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BRAIN Publication-derived

Brain Organoids for Regenerative Medicine with Vasculature

Full SOP with Academic or a plan

Source Daviaud et al., 2018 · Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai · 10.1523/eneuro.0219-18.2018

Authors: Nicolas Daviaud, Roland H. Friedel, Hongyan Zou Duration: 60 days 7 phases Cell source: Human ESC (H9 hES cells)

Abstract

This protocol describes the generation of cerebral organoids from human embryonic stem cells (ESCs) and their transplantation into lesioned mouse cortex. The organoids exhibit enhanced survival, robust vascularization from host brain, and multilineage neuronal differentiation compared to dissociated neural progenitor cells, providing a tissue-based alternative for modeling human neurodevelopment and CNS disease in vascularized cortical tissue.

Cell source
Human ESC (H9 hES cells)
Application
Neural transplantation and CNS repair

Protocol overview

34 steps across 7 phases

Phase 1 · hESC Culture and GFP Labeling 0-1
  1. a Prepare hESC culture plates
  2. b Plate and culture hESCs
  3. c Infect hESCs with lentivirus
  4. d Select GFP-positive hESCs
  5. e Verify pluripotency of GFP-labeled hESCs
Phase 2 · Generation of hNPC from hESCs 1-22
  1. a Generate embryoid bodies
  2. b Attach EBs to Matrigel-coated plates
  3. c Apply neural induction medium
  4. d Plate cells on laminin-coated plates
Phase 3 · Cerebral Organoid Generation 0-42
  1. a Detach and plate hES cells for EB formation
  2. b Supplement with ROCK inhibitor during early EB formation
  3. c Switch to neural induction medium
  4. d Embed organoids in Matrigel droplets
  5. e Prepare cerebral organoid differentiation media
  6. f Culture organoids on orbital shaker with vitamin A
  7. g Quality control and sizing of organoids pre-transplant
Phase 4 · Surgical Preparation and Transplantation 42-44
  1. a Anesthetize recipient mice
  2. b Prepare surgical site
  3. c Create cortical lesion
  4. d Implant cerebral organoid
  5. e Close craniotomy window
Phase 5 · Intracerebral Implantation of NPC (Parallel Transplant) 42-44
  1. a Prepare NPC for injection
  2. b Anesthetize and position recipient mouse
  3. c Inject NPC using stereotactic coordinates
  4. d Close surgical site and recover mouse
Phase 6 · Post-Transplantation Analysis and Histology 44-58
  1. a Terminal perfusion and tissue collection
  2. b Cryoprotect and freeze tissue
  3. c Section brain tissue
  4. d Block non-specific binding
  5. e Apply primary antibodies
  6. f Wash and apply secondary antibodies
Phase 7 · Image Acquisition and Quantification 58-60
  1. a Image immunofluorescence sections
  2. b Outline graft areas for analysis
  3. c Quantify cellular markers

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Attribution

This SOP was written by Organthis based on the published method in Daviaud et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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