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 x 10(7)) 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 intraportally 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.

Fetal rat hepatocytes: isolation, characterization, and transplantation in the Nagase analbuminemic rats.

BACKGROUND: In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative, less immunogenic, and resistant to cryopreservation and ischemic injury. These qualities could enhance FH engraftment, proliferation, and gene transfer requiring active DNA synthesis. METHODS: Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Free and cultured cells were studied using electron microscopy, fluorescence-activated cell sorting, and Northern analysis using alpha-fetoprotein and albumin as markers of hepatocyte lineage. DNA synthetic activity was measured in quiescent and mitogen-stimulated fetal and adult hepatocytes by [3H]thymidine incorporation. Susceptibility of cultured FH to retrovirally mediated gene transfer was studied using an amphotropic retroviral vector carrying the Escherichia coli lac-Z gene. Nagase analbuminemic rats were used as recipients to study the effects of intraportal FH transplantation. Analysis of serum albumin was carried out by enzyme-linked immunosorbent assay. RESULTS: In fetal liver, 87+/-2% of the cells showed morphological and molecular features of hepatocytes. DNA synthetic activity in nonstimulated cultured FH was 10 times greater than the maximal hepatocyte growth factor-driven response in adult rat hepatocytes. A total of 5-15% FH stained positive for X-gal; results of transduction in adult hepatocyte cultures were negative. In Nagase analbuminemic rat recipients, FH produced significant amounts of albumin only when a hepatic regenerative stimulus was applied. Immunohistochemistry confirmed presence of albumin-positive hepatocytes. CONCLUSIONS: Fetal rat liver from the late gestation period is highly enriched with hepatocyte progenitors. They are highly proliferative and susceptible to retroviral transduction and can engraft and function in the adult rat liver if transplanted under a hepatic regenerative stimulus.

Treatment of hypercholesterolemia in the Watanabe rabbit using allogeneic hepatocellular transplantation under a regeneration stimulus.

Numerous studies have reported successful allotransplantation of hepatocytes. However, none have shown long-term correction of a liver-related metabolic defect. In this study, we used a method of regional hepatocyte transplantation and subsequent induction of transplanted cell proliferation by regeneration response in the transplant-bearing liver lobes. New Zealand White rabbits were used as cell donors and Watanabe heritable hyperlipidemic (WHHL) rabbits were used as cell recipients (2 x 10(8) cells/rabbit). All recipient rabbits were maintained on daily cyclosporine. Two weeks after baseline serum cholesterol determination, group I WHHL rabbits (n = 7) received an infusion of cells into the right lateral liver lobe, and a loose ligature was placed around the portal venous branch supplying the anterior lobe. After 1 week, to allow engraftment, the portal venous branch was ligated, which resulted in the atrophy of the affected liver parenchyma and induction of hyperplasia in the transplant-bearing liver tissue. Group II rabbits (n = 6) were transplanted with New Zealand White hepatocytes without portal branch ligation (PBL) and group III rabbits (n = 4) were subjected to sham transplantation (saline) and PBL. The experimental period extended to 150 days after transplantation. All WHHL rabbits transplanted with normal hepatocytes showed reduction in serum cholesterol and low-density lipoprotein (LDL) levels. Group I (PBL-stimulated) recipients demonstrated a more pronounced and sustained effect than group II animals (P < 0.05). Group III controls showed only a slight, typical for aging decrease in serum cholesterol. Group I recipient livers perfused with LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) showed much higher numbers of DiI-LDL-positive hepatocytes than those of group II recipients. In conclusion, a liver regeneration stimulus enhanced the population of transplanted hepatocytes and their functional effect in a large animal model of inborn error of liver metabolism.

Intracranial pressure during liver transplantation for fulminant hepatic failure.

During orthotopic liver transplantation (OLT) for fulminant hepatic failure (FHF), some patients develop cerebral injury secondary to intracranial hypertension. We monitored intracranial pressure (ICP) and cerebral perfusion pressure (CPP) before and during OLT in 12 FHF patients undergoing transplantation. All four patients who had normal ICP preoperatively maintained normal ICP/CPP throughout OLT. During OLT, four of the eight patients with pretransplant intracranial hypertension had six episodes of ICP increase. These episodes of intracranial hypertension occurred during failing liver dissection (n=3) and graft reperfusion (n=3). At the end of the anhepatic phase, the ICP was lower than the preoperative ICP in all patients, and was below 15 mmHg in all but one patient. These data suggest that in FHF patients who develop intracranial hypertension before OLT, dissection of the native liver and graft reperfusion are associated with a risk of brain injury resulting from intracranial hypertension and cerebral hypoperfusion.

Serum bile acids in patients with liver failure supported with a bioartificial liver.

