Anthony M. Sun

Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immunosuppression.

Porcine pancreatic islets were microencapsulated in alginate-polylysine-alginate capsules and transplanted intraperitoneally into nine spontaneously diabetic monkeys. After one, two, or three transplants of 3-7 x 10(4) islets per recipient, seven of the monkeys became insulin independent for periods ranging from 120 to 804 d with fasting blood glucose levels in the normoglycemic range. Glucose clearance rates in the transplant recipients were significantly higher than before the graft administration and the insulin secretion during glucose tolerance tests was significantly higher compared with pretransplant tests. Porcine C-peptide was detected in all transplant recipients throughout their period of normoglycemia while none was found before the graft administration. Hemoglobin A1C levels dropped significantly within 2 mo after transplantation. While ketones were detected in the urine of all recipients before the graft administration, all experimental animals became ketone free 2 wk after transplantation. Capsules recovered from two recipients 3 mo after the restoration of normoglycemia were found physically intact with enclosed islets clearly visible. The capsules were free of cellular overgrowth. Examination of internal organs of two of the animals involved in our transplantation studies for the duration of 2 yr revealed no untoward effect of the extended presence of the microcapsules.

Prolonged survival of transplanted islets of Langerhans encapsulated in a biocompatible membrane

Prolonged survival of islet allografts in streptozotocin-induced diabetic rats was achieved by encapsulating individual islets in protective, biocompatible alginate-polylysine-alginate membranes. A single intraperitoneal transplant of encapsulated islets reversed the diabetic state for up to 1 year. In contrast, a single injection of unencapsulated islets was effective for less than 2 weeks. The microencapsulation procedure, by protecting transplanted tissue from the components of the immune system, has great clinical potential in the treatment of diseases requiring organ transplantation, such as diabetes and liver disease.

Reversal of Diabetes in BB Rats by Transplantation of Encapsulated Pancreatic Islets

Prolonged survival of pancreatic islet allografts implanted in diabetic BB rats was achieved by encapsulation of individual islets in a protective biocompatible alginate-polylysine-alginate membrane without immunosuppression. Intraperitoneal transplantation of the encapsulated islets reversed the diabetic state of the recipients within 3 days and maintained normoglycemia for 190 days. Normal body weight and urine volume were maintained during this period, and no cataracts were detected in the transplant recipients. In contrast, control rats receiving transplants of unencapsulated islets experienced normoglycemia for <2 wk. These results demonstrated that microencapsulation can protect allografted islets from both graft rejection and autoimmune destruction without immunosuppression in an animal model that mimics human insulin-dependent diabetes.

The Use, in Diabetic Rats and Monkeys, of Artificial Capillary Units Containing Cultured Islets of Langerhans (Artificial Endocrine Pancreas)

A unit was constructed that consisted of a core of hollow fibers through which low-molecular-weight substances, such as glucose and insulin, could pass freely but were impermeable to high-molecular-weight proteins, such as antibodies. Islets of Langerhans from normal rats were planted in the space surrounding the fibers, and either blood or nutrient medium was circulated through the fibers themselves. In experiments with animals, the units were attached to the vascular system of diabetic rats and monkeys. Blood glucose concentrations in the rats were reduced to nondiabetic levels within one hour and were maintained for the duration of the experiments. In monkeys the blood glucose level declined from 210 mg./100 ml. to 90 mg./100 ml. in four hours and insulin in the serum rose to 93 μU./ml. in one-half hour. Also, we have found that islets from monkeys cultivated in the artificial endocrine pancreas (AEP) continue to release insulin into circulating tissue culture medium for over eight months.

Prolonged Reversal of Diabetic State in NOD Mice by Xenografts of Microencapsulated Rat Islets

Transplantation of the islets of Langerhans could be the most promising approach to the clinical treatment of insulin-dependent (type I) diabetes mellitus. In this study, we report on a modified encapsulation technique that produces small alginate-polylysine capsules (0.25–0.35 mm diam). In an in vitro study, both encapsulated and unencapsulated islets showed comparable responses to glucose challenge in terms of insulin secretion. With the new capsules, 16 spontaneously diabetic NOD mice received transplants of 800 encapsulated rat islets/animal. Nonfasting blood glucose concentration decreased from 24.4 ± 1.4 to 4.0 ± 1.3 mM. At 4 and 5 mo posttransplantation, the capsules were removed from 2 recipients. Both animals regressed to a hyperglycemic state after capsule removal. However, after another islet transplantation, normoglycemia was again restored in these 2 animals. In control mice, which received unencapsulated islets, the xenografts remained functional for <10 days. A high mortality rate was observed among these animals within 2 mo of the recurrence of the hyperglycemic state. Our results clearly indicate that encapsulation of pancreatic islets in the improved capsules can effectively prolong xenograft survival without immunosuppression in an animal model that mimics human type I diabetes mellitus.

