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BRAIN Publication-derived

Cerebral Organoids for Infection Studies (2022)

Full SOP with Academic or a plan

Source Gumbs et al., 2022 · University Medical Center Utrecht, Department of Medical Microbiology · 10.3390/v14040829

Authors: Stephanie B. H. Gumbs, Amber Berdenis van Berlekom, Raphael Kübler, Pauline J. Schipper, Lavina Gharu, Marco P. Boks, Paul R. Ormel, Annemarie M. J. Wensing, Lot D. de Witte, Monique Nijhuis Duration: 28 days 8 phases Cell source: Human iPSC

Abstract

This protocol describes the generation and HIV infection of microglia-containing human cerebral organoids derived from induced pluripotent stem cells (iPSC). The organoids support productive HIV infection via the CCR5 co-receptor in intrinsically developed microglia, providing a 3D model to study HIV's molecular mechanisms in the central nervous system, HIV-associated neurocognitive disorders, and therapeutic strategies.

Cell source
Human iPSC
Application
Disease modeling; HIV neuropathogenesis and CNS reservoir study

Protocol overview

57 steps across 8 phases

Phase 1 · Generation of 3D Human Microglia-Containing Cerebral Organoids 0-28+
  1. a Prepare iPSC lines
  2. b Differentiate organoids from iPSC lines
  3. c Supplement medium with retinoic acid
  4. d Place petri dishes on belly dancer shaker
  5. e Perform qualitative selection
Phase 2 · Generation of 2D Human Cerebral Organoid Dissociates Weeks 3-9
  1. a Dissociate 3D organoids with accutase
  2. b Centrifuge and collect pellet
  3. c Plate cells on matrigel-coated 6-well plates
  4. d Maintain 2D cultures for 6 days
Phase 3 · Isolation and Culture of Primary and Organoid-Derived Microglia Weeks 6-9
  1. a Obtain postmortem human brain tissue
  2. b Isolate primary microglia from brain tissue
  3. c Dissociate organoids for microglia extraction
  4. d Perform CD11b magnetic cell sorting
  5. e Culture primary and organoid-derived microglia
Phase 4 · Viral Preparation Days -5 to -1
  1. a Prepare infectious plasmids
  2. b Transfect HEK-293T cells
  3. c Harvest supernatant containing replication-competent virus
  4. d Store viral supernatant
  5. e Determine p24 concentration
Phase 5 · HIV Infection of Microglia and Organoid Models Days 1-15
  1. a Infect primary and organoid-derived microglia
  2. b Replace medium the day after infection
  3. c Culture without medium refreshment
  4. d Optional: Add MVC on day 3 post-infection
  5. e Perform all microglia experiments in duplicate
  6. f Infect 2D organoid dissociates
  7. g Replace medium and continue culture
  8. h Refresh medium weekly
  9. i Perform 2D/3D organoid experiments in duplicate or triplicate
  10. j Prepare matrigel negative control
  11. k Infect 3D cerebral organoids
Phase 6 · Luminescence and Immunofluorescence Analysis Days 2-14 post-infection
  1. a Collect supernatant for luminescence assay
  2. b Perform Nano-Glo Luciferase Assay
  3. c Fix 2D organoid dissociates for immunostaining
  4. d Fix 3D cerebral organoids
  5. e Embed 3D organoids in tissue tek
  6. f Section organoids on cryostat
  7. g Perform immunostaining
  8. h Acquire fluorescence images
Phase 7 · Gene Expression Analysis with Real-Time PCR Days 1-15 post-infection
  1. a Isolate RNA from samples
  2. b Perform RNA quality assessment
  3. c Prepare samples for analysis
  4. d Perform cDNA synthesis
  5. e Perform quantitative Real-Time PCR (qPCR)
  6. f Analyze gene expression data
  7. g Generate graphs
Phase 8 · Single-Cell Sequencing of Cerebral Organoids (SORT-Seq) Week 9-10
  1. a Dissociate organoids for single-cell analysis
  2. b Prepare viability staining
  3. c FACS sort cells into cell capture plates
  4. d Centrifuge and freeze sorted plates
  5. e Perform cell lysis
  6. f Perform cDNA synthesis and second-strand synthesis
  7. g Pool and amplify barcoded cDNA
  8. h Perform library preparation
  9. i Sequence library
  10. j Demultiplex and map sequencing data
  11. k Perform bioinformatics analysis
  12. l Annotate cell types

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Attribution

This SOP was written by Organthis based on the published method in Gumbs et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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