Vascularized porcine liver on-a-chip modeling human xenoimmunity

About This Grant

PROJECT SUMMARY Liver transplantation remains the only treatment available for patients with end-stage organ failure. However, due to the shortage of human donors, patients are waiting progressively longer to receive a transplant, and a larger number are dying every year before an organ is available for transplantation. Porcine liver xenotransplantation (XTx) has the potential to address the current shortage of human donor organs and save the lives of patients with end-stage liver disease. Nonetheless, porcine liver xenograft survival remains extremely limited due to major unresolved clinical problems, including xenogeneic rejections, coagulation dysregulation, and thrombocytopenia. The xenoimmune responses include hyperacute rejection, acute vascular rejection, and chronic rejection, but their mechanisms are not fully elucidated. Despite significant advances in developing genetically modified pigs, a considerable gap in translating liver xenografts to the clinics remains. Pig-to-human liver XTx is still experimental and mainly studied using NHP, ex vivo perfusion, or simple 2D in vitro co-culture models. These methods present significant challenges to understanding pig-to-human liver XTx due to their costs, limited human physiological relevance, and clinical translation. Various innovative human organ-on-a-chip (OoC) models mimicking in vivo organ structures and functions have been developed, but they have never been used to model pig-to-human liver XTx. Therefore, the overarching goal of this proposal is to develop a vascularized porcine liver on-a-chip (vPLoC) platform capable of modeling human xenoimmunity towards porcine liver xenografts. Modeling liver XTx on-a-chip requires a microfluidic device that mimics the physiological function of in vivo pig liver tissues, including a functional microvasculature. The pig endothelium is the first contact site with the human immune system and is a crucial determinant in xenograft rejection. Despite recent OoC models including microvasculatures, no vPLoC platforms featuring perfusable porcine liver lobules have been established to study the mechanisms of xenoimmune responses. To bridge this gap, we propose to develop a functional vPLoC platform that models humoral and cellular xenoimmune responses. The purpose is to demonstrate whether the level of genetic modification will be correlated to the level of humoral and cellular xenoimmune-based injury. First, we will develop a microfluidic-based device in which primary porcine liver cells (hepatocytes, stellate cells, and ECs) from wild-type (WT) and genetically modified (KO) pig livers with different levels of genetic modification will be embedded in fibrin and co-cultured to generate vascularized and perfusable pig liver tissue. Then, we will model xenoimmune responses by perfusing human xenoantibodies and human immune cells through the vPLoC model. Humoral and cellular xenorejection markers will be assessed and correlated with the nature and level of genetic modification of the pig liver cells. This novel platform can significantly impact xenotransplant immunology research while accelerating the understanding of human xenoimmune responses to pigs.