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Mammalian liver possesses an extraordinary capacity for compensatory growth in response to conditions that induce cell loss by physical, infectious, or toxic injury. In normal animals and humans, it is a tightly regulated process of both hypertrophy and hyperplasia involving different liver cell populations and a finely tuned interplay between growth factors, cytokines, extracellular matrix components and other regulators. The regeneration response is maximal when two-thirds of the liver is resected. When a lesser amount of parenchyma is removed, residual liver grows more slowly. Resections exceeding two-thirds of the liver mass also retard and diminish both DNA synthesis and mitotic activity and subtotal (90%) hepatectomy invariably results in the death of rats without regeneration. The underlying mechanisms of liver growth inhibition are poorly understood. In particular, only a few studies exist that provide insight into the mechanisms that control regeneration after extensive hepatocyte loss. In this regard, the role of growth-regulatory factors and other compounds that accumulate in the blood circulation as a result of hepatic insufficiency and liver cell death remains unclear. We have initiated studies of these mechanisms and demonstrated that in rats with low (10%) hepatocyte mass, a marked and sustained elevation of blood IL-6, HGF and TGF-b1 levels was associated with lack of hepatocyte proliferation and suppression of Stat3 DNA binding. While searching for the possible cause of inhibited IL-6/Stat3 signaling, we found that IL-6 receptor (IL-6R & gp130) was preserved, that nuclear Stat3 protein content was lowered, and that IL-6/Stat3 pathway inhibitors (SOCS-1, PIAS3) were induced during the pre-replicative Go-G1 period.

Mechanisms that control function and repair of the injured liver remain unclear. We hypothesized that after liver injury, elevated blood TGF-beta1 levels may reflect an adaptive response to help maintain differentiated functions in surviving hepatocytes affected by excessive amounts of HGF. We thus studied the effect of HGF, EGF, TGF-beta1, HGF + TGF-beta1, or EGF + TGF-beta1 on the expression of liver-enriched transcription factors and genes which remain under their regulatory activity. The peak [3H]thymidine uptake induced by 20 ng/ml of either HGF or EGF was seen after 72 h; however, DNA binding of C/EBP and HNF1 decreased already after 6 h (electrophoretic mobility shift assay). Addition of TGF-beta1 antagonized these effects. Also at the mRNA level, TGF-beta1 counteracted at one point or another the decrease in C/EBPalpha, C/EBPbeta, HNF1beta, and HNF4 expression; HNF1alpha and COUP-TF showed similar responses and, additionally, were downregulated by TGF-beta1 at 24 h (Northern blot). Albumin and apolipoprotein B mRNA levels were decreased after 24-h treatment with HGF, whereas addition of TGF-beta1 increased their levels. The same pattern was found with EGF, but not until 48 h. PEPCK mRNA was dramatically lowered with either EGF or HGF, and TGF-beta1 did not counteract these effects. Id-1 was expressed only in cultures treated for 24 and 48 h with both the mitogen (EGF, HGF) and TGF-beta1 and in those treated for 48 h with TGF-beta1 alone.

To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.

Recently, researchers have focused on the use of bioartificial liver (BAL) to support patients with fulminant hepatic failure (FHF). We have developed a cell-based BAL, consisting of porcine hepatocytes in a hollow-fiber bioreactor. To better characterize BAL metabolic functions in vitro, bioreactors were inoculated with 48-h-cultured, microcarrier-attached hepatocytes and perifused with recirculating human plasma that contained either 1 microCi of [24-14C] plasma-enriched cholate or 1 microCi of [24-14C] plasma-enriched taurocholate. Bile acids were sampled hourly and separated into four fractions (unconjugated, glycoconjugated, tauroconjugated, and sulfated) for radioactivity determination. Following 3 h perifusion, the glycoconjugated and sulfated bile acid fractions in the bioreactor extrafiber space were significantly elevated when compared to the recirculating plasma. During perifusion with taurocholate-enriched plasma, a relative decrease in the tauroconjugated fraction and an increase in the glycoconjugated fraction were observed. Cholate was accumulated by hepatocytes to a level threefold lower than taurocholate; however, a significant proportion of radioactivity (<25%) was detected in the glycoconjugated fraction. Ultrastructural examination of microcarrier-attached hepatocytes illustrated that the features typical of metabolically active liver cells were maintained. Our data demonstrate the ability of BAL to clear bile acids from the circulation, to accumulate cholate and taurocholate, and to conjugate a substantial amount of cholic acid., (Copyright 1998 Academic Press.)

