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BRAIN Publication-derived

Choroid Plexus Organoid Generation and Bacterial Infection Assays for Streptococcus suis Translocation Studies at the Blood-Cerebrospinal Fluid Barrier

Source Zhao et al., 2025 · 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 📋 13 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human iPSC-derived choroid plexus organoids that form polarized epithelial barriers with CSF-like fluid production, enabling in vitro modeling of blood-cerebrospinal fluid barrier (BCSFB) function. The organoids are used to investigate bacterial translocation of Streptococcus suis and the role of plasminogen-mediated mechanisms in pathogen crossing of the barrier without disruption of barrier integrity.

Cell source
Human iPSC
Application
Disease modeling; Infection mechanism investigation

Protocol overview

61 steps across 13 phases

Bacterial Culture Preparation Day -1 to Day 0
  1. 1 Overnight Bacterial Agar Culture
  2. 2 Liquid Culture Inoculation
iPSC Maintenance and Preparation Ongoing until Day 1 of differentiation
  1. 1 Vitronectin Coating of Culture Plates
  2. 2 iPSC Culture Maintenance
Embryoid Body Formation (Days 1–5) Day 1–Day 5
  1. 1 iPSC Dissociation into Single Cells
  2. 2 EB Formation Day 1 Seeding
  3. 3 EB Expansion Days 2 and 4
  4. 4 EB Size Assessment (Day 5)
  5. 5 Medium Replacement with Induction Medium
Matrigel Embedding and Neuroepithelial Expansion (Days 6–10) Day 6–Day 10
  1. 1 Matrigel Droplet Preparation (Day 7)
  2. 2 Transfer to Ultralow Adherent Plates
  3. 3 Neuroepithelial Expansion Incubation (Days 7–10)
  4. 4 Medium Replacement on Day 10
Choroid Plexus Differentiation and Maturation (Days 10–40) Day 10–Day 40
  1. 1 Continued Differentiation with Rotating Platform (Days 10–15)
  2. 2 Differentiation Medium Refresh (Day 13)
  3. 3 Transition to Maturation Medium (Day 15 onwards)
  4. 4 Cyst Formation Monitoring (Days 15–30)
  5. 5 Organoid Selection for Experiments
Streptococcus suis Translocation Assay (6-hour Infection) Day 30–Day 40
  1. 1 Bacterial Preparation
  2. 2 Organoid Placement and Medium Preparation
  3. 3 Bacterial Inoculation
  4. 4 Infection Incubation
  5. 5 Antibiotic Treatment to Eliminate Extracellular Bacteria
  6. 6 CSF Collection and Bacterial Enumeration
Cytotoxicity Assay (LDH Release Measurement) Day 30–Day 40
  1. 1 Culture Medium Collection Post-Infection
  2. 2 LDH Reagent Preparation and Incubation
  3. 3 Stop Solution and Absorbance Measurement
  4. 4 Cytotoxicity Calculation
Barrier Permeability Assay (FITC-Dextran Influx) Day 30–Day 40
  1. 1 Post-Infection Organoid Washing
  2. 2 FITC-Dextran Incubation
  3. 3 CSF Collection and Fluorescence Measurement
  4. 4 Permeability Calculation
S. suis Growth Assay in Organoid-Derived CSF Day 30–Day 40
  1. 1 CSF Collection from Organoid Lumen
  2. 2 Bacterial Preparation for Growth Assay
  3. 3 Inoculation of CSF with S. suis
  4. 4 Growth Monitoring
  5. 5 Growth Curve Analysis
Immunofluorescent Staining and Confocal Imaging Day 30–Day 40
  1. 1 Organoid Fixation
  2. 2 PBS Washing and Sucrose Cryoprotection
  3. 3 Gelatin/Sucrose Embedding and Freezing
  4. 4 Cryosectioning
  5. 5 Slide Thawing and Blocking
  6. 6 Primary Antibody Incubation
  7. 7 Secondary Antibody Incubation
  8. 8 Mounting and Imaging
RNA Extraction and Preparation for Sequencing Day 30–Day 40
  1. 1 Organoid Dissociation
  2. 2 Cell Pelleting and Lysis
  3. 3 RNA Precipitation and Binding
  4. 4 Column-Based RNA Purification
  5. 5 Additional Genomic DNA Removal
  6. 6 RNA Quality and Quantity Assessment
Library Preparation and RNA Sequencing As needed for sequencing
  1. 1 mRNA Purification
  2. 2 cDNA Synthesis
  3. 3 Library Quality Control
  4. 4 Multiplexed Sequencing
RNA-seq Data Processing and Analysis Post-sequencing analysis
  1. 1 Raw Read Quality Control and Trimming
  2. 2 Genome Index and Read Alignment
  3. 3 Transcript Assembly and Gene Quantification
  4. 4 Differential Expression Analysis
  5. 5 Gene Set Enrichment Analysis (GSEA)
  6. 6 Data Visualization

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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