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BRAIN Publication-derived

Brain Organoids at Air-Liquid Interface

Full SOP with Academic or a plan

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

Authors: Giandomenico SL, Mierber SB, Giber GM, Lancaster MA Duration: 135 days 9 phases Cell source: 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

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

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Attribution

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

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