Immunoisolation Techniques for Islet Cell Transplantation

Expert Opinion on Biological Therapy. Jun, 2002  |  Pubmed ID: 12079486

Diabetes remains a devastating disease, with tremendous cost in terms of human suffering and healthcare expenditures. The burden of diabetes is primarily related to the multiple complications, including retinopathy, nephropathy, neuropathy and cardiovascular disease that can develop as the disease progresses. It has been shown that these complications can be prevented, and in some cases, reversed by islet cell transplantation, which, until recently, had remained elusive as a viable routine treatment modality. In recent studies, islet cell transplantation has shown great promise as a viable alternative to solid pancreas transplantation. However, severe shortage of human pancreases and the need to use immunosuppressive drugs to prevent transplant rejection, remain major obstacles to routine use of islet cell transplants for the treatment of patients with Type 1 diabetes. In the attempt to overcome these barriers, many procedures have been designed to immunoisolate islet cells for transplantation. The ultimate goal in islet cell transplantation is the availability of unlimited supply of cells to be transplanted in a simple procedure performed with little or no use of immunosuppressive drugs. The development of reliable procedures to immunoisolate islets by microencapsulation prior to transplantation has a great deal of potential to accomplish this objective.

Effects of Omeprazole and Ascorbate on Gastric Emptying and Antioxidant Levels in a Mouse Model of Glutathione Depletion

Digestive Diseases and Sciences. Nov, 2002  |  Pubmed ID: 12452384

Increased free radical production with depletion of the antioxidant, glutathione, is a suggested mechanism for the development of ulcer disease in patients with Helicobacter pylori. The effects of ascorbate and omeprazole as potential gut antioxidants are incompletely understood. We hypothesized that as antioxidants, ascorbate and omeprazole protect against glutathione depletion. This study was designed to determine the effects of ascorbate and omeprazole on gastric emptying and gastric antioxidant levels in a mouse model of glutathione depletion. In an acute (10-day) mouse model, glutathione depletion was induced by inhibiting the rate limiting enzyme, gamma-glutamylcysteine synthetase. Enzymatic blockade produced depletion of gastric glutathione (P < 0.05) without increasing gastric lipid hydroperoxides. Glutathione depletion was associated with accelerated liquid gastric emptying. These effects were not prevented by supplementation with ascorbate or omeprazole. Omeprazole induced increased (P < 0.05) gastric and colonic total antioxidant capacity. One of the beneficial effects of omeprazole in patients may involve increased total antioxidant capacity.

Total Antioxidant Capacity Following Extrinsic Denervation and Small Intestinal Transplantation in the Rat

Neuroscience Letters. May, 2003  |  Pubmed ID: 12727336

Transplantation of small intestine in a rat model has been shown to affect expression of neurochemicals within enteric inhibitory nerves. However, the mechanism for altered expression of inhibitory neurochemicals is uncertain. Based on our previous studies, we hypothesized that small intestinal transplantation would result in altered intestinal levels of antioxidant capacity. Glutathione, total antioxidant capacity, and lipid peroxide levels were measured at 3 months following (1) transection of rat small intestine, (2) transection and extrinsic denervation of rat intestine, and (3) isotransplantation of rat ileum or (4) allotransplantation of rat ileum with cyclosporine therapy to suppress rejection. Glutathione levels were not significantly different among the four groups. There were trends toward increased lipid peroxide levels following isografting and extrinsic denervation. Total antioxidant capacity was increased following extrinsic denervation (P=0.05). Increased intestinal total antioxidant capacity in response to extrinsic denervation may represent a compensatory mechanism for protection against oxidative stress. This result enhances our understanding of the relationship between tissue antioxidant levels and alteration of enteric nerves.

Role of Oxidative Stress in the Etiology of Type 2 Diabetes and the Effect of Antioxidant Supplementation on Glycemic Control

Journal of Investigative Medicine : the Official Publication of the American Federation for Clinical Research. Jan, 2004  |  Pubmed ID: 14989366

Oxidative stress is a situation in which the amount of reactive oxygen species (ROS) exceeds the levels of neutralizing substances referred to as antioxidants. Numerous studies have shown that oxidative stress is associated with type 2 diabetes, and there is compelling biochemical evidence that suggests that ROS may even play a role, if only secondary, in the pathogenesis of type 2 diabetes. These observations have provided sufficient impetus for the use of antioxidant supplements as adjunct therapy for control of blood sugar in diabetic patients. However, there is currently no optimum regimen of antioxidant supplementation for diabetic patients. Studies are required to determine appropriate doses of relevant individual micronutrients that perhaps should be used in combination to diminish oxidative stress and improve glycemic control in individuals afflicted with type 2 diabetes.

