In vivo evaluation of a hollow fiber liver assist device.

Orthotopic liver transplantation is the only effective form of therapy currently available for patients with fulminant hepatic failure (FHF). The use of an extracorporeal (EC) liver assist device (LAD) may result in improved presurgical clinical management. Alternatively, patients treated with LADs could avoid the transplantation procedure if they are able to regenerate a critical mass of hepatocytes that will sustain functional viability. In this study, the efficacy of a prototype hollow fiber LAD seeded with rabbit hepatocytes was assessed in vivo by the use of two different animal models: (1) normal rabbits injected with diazepam or lidocaine, and (2) a galactosamine (Gal)-intoxicated rabbit model of FHF. The EC LAD clearly decreased the blood levels of the two drugs and significantly generated diazepam and lidocaine metabolites indicating the maintenance of active P450 forms in the cellular component of the devices. A 6-hour EC treatment significantly increased the survival time and delayed the onset of hepatic encephalopathy (HE) in the Gal-intoxicated rabbits. Histological evaluations of postmortem livers showed greater hepatocyte regenerative activity in the animals treated with hepatocyte-seeded LADs than in the two control groups, e.g., rabbits not treated or treated with unseeded devices. These findings support the concept that a microporous hollow fiber LAD seeded with rabbit hepatocytes is able to sustain drug detoxification in vivo as well as to modify the course of FHF in a well-characterized animal model.

Treatment of severe diabetes mellitus for more than one year using a vascularized hybrid artificial pancreas.

We report the successful application of a hybrid artificial pancreas device for the treatment of severe diabetes mellitus induced by total pancreatectomy in two dogs. Control of the blood sugar was achieved for more than 1 year in these two animals without any immunosuppressive therapy. Although exogenous insulin was required therapy. Although exogenous insulin was required during the latter part of the study period, removal of the devices resulted in a rapid increase in the fasting blood sugar levels and the exogenous insulin requirements (P < 0.001 versus weeks 1-52 in both dogs). Metabolic studies, postexplant in vitro studies, and histologic analyses confirmed islet cell survival and insulin production by the devices. This hybrid artificial pancreas has a clear clinical potential for islet cell transplantation without immunosuppression.

Treatment of severely diabetic pancreatectomized dogs using a diffusion-based hybrid pancreas.

Long-term survival of dog islet allografts implanted in diabetic pancreatectomized dogs was achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes (nominal M(r) exclusion of 50,000-80,000). Dog islets were isolated from the pancreases of outbred mongrel dogs by collagenase digestion. Chambers containing mean +/- SE 316 +/- 63K islet equivalents (mean islet volume, 558 +/- 111 mm3, purity 90-95%) were peritoneally implanted into six totally pancreatectomized dogs. The dogs were monitored for glycemic control by fasting and postprandial blood glucose determinations, and responses to both intravenous glucose (intravenous glucose tolerance test 0.5 g/kg) and oral glucose (oral glucose tolerance test 1 g/kg). All of the dogs required appreciably lower dosages of exogenous insulin therapy for control of fasting blood glucose levels, with the mean daily insulin dose dropping from 38 +/- 7 to 5 +/- 1 U/day during the 1st wk. Three recipients required no insulin for greater than 82, greater than 68, and 51 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 and 2 mo postimplantation were 2.7 +/- 0.4 and 2.0 +/- 0.5, respectively compared with 3.5 +/- 0.5 before pancreatectomy. The glucose values during oral glucose tolerance tests at 2 wk, although returning to less than 125 mg/dl (less than 7.0 mM) by 2 h, exceeded the normal range, with peak values of 174 to 202 mg/dl (9.7 to 11.3 mM). These preliminary results are encouraging, and represent an important step in determining the feasibility of using this type of diffusion-based hybrid artificial pancreas as treatment for diabetes mellitus in humans.

Islet transplantation using an immunoprotective membrane in dogs that have undergone a pancreatectomy.

The use of a selectively permeable membrane to transplant nonsyngeneic tissue without accompanying immunosuppressive therapy has been investigated using two approaches. The first hybrid artificial pancreas is implanted as a vascular shunt in which blood circulates through the lumen of a tubular membrane. The islet tissue is distributed within a chamber surrounding the membrane enclosed by an acrylic housing. Studies with diabetic dogs that have had pancreatectomies have demonstrated that these devices could replace exogenous insulin therapy for at least 6 months in five animals. This report presents data on two of these dogs, demonstrating viability and function of the transplanted tissue after 1 year. As an alternative to the vascular device, islets sealed within cylindrical permselective membrane chambers have been implanted in the peritoneum. Preliminary data from three dogs indicate that the nonvascular implants can also regulate fasting glucose levels in the diabetic dog model.

Long-term cultures of adult mammalian hepatocytes in hollow fibers as the cellular component of extracorporeal (hybrid) liver assist devices.

A discussion of the treatment of liver insufficiency with extracorporeal (hybrid) liver assist devices (LADs) should address a definition of the types of liver failure susceptible to being treated by these devices as well as the modalities of in vivo and in vitro testing. Relevant to the first subject is the subject of pathogenesis of hepatic coma, which should be the target for the design of these LADs. Although this modality of therapy is new, it can be predicted that these devices will demand minimal safety conditions, i.e., the seeding with cells that are not tumorigenic or carrying viral particles. Among other topics to be considered in the development of LADs is the proper choice of hollow fiber to be used and the testing on proper animal models of hepatic failure. It is our philosophy that the long-term culture of adult mammalian hepatocytes in hollow fibers is the basis for appropriate designs of this type of temporary liver support.

