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SKIN Publication-derived

Patient-Derived Merkel Cell Carcinoma Organoid Development and Therapeutic Screening

Source Forsythe et al., 2022 · Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine · 10.1038/s41598-022-17921-6

👤 Steven D. Forsythe, Richard A. Erali, Preston Laney, Hemamylammal Sivakumar, Wencheng Li, Aleksander Skardal, Shay Soker, Konstantinos I. Votanopoulos ⏱ 10 days 📋 5 phases 🧫 Patient-Derived Tumor Cells

Abstract

This protocol describes the biofabrication of patient-derived organoids (PTOs) and immune-enhanced organoids (iPTOs) from Merkel cell carcinoma surgical specimens. Unsorted MCC tumor cells are suspended in a collagen-hyaluronan hydrogel with optional patient-matched immune cells (blood or lymph node) to generate models suitable for rapid preclinical testing of chemotherapy and immunotherapy agents within 10 days of specimen receipt, enabling personalized treatment efficacy assessment.

Cell source
Patient-Derived Tumor Cells
Application
Disease modeling and personalized chemotherapy/immunotherapy screening

Protocol overview

42 steps across 5 phases

Tissue Processing and Cell Isolation Day 0 (post-resection)
  1. 1 Tissue Transport and Initial Preparation
  2. 2 Specimen Washing
  3. 3 Tissue Mincing and Enzymatic Digestion Setup
  4. 4 Enzymatic Digestion Termination
  5. 5 Cell Filtration and Centrifugation
  6. 6 Red Blood Cell Lysis
  7. 7 Cell Counting
  8. 8 Lymphocyte Isolation from Patient Blood (for iPTOs)
  9. 9 Lymph Node Processing (Alternative Immune Source)
Organoid Fabrication and Culture Day 0–7
  1. 1 Hydrogel Preparation
  2. 2 Tumor Cell-Hydrogel Mixture Preparation
  3. 3 Seeding Patient-Derived Organoids (PTOs) into Wells
  4. 4 UV Photo-crosslinking of PTOs
  5. 5 Addition of Culture Media to PTOs
  6. 6 Immune-Enhanced Organoid (iPTO) Fabrication
  7. 7 Media Changes During Culture
  8. 8 Pre-Treatment Organoid Viability Assessment
Therapeutic Screening Day 7–10
  1. 1 Chemotherapy Treatment Preparation
  2. 2 Immunotherapy Treatment Preparation
  3. 3 Media Removal and Drug Treatment Initiation
  4. 4 Drug Incubation Period
  5. 5 Post-Treatment Sample Preparation for Viability Assays
Viability Assessment and Outcome Analysis Day 10
  1. 1 LIVE/DEAD Staining
  2. 2 Confocal Microscopy Imaging
  3. 3 CellTiter-Glo 3D Cell Viability Assay
  4. 4 LDH-GLO Cytotoxicity Assay
  5. 5 Luminometry Reading
  6. 6 Viability Data Normalization and Treatment Response Determination
Organoid Tissue Characterization Day 10 (in parallel with viability assays)
  1. 1 Organoid Fixation
  2. 2 Tissue Processing and Paraffin Embedding
  3. 3 Microtome Sectioning
  4. 4 Hematoxylin and Eosin (H&E) Staining
  5. 5 Chromogenic Immunohistochemistry (IHC) - Antigen Retrieval
  6. 6 Primary Antibody Incubation - Chromogenic IHC
  7. 7 Secondary Antibody Incubation - Chromogenic IHC
  8. 8 DAB Chromogen Detection and Counterstaining
  9. 9 Chromogenic IHC Image Acquisition
  10. 10 Fluorescent IHC - Antigen Retrieval and Blocking
  11. 11 Fluorescent Primary Antibody Incubation
  12. 12 Fluorescent Secondary Antibody Incubation
  13. 13 Nuclear Counterstain (DAPI) and Coverslipping
  14. 14 Fluorescent IHC Image Acquisition and Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Forsythe et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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