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BLADDER Publication-derived

Bladder Cancer Tumouroids

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, Mark V. Silva, Tomasz B. Owczarek, Kwanghee Kim, Chee Wai Chua, LaMont J. Barlow, Cyriac Kandoth, Alanna B. Williams, Sarah K. Bergren, Eugene J. Pietzak, Christopher B. Anderson, Mitchell C. Benson, Jonathan A. Coleman, Barry S. Taylor, Cory Abate-Shen, James M. McKiernan, Hikmat Al-Ahmadie, David B. Solit, Michael M. Shen Duration: 14 days 8 phases Cell source: Patient-Derived Urothelial Carcinoma (mixed: non-muscle invasive and muscle invasive bladder cancer)

Abstract

This protocol describes the establishment and characterization of a biobank of patient-derived bladder cancer organoid lines that recapitulate the histopathological and molecular diversity of human bladder cancer. The organoid lines enable analysis of tumor evolution, clonal heterogeneity, and drug response, with validation of therapeutic responses using orthotopic xenografts.

Cell source
Patient-Derived Urothelial Carcinoma (mixed: non-muscle invasive and muscle invasive bladder cancer)
Application
Disease modeling and drug screening

Protocol overview

52 steps across 8 phases

Phase 1 · Sample Collection and Transport Day 0
  1. a Collect fresh tumor samples
  2. b Prepare transport medium
  3. c Transport tissue to laboratory
Phase 2 · Tissue Dissociation Day 0
  1. a Wash tumor tissue
  2. b Mince tissue with scissors
  3. c Enzymatic digestion with collagenase/hyaluronidase
  4. d Centrifuge and wash with PBS
  5. e TrypLE Express incubation
  6. f Stop trypsinization
  7. g Secondary wash and cell straining
Phase 3 · Organoid Culture Initiation Day 0-Day 3
  1. a Centrifuge and count cell clusters
  2. b Prepare Matrigel suspension
  3. c Plate organoid suspension
  4. d Solidify Matrigel
  5. e Add organoid culture medium
  6. f Monitor organoid formation
Phase 4 · Organoid Passaging Day 14 onwards
  1. a Digest Matrigel with dispase
  2. b Collect organoids
  3. c Dissociate organoids with TrypLE Express
  4. d Mechanically dissociate into cell clusters
  5. e Passage organoids
  6. f Plate passaged organoids
Phase 5 · Cryopreservation Day 14 onwards
  1. a Prepare organoids for freezing
  2. b Prepare cryopreservation medium
  3. c Snap-freeze organoids
  4. d Store frozen stocks
  5. e Recover frozen organoids
Phase 6 · Histology and Immunostaining Variable (as needed)
  1. a Harvest organoids for processing
  2. b Fix organoids in formalin
  3. c Embed in collagen I matrix
  4. d Prepare paraffin sections
  5. e Perform H&E staining
  6. f Prepare slides for immunostaining
  7. g Block non-specific binding
  8. h Incubate with primary antibodies
  9. i Incubate with secondary antibodies
  10. j Mount slides
  11. k Image immunofluorescence
Phase 7 · Orthotopic Xenograft Implantation Variable
  1. a Prepare organoids for implantation
  2. b Prepare animal and anesthesia
  3. c Locate bladder with ultrasound
  4. d Delaminate lamina propria from detrusor muscle
  5. e Implant organoids into submucosal pocket
  6. f Monitor tumor growth
  7. g Harvest xenografts
Phase 8 · Drug Response Assays Variable (typically 6-10 days for assay)
  1. a Prepare organoids for drug testing
  2. b Plate organoids in 96-well format
  3. c Prepare drug dilution series
  4. d Add drugs to organoids
  5. e Incubate with drugs
  6. f Assess cell viability
  7. g Normalize data and calculate IC50

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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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