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BRAIN Publication-derived

COVID-19 Brain Organoids with Astrocytes

Full SOP with Academic or a plan

Source Kong et al., 2022 · Gladstone Institute of Virology, San Francisco, California, USA · 10.1128/mbio.02308-22

Authors: Weili Kong, Mauricio Montano, Michael J. Corley, Ekram Helmy, Hirofumi Kobayashi, Martin Kinisu, Rahul Suryawanshi, Xiaoyu Luo, Loic A. Royer, Nadia R. Roan, Melanie Ott, Lishomwa C. Ndhlovu, Warner C. Greene Duration: 129 days 9 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of iPSC-derived brain organoids and infection with SARS-CoV-2 to model viral infection of astrocytes and neuronal dysfunction. The procedure produces 4-month-old cerebral organoids containing astrocytes, neurons, and neural progenitor cells, which are then infected with SARS-CoV-2 to study receptor-mediated entry, transcriptomic changes, and consequent bystander neuronal death driven by astrocyte infection.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

39 steps across 9 phases

Phase 1 · iPSC Maintenance and Organoid Initiation Days 0-5
  1. a iPSC Culture Maintenance
  2. b Embryoid Body Formation
  3. c EB Medium Supplementation (Days 2 and 4)
  4. d Transfer to Neural Induction Medium
  5. e Matrigel Embedding and Organoid Formation
Phase 2 · Organoid Culture and Maturation Days 8-120
  1. a Transfer to Neural Culture Medium and Orbital Shaker
  2. b Monitor Organoid Development
Phase 3 · SARS-CoV-2 Infection of Brain Organoids Day 120-127
  1. a Virus Preparation and Biosafety Setup
  2. b SARS-CoV-2 Infection of 4-Month-Old Organoids
  3. c Monitor Infection and Organoid Integrity
  4. d Prepare Organoids for Immunostaining or Analysis
Phase 4 · Immunostaining of Brain Organoids Day 127-129 (post-infection + immunostaining)
  1. a Organoid Dehydration and Cryoprotection
  2. b OCT Embedding and Freezing
  3. c Cryosectioning
  4. d Permeabilization and Blocking
  5. e Primary Antibody Incubation
  6. f Washing and Secondary Antibody Incubation
  7. g Mounting and Imaging
Phase 5 · Primary Human Astrocyte Culture and Infection Variable (culture maintenance + infection)
  1. a Primary Astrocyte Culture Setup
  2. b SARS-CoV-2 Infection of Primary Astrocytes
  3. c Maintain Infected Cultures
  4. d Harvest Astrocytes for Analysis
Phase 6 · RT-qPCR Analysis of Viral and Host Gene Expression Post-harvest analysis
  1. a RNA Extraction
  2. b cDNA Synthesis
  3. c Real-Time qPCR Setup and Amplification
  4. d Data Analysis and Normalization
Phase 7 · Viral Titer Determination by Plaque Assay Post-harvest analysis
  1. a Supernatant Collection
  2. b Plaque Assay on Vero-E6 Cells
  3. c Titer Calculation
Phase 8 · siRNA Knockdown and Receptor Identification Variable (3-day knockdown + 3-day infection)
  1. a siRNA Transfection into Astrocytes
  2. b Knockdown Efficiency Assessment (24-48 h post-transfection)
  3. c SARS-CoV-2 Infection of Knockdown Cells
  4. d Viral Gene Expression Analysis
  5. e Statistical Analysis
Phase 9 · TUNEL Assay for Neuronal Cell Death Detection Post-infection (7 days post-infection of brain organoids)
  1. a Organoid Fixation and Preparation
  2. b TUNEL Staining Protocol
  3. c Immunofluorescence for Neuronal Markers and Viral Antigen
  4. d Image Acquisition and Analysis
  5. e Statistical Quantification

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Attribution

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

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