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SKIN Publication-derived

Skin Cancer Organoid Biobank for Personalized Drug Sensitivity

Source Pauli et al. · Weill Cornell Medicine, New York, USA · 10.1158/2159-8290.cd-16-1154

👤 Pauli C, Hopkins BD, Prandi D, Shaw R, Fedrizzi T, Sboner A, Rajber V, Chen B, Demichelis F, Rubin MA ⏱ 65 days 📋 13 phases 🧫 Patient-Derived (BCC, SCC, Melanoma, Multiple Patients)

Abstract

Comprehensive living biobank framework for skin cancer organoids integrating whole-exome sequencing, transcriptomics, and high-throughput drug sensitivity testing. Links genomic alterations to drug responses across BCC, SCC, and melanoma for precision oncology.

Cell source
Patient-Derived (BCC, SCC, Melanoma, Multiple Patients)
Application
Precision Oncology Platform

Protocol overview

47 steps across 13 phases

Specimen Procurement and Transport Day 0
  1. 1 Obtain patient consent and collect fresh tissue
  2. 2 Snap-freeze and transport tissue
Tissue Processing and Primary Culture Initiation Day 0–1
  1. 1 Wash and dissect tissue
  2. 2 Enzymatic tissue digestion
  3. 3 Stop digestion and cell harvesting
  4. 4 Cell washing and resuspension
  5. 5 Plate organoids in Matrigel drops
Organoid Culture Maintenance and Passaging Day 1–30+
  1. 1 Media replacement and monitoring
  2. 2 Mycoplasma screening
  3. 3 Passage organoids at passage number <20
  4. 4 Cryopreservation and biobanking
Organoid Viability Assessment Passages 2–4
  1. 1 Prepare organoid sample
  2. 2 Stain with LIVE/DEAD dyes
  3. 3 Confocal microscopy imaging
Organoid Characterization by Cytology and Histology After viability assessment
  1. 1 Prepare samples for cytology and histology
  2. 2 Morphological assessment
Genomic Characterization by Whole Exome Sequencing After organoid establishment
  1. 1 Extract DNA from organoids
  2. 2 DNA quality control
  3. 3 Whole exome sequencing
  4. 4 Targeted sequencing alternative (if insufficient DNA)
2D High-Throughput Single-Agent Drug Screening After organoid characterization (typically passages 2–4)
  1. 1 Cell line optimization and preparation
  2. 2 Plate cells
  3. 3 Add drugs
  4. 4 Cell viability assay
  5. 5 Data analysis and prioritization
3D Dose-Response Validation in Matrigel Organoids After single-agent screening prioritization
  1. 1 Prepare 3D culture
  2. 2 Add drugs in dose-response
  3. 3 Cell viability assay (3D)
  4. 4 Data analysis
2D High-Throughput Combination Drug Screening After single-agent prioritization
  1. 1 Select sensitizing agent
  2. 2 Perform combination screening
  3. 3 Analyze combination data
3D Validation of Selected Combinations After combination screening prioritization
  1. 1 Prepare 3D co-cultures
  2. 2 Test combination at fixed ratio
  3. 3 Assess viability and synergy
Establishment of Patient-Derived Xenografts (PDXs) After organoid characterization (passages <20)
  1. 1 Prepare cell suspension for injection
  2. 2 Inject into nude mice
  3. 3 Monitor xenograft growth
In Vivo Drug Efficacy Studies in PDX Models After xenograft establishment (tumor diameter ~0.65 cm)
  1. 1 Randomize mice into treatment groups
  2. 2 Administer drugs
  3. 3 Monitor tumor growth and mouse weight
  4. 4 Harvest tumors at endpoint
Immunohistochemistry and Histological Analysis After tumor harvest
  1. 1 Prepare tissue sections
  2. 2 Antigen retrieval
  3. 3 Block and incubate with primary antibodies
  4. 4 Detect and visualize
  5. 5 Image analysis and quantification

Full SOP

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Attribution

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

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