Preservation of human islet function in vitro through EMc reestablishment

Abstract

The microencapsulation of pancreatic islets is a promising treatment for type I diabetes. Although microcapsule biocompatibility has improved, islet cell survival is still problematic. During islet isolation, the extracellular matrix (ECM) around the islet is destroyed, which negatively affects islet function and survival. Also, smaller islets have been shown to have better function and survival. Since encapsulated islets are not re-vascularized, their oxygen supply is entirely dependent upon diffusion. The use of dispersed islet cell re-aggregates of smaller diameter now allows the investigation of microcapsules of further reduced size. aim: The aim of this study is to improve microencapsulated islet cell survival, using three approaches: restoring the ECM loss, reducing the microcapsule diameter from 300 μm to 100 μm and reducing the islet diameter, using islet cell re-aggregates. It is known that dissociated islet cells have the capacity re-assemble in clusters that have the same properties of a normal islet. The first steps were to 1) characterize ECM in islets, after isolation and encapsulation, 2) produce islet cell re-aggregates and 3) evaluate the feasibility of producing 100-150 μm diameter microcapsules. Methods: The presence of ECM molecules (collagen I and IV, fibronectin and laminin) was evaluated by immunohistochemistry in islets that were cultured for different time periods, comparing encapsulated to non-encapsulated islets. Secondly, islet-like clusters were formed after dissociation of islets with trypsin and rotational incubation during 5-6 days. Different conditions were tested to achieve an optimal size. Finally, alginate-poly-L-lysine microcapsules were produced using an electrostatic pulse generator and different fabrication parameters were tested in order to achieve specific capsule diameters. results: Collagen IV and laminin were found to be present in whole pancreases used as a control, but only a minimal amount was found in isolated islets for all of the tested conditions. The other proteins under study were present for all conditions(fibronectin, collagen I). Isletlike clusters were produced and encapsulated in small microcapsules. We were able to produce microcapsules with a diameter of 132,32 ± 6,29 μm by extruding alginate through a 30G needle and reducing the alginate flow rate from 3,5mL/min to 0,3mL/min. Discussion/conclusion: This data suggests that replacing collagen IV and/or laminin in the re-aggregates might be useful. We also showed the feasibility of producing APA microcapsules of approximately 130 μm and of encapsulating islet cell re-aggregates within these microcapsules. Further research will be conducted to determine whether the use of this system improves oxygen supply to islets and islet survival.