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BRAIN Publication-derived

Neonatal Hypoxic Injury Brain Organoids

Full SOP with Academic or a plan

Source Boisvert et al., 2019 · Yale University School of Medicine · 10.1038/s41419-019-1553-x

Authors: Erin M. Boisvert, Robert E. Means, Michael Michaud, Joseph A. Madri, Samuel G. Katz Duration: 72 days 7 phases Cell source: Human ESC (H9)

Abstract

This protocol describes the generation of self-directed human brain organoids from H9 hESCs over 6 months of culture. The organoids develop forebrain and cortical markers with proper layer organization. The protocol enables modeling of neonatal hypoxic injury and evaluation of therapeutic interventions such as minocycline.

Cell source
Human ESC (H9)
Application
Disease modeling

Protocol overview

29 steps across 7 phases

Phase 1 · hESC maintenance and EB formation Day 0 (pre-differentiation)
  1. a Maintain H9 hESCs on matrigel substrate
  2. b Prepare hESCs for dissociation
  3. c Wash and rinse cells from substrate
  4. d Transfer cells to ultra-low attachment flask
Phase 2 · bFGF weaning (Days 1-5) Days 1-5
  1. a Pool cells on day 1
  2. b Further reduce bFGF on day 3
  3. c Switch to neural induction media on day 5
Phase 3 · Neural induction and organoid development (Days 5-35+) Days 5-35+ (up to 6 months)
  1. a Maintain organoids in neural induction media
  2. b Harvest samples at designated time points
Phase 4 · Hypoxia exposure (8% oxygen, 25 days) Days 10-35
  1. a Set up hypoxia chamber
  2. b Transfer organoids to hypoxic conditions
  3. c Perform gene expression time course
Phase 5 · Severe hypoxia exposure with minocycline treatment (1% oxygen, 72 hours) Day 10 (72 hours)
  1. a Prepare minocycline stock solution
  2. b Set up severe hypoxia and treatment groups
  3. c Calibrate severe hypoxia chamber to 1% oxygen
  4. d Expose organoids to 1% oxygen for 72 hours
  5. e Harvest samples for analysis
Phase 6 · qRT-PCR analysis After harvest
  1. a Extract RNA from organoid samples
  2. b Quantify RNA and synthesize cDNA
  3. c Perform qRT-PCR
  4. d Calculate relative gene expression
  5. e Perform statistical analysis
Phase 7 · Immunohistochemistry and immunofluorescence After harvest
  1. a Fix organoid samples
  2. b Cryoprotect organoids with sucrose gradient
  3. c Embed organoids in OCT and section
  4. d Block and incubate with primary antibody
  5. e Wash and incubate with secondary antibody
  6. f Apply DAPI stain and final washes
  7. g Mount and image

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Attribution

This SOP was written by Organthis based on the published method in Boisvert et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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