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LUNG Publication-derived

Generation and Characterization of Human iPSC-Derived Lung and Cerebral Organoids for SARS-CoV-2 Infection Studies

Source Tiwari et al. · University of California, San Diego (UCSD)

👤 Shashi Kant Tiwari, Shaobo Wang, Davey Smith, Aaron F. Carlin, Tariq M. Rana ⏱ 80 days 📋 16 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation of human induced pluripotent stem cells (hiPSCs) into lung organoids (LORGs) and cerebral organoids (CORGs) for modeling tissue-specific SARS-CoV-2 infection and characterizing host responses. The lung organoids develop alveolar and airway epithelial cell types expressing viral entry receptors (ACE2) and proteases (TMPRSS2), enabling study of viral infection mechanisms and therapeutic interventions.

Cell source
Human iPSC
Application
Disease modeling; SARS-CoV-2 infection studies

Protocol overview

80 steps across 16 phases

hiPSC Maintenance and Preparation Ongoing until 50-75% confluency reached
  1. 1 Culture hiPSCs on Growth-Factor-Reduced Matrigel
  2. 2 Passage hiPSCs via Dispase Treatment
  3. 3 Dissociate and Plate hiPSC Clumps
  4. 4 Monitor Confluency
Definitive Endoderm Differentiation Days 1-4
  1. 1 Day 1 Medium: RPMI + Activin A
  2. 2 Day 2 Medium: RPMI + FBS + Activin A
  3. 3 Days 3-4 Medium: RPMI + Higher FBS + Activin A
Anterior Foregut Endoderm Differentiation Days 5-10
  1. 1 Prepare Foregut Basal Medium
  2. 2 Supplement Basal Medium with Growth Factors and Small Molecules
  3. 3 Replace Medium with Supplemented Foregut Medium
  4. 4 Daily Medium Changes
3D Lung Organoid Culture (Step 1) Days 10-18
  1. 1 Embed Foregut Spheroids in Matrigel
  2. 2 Prepare 3D Culture Basal Medium
  3. 3 Prepare and Add 3D Step 1 Medium
  4. 4 Change Medium Every Other Day for 8 Days (Step 1)
3D Lung Organoid Culture (Step 2) Days 18-33
  1. 1 Transition to 3D Step 2 Medium on Day 25 Onwards
  2. 2 Change Step 2 Medium Every 2 Days for 8 Days
  3. 3 Supplement with cAMP, IBMX, and Dexamethasone
  4. 4 Re-embed Organoids Every 2-3 Weeks for Long-Term Culture
  5. 5 Use 60-Day-Old Organoids for Analysis
Cerebral Organoid Generation Days 0-80+
  1. 1 Culture hiPSCs on Matrigel and Prepare for Embryoid Body Formation
  2. 2 Form Embryoid Bodies via Hanging Drop Method
  3. 3 Transfer Embryoid Bodies to Petri Dishes (Day 2)
  4. 4 Neural Induction (Days 2-11)
  5. 5 Matrigel Embedding and 3D Culture Initiation (Day 11)
  6. 6 Transition to Bioreactor Culture at Day 15
  7. 7 Long-Term Culture with Vitamin A (Day 15+)
  8. 8 Harvest at 80 Days
Astrocyte Differentiation from NPCs 0-30 days
  1. 1 Plate NPCs on Matrigel
  2. 2 Culture NPCs for 2 Days
  3. 3 Switch to Astrocyte Differentiation Medium
  4. 4 Change Medium Every 3-4 Days for 1 Month
Neuronal Differentiation from NPCs 0-14 days
  1. 1 Coat Culture Dishes with Polyornithine and Laminin
  2. 2 Plate hNPCs in Complete NPC Medium
  3. 3 Culture NPCs for 2 Days
  4. 4 Switch to Neural Differentiation Medium
  5. 5 Change Medium Every 3-4 Days for 2 Weeks
Cell Line Maintenance (293FT and Calu-3) Ongoing
  1. 1 Culture 293FT Cells in DMEM + 10% FBS
  2. 2 Culture Calu-3 Cells in EMEM + 10% FBS + Antibiotics
  3. 3 Maintain Standard Culture Conditions
Organoid Sectioning for Immunofluorescence Single preparation
  1. 1 Wash Organoids with PBS
  2. 2 Recover Organoids from Matrigel
  3. 3 Wash and Fix Organoids
  4. 4 PBS Wash and Hematoxylin Staining
  5. 5 Incubate in 30% Sucrose Overnight
  6. 6 Wash and Embed in OCT
  7. 7 Cryosection Organoids
  8. 8 Block and Incubate with Primary Antibodies
  9. 9 Wash and Incubate with Secondary Antibodies
  10. 10 Final Wash and Mounting
qRT-PCR Analysis Single preparation
  1. 1 Extract Viral RNA from Supernatant
  2. 2 Extract Cellular RNA
  3. 3 Quantify RNA
  4. 4 Perform qRT-PCR with Gene-Specific Primers
Western Blotting Single preparation
  1. 1 Lyse Organoids in RIPA Buffer
  2. 2 Extract Proteins and Determine Concentration
  3. 3 Resolve Proteins by SDS-PAGE
  4. 4 Block Membranes and Incubate with Primary Antibodies
  5. 5 Wash Membranes and Incubate with Secondary Antibodies
  6. 6 Detect Immunoreactive Proteins
SARS-CoV-2 Pseudovirus Production and Infection Production: 1-2 days; Infection: 1 day
  1. 1 Transfect 293FT Cells with SARS-CoV-2 Spike Expression Plasmid
  2. 2 Infect Transfected Cells with VSV Pseudovirus
  3. 3 Wash Cells and Culture for Pseudovirus Production
  4. 4 Harvest and Store Pseudovirus Supernatant
SARS-CoV-2 Pseudovirus Infection of Organoids with Protease Inhibitor Treatment Single infection experiment
  1. 1 Pre-treat Lung Organoids with TMPRSS2 Inhibitor
  2. 2 Infect Organoids with Pseudovirus at MOI=2
  3. 3 Remove Virus and Add Fresh Medium
  4. 4 Measure Luciferase Activity
  5. 5 Optional: Treat with Nafamostat or EK-1 Peptide
Live Pseudovirus Infection Imaging Single infection experiment
  1. 1 Infect Organoids with EGFP-Pseudovirus
  2. 2 Remove Virus and Maintain Culture
  3. 3 Image Organoids at 24 Hours Post-Infection
  4. 4 Quantify Infection by Image Analysis
SARS-CoV-2 USA-WA1/2020 Isolate Infection Single infection experiment (harvest at 2, 48, 72 hours)
  1. 1 Prepare SARS-CoV-2 Isolate USA-WA1/2020
  2. 2 Infect Organoids and Cell Types at MOI=2
  3. 3 Wash Cells and Replace with Fresh Medium
  4. 4 Harvest Supernatant at 2, 48, and 72 Hours
  5. 5 Harvest and Lyse Infected Organoids/Cells
  6. 6 Extract Viral RNA Using Direct-zol RNA Kit
  7. 7 Quantify Viral RNA by qRT-PCR Using SARS-CoV-2 N Primers

Full SOP

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Attribution

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

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