*Medical protocols, *Structural models, *Research funding, *Cancer patients, *Quality control, *Cell culture, *Tissue culture, *Lung tumors, *Research methodology

Simple Summary: Patient-derived organoids are 3D models useful to study cancer biology and may be implemented as predictive tools in therapeutic decisions. We set up a two-step protocol that allows for establishing both short-term and long-term organoid cultures from lung cancer patients. We set up an optimal medium formulation and implemented rigorous quality controls, leading to a substantial improvement in organoid establishment success rate. Our 3D models are suitable for drug screening and co-culture experiments. This protocol represents a key advance for the employment of lung cancer organoids both as models for research and as predictive tools in clinical practice. Background/Objectives: Despite the introduction of innovative therapeutics, lung cancer is still the leading cause of cancer-related death. For this reason, lung cancer still requires deep characterization to identify cellular and molecular targets that can be used to develop novel therapeutic strategies. Three-dimensional cellular models, including patient-derived organoids (PDOs), represent useful tools to study lung cancer biology and may be employed in the future as predictive tools in therapeutic decisions. However, the successful establishment of lung cancer organoids cultures that faithfully represent the respective patient tissues is still challenging due to low success rate and/or overgrowth of normal airway epithelial cells. Methods: We set up a two-step protocol that allows for establishing both short-term and long-term 3D cultures, with different characteristics and success rates. Results: Cancer tissue-originated spheroids (CTOSs) show a 100% success rate and allow for the concomitant isolation of autologous tumor infiltrating leukocytes (TILs). On the contrary, PDOs can be expanded for a medium-long term and bio-banked but retain a lower success rate and the possibility of contamination with normal airway epithelial cells. To overcome these problems, we set up an optimal medium formulation and we implemented rigorous quality controls, leading to a substantial improvement in the success rate of tumoral PDO establishment. Conclusions: Overall, this protocol guarantees flexibility and reliability, also providing useful guidelines for quality control checks to support different experimental settings. The setting up of a robust protocol for lung cancer PDO culture establishment and expansion is a key requirement for their employment both in cancer research and as predictive tools in clinical practice. [ABSTRACT FROM AUTHOR]

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