BACKGROUND: Serum bile acids are increased in liver failure, but the composition of the bile acid pool in this condition has not been studied in detail. This information is of interest because of dihydroxy bile acid toxicity. METHODS: We measured serum bile acids by gas chromatography-mass spectrometry in 13 patients with fulminant liver failure and five patients with acute-on-chronic liver failure. Furthermore, serum bile acids were analysed in the same patients after 6 h of treatment with a bioartificial liver, consisting of a hollow-fibre cartridge with microcarrier-attached porcine hepatocytes and a charcoal column. RESULTS: Pre-bioartificial liver serum bile acids demonstrated a high dihydroxy/trihydroxy ratio and were higher in patients with acute-on-chronic liver failure than in those with fulminant liver failure (452.8 +/- 98.6 vs. 182.1 +/- 39.7 micro mol/L; P < 0.05). Bioartificial liver treatment decreased significantly serum bile acids in patients with fulminant liver failure (-38.8%) and acute-on-chronic liver failure (-35.8%), with a decreased dihydroxy/trihydroxy ratio. In vitro, porcine hepatocytes in the bioreactor cleared most conjugated bile acid species from pooled patient plasma. CONCLUSIONS: Acute liver failure is associated with very high serum levels of toxic bile acids that could contribute to the pathogenesis of the syndrome. Bioartificial liver treatment reduces both serum bile acid concentrations and the hydrophobicity of the bile acid pool.

Treatment of severe liver failure with a bioartificial liver.

Orthotopic liver transplantation (OLT) is the definitive therapy for severe liver failure. However, many patients die before an organ becomes available, mostly from cerebral edema. To provide temporary liver support, we developed a bioartificial liver (BAL) based on porcine hepatocytes and a charcoal column. Fifty-four consecutive BAL treatments were carried out in three groups of patients: Group I (n = 15) patients presented with FHF were listed for emergent OLT, Group II (n = 3) patients with primary non-function (PNF) of their liver grafts required urgent re-transplantation and Group III (n = 10) patients with acute exacerbation of chronic liver disease were not candidates for OLT. Patients were managed in a critical care unit receiving maximal standard support. Each BAL treatment was conducted for 6 hours. In Group I, all patients showed significant neurologic improvement, intracranial pressure (ICP) decreased and cerebral perfusion pressure (CPP) increased; other significant improvements, included lowered plasma ammonia and liver enzymes and increased glucose. One patient recovered spontaneously without OLT, all other patients were "bridged" to OLT, and recovered. Group II: PNF patients showed similar benefits. Group III: Chronic liver patients demonstrated transient beneficial effects after BAL treatment(s), however, most (n = 8) eventually succumbed to sepsis and multiple organ failure as they were not candidates for OLT; two patients, recovered, later were successfully transplanted and survived. Our clinical experience demonstrates that the BAL can serve as a bridge to OLT in patients with acute liver failure.

Growth of intraportally transplanted islets under liver regeneration stimulus and restoration of normoglycemia in streptozocin-diabetic rats.

BACKGROUND: Limitation of beta-cell growth after intraportal islet transplantation plays an important role in graft failure. To induce transplanted beta-cell proliferation, we studied the effect of compensatory liver growth in diabetic rats that had a subtherapeutic islet mass previously injected into the liver. METHODS: Syngeneic rats were used as islet donors or recipients; diabetes was induced by streptozocin. Three groups of streptozocin-treated rats were studied. In group 1, 250 islets were selectively transplanted into the posterior liver lobes and 10 days later anterior portal branch ligation (PBL) was performed (n = 18); in group 2, 250 islets were transplanted into the posterior lobes and 10 days later sham PBL was performed (n = 13); in group 3, rats underwent a sham transplantation and PBL (n = 6). Nonfasting blood glucose levels and body weight were monitored. Six rats in groups 1 and 2 were killed 48 hours after PBL, liver sections were stained for proliferating cell nuclear antigen, and islet cell labeling index was calculated. The remaining rats were killed 30 days later. Liver compensatory growth or atrophy was calculated and morphometric determination of beta-cell area was assessed on insulin-immunostained sections of the liver. RESULTS: In group 1 rats killed 48 hours after PBL, islet cell labeling index was significantly higher than in group 2 (p < 0.0001). After PBL, we observed normalization of nonfasting blood glucose levels in 10 of 12 rats. At 30 days, posterior liver lobes showed compensatory growth (218.5% +/- 18.6%) accompanied by atrophy of the anterior lobes; morphometric study of liver-engrafted islets showed a significant increase of individual beta-cell area, compared with group 2 (p < 0.0001). In groups 2 and 3, normoglycemia was not achieved. CONCLUSIONS: In streptozocin-diabetic rats, normoglycemia was restored after transplantation of a sub-therapeutic islet mass, followed by PBL-induced liver regeneration. Histologic and morphometric results indicating islet cell proliferation suggest that compensatory liver growth might have induced a hypertrophic/hyperplastic response in the intraportally transplanted beta-cells.

