Patient-Derived Organoid (PDO) Establishment and Expansion

  • Principle
  • Experimental Materials
  • Experimental Procedure
  • Troubleshooting

Materials Required

• MCE Matrigel

Principle

Patient-derived organoids (PDOs) are 3D in vitro models derived from patient tumor tissues that recapitulate the histological, genetic, and functional heterogeneity of the original tumors. These models are established by isolating tumor cells or tissue fragments and culturing them in a 3D extracellular matrix (ECM), such as Matrigel or decellularized ECM, to support self-organization, proliferation, and differentiation. The culture system preserves key features of the tumor microenvironment, including cell-cell interactions, stromal components, and ECM signaling, enabling accurate modeling of tumor biology and drug response for personalized medicine applications.

MCE has not independently verified the accuracy of these methods. They are for reference only.

Experimental Materials

• Primary tumor tissues obtained from surgical resections or biopsies;
• Enzymatic digestion reagents (e.g., collagenase, dispase);
• Matrigel or decellularized extracellular matrix (dECM);
• Serum-free organoid culture media (e.g., Gibco OncoPro Tumoroid Medium, modified 5-factor medium);
• Ultra-low attachment plates;
• Growth factors (e.g., Wnt ligands, EGF, R-spondin);
• Antibiotics;
• Cryopreservation medium;
• Immunohistochemistry/immunofluorescence reagents;
• Phase-contrast microscope;
• Microfluidic devices (for tri-culture models).

Experimental Procedure

• 1.Collect fresh primary tumor tissue under sterile conditions.

• 2.Mechanically dissociate and enzymatically digest tissue into single-cell suspensions or small fragments.

• 3.Filter cell suspension to remove debris and clumps.

• 4.Resuspend cells in pre-warmed Matrigel or dECM and plate into culture wells or microfluidic arrays.

• 5.Add appropriate organoid culture medium supplemented with essential growth factors.

• 6.Incubate at 37°C with 5% CO2.

• 7.Monitor organoid formation weekly using phase-contrast microscopy.

• 8.Perform passaging by enzymatic digestion and re-embedding in new Matrigel/ECM.

• 9.Expand and maintain cultures over multiple passages.

• 10.Freeze viable organoids in cryopreservation medium for long-term storage.

• 11.Validate organoids via histology, immunofluorescence, RNA sequencing, and drug sensitivity assays.

Troubleshooting

Low organoid formation efficiency:

Optimize tissue sampling depth and area; increase Wnt ligand concentration; use co-culture with mesenchymal stromal cells (MSCs) or fibroblasts.

Contamination with normal epithelial cells:

Implement stringent quality controls and use selective media formulations.

Organoid overgrowth or loss of heterogeneity:

Adjust culture medium composition and passage frequency.

Poor viability after thawing:

Ensure optimal cryopreservation protocol and use high-quality freezing medium.

Limited scalability:

Adopt suspension culture formats or automated platforms for high-throughput screening.

References:

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