Polymeric Materials for Perm-Selective Coating of Alginate Microbeads.

Application of microencapsulation to the immunoisolation of pancreatic islets holds promise for expanding the use of islet transplantation as a treatment option for Type 1 diabetes. It is generally believed that successful development of a reliable methodology will ideally allow for transplantation of pancreatic islets that are protected from the immune system, thereby obviating the need for the use of immunosuppressive drugs and their attendant side effects. In addition, this technology has the potential to expand the donor pool as islets from nonhuman donors could be used as xenografts in human patients. The complex polysaccharide, alginate, has been the most widely used polymer for microencapsulation of islets. However, it is known that alginate lacks appreciable permselectivity to confer immunoisolation of encapsulated islets, thus necessitating the routine permselective coating of alginate microbeads with polymers of amino acids, mainly, poly-L-lysine (PLL) and poly-L-ornithine (PLO). The protocol described in this chapter outlines the steps we have used in our studies on perm-selective coating of alginate microbeads for islet transplantation.

A Method of Porcine Pancreatic Islet Isolation for Microencapsulation.

Since the discovery of insulin by Banting and Best in 1921, the prognosis and treatment options for individuals with diabetes have improved. The development of various insulin types, various oral agents, and insulin pumps have improved the available medical options for individuals afflicted with diabetes. The current need for frequent blood glucose monitoring imposed by multiple daily insulin injections, result in significant life-style challenges for in individuals afflicted with Type 1 diabetes (T1D). In contrast the use of surgical interventions, such as whole organ pancreas transplantation (PT) requires less-intensive glucose monitoring while the organ is viable. Also, isolated human pancreatic islet transplantation (IT) holds similar promise as PT; however, the limited availability of human pancreata exacerbated by, the need for multiple pancreata per individual IT recipient, and issues with prolonged viability, still hamper widespread successful, and routine use of IT. The use of porcine pancreata holds promise as a viable alternative to human pancreas to significantly increase the volume of islets available to meet the needs of millions of patients afflicted with T1D. This chapter outlines our protocol utilized to reliably isolate and microencapsulate porcine islets.

Effect of alginate composition and gelling cation on microbead swelling.

PURPOSE: The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability. METHOD: Using a 2-channel droplet generator, microspheres were generated with 1.5% solutions of low viscosity high-mannuronic acid (LVM), medium viscosity high-mannuronic acid (MVM), low viscosity high-guluronic acid (LVG) and medium viscosity high-guluronic acid (MVG) alginate. They were gelled by cross-linking with 1.1% solution of either BaCl2 or CaCl2. The diameters of the microbeads were measured and recorded on day 0. The microbeads were subsequently washed and incubated in saline at 37 degrees C for 2 weeks with size assessment every 2 days. The data were normalized by calculation of the percentage change from control (day 0) for all groups of microbeads. RESULTS: Diameters of all beads were between 550-700 microns on day 0. Viscosity had no effect on swelling of Ba++- and Ca++-alginate microbeads. Ca++-alginate microbeads were more prone to swelling than the corresponding Ba++-alginate beads. High G-Ba++ beads had only a modest increase in size over time, in contrast to the high M-Ba++. CONCLUSION: Alginate composition and the gelling cation have significant effects on bead swelling.

Effect of alginate composition and gelling cation on micro-bead swelling.

PURPOSE: The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability. METHOD: Using a 2-channel droplet generator, microspheres were generated with 1.5% solutions of low viscosity high-mannuronic acid (LVM), medium viscosity high-mannuronic acid (MVM), low viscosity high-guluronic acid (LVG) and medium viscosity high-guluronic acid (MVG) alginate. They were gelled by cross-linking with 1.1% solution of either BaCl2 or CaCl2. The diameters of the micro-beads were measured and recorded on day 0. The micro-beads were subsequently washed and incubated in saline at 37 degrees C for 2 weeks with size assessment every 2 days. The data were normalized by calculation of the percentage change from control (day 0) for all groups of micro-beads. RESULTS: Diameters of all beads were between 550 and 700 microm on day 0. Viscosity had no effect on swelling of Ba++- and Ca++-alginate micro-beads. Ca++-alginate micro-beads were more prone to swelling than the corresponding Ba++-alginate beads. High G-Ba++ beads had only a modest increase in size over time, in contrast to the high M-Ba++. CONCLUSION: Alginate composition and the gelling cation have significant effects on bead swelling.

Characteristics of Poly-L-Ornithine-coated alginate microcapsules.

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.

Polyvinyl pyrrolidone: a novel cryoprotectant in islet cell cryopreservation.

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.

Total antioxidant capacity following extrinsic denervation and small intestinal transplantation in the rat.

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.

Immunoisolation techniques for islet cell transplantation.

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.