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LIVER Publication-derived

Liver Organ-on-Chip Models for Disease Modelling

Full SOP with Academic or a plan

Source Natarajan et al. · Gladstone Institutes / UCSF · 10.1098/rsob.210320

Authors: Natarajan V, Simoneau CR, Erickson AL, Meyers NL, Baron JL, Cooper S, McDevitt TC, Ott M Duration: 56 days 8 phases Cell source: Human ASC Liver Organoid + HLA-matched CD8+ T Cells

Abstract

Natarajan, Simoneau und Ott (Gladstone/UCSF) entwickelten ein Mikrofluidik-Coculture-System für HCV-spezifische CD8+ T-Zell-Antworten gegen Leberorganoids. HLA-matched CD8+ T-Zellen, die auf NS3 KLVALGINAV-Peptid trainiert wurden, lysierten Leberorganoids nach NS3-Peptid-Pulsing in Echtzeit-Beobachtung. Das System rekapituliert adaptive Immunantworten gegen HCV im hepatischen Microenvironment — erstmals ohne Krebszelllinien.

Cell source
Human ASC Liver Organoid + HLA-matched CD8+ T Cells
Application
HCV Immunology

Protocol overview

40 steps across 8 phases

Phase 1 · Liver Organoid Culture Establishment Days 0-28
  1. a Isolate and seed EpCAM+ liver cells
  2. b Prepare organoid culture media
  3. c Change culture media regularly
  4. d Split and expand organoids
Phase 2 · CD8+ T cell Clone Generation and Characterization Days 7-56
  1. a Sort HCV-specific CD8+ T cells using tetramer
  2. b Establish clones by limiting dilution
  3. c Refeed clone culture plates
  4. d Expand growing clones to 24-well plates
  5. e Maintain clones with periodic restimulation
  6. f Verify antigen specificity with chromium release assay
Phase 3 · Media Optimization for Coculture Days 0-3
  1. a Set up T cell and organoid monocultures in microfluidic chips
  2. b Label T cells for viability assessment
  3. c Add viability dye to T cell cultures
  4. d Monitor T cell viability by fluorescence imaging
  5. e Assess organoid viability by phase contrast microscopy
  6. f Select optimal coculture medium
Phase 4 · Organoid Peptide Pulsing Day 1 (before coculture)
  1. a Dissociate organoids to smaller size
  2. b Remove residual BME from organoids
  3. c Pulse organoids with HCV peptide
  4. d Wash and resuspend pulsed organoids
Phase 5 · Microfluidic Chip Setup and Coculture Days 1-3
  1. a Prepare T cells for microfluidic loading
  2. b Load organoids into central channel of microfluidic chip
  3. c Load media into flanking channels for monoculture
  4. d Load T cells into top flanking channel for coculture
  5. e Maintain appropriate media volume gradient
  6. f Replace culture media daily
  7. g Incubate chips in humidified environment
Phase 6 · Live Imaging and Data Collection Days 1-3 (during coculture)
  1. a Perform phase contrast microscopy for organoid morphology
  2. b Perform fluorescence microscopy for T cell tracking
  3. c Stitch and process image tiles
  4. d Count viable T cells in central channel
Phase 7 · Organoid Viability Assessment and Analysis Days 1-3 (post-imaging)
  1. a Trace and measure organoid morphometry at baseline
  2. b Monitor morphometric changes over time
  3. c Classify organoid viability status
  4. d Quantify percent viable organoids at each time point
  5. e Calculate T cell-mediated killing
Phase 8 · Validation Characterizations (Parallel studies) Days 1-14 (independent experiments)
  1. a Measure HLA class I expression in organoids by RT-qPCR
  2. b Assess HLA class I protein localization by light sheet microscopy
  3. c Confirm HCV receptor expression in organoids by RT-qPCR
  4. d Test CD8+ T cell recognition of organoids in well-based assay

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Attribution

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

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