241.3: Generation of Prevascularized Endocrine Constructs to Treat Type 1 Diabetes

Abstract

Introduction: The goal of our study was to generate a functional, prevascularized endocrine constructs utilizing amniotic membrane derived hydrogel, islets and blood outgrowth endothelial cells (BOECs) to be transplanted in diabetic hosts. Methods: Human amniotic membranes were dissected from placentas decellularized, lyophilized, and solubilized to obtain hydrogels. DNA quantification and scanning electron microscopy showed complete removal of cellular components and preserved extracellular matrix. Characterization studies demonstrated widespread distribution of all essential structural proteins, including collagen, GAGs and laminin. The islets and BOECs were admixed in amnion derived hydrogels and cultured in specially designed media to enhance endothelial cell assembly into tubular, vascular-like structures. Constructs were kept under culture for 3 days. Glucose stimulated insulin secretion (GSIS) tests showed an adequate insulin secretion in response to high glucose stimulation, confirming the functional activity of the prevascularized endocrine constructs. Results: To test in vivo biocompatibility and function, the vascularized constructs generated from 500 IEQ rat islets and 2× 105 human derived BOECs were implanted under the skin of the diabetic NSG mice. Transplantation of the constructs led to rapid and long-term (90 days) normalization of blood glucose levels. Removal of the grafts led to recurrence of hyperglycemia within 24 hours, indicating that the transplanted constructs was responsible for normalized glucose levels. Histological analysis of the explanted grafts revealed healthy islet morphology and perfect revascularization. Grafts stained positive for insulin and human specific CD 31 indicating presence of human endothelial cells in newly formed intra-islet micro vessels. Conclusion: Our findings show that amnion derived hydrogel seeded with endocrine pancreatic tissue and endothelial cells could be used for functional, preveascularized endocrine construct bioengineering. This work is supported by grants from the European Commission (Horizon 2020 Framework Program; VANGUARD grant 874700), the European Foundation for the Study of Diabetes (EFSD), the Juvenile Diabetes Research Foundation (JDRF; grant 3-SRA-2020-926-S-B) and the Shota Rustaveli National Science Foundation (grant FR-19-19760).