Beneficial effects of coating alginate microcapsules with macromolecular heparin conjugates-in vitro and in vivo study.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated pancreatic islets. Modification of the microcapsule membrane aimed at preventing PFO should improve graft survival. This study investigated the effect of macromolecular Corline Heparin Conjugate (CHC) binding on intrinsic properties of alginate microcapsules and assessed the anti-fibrotic potential of this strategy both in vitro and in vivo. CHC was bound to alginate microcapsules using a layer-by-layer approach incorporating avidin. CHC binding to alginate microcapsule was visualized by confocal microscopy. Effects of CHC binding on microcapsule size, strength, and permeability were assessed, and the anti-clotting activity of bound CHC was determined by coagulation assay. Effect of CHC binding on the viability of encapsulated human islets was assessed in vitro, and their ability to function was assessed both in vitro and in vivo in diabetic immunodeficient mice. The potential of bound CHC to reduce PFO was assessed in vivo in different rat transplantation models. Confocal microscopy demonstrated a uniform coating of CHC onto the surface of microcapsules. CHC binding affected neither size nor permeability but significantly increased the tensile strength of alginate microcapsules by ~1.3-fold. The bound CHC molecules were stable and retained their anti-clotting activity for 3 weeks in culture. CHC binding affected neither viability nor function of the encapsulated human islets in vitro. In vivo CHC binding did not compromise islet function, and diabetes was reversed in all recipients with mice exhibiting lower blood glucose levels similar to controls in oral glucose tolerance tests. CHC binding was beneficial and significantly reduced PFO in both syngeneic and allogeneic rat transplantation models by ~65% and ~43%, respectively. In conclusion, our results show a new method to successfully coat CHC on alginate microcapsules and demonstrate its beneficial effect in increasing capsule strength and reduce PFO. This strategy has the potential to improve graft survival of encapsulated human islets.

The induction of cytokines by polycation containing microspheres by a complement dependent mechanism.

The cytokine-inducing potential of various microspheres were evaluated in a short-time screening assay of lepirudin-anticoagulated human whole blood utilizing the Bio-Plex Human cytokine 27-plex system. The inflammatory cytokines IL-1ß, TNF and IL-6; the anti-inflammatory mediators IL-1ra and IL-10; the chemokines IL-8, MIP-1α and MCP-1; and the growth factor VEGF were induced by polycation (poly-l-lysine or poly(methylene-co-guanidine)) containing microspheres. Alginate microspheres without polycations did not induce the corresponding cytokine panel, nor did soluble alginate. By inhibiting complement C3 using compstatin analog CP20, a total inhibition of complement activation as well as the inflammatory mediators was achieved, indicating that complement activation alone was responsible for the induced cytokines. A strong deposition of C3c on the poly-l-lysine containing surface, while not on the microspheres lacking polycations, also points to the formation of C3 convertase as involved in the biomaterial-induced cytokine induction. These results show that complement is responsible for the induction of cytokines by polycation containing microspheres. We point to complement as an important initiator of inflammatory responses to biomaterials and the lepirudin anticoagulated whole blood assay as an important tool to identify the most tolerable and safe materials for implantation to humans.

Survival of human islets in microbeads containing high guluronic acid alginate crosslinked with Ca2+ and Ba2+.

BACKGROUND: The main hurdles to the widespread use of islet transplantation for the treatment of type 1 diabetes continue to be the insufficient number of appropriate donors and the need for immunosuppression. Microencapsulation has been proposed as a means to protect transplanted islets from the host's immune system. METHODS: This study investigated the function of human pancreatic islets encapsulated in Ca(2+) /Ba(2+) -alginate microbeads intraperitoneally transplanted in diabetic Balb/c mice. RESULTS: All mice transplanted with encapsulated human islets (n = 29), at a quantity of 3000 islet equivalent (IEQ), achieved normoglycemia 1 day after transplantation and retained normoglycemia for extended periods of time (mean graft survival 134 ± 17 days). In comparison, diabetic Balb/c mice transplanted with an equal amount of non-encapsulated human islets rejected the islets within 2 to 7 days after transplantation (n = 5). Microbeads retrieved after 232 days (n = 3) were found with little to no fibrotic overgrowth and contained viable insulin-positive islets. Immunofluorescent staining on the retrieved microbeads showed F4/80-positive macrophages and alpha smooth muscle actin-positive fibroblasts but no CD3-positive T lymphocytes. CONCLUSIONS: The Ca(2+) /Ba(2+) -alginate microbeads can protect human islets from xenogeneic rejection in immunocompetent mice without immunosuppression. However, grafts ultimately failed likely secondary to a macrophage-mediated foreign body reaction.

Alginate microbeads are complement compatible, in contrast to polycation containing microcapsules, as revealed in a human whole blood model.

Alginate microbeads and microcapsules are presently under evaluation for future cell-based therapy. Defining their inflammatory properties with regard to humans is therefore essential. A lepirudine-based human whole blood model was used as an inflammation predictor by measuring complement and leukocyte stimulation. Alginate microbeads were complement-compatible since they did not activate complement as measured by the soluble terminal complement complex (sTCC), Bb or the anaphylatoxins C3a and C5a. In addition, alginate microbeads were free of surface adherent leukocytes. In contrast, microcapsules containing poly-L-lysine (PLL) induced elevated levels of sTCC, Bb, C3a and C5a, surface active C3 convertase and leukocyte adhesion. The soluble PLL induced elevated levels of sTCC and up-regulated leukocyte CD11b expression. PMCG microcapsules containing poly(methylene-co-guanidine) complexed with sodium alginate and cellulose sulfate triggered a fast sTCC response and C3 deposition. The PMCG microcapsules were still less activating than PLL-containing microcapsules as a function of time. The amounts of anaphylatoxins C3a and C5a were diminished by the PMCG microcapsules, whereas leukocyte adherence demonstrated surface activating properties. We propose the whole blood model as an important tool for measuring bioincompatibility of microcapsules and microbeads for future applications as well as determining the mechanisms leading to inflammatory reactions.

