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VASCULAR Publication-derived

COVID-19 Vascular Organoids

Full SOP with Academic or a plan

Source Monteil et al., 2020 · Institute of Molecular Biotechnology of the Austrian Academy of Sciences · 10.1016/j.cell.2020.04.004

Authors: Vanessa Monteil, Hyesoo Kwon, Patricia Prado, Astrid Hagelkrüys, Reiner A. Wimmer, Martin Stahl, Alexandra Leopoldi, Elena Garreta, Carmen Hurtado del Pozo, Felipe Prosper, Juan Pablo Romero, Gerald Wirnsberger, Haibo Zhang, Arthur S. Slutsky, Ryan Conder, Nuria Montserrat, Ali Mirazimi, Josef M. Penninger Duration: 24 days 9 phases Cell source: Human iPSC

Abstract

This protocol describes the use of clinical-grade human recombinant soluble ACE2 (hrsACE2) to inhibit SARS-CoV-2 infection in engineered human tissue organoids, including blood vessel and kidney organoids. The protocol demonstrates that hrsACE2 reduces viral RNA recovery and infectious progeny production in a dose-dependent manner, providing a model system for studying COVID-19 pathogenesis and evaluating therapeutic interventions.

Cell source
Human iPSC
Application
Disease modeling; Viral infection and drug screening

Protocol overview

48 steps across 9 phases

Phase 1 · Preparation of Human Recombinant Soluble ACE2 Prior to infection experiments
  1. a Production of clinical-grade hrsACE2
  2. b Production of murine recombinant soluble ACE2 (mrsACE2) for controls
  3. c Determine protein concentration
Phase 2 · Kidney Organoid Differentiation from Human Embryonic Stem Cells Day -1 to Day 16
  1. a Prepare and seed human embryonic stem cells
  2. b Day 0: Initiate mesoderm induction with CHIR
  3. c Day 3-4: Switch to FGF9 and heparin-containing medium
  4. d Day 4: Form organoid spheroids
  5. e Day 11-16: Culture in basal medium
Phase 3 · Preparation and Culture of Blood Vessel Organoids Prior to infection (established protocol)
  1. a Engineer blood vessel organoids from human iPSCs
  2. b Culture blood vessel organoids in growth-promoting medium
Phase 4 · SARS-CoV-2 Infection of Vero E6 Cells with hrsACE2 Treatment (Early Stage Inhibition) Day 0 (infection) to Day 0.5 + 15 hours
  1. a Prepare Vero E6 cell monolayers
  2. b Mix hrsACE2 with SARS-CoV-2 virus at different MOIs
  3. c Infect Vero E6 cells for 1 hour
  4. d Wash cells and replace with fresh medium
  5. e Incubate cells for 15 hours post-infection
  6. f Harvest cells and extract RNA
  7. g Quantify viral RNA by qRT-PCR
Phase 5 · SARS-CoV-2 Infection of Vero E6 Cells with hrsACE2 (Progeny Virus Assessment) Day 0 (infection) to Day 0.5 + 15 hours
  1. a Mix hrsACE2 or mrsACE2 with SARS-CoV-2 at different MOIs
  2. b Infect Vero E6 cells for 15 hours without washing
  3. c Harvest cells and extract RNA
  4. d Quantify viral RNA by qRT-PCR
Phase 6 · SARS-CoV-2 Infection of Blood Vessel Organoids Day 0 (infection) to Day 6
  1. a Prepare blood vessel organoids for infection
  2. b Infect organoids with SARS-CoV-2
  3. c Wash organoids and replace medium
  4. d Culture infected organoids for 3-6 days
  5. e Harvest organoids at day 3 post-infection for RNA quantification
  6. f Collect supernatant at day 6 post-infection and assess progeny virus
  7. g Infect Vero E6 cells with organoid supernatant
  8. h Quantify viral RNA in Vero E6 cells
Phase 7 · SARS-CoV-2 Infection of Kidney Organoids Day 20 of organoid differentiation through Day 6 post-infection
  1. a Prepare kidney organoids for infection (day 20 differentiation)
  2. b Infect kidney organoids with SARS-CoV-2
  3. c Wash organoids and replace medium
  4. d Culture infected kidney organoids for 6 days
  5. e Harvest organoids at day 6 and assess viral RNA
  6. f Extract and quantify viral RNA by qRT-PCR
  7. g Assess progeny virus infectivity
  8. h Quantify viral RNA in Vero E6 cells at 48 hours post-infection
Phase 8 · Treatment of Blood Vessel and Kidney Organoids with hrsACE2 Day of infection to Day 3 or Day 6 post-infection
  1. a Pre-incubate hrsACE2 with SARS-CoV-2 virus
  2. b Infect organoids with hrsACE2-treated virus
  3. c Wash organoids and replace with fresh medium
  4. d Culture organoids and assess viral RNA at day 3 post-infection
  5. e Culture kidney organoids and assess viral RNA at day 3 post-infection
  6. f Extract RNA and perform qRT-PCR on organoid lysates
  7. g Cytotoxicity assessment (optional quality control)
Phase 9 · Cytotoxicity Assessment of hrsACE2 and mrsACE2 Single-day assay (24-hour pre-treatment + 15-hour exposure)
  1. a Seed Vero E6 cells in 96-well plate
  2. b Add different concentrations of hrsACE2 or mrsACE2
  3. c Assess cell viability using CellTiter-Glo assay
  4. d Interpret cytotoxicity data

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Attribution

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

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