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BRAIN Publication-derived

Humanized Cerebral Organoids-Based Ischemic Stroke Model for Anti-Stroke Drug Discovery

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

👤 Shu-na Wang, Zhi Wang, Xi-yuan Wang, Xiu-ping Zhang, Tian-ying Xu, Chao-yu Miao ⏱ 86 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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