Tracing the Fate of Extracellular Matrix Proteins During Human Islet Isolation and Culture

Abstract

Introduction: The precise action of different collagenase blends on the digestion of the peri-islet extracellular matrix (ECM) during human islet isolation is largely unknown. It is also unclear how the remaining ECM of purified islets fares during culture. The presence of certain ECM proteins in purified islets may improve survival and promote islet engraftment. The aims of this pilot study were to characterize 1) the digestion of pancreatic ECM proteins during islet isolation, and 2) their expression in purified islets in culture prior to transplantation. Methods: With appropriate consent and ethical approval, human pancreases were retrieved from multiorgan donors (n = 3, age 44-59, CIT 5-7.5h, BMI 29-31). 0.5cm3 biopsies were taken from the head of the pancreas prior to islet isolation. 1ml samples were taken from the circulation fluid during the digestion phase of islet isolation, and an aliquot of islets taken following purification and after 24h and 48h in culture. Immunofluorescence labelling and Western blotting were used to identify specific ECM proteins. Results: Collagen I, IV, V, VI and Laminin expression were widespread throughout the pancreas, both around and within the islets. Western blotting of samples from the digestion circuit indicate variations in the release of digested fragments of these ECM proteins, most notably Collagen I. ECM proteins were present in purified islets, with Collagen VI expression being the most extensive. The expression of ECM proteins decreased over time in culture, with Laminin expression lost completely from islets by 48h. Conclusion: The total loss of Laminin and the decline of other ECM proteins suggests that the glycoprotein framework of the islet basement membrane is destroyed, raising concerns that islets may fragment following isolation, which could partially account for the reduction in islet yield commonly seen during culture. The reduction in Collagen I would suggest the loss of endothelial cells important for angiogenesis during engraftment. Furthermore, the degradation of Collagen IV may reveal cryptic regions with anti-angiogenic properties.