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BRAIN Publication-derived

Fragile X Syndrome Cortical Organoids

Full SOP with Academic or a plan

Source Kang et al., 2021 · Emory University School of Medicine · 10.1038/s41593-021-00913-6

Authors: Yunhee Kang, Ying Zhou, Yujing Li, Yanfei Han, Jie Xu, Weibo Niu, Ziyi Li, Shiying Liu, Hao Feng, Wen Huang, Ranhui Duan, Tianmin Xu, Nisha Raj, Feiran Zhang, Juan Dou, Chongchong Xu, Hao Wu, Gary J Bassell, Stephen T Warren, Emily G Allen, Peng Jin, Zhexing Wen Duration: 71 days 8 phases Cell source: Patient-Derived iPSC (Fragile X Syndrome)

Abstract

This protocol describes the generation of three-dimensional human forebrain organoids from patient-derived iPSCs with fragile X syndrome (FXS) and isogenic controls. The method produces forebrain organoids that exhibit dysregulated neurogenesis, altered neuronal differentiation, increased synapse formation, and enhanced neuronal excitability. These organoids serve as a platform to model human FXS-associated neurodevelopmental defects and test therapeutic interventions.

Cell source
Patient-Derived iPSC (Fragile X Syndrome)
Application
Disease modeling

Protocol overview

27 steps across 8 phases

Phase 1 · iPSC Culture and Embryoid Body Formation Days 0-6
  1. a iPSC Culture on MEF Feeder Layer
  2. b Collagenase Dissociation and EB Medium Suspension
  3. c Daily Medium Change for Embryoid Body Formation
  4. d Transition to Induction Medium (Day 5-6)
Phase 2 · Matrigel Embedding and Organoid Formation (Days 7-13) Days 7-13
  1. a Matrigel Embedding of Organoids
  2. b Mechanical Dissociation from Matrigel (Day 14)
Phase 3 · Bioreactor Culture and Differentiation (Days 14-70) Days 14-70
  1. a Transfer to Spinning Bioreactor with Differentiation Medium
  2. b Routine Medium Changes (Days 14-70)
  3. c Optional Pharmacological Treatment (Days 42-56)
Phase 4 · Maturation Phase (Days 71 onwards) Day 71 onwards
  1. a Transition to Maturation Medium (Day 71)
  2. b Continued Culture with Routine Medium Changes
Phase 5 · Organoid Harvesting and Preparation for Analysis Day 56 onwards
  1. a Organoid Fixation for Immunocytochemistry
  2. b PBS Washes and Sucrose Cryoprotection
  3. c Tissue Embedding and Cryosectioning
  4. d Optional: Retroviral GFP Labeling (Day 49)
  5. e Optional: EdU Pulse Labeling
Phase 6 · Immunocytochemistry and Analysis Variable
  1. a Tissue Permeabilization
  2. b Blocking and Primary Antibody Incubation
  3. c Secondary Antibody Incubation and DAPI Staining
  4. d Microscopy and Quantitative Image Analysis
Phase 7 · Electrophysiology (Optional) Day 56 onwards
  1. a Organoid Slice Preparation
  2. b Whole-Cell Patch-Clamp Recording Setup
  3. c Current-Clamp Recording for Action Potential Analysis
  4. d Voltage-Clamp Recording for Ionic Currents
Phase 8 · RNA and Molecular Analysis (Optional) Variable
  1. a RNA Extraction from Organoids
  2. b cDNA Synthesis and qRT-PCR
  3. c Optional: RNA-seq Library Construction

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Attribution

This SOP was written by Organthis based on the published method in Kang et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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