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LIVER Publication-derived

Liver Organoids for Infection Studies

Full SOP with Academic or a plan

Source Collett et al., 2021 · RMIT University, School of Science · 10.1016/j.jcis.2021.02.067

Authors: Simon Collett, Joseph Torresi, Linda Earnest Silveira, Vi Khanh Truong, Dale Christiansen, Bang M. Tran, Elizabeth Vincan, Paul A. Ramsland, Aaron Elbourne Duration: 30 days 10 phases Cell source: Human hepatocarcinoma cell line (Huh7)

Abstract

This protocol generates polarized Huh7 hepatocyte organoids in 3D culture and compares HCV virus-like particle (VLP) binding interactions between polarized organoid cells and non-polarized monolayer cells using confocal microscopy and atomic force microscopy. The work demonstrates that HCV VLP binding to host cell receptors is measurable only on polarized 3D cells, providing insights into the importance of cellular architecture for accurate study of hepatitis C virus pathogenesis.

Cell source
Human hepatocarcinoma cell line (Huh7)
Application
Disease modeling; virus-host cell interaction studies

Protocol overview

30 steps across 10 phases

Phase 1 · VLP Production and Labeling Days -30 to -1
  1. a Produce HCV VLPs by transfection
  2. b Label VLPs with FITC
Phase 2 · Huh7 Cell Culture Setup Days -5 to 0
  1. a Culture Huh7 cells in monolayer
  2. b Establish 3D organoid cultures
  3. c Prepare organoids on microscope slides for staining
Phase 3 · Cell Dissociation for Flow Cytometry and AFM Day 5
  1. a Dissociate monolayer cells
  2. b Dissociate organoid cells from Matrigel
  3. c Block non-specific binding
Phase 4 · Flow Cytometry: VLP Blocking Assays Day 5
  1. a Incubate VLP with blocking reagents
  2. b Add cells to VLP-antibody mixture
  3. c Wash and prepare cells for FACS analysis
Phase 5 · Flow Cytometry: Receptor Expression Analysis Day 5
  1. a Stain cells with receptor antibodies
  2. b Wash and analyze receptor expression
Phase 6 · Confocal Laser Scanning Microscopy: Whole Organoid and Single-Cell Imaging Day 5
  1. a Fix and stain whole organoid cultures
  2. b Block and apply primary antibodies
  3. c Apply secondary antibodies and mount
  4. d Prepare dissociated organoid cells for single-cell imaging
  5. e Block and incubate with antibodies for live-cell imaging
  6. f Acquire confocal images
Phase 7 · AFM Imaging of VLPs on Mica Day 5
  1. a Prepare VLP sample for AFM imaging
  2. b Acquire AFM height images
  3. c Process AFM images
Phase 8 · AFM Probe Functionalization Day 5
  1. a Coat AFM probes with Concanavalin A
  2. b Bind VLPs to functionalized probes
Phase 9 · AFM Force Spectroscopy: Cell Preparation and Measurement Day 5
  1. a Prepare monolayer cells for AFM
  2. b Prepare organoid cells for AFM
  3. c Conduct AFM force spectroscopy in QI mode
  4. d Extract and analyze adhesion data
Phase 10 · AFM Force Spectroscopy: Blocking Experiments Day 5
  1. a Incubate functionalized AFM tips with blocking IgG
  2. b Record height and adhesion maps on blocked tips

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Attribution

This SOP was written by Organthis based on the published method in Collett et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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