3D Spheroid Model for the Investigation of the Inflammatory Role of Smooth Muscle Cells in Cardiovascular Diseases

Background: Smooth muscle cells (SMCs) are a key factor in the development of various cardiovascular diseases. It is known that they can change their phenotype in response to external stimuli into a macrophage-like phenotype and create a proinflammatory environment, which could be a trigger for cardiovascular diseases. In vitro experiments with SMCs should help to clarify the molecular mechanisms behind these observations. However, primary cells tend to dedifferentiate after time under 2D conditions. Hence, in this study we cultivated SMCs in a 3D culture spheroid model with and without inflammatory stimulation, hypothesizing that this will reflect the in vivo situation better than 2D cell culture systems.

Method: Aortas of male Wistar rats were removed, cut into rings, and cultured in culture medium until the SMCs migrated to the cell culture dish. Here, they were propagated until confluence and seed to 96 well ultra-low attachment plates of 40,000 cells/well. A special surface coating prevents cells from adhering, so they attach to each other and form cell clusters (spheroids). Control SMCs were seeded to classical 48 well plates. Mass spectrometric analysis was used to compare proteomes between 2D and 3D. To stimulate a phenotype switch, 2D and 3D SMCs were treated with LPS for 1 hour. F4/80 antibody staining and confocal microscopy were used to visualize the phenotype switch.

Results: We successfully cultured primary SMCs as 3D spheroids in vitro, which were self-organized in 3D cellular networks. Already the short LPS stimulus allowed us to detect a proinflammatory phenotype switch in the spheroids in contrast to the 2D cultured cells and to observe F4/80 positive cells by confocal microscopy. The proteomic analysis (>1,000 analyzed proteins) of the 3D compared with the 2D culture revealed significant changes in their proteome (10% of total).

Conclusion: The proteomic analysis of the 3D compared with the 2D culture showed that culture conditions lead to significant changes in their proteome. Based on our data, we can verify that 3D-cultured SMCs, in contrast to 2D cultured SMCs, switch their phenotype to a macrophage-like SMC type in response to inflammatory stimulation. Therefore, this model may help investigate the inflammatory role of SMCs in cardiovascular diseases.

Publication History

Article published online:
28 January 2023