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BRAIN Publication-derived

Cortical Spheroids for Disease Modelling

Full SOP with Academic or a plan

Source Paşca et al. · Stanford University School of Medicine, Stanford, CA, USA · 10.1038/nmeth.3415

Authors: Paşca AM, Sloan SA, Clarke LE, Tian Y, Makinson CD, Huber N, Kim CH, Park JY, O'Rourke NA, Nguyen KD, Smith SJ, Bhatt NP, Barres BA, Paşca SP Duration: 180 days 5 phases Cell source: Human iPSC

Abstract

Paşca et al. established a Matrigel-free method for generating human cortical spheroids (hCS) from iPSCs using dual SMAD inhibition, producing functional deep- and upper-layer cortical neurons alongside astrocytes over a 90-day culture period. This guided differentiation approach enabled electrophysiological recordings of spontaneous network activity and formed the foundation for the assembloid technology subsequently developed by the Paşca laboratory.

Cell source
Human iPSC
Application
Cortical Development Modeling

Protocol overview

12 steps across 5 phases

Phase 1 · hiPSC Culture and Colony Dissociation Day -7 to Day 0
  1. a Culture hiPSCs on feeders
  2. b Dissociate intact hiPSC colonies
Phase 2 · Neural Induction and SMAD Inhibition Day 0 to Day 6
  1. a Transfer colonies to low-attachment plates
  2. b Apply SMAD signaling inhibitors
Phase 3 · Neural Expansion and Progenitor Specification Day 6 to Day 25
  1. a Transition to neural medium with growth factors
  2. b Perform daily medium changes (days 6-16)
  3. c Perform every-other-day medium changes (days 16-25)
Phase 4 · Neuronal Differentiation Day 25 to Day 43
  1. a Replace growth factors with neurotrophic factors
  2. b Maintain BDNF and NT3 supplementation
Phase 5 · Maturation and Long-Term Culture Day 43 to Day 180+
  1. a Switch to growth-factor-free neural medium
  2. b Maintain hCSs through long-term culture
  3. c Monitor cellular development milestones

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Attribution

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

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