Artificial endocrine pancreas

Diabetes and its complications are a significant cause of morbidity and mortality in the world. Patients with diabetes have an excess risk of mortality compared with the nondiabetic population. Mortality rates are 41.8% for the diabetic population and 10.1% for the nondiabetic population. Among the diabetic population, the most common cause of death is cardiovascular disease, which accounts for 49.1% of deaths. The amount of life-years lost for patients with diabetes is strongly related to age at diagnosis and is a means of expressing mortality without relying on accurate prevalence data. The prevalence of physician-diagnosed diabetes has been steadily increasing in the US population, based on national household interview surveys conducted during the past 40 years. The prevalence of diagnosed diabetes from 1988 to 1994 was estimated to be 5.1% for US adults 20 years of age and older (10.2 million people when extrapolated to the 1997 US population). There were similar rates of diabetes for men and women. Based on American Diabetes Association criteria, the prevalence of diabetes (diagnosed plus undiagnosed) in people 40 to 74 years of age increased from 8.9% in the period 1976 to 1980 to 12.3% by 1988 to 1994. The prevalence of diabetes rose from 4.9% in 1990 to 6.5% in 1998. Increases were observed in both genders, all ages, all ethnic groups, all education levels, and nearly all states in the US. Many factors influence the prevalence of diabetes, including the development of new cases of diabetes and those that affect mortality from diabetes. High rates of abnormal fasting and postchallenge glucose, together with the increasing frequency of obesity and sedentary lifestyles in the population, make it likely that diabetes will continue to be a major health problem in the US. The prevalence of hypertension, hyperlipidemia, and the incidence of and mortality from heart disease and stroke are markedly declining in the US, but the prevalence of diabetes remains high. A similar increase also has been found world-wide. Diabetic complications such as neuropathy, retinopathy, nephropathy, and cardiovascular disease continue to pose major health risks for diabetic patients. Longterm complications associated with diabetes may result from a lack of blood glucose control. Consequently, much effort has focused on approaches that could replace conventional insulin therapy and provide more precise regulation of blood glucose levels. Indeed, the Diabetes Control and Complications Trial has conclusively demonstrated that improved metabolic control of glucose leads to the reduction in the rate of microvascular complications of diabetes. Conventional insulin treatment of diabetes mellitus does not prevent the vascular complications associated with longterm disease. To solve these problems, much investigation is conducted toward developing a means to provide near-physiologic insulin administration. Table 1 shows various treatments for diabetic patients. There are advantages and disadvantages to these treatments, which include pancreas transplants, islet or beta-cell transplants, hybrid devices in which the islet or beta cells are contained within permeable membranes, and a totally artificial endocrine pancreas (electromechanical artificial pancreas) that consists of a glucose sensor, a computer, and a pumping device. Biologic approaches are expected to fulfill all the endocrine functions of the normal pancreas, but at great expense; mechanical approaches fall short of total endocrine replacement but offer the substantial advantages of mass production. Each approach has inherent problems, and none is available for widespread clinical use now. So in general, many diabetic patients receive daily injections of insulin and face a high degree of compliance with a strict regimen of diet and exercise. Intensive insulin therapy is also usually associated with clinical problems including severe hypoglycemia and inherent instability of glucose control from the open-loop control technique. The difficulty of achieving longterm effective glucose control has encouraged us to develop a closed-loop control system, the artificial endocrine pancreas. PancreNo competing interests declared.

Microencapsulation of recombinant cells: a new delivery system for gene therapy.

The therapeutic potential of somatic gene therapy has been extensively investigated in recent years, yet its slow progression into the clinical setting can be attributed to problems associated with the inability to achieve efficient gene transfers, to obtain sustained level of expression of the transfected gene, and the necessity to avoid immunorejection after transplantation. Here we report on an alternate strategy in gene therapy that overcomes all three problems by immunoisolating genetically modified cells in a biocompatible membrane, thereby introducing a system that can provide sustained delivery of the desired gene product. As a model, mouse fibroblasts transformed with the human growth hormone gene (Ltk‐GH) were encapsulated with an alginate‐poly‐L‐lysinealginate membrane. Long‐term in vitro studies showed that the encapsulation of the cells was physiologically compatible with growth and survival of the cells. Furthermore, there was a unique pattern of secretion of the human protein by the encapsulated cells: there was a phase of steady increase in the secretion of the human growth hormone by each cell, followed by a plateau phase. The most convincing evidence of the feasibility of this strategy was provided by the in vivo study: Balb‐c mice transplanted with encapsulated Ltk‐GH cells had detectable serum levels of human growth hormone (hGH) for the duration of the study (115 days). Moreover, encapsulated cells recovered from a recipient 1 year after the transplantation continued to secrete high levels of hGH in culture.—Tai, I. T., Sun, A. M. Microencapsulation of recombinant cells: a new delivery system for gene therapy. FASEB J. 7: 1061‐1069; 1993.

