Islet transplantation for the purpose of treating insulin-dependent diabetes is currently limited by several factors, most significantly, islet survival post transplantation. In the following dissertation, a tissue-engineered prevascularized pancreatic encapsulating device (PPED) was designed, developed, and evaluated. Microvessel fragments placed within a 3-dimensional collagen-based matrix produce and secrete vascular endothelial growth factor, and inosculate with the host circulation. Isolated islets placed within collagen gels exhibited four-fold more insulin release in response to glucose stimulation than islets in tissue culture. The insulin released by β-cells in islets encapsulated in collagen exhibited unobstructed diffusion within the collagen gels. Subsequent studies evaluated the ability to create a sandwich comprised of two layers of prevascularized collagen gels around a central collagen gel containing islets. In vitro characterization of the islets within these constructs showed that islets are functional and respond to glucose stimulation. The PPEDs were implanted subcutaneously into SCID mice. Islet survival was assessed after 7, 14, and 28 days. Immunohistochemical analysis was performed on the implants to detect insulin and the presence of intraislet endothelial cells. At all time points, insulin was localized in association with intact and partially dissociated islets. Moreover, cells that exhibited insulin staining were co-localized with intraislet endothelial cells. Lastly, dextran-perfused PPEDs showed host perfusion throughout the implant, including perfusion to structures that are morphologically consistent with pancreatic islets. These data indicate that the PPED enhances islet survival by supporting islet viability, by maintaining intraislet endothelial cells, and by enhancing reperfusion to the islets.

OBJECTIVE: In today’s world, diabetes has become an ever-growing crisis, with no definitive cure yet found. In a report conducted by the American Diabetes Association in March of 2018, it was noted that 1.5 million Americans are diagnosed with diabetes each year (ADA, 2018). Insulin is both a limited and expensive source, with prices of Lispro, a rapid-acting insulin, costing upwards of $306 per 1000 units, to Glargine, a basal-analog insulin, costing $298 per 1000 units (McEwen et. al, 2017). Because of this, many diabetics are left with no alternatives to properly treat their blood sugars and maintain a healthy HbA1c level, a laboratory measure of glucose bound to hemoglobin that indicates a diabetic’s blood sugar over a two to three-month period (Mayo Clinic, 2018). Even with insulin treatment, diabetics can suffer from microvascular complications ranging from nephropathy, retinopathy, or even death thereafter if not properly cared for (Klein et. al, 2005). In turn, many researchers have delved into analyzing and perfecting a potential treatment procedure known as islet transplantation that can serve to eliminate the necessity of insulin injections and pump devices, and replace the beta cells destroyed by complications from Type I diabetes. Islet transplantation is the process of extracting healthy islets from the pancreas of an organ donor, purifying the islets in cell culture media that works to recover islet cells, known as islet isolation, and injecting the isolated islets into diabetic recipients whose beta cells are nonfunctional (Alejandro et. al, 2018). The goal of this procedure is to restore proper function of endogenous islets in the body, which contain beta cells that work to secrete insulin and better regulate the body’s glucose metabolism. While pancreatic islet transplantation can reverse diabetes, the process is inefficient, with many islets lost to hypoxemia before the islets become vascularized (Kumatzu et. al, 2018). We hypothesize that by prevascularizing islets ex-vivo, and using a gelatinous scaffold seeded with endothelial cells, one can avoid ischemic induced loss. This will ensure that islets are delivered the necessary oxygen and nutrients they need in order to restore endogenous function. By inserting this prevascularized device into the subcutaneous space of C57 BL/6 mice, islets can be surrounded by a vast blood network, allowing them to function similarly to when they are in the pancreas. If completed properly, this could ease the difficulty of diabetics continuously having to self-regulate their blood sugar levels by multiple injections of exogenous insulin each day. METHODS: Prior to implanting a prevascularized device into the mouse model, we isolated and purified healthy islets from the pancreases of C57 B/L 6 mouse donors, using the steps outlined in the Edmonton Protocol (Shapiro et. al, 2006). Each device could house approximately 300-400 islets, so about two to three mouse donors were used per vascularized graft implanted. In conjunction with IVIVA Medical, the functionalized, three-dimensional islet graft was created and contained a perfusable vascular bed to better ensure islet survival and improve integration immediately after implantation. Mice were monitored daily to ensure the graft was stable inside the subcutaneous space and to ensure the mice were not experiencing any adverse reactions from the implant. On specified days post-implantation, the graft was explanted from the mouse, along with the surrounding tissue, to analyze the foreign body reaction experienced from the implantation and whether a vascular network formed. The tissue sample was then sent to the Histopathology Department for further processing and analysis. RESULTS: In all three groups in the study, foreign body reactions were expressed by the recruitment and presence of multiple cell lines, including macrophages, dendritic cells, and B and T lymphocytes. While immune cells proliferated, there were limited endothelial cells and islets present post-implantation, indicating the presence of hypoxemia, poor vascular formation, and a potent inflammatory response, ultimately leading to islet dysfunction. CONCLUSION: While prevascularizing the scaffolds helped them better perfuse while in the subcutaneous space, we found that the inflammatory reaction, coupled with improper islet seeding, did not initially lead to islet graft survival. With modifications, we plan to create a stronger vascular network to surround the islet cells that would ensure their durability and survival in the long-term. In utilizing this data, future research can work to better stabilize islet cells, with the end goal of translating this work into human models in the near future.