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BRAIN Publication-derived

Modeling HIV-1 Neuropathogenesis Using Three-Dimensional Human Brain Organoids (hBORGs) with HIV-1 Infected Microglia

Source dos Reis et al., 2020 · University of Pittsburgh, Department of Infectious Diseases and Microbiology, Graduate School of Public Health · 10.1038/s41598-020-72214-0

👤 Roberta S. dos Reis, Shilpa Sant, Hannah Keeney, Marc C. E. Wagner, Velpandi Ayyavoo ⏱ 180 days 📋 8 phases 🧫 Human fetal cortical NPCs (18-20 weeks gestation)

Abstract

This protocol describes the generation of three-dimensional human brain organoids (hBORGs) from fetal neural progenitor cells that differentiate into neurons and astrocytes. The method incorporates HIV-1-infected primary human microglia to model the neuroinflammatory microenvironment of HIV-associated neurocognitive disorder (HAND), recapitulating synaptodendritic damage, neuronal loss, and pro-inflammatory cytokine release observed in HIV-infected brains.

Cell source
Human fetal cortical NPCs (18-20 weeks gestation)
Application
Disease modeling - HIV-1 neuropathogenesis

Protocol overview

43 steps across 8 phases

Isolation and 2D Differentiation of NPCs Days 0-42
  1. 1 Tissue Dissociation and NPC Isolation
  2. 2 Expand NPCs in Suspension Culture
  3. 3 Prepare Mixed Differentiation Media for 2D Cultures
  4. 4 Plate NPCs and Begin 2D Mixed Differentiation
Fabrication of Hydrogel Microwell Arrays and Neurosphere Generation Days 0-4
  1. 1 Fabricate PDMS Molds
  2. 2 Generate Non-Adhesive PEGDMA Microwell Arrays
  3. 3 Sterilize Hydrogel Devices
  4. 4 Seed NPCs into Microwell Arrays to Form Neurospheres
  5. 5 Culture Neurospheres Until Compaction
Differentiation of Neurospheres into Brain Organoids Days 4-28
  1. 1 Prepare Four Treatment Conditions
  2. 2 Apply Matrigel Overlay to Specified Groups
  3. 3 Monitor Morphological Changes Days 1-14
  4. 4 Change Half of Culture Media Every Other Day
  5. 5 Confirm Differentiation by qRT-PCR and Immunofluorescence (Day 14)
Characterization of Mature hBORGs Days 14-180
  1. 1 Assess Neuronal and Glial Markers at Multiple Timepoints
  2. 2 Confirm Neuronal Lineage Identity
  3. 3 Assess Synaptic Maturation
  4. 4 Monitor Cell Viability Over Extended Culture
Production of HIV-1 Stocks and Microglia Infection Days 0-3 (virus preparation); Days 0-3 (microglia infection)
  1. 1 Prepare HIV-1 Viral Stocks
  2. 2 Harvest and Concentrate Viral Particles
  3. 3 Titer Viral Stocks
  4. 4 Infect Microglia with HIV-1
  5. 5 Maintain Infected Microglia Until EGFP Expression
Incorporation of HIV-Infected Microglia into hBORGs (MG-hBORG Model) Days 15-45 of organoid culture
  1. 1 Prepare Labeled Microglia
  2. 2 Label hBORGs with Hoechst
  3. 3 Establish Optimal Microglia Density
  4. 4 Co-Culture Microglia with hBORGs Without Agitation
  5. 5 Transfer MG-hBORGs to Fresh Culture Medium
  6. 6 Maintain MG-hBORG Cultures
  7. 7 Monitor Microglia Infiltration by Confocal Microscopy
Assessment of Viral Replication and Inflammatory Response in MG-hBORGs Days 15-45 of organoid culture (post-microglia incorporation)
  1. 1 Extract RNA and Quantify HIV gag mRNA by qRT-PCR
  2. 2 Prepare cDNA from Isolated RNA
  3. 3 Perform qRT-PCR for HIV-1 gag mRNA Quantification
  4. 4 Measure Infectious Viral Particles in Culture Supernatants
  5. 5 Measure TNF-α and IL-1β Cytokine Release by ELISA
Assessment of HIV-1-Induced Neuronal Damage and Pathological Changes Days 15-45 of organoid culture (post-microglia incorporation)
  1. 1 Assess Cell Viability and Cytotoxicity by Live/Dead Staining
  2. 2 Quantify Cell Death by LDH Release Assay
  3. 3 Assess Neuronal Loss by qRT-PCR
  4. 4 Assess Astrocytosis by qRT-PCR
  5. 5 Confirm Microglial Persistence by qRT-PCR
  6. 6 Assess Synaptic Damage by Immunofluorescence
  7. 7 Quantify Synaptic Marker Intensity
  8. 8 Assess Synaptic Contact Loss by PSD95/SYN Co-localization

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in dos Reis et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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