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Pancreatic islet transplantation is limited by shortage of donor organs. Although β-cell lines could be used, their secretion of insulin is characteristically glucose independent and immunoisolation is required. Here we show that intrasplenic transplantation of encapsulated glucose-responsive mouse insulinoma cells reversed streptozotocin (STZ)-induced diabetes in rats. MIN-6 cells derived from a transgenic mouse expressing SV 40 large T antigen in pancreatic β-cells were transfected with minigene encoding for human glucagon-like-peptide-1 under the control of rat insulin promoter. The cells were encapsulated in alginate/poly-L-lysine and used for cell transplantation in STZ-diabetic rats. Rats with nonfasting blood glucose (n-FBG) greater than 350 mg/dl were used. In group I rats (n = 6) 20 million encapsulated cells were injected into the spleen. Group II rats (n = 6) received empty capsules. n-FBG was measured biweekly. After 4 and 8 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed in group I; normal rats served as controls. Plasma insulin level was measured every other week (RIA). After 8 weeks, spleens were removed 1 day before sacrifice. In rats transplanted with cells the n-FBG was 100—150 mg/dl until the end of the study. After splenectomy, all cell recipients became diabetic (glucose 400 ± 20 mg/dl). Transplanted rats showed increase in body weight and insulin production (3.3 ± 1.0 ng/ml versus 0.92 ± 0.3 ng/ml; p < 0.01) and had normal IPGTT. Spleens contained capsules with insulin-positive cells. Overall, data from this work indicate that intrasplenic transplantation of xenogeneic encapsulated insulin-producing cells without immunosuppression reversed diabetes in rats. Excellent survival and function of the transplanted cells was due to the fact that the cells were separated from the bloodstream by the immunoisolatory membrane only and insulin was delivered directly to the liver (i.e., in a physiological manner).

Hepatocyte transplantation and use of bioartificial liver support systems have been suggested as potential therapies for fulminant hepatic failure. Cryopreservation in liquid nitrogen is presently the major method of long-term storage of isolated hepatocytes. However, cryopreservation can result in low cell recovery and reduction in differentiated function. Several possible mechanisms of cell death during cryopreservation have been proposed. The most important mechanisms appear to be oxidative stress and apoptosis. In this study, we isolated fresh rat hepatocytes and cryopreserved them in three media: University of Wisconsin (UW) solution, an antioxidant-containing medium, and medium containing a caspase inhibitor. Viability and function of hepatocytes cryopreserved in these media were examined. Cryopreservation conditions had no effect on hepatocyte viability after thawing. However, after culture we found significant improvements in viability and function in both antioxidant- and caspase inhibitor-treated hepatocytes at 6 and 24 h.

Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained 1 × 10 7 rat hepatocytes. Human HepG2 cells were encapsulated using alginate/poly-l-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6–10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.

Clinical islet transplantation (Tx) in type I diabetic patients has been successful so far only in a minority of cases, probably because of multiple factors, partly immunologic and partly nonimmunologic in nature. Pre-clinical studies of islet Tx in large animals are still needed to clarify the reasons and find possible solutions. In this study, we tested the feasibility of noninvasive, repeated intrahepatic allo-Tx of porcine pancreatic islets obtained from multiple donors, in pigs rendered diabetic by total pancreatectomy (Pct). In group I Yucatan miniature swine (n = 6), after induction of diabetes by Pct, repeated islet allo-Tx of ≥80% pure islets was performed. Islets obtained from two pigs of the Hanford breed were injected twice a week, half freshly isolated and half 48-h cultured, over a period of 11 days, for a total of 23,647 ± 1617 islet equivalents (IE)/kg recipient body weight (BW). In group II Yucatan miniature swine (n = 3), after Pct, a single allo-Tx of ≥80% pure islets, previously obtained from two donors of the Hanford breed, was performed, using a total of 22,416 ± 1124 IE/kg BW. In group III Yucatan miniature swine (n = 3), auto-Tx of 60–75% pure islets, averaging 2980 ± 424 IE/kg BW, was performed a few hours after Pct. Group IV Yucatan mini pigs (n = 3) underwent Pct and were used as diabetic controls. Group V animals (n = 3) were normal control Yucatan mini pigs. Porcine islets were isolated by a modification of the standard collagenase digestion and Ficoll gradient purification method. Donors and recipients were chosen on the basis of moderate to high mutual alloreactivity in mixed lymphocyte culture (MLC). In groups I and II, cyclosporine A (CsA) was started 4 days before allo-Tx, at the dose of 15 mg/kg IM, and then gradually reduced to 4 mg/kg IM. In all group I animals, normal fasting blood glucose (FBG) was restored within 2–3 weeks. Two normoglycemic pigs died of acute pneumonia at 33 and 112 days, respectively, and one animal became progressively hyperglycemic at 100 days. After 3 months, discontinuation of CsA treatment resulted in FBG increase in two group I animals. In one pig, CsA was stopped after 151 days, and normoglycemia persisted until euthanasia, after 8 months. In group II pigs, normoglycemia lasted 4–20 days, with a progressive increase of insulin requirement thereafter. In group III animals, after islet auto-Tx, normoglycemia lasted 7–10 days, while insulin daily requirement progressively increased thereafter, stabilizing at 0.4 IU/kg/day, corresponding to about one third of the amount required in diabetic controls. The single most important result in this series of experiments is that intraportal allo-Tx of a sufficient islet mass, divided in multiple subtherapeutic doses, produced a better metabolic long-term control in comparison to a single injection of the same amount of islets. The technique of multiple-donor repeated islet Tx may prove useful to overcome the problem of primary nonfunction or early graft failure, currently limiting the success of clinical islet Tx in most cases.

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 × 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 [ 3 H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about × 100 higher and after 10 days in culture ×20 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 × 13.8 with EGF, ×18.5 with transforming growth factor alpha (TGF-α), and ×7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-α or HGF. EGF-, TGF-α- and HGF-dependent DNA synthesis was inhibited by transfroming growth factor beta-1 (TGF-β1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-α- 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-β1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.

Poor engraftment and consequent loss of β-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, K G (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. Copyright © 1997 Elsevier Science Inc.

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 × 107 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.

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 intra-portal infusion of 2 × 107 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 × 106 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.

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).