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BRAIN Publication-derived

Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Brain Organoids (2021)

Full SOP with Academic or a plan

Source Szebényi et al., 2021 · John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge · 10.1038/s41593-021-00923-4

Authors: Kornélia Szebényi, Léa M. D. Wenger, Yu Sun, Alexander W. E. Dunn, Colleen A. Limegrover, George M. Gibbons, Elena Conci, Ole Paulsen, Susanna B. Mierau, Gabriel Balmus, András Lakatos Duration: 240 days 10 phases Cell source: Human iPSC

Abstract

This protocol describes the generation and culture of three-dimensional human cortical organoid slices derived from patient-specific induced pluripotent stem cells (iPSCs) carrying the C9ORF72 mutation. The method produces organoid slice cultures (ALI-COs) cultured at the air–liquid interface up to 240 days in vitro, enabling investigation of early molecular pathology of ALS/FTD in relevant cell types without requiring tissue from presymptomatic patients.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

36 steps across 10 phases

Phase 1 · hiPSC Culture and Maintenance Ongoing
  1. a Culture hiPSCs in basal medium
  2. b Freeze hiPSCs for banking
Phase 2 · Cerebral Organoid Generation (Days 0-50) 0-50 DIV
  1. a Initiate organoid formation
  2. b Continue media feeding and morphogenesis
  3. c Add Matrigel for polarized cortical plate formation
Phase 3 · Organoid Slicing and Air–Liquid Interface Culture (Days 50-240) 50-240 DIV
  1. a Prepare organoid slices
  2. b Transfer slices to air–liquid interface (ALI) inserts
  3. c Daily feeding with slice medium
  4. d Monitor ALI-CO development and morphology
Phase 4 · Characterization: Single-Cell RNA Sequencing (At 150 DIV) 150 DIV
  1. a Dissociate organoid slices for scRNA-seq
  2. b Prepare cells for scRNA-seq library preparation
  3. c Perform single-cell RNA sequencing
Phase 5 · Characterization: Immunohistochemistry and Immunofluorescence (At specified timepoints) 30-240 DIV
  1. a Fix organoid slices or whole organoids
  2. b Prepare frozen sections
  3. c Permeabilize and block tissue sections
  4. d Apply primary antibodies
  5. e Apply secondary antibodies and counterstain
Phase 6 · Characterization: Western Blotting for Stress Markers (At 150-240 DIV) 150-240 DIV
  1. a Prepare whole-organoid lysates
  2. b Quantify protein concentration
  3. c Resolve proteins by SDS-PAGE
  4. d Transfer proteins to PVDF membrane
  5. e Block and incubate with primary antibodies
  6. f Apply secondary antibodies and detect signal
Phase 7 · Characterization: DNA Damage Assessment (At 150 DIV and after pharmacological treatment) 150-240 DIV
  1. a Treat samples with topoisomerase inhibitor (optional stress induction)
  2. b Fix slices and prepare cryostat sections
  3. c Immunostain for γ-H2AX (DNA damage marker)
  4. d Image and quantify γ-H2AX foci
Phase 8 · Pharmacological Rescue: Short-Term Treatment (12 hours) 200-220 DIV
  1. a Pre-treat ALI-COs with GSK2606414
  2. b Immerse organoid slices in treatment medium
  3. c Harvest samples for Western blotting
Phase 9 · Pharmacological Rescue: Long-Term Treatment (14 days) 200-240 DIV
  1. a Prepare long-term treatment setup
  2. b Daily medium replacement
  3. c Harvest samples after 14-day treatment
Phase 10 · Characterization: Multielectrode Array (MEA) Recordings (At 150-193 DIV) 150-193 DIV
  1. a Prepare 3D MEA chip
  2. b Record spontaneous neuronal activity
  3. c Analyze spike rates and connectivity

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Attribution

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

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