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LIVER Publication-derived

Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection

Source Baktash et al., 2018 · Department of Microbiology, The University of Chicago · 10.1016/j.chom.2018.02.005

👤 Yasmine Baktash, Anisha Madhav, Kelly E. Coller, Glenn Randall ⏱ 8 days 📋 9 phases 🧫 Human Hepatoma (Huh-7.5)

Abstract

This protocol describes the generation of three-dimensional polarized hepatoma organoids embedded in Matrigel and their use for single-particle imaging of HCV entry. The organoids display functional polarity with distinct basolateral and apical membrane compartments, enabling visualization of the sequential and ordered HCV entry process involving receptor trafficking to the tight junction, receptor engagement, and EGFR-dependent internalization via clathrin-mediated endocytosis.

Cell source
Human Hepatoma (Huh-7.5)
Application
Disease modeling; Viral entry mechanism characterization

Protocol overview

40 steps across 9 phases

Polarized Hepatoma Organoid Generation Days 0–8
  1. 1 Prepare Matrigel-cell mixture
  2. 2 Seed organoids on coverslips
  3. 3 Culture organoids
Polarity Validation Day 6–8
  1. 1 Immunofluorescence staining for polarity markers
  2. 2 Secondary antibody incubation and visualization
  3. 3 Functional polarity assessment (CMFDA retention)
HCV Labeling and Purification Days 0–3 (parallel to organoid generation)
  1. 1 Generate and concentrate HCV virions
  2. 2 Label virus with DiD
  3. 3 Gradient purification
  4. 4 Concentration and buffer exchange
  5. 5 Quality control via electron microscopy
Fixed-Cell Infection and Imaging Time Course Day 7–8 (organoid infection and processing)
  1. 1 Pre-cool organoids and prepare infection mixture
  2. 2 Apply virus and synchronize entry
  3. 3 Fix organoids
  4. 4 Permeabilization and blocking
  5. 5 Primary antibody incubation
  6. 6 Matrigel reformation and washing
  7. 7 Secondary antibody incubation
  8. 8 Mounting and microscopy
Live-Cell Single-Particle Tracking Day 7–8
  1. 1 Extract organoids and resuspend in imaging medium
  2. 2 Plate organoids on imaging dishes
  3. 3 Synchronize infection with temperature shift
  4. 4 Live-cell confocal imaging
  5. 5 Image processing and particle tracking
Pharmacological Perturbation and Internalization/Uncoating Assays Day 7–8
  1. 1 CD81 blocking antibody treatment
  2. 2 Actin polymerization inhibition (cytochalasin D)
  3. 3 EGFR kinase inhibition (erlotinib treatment)
  4. 4 DiD-HCV uncoating assay
  5. 5 Endosomal acidification inhibition (ammonium chloride treatment)
  6. 6 Clathrin and Rab5a colocalization imaging
Genetic Manipulation and Complementation Studies Days 0–7 (parallel to organoid generation)
  1. 1 Generate shRNA-mediated EGFR knockdown Huh-7.5 cells
  2. 2 Generate shRNA-resistant EGFR complementation constructs
  3. 3 Produce lentiviral particles and transduce shEGFR cells
  4. 4 Validate entry phenotype in shEGFR organoids
  5. 5 Assess HCV replication in complemented cells
EGFR Phosphorylation and Signaling Analysis Day 8
  1. 1 Western blot analysis of EGFR phosphorylation kinetics
  2. 2 SDS-PAGE and immunoblotting
Immunofluorescence Visualization of Phospho-EGFR Day 7–8
  1. 1 Fixed-cell phospho-EGFR and total EGFR staining
  2. 2 Secondary staining and z-stack imaging
  3. 3 Interpretation of subcellular localization

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Baktash et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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