The isolation and function of porcine islets from market weight pigs.

The efficacy of clinical islet transplantation has been demonstrated with autografts, and although islet allografts have established insulin independence in a small number of IDDM patients, the treatment is confounded by the necessity of immunosuppression. the lack of donor tissue, and recurring islet immunogenicity. These limitations underscore a need to develop therapies to serve the large population of diabetic patients. Porcine islet xenotransplantation, together with a successful immune intervention strategy, may provide the necessary clinical alternative. However, a major obstacle in evaluating this approach has been the difficulty of obtaining adequate volumes of functional islet tissue from pigs. Donors of market weight are preferable to retired breeders due to their abundance, lower animal and husbandry costs. and are more suitable to meet regulatory guidelines for donor tissue for xenotransplantation. We describe a simple isolation procedure that following purification yields a mean of 350,000 IE, corresponding to 179 units of insulin and 1.8 mg of DNA with an islet purity and viability in excess of 85% (n = 317 isolations). In both short- and long-term cell cultures, porcine islets demonstrated glucose-responsive insulin secretion. However, this secretion is density dependent, which may have significant consequences in the development of immunoisolation technologies to support porcine islet xenotransplantation. Following implantation into diabetic nude mice, porcine islets remained functional in excess of 1 year. Implantation of a bioartificial pancreas containing porcine islets into pancreatectomized dogs provided significant clinical benefit with an improved diabetic condition. Finally, secretagogue-induced insulin release was demonstrated in vitro from these devices after removal from immunocompetent recipients. Immunohistochemical staining identified well-granulated islets following long-term implantation in both the rodent and canine models. This study demonstrates the ability to isolate porcine islets in clinically relevant numbers from market animals, which survive and remain functional for prolonged periods of time in an immune-deficient or immunoprotected environment.

Improved assessment of isolated islet tissue volume using digital image analysis.

Accurate and consistent measurement of tissue volume is critical to performing many types of islet research; however, conventional visual determination of isolated islet yields through a microscope is heavily operator dependent. An improved method of islet volume determination using digital image analysis (DIA) was developed to remove operator bias and automate the islet counting process. A series of 140 porcine islet isolations were used to evaluate the DIA method in three separate stages. In Stage 1 (n = 29 isolations), the conventional and DIA methods were correlated with two other independent islet quantitation methods: insulin extraction, and DNA extraction. It was found that volumes determined by DIA correlated more closely with insulin content and DNA content than did conventionally determined volumes. In Stages 2 and 3 (n = 54 and 57 isolations, respectively), it was shown that an increase in the number of fields analyzed by DIA did not significantly improve the quality of the correlations. Inclusion of very small tissue (<50 microm in diameter), which is ignored in the conventional protocol affected yields by less than 10% and did not significantly improve the correlation with insulin or DNA content. Quantitation of isolated islet tissue volume using DIA has been shown to be rapid, consistent, and objective. In the laboratory, use of this method as the standard for islet volume measurement will allow more meaningful comparison of experimental results between centers. In the clinic, its use will allow more accurate dosing of transplanted tissue.

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

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

Oxygen delivery to and use by primary porcine hepatocytes in the HepatAssist 2000 system for extracorporeal treatment of patients in end-stage liver failure.

