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PROSTATE Publication-derived

Foundational Human Prostate Cancer Organoid Culture (MSKCC

Source Human Oncology and Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer Center (MSKCC), New York, USA · 10.1016/j.cell.2014.08.017

⏱ 56 days 📋 6 phases 🧫 Patient-Derived

Abstract

Cell source
Patient-Derived
Application
Basic Research

Protocol overview

32 steps across 6 phases

Tissue Dissociation and Cell Isolation Day 0–1
  1. 1 Tissue Collection and Processing
  2. 2 Enzymatic Digestion with Collagenase
  3. 3 Enzymatic Digestion with TrypLE
  4. 4 Cell Wash and Suspension Preparation
  5. 5 FACS Sorting – Cell Surface Staining
  6. 6 FACS Sorting – Cell Sorting
Organoid Establishment in Matrigel Day 1–14
  1. 1 Matrigel Embedding
  2. 2 Organoid Medium Overlay
  3. 3 Initial Culture and Morphology Monitoring
Organoid Characterization – Immunohistochemistry and Immunofluorescence Day 7–21
  1. 1 Organoid Fixation
  2. 2 Preparation for IHC – Paraffin Embedding
  3. 3 Immunohistochemistry (IHC) – Antigen Retrieval
  4. 4 IHC – Blocking and Antibody Staining
  5. 5 IHC – Detection and Visualization
  6. 6 Immunofluorescence (IF) – Organoid Preparation
  7. 7 IF – Primary Antibody Staining
  8. 8 IF – Secondary Antibody Staining and Imaging
Gene Expression Analysis – RT-qPCR Day 7–21
  1. 1 RNA Isolation
  2. 2 cDNA Preparation
  3. 3 Quantitative RT-PCR (qPCR) Setup
  4. 4 qPCR Cycling Protocol
  5. 5 DHT Response Assay (optional)
Organoid Passaging and Long-term Maintenance Day 14+ (weekly to bi-weekly)
  1. 1 Organoid Harvesting
  2. 2 Organoid Dissociation – Enzymatic or Mechanical
  3. 3 Cell Wash and Suspension
  4. 4 Replating in Fresh Matrigel
  5. 5 Ongoing Culture and Monitoring
Optional: UGSM Tissue Recombination Assay Day 56+ (8 weeks post-transplantation)
  1. 1 Organoid Cell Preparation for Recombination
  2. 2 UGSM Cell Preparation
  3. 3 Cell Mixing and Recombination
  4. 4 In Vivo Growth and Harvest
  5. 5 Histological Analysis

Full SOP

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Attribution

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

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