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BRAIN Publication-derived

3D Human Blood-Brain Barrier (BBB) Organoid Model of Cryptococcus neoformans Neuroinvasion

Source Bennett et al. · University of California, Davis, Department of Pharmacology, School of Medicine · 10.1101/2025.09.23.678106;

👤 Amelia B. Bennett, Dylan M. Lanser, Kiem Vu, Amita R. Sahoo, Matthias Buck, Angie Gelli ⏱ 30 days 📋 8 phases 🧫 Human immortalized brain endothelial cells (iBMEC/D3), primary human brain microvascular endothelial cells, human primary astrocytes, human primary brain vascular pericytes

Abstract

This protocol generates a 3D human blood-brain barrier (BBB) organoid model comprising immortalized or primary human brain endothelial cells, astrocytes, and pericytes in a 1:1:1 ratio. The organoid spontaneously self-organizes into a functional BBB structure with intact tight junctions (Claudin-5+) and an impermeable endothelial barrier that can be used to study transcellular mechanisms of Cryptococcus neoformans entry into the central nervous system via macropinocytosis.

Cell source
Human immortalized brain endothelial cells (iBMEC/D3), primary human brain microvascular endothelial cells, human primary astrocytes, human primary brain vascular pericytes
Application
Disease modeling; investigation of pathogenic fungal neuroinvasion mechanisms and blood-brain barrier transcytosis

Protocol overview

63 steps across 8 phases

Cell Culture Preparation and Organoid Formation Days 0-3
  1. 1 Maintain immortalized brain endothelial cells (iBMECs)
  2. 2 Prepare astrocytes and pericytes
  3. 3 Trypsinize and prepare single-cell suspensions
  4. 4 Seed astrocytes and pericytes into low-adhesion 96-well plates
  5. 5 Allow organoid formation for 48 hours
  6. 6 Seed endothelial cells on top of organoid cores
  7. 7 Culture organoids until maturation
Organoid Validation and Barrier Integrity Assessment Days 7-10
  1. 1 Assess baseline permeability using FITC-dextran
  2. 2 Image organoids for permeability quantification
  3. 3 Quantify permeability
  4. 4 Fix and section organoids for immunofluorescence validation
  5. 5 Cryoprotect and prepare organoids for sectioning
  6. 6 Flash-freeze organoids and store
  7. 7 Section organoids in cryostat
  8. 8 Perform immunofluorescence to validate tight junctions
  9. 9 Apply secondary antibodies and nuclear stain
  10. 10 Image and confirm barrier integrity
Cryptococcal Infection Assay and Macropinocytosis Inhibition Days 11-13
  1. 1 Prepare Cryptococcus neoformans inoculum
  2. 2 Label fungi with CFDA-SE fluorescent dye
  3. 3 Treat organoids with amiloride (macropinocytosis inhibitor)
  4. 4 Infect organoids with Cryptococcus neoformans
  5. 5 Harvest and prepare organoids for imaging
  6. 6 Quantify fungal internalization
  7. 7 Compare amiloride-treated vs. control organoids
Verification in 2D Transwell BBB Model Days 14-15
  1. 1 Culture iBMECs on collagen-coated transwells
  2. 2 Prepare Cryptococcus neoformans inoculum for transwell assay
  3. 3 Add fungi to transwell upper chamber with amiloride treatment conditions
  4. 4 Quantify fungal crossing at 12 hours
  5. 5 Compare amiloride inhibition of transcytosis
  6. 6 Verify clathrin-mediated endocytosis is not inhibited (negative control)
Macropinocytosis Verification via Dextran Uptake Assay Days 16-17
  1. 1 Prepare brain endothelial cells for dextran uptake assay
  2. 2 Prepare FITC-labeled dextran solution
  3. 3 Test serum starvation as positive control for macropinocytosis
  4. 4 Expose cells to Cryptococcus neoformans and FITC-dextran
  5. 5 Wash and fix cells
  6. 6 Image cells to visualize internalized dextran
  7. 7 Quantify internalized dextran signal
  8. 8 Test amiloride inhibition of Cn-induced dextran uptake
EphA2 Knockout and Organoid Infection Studies Days 18-25
  1. 1 Generate EphA2 knockout cell line using CRISPR/Cas9
  2. 2 Select EphA2 knockout cells with puromycin
  3. 3 Enrich RFP+ EphA2 KO cells using flow cytometry
  4. 4 Verify EphA2 knockout by Western blot
  5. 5 Generate BBB organoids with EphA2 KO endothelial cells
  6. 6 Infect EphA2 KO and WT organoids with Cryptococcus neoformans
  7. 7 Harvest, fix, and section organoids
  8. 8 Quantify fungal internalization in EphA2 KO vs. WT organoids
  9. 9 Assess astrocyte activation (GFAP) in EphA2 KO vs. WT organoids
Cdc42 Activation Assay and Molecular Complex Studies Days 26-28
  1. 1 Prepare iBMECs for Cdc42 activation assay
  2. 2 Expose cells to Cryptococcus neoformans
  3. 3 Lyse cells and prepare lysates
  4. 4 Perform Cdc42 G-LISA activation assay
  5. 5 Verify Cdc42 protein expression by Western blot
  6. 6 Test Cdc42 activation with CPS1-deficient Cryptococcus neoformans strain
  7. 7 Compare Cdc42 activation across conditions
EphA2-CD44 Protein-Protein Interaction Detection via Proximity Ligation Assay Days 29-30
  1. 1 Culture brain endothelial cells on chamber slides and differentiate
  2. 2 Prepare Cryptococcus neoformans and Cryptococcus deuterogattii inocula
  3. 3 Co-incubate cells with fungi
  4. 4 Fix cells for proximity ligation assay
  5. 5 Perform proximity ligation assay (PLA) using DuoLink system
  6. 6 Add coverslips and image
  7. 7 Quantify PLA signal
  8. 8 Compare PLA signal between Cn and Cd treatments
  9. 9 Include negative controls

Full SOP

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Attribution

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

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