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BRAIN Publication-derived

Ischaemic Stroke Brain Organoids with Single-Cell Profiling

Full SOP with Academic or a plan

Source Zhu et al., 2025 · Stem Cell and Regenerative Medicine Lab, Institute of Clinical Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Translational Medicine Center, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College · 10.1016/j.bioactmat.2024.09.038

Authors: Wendi Zhu, Yue Wu, Xiao Li, Hongjun Yang, Fuchu He, Jie Ma, Junying Wei, Ling Leng Duration: 45 days 6 phases Cell source: Human iPSC

Abstract

This protocol establishes human iPSC-derived brain organoids exposed to hypoxic conditions to model ischemic stroke in vitro. The organoids are analyzed using proteomics, single-cell transcriptomics, and histopathology to identify pathological changes and test the therapeutic efficacy of traditional Chinese medicine (DengZhanShengMai capsule) on stroke-induced injury.

Cell source
Human iPSC
Application
Disease modeling - Stroke/Ischemic injury study

Protocol overview

22 steps across 6 phases

Phase 1 · Human iPSC maintenance and embryoid body formation Day 0-2
  1. a Dissociate hiPSC colonies into single cells
  2. b Feed embryoid bodies with fresh medium
Phase 2 · Embryoid body expansion and neural induction Day 4-10
  1. a Replace medium without Y27632
  2. b Transfer EBs to neural induction media
  3. c Feed EBs with additional neural induction medium
Phase 3 · Matrigel embedding and cerebral organoid differentiation Day 11-45
  1. a Embed EBs in Matrigel and initiate organoid differentiation
  2. b Feed organoids and continue static culture
  3. c Switch to media with vitamin A and begin orbital shaking
  4. d Maintain organoid culture with regular media changes
Phase 4 · Hypoxia treatment of brain organoids Day 40+
  1. a Prepare organoids for hypoxia treatment
  2. b Incubate organoids under hypoxic conditions
Phase 5 · Tissue preparation and histological analysis Post-hypoxia treatment
  1. a Fix organoids and brain tissue
  2. b Dehydrate mouse brain tissue
  3. c Dehydrate cerebral organoid tissue
  4. d Embed tissue in paraffin and section
  5. e Perform H&E staining
  6. f Perform immunofluorescence staining
Phase 6 · Single-cell RNA sequencing of brain organoids Post-hypoxia treatment
  1. a Collect organoids for single-cell analysis
  2. b Dissociate organoids into single-cell suspension
  3. c Filter and wash cells
  4. d Count cells and assess viability
  5. e Perform single-cell 3′ RNA-seq

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Attribution

This SOP was written by Organthis based on the published method in Zhu et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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