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

摘要

Loss of extracellular matrix (ECM) is known to result in low pancreatic islet survivalrates post-isolation and result in poor transplantation outcomes in the treatment ofdiabetes. Several approaches to ECM supplementation have been used to helpimprove islet longevity, however, long-term culture of isolated islets is currentlyunachievable and rates of clinical insulin independence from islet transplantation isstill less than 50%. The causes of this are multi-faceted, however, the kind of ECMcomponents that constitutes a healthy islet is still not known. Healthy, adult rodentECM was characterized by immunohistochemistry and immunoblotting. Thebasement membrane was distributed throughout the islet in a pattern that was tightlyassociated with the interweaving endothelium. In contrast, heparan sulfate (HS) wasthe predominant glycosaminoglycan found in the islet and was mainly on or in thebeta-cells. The novel localization of the HS proteoglycan, syndecan-4, showed asimilar distribution pattern to the HS within the rodent islet, thus suggesting that theislet HS is attached to syndecan-4. Human umbilical cord vein endothelial cells(HUVECs) were found to be capable of producing ECM components that includedcollagen IV, fibronectin, laminin, perlecan and HS. Macromolecular crowdingenhanced the deposition of ECM components by 2-6 fold. These results showed thatHUVECs could produce ECM similar in composition to those observed in healthy,adult islets and macromolecular crowding could increase the quantity of thesedeposited components.The crowded and decellularized HUVEC ECM facilitated the maintenance ofglucose stimulated insulin secretion and did not increase the occurrence of apoptosisand 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 hormoneexpression and genetic expression of important -cell-specific markers. In additionto this, primary islets cultured on the crowded HUVEC ECM tended to maintaintheir spherical morphology. This work demonstrated that in vitro endothelial cellscan simulate ECM produced by islet vasculature. Furthermore, enhancing ECM bymacromolecular crowding can produce better quality and quantity of ECM tosupport and maintain islet function and structure in extended culture. These methodscan be used to increase longevity of islets in culture and better prepare isolated isletsfor transplantation.