Skip to content
← Back to browse
BRAIN Publication-derived

Minocycline mitigates the effect of neonatal hypoxic insult on human brain organoids

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

👤 Erin M. Boisvert, Robert E. Means, Michael Michaud, Joseph A. Madri, Samuel G. Katz ⏱ 72 days 📋 7 phases 🧫 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

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

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Boisvert et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence