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BRAIN Publication-derived

COVID-19 Cortical Organoids with Astrocytes

Full SOP with Academic or a plan

Source Andrews et al., 2022 · University of California, San Francisco · 10.1073/pnas.2122236119

Authors: Madeline G. Andrews, Tanzila Mukhtar, Ugomma C. Eze, Camille R. Simoneau, Jayden Ross, Neelroop Parikshak, Shaohui Wang, Li Zhou, Mark Koontz, Dmitry Velmeshev, Clara-Vita Siebert, Kaila M. Gemenes, Takako Tabata, Yonatan Perez, Li Wang, Mohammed A. Mostajo-Radji, Martina de Majo, Kevin C. Donohue, David Shin, Jahan Salma, Alex A. Pollen, Tomasz J. Nowakowski, Erik Ullian, G. Renuka Kumar, Ethan A. Winkler, Elizabeth E. Crouch, Melanie Ott, Arnold R. Kriegstein Duration: 5 days 9 phases Cell source: Human iPSC and primary cortical tissue (developmental and adult)

Abstract

This protocol evaluates SARS-CoV-2 infection of human cortical cells using organotypic slice cultures and iPSC-derived cortical organoids at multiple developmental stages. The method demonstrates robust infection and viral replication in cortical astrocytes (both immature and mature) via CD147 and DPP4 entry factors, with minimal infection in neurons. Infected astrocytes display inflammatory gene expression, reactivity markers (SYNM, EGFR, VIM), and endoplasmic reticulum stress, providing a model of viral neurotropism relevant to COVID-19 neurological symptoms.

Cell source
Human iPSC and primary cortical tissue (developmental and adult)
Application
Disease modeling (COVID-19 neuropathology and astrocyte tropism)

Protocol overview

34 steps across 9 phases

Phase 1 · Primary Cortical Tissue Preparation and Viral Exposure Day 0
  1. a Prepare organotypic cortical tissue slices
  2. b Expose tissue slices to SARS-CoV-2
  3. c Wash and change media
Phase 2 · Cortical Organoid Preparation and Viral Exposure Day 0 (varies by differentiation timepoint)
  1. a Differentiate iPSC-derived cortical organoids
  2. b Expose organoids to SARS-CoV-2
  3. c Wash organoids and change media
Phase 3 · Viral Replication and Infection Assessment (72 hours post-exposure) Day 3
  1. a Culture infected tissue/organoids
  2. b Collect supernatant for plaque assays
  3. c Perform plaque assay on Vero cells
  4. d Extract tissue and prepare for downstream analysis
Phase 4 · Viral RNA Detection and Quantification Day 0-3 (concurrent with culture)
  1. a Extract RNA from infected tissues
  2. b Perform quantitative RT-PCR (qRT-PCR) for viral RNA
Phase 5 · Loss-of-Function and Gain-of-Function Studies Day -5 to Day 3
  1. a Prepare lentiviral shRNA constructs
  2. b Transduce primary tissue with lentiviral shRNAs (knockdown)
  3. c Expose knockdown tissue to SARS-CoV-2
  4. d Overexpress CD147 or DPP4 in early organoids (gain-of-function)
Phase 6 · Pharmacological Inhibition Studies Day -1 to Day 3
  1. a Treat tissue with DPP4 inhibitor
  2. b Expose inhibitor-treated tissue to SARS-CoV-2
  3. c Quantify infection and cellular stress in inhibitor-treated samples
Phase 7 · Immunofluorescence Analysis Day 3 (post-infection)
  1. a Fix infected tissue samples
  2. b Prepare sections for staining
  3. c Perform immunostaining for viral and cellular markers
  4. d Mount and image samples
  5. e Quantify cell populations and colocalization
Phase 8 · RNA-Sequencing and Gene Expression Analysis Day 3 (post-infection)
  1. a Extract RNA from infected primary cultures
  2. b Perform bulk RNA-sequencing (RNA-seq)
  3. c Perform differential gene expression analysis
  4. d Perform pathway enrichment analysis
  5. e Correlate gene expression with viral load
  6. f Analyze cell state and reactivity signatures
Phase 9 · Receptor Expression and Apoptosis Assays Day 3 (post-infection)
  1. a Perform in situ hybridization (ISH) for ACE2 RNA
  2. b Perform immunohistochemistry (IHC) for ACE2 protein
  3. c Assess apoptosis by cleaved caspase-3 and TUNEL staining
  4. d Quantify endoplasmic reticulum stress marker ARCN1

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Attribution

This SOP was written by Organthis based on the published method in Andrews et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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