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BLADDER Publication-derived

Bladder Cancer Tumouroids for Drug Screening

Full SOP with Academic or a plan

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

Authors: Suk Hyung Lee, Wenhuo Hu, Justin T. Matulay, Michael M. Shen Duration: 50 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Lee et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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