Skip to content
← Back to browse
INTESTINAL Publication-derived

3D Model for CAR-Mediated Cytotoxicity Using Patient-Derived Colorectal Cancer Organoids

Source Schnalzger et al., 2019 · Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany · 10.15252/embj.2018100928

👤 Theresa E Schnalzger, Marnix HP de Groot, Congcong Zhang, Mohammed H Mosa, Birgitta E Michels, Jasmin Röder, Tahmineh Darvishi, Winfried S Wels, Henner F Farin ⏱ 60 days 📋 8 phases 🧫 Patient-Derived iPSC (colorectal cancer)

Abstract

This protocol establishes a quantitative 3D platform to study chimeric antigen receptor (CAR)-engineered NK-92 cell-mediated cytotoxicity against patient-derived colorectal cancer organoids. The assay employs luciferase-based endpoint measurement for high-throughput testing and confocal live-cell imaging for dynamic, single-organoid-level analysis of effector cell recruitment and target cell lysis.

Cell source
Patient-Derived iPSC (colorectal cancer)
Application
Disease modeling; Drug screening; CAR-T/NK cell cytotoxicity assay development

Protocol overview

46 steps across 8 phases

Organoid culture preparation and NK-92 compatibility testing Days 1-5
  1. 1 Establish and maintain colorectal organoids
  2. 2 Test NK-92 cell growth in organoid medium
  3. 3 Identify inhibitory factors in organoid medium
  4. 4 Confirm organoid viability in nicotinamide-free medium
Lentiviral transduction of organoids with luciferase and fluorescent reporters Days 6-15
  1. 1 Generate luciferase/GFP-expressing lentiviral vector
  2. 2 Produce VSV-G pseudotyped lentiviral particles
  3. 3 Transduce organoids with luciferase/GFP lentiviral vector
  4. 4 Validate luciferase expression and sensitivity
CAR-NK-92 cell generation and characterization Days 16-25
  1. 1 Prepare CAR-NK-92 constructs (EPCAM, EGFRvIII, FZD-targeting)
  2. 2 Produce lentiviral particles for CAR-NK-92 transduction
  3. 3 Transduce NK-92 cells with CAR lentiviral vectors
  4. 4 Confirm CAR surface expression
Optimization of organoid-NK-92 co-culture conditions Days 26-30
  1. 1 Test organoid embedding strategies
  2. 2 Confirm CAR cell recruitment and cytotoxicity kinetics
  3. 3 Determine optimal effector-to-target (E:T) ratio
  4. 4 Test co-culture with stromal cells
Luciferase-based quantitative cytotoxicity assay Days 31-35
  1. 1 Determine organoid cell number by image-based counting
  2. 2 Prepare organoid-coated assay plates
  3. 3 Seed organoids and incubate before CAR-NK-92 addition
  4. 4 Add CAR-NK-92 or control cells
  5. 5 Measure luciferase activity at endpoint
  6. 6 Calculate target cell lysis and CAR-specific lysis
Live-cell imaging-based cytotoxicity assay Days 36-40
  1. 1 Prepare organoids with stable GFP or DsRED expression
  2. 2 Seed organoids on imaging plates
  3. 3 Label and add NK-92 cells
  4. 4 Perform spinning-disk confocal live-cell imaging
  5. 5 Process images and create maximum intensity projections
  6. 6 Automatically detect organoids and measure area loss
  7. 7 Measure NK-92 cell recruitment to organoids
  8. 8 Analyze single-organoid dynamics and track over time
  9. 9 Validate image-based readout against luciferase assay
CAR targeting of tumor neoantigens (EGFRvIII model) Days 41-50
  1. 1 Generate EGFRvIII-expressing organoid lines
  2. 2 Validate EGFRvIII expression at RNA level
  3. 3 Transduce EGFRvIII organoids with luciferase/GFP vector
  4. 4 Test EGFRvIII-CAR NK-92 cytotoxicity against EGFRvIII-expressing organoids
  5. 5 Perform competitive co-cultures with mixed tumor and normal organoids
  6. 6 Perform long-term co-culture with organoid replating assay
  7. 7 Monitor tumor-specific killing by live imaging
  8. 8 Analyze CAR cell recruitment to tumor vs. normal organoids
Evaluation of CAR targeting of FRIZZLED receptors (FZD-CAR) Days 51-60
  1. 1 Validate FZD-CAR NK-92 activity on FZD-overexpressing 2D cell line
  2. 2 Establish mouse organoid models with differential FZD expression
  3. 3 Transduce mouse organoids with luciferase/GFP vector
  4. 4 Test FZD-CAR NK-92 cytotoxicity against mouse organoids
  5. 5 Test FZD-CAR NK-92 at reduced E:T ratios
  6. 6 Evaluate FZD-CAR activity against primary human CRC organoids
  7. 7 Test FZD-CAR activity against primary human CRC organoid panel

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Schnalzger et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence