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PROSTATE Publication-derived

Prostate Cancer Tumouroids

Full SOP with Academic or a plan

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

Duration: 56 days 6 phases Cell source: Patient-Derived

Abstract

Cell source
Patient-Derived
Application
Basic Research

Protocol overview

32 steps across 6 phases

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

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Attribution

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

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