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.

Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in experimental animal models with genetic disorders of liver metabolism and liver failure. Fetal hepatocytes have several characteristics that make them potentially suitable as donor cells. In contrast to adult hepatocytes, fetal hepatocytes are thought to be highly proliferative, which may facilitate engraftment, expansion of transplanted cell population, and gene transfer requiring active DNA synthesis. The present study was undertaken to evaluate the proliferative capacity of fetal and adult rat hepatocytes under standardized culture conditions. Fetal (20 days of gestation) and adult hepatocytes were cultured in serum-free media at low densities and treated with growth factors. Proliferation was assessed by [3H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about x100 higher and after 10 days in culture x20 higher in fetal compared to adult hepatocytes. When epidermal growth factor (EGF) was added, maximal DNA synthesis in fetal hepatocytes was seen at 48 h, whereas in adult hepatocytes at 72 h. For adult hepatocytes, the average increase compared to untreated cells was x13.8 with EGF, x18.5 with transforming growth factor alpha (TGF-alpha), and x7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-alpha or HGF. EGF-, TGF-alpha- and HGF-dependent DNA synthesis was inhibited by transforming growth factor beta-1 (TGF-beta1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-alpha- and HGF-dependent DNA synthesis in fetal hepatocyte cultures decreased by 36-46%, whereas in adult hepatocytes by 19-27 %. These results show that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independently of growth factors and are relatively resistant to the inhibitory effect of TGF-beta1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.

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.

We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobes necrosis. After this procedure, lack of regenerative response in the residual viable liver tissue (omental lobes) was associated with elevated plasma hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta1) levels and delayed expression of HGF and c-met mRNA in the remnant liver. Here, we investigated whether syngeneic isolated hepatocytes transplanted in the spleen will prolong survival and facilitate liver regeneration in FHF rats. Inbred male Lewis rats were used. Group I rats (n = 46) received intrasplenic injection of 2 x 10(7) hepatocytes and 2 days later FHF was induced. Group II FHF rats (n = 46) received intrasplenic injection of saline. Rats undergoing partial hepatectomy of 68% (PH; n = 30) and a sham operation (SO; n = 30) served as controls. In 20 FHF rats (10 rats/group), survival time was determined. The remaining 72 FHF rats (36 rats/group) were used for physiologic studies (liver function and regeneration and plasma growth factor levels). In Group I rats survival was longer than that of Group II controls (73 +/- 22 hr vs. 33 +/- 9 hr; P < 0. 01). During the first 36 hr, Group I rats had lower blood ammonia, lactate, total bilirubin, PT, and PTT values, lower activity of liver enzymes, and higher monoethylglycinexylidide (MEGX) production than Group II rats. In Group I rats, livers increased in weight at a rate similar to that seen in PH controls and showed distinct mitotic and DNA synthetic activity (incorporation of bromodeoxyuridine and proliferation cell nuclear antigen expression). Plasma HGF and TGF-beta1 levels in these rats decreased and followed the pattern seen in PH rats; additionally, c-met expression in the remnant liver was accelerated. Hepatocyte transplantation prolonged survival in FHF rats and facilitated liver regeneration. Even though the remnant liver increased in weight four times reaching 30% of the original liver mass, the transplant-bearing rats expired due to inability of the regenerating liver to support the rat., (Copyright 1997 Academic Press.)

Unlabelled: 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 (mumol/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 (mumol/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.

