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BRAIN Publication-derived

Multicellular 3D Neurovascular Unit Model for Assessing Hypoxia and Neuroinflammation Induced Blood-Brain Barrier Dysfunction

Source Nzou et al., 2020 · Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine · 10.1038/s41598-020-66487-8

👤 Goodwell Nzou, Robert T. Wicks, Nicole R. Vanostrand, Gehad A. Mekky, Stephanie A. Seale, Aya El-taibany, Elizabeth E. Wicks, Carl M. Nechtman, Eric J. Marotte, Vishruti S. Makani, Sean V. Murphy, M. C. Seeds, John D. Jackson, Anthony J. Atala ⏱ 21 days 📋 12 phases 🧫 Human iPSC-derived cells (oligodendrocytes, microglia, neural stem cells) and primary human cells (brain microvascular endothelial cells, pericytes, astrocytes)

Abstract

This protocol describes the generation and culture of a six-cell-type 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes, and neurons to model blood-brain barrier dysfunction under hypoxic (0.1% O2) and neuroinflammatory conditions. The organoid model recapitulates BBB permeability changes, altered protein expression, oxidative stress, and pro-inflammatory cytokine production observed in hypoxic conditions and can be used for assessment of neuroprotective drug candidates.

Cell source
Human iPSC-derived cells (oligodendrocytes, microglia, neural stem cells) and primary human cells (brain microvascular endothelial cells, pericytes, astrocytes)
Application
Disease modeling

Protocol overview

73 steps across 12 phases

Cell Expansion and Preparation Pre-culture (variable duration)
  1. 1 Expand primary human brain microvascular endothelial cells (HBMEC)
  2. 2 Expand human brain microvascular pericytes (HBVP)
  3. 3 Expand human astrocytes (HA)
  4. 4 Propagate human iPSC-derived oligodendrocyte progenitor cells (HO)
  5. 5 Differentiate oligodendrocyte progenitor cells (HO) prior to organoid formation
  6. 6 Propagate human iPSC-derived microglia (HM)
  7. 7 Propagate human iPSC-derived neural stem cells (HN)
Organoid Assembly and Culture Days 0–5 in vitro
  1. 1 Harvest cells using appropriate dissociation enzymes
  2. 2 Prepare hanging drop culture for neuro-glial core organoid
  3. 3 Coat neuro-glial organoid with endothelial cells and pericytes
  4. 4 Culture organoids in organoid media and allow maturation
  5. 5 Transfer mature organoids to 96-well plate for long-term culture
Hypoxic Stress Application Day 6 in vitro
  1. 1 Culture organoids under hypoxic conditions
  2. 2 Maintain normoxic control organoids
BBB Permeability Assessment (FITC-IgG and FITC-Albumin) Pre-hypoxia and post-hypoxia (Day 6 and 7 in vitro)
  1. 1 Assess baseline BBB integrity prior to hypoxic exposure
  2. 2 Wash organoids after FITC-IgG/albumin incubation
  3. 3 Image pre-hypoxia organoids via confocal microscopy
  4. 4 Assess BBB permeability after hypoxic stress
  5. 5 Quantify albumin and IgG penetration depth
Cell Viability Assessment Days 4, 5, 7, 10, and 21 in vitro
  1. 1 Prepare live-dead cell staining solution
  2. 2 Incubate organoids in live-dead stain
  3. 3 Wash organoids after staining
  4. 4 Image organoids via confocal microscopy
  5. 5 Quantify cell viability
Hypoxia and Oxidative Stress Detection Day 7 in vitro
  1. 1 Pool organoids into eppendorf tubes
  2. 2 Prepare hypoxia/oxidative stress detection mix
  3. 3 Incubate organoids with detection reagents
  4. 4 Wash organoids after staining
  5. 5 Image organoids via confocal microscopy
  6. 6 Prepare positive controls
Metabolic Activity Assessment (ATP Production) Day 7 in vitro
  1. 1 Transfer organoids to opaque 96-well plate
  2. 2 Add CellTiter-Glo 3D reagent
  3. 3 Lyse cells by orbital mixing
  4. 4 Allow luminescent signal to stabilize
  5. 5 Measure luminescence
  6. 6 Calculate ATP levels
Protein and Cytokine Quantification (ELISA) Day 7 in vitro (post-hypoxia/treatment)
  1. 1 Pool organoids for protein extraction
  2. 2 Dissociate organoids with dispase
  3. 3 Collect organoid cell lysate
  4. 4 Quantify total protein concentration (BCA assay)
  5. 5 Collect supernatant for cytokine quantification
  6. 6 Perform ELISA for target proteins
  7. 7 Read and analyze ELISA plates
  8. 8 Normalize protein quantification results
Treatment with Neuroprotective Compounds (SDG and 2-AG) Days 4–7 in vitro
  1. 1 Prepare secoisolariciresinol diglycoside (SDG) treatment groups
  2. 2 Apply SDG pretreatment (48 hours prior to hypoxia)
  3. 3 Transition SDG-pretreated organoids to hypoxic chamber
  4. 4 Apply SDG treatment during hypoxia only
  5. 5 Culture hypoxia-only and re-oxygenation control groups
  6. 6 Prepare 2-arachidonyl glycerol (2-AG) treatment groups
  7. 7 Harvest organoids and supernatant after treatment
Exogenous Cytokine Treatment (IL-6 and TNF-α) Day 6 in vitro
  1. 1 Prepare exogenous cytokine solutions
  2. 2 Treat organoids with individual cytokines
  3. 3 Treat organoids with cytokine mixture
BBB Permeability Assessment After Cytokine Treatment (FITC Dextran and FITC-IgG) Day 6 in vitro (post-12-hour cytokine treatment)
  1. 1 Prepare FITC-labeled permeability tracers
  2. 2 Incubate cytokine-treated organoids with FITC tracers
  3. 3 Wash organoids after tracer incubation
  4. 4 Image organoids via confocal microscopy
  5. 5 Qualitatively assess BBB permeability to tracers
Immunohistochemistry: Tight Junction Protein Localization Day 7 in vitro (post-hypoxia/treatment)
  1. 1 Collect organoids into eppendorf tubes
  2. 2 Fix organoids in formaldehyde
  3. 3 Wash fixed organoids with cold PBS
  4. 4 Permeabilize organoids with Tween-20
  5. 5 Wash organoids after permeabilization
  6. 6 Block non-specific antibody binding
  7. 7 Incubate organoids with primary antibodies
  8. 8 Wash organoids after primary antibody incubation
  9. 9 Incubate organoids with secondary antibodies
  10. 10 Wash organoids after secondary antibody incubation
  11. 11 Perform nuclear counterstaining with DAPI
  12. 12 Final wash and preparation for imaging
  13. 13 Image stained organoids via confocal microscopy
  14. 14 Analyze tight junction protein distribution

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Nzou et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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