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BRAIN Publication-derived

Ischaemic Stroke Cerebral Organoids

Full SOP with Academic or a plan

Source Cao et al., 2023 · Department of Pharmacology, School of Pharmacy, Nanjing Medical University · 10.1038/s41536-023-00301-7

Authors: Shi-Ying Cao, Di Yang, Zhen-Quan Huang, Yu-Hui Lin, Hai-Yin Wu, Lei Chang, Chun-Xia Luo, Yun Xu, Yan Liu, Dong-Ya Zhu Duration: 180 days 9 phases Cell source: Human iPSC (IMR-90-4), Human ESC (H9), Human iPSC (H9-CAG-ChR2-EYFP)

Abstract

This protocol describes the generation of human cerebral organoids from pluripotent stem cells and their transplantation into photothrombotic stroke-injured mouse brain. Grafted organoids survive long-term in the infarcted core, differentiate into cortical neurons, repair tissue structure, establish synaptic connections with host circuits, and restore sensorimotor function.

Cell source
Human iPSC (IMR-90-4), Human ESC (H9), Human iPSC (H9-CAG-ChR2-EYFP)
Application
Disease modeling (stroke), Tissue repair, Functional restoration

Protocol overview

34 steps across 9 phases

Phase 1 · Pluripotent Stem Cell Culture Days -30 to 0 (preparation)
  1. a Culture human pluripotent stem cells
Phase 2 · Embryoid Body Formation and Rosette Induction Days 0-15 in vitro
  1. a Detach pluripotent stem cells and form embryoid bodies
  2. b Culture embryoid bodies in neural induction medium
  3. c Allow EBs to attach and form rosettes
Phase 3 · Cerebral Organoid Formation Days 16-20+ in vitro
  1. a Dissect and culture rosette structures
  2. b Allow organoids to develop neural tube structures
  3. c Continue long-term culture and medium refresh
Phase 4 · Lentivirus Transduction of Organoids Days 40-50 in vitro (10 days prior to transplantation)
  1. a Prepare lentiviral infection solution for EGFP labeling
  2. b Incubate organoids with lentiviral solution
  3. c Transfer infected organoids to culture flask
  4. d Culture infected organoids before transplantation
Phase 5 · Photothrombotic Stroke Model in Mice Surgical day (day 0)
  1. a Prepare mouse for surgery
  2. b Inject rose bengal dye
  3. c Illuminate brain to induce focal stroke
  4. d Close surgical site and recover animal
Phase 6 · Organoid Transplantation Day 7 post-stroke
  1. a Prepare mouse for stereotaxic transplantation
  2. b Transplant three cerebral organoids
  3. c Close surgical site and recover animal
Phase 7 · Long-term Assessment and Functional Studies Days 30-180 post-transplantation
  1. a Perform behavioral testing
  2. b Prepare acute brain slices for electrophysiology
  3. c Recover and equilibrate brain slices
  4. d Perform whole-cell patch-clamp electrophysiology on organoid neurons
  5. e Test organoid neuron responses to optogenetic stimulation
  6. f Perform local field potential (LFP) recordings in distant brain targets
Phase 8 · Immunohistochemistry and Cell Composition Analysis Days 45-180 post-transplantation
  1. a Prepare organoids or brain tissue for immunostaining
  2. b Section tissue and prepare slides
  3. c Block nonspecific binding and permeabilize
  4. d Incubate sections with primary antibodies
  5. e Apply secondary antibodies and fluorescent detection
  6. f Mount and image sections
  7. g Perform cell counting and quantitative analysis
Phase 9 · Chemogenetic Modulation Studies Days 150-180 post-transplantation
  1. a Transplant chemogenetically-modified organoids
  2. b Validate hM4Di/CNO system in vitro
  3. c Perform behavioral testing with CNO treatment

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Attribution

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

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