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BRAIN Publication-derived

COVID-19 Choroid Plexus Organoids

Full SOP with Academic or a plan

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

Authors: Fadi Jacob, Sarshan R. Pather, Wei-Kai Huang, Hongjun Song, Juan Carlos de la Torre, Guo-li Ming Duration: 72 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Jacob et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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