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BRAIN Publication-derived

Alzheimer's Disease Cerebral Organoids (2018)

Full SOP with Academic or a plan

Source Gonzalez et al., 2018 · University of Texas Medical School at Houston, Department of Neurology, Mitchell Center for Alzheimer's Disease and Related Brain Disorders · 10.1038/s41380-018-0229-8

Authors: Cesar Gonzalez, Enrique Armijo, Javiera Bravo-Alegria, Andrea Becerra-Calixto, Charles E. Mays, Claudio Soto Duration: 110 days 6 phases Cell source: Human iPSC (familial Alzheimer's disease and Down syndrome patient-derived; healthy control iPSC)

Abstract

This protocol describes the generation of cerebral organoids (COs) from human iPSCs derived from Alzheimer's disease (AD) and Down syndrome patients to model AD pathology in a 3D cortical-like tissue structure. The COs spontaneously develop amyloid-beta plaques and tau neurofibrillary tangles characteristic of AD, providing a human cellular model for studying disease mechanisms and screening therapeutic candidates.

Cell source
Human iPSC (familial Alzheimer's disease and Down syndrome patient-derived; healthy control iPSC)
Application
Disease modeling

Protocol overview

31 steps across 6 phases

Phase 1 · Fibroblast Culture and iPSC Generation Days 0-30
  1. a Culture of human dermal fibroblasts
  2. b Prepare lentiviral plasmids
  3. c Produce lentiviral particles
  4. d Transduce fibroblasts with lentivirus
  5. e Culture reprogrammed cells on MEF feeder layers
  6. f Select and expand iPSC colonies
Phase 2 · iPSC Characterization Days 0-30+ (parallel to expansion)
  1. a RT-PCR analysis of pluripotent markers
  2. b Alkaline phosphatase (AP) staining
  3. c Embryoid body assay
Phase 3 · Cerebral Organoid Generation and Maturation Days 0-110
  1. a Prepare single-cell suspension from iPSCs
  2. b Seed cells in ultra-low attachment 96-well plates
  3. c Aggregate formation and expansion (Days 0-6)
  4. d Neural induction (Day 6)
  5. e Embedding in Geltrex and transfer to 6-well plates (Days 11-12)
  6. f Maturation and long-term culture (Days 16+)
Phase 4 · Sample Preparation and Immunofluorescence Analysis At 30 DIV and 110 DIV
  1. a Fix samples for microscopy
  2. b Incubate with primary antibodies
  3. c Incubate with secondary antibodies and mount
  4. d BTA-1 staining for amyloid aggregates
  5. e Hematoxylin staining
  6. f Gallyas silver staining for neurofibrillary tangles
  7. g Image acquisition
Phase 5 · Biochemical Analysis: Western Blot and ELISA At 110 DIV
  1. a Collect and prepare organoid homogenates
  2. b Quantify protein concentration
  3. c Western blot sample preparation
  4. d Transfer to membrane and blocking
  5. e Incubate with primary and secondary antibodies (phospho-tau detection)
  6. f Develop and visualize blots
  7. g ELISA sample preparation: Fractionation of Aβ and tau
  8. h ELISA for Aβ and tau quantification
Phase 6 · Statistical Analysis During data analysis
  1. a Data analysis for Aβ and tau aggregates

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Attribution

This SOP was written by Organthis based on the published method in Gonzalez et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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