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BRAIN Publication-derived

Identification of neural oscillations and epileptiform changes in human brain organoids

Source Samarasinghe et al., 2021 · David Geffen School of Medicine at UCLA · 10.1038/s41593-021-00906-5

👤 Ranmal A. Samarasinghe, Osvaldo A. Miranda, Jessie E. Buth, Simon Mitchell, Isabella Ferando, Momoko Watanabe, Thomas F. Allison, Arinnae Kurdian, Namie N. Fotion, Michael J. Gandal, Peyman Golshani, Kathrin Plath, William E. Lowry, Jack M. Parent, Istvan Mody, Bennett G. Novitch ⏱ 107 days 📋 7 phases 🧫 Human iPSC (Rett syndrome patient-derived), Human ESC (H9)

Abstract

This protocol describes the generation of cerebral cortex-ganglionic eminence (Cx+GE) fusion brain organoids from human pluripotent stem cells and their characterization using calcium imaging and extracellular recording techniques. The protocol enables assessment of neural network-level activities including oscillatory rhythms and identification of pathological epileptiform changes, particularly relevant for modeling Rett syndrome and other neurological disorders affecting network function without overt anatomical abnormalities.

Cell source
Human iPSC (Rett syndrome patient-derived), Human ESC (H9)
Application
Disease modeling, Drug screening, Neural network characterization

Protocol overview

49 steps across 7 phases

hESC/hiPSC Culture and Organoid Generation Day 0–56
  1. 1 Conduct ESCRO and IRB approval
  2. 2 Culture H9 hESC or hiPSC lines
  3. 3 Generate cortex (Cx) organoids without Shh signaling
  4. 4 Generate ganglionic eminence (GE) organoids with Shh pathway stimulation
  5. 5 Prepare AAV1-tdTomato labeling (optional; for migration tracking)
Organoid Fusion and Early Culture Day 56–100
  1. 1 Prepare fusion setup
  2. 2 Combine organoid halves for fusion
  3. 3 Incubate in hyperoxic environment
  4. 4 Transfer fused organoids to oxygen-permeable dishes
  5. 5 Culture fused organoids with media changes
Single-Cell RNA Sequencing Sample Preparation Day 56–100
  1. 1 Collect organoids for dissociation
  2. 2 Prepare Papain dissociation solution
  3. 3 Dice organoid tissue
  4. 4 Incubate organoid chunks in Papain
  5. 5 Triturate organoid tissue (first round)
  6. 6 Triturate organoid tissue (second round)
  7. 7 Filter and wash cell suspension
  8. 8 Count and prepare cells for sequencing
Immunohistochemistry and Cell Quantification Day 84–106
  1. 1 Fix organoids
  2. 2 Cryoprotect and freeze organoids
  3. 3 Cryosection organoids
  4. 4 Perform immunostaining
  5. 5 Image stained sections
  6. 6 Quantify GAD65+ and SATB2+ cells
  7. 7 Quantify synaptic puncta
Live Calcium Imaging and Microcircuit Identification Day 88–106
  1. 1 Infect organoids with AAV1-GCaMP6f
  2. 2 Prepare organoid for imaging
  3. 3 Perform baseline calcium imaging
  4. 4 Apply GABAergic receptor antagonist
  5. 5 Re-image after drug application
  6. 6 Extract raw calcium imaging data
  7. 7 Process calcium imaging data with CNMF-E
  8. 8 Identify neuronal microcircuit clusters
  9. 9 Quantify synchronization metrics
Extracellular Local Field Potential (LFP) Recordings Day 100–107
  1. 1 Prepare aCSF recording solution
  2. 2 Prepare kainate solution
  3. 3 Prepare patch pipettes for LFP recording
  4. 4 Position organoid and pipette
  5. 5 Perfuse organoid with kainate-containing aCSF
  6. 6 Acquire extracellular LFP recordings
  7. 7 Analyze power spectral density (PSD)
  8. 8 Analyze high-frequency activity
  9. 9 Quantify spike frequency and interspike intervals
Drug Treatment and Efficacy Testing Day 100–106
  1. 1 Prepare drug solutions
  2. 2 Establish baseline LFP recordings
  3. 3 Incubate organoid with drug
  4. 4 Acquire LFP recordings after drug treatment
  5. 5 Analyze drug effects on LFP activity
  6. 6 Optional: assess cell death following drug treatment

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Samarasinghe 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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