A system for the extracorporeal treatment of end-stage liver failure patients has been developed and is now in clinical studies. This HepatAssist 2000 system consists of perfusing the plasma of the patient through a hollow-fiber bioreactor containing primary porcine hepatocytes. The goal of the system is to assist the patient while the liver regenerates or a suitable liver is found for transplantation. During the development of this system in the laboratory, an experimental protocol has been developed to simulate patient treatments. The protocol substitutes donor calf serum for patient plasma, but is otherwise similar to clinical conditions. The protocol uses a blood gas analyzer to monitor dissolved oxygen and carbon dioxide tensions at the entrance to and exit from the bioreactor. Samples are withdrawn using a standard 1-ml syringe and analyzed off-line immediately. The blood gas measurements have been used to verify that the oxygenator, placed immediately prior to the bioreactor in the plasma circuit, is properly sized to allow complete equilibration with feed gas and to maintain a physiologic level of carbon dioxide. The measurements were also used to calculate overall bioreactor oxygen consumption rates under various conditions. In one set of studies, composition of the feed gas to the oxygenator was varied from 20% to 70% oxygen, while maintaining 5% carbon dioxide and balance nitrogen. Rates of oxygen consumption and liver cell function (metabolism of diazepam) were unchanged, and no signs of oxygen toxicity were observed. In another set of studies, oxygen consumption increased linearly with number of hepatocytes used in the clinically used range of 5-10 billion hepatocytes, whether all hepatocytes were placed in one device or split between two devices operating in series or in parallel. In a third set of studies, oxygen consumption was a weak function of flowrate through the bioreactor but was highest at the clinically used recirculation rate of 400 ml/min. These data indicate that blood gas meters--readily available in hospital clinical laboratories--can be used to assist in the design of extracorporeal cell therapies, to monitor the effectiveness of system components, and to assess the effects of system modifications. These same measurements can be made noninvasively in the clinical setting to monitor oxygen consumption rates during patient treatment.

Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems.

The provision of an immediate supply of isolated porcine hepatocytes for artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions. By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatment facilities. Additionally, cryopreservation provides an adequate period for quality control testing to be completed prior to use of any specific cell lot. We are reporting a dual approach, namely the preservation of porcine hepatocytes, at 4 degrees C and at -196 degrees C in liquid nitrogen (LN2). Using a combination of cryoprotectant agents with Chee's modified Eagle's culture media (CEM), collagenase isolated hepatocytes stored at 4 degrees C for 24 h maintained 80% of the initial diazepam metabolism measured in freshly isolated cells and nearly 100% of initial function was preserved in hepatocytes stored up to 6 mo at -196 degrees C. University of Wisconsin solution (UW) was also tested and while adequate for 4 degrees C storage, it certainly did not match the performance of the CEM formulations for preservation of metabolic function of cells stored in liquid nitrogen. Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN2 frozen cells. Other findings in these studies underlined the importance of the gradual introduction of DMSO in the prefreezing process, the period of osmotic equilibration, and the rapid postthaw withdrawal of this agent to minimize cytotoxic effects at these critical stages. Our freezing methodology provides the foundation for further technological developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices.

Evaluation of anti-AIDS drugs in conventional mice implanted with a permeable membrane device containing human T cells infected with HIV.

We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.

Clinical experience with a bioartificial liver in the treatment of severe liver failure. A phase I clinical trial.

OBJECTIVE: The purpose of this study was to develop a bioartificial liver (BAL) to treat patients with severe liver failure until they can be either transplanted or recover spontaneously. SUMMARY BACKGROUND DATA: Severe acute liver failure is associated with high mortality. Liver transplantation has emerged as an effective therapy for patients who did not respond to standard management. However, because of the donor organ shortage and urgent need for transplantation, many patients die before they can be transplanted and others do not survive after transplantation, primarily because of intracranial hypertension. METHODS: Three groups of patients with severe acute liver failure were treated with the BAL. In group 1 (n = 18) were patients with fulminant hepatic failure (FHF), in group 2 (n = 3) were patients with primary nonfunction (PNF) of a transplanted liver, and in group 3 (n = 10) were patients with acute exacerbation of chronic liver disease. Patients in groups 1 and 2 were candidates for transplantation at the time they entered the study, whereas patients in group 3 were not. RESULTS: In group 1, 16 patients were "bridged" successfully to transplantation, 1 patient was bridged to recovery without a transplant, and 1 patient died because of concomitant severe pancreatitis. In group 2, all patients were bridged successfully to retransplantation. In group 3, two patients were supported to recovery and successful transplants at later dates; the other eight patients, although supported temporarily with the BAL, later died because they were not candidates for transplantation. CONCLUSIONS: The authors' clinical experience with the BAL has yielded encouraging results. A randomized, controlled, prospective trial (phase II-III) is being initiated to determine the efficacy of the system.

