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BRAIN Publication-derived

Alzheimer's Disease Cerebral Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Pavoni et al., 2018 · Service d'Etude des Prions et Infections Atypiques (SEPIA), Institut François Jacob, CEA, Université Paris-Saclay, Fontenay-aux-Roses, France · 10.1371/journal.pone.0209150

Authors: Serena Pavoni, Rafika Jarray, Ferid Nassor, Anne-Cécile Guyot, Steve Cottin, Jessica Rontard, Jacqueline Mikol, Aloïse Mabondzo, Jean-Philippe Deslys, Frank Yates Duration: 5 days 11 phases Cell source: Human iPSC

Abstract

This protocol describes the generation and chemical induction of human cerebral organoids (mini-brains) using Aftin-5 to produce elevated Aβ-42 peptide and reproduce Alzheimer's disease-associated phenotypes. Mini-brains differentiated from control iPSCs demonstrate time-dependent Aβ accumulation and, following Aftin-5 treatment, show increased Aβ42/Aβ40 ratios characteristic of sporadic Alzheimer's disease.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

30 steps across 11 phases

Phase 1 · iPSC Maintenance and Feeder-Free Adaptation Ongoing until EB generation
  1. a Maintain iPSC cultures in feeder-free conditions
Phase 2 · Embryoid Body (EB) Generation via Hanging Drop Method Day 0-2
  1. a Dissociate iPSC colonies and prepare cell suspension
  2. b Culture hanging drops on petri dish cover
  3. c Harvest embryoid bodies
Phase 3 · Neural Induction Day 2 onwards (exact duration per Lancaster protocol)
  1. a Perform neural induction on EBs
Phase 4 · Neural Differentiation and Organoid Formation Following neural induction through 2 months of culture
  1. a Embed neural induced EBs in Matrigel droplets
  2. b Culture organoids for expansion and regionalization
Phase 5 · Aftin-5 Chemical Induction At 1-month and/or 2-month-old organoids
  1. a Prepare Aftin-5 treatment solution
  2. b Treat organoids with Aftin-5
  3. c Harvest conditioned media
Phase 6 · Sample Preparation for Histological Analysis Upon completion of treatment or at specified timepoints
  1. a Fix organoids in formaldehyde
  2. b Prepare organoids for paraffin embedding
  3. c Section paraffin-embedded organoids
Phase 7 · Immunohistochemical Analysis Upon completion of paraffin sectioning
  1. a Dewax and perform epitope retrieval
  2. b Block and wash slides
  3. c Incubate with primary antibodies
  4. d Wash and apply secondary antibody
  5. e Develop chromogenic signal and counterstain
  6. f Dehydrate and mount slides
Phase 8 · Gene Expression Analysis via Real-Time qPCR Upon sample collection at specified timepoints
  1. a Extract total RNA from organoids
  2. b Perform reverse transcription
  3. c Perform real-time qPCR amplification
  4. d Analyze qPCR data
Phase 9 · Western Blot Analysis of APP and PrP(C) Upon sample collection at specified timepoints
  1. a Prepare organoid lysates
  2. b Perform SDS-PAGE and transfer
Phase 10 · Aβ Quantification by ELISA 4 days post-Aftin-5 treatment (or at specified timepoints)
  1. a Prepare conditioned media samples
  2. b Perform multiplex Aβ ELISA
  3. c Calculate Aβ42/Aβ40 ratio
Phase 11 · Statistical Analysis Upon completion of all measurements
  1. a Organize and analyze data
  2. b Perform statistical significance testing

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Attribution

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

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