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BRAIN Publication-derived

Alzheimer's Disease Cerebral Organoids for Infection Studies

Full SOP with Academic or a plan

Source Zhao et al. · Department of Neuroscience, Mayo Clinic, Jacksonville, FL · 10.1101/2022.06.30.498241

Authors: Jing Zhao, Tadafumi C. Ikezu, Wenyan Lu, Jesse R. Macyczko, Yonghe Li, Laura J. Lewis-Tuffin, Yuka A. Martens, Yingxue Ren, Yiyang Zhu, Yan W. Asmann, Nilüfer Ertekin-Taner, Takahisa Kanekiyo, Guojun Bu Duration: 90 days 6 phases Cell source: Human iPSC

Abstract

This protocol generates three-dimensional cerebral organoids from human iPSCs (parental and APOE-deficient lines) to model neural differentiation and cholesterol biosynthesis dysregulation relevant to Alzheimer's disease. The organoids are characterized by single-cell RNA sequencing, immunostaining, and molecular validation to define cell-type-specific effects of APOE deficiency on neurogenesis, lipid metabolism, and stress pathways.

Cell source
Human iPSC
Application
Disease modeling; Alzheimer's disease pathogenesis

Protocol overview

27 steps across 6 phases

Phase 1 · Embryoid Body Formation Day 0-4
  1. a Dissociate iPSC colonies into single-cell suspension
  2. b Dispense cells into a 96-well plate with ultra-low-attachment surfaces and U-shaped bottom wells.
  3. c Add medium supplementation on Day 2
  4. d Add medium supplementation on Day 4
Phase 2 · Embryoid Body Maturation in Low-Attachment Plates Day 5-9
  1. a Transfer embryoid bodies to 48-well low-attachment plates
  2. b Culture in Medium B for 3-5 days
Phase 3 · Matrigel Embedding and Organoid Initiation Day 10-13
  1. a Embed embryoid bodies in Matrigel
  2. b Culture embedded organoids in Medium C+D
Phase 4 · Orbital Shaker Culture and Neuronal Maturation Day 13-60 (and beyond for ISRIB treatment)
  1. a Transfer organoids to orbital shaker culture
  2. b Switch to neuronal maturation medium after 4 weeks
  3. c Continue culture until Day 90
Phase 5 · ISRIB Treatment (Optional, for EIF2 Pathway Inhibition) Day 60-90
  1. a Initiate ISRIB treatment at Day 60
  2. b Replace medium every 3 days
  3. c Harvest organoids at Day 90
Phase 6 · Single-Cell Dissociation and RNA-Sequencing Library Preparation Day 90 (harvest day)
  1. a Prepare Papain + DNase solution
  2. b Transfer organoids to dissociation dish
  3. c Mince organoids into small pieces
  4. d Enzymatic dissociation in shaking incubator (first incubation)
  5. e Mechanical dissociation by pipetting
  6. f Enzymatic dissociation in shaking incubator (second incubation)
  7. g Complete mechanical dissociation and stop enzymatic reaction
  8. h Transfer cell suspension to stop solution
  9. i Resuspend cells in PBS-BSA buffer
  10. j Assess cell viability
  11. k Load cells onto 10x Chromium chip
  12. l Construct single-cell libraries
  13. m Sequence libraries on Illumina HiSeq 4000

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Attribution

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

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