Use of a novel bioartificial liver in a patient with acute liver insufficiency.

We have developed a bioartificial liver support system (BAL) using porcine hepatocytes attached to microcarriers and placed on the outer surface of hollow fibers. The BAL system was attached to a plasmapheresis device that was then used to treat the plasma of a patient with acute liver failure. Our aim was to test the efficacy and safety of this system after a single short treatment period. A patient with alcohol-induced, severe, acute liver failure manifested by coagulopathy, rising plasma ammonia level, and deteriorating mental status was studied. The procedure was well tolerated by the patient, who remained hemodynamically stable throughout the treatment period. A marked increase in coagulation factor V, VII, VIII, and IX activities, a decrease in serum ammonia level (120 to 32 mumol/L), a twofold increase in all serum amino acids except for aminobutyric acid, and an improvement in mental status were noted after a 6-hour treatment period. This preliminary report of the first use of this novel BAL system in conjunction with plasmapheresis appears promising. A clinical study is now in progress to prove its efficacy.

In vivo regional delivery of retrovirally mediated foreign genes to rat liver cells: need for partial hepatectomy for successful foreign gene expression.

A novel surgical technique was developed to deliver retroviral gene vectors directly to a rat liver lobe in vivo. It was observed that viral infection efficiency was enhanced by inducing hepatocyte DNA synthesis by prior partial hepatectomy. Two retroviral vectors were used to integrate specific bacterial genes: an amphotropic virus expressing the hph gene for hygromycin B phosphotransferase and an ecotropic virus expressing the lac-Z gene for beta-galactosidase. The vectors were directed to the liver by in situ selective perfusion of the posterior liver lobes with a viral suspension with inflow and outflow catheters. Male Sprague-Dawley rats were divided into three groups. Animals in the first group underwent 70% partial hepatectomy and the remnant liver lobes were allowed to regenerate for 20 hours before perfusion with the viral supernatant. Group 2 rats were perfused with viral supernatant and 2 hours later underwent 70% partial hepatectomy. Animals in the third group were perfused with the viral supernatant without partial hepatectomy. Viral transduction of hepatocytes was assessed 4 or 6 days after treatment. Hygromycin B-resistant hepatocytes were isolated from the liver remnants of rats in group 1 (21.6%) and group 2 (26.9%). No resistant hepatocytes could be detected in hepatocytes from either control rats perfused with medium alone or those from rats that did not undergo hepatectomy (group 3). In animals that received the ecotropic virus, only those that underwent hepatectomy before virus exposure (group 1) showed a small number of hepatocytes expressing beta-galactosidase in liver sections.

Repeated intraportal injections of subtherapeutic islet cell isografts restore normoglycemia in streptozotocin-diabetic rats.

Poor engraftment and consequent loss of beta-cell mass could be one of the factors that are responsible for function loss after intraportal islet transplantation (Tx). Streptozotocin-diabetic rats were transplanted with syngeneic islets, which were injected into the portal vein via an indwelling catheter connected to a subcutaneous port. In Group I (n = 6), 1,000 islets were injected in a single dose into the liver. In Group II (n = 6), five doses of 200 islets were repeatedly injected over a period of 14 days, for a total of 1,000 islets. In Group III (n = 4), five decreasing doses of islets were injected over a period of 14 days, for a total of 750 islets. Nonfasting blood glucose (n-FBG) and body weight (b.wt.) were determined twice a week and an intravenous glucose tolerance test (IVGTT) was performed at 30 and 90 days. In Group I, n-FBG decreased in 2 wk from the time of first islet injection, averaging 110 +/- 21.9 mg/dl at 1 mo (p < 0.05 vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group II, n-FBG was normalized in 2 wk averaging 90.2 +/- 25 mg/dL on day 12 (p = NS vs. normal controls) and 75.8 +/- 14.6 mg/dL at 1 month (p = NS vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group III, n-FBG decreased to normal values in 2 wk, averaging 77 +/- 15.7 mg/dL at 1 mo (p = NS vs. normal controls), but normoglycemia was maintained for 40 days and then followed by a progressive increase. Only in Group II, KG (percent/min decline in glucose level) was not significantly different from that of normal controls (1.702 +/- 0.531 at 1 mo and 1.676 +/- 0.891 at 3 mo), while it was significantly lower than normal controls in both Group I and III animals. Body weight increase after Tx correlated with the number of transplanted islets and at 90 days, Group III rats showed less increase than Groups I and II (p < 0.05), while no significant differences in b.wt. were recorded between Group I and II. The findings indicate that intraportal islet Tx, injected repeatedly and in small doses, produced better metabolic effects than injection of the same total number of islets in a single dose.