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CARDIAC Publication-derived

COVID-19 Cardiac 2D Cell Cultures

Full SOP with Academic or a plan

Source Marchiano et al. · Department of Pathology, University of Washington, Seattle, USA · 10.1016/j.stemcr.2021.02.005

Authors: Marchiano S, Hsiang TY, Khanna A, Bhatt S, Higashi T, Whitmore LS, Bargehr J, Davaapil H, Chang J, Smith E, Ong LP, Colzani M, Murry CE Duration: 82 days 8 phases Cell source: iPSC-Derived (Human iPSCs)

Abstract

Marchiano et al. demonstrated that SARS-CoV-2 directly infects iPSC-derived cardiomyocytes via ACE2, causing sarcomeric disruption, beating cessation, and innate immune activation. The model confirmed direct cardiac tropism independent of immune infiltration and validated remdesivir as a cardioprotective antiviral agent.

Cell source
iPSC-Derived (Human iPSCs)
Application
COVID-19 Cardiac Research

Protocol overview

44 steps across 8 phases

Phase 1 · iPSC Preparation and Plating Day 0
  1. a Dispase Treatment and Colony Dissociation
  2. b Generate Smaller Colony Clumps
  3. c Plate on Matrigel-Coated Wells
Phase 2 · Definitive Endoderm (DE) and Anterior Foregut Endoderm (AFE) Differentiation Days 1-12
  1. a Initiate DE Differentiation
  2. b Specify to Anterior Foregut Endoderm (AFE)
  3. c Characterize AFE by Immunostaining and qRT-PCR
Phase 3 · Lung Organoid (LORG) Development Days 12-60
  1. a Differentiate AFE to Lung Progenitor Cells
  2. b Generate 3D Lung Organoids (LORGs)
  3. c Confirm LORG Morphology by H&E Staining
  4. d Characterize Cell Type Composition by Immunostaining
  5. e Confirm Gene Expression by qRT-PCR
  6. f Confirm Protein Expression by Western Blot
Phase 4 · Cerebral Organoid (CORG) and Neural Cell Differentiation Days 1-80
  1. a Generate Cerebral Organoids (CORGs)
  2. b Characterize CORG Cell Type Composition
  3. c Differentiate iPSCs into Neural Progenitor Cells (NPCs)
  4. d Differentiate NPCs into Neurons
  5. e Differentiate iPSCs into Astrocytes
  6. f Quantify ACE2 and TMPRSS2 Expression in Neural Cells
Phase 5 · SARS-CoV-2 Pseudovirus Infection Studies Days 60-65 (LORGs); Day 80+ (CORGs/Neural Cells)
  1. a Prepare SARS-CoV-2 Pseudovirus
  2. b Infect LORGs with SARS-CoV-2 Pseudovirus (GFP)
  3. c Visualize GFP Signal in LORGs
  4. d Confirm Viral Co-localization with Receptors
  5. e Quantify Pseudovirus Infection by Luciferase Activity
  6. f Infect CORGs, NPCs, and Neurons with GFP Pseudovirus
  7. g Assess Cell-Type-Specific GFP Infection
  8. h Compare Pseudovirus Infection Efficiency: LORGs vs. CORGs
Phase 6 · SARS-CoV-2 Virus Infection and Drug Inhibition Studies Days 62-66 (LORGs for drug testing); Day 80+ (CORGs/Neural Cells for infection)
  1. a Treat LORGs with Viral Entry Inhibitors
  2. b Infect Inhibitor-Treated LORGs with SARS-CoV-2 Pseudovirus
  3. c Quantify Inhibition of Viral Infection
  4. d Test Inhibitors in Lung Epithelial Cell Line (Calu-3)
  5. e Assess Nafamostat in Patient-Derived SARS-CoV-2 Isolate
Phase 7 · Live SARS-CoV-2 USA-WA1/2020 Infection and Host Response Analysis Days 62-65 (LORGs); Day 80-82 (NPCs/Neurons/Astrocytes)
  1. a Prepare Patient-Derived SARS-CoV-2 USA-WA1/2020 Inoculum
  2. b Infect Day 60 LORGs with USA-WA1/2020 Virus
  3. c Quantify Viral RNA in LORG Supernatant
  4. d Quantify Viral RNA in LORG Cellular Fraction
  5. e Analyze Immune Response Gene Expression in Infected LORGs
  6. f Analyze Inflammasome Pathway Gene Expression in Infected LORGs
  7. g Infect NPCs, Neurons, and Astrocytes with USA-WA1/2020
  8. h Quantify Viral RNA in Neural Cell Supernatant and Cellular Fractions
  9. i Analyze Innate Immunity Gene Expression in Infected Neurons
  10. j Analyze Complement System Gene Expression in Infected Neurons
  11. k Analyze Neuronal Dysfunction and Apoptosis Gene Expression
Phase 8 · Optional: Large-Scale Organoid Culture in Bioreactors Days 80+
  1. a Transfer Organoids to Bioreactor System
  2. b Maintain Bioreactor Culture

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Attribution

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

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