Spheroid-based smooth muscle cell contraction assays for the analysis of vasoactive agonists

To exploit the contractile phenotype for analysis of vasoactive agonists, VSMC spheroids were attached to collagen type I hydrogels and morphological changes were traced by time-lapse recording. The model was tested by treating the spheroids with potassium chloride to induce VSMC depolarization and subsequent contraction triggered by an increase in intracellular calcium levels via activation of L-type calcium channels. In addition, we tested the response to caffeine, which predominantly supports relaxation. Quantification of the morphological changes were done by automated segmentation analyses to identify cellular structures. While stimulation with solvent did not induce major changes in the tone of VSMC, potassium chloride induced a contraction and caffeine relaxed the spheroid structure mimicking the responses of arterial VSMC in vivo. 

_{In vitro contraction/relaxation assay. To trace agonist-mediated contraction or relaxation of VSMC spheroids, we developed an assay by adhering VSMC spheroids to collagen hydrogels and recording structural changes by time-lapse microscopy. Under these conditions, both mouse and human VSMC spheroids contracted/relaxed upon treatment with vasoactive agonists (Click on the images for control, KCl and Caffeine to start the videos). Representative images showing structural segments (computer-assisted detection) of mouse aortic SMC spheroids attached to collagen type I hydrogels before (red) and after (green) being stimulated with solvent (control), potassium chloride (KCl, 60 mM) or caffeine (10 mM). The dotted circle defines the area that serves as ‘anchor’ for the contracting/relaxing spheroid. Colored arrows indicate the accumulated movement vector of the cellular segments in relation to the anchor point. Finally, area changes upon exposure to the stimuli were quantified (n=4-5, *p<0.05, **p<0.01).}