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BRAIN Publication-derived

Generation of hiPSC-Derived Choroid Plexus Organoids and SARS-CoV-2 Infection Protocol

Source Jacob et al., 2020 · University of Pennsylvania, Perelman School of Medicine · 10.1016/j.stem.2020.09.016

👤 Fadi Jacob, Sarshan R. Pather, Wei-Kai Huang, Hongjun Song, Juan Carlos de la Torre, Guo-li Ming ⏱ 72 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human induced pluripotent stem cell (hiPSC)-derived choroid plexus organoids (CPOs) and their infection with SARS-CoV-2 to model viral neurotropism and cellular consequences of infection. CPOs exhibit morphology and transcriptome similar to adult human choroid plexus tissue and express high levels of SARS-CoV-2 receptors, enabling investigation of productive viral infection, cell death, and inflammatory dysregulation.

Cell source
Human iPSC
Application
Disease modeling; SARS-CoV-2 infection susceptibility study

Protocol overview

27 steps across 6 phases

hiPSC Preparation and Embryoid Body Formation Day 0–1
  1. 1 Culture hiPSCs in feeder-free conditions
  2. 2 Dissociate hiPSC colonies into clusters
  3. 3 Prepare hiPSC clusters for aggregation
  4. 4 Form embryoid bodies using Aggrewell plate
Neural Induction and Choroid Plexus Patterning Day 1–8
  1. 1 Transfer EBs to neural induction medium
  2. 2 Maintain EBs in neural induction medium
  3. 3 Transfer EBs to choroid plexus induction medium
  4. 4 Begin orbital shaker culture
Choroid Plexus Organoid Maturation and Expansion Day 8–30
  1. 1 Perform half-medium replacements during induction phase
  2. 2 Monitor organoid morphology
  3. 3 Transition to differentiation medium on Day 30
Choroid Plexus Organoid Differentiation and Maturation Day 30–50+
  1. 1 Continue orbital shaker culture in differentiation medium
  2. 2 Monitor maturation and morphology
  3. 3 Maintain CPOs for extended culture (Day 50–100+)
SARS-CoV-2 Infection of Choroid Plexus Organoids Day 47–67+
  1. 1 Prepare SARS-CoV-2 virus stock
  2. 2 Prepare infection medium
  3. 3 Transfer CPOs to infection plates
  4. 4 Infect CPOs with SARS-CoV-2
  5. 5 Wash infected CPOs
  6. 6 Return infected CPOs to culture
Post-Infection Analysis and Sampling 24–72 hours post-infection (hpi)
  1. 1 Collect samples for immunostaining at 24 hpi
  2. 2 Cryoprotect fixed organoids
  3. 3 Freeze organoids for cryosectioning
  4. 4 Prepare tissue sections for immunostaining
  5. 5 Collect supernatant and lysate for viral titer at 24 and 72 hpi
  6. 6 Collect samples for RNA extraction at 24 and 72 hpi
  7. 7 Repeat sampling at 72 hpi

Full SOP

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Attribution

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

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