A Comparative Study of 3D Culture Methods in Lung Cancer Research: Finding the Optimal Path for Spheroid Formation

ABSTRACT Objective: Cancer, characterized by uncontrolled cell proliferation and invasion into surrounding tissues, is a leading cause of global mortality. Traditional two-dimensional (2D) cell culture systems fail to adequately replicate the tumor microenvironment (TME). In contrast, three-dimensional (3D) culture systems, which better simulate cell–cell and cell–extracellular matrix (ECM) interactions, have become powerful tools in biomedical research. This study aims to compare the spheroid formation capacity of A549 lung cancer cells using three different 3D culture methods: ultra-low attachment (ULA) plates, agarose hydrogel, and the hanging drop technique. The primary objective is to identify the most effective spheroid formation method for A549 cells and to provide findings that can guide future biomedical research, particularly in cancer modeling, drug screening studies, and investigations of the tumor microenvironment.Materials and Methods: A549 cells were cultured using three different 3D culture methods: ultra-low attachment plates, agarose hydrogel, and the hanging drop method. In the ultra-low attachment method, spheroid formation was observed at cell densities of 5,000, 10,000, and 30,000 cells/ml. In the agarose hydrogel method, agarose concentrations of 1%, 1.5%, and 2% were used to evaluate cell aggregation and spheroid stability. In the hanging drop method, cells aggregated under the influence of gravity. Spheroid diameter and area were analyzed using ImageJ software.Results: In this study, the spheroid formation capacity of A549 lung cancer cells was evaluated using three different three-dimensional (3D) culture methods. The ultra-low attachment (ULA) plate method allowed cell aggregation; however, the resulting structures were not large or compact enough to be classified as spheroids. The hanging drop method showed that cells formed small clusters by day 3 but failed to develop a compact and stable spheroid structure by day 7. The agarose hydrogel method, particularly at a 2% agarose concentration, demonstrated the highest spheroid formation capacity compared to the other methods. In this method, spheroid formation began at 72 hours depending on cell density, with significant growth observed at a density of 30,000 cells/ml (p < 0.0001). Trypan Blue staining results indicated that 2% agarose and cell densities of 10,000–30,000 cells/ml provided the highest cell viability. Specifically, 4,800 viable cells were counted at a density of 30,000 cells/ml, while 3,600 viable cells were observed at 10,000 cells/ml. These findings suggest that the agarose hydrogel method, especially at 2% agarose concentration and higher cell densities, offers optimal spheroid formation and cell viability for A549 lung cancer cells.Conclusion: This study demonstrated that the agarose hydrogel method effectively promoted stable and organized spheroid formation in A549 lung cancer cells. Notably, the 2% agarose concentration was identified as the most effective condition for maintaining cell viability and optimizing spheroid size. In contrast, the ultra-low attachment (ULA) plate and hanging drop methods exhibited limited spheroid formation capacity, resulting in less compact and disorganized structures. These findings emphasize the critical role of three-dimensional (3D) cell culture methods in biomedical research, particularly for experimental tumor modeling and drug screening studies. In this context, the agarose hydrogel method, with its high spheroid formation capacity and ability to support cell viability, emerges as a promising 3D culture model that warrants further exploration in cancer research.