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BLADDER Publication-derived

Patient-Derived Bladder Tumor Organoid Establishment, Culture, and Characterization

Source Lee et al., 2018 · Columbia University Medical Center · 10.1016/j.cell.2018.03.017

👤 Suk Hyung Lee, Wenhuo Hu, Justin T. Matulay, Michael M. Shen ⏱ 50 days 📋 7 phases 🧫 Patient-Derived Bladder Tumor (Urothelial Carcinoma)

Abstract

This protocol describes the efficient establishment and maintenance of patient-derived bladder tumor organoid lines from fresh transurethral resection specimens. The organoid lines recapitulate the histopathological and molecular diversity of human bladder cancer, including both non-muscle-invasive and muscle-invasive disease, and can be used for clonal evolution studies and preclinical drug response testing validated by orthotopic xenografts.

Cell source
Patient-Derived Bladder Tumor (Urothelial Carcinoma)
Application
Disease modeling and drug screening for bladder cancer

Protocol overview

53 steps across 7 phases

Tumor Sample Collection and Preparation Day 0
  1. 1 Collect fresh tumor samples
  2. 2 Transport tissue to laboratory
  3. 3 Divide tissue for processing
Tissue Dissociation Day 0-1
  1. 1 Prepare dissociation media
  2. 2 Mince tumor tissue
  3. 3 Enzymatic digestion with collagenase/hyaluronidase
  4. 4 Wash dissociated tissue
  5. 5 Trypsin digestion
  6. 6 Halt trypsinization
  7. 7 Final wash and cell straining
  8. 8 Count and quantify dissociated cells
Organoid Culture Initiation Day 1-3
  1. 1 Prepare Matrigel-media mixture
  2. 2 Resuspend dissociated cells in Matrigel
  3. 3 Plate organoid drops
  4. 4 Solidify Matrigel drops
  5. 5 Add culture medium
  6. 6 Incubate organoids
Organoid Maintenance and Passaging Day 7 onwards
  1. 1 Monitor organoid growth
  2. 2 Change culture medium
  3. 3 Digest Matrigel for passaging (around day 14-21)
  4. 4 Collect organoids
  5. 5 Enzymatic dissociation with TrypLE
  6. 6 Passage organoids
  7. 7 Define successful line establishment
  8. 8 Cryopreserve organoid stocks
Histological and Molecular Characterization Throughout culture (Day 0 onwards)
  1. 1 Harvest organoids for histology
  2. 2 Prepare paraffin sections
  3. 3 Perform H&E staining
  4. 4 Evaluate histology by pathologist
  5. 5 Perform targeted exome sequencing
  6. 6 Perform RNA sequencing (RNA-seq)
  7. 7 Perform immunofluorescence staining
Orthotopic Xenograft Generation (Optional) Day 30+ (when organoids are well-established)
  1. 1 Prepare organoid cells for xenografting
  2. 2 Count and prepare cell suspension
  3. 3 Anesthetize mice
  4. 4 Prepare surgical site
  5. 5 Expose bladder
  6. 6 Perform ultrasound-guided submucosal injection
  7. 7 Inject organoid suspension
  8. 8 Close the surgical incision
  9. 9 Post-operative monitoring
  10. 10 Monitor tumor growth by ultrasound
  11. 11 Harvest tumors
Drug Response Testing (Optional) Day 30-50 (at passage 5-12)
  1. 1 Prepare organoids for drug assay
  2. 2 Passage organoids through 100 μm cell strainer
  3. 3 Plate organoids in ultra-low-attachment 96-well plates
  4. 4 Allow organoids to settle (24 hours)
  5. 5 Prepare drug dilution series
  6. 6 Add drug to organoid cultures
  7. 7 Incubate with drug treatment
  8. 8 Assess cell viability with CellTiter-Glo assay
  9. 9 Normalize and calculate IC50 values
  10. 10 Interpret drug response results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Lee et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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