A recommended laparoscopic procedure for implantation of microcapsules in the peritoneal cavity of non-human primates.

BACKGROUND: The anatomical spatial distribution of microencapsulated islets transplanted into the peritoneal cavity of large animals remains a relatively unexplored area of study. In this study, we developed a new implantation approach using laparoscopy in order to avoid microcapsule amalgamation. This approach constitutes a clinically relevant method, which can be used to evaluate the distribution and in vivo biocompatibility of various types of transplanted microcapsules in the future. MATERIALS AND METHODS: Two healthy baboons were implanted intraperitoneally with microencapsulated islets through mini-laparotomy and observed at 76 d after implantation. Nine baboons underwent laparoscopic implantation of approximately 80,000 empty microcapsules. Microcapsule distribution was observed by laparoscopic camera during and after implantation at 1, 2, and 4 wk. At each time point, microcapsules were retrieved and evaluated with brightfield microscopy and histologic analysis. RESULTS: Mini-laparotomic implantation resulted in microcapusle aggregation in both baboons. In contrast, laparoscopic implantation resulted in even distribution of microcapsules throughout the peritoneum without sedimentation to the Douglas space in all animals. In eight out of nine animals, retrieved microcapsules were evenly distributed in the peritoneal cavity and presented with no pericapsular overgrowth and easily washed out during laparoscopic procedure. The one exception was attributed to microcapsule contamination with blood from the abdominal wall following trocar insertion. CONCLUSIONS: Laparoscopic implantation of microcapsules in non-human primates can be successfully performed and prevents microcapsule aggregation. Given the current widespread clinical application of laparoscopy, we propose that this presented laparoscopy technique could be applied in future clinical trials of microencapsulated islet transplantation.

Mechanism of ethanol-induced insulin secretion from INS-1 and INS-1E tumor cell lines.

UNLABELLED: Alcohol causes reactive hypoglycemia by attenuating the release of counter regulatory hormones, redistribution of pancreatic blood flow and direct stimulation of insulin secretion. Objective of this study was characterization of ethanol-induced insulin secretion. Signaling of ethanol- and glucose-induced insulin release from INS-1 and INS-1E cells was compared. Both cell lines responded similarly to all experimental interventions. In contrast to glucose, ethanol-induced insulin secretion was not hindered in calcium depleted medium or by addition of 10 microM BAPTA/AM (intracellular chelator). Inhibitor of protein kinase C Bisindolylmaleimide (3 microM) abolished glucose- but not ethanol-induced insulin secretion. Tetanus toxin (20 nM), inhibitor of SNARE proteins complex formation, blocked ethanol-induced insulin secretion. Both 5 mM N-ethylamaleimide and 10 microM ZnCl(2) (inhibitor of protein tyrosine phosphatases), which block disassembly of SNARE complexes and their further participation in exocytosis, increased basal insulin secretion. In contrast to glucose, already high insulin secretion was further increased after ethanol stimulation in either treatment. CONCLUSION: Signaling of ethanol-induced insulin secretion from INS-1 and INS-1E cell lines bypasses calcium and PKC involving steps, is sensitive to tetanus toxin but resistant to N-ethymaleimide and ZnCl(2). An extra pool of secretory vesicles not available for glucose is exploited for exocytosis after ethanol stimulation.

Multiscale requirements for bioencapsulation in medicine and biotechnology.

Bioencapsulation involves the envelopment of tissues or biological active substances in semipermeable membranes. Bioencapsulation has been shown to be efficacious in mimicking the cell's natural environment and thereby improves the efficiency of production of different metabolites and therapeutic agents. The field of application is broad. It is being applied in bioindustry and biomedicine. It is clinically applied for the treatment of a wide variety of endocrine diseases. During the past decades many procedures to fabricate capsules have been described. Unfortunately, most of these procedures lack an adequate documentation of the characterization of the biocapsules. As a result many procedures show an extreme lab-to-lab variation and many results cannot be adequately reproduced. The characterization of capsules can no longer be neglected, especially since new clinical trials with bioencapsulated therapeutic cells have been initiated and the industrial application of bioencapsulation is growing. In the present review we discuss novel Approached to produce and characterize biocapsules in view of clinical and industrial application. A dominant factor in bioencapsulation is selection and characterization of suitable polymers. We present the adequacy of using high-resolution NMR for characterizing polymers. These polymers are applied for producing semipermeable membranes. We present the pitfalls of the currently applied methods and provide recommendations for standardization to avoid lab-to-lab variations. Also, we compare and present methodologies to produce biocompatible biocapsules for specific fields of applications and we demonstrate how physico-chemical technologies such as FT-IR, XPS, and TOF-SIMS contribute to reproducibility and standardization of the bioencapsulation process. During recent years it has become more and more clear that bioencapsulation requires a multidisciplinary approach in which biomedical, physical, and chemical technologies are combined. For adequate reproducibility and for understanding variations in outcome of biocapsules it is advisable if not mandatory to include the characterization processes presented in this review in future studies.