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BRAIN Publication-derived

Brain Embryo Models for Drug Screening

Full SOP with Academic or a plan

Source Durens et al., 2020 · Hussman Institute for Autism, University of Maryland School of Medicine · 10.1016/j.jneumeth.2020.108627

Authors: Madel Durens, Jonathan Nestor, Madeline Williams, Kevin Herold, Robert F. Niescier, Jason W. Lunden, Andre W. Phillips, Yu-Chih Lin, Derek M. Dykxhoorn, Michael W. Nestor Duration: 60 days 6 phases Cell source: Human iPSC

Abstract

This protocol describes the generation and characterization of serum-free embryoid bodies (SFEBs) derived from human iPSCs and their assessment using high-content imaging (HCI), multi-electrode arrays (MEA), and calcium imaging. SFEBs are thinned 3D cortical organoids amenable to automated screening for neurite morphology, cellular composition, and electrophysiological activity without requiring tissue sectioning.

Cell source
Human iPSC
Application
Disease modeling, Drug screening, High-throughput organoid analysis

Protocol overview

43 steps across 6 phases

Phase 1 · hiPSC maintenance and SFEB formation Day 0 to Day 14
  1. a Maintain hiPSCs on MEF feeder layer
  2. b Prepare hiPSCs for aggregation
  3. c Seed cells for SFEB aggregation
  4. d Promote SFEB aggregation via centrifugation
  5. e Culture SFEBs for 14 days
Phase 2 · SFEB transfer and maturation on PTFE inserts Day 15 to Day 54
  1. a Transfer SFEBs to PTFE cell culture inserts
  2. b Culture in Neural Formation medium with neurotrophic factors
  3. c Induce terminal differentiation at DIV30
  4. d Maintain cultures until DIV45 with DAPT
  5. e Withdraw DAPT at DIV45
Phase 3 · Neuronal morphology assessment (DIV60) Day 60
  1. a Fix SFEBs with paraformaldehyde
  2. b Transfer to optically clear imaging plates
  3. c Wash and block SFEBs
  4. d Primary antibody incubation
  5. e Wash after primary antibody
  6. f Secondary antibody incubation
  7. g Wash after secondary antibody
  8. h Nuclear staining with Hoechst
  9. i Prepare plates for imaging
  10. j High-content image acquisition
  11. k Neurite morphology analysis
Phase 4 · Multi-electrode array (MEA) electrophysiology Day 54 onwards (after SFEB transfer to MEA plates)
  1. a Prepare MEA plates with coating
  2. b Wash MEA plates
  3. c Air-dry and add laminin
  4. d Transfer SFEBs to MEA plates
  5. e Recovery period before recording
  6. f Configure MEA recording parameters
  7. g Record baseline spontaneous activity
  8. h Pharmacological manipulation—glutamate receptor blockade
  9. i Pharmacological manipulation—GABA receptor blockade
  10. j Chemical long-term potentiation (cLTP) induction
  11. k Generate continuous traces
Phase 5 · Immunocytochemistry for MEA samples After MEA recording
  1. a Fix SFEBs on MEA plates
  2. b Immunostaining on MEA plates
  3. c Image stained SFEBs on MEA plates
Phase 6 · Calcium imaging (GCaMP6s) Day 54 onwards (parallel to or after MEA recordings)
  1. a Prepare SFEBs for viral transduction
  2. b Transduce SFEBs with GCaMP6s virus
  3. c Complete media change post-transduction
  4. d Allow GCaMP6s expression maturation
  5. e High-throughput calcium imaging acquisition
  6. f Analyze calcium transients
  7. g Identify cells near active electrodes
  8. h High-magnification validation of individual cell activity

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Attribution

This SOP was written by Organthis based on the published method in Durens et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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