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BRAIN Publication-derived

Ischaemic Stroke Cerebral Organoids for Transplantation

Full SOP with Academic or a plan

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

Authors: Shu-Na Wang, Zhi Wang, Tian-Ying Xu, Ming-He Cheng, Wen-Lin Li, Chao-Yu Miao Duration: 55 days 11 phases Cell source: 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

Phase 1 · Human Embryonic Stem Cell Culture and Maintenance Ongoing maintenance
  1. a hESC Culture Setup
  2. b Y-27632 ROCK Inhibitor Addition
Phase 2 · Embryonic Body (EB) Formation Days 0-8 of induction
  1. a Single Cell Digestion and Seeding
  2. b EB Culture in Low bFGF Medium
  3. c Medium Change to hES Medium
  4. d EB Transfer to 24-Well Plates
Phase 3 · Neural Induction and Neuroepithelium Formation Days 8-15 of induction (4-6 days in neural induction medium)
  1. a Neural Induction Medium Culture
  2. b Identification of Neuroepithelium Formation
Phase 4 · Cerebral Organoid Differentiation in Matrigel Droplets Days 15-55+ of induction
  1. a Matrigel Embedding and Stationary Culture
  2. b Transfer to Spinning Bioreactor
  3. c Continued Culture with Vitamin A-Containing B27
  4. d Quality Control of Organoid Morphology
Phase 5 · Cerebral Organoid Processing for Transplantation Day 55 of induction (preparation for surgery)
  1. a Organoid Collection
  2. b Organoid Preparation for Transplantation
Phase 6 · Rat Middle Cerebral Artery Occlusion (MCAO) Model and Organoid Transplantation Day 0 (MCAO surgery)
  1. a Anesthesia Induction
  2. b Cyclosporin A Immunosuppression (Pre-surgery)
  3. c Craniotomy
  4. d Middle Cerebral Artery Occlusion (MCAO)
  5. e Transplantation Cavity Creation
  6. f Cerebral Organoid Transplantation
  7. g Skull Closure
  8. h Incision Closure and Infection Prevention
Phase 7 · Post-transplantation Care and Monitoring Days 1-28 post-implantation
  1. a Cyclosporin A Continuation
  2. b BrdU Administration for Neurogenesis Tracking
  3. c Behavioral Testing (mNSS)
  4. d Behavioral Testing (Beam Walking)
  5. e Animal Monitoring
Phase 8 · Brain Tissue Collection and Processing Days 7, 14, and 28 post-implantation
  1. a TTC Staining for Infarct Volume Measurement
  2. b Paraformaldehyde Fixation
  3. c Brain Image Acquisition
  4. d Brain Perfusion for Immunofluorescence
  5. e Cryoprotection and Sectioning
Phase 9 · Immunofluorescence Staining of Brain Tissue After brain tissue collection
  1. a Tissue Permeabilization
  2. b Blocking
  3. c Primary Antibody Incubation
  4. d Secondary Antibody Incubation
  5. e Nuclear Staining
  6. f Microscopy Examination
Phase 10 · BrdU Immunostaining After tissue collection and processing
  1. a BrdU Staining Protocol
Phase 11 · Histology and Additional Staining After tissue collection
  1. a Hematoxylin and Eosin (HE) Staining
  2. b Immunohistochemistry (IHC) Staining
  3. c Nissl's Staining

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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