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LIVER Publication-derived

Hepatitis C Liver Organoids for Infection Studies

Full SOP with Academic or a plan

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

Authors: Yasmine Baktash, Anisha Madhav, Kelly E. Coller, Glenn Randall Duration: 8 days 9 phases Cell source: 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

Phase 1 · Polarized Hepatoma Organoid Generation Days 0-8
  1. a Prepare Matrigel-cell mixture
  2. b Seed organoids on coverslips
  3. c Culture organoids
Phase 2 · Polarity Validation Day 6-8
  1. a Immunofluorescence staining for polarity markers
  2. b Secondary antibody incubation and visualization
  3. c Functional polarity assessment (CMFDA retention)
Phase 3 · HCV Labeling and Purification Days 0-3 (parallel to organoid generation)
  1. a Generate and concentrate HCV virions
  2. b Label virus with DiD
  3. c Gradient purification
  4. d Concentration and buffer exchange
  5. e Quality control via electron microscopy
Phase 4 · Fixed-Cell Infection and Imaging Time Course Day 7-8 (organoid infection and processing)
  1. a Pre-cool organoids and prepare infection mixture
  2. b Apply virus and synchronize entry
  3. c Fix organoids
  4. d Permeabilization and blocking
  5. e Primary antibody incubation
  6. f Matrigel reformation and washing
  7. g Secondary antibody incubation
  8. h Mounting and microscopy
Phase 5 · Live-Cell Single-Particle Tracking Day 7-8
  1. a Extract organoids and resuspend in imaging medium
  2. b Plate organoids on imaging dishes
  3. c Synchronize infection with temperature shift
  4. d Live-cell confocal imaging
  5. e Image processing and particle tracking
Phase 6 · Pharmacological Perturbation and Internalization/Uncoating Assays Day 7-8
  1. a CD81 blocking antibody treatment
  2. b Actin polymerization inhibition (cytochalasin D)
  3. c EGFR kinase inhibition (erlotinib treatment)
  4. d DiD-HCV uncoating assay
  5. e Endosomal acidification inhibition (ammonium chloride treatment)
  6. f Clathrin and Rab5a colocalization imaging
Phase 7 · Genetic Manipulation and Complementation Studies Days 0-7 (parallel to organoid generation)
  1. a Generate shRNA-mediated EGFR knockdown Huh-7.5 cells
  2. b Generate shRNA-resistant EGFR complementation constructs
  3. c Produce lentiviral particles and transduce shEGFR cells
  4. d Validate entry phenotype in shEGFR organoids
  5. e Assess HCV replication in complemented cells
Phase 8 · EGFR Phosphorylation and Signaling Analysis Day 8
  1. a Western blot analysis of EGFR phosphorylation kinetics
  2. b SDS-PAGE and immunoblotting
Phase 9 · Immunofluorescence Visualization of Phospho-EGFR Day 7-8
  1. a Fixed-cell phospho-EGFR and total EGFR staining
  2. b Secondary staining and z-stack imaging
  3. c Interpretation of subcellular localization

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Attribution

This SOP was written by Organthis based on the published method in Baktash et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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