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BRAIN Publication-derived

Alzheimer's Disease Brain Organoids for Infection Studies (2022)

Full SOP with Academic or a plan

Source Lee et al., 2022 · Seoul National University, Konkuk University · 10.1038/s41420-022-00958-x

Authors: Seung-Eun Lee, Hanul Choi, Nari Shin, Dasom Kong, Nam Gyo Kim, Hee-Yeong Kim, Min-Ji Kim, Soon Won Choi, Young Bong Kim, Kyung-Sun Kang Duration: 98 days 12 phases Cell source: Human iPSC (Wild-type), Patient-Derived iPSC (PSEN1 mutation)

Abstract

This protocol describes the generation of 3D cerebral brain organoids from human iPSCs (wild-type and Alzheimer's disease patient-derived), characterization of cortex-like structures, infection with Zika virus (ZIKV), and treatment with PERK inhibitors. The protocol demonstrates that ZIKV infection accelerates AD pathological features including β-amyloid and phosphorylated-Tau accumulation through endoplasmic reticulum stress and PERK-eIF2α pathway activation.

Cell source
Human iPSC (Wild-type), Patient-Derived iPSC (PSEN1 mutation)
Application
Disease modeling, Viral infection study, Alzheimer's disease pathogenesis

Protocol overview

45 steps across 12 phases

Phase 1 · hiPSC Maintenance and Preparation Ongoing, passage every 4-6 days
  1. a Maintain wild-type hiPSCs in Essential 8 medium
  2. b Maintain AD patient-derived hiPSCs in mTESR plus medium
Phase 2 · Embryoid Body Formation Days 0-5
  1. a Dissociate and aggregate iPSCs
  2. b Change medium on day 3
  3. c Transfer embryoid bodies to 24-well plates when diameter exceeds 500 µm
Phase 3 · Neural Induction and Early Organoid Formation Days 5-11
  1. a Culture in neural induction medium
  2. b Embed neuroepithelial tissue in Matrigel
Phase 4 · Organoid Differentiation and Maturation Days 11-75+
  1. a Culture in cerebral organoid differentiation medium (CODM) – stationary phase
  2. b Transfer to orbital shaker with modified CODM
  3. c Monitor organoid size and morphology
Phase 5 · Viral Stock Preparation Prior to infection
  1. a Obtain and propagate ZIKV stock
  2. b Perform plaque assay for ZIKV titration
  3. c Obtain and propagate vaccinia virus (VACV) control stock
Phase 6 · ZIKV and VACV Infection of Brain Organoids Day 60 to 14 dpi (Day 75)
  1. a Prepare organoids for infection
  2. b Infect organoids with ZIKV
  3. c Replace virus-containing medium with fresh CODM
  4. d Maintain infected organoids in culture with regular medium changes
  5. e Collect supernatant at designated time points for plaque assay
Phase 7 · PERK Inhibitor Treatment Day 60 to 14 dpi (Day 75)
  1. a Prepare GSK2656157 (PERK inhibitor) solution
  2. b Infect organoids with ZIKV (0.2 MOI) and treat with PERK inhibitor
  3. c Continue inhibitor treatment for 14 days
Phase 8 · Viral Confirmation by RT-qPCR Days 64, 67, 74 (4 dpi, 7 dpi, 14 dpi)
  1. a Extract total RNA from organoids
  2. b Synthesize complementary DNA (cDNA)
  3. c Perform qRT-PCR for ZIKV NS5
Phase 9 · Protein Analysis by Western Blotting 14 dpi (Day 75)
  1. a Prepare organoid lysates
  2. b Quantify total protein
  3. c Perform SDS-PAGE and transfer
  4. d Block and probe with primary antibodies
  5. e Incubate with secondary antibodies and detect
Phase 10 · Amyloid-Beta ELISA Day 60 (WT/AD characterization) or 14 dpi (post-infection)
  1. a Prepare RIPA lysates for Aβ measurement
  2. b Quantify total protein by BCA assay
  3. c Measure Aβ₄₀ and Aβ₄₂ by ELISA
Phase 11 · Immunofluorescence Staining and 3D Imaging Day 60 (characterization) or 14 dpi (post-infection)
  1. a Fix organoids in paraformaldehyde
  2. b Cryoprotect and embed organoids
  3. c Prepare cryosections
  4. d Perform antigen retrieval
  5. e Block and incubate with primary antibodies
  6. f Incubate with secondary antibodies
  7. g Stain nuclei and mount
  8. h Image and analyze immunofluorescence
  9. i Perform 3D clearing and imaging of whole organoids
Phase 12 · RNA Extraction and qRT-PCR for ER Stress and UPR Markers Day 38 post-infection (Day 98) or Day 60
  1. a Extract total RNA from organoids
  2. b Synthesize cDNA
  3. c Perform qRT-PCR for ER stress/UPR genes
  4. d Expected results for ZIKV-infected organoids

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Attribution

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

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