Abstract
We have established and optimized a robust protocol to isolate, culture, and study primary human brain myeloid cells (BrMCs) from rapid-autopsy postmortem brain tissues obtained from people with or without HIV-1 (HIV), in which >97.3% of BrMCs are brain microglia (MG). Therefore, we refer to these isolated BrMCs as MG or BrMCs/MG to better represent the cell populations. This method enables comprehensive ex vivo investigation of microglial biology, including their roles in maintaining HIV reservoirs in the brain, mediating neuroinflammation, and contributing to neuronal injury, as well as the evaluation of HIV cure strategies. To preserve cellular viability and RNA/protein integrity, brain tissues were collected within 6 h after death, following rapid research autopsy. Multiple regions, including the frontal, parietal, and occipital cortices, the hippocampus, and the basal ganglia, were processed using enzymatic digestion and controlled mechanical dissociation, followed by density gradient centrifugation to generate viable single-cell suspensions with efficient removal of myelin debris and erythrocyte lysis. BrMCs/MG were then purified using a two-step isolation approach, consisting of CD3⁺ T cell depletion followed by CD11b⁺ magnetic bead enrichment to ensure purity and specificity. Purified myeloid cells were cultured under defined conditions supplemented with M-CSF to maintain viability and functional integrity during prolonged ex vivo culture. This optimized platform provides a reliable, scalable platform for downstream molecular, immunological, and virological analyses of brain-resident HIV reservoirs and related mechanisms in BrMCs/MG.