Background: The preferred therapy for acute and chronic liver insufficiency and severe heritable disorders of liver metabolism is whole-organ transplantation. However, due to the shortage of organ donors and high cost, alternative therapeutic approaches have been proposed, including transplantation of normal allogeneic hepatocytes. Recently, it has been reported that many hepatocytes transplanted into the spleen migrated to the liver. We therefore carried out a series of large-animal experiments to reexamine the intrasplenic route and to develop a method for large-scale hepatocellular transplantation in pigs., Methods: Allogeneic porcine hepatocytes were transplanted using the following routes: (1) retrograde injection of cells via the splenic vein, (2) intraarterial injection of cells, (3) direct intrasplenic injection of cells after laparotomy, (4) percutaneous intrasplenic injection of cells under laparoscopic control, (5) laparoscopic intrasplenic injection of cells. The number of cells injected varied from 2 x 10(9) to 10 x 10(9) cells., Results: Of all the methods tested, only direct intrasplenic injection of 2 bln of cells was found to be compatible with survival. However, even with this "small" number of cells (2% original liver mass), there was a significant risk of spleen infarction, perisplenic adhesion formation, and portal vein thrombosis. The laparoscopic approach was found to be reliable, simple, and safe., Conclusion: Even though the spleen is considered by many authors the optimal site for hepatocellular transplantation, transplantation of cells in a number needed to support the failing liver may be associated with significant complications, morbidity, and mortality.

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.

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.

Ex vivo reproduction of liver microstructure using isolated hepatocytes is critical for bioartificial liver use. We have developed a method of producing matrix-induced liver cell aggregates (MILCA) using a small number of collagen-coated beads as a nidus for formation of hepatocyte aggregates. Porcine hepatocytes were obtained by EDTA/collagenase digestion. Cell viability was assessed by trypan blue exclusion and LDH release. Cytochrome P-450 activity was determined at 4 and 24 hours by measuring the formation of 7-hydroxycoumarine (7-HC) from 7-ethoxycoumarine (7-EC). At 4 hours, the viability of MILCA was 92 +/- 2%, LDH release was 100 +/- 22 U/L and 7-HC formation was 140 +/- 34 nM/g cells. At 24 hours, MILCA viability remained greater than 90%, but 7-HC formation was lower than that of parallel control monolayer hepatocyte cultures (194 +/- 43 vs 481 +/- 78 nM/g cells; p < 0.002). On transmission electron microscopy, MILCA ultrastructure resembled that of a normal liver (maintenance of cell polarity, gap junctions, bile canaliculi, intact organellae, glycogen granules). MILCA were subsequently inoculated into hollow-fiber bioreactors which were perfused for 6 hours with plasma recovered from patients with fulminant hepatic failure (n = 6; 5 x 10(9) cells/cartridge, recirculation of 350 ml of plasma at 400 ml/min). In these studies, lidocaine (20 micrograms/ml) was cleared in less than 3 hours and 7-HC production at 6 hours was 71 +/- 8 nM/g cells. Other MILCA effects noted in this system included lowering of plasma lactate, bilirubin and ammonia and increase in the level of several non-essential amino acids.

Whole organ transplantation is the only clinically effective method of treating fulminant hepatic failure and chronic liver failure due to specific genetic, hepatocellular, and anatomic defects of liver function. However, wider application of liver transplantation is limited by shortage of organ donors, high cost, a relatively high morbidity rate, and need for life long immunotherapy. As a result, investigators have attempted to develop alternative methods to treat liver insufficiency. These ranged from use of plasma exchange to use of detoxification columns and extracorporeal devices loaded with various liver tissue preparations. Several liver support systems were developed in the 1950s and 1960s, but it was not until recently that advances in hepatocyte isolation and culture, improved understanding of hepatocyte-matrix interactions, availability of new biomaterials, improved hollow fiber technology, and better understanding of flow and mass transport across semipermeable membranes resulted in the development of a new generation of liver assist devices. Some of these devices are being tested in the clinical setting. In this article, the authors review past experience with liver support systems, critically examine the current status of the field by drawing primarily on their own experience, and attempt to speculate on the future direction of liver assist system development.

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 ethylenediaminetetraacetate 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 x 10(10) viable hepatocytes/liver; Procedure B: 1.38 +/- 0.32 x 10(10) 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.

