Alginate-poly-L-lysine (PLL) microcapsules can be used for transplantation of insulin-producing cells for treatment of type I diabetes. In this work we wanted to study the inflammatory reactions against implanted microcapsules due to PLL. We have seen that by reducing the PLL layer, less overgrowth of the capsule is obtained. By incubating different cell types with PLL and afterwards measuring cell viability with MTT, we found massive cell death at concentrations of PLL higher than 10 microg/ml. Staining with annexin V and propidium iodide showed that PLL induced necrosis but not apoptosis. The proinflammatory cytokine, tumor necrosis factor (TNF), was detected in supernatants from monocytes stimulated with PLL. The TNF response was partly inhibited with antibodies against CD14, which is a well-known receptor for lipopolysaccharide (LPS). Bactericidal permeability increasing protein (BPI) and a lipid A analogue (B-975), which both inhibit LPS, did not inhibit PLL from stimulating monocytes to TNF production. This indicates that PLL and LPS bind to different sites on monocytes, but because they both are inhibited by a p38 MAP kinase inhibitor, they seem to have a common element in the signal transducing pathway. These results suggest that PLL may provoke inflammatory responses either directly or indirectly through its necrosis-inducing abilities. By combining soluble PLL and alginate both the toxic and TNF-inducing effects of PLL were reduced. The implications of these data are to use alginate microcapsules with low amounts of PLL for transplantation purposes.

Alginate-polylysine-alginate capsules containing insulin-producing cells have been used as a bio-artificial pancreas in the treatment of diabetes mellitus. In a search for microcapsules with improved diffusion characteristics, a high voltage system was developed that produces 250,000 beads/min with a diameter of 160 microm +/- 3-5%. The diameter of the beads could be varied between 160-700 microm depending on the needle diameter and construction, the voltage, the distance between the electrodes and the flow of alginate solution. Ca-alginate beads with diameters of 200 and 500 microm were produced by the high voltage electrostatic system. The 200 microm beads were sensitive to poly-L-lysine (PLL) exposure and had to be washed in ion-free solution to avoid collapse. The 200 microm beads swelled more than the 500 microm beads in the washing and PLL treatment. Also, the porosity of the capsules changed with size, but capsules impermeable to tumour necrosis factor (TNF) could be made by exchanging PLL with poly-D-lysine (PDL) for the 500 microm beads. The 200 microm beads were impermeable to IgG after PLL exposure. Islets of Langerhans were encapsulated in alginate-PLL-alginate capsules and evaluated by measuring protruding islets and insulin production. Islets in microcapsules made by the high voltage electrostatic system did not function differently from islets in larger microcapsules made by an air jet system. In conclusion, alginate capsules made by a high voltage electrostatic system enable large-scale production of small capsules with a narrow size distribution that can meet the functional properties of larger capsules by small changes in the encapsulation procedure.

Background. An active device that downregulates abdominal vagal signalling has resulted in significant weight loss in feasibility studies. Objective. To prospectively evaluate the effect of intermittent vagal blocking (VBLOC) on weight loss, glycemic control, and blood pressure (BP) in obese subjects with DM2. Methods. Twenty-eight subjects were implanted with a VBLOC device (Maestro Rechargeable System) at 5 centers in an open-label study. Effects on weight loss, HbA1c, fasting blood glucose, and BP were evaluated at 1 week to 12 months. Results. 26 subjects (17 females/9 males, 51 ± 2 years, BMI 37 ± 1 kg/m2, mean ± SEM) completed 12 months followup. One serious adverse event (pain at implant site) was easily resolved. At 1 week and 12 months, mean excess weight loss percentages (% EWL) were 9 ± 1% and 25 ± 4% (P < 0.0001), and HbA1c declined by 0.3 ± 0.1% and 1.0 ± 0.2% (P = 0.02, baseline 7.8 ± 0.2%). In DM2 subjects with elevated BP (n = 15), mean arterial pressure reduced by 7 ± 3 mmHg and 8 ± 3 mmHg (P = 0.04, baseline 100 ± 2 mmHg) at 1 week and 12 months. All subjects MAP decreased by 3 ± 2 mmHg (baseline 95 ± 2 mmHg) at 12 months. Conclusions. VBLOC was safe in obese DM2 subjects and associated with meaningful weight loss, early and sustained improvements in HbA1c, and reductions in BP in hypertensive DM2 subjects. This trial is registered with ClinicalTrials.gov NCT00555958.

Microencapsulation of genetically engineered cells may have important applications as delivery systems for therapeutic proteins. However, optimization of the microcapsules with regard to mechanical stability, cell growth, and secretion of proteins is necessary in order to evaluate the future use of this delivery technology. We have explored the growth, survival, and secretion of therapeutic proteins from 293-EBNA cells producing endostatin (293 endo cells) and JJN3 myeloma cells producing hepatocyte growth factor (HGF) that have been embedded in various types of alginate capsules. Parameters that affect capsule integrity such as homogenous and inhomogenous gel cores and addition of an outer poly-L-lysine (PLL)-alginate coating were evaluated in relation to cell functions. When cells were encapsulated, the PLL layer was found to be absolutely required for the capsule integrity. The JJN3 and 293 endo cells displayed completely different growth and distribution patterns of live and dead cells within the microcapsules, as shown by 3D pictures reconstructed from images taken with confocal laser scanning microscopy (CLSM). Encapsulated JJN3 cells showed a bell-shaped growth and HGF secretion curve over a time period of 5 months. The 293 endo cells reached a plateau phase in growth after 23 days postencapsulation; however, after around 30 days a fraction of the microcapsules started to disintegrate. Microcapsule disintegration occurred with time irrespective of capsule and cell type, showing that alginate microcapsules possessing relatively high gel strength are not strong enough to keep proliferating cells within the microcapsules for prolonged time periods. Although this study shows that the stability of an alginate-based cell factory can be increased by a PLL-alginate coating, further improvement is necessary with regard to capsule integrity as well as controlling the cell growth before this technology can be used for therapy.

Alginate/poly-L-lysine(PLL)/alginate capsules are used widely for the microencapsulation of cells. Alginate consists of guluronic acid and mannuronic acid, the ratio and sequence of which affect the properties of the alginate. Using C5-epimerases, mannuronic acid can be converted to guluronic acid in the alginate polymer. Such an enzyme, AlgE4, was used to convert blocks of mannuronic acid (M-blocks) to blocks of alternating sequence (MG-blocks). The aims of this study were 1) to investigate whether the use of epimerized alginate as a coating could improve the biocompatibility of alginate/PLL/alginate capsules and 2) to study the biocompatibility of simple alginate beads prepared with epimerized alginate. Four different capsules, two of which contained epimerized alginate, were investigated after implantation in C57BL/6 mice for 1 week. The biocompatibility of alginate/PLL/alginate capsules, as measured by retrieval rates of the capsules and DNA contents and glucose oxidation rates of the cellular overgrowth, was improved when an epimerized coating alginate was used. There were, however, no statistically significant differences in the biocompatibility of simple alginate beads made from epimerized alginate when compared with non-epimerized alginate beads. In general, such beads produced without a PLL coating swelled to a higher extent than the conventional alginate/PLL/alginate capsules. In conclusion, the use of an epimerized coating on alginate-PLL-alginate can improve the biocompatibility of such capsules but still cannot completely eliminate the detrimental effects of PLL on the biocompatibility of the capsules.