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BRAIN Publication-derived

Cerebral Organoids Transplantation for Ischemic Stroke Repair

Source Wang et al., 2020 · Second Military Medical University / Naval Medical University, Shanghai, China · 10.1007/s12975-019-00773-0

👤 Shu-Na Wang, Zhi Wang, Tian-Ying Xu, Ming-He Cheng, Wen-Lin Li, Chao-Yu Miao ⏱ 55 days 📋 11 phases 🧫 Human ESC (H1)

Abstract

This protocol describes the generation of cerebral organoids (COs) from human H1 embryonic stem cells and their transplantation into a rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. Transplanted COs at 55 days of differentiation reduce brain infarct volume, improve neurological motor function, enhance neurogenesis and synaptic reconstruction, and promote angiogenesis and axonal regeneration.

Cell source
Human ESC (H1)
Application
Disease modeling and therapeutic transplantation for ischemic stroke

Protocol overview

42 steps across 11 phases

Human Embryonic Stem Cell Culture and Maintenance Ongoing maintenance
  1. 1 hESC Culture Setup
  2. 2 Y-27632 ROCK Inhibitor Addition
Embryonic Body (EB) Formation Days 0–8 of induction
  1. 1 Single Cell Digestion and Seeding
  2. 2 EB Culture in Low bFGF Medium
  3. 3 Medium Change to hES Medium
  4. 4 EB Transfer to 24-Well Plates
Neural Induction and Neuroepithelium Formation Days 8–15 of induction (4–6 days in neural induction medium)
  1. 1 Neural Induction Medium Culture
  2. 2 Identification of Neuroepithelium Formation
Cerebral Organoid Differentiation in Matrigel Droplets Days 15–55+ of induction
  1. 1 Matrigel Embedding and Stationary Culture
  2. 2 Transfer to Spinning Bioreactor
  3. 3 Continued Culture with Vitamin A-Containing B27
  4. 4 Quality Control of Organoid Morphology
Cerebral Organoid Processing for Transplantation Day 55 of induction (preparation for surgery)
  1. 1 Organoid Collection
  2. 2 Organoid Preparation for Transplantation
Rat Middle Cerebral Artery Occlusion (MCAO) Model and Organoid Transplantation Day 0 (MCAO surgery)
  1. 1 Anesthesia Induction
  2. 2 Cyclosporin A Immunosuppression (Pre-surgery)
  3. 3 Craniotomy
  4. 4 Middle Cerebral Artery Occlusion (MCAO)
  5. 5 Transplantation Cavity Creation
  6. 6 Cerebral Organoid Transplantation
  7. 7 Skull Closure
  8. 8 Incision Closure and Infection Prevention
Post-transplantation Care and Monitoring Days 1–28 post-implantation
  1. 1 Cyclosporin A Continuation
  2. 2 BrdU Administration for Neurogenesis Tracking
  3. 3 Behavioral Testing (mNSS)
  4. 4 Behavioral Testing (Beam Walking)
  5. 5 Animal Monitoring
Brain Tissue Collection and Processing Days 7, 14, and 28 post-implantation
  1. 1 TTC Staining for Infarct Volume Measurement
  2. 2 Paraformaldehyde Fixation
  3. 3 Brain Image Acquisition
  4. 4 Brain Perfusion for Immunofluorescence
  5. 5 Cryoprotection and Sectioning
Immunofluorescence Staining of Brain Tissue After brain tissue collection
  1. 1 Tissue Permeabilization
  2. 2 Blocking
  3. 3 Primary Antibody Incubation
  4. 4 Secondary Antibody Incubation
  5. 5 Nuclear Staining
  6. 6 Microscopy Examination
BrdU Immunostaining After tissue collection and processing
  1. 1 BrdU Staining Protocol
Histology and Additional Staining After tissue collection
  1. 1 Hematoxylin and Eosin (HE) Staining
  2. 2 Immunohistochemistry (IHC) Staining
  3. 3 Nissl's Staining

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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