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LIVER Publication-derived

Liver Organoid + CD8 T Cell Microfluidic Coculture

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

👤 Natarajan V, Simoneau CR, Erickson AL, Meyers NL, Baron JL, Cooper S, McDevitt TC, Ott M ⏱ 56 days 📋 8 phases 🧫 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

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

Full SOP

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Attribution

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

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