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BRAIN Publication-derived

Midbrain Organoids for Disease and Development

Full SOP with Academic or a plan

Source Jo et al. · National University of Singapore; National Institute of Mental Health, USA

Authors: Junghyun Jo, Yixin Xiao, Alfred Xuyang Sun, Huck-Hui Ng Duration: 60 days 3 phases Cell source: Human ESC (H1, H9)

Abstract

This protocol describes the generation of midbrain-like organoids (hMLOs) from human pluripotent stem cells that develop functional dopaminergic and neuromelanin-producing neurons. hMLOs are patterned using SHH and FGF8 signaling, embedded in Matrigel, and cultured in differentiation medium to produce mature midbrain tissue with dopaminergic neurons expressing TH, MAP2, and neuromelanin by day 60-146.

Cell source
Human ESC (H1, H9)
Application
Disease modeling; developmental neurobiology

Protocol overview

16 steps across 3 phases

Phase 1 · EB Formation and Neuronal Induction Days 0-4
  1. a Dissociate hPSCs to single cells
  2. b Plate cells in low-adhesion 96-well plates
  3. c Add ROCK inhibitor Y27632
  4. d Change neuronal induction medium
  5. e Add midbrain patterning factors
Phase 2 · Matrigel Embedding and Neuroepithelium Expansion Days 7-9
  1. a Monitor neuroectodermal bud extrusion
  2. b Remove culture medium completely
  3. c Embed in reduced growth factor Matrigel
  4. d Allow Matrigel to solidify
  5. e Culture in tissue growth induction medium
  6. f Transfer organoids to ultra-low-attachment plates
Phase 3 · Final Differentiation and Maturation Days 9-60+
  1. a Culture in final differentiation medium
  2. b Use orbital shaker for culture
  3. c Include antibiotics to prevent contamination
  4. d Change medium every 3 days
  5. e Culture until day 60 or beyond

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Attribution

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

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