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INTESTINAL Publication-derived

Colorectal Cancer Colonic Tumouroids for Biobanking

Full SOP with Academic or a plan

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

Authors: 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 Duration: 60 days 8 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Schnalzger et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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