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BRAIN Publication-derived

Ischaemic Stroke Cerebral Organoids for Drug Screening

Full SOP with Academic or a plan

Source Wang et al., 2023 · Second Military Medical University/Naval Medical University, Shanghai · 10.1038/s41401-022-00986-4

Authors: Shu-na Wang, Zhi Wang, Xi-yuan Wang, Xiu-ping Zhang, Tian-ying Xu, Chao-yu Miao Duration: 86 days 7 phases Cell source: Human ESC (H1)

Abstract

This protocol establishes a three-dimensional cerebral organoid-based model of ischemic stroke induced by oxygen-glucose deprivation (OGD). The model recapitulates human brain cell composition, structure, and neural connectivity, enabling screening of potential anti-stroke agents with improved translational relevance over rodent models.

Cell source
Human ESC (H1)
Application
Disease modeling; Drug screening for ischemic stroke neuroprotection

Protocol overview

42 steps across 7 phases

Phase 1 · Human Embryonic Stem Cell Culture and Maintenance Ongoing; passages 45-55
  1. a Prepare feeder-free culture plates
  2. b Culture hESCs in mTesR™1 medium
  3. c Passage hESCs
  4. d Add Y-27632 at first passage day
Phase 2 · Cerebral Organoid Generation: Embryonic Body Formation and Neuroectoderm Induction Days 1-14
  1. a Prepare single cell suspension and seed in ultra-attachment 96-well plate
  2. b Embryonic body formation (Day 1)
  3. c Monitor EB growth and expand to larger well plates
  4. d Continue EB growth in 24-well plates
  5. e Initiate neural induction
  6. f Identify neuroepithelial buds and embed in Matrigel
  7. g Continue stationary culture in Matrigel
Phase 3 · Cerebral Organoid Culture and Maturation in Bioreactor Days 17-85 and beyond
  1. a Transfer organoids to spinning bioreactor
  2. b Perform medium changes during bioreactor culture
  3. c Monitor organoid growth and morphology through culture
  4. d Select 85-day organoids with diameter > 2.0 mm for downstream applications
Phase 4 · Characterization of Cerebral Organoids: Immunofluorescence and Flow Cytometry At days 15, 35, 75, and 85
  1. a Prepare organoid cryosections
  2. b Perform immunofluorescence staining of cryosections
  3. c Image stained sections by confocal microscopy
  4. d Quantify immunofluorescence staining
  5. e Prepare single cell suspension for flow cytometry
  6. f Perform surface flow cytometry staining
  7. g Perform intracellular flow cytometry staining
  8. h Set up flow cytometer and acquire data
Phase 5 · Oxygen-Glucose Deprivation (OGD) Model and Drug Treatment Days 85-86 (2 h, 4 h, 8 h, or 12 h OGD exposure depending on experimental design)
  1. a Prepare glucose-free medium
  2. b Prepare organoids for OGD
  3. c Establish OGD condition
  4. d Maintain normoxic control condition
  5. e Add test compounds during OGD exposure
  6. f End OGD exposure and harvest organoids
Phase 6 · Assessment of Cell Damage: Caspase-3 Activity and LDH Release Assays Immediately after OGD exposure or drug treatment
  1. a Harvest organoid lysates for protein analysis
  2. b Measure protein concentration
  3. c Measure Caspase-3 activity
  4. d Calculate Caspase-3 activity per unit protein
  5. e Collect culture supernatants for LDH assay
  6. f Perform LDH release assay
  7. g Measure LDH absorbance
Phase 7 · Histological Analysis: H&E, Immunohistochemistry, TUNEL, and Nissl Staining Immediately after OGD exposure or drug treatment
  1. a Fix organoid tissue
  2. b Dehydrate and embed in paraffin
  3. c Perform haematoxylin and eosin (H&E) staining
  4. d Perform immunohistochemistry staining
  5. e Quantify immunohistochemistry staining
  6. f Perform Nissl staining

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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