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Background: Human malignant cell models that reflect the structural and physiological complexity of tumor tissue are of great importance for preclinical oncology research. Spheroids and tumoroids derived from solid tumors are of particular interest as cellular models mimicking the initial avascular growth phase of a tumor node. However, the degree of similarity between artificially created tumor multicellular aggregates and real tumor tissue requires further clarification and validation to determine how closely spheroids resemble malignized tissues in vivo compared to traditionally used monolayer cell cultures. Patients and Methods For the creation of two-dimensional (2D) and three-dimensional (3D) cell models, tumor cells isolated from solid tumor fragments obtained intraoperatively were used. The study included 15 samples of skin melanoma, 20 samples of soft tissue and bone sarcomas (STBS), and 9 samples of epithelial tumors (ET). All tumor cells were cultivated for at least ten passages. Phase-contrast microscopy, confocal microscopy, and immunohistochemistry were used to investigate spheroids and monolayer cultures. Supernatants from tumor cells grown in 2D and 3D cultures were analyzed using ELISA and multiplex assays to evaluate the production of chemokines and cytokines associated with immunosuppression, invasion, and metastasis. Results: Seventy-five percent of tumor specimens were of metastatic origin. Two-dimensional cultures were obtained in 100% of cases, while spheroids were successfully generated in 88.6% of cases (39 of 44). There was no direct correlation between tumoroid formation efficiency and tumor histogenesis or disease stage (primary tumor, recurrence, or metastasis). The median spheroid size after 4–5 days of cultivation at an initial concentration of 10,000 cells per well was: 657.14 μm for melanoma (range 400–1000 μm), 571.42 μm (range 400–700 μm) and 507.14 μm (range 300–600 μm) for soft tissue sarcomas, 650.0 μm (range 400–900 μm) for osteogenic sarcomas. Immunochemical analysis of Ki-67, GLUT1, and E-cadherin was performed on tumor tissue samples, monolayer cultures, and tumoroids. Ki-67-positive cells were more abundant in spheroids, reflecting increased proliferative activity. No dependence of Ki-67 or GLUT1 localization on spheroid size was detected. E-cadherin expression was identified in tumor tissue and tumoroids of breast carcinoma and one melanoma culture but was absent in monolayer cultures. A significant increase in secretory activity was observed in 3D cultures, with elevated levels of CCL2, CCL3, CXCL1, CXCL16, MIF, IL-10, and MICA (p < 0.01), indicating enhanced immunomodulatory capacity of spheroids. Conclusion: Three-dimensional tumor spheroid models more accurately reproduce proliferative, metabolic, and immunological characteristics of solid tumors than conventional monolayer cultures. Construction of spheroids from tumors of different histogenesis requires an individualized approach and further investigation but provides valuable insight into tumor microenvironment formation and tumor–immune system interactions.