An encapsulation system for the immunoisolation of pancreatic islets.

Over a thousand combinations of polyanions and polycations were tested to search for new polymer candidates that would be suitable for encapsulation of living cells. The combination of sodium alginate, cellulose sulfate, poly (methylene-co-guanidine) hydrochloride, calcium chloride, and sodium chloride was most promising. In parallel, a novel multiloop chamber reactor was developed to control the time of complex formation and to negate gravitational effects such as pancreatic islet sedimentation and droplet deformation during the encapsulation process. Encapsulated rat islets demonstrated glucose-stimulated insulin secretion in vitro, and reversed diabetes in mice. This new capsule formulation and encapsulation system allows independent adjustments of capsule size, wall thickness, mechanical strength, and permeability, which may offer distinct advantages for immunoisolating cells.

Permeability assessment of capsules for islet transplantation.

Despite considerable progress in the development of immunoisolation devices, the optimal permeability of such devices is not known. This limitation stems partly from deficits in knowledge about which molecules should be allowed to traverse the semipermeable membrane and which molecules should be excluded, and also partly from experimental obstacles that have prevented a systematic study of permeability. To determine the optimal permeability of immunoisolation devices, we have created a series of microcapsules (800 microM diameter) that span a broad range of molecular exclusion limits yet are identical in wall thickness and chemical composition. Capsule permeability was precisely defined by two complementary methods--size exclusion chromatography (SEC) and a newly developed methodology to assess permeability of biologically relevant proteins. The entry of interleukin-1 beta-125I was significantly delayed, but not prevented, when the capsule exclusion limit was decreased from 230 kD to 3.2 kD, as determined by SEC with dextran standards. The influx of IgG was as predicted, based on the viscosity radius R eta of IgG and the capsule exclusion limit defined by SEC. Glucose-stimulated insulin secretion by encapsulated pancreatic islets did not differ as capsule permeability was decreased from a molecular exclusion limit of 230 kD to 120 kD. These studies should assist in the design of immunoisolation devices by defining the permeability optimal for cell function and also should be applicable to any cell type or immunoisolation device.

A novel reactor for making uniform capsules.

A novel chemical reactor was designed and developed for the continuous high-rate production of uniform capsules. This reactor helps to control precisely the reaction time between the reacting liquids (anion drops and the cation bath, or vice versa), thereby leading to the formation of uniform capsules with walls of identical thickness. In addition, mild tumbling of the capsules during transit through the reactor ensures that every capsule wall is uniformly thick all around.

A two-step process for controlling the surface smoothness of polyelectrolyte-based microcapsules.

Biocompatibility is one of the crucial requirements to be fulfilled when designing devices for immunoisolation of transplanted cells. The quality of the capsule surface (smoothness/roughness) influences the nature of cell overgrowth on it by immunocytes, which eventually may lead to the transplant failure. A microcapsule has been developed based on the polyelectrolyte complexation of the polyanions sodium alginate and cellulose sulphate with the polycation poly(methylene-co-guanidine), which was successfully tested in rodent animal models. Recently, the principles for controlling the surface smoothness of these capsules has been identified. This paper reports on a two-step process used for production of stable capsules with improved surface properties. The methodology involves separating the process of drop shape recovery and precursor capsule formation from the process of membrane formation by applying a two-reactor design. The multi-loop reactors are connected in series, and the process separation is given by the different composition of cation solutions flowing in each reactor. This process enables one to prepare the microcapsule immunoisolation device, which can differ in the extent of surface roughness and, thus, is suitable for studying the effect of surface morphology of the immunoisolation device on cell overgrowth. The effect of this process on the capsule permeability has also been evaluated.

Evaluation of microcapsule permeability via inverse size exclusion chromatography.

