Characterisation and production of pancreatic islet extracellular matrix for sustaining islet function after isolation

Abstract

Loss of extracellular matrix (ECM) is known to result in low pancreatic islet survival rates post-isolation and result in poor transplantation outcomes in the treatment of diabetes. Several approaches to ECM supplementation have been used to help improve islet longevity, however, long-term culture of isolated islets is currently unachievable and rates of clinical insulin independence from islet transplantation is still less than 50%. The causes of this are multi-faceted, however, the kind of ECM components that constitutes a healthy islet is still not known. Healthy, adult rodent ECM was characterized by immunohistochemistry and immunoblotting. The basement membrane was distributed throughout the islet in a pattern that was tightly associated with the interweaving endothelium. In contrast, heparan sulfate (HS) was the predominant glycosaminoglycan found in the islet and was mainly on or in the beta-cells. The novel localization of the HS proteoglycan, syndecan-4, showed a similar distribution pattern to the HS within the rodent islet, thus suggesting that the islet HS is attached to syndecan-4. Human umbilical cord vein endothelial cells (HUVECs) were found to be capable of producing ECM components that included collagen IV, fibronectin, laminin, perlecan and HS. Macromolecular crowding enhanced the deposition of ECM components by 2-6 fold. These results showed that HUVECs could produce ECM similar in composition to those observed in healthy, adult islets and macromolecular crowding could increase the quantity of these deposited components. The crowded and decellularized HUVEC ECM facilitated the maintenance of glucose stimulated insulin secretion and did not increase the occurrence of apoptosis and necrosis in the MIN6 beta-cell line more than that of tissue culture plastic. The crowded HUVEC ECM extended the life of primary islets by maintaining hormone expression and genetic expression of important -cell-specific markers. In addition to this, primary islets cultured on the crowded HUVEC ECM tended to maintain their spherical morphology. This work demonstrated that in vitro endothelial cells can simulate ECM produced by islet vasculature. Furthermore, enhancing ECM by macromolecular crowding can produce better quality and quantity of ECM to support and maintain islet function and structure in extended culture. These methods can be used to increase longevity of islets in culture and better prepare isolated islets for transplantation.