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BRAIN Publication-derived

Hypoxic Brain Injury Organoids

Full SOP with Academic or a plan

Source Kim et al., 2021 · Adult Stem Cell Research Center and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University · 10.3390/cells10020234

Authors: Min Soo Kim, Da-Hyun Kim, Hyun Kyoung Kang, Myung Geun Kook, Soon Won Choi, Kyung-Sun Kang Duration: 86 days 4 phases Cell source: Human adult dermal fibroblast-derived neural stem cells (iNSCs)

Abstract

This protocol generates self-organized 3D human neural organoids from adult dermal fibroblast-derived induced neural stem cells (iNSCs), which recapitulate the structure of the human cerebral cortex including ventricular and cortical plate zones. The organoids are then subjected to hypoxic injury followed by reoxygenation to model hypoxic-ischemic brain damage, enabling study of neuronal proliferation and maturation responses.

Cell source
Human adult dermal fibroblast-derived neural stem cells (iNSCs)
Application
Disease modeling, drug screening, and development of personalized medicine approaches for hypoxic brain injury

Protocol overview

19 steps across 4 phases

Phase 1 · Culture of Human Induced Neural Stem Cells (iNSCs) Days 1-40+
  1. a Transduce human dermal fibroblasts with retroviral factors
  2. b Expand transduced cells
  3. c Change to NSC maintenance medium
  4. d Neural induction and neurosphere collection
  5. e Establish iNSC line by alternating culture conditions
  6. f Passaging iNSCs using enzymatic dissociation
Phase 2 · Generation of Human Neural Organoids from iNSCs Days 7-84
  1. a Embed neurospheres in Matrigel (Day 7)
  2. b Transfer to ultra-low attachment dishes with CODM (Day 7)
  3. c Replace CODM every other day (Days 7-21)
  4. d Transfer to spinner flasks with modified CODM (Day 21)
  5. e Continue culture in spinner flasks with agitation for 35 days (Days 21-56)
  6. f Continue culture for mature neural organoid generation (Days 56-84)
Phase 3 · Sample Preparation As needed for analysis
  1. a Fix neural organoids with paraformaldehyde
  2. b Wash organoids in PBS
  3. c Immerse in 30% sucrose solution
  4. d Embed in sucrose-gelatin mixture and freeze
Phase 4 · Hypoxic Brain Injury Modeling Days 84-86
  1. a Transfer organoids to hypoxic chamber (Day 84)
  2. b Maintain CODM during hypoxia
  3. c Transfer to reoxygenation chamber (Day 86)

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Attribution

This SOP was written by Organthis based on the published method in Kim et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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