Isolation of Brain Microglia to Study Persistence of Human Immunodeficiency Virus-1

Nikesh Katuwal; Xiaoyi Li; Yuyang Tang; Guochun Jiang

doi:10.3791/71673

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Method Article

Isolation of Brain Microglia to Study Persistence of Human Immunodeficiency Virus-1

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DOI:

10.3791/71673

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September 18th, 2026

Nikesh Katuwal¹ , Xiaoyi Li¹ , Yuyang Tang^*¹^,² , Guochun Jiang^*¹^,²^,³

¹UNC HIV Cure Center, University of North Carolina at Chapel Hill, ²Division of Infectious Diseases at the Department of Medicine, University of North Carolina at Chapel Hill, ³Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill

^* These authors contributed equally

Summary

This protocol describes the isolation, purification, and culture of highly pure human myeloid cells/microglia from rapid-autopsy postmortem brain tissue obtained from people with and without human immunodeficiency virus-1 (HIV-1). It provides a robust ex vivo platform for investigating myeloid cell/microglial biology, brain HIV reservoirs, neuroinflammation, and HIV cure strategies.

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.

Introduction

Brain myeloid cells (BrMCs)/microglia are the main innate immune cells in the central nervous system (CNS)¹^,²^,³. In healthy conditions, they constantly monitor the brain and help with tasks such as synaptic pruning, clearing debris, and supporting neurons⁴^,⁵^,⁶. When disease is present, microglia are immunologically activated, leading to neuroinflammation, which plays an essential role in neurodegeneration in diverse CNS disorders⁷^,^....