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BRAIN Publication-derived

Generation of Cerebral Organoids from Human Pluripotent Stem

Source Lancaster et al. · IMBA — Institute of Molecular Biotechnology, Vienna, Austria · 10.1038/nprot.2014.158

👤 Lancaster MA, Knoblich JA ⏱ 15 days 📋 6 phases 🧫 Human iPSC / Human ESC

Abstract

The definitive step-by-step protocol for generating cerebral organoids from human pluripotent stem cells, published as a Nature Protocols companion to the original Lancaster et al. 2013 study. This protocol provides detailed instructions for EB formation, neural induction, Matrigel embedding, and spinning bioreactor culture with comprehensive troubleshooting guidance.

Cell source
Human iPSC / Human ESC
Application
Standard Cerebral Organoid Production

Protocol overview

34 steps across 6 phases

Making embryoid bodies Day 0
  1. 1 Prepare hESC or iPSC colonies
  2. 2 Dissociate feeder-dependent PSCs (Option A.i)
  3. 3 Remove Dispase and recover colonies (Option A.ii)
  4. 4 Wash colonies to remove single cells and MEFs (Option A.iii)
  5. 5 Dissociate colonies to single cells (Option A.iv)
  6. 6 Count cells and centrifuge (Option A.v)
  7. 7 Resuspend cells in ROCK inhibitor medium (Option A.vi)
  8. 8 Plate cells in 96-well U-bottom plate (Option A.vii)
  9. 9 Dissociate feeder-independent hESCs (Option B.i)
  10. 10 Treat with Accutase (Option B.ii)
  11. 11 Recover and triturate feeder-independent cells (Option B.iii)
  12. 12 Count feeder-independent cells and centrifuge (Option B.iv)
  13. 13 Resuspend feeder-independent cells in ROCK inhibitor medium (Option B.v)
  14. 14 Plate feeder-independent cells (Option B.vi)
Feeding EBs and initiation of germ layer differentiation Days 1-6
  1. 1 Observe EBs at 24 hours
  2. 2 Feed EBs every other day (early stage)
  3. 3 Feed EBs without ROCK inhibitor and bFGF
Induction of primitive neuroepithelia Days 6-11
  1. 1 Transfer EBs to neural induction media
  2. 2 Feed EBs in neural induction media
  3. 3 Assess neuroepithelium formation
Transferring neuroepithelial tissues to Matrigel droplets Day 11
  1. 1 Thaw Matrigel on ice
  2. 2 Prepare dimpled Parafilm substrate
  3. 3 Create grid of Matrigel droplet dimples
  4. 4 Transfer neuroepithelial tissues to Matrigel dimples
  5. 5 Remove excess media from tissues
  6. 6 Add Matrigel droplets to aggregates
  7. 7 Position aggregates in Matrigel droplets
  8. 8 Allow Matrigel to polymerize
  9. 9 Add cerebral organoid differentiation media
  10. 10 Remove Matrigel droplets from Parafilm
Stationary culture of expanding neuroepithelial buds Days 12-15
  1. 1 Observe organoid development
  2. 2 Feed organoids in differentiation media (first feed)
Growth of cerebral tissue Day 15 onwards (up to 1 year)
  1. 1 Transfer organoids to spinning bioreactor or orbital shaker
  2. 2 Change media and monitor organoid development

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Lancaster et al.. 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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