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LIVER Publication-derived

Liver Organoid and T Cell Coculture Models Cytotoxic T Cell Responses against Hepatitis C Virus

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

👤 Vaishaali Natarajan, Camille R Simoneau, Ann L Erickson, Nathan L Meyers, Jody L Baron, Stewart Cooper, Todd C McDevitt, Melanie Ott ⏱ 4 days 📋 11 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Natarajan et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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