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SKIN Publication-derived

Skin Cancer Tumouroids for Biobanking

Full SOP with Academic or a plan

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

Authors: Pauli C, Hopkins BD, Prandi D, Shaw R, Fedrizzi T, Sboner A, Rajber V, Chen B, Demichelis F, Rubin MA Duration: 65 days 13 phases Cell source: 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

Phase 1 · Specimen Procurement and Transport Day 0
  1. a Obtain patient consent and collect fresh tissue
  2. b Snap-freeze and transport tissue
Phase 2 · Tissue Processing and Primary Culture Initiation Day 0-1
  1. a Wash and dissect tissue
  2. b Enzymatic tissue digestion
  3. c Stop digestion and cell harvesting
  4. d Cell washing and resuspension
  5. e Plate organoids in Matrigel drops
Phase 3 · Organoid Culture Maintenance and Passaging Day 1-30+
  1. a Media replacement and monitoring
  2. b Mycoplasma screening
  3. c Passage organoids at passage number <20
  4. d Cryopreservation and biobanking
Phase 4 · Organoid Viability Assessment Passages 2-4
  1. a Prepare organoid sample
  2. b Stain with LIVE/DEAD dyes
  3. c Confocal microscopy imaging
Phase 5 · Organoid Characterization by Cytology and Histology After viability assessment
  1. a Prepare samples for cytology and histology
  2. b Morphological assessment
Phase 6 · Genomic Characterization by Whole Exome Sequencing After organoid establishment
  1. a Extract DNA from organoids
  2. b DNA quality control
  3. c Whole exome sequencing
  4. d Targeted sequencing alternative (if insufficient DNA)
Phase 7 · 2D High-Throughput Single-Agent Drug Screening After organoid characterization (typically passages 2-4)
  1. a Cell line optimization and preparation
  2. b Plate cells
  3. c Add drugs
  4. d Cell viability assay
  5. e Data analysis and prioritization
Phase 8 · 3D Dose-Response Validation in Matrigel Organoids After single-agent screening prioritization
  1. a Prepare 3D culture
  2. b Add drugs in dose-response
  3. c Cell viability assay (3D)
  4. d Data analysis
Phase 9 · 2D High-Throughput Combination Drug Screening After single-agent prioritization
  1. a Select sensitizing agent
  2. b Perform combination screening
  3. c Analyze combination data
Phase 10 · 3D Validation of Selected Combinations After combination screening prioritization
  1. a Prepare 3D co-cultures
  2. b Test combination at fixed ratio
  3. c Assess viability and synergy
Phase 11 · Establishment of Patient-Derived Xenografts (PDXs) After organoid characterization (passages <20)
  1. a Prepare cell suspension for injection
  2. b Inject into nude mice
  3. c Monitor xenograft growth
Phase 12 · In Vivo Drug Efficacy Studies in PDX Models After xenograft establishment (tumor diameter ~0.65 cm)
  1. a Randomize mice into treatment groups
  2. b Administer drugs
  3. c Monitor tumor growth and mouse weight
  4. d Harvest tumors at endpoint
Phase 13 · Immunohistochemistry and Histological Analysis After tumor harvest
  1. a Prepare tissue sections
  2. b Antigen retrieval
  3. c Block and incubate with primary antibodies
  4. d Detect and visualize
  5. e Image analysis and quantification

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Attribution

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

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