PROJECT SUMMARY Organ transplantation remains the best and most cost-effective clinical solution for end-stage organ failure; however, it comes with the burden of lifelong immunosuppression. Although effective in preventing allograft rejection, immunosuppressive drugs may not prevent the development of donor-specific antibodies (DSA). The presence of DSA in transplant recipients is associated with an increased risk of antibody-mediated rejection (ABMR), leading to chronic rejection and allograft failure. DSA is a key component of diagnosing ABMR in kidney, lung, liver, and heart transplants; however, the underlying mechanism is still poorly understood and remains highly variable between transplanted organs. Since DSA are not equally pathogenic, there is an urgent need to identify the specific DSA parameters that increase the risk of ABMR. Conventional transplant research to study the role of DSA in ABMR is performed in clinical settings or animal models but rarely uses in vitro platforms. With the recent passing of the FDA Modernization Act 2.0, organs-on-a-chip (OoC) technologies have gained tremendous attraction for their capability to generate clinically translatable data. Several OoC models of human organs, such as the lungs, heart, and liver, have been manufactured on chips; however, no existing model allows for studying ABMR. Therefore, this proposal aims to develop and validate a multi-organ-on-a- chip (MoC) platform comprised of a vascularized liver-on-a-chip (vLoC) and heart-on-a-chip (vHoC) with a fully integrated microfluidic label-free electrochemical (EC) biosensor system to study the underlying mechanism of ABMR. Modeling ABMR on-a-chip requires a microfluidic device that mimics the physiological function of in vivo tissues. The donor’s endothelium is a crucial determinant in allograft rejection mechanisms because it is the first contact site with the recipient’s immune system. Despite recent OoC models including microvasculatures, no vascularized on-a-chip platforms have been established to study DSA-mediated damage of tissue organoids. To bridge this gap, we propose to develop a functional and automated MoC platform that models and monitors ABMR. The scientific premise is to demonstrate that not all DSA can induce ABMR and chronic rejection equally. First, we will engineer a vLoC and a vHoC model using blood-derived endothelial cells to generate a microvascular bed that mimics the allograft endothelium. Then, we will model ABMR by perfusing allogeneic anti-HLA antibodies (DSA and non-DSA) through the vLoC and vHoC models. ABMR markers will be assessed and correlated with DSA parameters (HLA specificity, epitopes, isotype (IgG and IgM), subclass (1-4), C1q binding ability, and titer). Last, we will develop and validate a fully automated MoC platform for long-term, accurate, label-free ABMR monitoring through the seamless integration of the microfluidic EC biosensors with the vLoC and vHoC. This platform can significantly impact the...