Polyvinyl Pyrrolidone: a Novel Cryoprotectant in Islet Cell Cryopreservation

Cell Transplantation. 2004  |  Pubmed ID: 15191161

The present study was performed on the basis of the hypothesis that the low molecular weight (MW) compounds, DMSO and glycerol, permeate the cell and interact hydrophobically with intracellular proteins, thereby perturbing the cytoskeletal architecture of frozen cells and diminishing islet cell integrity and function. Isolated rat islets were cultured overnight (18-24 h) at 37 degrees C in RPMI medium supplemented with 10% fetal calf serum and 1% mixture of penicillin/streptomycin. Using a programmable temperature controller, samples of precounted islets were then frozen under liquid nitrogen, in the presence of either 2 M DMSO (MW = 0.078 kDa), 3 M glycerol (MW = 0.092 kDa), 5% polyethylene glycol (PEG, MW = 20 kDa), or 10% polyvinylpyrrolidone (PVP, MW = 40 kDa), and stored at -80 degrees C for 1 week. Following thawing and overnight (18-24 h) culture, intact islet recovery was determined by islet counting after dithizone staining. Islet function was assessed by determination of glucose-stimulated insulin secretion in perifusion experiments with Krebs-Ringer bicarbonate buffer, pH 7.4, containing either basal (3.3 mM) or high (16.7 mM) glucose concentrations. The assessment of islet recovery and function of all cryopreserved samples was performed only after thawing and overnight culture (18-24 h) of islets. The mean +/- SEM percent intact islet recovery was higher with PVP compared with DMSO (82 +/- 4.6 vs. 62.7 +/- 3.1%, respectively, p < 0.005, n = 9). Furthermore, the glucose stimulation index of insulin secretion by islets taken from samples frozen with PEG and PVP, after thawing and overnight culture, was comparable to that of freshly isolated islets, in contrast to DMSO and glycerol. There was no significant difference in intact islet recovery and function between samples frozen with PVP and those frozen with PEG. Samples frozen with DMSO and glycerol had similar results in islet recovery and function. These data show that PVP is a new and potent cryoprotectant for islet cell freezing.

Effect of the H, K-ATPase Inhibitor, Esomeprazole Magnesium, on Gut Total Antioxidant Capacity in Mice

The Journal of Nutritional Biochemistry. Sep, 2004  |  Pubmed ID: 15350983

Antioxidant depletion is believed to be a mechanism involved in the pathophysiology of several upper gastrointestinal disorders, and H, K-ATPase inhibitors can alter free radical production by neutrophils. We hypothesized that the H, K-ATPase inhibitor esomeprazole magnesium would decrease gut free radical production with a concomitant increase in gut total antioxidant capacity. A/J mice (n = 10/group) received either vehicle (control) or one of three concentrations of esomeprazole magnesium in vehicle by once-daily gavage for 10 days. Using tissue extracts from stomach and colon, total antioxidant capacity, lipid peroxide levels, and constitutive Cu/Zn-superoxide dismutase were measured using validated assays. There was a dose-related increase in total antioxidant capacity (analysis of variance, P < 0.001) in stomach, but there was no change in the colon. In the assessment of free radical production, there was a trend toward decreased lipid peroxide levels in stomach from mice receiving esomeprazole. In stomach, Cu/Zn-superoxide dismutase activity was increased (ANOVA: p=.03) in mice receiving esomeprazole. In conclusion, gastric total antioxidant capacity and Cu/Zn-superoxide dismutase activity are increased by esomeprazole, and these changes may result in part from decreased free radical production. The present results support the notion that the pharmacological effects of this agent on upper intestinal tissue are more complex than previously thought, and appear to involve both enzymatic and nonenzymatic tissue antioxidants.