We have recently conducted a series of experiments to characterize the pattern of reaction of human natural antibodies (NA) with individual pig liver cells. Pooled normal human serum (PHS) was incubated with cultured pig hepatocytes (HEP), aortic endothelial cells (AEC), and portal endothelial cells (PEC), and the reaction of NA to different cell types was measured by antibody-mediated cytotoxic (MTT assay), antibody binding (ELISA), and flow cytometric analysis. The human NA displayed a differential pattern of binding with hepatocytes exhibiting a more limited expression of xenoantigen expression than either aortic or portal endothelial cells. These differences in reaction patterns were also noted for Western blot analysis of individual cell membrane extracts. Preincubation of the pig cells with anti-pig MHC antibodies did not inhibit the binding of human IgM natural antibodies to the pig cells. Comparison of the pattern of NA absorption following the use of bioartificial liver support in patients with acute hepatic failure demonstrated limited ability of pig hepatocytes to absorb substantial amounts of NA. These studies indicate that pig hepatocytes are less vulnerable to NA cytotoxicity than pig vascular endothelial cells and that pig vascular endothelial cells express xenoantigens that are unique and not found on hepatocytes.

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.

A bioartificial liver (BAL) support system, using plasma separation, has been developed to support acute liver failure patients. This study examined 14 consecutive BAL treatments in nine patients with severe acute liver failure. We report methods to achieve and manage plasma separation for an extended period of time. The mean duration of a BAL treatment was 435 minutes, with 26-59 liters of blood processed. Ionized hypocalcemia resulting in muscle twitching was a side effect of the therapy. Ionized calcium levels decreased significantly (P < .02) after BAL treatment; however, total calcium levels increased (P < .05). No significant changes were noted in heart rate, electrocardiogram [Q-T (Q-Tc) interval], blood pressure, prothrombin time, partial thromboplastin time, hematocrit, platelet count and serum phosphorous, magnesium, glucose, and pH. Plasma fibrinogen levels decreased significantly (P < .002). Ionized hypocalcemia due to the chelating effect of sodium citrate was controlled by calcium chloride administration, adjustment of blood separation rates, and reduction of the blood-to-citrate ratio. This report demonstrates that intensive, large-volume plasma separation for long periods of time can be achieved safely in critically ill patients without serious adverse effects.

Background: Severe liver failure is associated with high mortality. Orthotopic liver transplantation (OLT) is the only effective therapeutic modality; there is a need for a 'bridge' system to support patients until an organ becomes available., Methods: A bioartificial liver (BAL) was used to treat 10 patients with severe liver failure. A plasmapheresis system was used to pump patient plasma through a module with porcine hepatocytes. Each treatment lasted 6-7 h., Results: All patients tolerated the procedure(s) well. Eight patients underwent OLT following BAL treatment(s). There were two late deaths after recovery from liver failure. Five patients with increased intracranial pressure (ICP) and decerebration had ICP normalization, increased cerebral perfusion pressure and full neurologic recovery after OLT. There was improvement in the level of encephalopathy and a significant decrease in serum ammonia after BAL treatment(s)., Conclusions: BAL treatment is safe and beneficial and can be successfully used as a 'bridge' to 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 x 10(6) cells/mL) in 20.0 microM CFSE incubated for 15 min at 37 degrees 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 x 10(7) 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).

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.

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.

We have developed a bioartificial liver support system utilizing hollow-fiber bioreactor, plasmapheresis and microcarrier cell culture technologies. Liver cells were obtained through portal vein perfusion with ethylenediaminetetraacetate or ethylenediaminetetraacetate/collagenase. A mathematical model of mass transport in a hollow-fiber module, at various plasma flow velocities and system configurations, was developed. The bioartificial liver's ability to carry out specific differentiated metabolic liver functions was tested in vitro and in vivo. A reproducible large-animal model of acute ischemic liver failure was developed. Most major first-generation cyclosporine and 19-norterstosterone metabolites were isolated after substrate addition to the bioartificial liver in vitro. After bioartificial liver treatment for 6 hr (with dog or pig liver cells), dogs with acute liver failure had significantly lower serum ammonia and lactate levels and significantly higher serum glucose levels than did control animals treated with a bioartificial liver system inoculated with microcarriers alone. In addition, bioartificial liver-treated animals had significantly higher mean systolic blood pressures than did controls. Liver cell viability at the end of the 6-hr in vivo experiment was greater than 90%.

