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Organoid cultures generated from gastrointestinal tissues have been an invaluable advancement for in vitro studies of physiological function and disease. Here we present a comprehensive protocol for the establishment and culture of human- and mouse-derived 3-dimensional gastric organoids transferred to 2-dimensional gastric epithelial cell monolayers. We introduce two methods that include the establishment of monolayers from: (1) intact organoids, and (2) single cells dissociated from intact organoids.

The interaction between the receptor, programmed cell death protein 1 (PD-1) and ligand, programmed cell death 1 (PD-L1) is known to inhibit CD8+ cytotoxic T lymphocyte proliferation, survival, and effector function. The result of this interaction leads to evasion of immune surveillance by tumors and subsequently cancer cell proliferation. Immunotherapy via PD-L1 blockade is used for a variety of malignancies, yet the prognostic value of immune checkpoint inhibition for the treatment of gastric cancer remains controversial. Thus, preclinical models that would predict the efficacy of such therapy in a subgroup of gastric cancer patients would be an advancement in the personalized treatment of this disease. Three-dimensional organoid cultures have not only been used to investigate the mechanisms regulating development and disease, but have also been used for high-throughput drug screening for targeted personalized therapy. Here we present the methodology for the co-culture of mouse-derived gastric cancer organoids with autologous immune cells specifically for the study of PD-L1/PD-1 interactions within the tumor microenvironment in vitro.