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BRAIN Publication-derived

A human-derived 3D brain organoid model to study JC virus infection

Source Barreras et al., 2022 · Johns Hopkins University · 10.1007/s13365-022-01062-7

👤 Paula Barreras, David Pamies, Maria Chiara Monaco, Laura S. Muñoz, Xiali Zhong, Eugene O. Major, Helena T. Hogberg, Thomas Hartung, Carlos A. Pardo ⏱ 56 days 📋 7 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human-derived 3D brain organoids containing neurons, astrocytes, and oligodendrocytes, and their infection with JC virus (JCV) for studying progressive multifocal leukoencephalopathy (PML) pathogenesis. The organoids sustain productive JCV infection for up to 3 weeks, providing a human tissue model suitable for investigating viral pathogenic mechanisms and screening therapeutic compounds.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

36 steps across 7 phases

iPSC Generation and Culture Days 1–42
  1. 1 Reprogram fibroblasts to iPSC
  2. 2 Culture iPSCs on irradiated mouse embryonic fibroblasts
  3. 3 Passage iPSCs using collagenase
Differentiation to Neural Progenitor Cells (NPCs) Days 1–14 (after iPSC establishment)
  1. 1 Differentiate iPSCs into neural progenitor cells
  2. 2 Expand NPCs on poly-L-ornithine and laminin-coated flasks
Brain Organoid Formation and Differentiation Days 1–56 (2 days in NPC medium + 8 weeks in differentiation medium)
  1. 1 Prepare NPC suspension for organoid formation
  2. 2 Plate cells in non-treated 6-well plates under gyratory shaking
  3. 3 Culture in NPC medium for 2 days
  4. 4 Switch to differentiation medium
  5. 5 Culture under constant gyratory shaking for up to 8 weeks
JC Virus Exposure (Infection Protocol) Week 5 post-organoid formation (Day 1 of viral exposure protocol)
  1. 1 Prepare JCV-MAD4 isolate or JCV(+) CSF for infection
  2. 2 Expose organoids to JCV-MAD4 isolate or JCV(+) CSF
  3. 3 Maintain organoids under gyratory shaking for 48 hours
  4. 4 Remove virus inoculum and replace with fresh media
  5. 5 Maintain culture and harvest at designated timepoints
  6. 6 Collect media supernatant for viral quantification
Nucleic Acid Extraction and Multiplex qPCR Assay Post-harvest (Days 0, 7, 14, 21 post-exposure)
  1. 1 Resuspend organoid cell pellets in normal saline
  2. 2 Extract DNA using QIAamp MinElute Virus Spin kit
  3. 3 Prepare qPCR master mix
  4. 4 Set up real-time qPCR reactions
  5. 5 Run real-time PCR program
  6. 6 Calculate and analyze viral load
Immunocytochemistry and Confocal Imaging Post-harvest (Days 0, 7, 14, 21 post-exposure)
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Prepare blocking solution
  3. 3 Incubate with primary antibodies
  4. 4 Wash and incubate with secondary antibodies
  5. 5 Counterstain nuclei and mount slides
  6. 6 Acquire confocal microscopy images
  7. 7 Analyze images and quantify infected cells
Electron Microscopy Post-harvest (Days 0, 7, 14, 21 post-exposure)
  1. 1 Prepare samples for electron microscopy
  2. 2 Post-fix with osmium and stain with uranyl acetate
  3. 3 Dehydrate in graded ethanol series
  4. 4 Embed in Embed 812 resin
  5. 5 Section and prepare grids
  6. 6 Stain grids with uranyl acetate and lead citrate
  7. 7 Examine with electron microscope

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Barreras et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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