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BRAIN Publication-derived

Human ALS/FTD Brain Organoid Slice Cultures: Generation and Characterization

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

👤 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 ⏱ 240 days 📋 10 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Szebényi et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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