(275a) Injection Molded Hydrogel Design for Macroencapsulated Therapeutic-Delivering Cell Therapies

Allogeneic cell therapies are promising potential treatments for chronic diseases such as type 1 diabetes. Stem cell-derived allogeneic cell sources have shown early promise in clinical trials; however, these strategies have limited patient markets due to immune rejection and the acute risks associated with ineffective and toxic immunosuppressive drug regimens. The potent immune response to allogeneic cell therapies remains the greatest challenge to long-term engraftment and function, and necessitates larger numbers of cells. Methods to eliminate graft rejection without chronic systemic immunosuppression will vastly expand the eligible patient population and reduce the risks associated with allogeneic cell therapy. Additionally, stem cell-derived cell sources pose significant potential safety concerns as evidenced by a recent case study of teratoma formation in an immune competent individual who received stem cell-derived beta cell therapy. Given these potential risks, encapsulation devices that isolate cell grafts from patients and enable full graft retrievability are critical to the safe translation of stem cell-based cell therapies.

Cell encapsulation within a nondegradable biomaterial has long been proposed as a means for reducing immune responses to transplanted grafts via a physical barrier to direct antigen recognition by immune cells, with decades of promising research in preclinical studies; however, translation of this technique has been hampered by poor clinical outcomes. Primary obstacles to clinical translation of cell encapsulation include (1) safety concerns with delivering traditional hydrogel microcapsules to the intraperitoneal (IP) cavity, (2) fibrosis at the capsule surface, limiting nutrient and oxygen transport and reducing long-term graft survival, and (3) indirect antigen recognition of the encapsulated cell graft, leading to delayed graft destruction. As such, macroencapsulation devices for cell encapsulation have been explored in preclinical and clinical studies as they confer the safety benefit of a single, retrievable device, though function is often hampered by poor oxygen and nutrient transport. Addressing these specific limitations facing macroencapsulation devices, we use computational modeling-guided device design for improved oxygen transport, and degradable hydrogel-guided enhanced vascularization at the device surface to maximize oxygen access and mitigate fibrosis. Further, we use synergistic approaches with encapsulation to induce localized tolerance within the cell graft site.

Here, we describe a method to macroencapsulate insulin secreting cells using a hydrogel injection molding-based approach that is amenable to high-throughput biomanufacturing of encapsulated cell products. We evaluate primary islet and stem cell-derived insulin secreting cell function in vitro and in vivo. Finally, we describe efforts to combat indirect antigen recognition of encapsulated cell grafts via the local delivery of soluble factors secreted by tolerogenic cells.

Methods and Results

We first designed alginate-based (1.5% w/v) hydrogel macroencapsulation devices with high surface area-to-volume (SA:V) ratios to maximize oxygen transport to encapsulated cells. Finite element modeling of oxygen transport demonstrated that a spiral geometry design (spiral arm diameter = 1 mm) and 10 islet equivalent (IEQ) per µL loading density resulted in the greatest number of surviving and functional insulin-secreting cells, and significantly improved oxygen concentration within the device relative to lower SA:V gemoetries such as the cylinder (1 mm height). In vitro evaluation of spiral alginate injection molded human islets demonstrated comparable islet survival (live/dead imaging and metabolic activity) and insulin responsiveness to unencapsulated islets.

We next evaluated in vivo function of spiral injection molded insulin secreting cells. We hypothesized that delivery of macroencapsulated islets to a highly vascularized transplant site could result in greater long-term cell function than traditional IP delivery of encapsulated cells, if vasculature could be generated at the alginate surface. We used a syngeneic rat islet omentum transplant model, as rat fibrotic responses are more severe than mouse and closer to non-human primate and human fibrotic responses; additionally, we use a vasculogenic degradable hydrogel at the alginate macroencapsulation device surface to maximize vasculogenesis and local oxygen availability. Transplantation of a marginal islet mass (6000 IEQ/kg), which reversed only 25% of unencapsulated transplant recipients, resulted in comparable 25% reversal in spiral alginate encapsulated islets and 0% reversal in cylindrical encapsulated islets. Additionally, glucose responsiveness was improved in spiral vs. cylinder recipients at 100 days post-transplant, and spiral grafts exhibited robust insulin staining at the experimental endpoint.

Next, we investigated the immune protective capacity of alginate spiral macroencapsulated cells. Luciferase-expressing HLA-A2+ human stem cell-derived beta cell clusters (sBC) were encapsulated or unmodified and transplanted in the omentum-equivalent epididymal fat pad (EFP) of nondiabetic immune compromised NSG mice. Quantitative longitudinal IVIS in vivo imaging of sBCs demonstrates stable equivalent survival of encapsulated or unmodified sBCs out to at least 60 days. To evaluate cell survival in the presence of human immune cells, human HLA-A2-targeting CAR-T cells were adoptively transferred into recipients on day 52 post-transplant. Unencapsulated sBCs exhibited a more significant reduction in luciferase signal (85.3% average signal prior to adoptive transfer, 4.9% average after) relative to encapsulated sBCs (74% average signal prior to adoptive transfer, 19.6% average after). This demonstrates a significant protection from direct antigen recognition, but a significant reduction in signal via indirect antigen recognition.

Finally, we evaluated the capacity of tolerogenic trophoblast-secreted soluble factors to prevent the indirect antigen recognition-mediated destruction of encapsulated cells in a xenogeneic transplant model. Human luciferase-expressing HEK cells were spiral encapsulated and transplanted within the subcutaneous site in immune competent C57BL/6J mice. Quantitative longitudinal IVIS in vivo imaging demonstrated rapid rejection of HEK cells, with 1% of HEK signal remaining by day 14 post-transplant. Conversely, 31.8% of HEK signal remained in recipients that received co-delivery of encapsulated human trophoblast cells.

Implications

Chronic systemic immunosuppression remains the most significant barrier to allogeneic cell therapy translation due to significant acute risks. Methods to eliminate the need for systemic immunosuppression in cell transplantation would vastly widen the eligible patient population. Here we demonstrate that a high-throughput method of cell macroencapsulation can generate complex geometries that improve encapsulated cell survival and function relative to traditional geometries, and reduce immune destruction. Further, we demonstrate that local delivery of tolerogenic factors can synergize with encapsulation to significantly prolong xenogeneic encapsulated cell survival in an immune competent mouse model.