Modeling 3D Glioblastoma: A review of spheroid culture techniques and standardization challenges

Glioblastoma (GB) is among the most aggressive and treatment-resistant cancers, highlighting the urgent need for physiologically relevant preclinical models. Traditional two-dimensional (2D) cultures fall short in replicating the complex microenvironment of GB, whereas three-dimensional (3D) culture models offer improved structural, biochemical, and functional resemblance to in vivo tumors. However, the lack of a standardized protocol for GB spheroid formation leads to significant inter-study variability and limited reproducibility. This review summarizes the key variables affecting spheroid generation, such as cell line selection, passage number, seeding density, matrix composition, and culture duration, and compares the effectiveness of commonly used methods like low-attachment plates, scaffold-based systems, and hydrogel embedding. Additionally, emerging trends in co-culture strategies and tumor microenvironment (TME) simulation are also highlighted. By outlining current strengths, limitations, and gaps, we provide a framework for advancing standardized, high-fidelity GB spheroid systems to accelerate translational research and therapeutic discovery.