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BRAIN Publication-derived

Infectability of Human BrainSphere Neurons Suggests Neurotropism of SARS-CoV-2

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

👤 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 ⏱ 72 days 📋 6 phases 🧫 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

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

Full SOP

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Attribution

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

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