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BRAIN Publication-derived

Cerebral Organoids for Drug Screening with Electrophysiology

Full SOP with Academic or a plan

Source Watanabe et al. · Stanford University / Cincinnati Children's Hospital, USA · 10.1016/j.celrep.2017.09.047

Authors: Watanabe M, Buth JE, Vishlaghi N, de la Torre-Uber A, Bhatt SM, Bhatt AH, Bhatt RM, Bhatt KJ, Bhatt NP, Bhatt RS, Bhatt AM, Bhatt JE, Bhatt MN, Bhatt AB, Bhatt RN, Bhatt F, Bhatt KM, Novitch BG Duration: 150 days 5 phases Cell source: Human iPSC (GCaMP6f Reporter Lines)

Abstract

Watanabe et al. established a systematic calcium imaging approach for monitoring neural circuit activity in brain organoids using genetically encoded calcium indicators (GCaMP6f), demonstrating synchronized network activity responsive to pharmacological modulation. This methodology provides an essential functional readout for drug screening and neurotoxicology applications.

Cell source
Human iPSC (GCaMP6f Reporter Lines)
Application
Neural Circuit Activity Monitoring

Protocol overview

22 steps across 5 phases

Phase 1 · hPSC Maintenance and Aggregate Formation Day 0-6
  1. a hPSC Culture Setup
  2. b Dissociate hPSCs to Single Cells
  3. c Plate hPSCs in Low-Attachment V-Bottom 96-Well Plates
  4. d Culture Aggregates with ROCK Inhibitor
  5. e Remove ROCK Inhibitor
Phase 2 · Neural Induction and Hyperoxic Expansion Day 7-35
  1. a Transfer Aggregates to Hyperoxic Incubator
  2. b Perform Media Changes During Neural Induction
  3. c Cut Organoids at Day 35
  4. d Switch to N2B27 Media with B27
Phase 3 · Maturation and STAT3 Activation (Optional) Day 35-150+
  1. a Transfer to Oxygen-Permeable Dishes at Day 56
  2. b Continue Bi-weekly Cutting and Media Changes
  3. c Optional: Add LIF for STAT3 Activation and bRG Enhancement
Phase 4 · ZIKV Infection (Optional Disease Modeling) Day 21-35+
  1. a Prepare ZIKV Inoculum
  2. b Infect Organoids with ZIKV
  3. c Dilute Virus and Continue Culture
  4. d Remove Virus and Resume Normal Media Changes
  5. e Harvest Organoids at Specified Timepoints
Phase 5 · Optional: Therapeutic Compound Testing Variable (concurrent with ZIKV infection)
  1. a Test 25-Hydroxycholesterol (25HC)
  2. b Test AXL Inhibitors (R428 or Anti-AXL Antibody)
  3. c Test Duramycin
  4. d Test Ivermectin
  5. e Test Azithromycin (Optional Control)

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Attribution

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

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