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BLADDER Publication-derived

Patient-Derived Bladder Tumor Organoid Establishment and Culture

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 ⏱ 21 days 📋 6 phases 🧫 Patient-Derived Urothelial Carcinoma

Abstract

This protocol establishes a biobank of patient-derived bladder tumor organoid lines that recapitulate the histopathological and molecular diversity of human urothelial carcinoma. Organoids are cultured in three-dimensional Matrigel culture and can be serially passaged, cryopreserved, and interconverted with orthotopic xenografts to model tumor evolution and evaluate drug responses.

Cell source
Patient-Derived Urothelial Carcinoma
Application
Disease modeling and drug screening

Protocol overview

39 steps across 6 phases

Sample Collection and Tissue Preparation Day 0
  1. 1 Collect tumor tissue from patient
  2. 2 Prepare transport medium
  3. 3 Place tumor tissue in transport medium
Tissue Dissociation Day 0
  1. 1 Prepare organoid culture medium
  2. 2 Wash tissue
  3. 3 Mince tissue
  4. 4 Enzymatic digestion with collagenase/hyaluronidase
  5. 5 First centrifugation
  6. 6 Second centrifugation
  7. 7 Trypsin dissociation
  8. 8 Stop trypsinization
  9. 9 Centrifuge dissociated cells
  10. 10 Wash dissociated cells
  11. 11 Filter dissociated cells
  12. 12 Count and assess cell clusters
Organoid Culture Initiation Day 0
  1. 1 Prepare Matrigel-media mixture
  2. 2 Plate organoid drops
  3. 3 Solidify Matrigel drops
  4. 4 Add culture medium
  5. 5 Initial culture period
Medium Changes and Early Passaging Days 3–21
  1. 1 First medium change
  2. 2 Continue regular medium changes
  3. 3 First passage preparation
  4. 4 Collect organoids after dispase treatment
  5. 5 Wash organoids
  6. 6 Trypsin dissociation of organoids
  7. 7 Mechanical dissociation
  8. 8 Assess dissociation
Serial Passaging and Maintenance Days 21+
  1. 1 Re-plate dissociated organoids
  2. 2 Solidify Matrigel and add medium
  3. 3 Continue culture
  4. 4 Define successful establishment
Cryopreservation and Long-Term Storage Any passage ≥6
  1. 1 Prepare organoids for freezing
  2. 2 Prepare cryopreservation medium
  3. 3 Resuspend cells in cryopreservation medium
  4. 4 Aliquot into cryovials
  5. 5 Controlled-rate freezing
  6. 6 Transfer to liquid nitrogen
  7. 7 Thawing and recovery

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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