Porcine islets for xenotransplantation.

Long-term function of isolated porcine islets was investigated in diabetic nude mice. Seven of eight mice that received transplants of porcine islets remained normoglycemic for 1 year with progressive weight gain. Circulating porcine C-peptide was detected throughout the study period. Intravenous glucose tolerance tests showed a rapid glucose clearance rate. Together with our recent finding that porcine islets contained within an immunoexclusion device achieved glycemic control in a totally pancreatectomized dog, these results clearly demonstrate that isolated porcine islets are capable of functioning for prolonged periods in xenogeneic hosts and are suitable for long-term use in an immunoexclusion device in a discordant host.

Treatment of diabetes by xenogeneic islets without immunosuppression. Use of a vascularized bioartificial pancreas.

Tight glycemic control by intensive insulin therapy effectively delays the onset and slows the progression of diabetic complications but is associated with frequent dose adjustments and a high incidence of hypoglycemia. Successful pancreas transplantation corrects abnormal glucose metabolism but subjects patients to morbidity and mortality associated with chronic immunosuppression. A vascularized artificial pancreas device containing pancreatic islets is designed to provide glycemic control without immunosuppression. We report here that devices seeded with porcine islets implanted into pancreatectomized severely diabetic dogs maintained a marked improvement in glycemic control with reduced exogenous insulin requirements for up to 9 months with improved glucose tolerance and a reduction in glycosylated hemoglobin levels. No immunosuppression was used. Thus, use of a vascularized artificial pancreas containing xenogeneic porcine islets could be an alternative to intensive insulin therapy and pancreatic transplantation in treating diabetic patients before the development of severe diabetic complications.

Early treatment of diabetes with porcine islets in a bioartificial pancreas.

Successful pancreas transplantation is an effective therapy for insulin-dependent diabetes mellitus (IDDM) but subjects patients to morbidity and mortality associated with chronic immunosuppression. Bioartificial pancreas devices containing pancreatic islets provide glycemic control without immunosuppression by physically separating the islet grafts from immune lymphocytes and immunoglobulins. Because immunosuppression is not required, the bioartificial pancreas may offer early treatment of IDDM prior to the development of debilitating diabetic complications. Use of xenogeneic islets (i.e., porcine islets) in the device also provides a solution to the limited availability of human donor organs. This report provides a brief summary of our experience with vascularized bioartificial pancreas devices containing xenogeneic porcine islets used for treatment of experimental diabetes in dogs and describes our plans for a clinical phase I/II trial of the vascularized bioartificial pancreas in patients with IDDM.

Novel delivery of pancreatic islet cells to treat insulin-dependent diabetes mellitus.

Immune protective devices containing pancreatic islets are designed to treat insulin-dependent diabetes mellitus by providing glycaemic control without immunosuppression. The immune protection is achieved by separating allogeneic or xenogeneic islets from the host by semipermeable membranes that allow only small molecules such as glucose, insulin and nutrients to pass through. Lymphocytes and immunoglobulins are excluded by the membrane and unable to cause rejection of the islets. Three types of immune protective devices, i.e. microcapsules, diffusion chambers and perfusion devices (vascularised artificial pancreas), have been studied. Microcapsules injected into the abdominal cavity in a large quantity achieved glycaemic control, but required a small amount of immunosuppression to prevent fibrosis around the the capsules. A clinical attempt to use microcapsulated human islets in a diabetic patient who has maintained functional kidney allografts has been reported. Intra-abdominal placement of diffusion chambers containing allogeneic islets achieved excellent glycaemic control without immunosuppression in diabetic dogs. However, their use was limited by the eventual breakage of tubular chambers. We have extensively used the vascularized artificial pancreas for treatment of experimental diabetes mellitus. Excellent biocompatibility of the device was evidenced by the extraordinary longevity of the patency of the device in healthy dogs. Long term control of severe diabetes mellitus was achieved in totally pancreatectomised dogs without immunosuppression by devices seeded with allogeneic (canine) and xenogeneic (porcine) islets. The vascularised artificial pancreas could be an excellent alternative to Diabetes Control and Complication Trial (DCCT)-type intensive insulin therapy or pancreatic transplantation by providing tight glycaemic control with minimal exogenous insulin therapy without immunosuppression.

