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LIVER Publication-derived

Hepatitis C Liver Organ-on-Chip Models

Full SOP with Academic or a plan

Source Natarajan et al., 2021 · The Gladstone Institutes, San Francisco, CA, USA · 10.1101/2021.08.10.455738

Authors: Vaishaali Natarajan, Camille R Simoneau, Ann L Erickson, Nathan L Meyers, Jody L Baron, Stewart Cooper, Todd C McDevitt, Melanie Ott Duration: 4 days 11 phases Cell source: Patient-Derived Adult Stem Cells (ASC), HCV spontaneous resolver CD8+ T cells

Abstract

This protocol establishes a microfluidic coculture system combining adult stem cell-derived liver organoids with HCV-specific CD8+ T cells to model cytotoxic T cell responses against hepatitis C virus. The system enables real-time monitoring of T cell killing of peptide-pulsed organoids, recapitulating immune dynamics in a physiologically relevant in vitro setting.

Cell source
Patient-Derived Adult Stem Cells (ASC), HCV spontaneous resolver CD8+ T cells
Application
Disease modeling and immune response characterization

Protocol overview

66 steps across 11 phases

Phase 1 · Liver Organoid Culture Initiation Day 0 onwards
  1. a Isolate and sort EpCAM+ cells from liver tissue
  2. b Mix sorted cells with basement membrane extract
  3. c Prepare organoid basal media
  4. d Culture organoids in prepared media
  5. e Replace culture media
  6. f Split and expand organoids
Phase 2 · RNA Isolation and Characterization of Organoids Before coculture experiments
  1. a Harvest organoids for RNA extraction
  2. b Extract total RNA
  3. c Transcribe RNA to cDNA
  4. d Perform quantitative RT-PCR (qPCR)
  5. e Analyze qPCR data
Phase 3 · Immunofluorescence and Light Sheet Microscopy Before coculture experiments
  1. a Fix organoids
  2. b Wash and store fixed organoids
  3. c Block organoids
  4. d Incubate primary antibody
  5. e Wash organoids after primary antibody
  6. f Incubate secondary antibody
  7. g Wash organoids after secondary antibody
  8. h Stain nuclei with Hoechst
  9. i Image organoids on light sheet microscope
  10. j Process microscopy images
Phase 4 · Generation of HCV-Specific CD8+ T Cell Clone Weeks 1-4 before coculture
  1. a Stain CD8+ enriched PBMC with HLA tetramer
  2. b Sort tetramer-positive T cells
  3. c Set up limiting dilution cloning
  4. d Culture cloning plates
  5. e Expand growing clones
  6. f Maintain T cell clones
Phase 5 · Validation of T Cell Clone Antigen Specificity Weeks 2-4 after clone establishment
  1. a Prepare target cells for chromium release assay
  2. b Load exogenous peptide and chromium label
  3. c Wash and resuspend peptide-pulsed targets
  4. d Prepare vaccinia virus-infected targets
  5. e Set up chromium release assay
  6. f Harvest supernatants
  7. g Count radioactivity
  8. h Calculate percent specific lysis
Phase 6 · Peptide Pulsing of Organoids Day before microfluidic coculture
  1. a Dissociate organoids from BME
  2. b Prepare peptide solutions
  3. c Incubate organoids with peptide
  4. d Wash pulsed organoids
  5. e Re-embed organoids in BME for microfluidic loading
Phase 7 · CD8+ T Cell Preparation for Microfluidic Coculture Day of microfluidic coculture
  1. a Prepare T cell culture for labeling
  2. b Wash T cells to remove serum
  3. c Prepare CellTracker staining solution
  4. d Stain T cells with CellTracker Green
  5. e Wash labeled T cells
  6. f Count labeled T cells
Phase 8 · Microfluidic Chip Preparation and Organoid Loading Day of coculture
  1. a Prepare organoid suspension for chip loading
  2. b Mix organoids with BME
  3. c Load organoid suspension into microfluidic chip
  4. d Verify organoid distribution
  5. e Allow BME polymerization
Phase 9 · Media Channel Preparation and T Cell Introduction Day of coculture (after BME polymerization)
  1. a Load media into flanking channels (monoculture control)
  2. b Fill media reservoirs (monoculture)
  3. c Load T cells into media channel (coculture)
  4. d Fill coculture media reservoirs
Phase 10 · Microfluidic Coculture and Monitoring Days 0-3 of coculture
  1. a Place chips in incubation chamber
  2. b Replace culture media daily
  3. c Acquire phase contrast microscopy images
  4. d Acquire fluorescence microscopy images
  5. e Stitch microscopy tiles together
Phase 11 · Image Analysis and Organoid Viability Assessment After coculture completion
  1. a Trace and measure organoid morphology
  2. b Monitor organoids over time
  3. c Classify organoid viability
  4. d Count T cells in central channel
  5. e Quantify T cell viability
  6. f Perform statistical analysis

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Attribution

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

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