Cytokine supplementation for improved tumoroid-immune cell co-culture

Immunotherapies for solid cancers rely on robust engagement between immune and epithelial tumor cells within the tumor microenvironment. However, culture conditions that simultaneously sustain epithelial and tumor-infiltrating immune cell viability during short-term ex vivo culture remain poorly defined. Patient-derived tumoroids, multicellular organoid systems that recapitulate cancer cells in vitro but lack the immune cell component of tumors, enable interrogation of tumor-immune interactions in vitro via addback of immune cells. To explore an alternative approach, namely short-term co-culture of tumor and immune cells, we optimized OncoPro™ Tumoroid Culture Medium to promote immune cell survival and composition stability during culture of patient-derived dissociated tumor cell (DTC) digests. Initial studies showed that healthy donor PBMCs cultured in OncoPro medium alone lost approximately 80% of viable cells after 5 days. A design of experiments (DOE) screen of 13 cytokines and growth factors identified combinations that enhanced PBMC survival while maintaining balanced immune subpopulations. Among these, IL-2, GM-CSF, IL-15, IL-4, and BAFF emerged as candidates that increased PBMC cell counts relative to non-supplemented medium across multiple donors and helped preserve lymphocyte, monocyte, NK, helper T, and cytotoxic T cell proportions within 10% of baseline. Using this list of cytokine cocktails, we next assessed culture conditions supporting both immune (CD45⁺) and epithelial (EpCAM⁺) cell populations in colorectal cancer DTCs. Three donors were evaluated in a blocked DOE framework to control for donor variability. We evaluated both embedded culture where cells were encapsulated in Geltrex™ Flex matrix before media overlay, as well as suspension culture where cells were plated in OncoPro medium supplemented with Y-27632, 2% (v/v) Geltrex Flex matrix, and cytokines, and analyzed after 5 days by flow cytometry. Tube-based dissociation yielded ~25% higher recovery than in-plate dissociation, and bead-based enumeration cytometry using CountBright™ absolute counting beads agreed within ~25% of Trypan Blue-based counts made prior to staining samples for flow cytometry. Suspension culture favored immune cell viability, whereas embedding in Geltrex Flex matrix modestly improved tumoroid recovery but reduced CD45⁺ cell survival. Across donors, cultures supplemented with IL-2, IL-15, GM-CSF, and BAFF achieved the highest total viable cell recovery and the lowest change in immune composition. Together, these results establish a cytokine-optimized medium that enhances immune cell viability and stability during short-term tumoroid co-culture, enabling improved modeling of the microenvironment and facilitating downstream studies of tumor-immune interactions and therapeutic response.