functional recovery from massive liver resection or to assist patients with decompensated chronic liver disease. Over the years, various methods utilizing charcoal and resin hemoperfusion, dialysis, plasma exchange, and other methods of blood detoxification have been developed and tested, but none have gained wide acceptance. This was due to: (i) incomplete understanding of the pathophysiology of liver failure; (ii) lack of accurate methods of assessment, quantitation, and stratification of the degree of liver dysfunction; and (iii) inadequate numbers of prospective controlled clinical trials examining the effects of specific therapeutic modalities. Liver support systems utilizing liver tissue preparations were developed in the 1950s, but it was not until recently that advances in hepatocyte isolation and culture, better understanding of hepatocyte‐matrix interactions, and improved hollow‐fiber technology have resulted in the development of a new generation of liver assist devices. Some of these devices are currently being tested in the clinical setting. In a preliminary clinical study, we have used a porcine hepatocyte‐based liver support system to treat patients with acute liver failure as well as patients with acute exacerbation of chronic liver disease. Patients in the first group, who were candidates for transplantation, were successfully bridged to a transplant with excellent survival. No obvious benefit from bioartifical liver treatments was seen in the second group. It is possible that, in this group, patients will have to be treated earlier and for longer periods of time. Prospective controlled trials will be initiated as soon as the current phase I study is concluded to determine the efficacy of this system in both patients populations.

Liver assist systems: state of the art.

Attempts to develop liver support systems for the treatment of patients with liver failure have ranged from use of plasma exchange to utilization of charcoal columns and extracorporeal devices loaded with liver tissue. However, no system has achieved wide clinical use and - in the absence of liver transplantation - severe hepatic failure continues to be associated with significant morbidity and mortality. In this paper, the authors review the current status of liver assist systems and summarize their clinical experience with a xenogeneic cell based-bioartificial liver.

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.

Transplantation of hepatocytes for prevention of intracranial hypertension in pigs with ischemic liver failure.

Intracranial hypertension leading to brain stem herniation is a major cause of death in fulminant hepatic failure (FHF). Mannitol, barbiturates, and hyperventilation have been used to treat brain swelling, but most patients are either refractory to medical management or cannot be treated because of concurrent medical problems or side effects. In this study, we examined whether allogeneic hepatocellular transplantation may prevent development of intracranial hypertension in pigs with experimentally induced liver failure. Of the two preparations tested--total hepatectomy (n = 47), and liver devascularization (n = 16)--only pigs with liver ischemia developed brain edema provided, however, that animals were maintained normothermic throughout the postoperative period. This model was then used in transplantation studies, in which six pigs received intrasplenic injection of allogeneic hepatocytes (2.5 x 10(9) cells/pig) and 3 days later acute liver failure was induced. In both models (anhepatic state, liver devascularization), pigs allowed to become hypothermic had significantly longer survival compared to those maintained normothermic. Normothermic pigs with liver ischemia had, at all time points studied, ICP greater than 20 mmHg. Pigs that received hepatocellular transplants had ICP below 15 mmHg until death; at the same time, cerebral perfusion pressure (CPP) in transplanted pigs was consistently higher than in controls (45 +/- 11 mmHg vs. 16 +/- 18 mmHg; p < 0.05). Spleens of transplanted pigs contained clusters of viable hepatocytes (hematoxylin-eosin, CAM 5.2). It was concluded that removal of the liver does not result in intracranial hypertension; hypothermia prolongs survival time in both anhepatic pigs and pigs with liver devascularization, and intrasplenic transplantation of allogeneic hepatocytes prevents development of intracranial hypertension in pigs with acute ischemic liver failure.