Development and characterization of a 3D printed implantable niche with local immunosuppression for cell transplantation

Cell transplantation is an attractive strategy to treat endocrine disorders as it is a dynamic and stimulus-responsive system where cells serve as sensors that secrete molecules in response to their environment to maintain homeostasis. The main challenge of cell encapsulation is attaining appropriate vascularization of the graft, which is imperative for preserving cell function and viability, while maintaining sufficient immune evasion to ensure graft survival. To meet this need, we developed a 3D-printed cell encapsulation system comprised of a cell reservoir separated from a drug reservoir by nanoporous membranes for sustained local release of immunosuppressant with loading ports for transcutaneous drug and cell loading. The device was 3D printed through selective laser sintering using biocompatible polyamide (nylon). Two 100 nm pores nylon membrane were assembled between the reservoirs and nylon meshes were used to separate the cell reservoir from the subcutaneous environment, having the goal to retain the cells whilst allowing blood vessel and tissue ingrowth. The device was characterized to verify the meeting of the requirements of an encapsulation platform such as mechanical stability, biocompatibility, scalability, clinical feasibility and utility, efficient mass transport and immune system evasion. Several in vitro and in vivo experiments were performed leading to an animal experiment carried to verify the overall efficacy of the device. To maximize tissue growth and vascularization, immediately prior to implantation, a mesenchymal stem cell-pluronic acid hydrogel was loaded inside the cell reservoir. Immune-competent Sprague-Dawley rats were subcutaneously implanted with our cell encapsulation system. Three weeks after implantation, a vascularized tissue ingrowth formed within the cell reservoir conducive for cell transplantation. Two days prior to cell transplantation, rats were randomly segregated into five immunosuppression regimes (n=6 per group): 1) no treatment, 2) systemic CTLA4-Ig (300 ug/day i.p.), or local CTLA4-Ig (300 ul loaded in drug reservoir) at 3 different concentration that are 3) low dose (1.45 mg/ml), 4) medium dose (3.4 mg/ml) and 5) high dose (11 mg/ml). Rat Leydig cells were transcutaneously injected through the nylon meshes. Throughout the span of the study, rats in the systemic group received daily CTLA4-IG injections while those in the local groups had the drug reservoir refilled twice. Blood was collected weekly and plasma was used to assess circulating CTLA4-Ig via ELISA. 30 days post-cell injection, all devices were explanted and the tissue contained within the cell reservoir was used for H&E and immunohistochemistry analysis to assess vascularization as well as Leydig cell colonization. 7 weeks post-implantation, H&E staining revealed the cell reservoir was completely filled with vascularized tissue at the time of implant removal. In the local high dose group the local immunosuppression allowed cell survival up to 23 days post cell transplantation whereas in the control group cells died after 7 days, showing also plasma levels of CTLA4-Ig 10-fold lower compared to the systemic group. This study presents a promising cell transplantation strategy using a system that allows for both graft vascularization and local immunosuppression to prevent immune rejection. Overall, our dual reservoir system offers a potential solution for successful long-term cell transplantation to treat endocrine diseases.