Oxidative Stress in Nonalcoholic Fatty Liver Disease: Pathogenesis and Antioxidant Therapies

Journal of Investigative Medicine : the Official Publication of the American Federation for Clinical Research. Dec, 2004  |  Pubmed ID: 15682682

Nonalcoholic fatty liver disease is a common cause of chronic liver disease, a common finding on liver biopsy in those patients with abnormal blood transaminase levels, and a common cause of cryptogenic cirrhosis in the United States. The prevalence of this disorder is expected to rise with the increase in obesity, and the clinical spectrum can range from simple steatosis (fatty liver) to cirrhosis of the liver. Insulin resistance is thought to be pivotal for the development of steatosis, and oxidative stress may be a potential factor that can promote hepatic necroinflammation and fibrosis. Preliminary studies have examined the role of oxidative stress and antioxidants in animal and human studies of this disorder. Efforts to improve the hepatic antioxidant system could be achieved by optimizing the patient's diet, by supplementation with precursors for antioxidants, or by supplementation with essential metals and/or antioxidants. Randomized, controlled trials are required to examine these potential approaches using patients with this disorder.

Effects of Esomeprazole Magnesium on Nonsteroidal Anti-inflammatory Drug Gastropathy

Digestive Diseases and Sciences. Jan, 2005  |  Pubmed ID: 15712643

It has been proposed that tissue damage induced by nonsteroidal anti-inflammatory drugs is related to increased tissue free radical production with antioxidant depletion. We have shown that esomeprazole increases gastric total antioxidant capacity in mice and, therefore, hypothesized that the protective effect of esomeprazole during treatment with a nonsteroidal anti-inflammatory drug is related to increased gastric antioxidant capacity and decreased tissue free radical production. A/J mice received one of four treatments by daily gavage: saline in vehicle (control), indomethacin, esomeprazole, or indomethacin and esomeprazole. After 10 days, all mice were sacrificed and validated assays were used to measure gastric total antioxidant capacity, lipid peroxide levels, and myeloperoxidase activity. Indomethacin-treated mice developed weight loss and melena. No mice receiving indomethacin and esomeprazole died, but the death rate while receiving indomethacin was 38% (chi2, P = 0.05). Gastric lipid peroxide levels increased in mice receiving indomethacin treatment compared to treatment with esomeprazole and indomethacin (P = 0.03). There was a strong trend (P = 0.08) toward increased gastric total antioxidant capacity in mice receiving esomeprazole and indomethacin compared to mice receiving indomethacin. Gastric myeloperoxidase activities were not different among the four groups. Esomeprazole significantly improved survival in mice that received indomethacin, reduced free radical production, as estimated by lipid peroxide levels, and appeared to increase gastric total antioxidant capacity. The mechanisms for the beneficial effects of esomeprazole in the treatment of gastropathy are more complex than previously thought.

Characteristics of Poly-L-Ornithine-coated Alginate Microcapsules

Biomaterials. Dec, 2005  |  Pubmed ID: 15955558

Poly-L-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-L-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean +/- SEM diameter of PLL-coated microcapsules increased from 718+/-17 to 821 +/- 17 microm (p < 0.05) during 14 days incubation at 37 degrees C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.7 +/- 4.9%) compared to 3.1 +/- 2.05% for PLO capsules (p < 0.0001,n = 6). We conclude that PLO-coated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules.

Antioxidants and Micronutrients

Disease-a-month : DM. Apr, 2006  |  Pubmed ID: 16828355

Oxidative Stress

Disease-a-month : DM. May, 2006  |  Pubmed ID: 16828360

Effect of Alginate Composition and Gelling Cation on Micro-bead Swelling

Journal of Microencapsulation. Feb, 2006  |  Pubmed ID: 16830975

The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability.

Effect of Alginate Composition and Gelling Cation on Microbead Swelling

Journal of Microencapsulation. Sep, 2006  |  Pubmed ID: 17118877

The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability.

