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BRAIN Publication-derived

Derivation and SARS-CoV-2 Infection of Human Cortical Organoids and Organotypic Cultures

Source University of California, San Francisco

👤 M.G.A., T.M., C.R.S., G.R.K, M.O., A.R.K. ⏱ 70 days 📋 18 phases 🧫 Human iPSC, Human ESC, Patient-Derived Primary Cortical Tissue

Abstract

This protocol describes the differentiation of human pluripotent stem cells (iPSCs and ESCs) into cortical organoids, maintenance of primary cortical tissue in organotypic slice cultures, and subsequent infection with SARS-CoV-2 to model viral tropism and cellular responses in developing and adult human cortex. Organoids and slices are analyzed for viral infection patterns, inflammatory responses, and receptor/protease expression.

Cell source
Human iPSC, Human ESC, Patient-Derived Primary Cortical Tissue
Application
Disease modeling, Viral infection study, Drug screening

Protocol overview

110 steps across 18 phases

Pluripotent Stem Cell Expansion Passage-based (P25–P40)
  1. 1 Prepare Matrigel-coated plates
  2. 2 Thaw pluripotent stem cell lines
  3. 3 Plate cells on Matrigel
  4. 4 Culture and passage cells
  5. 5 Validate cell lines
Cortical Organoid Differentiation Day 0–70 (22 weeks of culture)
  1. 1 Dissociate PSC lines to single cells
  2. 2 Prepare neural induction media and seed organoids
  3. 3 Apply Rock inhibitor for initial 6 days
  4. 4 Transfer organoids to larger plates (day 18)
  5. 5 Change media at day 35
  6. 6 Supplement media at day 70
  7. 7 Maintain feeding schedule
  8. 8 Collect organoids for analyses
Organotypic Slice Culture of Developing Tissue Day 0–10 (culture duration)
  1. 1 Prepare dissection medium
  2. 2 Obtain and section developing cortical tissue
  3. 3 Plate slices on Millicell inserts
  4. 4 Culture at air-liquid interface
  5. 5 Maintain culture and feeding
Organotypic Slice Culture of Adult Tissue Day 0–5 (culture duration)
  1. 1 Obtain human tissue and prepare NMDG-ACSF
  2. 2 Transport tissue with continuous carbonation
  3. 3 Acute section tissue
  4. 4 Plate slices on Millicell inserts
  5. 5 Prepare culture media
  6. 6 Culture at air-liquid interface
  7. 7 Maintain slices and feed
Supporting Cell Cultures (NHA, Vero, Astrocyte Spheroids) Variable (depends on cell type)
  1. 1 Culture Normal Human Astrocytes (NHA)
  2. 2 Culture Vero cells
  3. 3 Isolate primary radial glia
  4. 4 Generate and expand astrocyte spheroids
  5. 5 Validate astrocyte progenitor cells (day 160)
  6. 6 Differentiate astrocytes for ICC (day 0 of ICC)
  7. 7 Fix and process for ICC (day 7)
SARS-CoV-2 Infection Protocol Day 0–3 (infection and collection)
  1. 1 Prepare SARS-CoV-2 inoculum
  2. 2 Determine MOI and cell count
  3. 3 Incubate tissue with virus
  4. 4 Remove inoculum and refresh media
  5. 5 Incubate infected tissue
  6. 6 Collect culture media for viral titering
  7. 7 Wash and fix tissue
DPP4 Inhibitor Experiment Day -1 to Day 3 (relative to infection)
  1. 1 Prepare Vildagliptin inhibitor solution
  2. 2 Pre-treat tissue slices
  3. 3 Maintain inhibitor during infection
Plaque Assay for Viral Titering Day 0–3 (post-infection culture media collection)
  1. 1 Collect and dilute culture media
  2. 2 Inoculate Vero E6 cells
  3. 3 Apply overlay
  4. 4 Incubate for plaque formation
  5. 5 Fix and stain plaques
  6. 6 Count and calculate titer
Immunohistochemistry on Organoids Day 1–4 (post-infection)
  1. 1 Fix organoids
  2. 2 Wash and cryopreserve
  3. 3 Embed in cryomolds
  4. 4 Cryosection organoids
  5. 5 Antigen retrieval
  6. 6 Block tissue
  7. 7 Apply primary antibodies
  8. 8 Wash slides
  9. 9 Apply secondary antibodies
  10. 10 Final wash and mount
Immunohistochemistry on Organotypic Slices Day 1–5 (post-infection or collection)
  1. 1 Fix tissue slices
  2. 2 Wash with PBS
  3. 3 Cryopreserve in sucrose
  4. 4 Embed in cryomolds
  5. 5 Cryosection slices
  6. 6 Antigen retrieval
  7. 7 Permeabilize and block
  8. 8 Apply primary antibodies
  9. 9 Wash slides
  10. 10 Apply secondary antibodies
  11. 11 Final wash and imaging
RNA Isolation and qPCR Variable (sample-dependent)
  1. 1 Collect tissue samples
  2. 2 Extract RNA
  3. 3 QC RNA quality
  4. 4 Synthesize cDNA
  5. 5 Perform qPCR
  6. 6 Analyze qPCR data
Bulk RNA Sequencing and Analysis Variable (sample-dependent)
  1. 1 Collect and extract RNA from primary tissue
  2. 2 Prepare library and sequence
  3. 3 Align and quantify reads
  4. 4 Perform differential expression analysis
  5. 5 Perform pathway analysis
TUNEL Assay for Cell Death Detection Day 0–1 (post-fixation)
  1. 1 Post-fix tissue on slides
  2. 2 Wash with TBS
  3. 3 Permeabilize tissue
  4. 4 Wash with TBS
  5. 5 Apply TUNEL reagent
  6. 6 Final wash
  7. 7 Mount and visualize
In Situ Hybridization (RNAscope) Day 0–1 (post-fixation)
  1. 1 Prepare fixed tissue samples
  2. 2 Perform RNAscope multiplex fluorescence assay
  3. 3 Perform antibody poststaining
Western Blotting Day 0–1
  1. 1 Extract protein lysates
  2. 2 Prepare samples
  3. 3 Separate proteins by SDS-PAGE
  4. 4 Block and incubate with primary antibodies
  5. 5 Incubate with secondary antibodies
  6. 6 Detect signal by chemiluminescence
Lentiviral Knockdown of Receptor Genes Variable (virus generation and infection)
  1. 1 Clone shRNA constructs
  2. 2 Generate lentiviruses
  3. 3 Concentrate lentiviral particles
  4. 4 Infect primary astrocytes
  5. 5 Validate knockdown efficiency
Lentiviral Overexpression of Receptor Genes Variable (virus generation and infection)
  1. 1 Subclone receptor cDNAs
  2. 2 Generate overexpression lentiviruses
  3. 3 Concentrate lentiviruses
  4. 4 Infect organoid samples
  5. 5 Validate overexpression
Quantification and Image Analysis Variable (post-staining)
  1. 1 Image infected cells/tissue
  2. 2 Quantify infected cell types
  3. 3 Quantify reactivity and stress markers
  4. 4 Quantify inhibitor experiments

Full SOP

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Attribution

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

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