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BLADDER Publication-derived

Bladder Cancer Organoids for Transplantation

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: 21 days 6 phases Cell source: 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

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

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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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