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BRAIN Publication-derived

Cortical 2D Cell Cultures for Developmental Studies

Full SOP with Academic or a plan

Source Kadoshima et al. · RIKEN Center for Developmental Biology, Kobe, Japan · 10.1073/pnas.1315710110

Authors: Kadoshima T, Sakaguchi H, Nakano T, Soen M, Ando S, Eiraku M, Sasai Y Duration: 112 days 7 phases Cell source: Human ESC

Abstract

Kadoshima et al. demonstrated that human ESC-derived cortical neuroepithelium can self-organize into layered structures recapitulating the ventricular zone, subventricular zone, and cortical plate using the SFEBq method. This RIKEN/Sasai lab protocol generated human-specific outer radial glia cells and represented a parallel foundational advance alongside Lancaster's cerebral organoid work.

Cell source
Human ESC
Application
Cortical Layering Studies

Protocol overview

29 steps across 7 phases

Phase 1 · hESC Maintenance on Feeder Layer Ongoing maintenance prior to differentiation
  1. a Prepare feeder layer with inactivated MEFs
  2. b Culture hESCs on MEF feeder in maintenance medium
  3. c Passage hESCs by enzymatic digestion
  4. d Re-plate hESCs at optimal split ratio
Phase 2 · Initiation of SFEBq Culture and Cortical Specification (Days 0-18) Days 0-18
  1. a Dissociate hESCs to single cells
  2. b Quick reaggregate hESCs in low-adhesion plates
  3. c Prepare cortex differentiation medium
  4. d Add morphogens for cortical specification (Days 0-18)
  5. e Monitor aggregate morphology
Phase 3 · Transfer to Suspension Culture and Early Expansion (Days 18-35) Days 18-35
  1. a Transfer aggregates to non-adhesive Petri dish
  2. b Switch to N2-supplemented medium
  3. c Culture under elevated oxygen (40% O₂/5% CO₂)
  4. d Add FBS, heparin, and Matrigel (from Day 35)
  5. e Perform medium changes every 2-3 days
Phase 4 · Mechanical Dissection and Long-Term Culture (Days 35-56) Days 35-56
  1. a Cut aggregates to prevent central cell death
  2. b Maintain medium composition
  3. c Prepare for transition to high-O₂ dishes (Day 56+)
Phase 5 · Maturation and Extended Culture (Days 56-112+) Days 56-112+
  1. a Switch to lumox dishes for enhanced oxygen supply
  2. b Increase Matrigel concentration (Day 70)
  3. c Continue mechanical dissection and medium changes
  4. d Maintain final medium composition
Phase 6 · Optional Treatments: Rostralization, Ventralization, and ROCK Inhibition Days 15-42 (rostralization); Days 15-21 (ventralization); Days 26-30 (ROCK inhibition)
  1. a FGF8b treatment for rostralization (Days 24-42)
  2. b Smoothened agonist (SAG) treatment for ventralization (Days 15-21)
  3. c Y-27632 treatment for ROCK inhibition (Days 26-30)
Phase 7 · Generation of Knock-In hESC Lines (foxg1::Venus and pax6::Venus) Variable; performed prior to experimental use
  1. a Design zinc finger nucleases (ZFNs) for gene targeting
  2. b Construct targeting vector with homology arms and venus
  3. c Dissociate hESCs and prepare for electroporation
  4. d Electroporate targeting vector and ZFN-coding mRNAs
  5. e Select homologous recombinant clones

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Attribution

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

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