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BRAIN Publication-derived

Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

Source Bennett et al., 2025 · Department of Pharmacology, School of Medicine, University of California, Davis · 10.1101/2025.09.23.678106;

👤 Amelia B. Bennett, Dylan M. Lanser, Kiem Vu, Amita R. Sahoo, Matthias Buck, Angie Gelli ⏱ 18 days 📋 7 phases 🧫 Human iPSC-derived brain microvascular endothelial cells (immortalized HCMEC/D3), primary human brain endothelial cells, primary human astrocytes, primary human brain vascular pericytes

Abstract

This protocol describes the generation and use of a 3D human blood-brain barrier organoid model consisting of brain endothelial cells, astrocytes, and pericytes to study the mechanism by which Cryptococcus neoformans enters the central nervous system. The protocol demonstrates that C. neoformans exploits macropinocytosis as the endocytic mechanism for transcellular entry across the blood-brain barrier by recruiting CD44 and EphA2 as a molecular complex.

Cell source
Human iPSC-derived brain microvascular endothelial cells (immortalized HCMEC/D3), primary human brain endothelial cells, primary human astrocytes, primary human brain vascular pericytes
Application
Disease modeling

Protocol overview

35 steps across 7 phases

BBB Organoid Formation and Preparation Days 1-3
  1. 1 Culture and Expand Cell Types
  2. 2 Trypsinize and Prepare Astrocytes and Pericytes
  3. 3 Seed Astrocytes and Pericytes into Low-Adhesion Plates
  4. 4 Trypsinize and Seed Endothelial Cells
Organoid Maturation and Validation Days 4-14
  1. 1 Culture Organoids to Maturity
  2. 2 Verify Organoid Barrier Integrity Using FITC-Dextran
  3. 3 Fix and Cryo-section Organoids for Immunofluorescence
  4. 4 Perform Immunofluorescence Staining for Barrier Markers
BBB Crossing and Macropinocytosis Assays Days 15-18
  1. 1 Prepare Cryptococcus neoformans Inoculum
  2. 2 Expose Organoids to Cryptococcus and Optional Amiloride Treatment
  3. 3 Fix Organoids and Prepare for Imaging
  4. 4 Quantify Fungal Internalization
FITC-Dextran Uptake Assay (2D Culture) Days 15-16
  1. 1 Culture iBMECs on Chamber Slides
  2. 2 Serum Starvation (Optional Control)
  3. 3 Expose Cells to C. neoformans and FITC-Dextran
  4. 4 Remove Surface-Bound Dextran
  5. 5 Fix and Image Cells
  6. 6 Amiloride Inhibition Control
EphA2 Knockout and Validation Days 1-14
  1. 1 CRISPR/Cas9 Transfection of EphA2 KO Plasmids
  2. 2 Incubate Transfected Cells
  3. 3 Puromycin Selection
  4. 4 FACS Purification and Enrichment
  5. 5 Verify EphA2 Knockout by Western Blot
Cdc42 Activation Assay Days 15-16
  1. 1 Culture Wild-Type and EphA2 KO iBMECs
  2. 2 Prepare C. neoformans Inoculum
  3. 3 Expose Cells to Fungal Inoculum
  4. 4 Lysate Preparation
  5. 5 Cdc42 Activation G-LISA Assay
  6. 6 Verification of Cdc42 Protein Expression
Proximity Ligation Assay (PLA) for EphA2-CD44 Complex Detection Days 15-16
  1. 1 Culture Brain Endothelial Cells on Chamber Slides
  2. 2 Promote BBB Phenotype by Growth Factor Reduction
  3. 3 Prepare C. neoformans Inoculum
  4. 4 Co-incubate Fungal Cells with Brain Endothelial Cells
  5. 5 Fix Cells and Perform Proximity Ligation Assay
  6. 6 Image and Quantify PLA Signal

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Bennett et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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