LP-52 Development of 3D Spheroids from Gastric Tumors as a Tool for Drug Toxicity Testing

Gastric adenocarcinoma remains a major global health concern highlighting the urgent need for development of reliable in vitro models serving as a tool for drug screening and personalized treatment strategies. In this study, we established a robust protocol for isolating and cultivating viable human gastric epithelial cells from surgically resected tissues in both two-dimensional (2D) and three-dimensional (3D) formats. Gastric specimens from the patients were processed to derive epithelial cells from both tumor and adjacent non-tumor tissue using enzymatic dissociation with collagenase and dispase. Isolated cells were cultured on collagen-coated surfaces in DMEM/F12 medium enriched with growth factors (EGF, FGF), antibiotics, and either 10% FBS (healthy cells) or 2% FBS (tumor cells) to limit fibroblast overgrowth. Initial cell proliferation was modest during the first week, followed by a marked increase in epithelial cell numbers around day 12. Tumorderived cells exhibited more rapid proliferation (doubling times: 23–52 h) compared to normal cells (32–64 h). The established cultures were characterized by morphological analysis, immunocytochemistry, periodic acid-Schiff (PAS) staining, and cell viability assays. Cytokeratin 18 and mucin markers (MUC5AC, MUC1) confirmed epithelial identity and secretory function of gastric cells. CEA staining verified the high purity of both tumor and non-tumor populations. The NCI-N87 gastric cancer cell line served as a reference for optimization and quality control. Our findings demonstrate the feasibility of generating patientderived 2D and 3D gastric epithelial models directly from surgical material. These models represent a valuable platform for preclinical drug testing, mechanistic studies of gastric disease, and the development of patient-specific therapeutic approaches that closely mimic the native tumor microenvironment. The study was supported by the University Pardubice, Holecek Family Foundation, and Institutional Support DZRVO MO 1012 of the Ministry of Defence of the Czech Republic.