Device design and materials optimization of conformal coating for islets of Langerhans

Encapsulation of islets of Langerhans may represent a way to transplant islets in the absence of immunosuppression. Traditional methods for encapsulation lead to diffusional limitations imposed by the size of the capsules (600-1,000 m in diameter), which results in core hypoxia and delayed insulin secretion in response to glucose. Moreover, the large volume of encapsulated cells does not allow implantation in sites that might be more favorable to islet cell engraftment. To address these issues, we have developed an encapsulation method that allows conformal coating of islets through microfluidics and minimizes capsule size and graft volume. In this method, capsule thickness, rather than capsule diameter, is constant and tightly defined by the microdevice geometry and the rheological properties of the immiscible fluids used for encapsulation within the microfluidic system. We have optimized the method both computationally and experimentally, and found that conformal coating allows for complete encapsulation of islets with a thin (a few tens of micrometers) continuous layer of hydrogel. Both in vitro and in vivo in syngeneic murine models of islet transplantation, the function of conformally coated islets was not compromised by encapsulation and was comparable to that of unencapsulated islets. We have further demonstrated that the structural support conferred by the coating materials protected islets from the loss of function experienced by uncoated islets during ex vivo culture

Fibrin gels engineered with pro-angiogenic growth factors promote engraftment of pancreatic islets in extrahepatic sites in mice

With a view toward reduction of graft loss, we explored pancreatic islet transplantation within fibrin matrices rendered pro-angiogenic by incorporation of minimal doses of vascular endothelial growth factor-A165 and platelet-derived growth factor-BB presented complexed to a fibrin-bound integrin-binding fibronectin domain. Engineered matrices allowed for extended release of pro-angiogenic factors and for their synergistic signaling with extracellular matrix-binding domains in the post-transplant period. Aprotinin addition delayed matrix degradation and prolonged pro-angiogenic factor availability within the graft. Both subcutaneous (SC) and epididymal fat pad (EFP) sites were evaluated. We show that in the SC site, diabetes reversal in mice transplanted with 1,000 IEQ of syngeneic islets was not observed for islets transplanted alone, while engineered matrices resulted in a diabetes median reversal time (MDRT) of 38 days. In the EFP site, the MDRT with 250 IEQ of syngeneic islets within the engineered matrices was 24 days, versus 86 days for islets transplanted alone. Improved function of engineered grafts was associated with enhanced and earlier (by day 7) angiogenesis. Our findings show that by engineering the transplant site to promote prompt re-vascularization, engraftment and long-term function of islet grafts can be improved in relevant extrahepatic sites

CCL21 Expression in β-Cells Induces Antigen-Expressing Stromal Cell Networks in the Pancreas and Prevents Autoimmune Diabetes in Mice

Tumors induce tolerance toward their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, nonobese diabetic (NOD) mice express CCL21 in pancreatic β-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes. Ins2-CCL21 mice develop TLOs by 4 weeks of age, consisting of naive CD4 T cells compartmentalized within networks of CD45 gp38 CD31 fibroblastic reticular cell (FRC)-like cells. Importantly, 12-week-old Ins2-CCL21 TLOs contained FRC-like cells with higher contractility, regulatory, and anti-inflammatory properties and enhanced expression of β-cell autoantigens compared with nontransgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and a higher proportion of regulatory T cells in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for the regulation of autoimmunity, and although the effect is systemic, the induction is mediated locally likely by lymphocyte trafficking through TLOs. Overall, our findings suggest that CCL21 promotes TLOs that differ from inflammatory TLOs found in type 1 diabetic islets in that they resemble lymph nodes, contain FRC-like cells expressing β-cell autoantigens, and are able to induce systemic and antigen-specific tolerance leading to diabetes prevention