Injectable microencapsulated islet cells as a bioartificial pancreas

Rat islets encapsulated in semipermeable membranes remained viable in culture for 4 months. Multiple allotransplants of islets encapsulated in alginate-polylysine-polyethyleneimine membranes restored normoglycemia in recipient diabetic rats for most of a 90-day experimental period. Each individual transplant restored normal fasting plasma glucose levels for 15–20 d. The failure of the encapsulated islets was caused by an inflammatory response induced by polyethyleneimine. In contrast a single transplant of islets encapsulated in a biocompatible alginate-polylysine-alginate membrane restored normoglycemia in recipient animals for up to 10 months. Capsules with intact membranes and containing viable islets were recovered from the abdominal cavity 5 months post-transplantation. SEM studies on capsule membranes revealed essentially smooth surfaces. Differences between wet and dry wall thicknesses indicated that the membrane is a hydrogel, 4.00±0.28 μm thick in an aqueous environment.The clinical potential of transplanting cells encapsulated in biocompatible semipermeable hydrogel membranes is demonstrated by this study.

Generation of Alginate-Poly-L-Lysine-Alginate (APA) Biomicrocapsules: the Relationship Between the Membrane Strength and the Reaction Conditions

Alginate-poly-l-lysine-alginate (APA) microcapsules have proven effective in protecting enclosed live cells from immune rejection following transplantation into experimental animals, thereby eliminating the need for immunosuppressive therapy. However, in order for the capsules to remain intact for extended periods in vivo, the thickness of the membrane material must be optimized. In this study, the membrane thickness was examined as an indicator of membrane strength and measured under different reaction conditions. The thickness was found to increase 1) from 4.6 microns to 6.6 microns with an increase in the concentration of sodium alginate from 1.25 (w/v) to 2.0% (w.v); 2) from 4.2 microns to 6.2 microns with an increase in the concentration of the calcium solution from 20 mM to 100mM; 3) from 3.9 microns to 10.3 microns with an increase in the concentration of poly-l-lysine (PLL) from 0.02% (w/v) to 0.08% (w/v); and 4) from 2.3 microns to 7.4 microns with an increase in the reaction time with the PLL from two to seven minutes. On the other hand, membrane thickness decreased 1) from 9.8 microns to 8.6 microns with an increase of the pH in the PLL solution from 5.8 to 9.2; 2) from 13.2m to 5.8 microns with an increase in the molecular weight of PLL from 14,000 to 57,000; 3) from 8.4 microns to 6.0 microns with an increase in the treatment time with 0.9 (w/v) NaCl solution from zero to fifteen minutes and; 4) from 7.5 microns to 6.1 microns with an increase in the treatment time of the second sodium alginate coating from zero to ten minutes. Membrane thickness was inversely proportional to capsule volume expansion during membrane synthesis. By replacing calcium chloride by calcium lactate and eliminating the use of CHES in the construction of capsule membranes, we improved the strength and biocompatibility of our capsules, as evidenced by marked improvements in the survival rates of diabetic mice treated with islet transplants enclosed in the new capsules. These results indicate that it is possible to obtain optimal membrane thickness for a given purpose by creating specific reaction conditions under which membranes are synthesized.

Xenografts of rat islets into diabetic mice : an evaluation of new smaller capsules

Healthy rat islets were encapsulated in alginate-polylysine-alginate capsules measuring 0.25-0.35 mm in diameter using a modified encapsulation technique. The encapsulated islets were transplanted intraperitoneally in nonimmunosuppressed streptozotocin-induced diabetic BALB/c mice. The diabetic condition of the experimental animals was reversed within two days following the transplantation and the animals remained normoglycemic for up to 308 days, with a mean xenograft survival of 219.8 +/- 46.2 days. Four and six months posttransplant the capsules were removed from two recipients. This resulted in regression to a hyperglycemic state. After a second transplant of encapsulated islets, the animals returned to normoglycemia. In control mice that received free unencapsulated islets, the xenografts remained functional for no more than 12 days. Our study clearly demonstrates that the encapsulation of islets in the new smaller capsules can effectively prolong xenograft survival without immunosuppression.