Skip to content
← Back to browse
BRAIN Publication-derived

Human 3D Cellular Model of Hypoxic Brain Injury of Prematurity

Source Paşca et al., 2019 · Stanford University School of Medicine · 10.1038/s41591-019-0436-0

👤 Anca M. Paşca, Jin-Young Park, Hyun-Woo Shin, Qihao Qi, Omer Revah, Rebecca Krasnoff, Ruth O'Hara, A. Jeremy Willsey, Theo D. Palmer, Sergiu P. Paşca ⏱ 120 days 📋 13 phases 🧫 Human iPSC

Abstract

This protocol generates human cortical spheroids (hCS) from induced pluripotent stem cells and exposes them to hypoxia to model encephalopathy of prematurity. The hCS are differentiated over 74–78 days and then exposed to <1% O2 for 48 hours to induce a hypoxic response, allowing investigation of oxygen deprivation effects on cortical progenitor populations.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

62 steps across 13 phases

hiPS Cell Culture Ongoing maintenance
  1. 1 Culture hiPS cells on feeder layer
  2. 2 Prepare hiPS cell medium
Generation of Human Cortical Spheroids (hCS) from hiPS Cells Days 0–42
  1. 1 Initiate neural spheroid formation
  2. 2 Change medium for first five days
  3. 3 Transfer to neural medium (Day 6)
  4. 4 Maintain with EGF and FGF2 (Days 6–24)
  5. 5 Switch to BDNF and NT3 (Days 25–42)
  6. 6 Maintain in unsupplemented neural medium (Day 43 onwards)
Exposure of hCS to Low Oxygen (Hypoxia) Days 74–78 (hypoxia) + Days 79–120 (re-oxygenation, optional)
  1. 1 Pre-equilibrate media at low oxygen
  2. 2 Transfer hCS to hypoxia chamber
  3. 3 Maintain hypoxia for 48 hours
  4. 4 Collect samples or initiate re-oxygenation
Optional: Exposure to Tunicamycin (UPR Induction Control) Days 74–78
  1. 1 Expose hCS to tunicamycin
  2. 2 Collect samples
Optional: ISRIB Treatment during Hypoxia Days 74–78
  1. 1 Prepare ISRIB solution
  2. 2 Co-expose hCS to hypoxia and ISRIB
  3. 3 Collect samples
Sample Preparation: Cryopreservation for Immunocytochemistry Immediate after sample collection
  1. 1 Fix hCS in paraformaldehyde
  2. 2 Wash in PBS
  3. 3 Cryoprotect in sucrose
  4. 4 Embed in OCT compound
  5. 5 Cut cryosections
Immunocytochemistry on hCS Cryosections After cryosection preparation
  1. 1 Remove OCT and block
  2. 2 Incubate with primary antibodies (standard markers)
  3. 3 Incubate with primary antibodies (TBR2 with antigen retrieval)
  4. 4 Wash and apply secondary antibodies
  5. 5 Stain nuclei and mount
  6. 6 Image
Immunocytochemistry Quantification After imaging
  1. 1 Quantify cell density in proliferative zones
  2. 2 Quantify cell proportions in whole cryosections
  3. 3 Analyze co-expression data
Western Blot Analysis Immediate after sample collection
  1. 1 Lyse hCS
  2. 2 Separate proteins on gel
  3. 3 Transfer to PVDF membrane
  4. 4 Incubate with primary antibodies
  5. 5 Apply secondary antibodies and detect
  6. 6 Quantify bands
RNA Extraction and Quantitative PCR (qPCR) After sample collection
  1. 1 Extract mRNA
  2. 2 Synthesize cDNA
  3. 3 Perform qPCR
  4. 4 Analyze qPCR data
RNA-Sequencing At 24, 48 hours (hypoxia), and 120 hours (after re-oxygenation)
  1. 1 Extract RNA for RNA-Seq
  2. 2 Prepare RNA-Seq library
  3. 3 Sequence on Illumina HiSeq
  4. 4 Align reads and generate count data
  5. 5 Normalize and filter gene expression data
  6. 6 Identify confounding variables
  7. 7 Perform differential expression analysis
  8. 8 Perform WGCNA (co-expression analysis)
  9. 9 Perform enrichment analysis
Optional: Oxygen Tension Measurements with Fiber Optic Microsensor At baseline and during hypoxia exposure
  1. 1 Calibrate oxygen microsensor
  2. 2 Mount sensor on micromanipulator
  3. 3 Measure oxygen partial pressure at various depths
Validation in Primary Human Cortical Tissue Days 0–2 (tissue culture)
  1. 1 Obtain and prepare human fetal cortical tissue
  2. 2 Section tissue in agarose
  3. 3 Pre-incubate tissue sections
  4. 4 Expose tissue to hypoxia or maintain in normoxia
  5. 5 Optional: Co-expose with ISRIB
  6. 6 Prepare tissue for immunohistochemistry
  7. 7 Cut tissue sections for immunohistochemistry
  8. 8 Perform immunohistochemistry on tissue sections
  9. 9 Image and quantify tissue sections

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 Paşca 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