Selenium is readily absorbed from the gastrointestinal tract and utilized for synthesis of selenoproteins. Roles of intestine, liver, and selenoprotein P in this process were evaluated. Rats were given 75Se-selenite by stomach tube, and distribution of 75Se was followed for 3 h. A high portal vein plasma-to-hepatic vein plasma ratio of 75Se 15 min after 75Se administration and earlier uptake by liver than by other tissues indicated avid hepatic extraction of absorbed selenium from portal vein blood. The results of gel filtration of plasma taken 15 min after 75Se administration suggested that the 75Se was in the form of small molecules with some affinity for protein. Immunoprecipitation studies using plasma indicated that 75Se began to appear in selenoprotein P between 15 and 30 min after intragastric administration. To evaluate the role of the liver in the fate of absorbed selenium, rats with portacaval shunts, in which absorbed selenium bypasses the liver, were compared with sham-operated rats. After intragastric administration of selenium, uptake by the liver and incorporation into selenoprotein P were diminished in rats with portacaval shunts but kidney uptake and urinary excretion were increased. This suggests that hepatic extraction of absorbed selenium from portal vein blood decreases its entrance into the systemic circulation. The results of this study indicate that intestine releases absorbed selenium into portal blood in a small-molecule form, designated A-Se, which is highly extracted by the liver. The liver takes up A-Se better than other tissues because of a high extraction capacity and the fact that it is the first organ through which the blood from the intestine passes.

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.

A common model for producing experimental liver cirrhosis is the administration of CCl4 to phenobarbital (PhB)-stimulated rats. However, concern may arise due to the complex actions of PhB upon liver metabolism. This study examined the role of PhB in the production of CCl4-induced liver cirrhosis in the rat. In addition, regenerative capacity of the liver after partial hepatectomy (PH) or portal branch ligation (PBL) was studied in cirrhotic rats, rats treated with CCl4 alone, and in PhB-treated controls. In rats given PhB throughout the CCl4-induction period, ascitic form of micronodular cirrhosis was found in 93% with only 3% mortality. In contrast, rats pretreated with PhB for only 2 weeks followed by CCl4 alone for 18 weeks did not develop liver cirrhosis. In most of the cirrhotic rats, PH induced hepatic regeneration associated with improved liver histology. PBL was less effective. Treatment with PhB alone for 10 weeks resulted in liver atrophy and reduced hepatic regenerative capacity. Impaired regeneration response was also found in rats treated with CCl4 alone. In conclusion, treatment with PhB throughout the CCl4-induction period seems necessary for the production of liver cirrhosis in rats. However, prolonged treatment with PhB alone results in liver atrophy and an impaired regenerative response. Therefore, though necessary for the cirrhotic model, PhB by itself has negative hepatotrophic influences which questions the thoroughness of the PhB/CCl4 experimental model.

Portal hypertension was mechanically induced in rats by acute constriction of the portal vein. Using a new "button" technique, a stricture 0.9 mm in diameter was found to be compatible with life in more than 90% of the rats. Angiographic and anatomic studies of portosystemic collaterals confirmed observations in earlier experiments. Oesophageal varices were not seen, despite sustained elevation of portal venous pressure during the four weeks following induction of portal hypertension. Development of paraportal veins bridging the obstacle is suggested to be responsible for maintenance of normal liver structure and for recovery from the transient hepatic dysfunction. Increase of phagocytosis in the germinal centres of the enlarged spleens was found after four weeks, suggesting immunologic changes caused by portal hypertension and/or portosystemic shunt circulation.

A method for portal stump pressure monitorization following arterialization is presented. Both intra and postoperative pressure determinations were performed and a significant difference between intra and postoperative values was observed. The pressure of the arterialized portal stump either using the left gastric artery or one of its branches, when compared to the free portal pressure did not reveal significant differences. According to these data, the proposed method of arterialization seems to create a "physiologic" arterialized stump pressure. This information while contributing to a better understanding of the complex hemodynamics of the arterialized liver represents essential guidelines for further studies on liver trophism and experimental portal hypertension.

The stimulatory effect of a growing kidney upon the liver was studied in rats with renoportal anastomosis (RPA) and contralateral nephrectomy. DNA synthesis, mitotic activity, and index of growth in the renoprival kidneys were independent of the shunt. Hepatic uptake of [Me-3H]thymidine was increased in both RPA-nephrectomized and sham-RPA-nephrectomized rats; however, in the latter group of animals the response was significantly greater. Results of the study suggest that stimulatory substance of renal origin is carried in the venous blood of a growing kidney. A nonspecific interaction between the renotropic and portal-borne hepatotrophic factors seemed to be responsible for stimulation of hepatic DNA synthesis. The study supports the view that mechanisms humoral in nature but not common promote reparative growth in different organs such as the liver and kidney.

The effects of portal occlusion on the liver have been differently reported in different studies. The authors therefore reevaluated a model of portal branch ligation (PBL) in the rat. Histologic appearance, DNA synthetic activity, labeling count, and mitotic index were serially evaluated in both ligated and nonligated parts of the liver after interruption of the portal flow to one fourth, one third, and two thirds of the liver mass. The authors confirmed the presence of compensatory hyperplasia induced in the nonligated liver lobe(s) by PBL, and its intensity was roughly proportional to the amount of liver tissue devoid of portal perfusion. Portal-deprived liver tissue underwent a rapid and progressive atrophy, and, by the end of the first week, the weight of this part had decreased 10-fold. By a balance between atrophy and compensatory growth, the total liver weight was maintained at the level of sham-operated animals throughout the experiment. PBL invariably resulted in early centrilobular necrosis, which occupied 15-24% of the ligated lobe(s). However, already after 4 days it was almost totally resorbed and did not appear de novo. PBL was not followed by local collateralization.

Despite vast knowledge on liver regeneration, little is known about the effect of active liver atrophy and regeneration on tumor growth. Ligation of a branch of the portal vein to the one or two anterior lobes was performed in inbred Wistar rats. This induces acute atrophy of the anterior and regeneration of the other lobes. During the same operation a tumor cell suspension (NGW1--adenocarcinoma) was inoculated in liver lobes undergoing atrophy and regeneration. Tumor volume and weight were measured and the histologic appearance was assessed. During the early and active phases the tumor growth was significantly accelerated in regenerating lobes and partially inhibited in rapidly atrophied segments. After the regeneration and atrophy was completed the normal pattern of growth was re-established in both parts of the liver. The results suggest that tumor growth is affected in proportion to regenerative response. They further suggest that portal branch ligation is of limited value in surgical palliation of liver tumors. The risk for further induction of growth of clinically undetected tumor foci in the remaining liver tissue appears to be small, although a significant, but short-lasting, stimulatory response was found.

The hepatotrophic effect of portal blood was studied in rats with portal branch ligation (PBL) simultaneously subjected to mesocaval shunt or total hepatic arterialization. A direct supply of the nonligated liver lobes with pancreatic effluent did not improve DNA synthesis or mitotic response compared to arterialized rats, in which the portal-borne hepatotrophic factors reached the same lobes after systemic recirculation. A comparison with shunted but Sham-PBL rats showed that restorative capacity of the liver was not seriously impaired. In rats with PBL alone the lobes with portal occlusion showed extensive necrosis. In contrast, in PBL + shunt rats necrosis was significantly inhibited, indicating a hepatoprotective role of portal blood during hepatic arterial recirculation. Thus, the study suggests that quality of hepatic blood supply is of vital importance in maintenance of hepatocellular integrity. Hemodynamical factors seem to be of importance, but more in a sense of increased or decreased accessibility of humoral hepatotrophic factors in the blood.

Portal branch ligation (PBL) is known to induce a rapid and progressive atrophy in the liver parenchyma without portal blood flow and compensatory hyperplasia in the segments receiving the whole portal flow. In this study, the hepatotrophic effect of portal blood was studied in rats with PBL and after this procedure was combined with different portosystemic shunts. After 2 weeks, the most severe atrophy was found in ligated lobes of rats with PBL alone. In shunted animals, the atrophy was significantly inhibited and in relation to the magnitude of portal flow bypassed the liver. This suggests that in shunted rats, the portal-bone hepatotrophic factors undergo systemic recirculation and affect the liver by way of the hepatic artery. Simultaneously, in PBL + shunt rats, the rate of atrophy normally induced by a shunt was also dependent on the amount of portal blood available to this part of the liver. By a balance between these 2 processes, the total liver mass was maintained at the level found in sham PBL + shunt control rats.