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BRAIN Publication-derived

COVID-19 Brain Organoids for Infection Studies

Full SOP with Academic or a plan

Source Bullen et al., 2020 · Johns Hopkins University, Bloomberg School of Public Health, Center for Alternatives to Animal Testing (CAAT) · 10.14573/altex.2006111

Authors: C. Korin Bullen, Helena T. Hogberg, Asli Bahadirli-Talbott, William R. Bishai, Thomas Hartung, Casey Keuthan, Monika M. Looney, Andrew Pekosz, J. Carolina Romero, Fenna C. M. Sillé, Peter Um, Lena Smirnova Duration: 72 days 6 phases Cell source: Human iPSC (NIBSC-8 and CRL-2097 lines)

Abstract

This protocol establishes a human iPSC-derived 3D BrainSphere model to investigate SARS-CoV-2 infectability of neural cells. BrainSpheres are differentiated over 8 weeks to contain neurons, astrocytes, and oligodendrocytes, then infected with SARS-CoV-2 at MOI 0.1 to detect viral replication and characterize infected neuronal populations through RT-qPCR and immunohistochemistry.

Cell source
Human iPSC (NIBSC-8 and CRL-2097 lines)
Application
Disease modeling - SARS-CoV-2 neurotropism and CNS infectability

Protocol overview

29 steps across 6 phases

Phase 1 · BrainSphere Differentiation Days 0-56 (8 weeks)
  1. a Neural induction from iPSCs
  2. b NPC expansion
  3. c BrainSphere formation initiation
  4. d BrainSphere differentiation induction
  5. e Extended differentiation culture
Phase 2 · Receptor Expression Assessment (ACE2 and TMPRSS2) Days 0-56 (parallel with differentiation)
  1. a RNA extraction from samples
  2. b Reverse transcription
  3. c TaqMan qPCR for ACE2 and TMPRSS2
  4. d Data analysis
Phase 3 · SARS-CoV-2 Infection Protocol Day 56 onwards (post-maturation)
  1. a BrainSphere plating for infection
  2. b Transfer to BSL-3 facility
  3. c Viral inoculation
  4. d Early timepoint sampling (6 hpi)
  5. e Extended culture and late timepoint sampling (72 hpi)
  6. f Control conditions
Phase 4 · Viral RNA Quantification by RT-qPCR Post-infection (6 hpi and 72 hpi sampling)
  1. a RNA extraction from cell lysates
  2. b RNA extraction from supernatants
  3. c cDNA synthesis from viral RNA
  4. d Quantitative RT-qPCR amplification
  5. e Standard curve generation
  6. f Normalization to housekeeping gene (for cell lysates)
  7. g Data analysis and interpretation
Phase 5 · Immunohistochemistry and Viral Localization At 72 hpi
  1. a Fixation of BrainSpheres
  2. b Antibody staining – primary antibodies
  3. c Nuclear counterstain
  4. d Confocal microscopy imaging
  5. e Image analysis
Phase 6 · Optional Extended Culture and Rigorous Washing Variant Post-infection (alternative protocol for high-background reduction)
  1. a Rigorous washing at 6 hpi (alternative method)
  2. b Extended incubation and sampling

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Attribution

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

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