Inverse aqueous size exclusion chromatography (SEC) was adopted to measure the permeability of microcapsules (hollow hydrogel spheres with diameter < 1 mm) using dextran molecular weight standards. Alginate/poly(L-lysine)/alginate microcapsules were chosen as a column substrate. Data from column SEC experiments were verified by kinetic studies of solute size exclusion. The permeability of tested microcapsules was modified by the reaction time with 0.05wt.% poly(L-lysine) (PLL). The exclusion limit of the microcapsules prepared at 5-min reaction time was found to be 100,000, while the microcapsules that were allowed to react with PLL for 20 min became less permeable and their exclusion limit was approximately 50,000. Based on relationships between solute size and molecular weight, the exclusion limits determined with dextrans were converted to the size and approximate molecular weight of protein presumably excluded by the capsular membrane at "ideal" conditions. The results from both column SEC and batch experiments suggest that the standard alginate/PLL/alginate capsules are permeable to immunoglobulins of IgG class. Unlike other techniques which utilize only a limited number of solutes, inverse SEC enables one to examine the capsule permeability to a homologous series of molecular weight standards. Inverse SEC also provides an opportunity to evaluate the properties of a large series of capsules directly by comparing their calibration curves. In addition, undesirable enthalpic effects in permeability studies with globular proteins as test solutes can be minimized or eliminated by using the inert molecular weight standards such as polysaccharides.

New capsule with tailored properties for the encapsulation of living cells.

A new capsule for the encapsulation and transplantation of pancreatic islets has been developed. Five active ingredients are involved in the capsule formation process: high viscosity sodium alginate (SA-HV), cellulose sulfate (CS), poly(methylene-co-guanidine) hydrochloride (PMCG), calcium chloride, and sodium chloride. Complexation reaction exhibits several unique features: (1) solution of SA-HV with CS represents a physical mixture of two entangled polyanions that provide both pH-sensitive (carboxylic) and permanently charged (sulfate) groups; (2) presence of CaCl2 in the cation solution ensures formation of the gelled bead after the drop of polyanion solution is immersed in the cation solution; (3) character of the polycation (PMCG), i.e., low molecular weight and unusually high charge density, combines both high mobility and reactivity; (4) presence of PMCG in cation solution, together with CaCl2, gives rise to the competitive binding of these two cations based on their diffusion and affinity towards the anion groups; and (5) NaCl provides the anti-gelling sodium ions that significantly affect the reaction of CaCl2 with the polyanion matrix, thus altering the final properties of the capsule surface, shape, and permeability. The capsule size, mechanical strength, membrane thickness, and permeability can be precisely adjusted and quantified. Detailed information on the permeability aspects is given in another paper by Brissová et al. [J. Biomed. Mater. Sci., 39, 61 (1998)]. The new features concerning capsule processing and testing are presented. We believe that the capsule characteristics can be optimized in the next step to meet the biological criteria. The initial transplantation results suggest that this capsule is biocompatible and noncytotoxic and is a promising candidate for the immunoisolation of cells such as pancreatic islets.

Control and measurement of permeability for design of microcapsule cell delivery system.

Transplantation of immunoisolated islets of Langerhans has been proposed as a promising approach to treating insulin-dependent diabetes mellitus. Recently, a cell delivery system based on a multicomponent microcapsule has been designed for the immunoisolation of insulin-secreting pancreatic islets. The capsule, formed by polyelectrolyte complexation of sodium alginate and cellulose sulfate with poly(methylene-co-guanidine), markedly has improved mechanical strength compared with the widely used alginate/poly(L-lysine) capsules. It also provides a flexibility for readily adjusting membrane thickness and capsule size, and, more important, the membrane permeability can be altered over a wide range of molecular sizes. To rigorously test the capsule diffusion properties, we have improved capsule permeability measurement by using two complementary methods: (1) size exclusion chromatography with dextran standards; and (2) newly developed methodology for assessing permeability to a series of biologically relevant proteins. Viability and function of rat pancreatic islets enclosed in the capsules with different permeability were tested in vitro. The insulin secretion of encapsulated islets was well preserved even though slightly delayed in comparison with a control group of free islets. We believe that the unique features of this encapsulation system together with the precise characterization of its physical parameters will enable us to find the optimal range of capsule permeability for in vitro and in vivo survival and function of encapsulated pancreatic islets.

Atomic force microscopy used for the surface characterization of microcapsule immunoisolation devices.

The surface morphology of the microcapsule used as a bioartificial pancreas was examined by atomic force microscopy (AFM) under ambient conditions in a liquid environment. The standard contact mode was used for imaging. The capsules exhibited different morphologies and surface roughness depending on the composition of the cation solution: namely, the mole ratio of antigelling and gelling cations [Na+]/[Ca2+]. Surface roughness parameters obtained by AFM measurements provide quantitative information on the surface properties of the capsular membrane. In this respect, AFM can be considered a valuable technique complementary to optical microscopy in providing feedback for capsule optimization.