<p>Gliomas are the most frequent tumors of the central nervous system, with an extremely low efficiency of treatment. Primary glioma cell cultures may provide an in vitro model for studying these tumors and the development of therapeutic approaches. In this review, we assess different factors that may contribute to glioma malignancy, such as the presence of glioma stem cells, cellular heterogeneity, and selection for specific genotypes. We discuss approaches for primary glioma cell culture establishment and the role of particular components of the cultivation media: culture in monolayer, neurospheres, and glioblastoma organoids; the influence of serum, growth factors, and surface coating; and the presence of glioma stem cells. Different cell culture protocols have various drawbacks – loss of the parental tumor cellular composition, loss of glioma stem cells, or loss of the glioma microenvironment. We argue that to produce primary glioma cell culture, researchers shall use a combination of standardized protocols: serum-free neurosphere culture, serum-based monolayer culture, and glioblastoma organoids.</p>