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BRAIN Publication-derived

HIV-Associated Neurocognitive Disorder Brain Organoids with Microglia

Full SOP with Academic or a plan

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

Authors: Roberta S. dos Reis, Shilpa Sant, Hannah Keeney, Marc C. E. Wagner, Velpandi Ayyavoo Duration: 180 days 8 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in dos Reis et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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