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.

Extracorporeal enzymatic removal of low density lipoproteins in rabbits: efficacy and safety.

An extracorporeal circuit incorporating a plasma separator reactor (PSR) was designed to modify low density lipoproteins (LDL). The PSR was tested in vivo with hypercholesterolemic New Zealand White rabbits. The bioreactor enzymatically converts LDL to a form that can be removed by the body at an enhanced rate. The physiological response of hypercholesterolemic New Zealand White rabbits to 90 minute extracorporeal treatments was monitored. The total plasma cholesterol concentration in the treated rabbits fell sharply (up to 40% decrease) during and following the treatment. Results of safety tests indicate no significant enzyme leaching from the device, no disruption or damage to erythrocytes, no increase in white blood cell count and no liver damage as indicated by five enzyme assays. All safety measurements suggest that the treatment is safe.

Enzymatic modification of plasma low density lipoproteins in rabbits: a potential treatment for hypercholesterolemia.

Phospholipase A2 (EC 3.1.1.4) hydrolyzes certain phospholipids of low density lipoprotein (LDL). Plasma clearance of phospholipase A2-modified human LDL is up to 17 times faster than that of native human LDL in hypercholesterolemic rabbits. Modification of blood lipoproteins of hypercholesterolemic rabbits was performed by using an extracorporeal circuit containing immobilized phospholipase A2. After 90-min treatments, nearly 30% decreases in plasma cholesterol concentrations were observed. Erythrocyte, leukocyte, and platelet counts showed no net change after treatment. This technique does not require any fluid replacement or sorbent regeneration and offers a potential approach for lowering serum cholesterol and LDL levels.

A mathematical analysis of the U-shaped hybrid artificial pancreas--a novel insulin release rate equation.

While most artificial pancreases rely mainly on diffusive transport of solutes, Reach et al. (1) proposed a U-shaped device which combines diffusive and convective transports to shorten the lag time between insulin release and a rise in blood glucose concentration. This paper presents a mathematical model which predicts well the performance of the U-shaped hybrid artificial pancreas for both square-wave and ramp glucose stimulations. The model uses a novel insulin secretion equation, and a possible inhibition mechanism regulating insulin secretion is also discussed.

Simulation of bilirubin detoxification in the newborn using an extracorporeal bilirubin oxidase reactor.

Jaundice, which is characterized by an excessive accumulation of bilirubin in the blood and tissues, occurs in 13% of newborns. The common treatments for neonatal jaundice are phototherapy and blood exchange transfusion. A novel approach using an extracorporeal blood filter containing immobilized bilirubin oxidase was recently proposed to detoxify jaundiced blood, and a prototype device markedly reduced serum bilirubin in genetically jaundiced Gunn rats. The primary toxicologic effect in that study was a 20% reduction in red blood cell count. Using a compartmental model for bilirubin metabolism, a mathematical simulation of the extracorporeal treatment's ability to reduce serum bilirubin levels in jaundiced infants is presented. Using a 10-mL reactor volume containing immobilized bilirubin oxidase, the simulation predicts a 32 to 65% decrease in plasma bilirubin concentration over a 4-h treatment for a 2 kg preterm hyperbilirubinemic newborn. In addition, a new approach to altering support material has essentially eliminated red blood cell lysis in vivo using Gunn rats and in vitro using adult blood.

The influence of bond chemistry on immobilized enzyme systems for ex vivo use.

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50-80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25-30%) with tresyl chloride activated agarose and oxirane activated acrylic.The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in protein leaching was reached after a few hours of incubation and the transient leaching period was well represented by a logarithmic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunosorbant assay (ELISA), no immune response was detected. The transient leaching profile was shortened by washing the enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.