Sustained Delivery of FGF-1 Increases Vascular Density in Comparison to Bolus Administration

Microvascular Research. Sep, 2009  |  Pubmed ID: 19555698

The use of growth factors for the therapeutic stimulation of neovascularization in regenerative medicine has been extensively investigated, but the inability to control their temporal delivery may limit clinical success. A strategy that delivers continuous therapeutic concentrations of growth factors may increase the protein's efficacy. The present study investigates the ability of sustained delivery of fibroblast growth factor-1 (FGF-1), to induce neovascularization in vivo. Alginate microbeads were synthesized to release active FGF-1 for three weeks. Microbeads loaded with FGF-1 (total amount 150 ng) were implanted into a surgically created omentum pouch in rats and were compared to control empty microbead implants and a single bolus injection of 150 ng of FGF-1 with empty microbead implant. Animals were sacrificed at either 3 or 6 weeks post implantation and omenta were analyzed for vascular density and mural cell interactions. Vascular area for bolus FGF-1 and FGF-1 loaded microbeads was higher than control at 3 weeks. At 6 weeks, vascular density in the group with FGF-1 loaded microbeads was significantly higher than the group with bolus administration of FGF-1, primarily due to an increase in the number of vessels less than 20 microm in diameter. Vascular density in omenta of the group receiving the bolus FGF-1 returned to control levels by 6 weeks. Staining for smooth muscle actin showed that 50% of vessels had associated mural cells. There was a trend of increased mural cell staining at 6 weeks for the FGF-1 loaded beads compared to bolus FGF-1 and control levels. Results in these studies suggest that sustained release of FGF-1 increases the duration of the vascular response in contrast to a bolus injection of FGF-1.

Fibroblast Growth Factor-1 (FGF-1) Loaded Microbeads Enhance Local Capillary Neovascularization

The Journal of Surgical Research. May, 2010  |  Pubmed ID: 19959194

Growth of new blood vessels (neovascularization) occurs naturally in the body, but the slow rate of the process may not be sufficient for survival of engineered tissues and transplanted cells, such as pancreatic islets. For transplanted islets, it is crucial that the transplantation site has sufficient vasculature to support the needs of the islets. Therefore, the specific aim of this research was quantify the effect of FGF-1 incorporation into alginate microbeads on neovascularization of such capsules in an in vivo rat transplant model.

The Effect of FGF-1 Loaded Alginate Microbeads on Neovascularization and Adipogenesis in a Vascular Pedicle Model of Adipose Tissue Engineering

Biomaterials. Apr, 2010  |  Pubmed ID: 20080298

Engineered vascularized adipose tissue could serve as an alternative to traditional tissue reconstruction procedures. Adipose formation occurs in a coordinated fashion with neovascularization. Previous studies have shown that extracellular matrix-based materials supplemented with factors that stimulate neovascularization promote adipogenesis in a number of animal models. The present study examines the ability of fibroblast growth factor (FGF-1) delivered from alginate microbeads to induce neovascularization and adipogenesis in type I collagen gels in a vascular pedicle model of adipose tissue engineering. FGF-1 loaded microbeads stimulated greater vascular network formation in an in vitro 3D co-culture model than a single bolus of FGF-1. In in vivo studies, FGF-1 loaded beads suspended in collagen and implanted in a chamber surrounding the exposed femoral pedicle of a rat resulted in a significant increase in vascular density at 1 and 6 weeks in comparison to bolus administration of FGF-1. Staining for smooth muscle actin showed that over 48% of vessels had associated mural cells. While an increase in neovascularization was achieved, there was less than 3% adipose under any condition. These results show that delivery of FGF-1 from alginate beads stimulated a more persistent neovascularization response than bolus FGF-1 both in vitro and in vivo. However, unlike previous studies, this increased neovascularization did not result in adipogenesis. Future studies need to provide a better understanding of the relationship between neovascularization and adipogenesis in order to design advanced tissue engineering therapies.

Design of a Bioartificial Pancreas(+)

Journal of Investigative Medicine : the Official Publication of the American Federation for Clinical Research. Oct, 2010  |  Pubmed ID: 20683347

In type 1 diabetes, the β-cells that secrete insulin have been destroyed such that daily exogenous insulin administration is required for the control of blood glucose in individuals with the disease. After the development of reliable techniques for the isolation of islets from the human pancreas, islet transplantation has emerged as a therapeutic option, albeit for only a few selected patients largely because there are not enough islets for the millions of patients requiring the treatment, and there is also the need to use immunosuppressive drugs to prevent transplant rejection. In 1980, the concept of islet immunoisolation by microencapsulation was introduced as a technique to overcome these 2 major barriers to islet transplantation. Microencapsulation of islets and transplantation in the peritoneal cavity was then described as a bioartificial pancreas. However, it is difficult to retrieve encapsulated islets transplanted in the peritoneal cavity, thus making it difficult to meet all the criteria for a bioartificial pancreas. A new design of a bioartificial pancreas comprising islets co-encapsulated with angiogenic protein in permselective multilayer alginate-poly-L-ornithine-alginate microcapsules and transplanted in an omentum pouch is described in this paper.