Evaluation of the hybrid artificial pancreas in diabetic dogs.

Immunoisolation of nonsyngeneic tissue using a selectively permeable membrane is designed to facilitate transplantation without the use of immunosuppression. The authors' studies have evaluated a hybrid artificial pancreas device that is implanted as an arteriovenous vascular shunt. Devices containing allogeneic or xenogeneic islets were implanted in diabetic dogs who had undergone pancreatectomies, and the devices eliminated the requirement for exogenous insulin for control of fasting glycemia in 11 animals for periods ranging from 1 to 8 months. Furthermore, unseeded devices in normal dogs have been shown to remain patent for over 2 years with low doses of aspirin as the only anticoagulant. These results indicate that this approach has potential as a therapy for diabetes.

Xenotransplantation of canine, bovine, and porcine islets in diabetic rats without immunosuppression.

Permselective acrylic membranes were employed to prevent immune rejection of discordant islet xenografts isolated from various large animals. Canine, porcine, and bovine islets were seeded into tubular diffusion chambers and transplanted into the peritoneum of 27 nonimmunosuppressed streptozotocin-induced diabetic Lewis rats. Six recipients received islet grafts from bovine calves, 7 received grafts from pigs, and 14 received grafts from dogs. Four of the latter were removed at 1 month. In the control group of 10 diabetic rats, 4 received nonencapsulated canine islets, 3 received nonencapsulated bovine islets, and 3 received nonencapsulated porcine islets. Recipients of encapsulated islets promptly dropped from a pretransplantation plasma glucose level of 487 +/- 36 (mean +/- SEM) to 84 +/- 2 (canine), 81 +/- 4 (bovine), and 81 +/- 3 mg/dl (porcine) during the first week. All of the animals sustained these levels for at least 1 month. One rat spontaneously reverted to diabetes at 54 days posttransplantation; 4 other rats became hyperglycemic (glucose, greater than 600 mg/dl) after membrane removal on day 30. The remaining 22 rats maintained fasting euglycemia for greater than 10 weeks. In contrast, rats that received nonencapsulated islets became hyperglycemic in less than 7 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 month for the canine and bovine encapsulated islet transplant group were 3.5 +/- 0.3 and 3.3 +/- 0.1 compared with 3.3 +/- 0.1 (P = 0.63) and 0.91 +/- 0.1 (P less than 0.0001) for normal (n = 4) and diabetic (n = 4) control groups. Morphologic studies of long-term functioning grafts (30-130 days) revealed well-preserved alpha, beta, and delta cells, with varying degrees of granulation. These results demonstrate that immune isolation of islet tissue using permselective artificial membranes can protect discordant islet xenografts from immune rejection in the absence of any immunosuppressive drugs.

Transplantation of islet allografts and xenografts in totally pancreatectomized diabetic dogs using the hybrid artificial pancreas.

Previously the authors reported on a Hybrid Artificial Pancreas device that maintained patent vascular anastomoses in normal dogs and, when seeded with allogeneic canine islets, maintained normal fasting blood sugars (FBS) in diabetic pancreatectomized dogs. Eventual failure of these devices was believed to be related to loss of islet viability and/or insufficient islet mass. The current study evaluates the effect of increased islet mass produced by implantation of two islet-seeded devices in pancreatectomized dogs and compares the results with those from dogs that received a single device. Twelve of fifteen dogs receiving single devices showed initial function as determined by elimination or reduction of exogenous insulin requirement; four showed initial function and seven showed extended function (100 to 284 days). Excessive weight loss (more than 20%), despite normal FBS and insulin dependence, required that four animals in this latter group be killed. Devices seeded with xenogeneic islets have met with limited success. One dog that received two bovine islet-seeded devices achieved function for more than 100 days; the remaining bovine-seeded devices (n = 8) functioned for only 3 to 16 days. Porcine islet-seeded devices were assessed by intravenous glucose tolerance tests (IVGTT). Recipients of two devices seeded with allogeneic islets demonstrated improved IVGTT results when compared to those from pancreatectomized dogs and recipients of single devices but were abnormal when compared to intact animals. Histologic examination of device and autopsy material from all failed experiments was performed and showed no mononuclear cell infiltration of the islet chamber or vascular graft material, only a few incidence of device thrombosis, and varying degrees of islet viability as judged by morphologic and immunohistochemical evaluation. The authors believe they have demonstrated progress toward the development and clinical applicability of the Hybrid Artificial Pancreas.

Biohybrid artificial pancreas: long-term implantation studies in diabetic, pancreatectomized dogs.

Diabetic complications such as neuropathy, retinopathy, and renal and cardiovascular disease continue to pose major health risks for diabetic patients. Consequently, much effort has focused on approaches that could replace conventional insulin therapy and provide more precise regulation of blood glucose levels. The biohybrid perfused artificial pancreas was designed to incorporate islet tissue and a selectively permeable membrane that isolates this tissue from the immune system of the recipient. Biohybrid pancreas devices containing canine islet allografts were implanted in ten pancreatectomized dogs requiring 18 to 32 units of injected insulin daily. These implants resulted in good control of fasting glucose levels in six of these animals without further exogenous insulin for periods of up to 5 months.