Investigating the Impact of Various 2D and 3D Cell Culture Platforms on the Production of Extracellular Vesicles

Extracellular vesicles (EVs) play a critical role in intercellular communication and hold great promise as diagnostic biomarkers and therapeutic agents. Due to the limited availability of patient samples, in vitro cell culture models have become indispensable tools for generating EVs under controlled conditions and investigating their biological roles. While conventional 2D cultures are widely used, they lack the complexity of native tissues. In contrast, 3D culture platforms better mimic in vivo conditions and may influence EV secretion dynamics and characteristics. However, there is a lack of research directly comparing these various 2D and 3D platforms for EV production. In this study, we temporarily compared 2D culture with three 3D platforms composed of distinct biomaterials: ultralow attachment (ULA) plates with a nonadherent surface, collagen-coated plates with a biologically active matrix, and AlgiMatrix plates with porous alginate sponges. Cell growth and EV production were evaluated over multiple time points using the human mammary epithelial cell (HMEC) as a model, including assessments of cell morphology, EV yield, size distribution, and morphology. The results showed that both ULA and collagen-based platforms effectively produced smaller and more uniform EVs compared to the 2D platform, with yields exceeding those observed in 2D. In contrast, the AlgiMatrix system was unsuitable for size-based EV quantification due to contamination from scaffold-related materials. These results demonstrate distinct EV production shaped by the physical and biochemical features of each culture platform, highlighting the importance of biomaterial selection and time-course analysis when optimizing EV production for downstream applications, such as diagnostics and therapeutic development.