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LIVER Publication-derived

Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle

Source Molina-Jimenez et al., 2012 · Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa, Madrid, Spain · 10.1016/j.virol.2011.12.021

👤 Francisca Molina-Jimenez, Ignacio Benedicto, Viet Loan Dao Thi, Virginia Gondar, Dimitri Lavillette, Jose J. Marin, Oscar Briz, Ricardo Moreno-Otero, Rafael Aldabe, Thomas F. Baumert, François-Loïc Cosset, Manuel Lopez-Cabrera, Pedro L. Majano ⏱ 96 days 📋 6 phases 🧫 Human Huh-7 hepatocyte-derived cell line

Abstract

This protocol describes the establishment of Matrigel-embedded 3D cultures of Huh-7 cells that develop hepatocyte-like polarity with functional bile canaliculi-like structures. These 3D cultures support hepatitis C virus (HCV) infection by JFH-1 virus, permit robust virion production, and provide a more physiologically relevant model than standard 2D cultures for studying the entire HCV cycle.

Cell source
Human Huh-7 hepatocyte-derived cell line
Application
HCV infection and virion production study in polarized hepatocyte-like context

Protocol overview

23 steps across 6 phases

Cell Culture Setup and 3D Embedding Day 0–1
  1. 1 Prepare cell culture media
  2. 2 Prepare Matrigel solution
  3. 3 Embed cells in Matrigel for standard 3D culture
  4. 4 Polymerize Matrigel
  5. 5 Cover with complete medium
  6. 6 Culture for 6 or 9 days with medium changes
HCV Preparation and Infection Setup Prior to Day 6 (or as needed)
  1. 7 Generate HCV replicon-containing cell lines
  2. 8 Produce JFH1 HCVcc
HCV Infection (Day 6) Day 6
  1. 9 Prepare 2D and 3D cultures for infection
  2. 10 Apply blocking antibodies (if required)
  3. 11 Infect with HCVcc overnight
  4. 12 Replace medium post-infection
  5. 13 Add IFN-α (if applicable)
Viral RNA Replication and Production Monitoring (Days 6–9) Day 6–9 post-infection (Days 12–15 of protocol)
  1. 14 Harvest cells for real-time RT-PCR at multiple time points
  2. 15 Perform immunofluorescence for viral core protein
Viral Supernatant Collection and Titration Days 9–10 (96 h post-infection)
  1. 16 Harvest culture supernatants
  2. 17 Filter and concentrate supernatants
  3. 18 Infect naïve 2D-cultured Huh-7.5 cells for titration
Characterization of Viral Particles by Density Gradient Day 9–10 (post-96 h incubation)
  1. 19 Scale up 2D and 3D cultures for viral production
  2. 20 Harvest and concentrate viral supernatants
  3. 21 Prepare iodixanol gradients
  4. 22 Load sample and ultracentrifuge
  5. 23 Collect and analyze fractions

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Molina-Jimenez et al., 2012. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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