Synthesis of Multilayered Alginate Microcapsules for the Sustained Release of Fibroblast Growth Factor-1

Journal of Biomedical Materials Research. Part A. Nov, 2010  |  Pubmed ID: 20725969

Alginate microcapsules coated with a permselective poly-L-ornithine (PLO) membrane have been investigated for the encapsulation and transplantation of islets as a treatment for type 1 diabetes. The therapeutic potential of this approach could be improved through local stimulation of microvascular networks to meet mass transport demands of the encapsulated cells. Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor with optimal effect occurring when it is delivered in a sustained manner. In this article, a technique is described for the generation of multilayered alginate microcapsules with an outer alginate layer that can be used for the delivery of FGF-1. The influence of alginate concentration and composition (high mannuronic acid (M) or guluronic acid (G) content) on outer layer size and stability, protein encapsulation efficiency, and release kinetics was investigated. The technique results in a stable outer layer of alginate with a mean thickness between 113 and 164 μm, increasing with alginate concentration and G-content. The outer layer was able to encapsulate and release FGF-1 for up to 30 days, with 1.25% of high G alginate displaying the most sustained release. The released FGF-1 retained its biologic activity in the presence of heparin, and the addition of the outer layer did not alter the permselectivity of the PLO coat. This technique could be used to generate encapsulation systems that deliver proteins to stimulate local neovascularization around encapsulated islets.

Multilayered Microcapsules for the Sustained-release of Angiogenic Proteins from Encapsulated Cells

American Journal of Surgery. Nov, 2010  |  Pubmed ID: 21056148

Multilayered alginate microcapsules with a permselective poly-L-ornithine membrane can be used for the dual purpose of encapsulating cells in the inner core and sustained release of angiogenic proteins from the outer layer. The aim of this study was to examine the encapsulation and release of a novel chimeric form of fibroblast growth factor-1 (FGF-1) from the outer layer of alginate microcapsules.

A Three-dimensional Microfluidic Approach to Scaling Up Microencapsulation of Cells

Biomedical Microdevices. Jun, 2012  |  Pubmed ID: 22245953

Current applications of the microencapsulation technique include the use of encapsulated islet cells to treat Type 1 diabetes, and encapsulated hepatocytes for providing temporary but adequate metabolic support to allow spontaneous liver regeneration, or as a bridge to liver transplantation for patients with chronic liver disease. Also, microcapsules can be used for controlled delivery of therapeutic drugs. The two most widely used devices for microencapsulation are the air-syringe pump droplet generator and the electrostatic bead generator, each of which is fitted with a single needle through which droplets of cells suspended in alginate solution are produced and cross-linked into microbeads. A major drawback in the design of these instruments is that they are incapable of producing sufficient numbers of microcapsules in a short-time period to permit mass production of encapsulated and viable cells for transplantation in large animals and humans. We present in this paper a microfluidic approach to scaling up cell and protein encapsulations. The microfluidic chip consists of a 3D air supply and multi-nozzle outlet for microcapsule generation. It has one alginate inlet and one compressed air intlet. The outlet has 8 nozzles, each having 380 micrometers inner diameter, which produce hydrogel microspheres ranging from 500 to 700 μm in diameter. These nozzles are concentrically surrounded by air nozzles with 2 mm inner diameter. There are two tubes connected at the top to allow the air to escape as the alginate solution fills up the chamber. A variable flow pump 115 V is used to pump alginate solution and Tygon® tubing is used to connect in-house air supply to the air channel and peristaltic/syringe pump to the alginate chamber. A pressure regulator is used to control the flow rate of air. We have encapsulated islets and proteins with this high throughput device, which is expected to improve product quality control in microencapsulation of cells, and hence the outcome of their transplantation.

Stability of Alginate Microbead Properties in Vitro

Journal of Materials Science. Materials in Medicine. Apr, 2012  |  Pubmed ID: 22350778

Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3 weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy.