Abstract HIV-1 infects resident cells of the central nervous system (CNS) leading to neuropathogenesis. HIV- neuropathogenesis is likely caused by direct and indirect viral and host factors. However, the exact underlying mechanisms remain unclear. Despite the success of antiretroviral therapy (ART) in suppressing HIV replication, near half of people living with HIV (PLWH) still have varying degrees of HIV-associated neurological disorders (HAND). There is also evidence that the CNS serves as an HIV reservoir and sanctuary site that may allow low level viremia, contributing to persistent neuroinflammation. The evolving molecular events underlying HIV neuropathogenesis are difficult to delineate, partially due to the lack of realistic HIV animal models and because human brain tissues rarely become available for studies until patients die, often due to advanced diseases. Human brain cortical organoids (BCO) are an emerging, cutting-edge technology for studying neuropathological disorders; because of their human origin, they better match the genomic and structural features of the developing human brain compared to animal models. This model consists of a self-assembled dynamic 3-D structure that provides an interplay of different cell types, which is limited in traditional monolayer cultures. We optimized protocols to generate long-term viable and functional BCO. Our BCO model has an unprecedented cell type diversity, via a dynamic development from progenitor cells to neuronal cells, that become interspersed with quiescent astrocytes over time; a difficult phenotype to obtain ex vivo. With the cellular components for generation of a functional neural network in place, our BCO model shows a robust extracellular electrical activity at early stages and progressively develops into an organized oscillatory network. Additionally, we have previously established methods for integration of iPSC-derived microglia into the BCO forming an assembloid, which is crucial tor this study. A BCO assembloid model containing relevant immune cell types will enable susceptibility to HIV and the study of the contributions of different cell types to the neurological consequences of infection. Using this robust and functional BCO assembloid, we propose to develop a new human model to study the cellular and molecular mechanisms underlying HIV neuropathogenesis, and the potential interactive, additive, or synergistic effects of antiretroviral treatment (ART) and opioid exposure. This microglia-infused BCO with endogenous astrocytes will allow HIV infection and its related pathophysiological events and help to disentangle the contribution and interplay of relevant immune cells to neuropathogenesis.