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BRAIN Publication-derived

Choroid Plexus Organoids for Streptococcus suis Translocation Studies at the Blood-Cerebrospinal Fluid Barrier

Source Zhao et al., 2025 · Host-Microbe Interactomics, Department Animal Science, Wageningen University & Research · 10.1186/s12987-025-00627-y

👤 Tiantong Zhao, Laura Pellegrini, Bart van der Hee, Jos Boekhorst, Aline Fernandes, Sylvia Brugman, Peter van Baarlen, Jerry M. Wells ⏱ 40 days 📋 10 phases 🧫 Human iPSC (EDi002-A line)

Abstract

This protocol describes the generation of human iPSC-derived choroid plexus (ChP) organoids that recapitulate the blood-cerebrospinal fluid barrier (BCSFB) in vitro. The organoids form polarized epithelial structures with tight junctions and secrete CSF-like fluid, enabling investigation of bacterial translocation mechanisms and host responses to infection with Streptococcus suis and other pathogens.

Cell source
Human iPSC (EDi002-A line)
Application
Disease modeling and bacterial infection studies

Protocol overview

86 steps across 10 phases

Bacterial Strain Preparation 1 day prior to experiments
  1. 1 Plate bacterial strain on agar media
  2. 2 Prepare liquid culture from overnight colonies
ChP Organoid Generation from Human iPSC Days 1-7 (embryoid body formation and embedding)
  1. 1 Coat 6-well plates with Vitronectin
  2. 2 Maintain human iPSC line EDi002-A
  3. 3 Dissociate iPSC into single cells (Day 1)
  4. 4 Seed cells for embryoid body formation (Day 1)
  5. 5 Add fresh EB Formation Medium (Days 2 and 4)
  6. 6 Assess embryoid body size (Day 5)
  7. 7 Replace medium with Induction Medium (Day 5)
  8. 8 Embed EB in Matrigel droplets (Day 7)
  9. 9 Transfer Matrigel droplets to 6-well plate (Day 7)
  10. 10 Culture in Expansion Medium (Days 7-10)
ChP Organoid Differentiation and Maturation Days 10-40
  1. 1 Replace medium with Choroid Plexus Differentiation Medium (Day 10)
  2. 2 Place plate on rotator platform (Day 10 onwards)
  3. 3 Refresh Choroid Plexus Differentiation Medium (Day 13)
  4. 4 Switch to Maturation Medium (Day 15 onwards)
  5. 5 Monitor cyst formation and morphology (Days 20-30)
  6. 6 Verify mature ChP organoid phenotype (Day 30-40)
ChP Organoid Embedding, Cryosectioning and Immunostaining Variable (post-experimental, for analysis)
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Cryoprotect in sucrose buffer
  3. 3 Pre-warm organoids in gelatin/sucrose solution
  4. 4 Prepare embedding mold with gelatin base
  5. 5 Embed organoid in gelatin block
  6. 6 Prepare gelatin block for freezing
  7. 7 Freeze gelatin block in 2-Methylbutane with dry ice
  8. 8 Prepare cryostat and cut sections
  9. 9 Collect sections on adhesion slides
  10. 10 Thaw slides for immunostaining
  11. 11 Draw hydrophobic borders and rehydrate
  12. 12 Block and permeabilize
  13. 13 Incubate with primary antibodies
  14. 14 Wash and incubate with secondary antibodies
  15. 15 Final wash and mount
  16. 16 Image organoid sections
S. suis Growth in ChP Organoid-derived CSF 1-2 days
  1. 1 Prepare for CSF collection
  2. 2 Collect CSF from organoid lumen
  3. 3 Dispense CSF into assay plate
  4. 4 Prepare S. suis inoculum
  5. 5 Inoculate CSF with S. suis
  6. 6 Monitor S. suis growth in CSF
  7. 7 Analyze growth kinetics
S. suis Translocation Assay 1 day
  1. 1 Prepare ChP organoids and estimate cell numbers
  2. 2 Add culture medium to organoid wells
  3. 3 Prepare S. suis inoculum at MOI 10
  4. 4 Inoculate organoids with S. suis
  5. 5 Wash organoids with warm PBS
  6. 6 Treat with antibiotics to kill extracellular bacteria
  7. 7 Final wash with PBS
  8. 8 Collect CSF from organoid lumen
  9. 9 Enumerate translocated S. suis
Cytotoxicity Assay (LDH Release) Parallel to translocation assay (6-hour S. suis challenge)
  1. 1 Inoculate organoids with S. suis and incubate
  2. 2 Collect culture medium from infected organoids
  3. 3 Add CytoTox 96 Reagent
  4. 4 Incubate assay plate in the dark
  5. 5 Prepare positive control (maximum lysis)
  6. 6 Add Stop Solution and measure absorbance
  7. 7 Calculate cytotoxicity percentage
ChP Organoid Permeability Assay (FITC-dextran) Parallel to translocation assay (6-hour S. suis challenge + 2-hour dextran incubation)
  1. 1 Inoculate organoids with S. suis and incubate
  2. 2 Wash organoids with warm PBS
  3. 3 Incubate organoids with FITC-dextran
  4. 4 Collect CSF from organoid lumen
  5. 5 Measure fluorescence intensity
  6. 6 Calculate FITC intensity percentage
  7. 7 Compare permeability between conditions
RNA Isolation and RNA-seq 1-2 days from organoid harvest
  1. 1 Prepare organoid samples for RNA extraction
  2. 2 Dissociate organoids into single cells
  3. 3 Pellet cells
  4. 4 Lyse cells in Buffer RLT
  5. 5 Add ethanol and transfer to RNeasy column
  6. 6 Complete RNeasy Mini Kit extraction protocol
  7. 7 Remove genomic DNA
  8. 8 Quantify and assess RNA quality
  9. 9 Prepare cDNA library
  10. 10 Quality check library
  11. 11 Pool libraries and sequence
  12. 12 Process raw sequencing data
  13. 13 Align reads to reference genome
  14. 14 Assemble and quantify gene expression
RNA-seq Data Analysis and Gene Set Enrichment Variable (post-sequencing bioinformatics)
  1. 1 Perform differential expression analysis
  2. 2 Generate gene expression heatmaps
  3. 3 Perform Gene Set Enrichment Analysis (GSEA)
  4. 4 Identify significantly enriched GO terms
  5. 5 Visualize GO term networks with Cytoscape
  6. 6 Identify key upregulated and downregulated genes
  7. 7 Analyze gene expression correlations
  8. 8 Perform principal component analysis (PCA)

Full SOP

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Attribution

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

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