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LIVER Publication-derived

Hepatitis C Liver Models in Matrigel

Full SOP with Academic or a plan

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

Authors: 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 Duration: 96 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Molina-Jimenez et al., 2012. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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