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BRAIN Publication-derived

Patch-Clamp Electrophysiology of Brain Organoid Slice Cultures

Source Giandomenico et al. · MRC Laboratory of Molecular Biology, Cambridge, UK · 10.1038/s41593-019-0350-2

👤 Giandomenico SL, Mierber SB, Giber GM, Lancaster MA ⏱ 135 days 📋 9 phases 🧫 Human iPSC / ESC

Abstract

Giandomenico et al. developed an air-liquid interface organoid slice culture system that overcomes diffusion limitations, enabling long axonal tract formation and detailed whole-cell patch-clamp electrophysiological characterization. This Nature Neuroscience 2019 Lancaster lab protocol provides single-neuron resolution functional data from brain organoids.

Cell source
Human iPSC / ESC
Application
Detailed Electrophysiology of Brain Organoids

Protocol overview

48 steps across 9 phases

Human Pluripotent Stem Cell Culture and Preparation Days 0–35
  1. 1 Maintain hESC culture on Matrigel-coated plates
  2. 2 Prepare plasmid constructs for fluorescent labeling
Cerebral Organoid Generation (enCOR Method) Days 0–60
  1. 1 Generate cerebral organoids using enCOR protocol
  2. 2 Supplement medium with Matrigel for cortical plate establishment
  3. 3 Electroporate plasmids for fluorescent labeling
  4. 4 Allow organoid maturation after electroporation
Air-Liquid Interface Cerebral Organoid (ALI-CO) Culture Setup Day 55–60 onwards
  1. 1 Prepare organoids for embedding and sectioning
  2. 2 Embed organoids in low-gelling-temperature agarose
  3. 3 Cool agarose blocks and prepare for sectioning
  4. 4 Collect organoid sections
  5. 5 Equilibrate sections in serum-supplemented culture medium
  6. 6 Transition to serum-free slice culture medium
  7. 7 Maintain ALI-CO cultures at the air-liquid interface
Live Imaging of Axon Outgrowth Dynamics (Optional) Days 5–24 at ALI
  1. 1 Prepare ALI-COs for confocal live imaging
  2. 2 Acquire time-lapse confocal images
  3. 3 Analyze axon growth cone dynamics
Histological Analysis and Immunostaining Throughout ALI culture (any timepoint)
  1. 1 Fix ALI-CO sections for histology
  2. 2 Prepare samples for cryosectioning
  3. 3 Perform immunohistochemical staining
  4. 4 Perform TUNEL assay to assess cell death
Electrophysiological Recordings Days 20+ at ALI
  1. 1 Prepare ALI-COs for multi-electrode array (MEA) recordings
  2. 2 Transfer ALI-CO to MEA device
  3. 3 Record spontaneous neural activity
  4. 4 Optionally apply tetrodotoxin (TTX) to block activity
  5. 5 Analyze MEA data for network connectivity
  6. 6 Perform whole-cell patch-clamp recordings
Single-Cell RNA Sequencing (scRNA-seq) Days 16–22 at ALI (53–75 days organoid age)
  1. 1 Dissociate organoid slices into single cells
  2. 2 Filter and prepare single-cell suspension
  3. 3 Prepare single-cell RNA-seq libraries
  4. 4 Perform reverse transcription and cDNA amplification
  5. 5 Quality control and library validation
  6. 6 Sequence on Illumina platform
  7. 7 Analyze single-cell data computationally
Retrograde Tracing and Tract Identification Days 15–25 at ALI
  1. 1 Perform retrograde labeling with cholera toxin subunit B (CTB)
  2. 2 Perform DiI lipophilic dye tracing (alternative)
  3. 3 Allow dye transport and labeling
  4. 4 Fix and analyze labeled tissues
Co-culture with Mouse Spinal Cord and Functional Testing Days 50–75 at ALI (total organoid age 105–135 days)
  1. 1 Prepare embryonic mouse spinal cord tissue
  2. 2 Prepare organoid and mouse tissue for co-culture embedding
  3. 3 Section co-culture blocks
  4. 4 Culture organoid-mouse co-cultures
  5. 5 Record spontaneous muscle contractions
  6. 6 Analyze muscle contraction displacement
  7. 7 Perform electrical stimulation of ALI-CO axon tracts
  8. 8 Measure evoked response latency
  9. 9 Perform axotomy (tract lesion)
  10. 10 Analyze muscle contraction data post-axotomy
  11. 11 Test pharmacological blockade with tetrodotoxin (TTX)

Full SOP

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Attribution

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

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