Assistant Professor Auburn University AUBURN, Alabama, United States
Introduction:: Smooth muscle cells (SMCs) are essential for controlling blood pressure by contracting or relaxing. It is widely established that the microenvironment of SMCs, which includes physical and chemical cues, impacts their behavior. Previous studies have emphasized the significance of biochemical and mechanical cues, such as growth factors, stiffness, shear stress, and cyclic stretching, in the regulation of SMC phenotype and contraction; however, the potential influence of 3D confinement on these processes has been largely overlooked. In vivo, extracellular matrix (ECM) proteins and neighboring cells physically confine SMCs, limiting their (W)idth from 2 to 7 µm, (H)eight from 3 to 10 μm, and (L)ength from 30 to 100 µm. Moreover, compressive forces due to confinement increase with substrate stiffening, a phenomenon that is particularly evident during vascular aging and hypertension. In this study, we investigated the effects of long-term 3D confinement on SMC behavior using microfabricated channels with dimensions that mimic the space in which SMCs reside.
Materials and Methods:: To evaluate SMC behavior in long-term 3D confinement, we developed a novel high-throughput cell confinement assay that enabled continuous monitoring of cell function in physiologically relevant confined microenvironments. This device was fabricated by combining standard multilayer photolithography and photopatterning. Using standard multilayer photolithography and replica molding, we first fabricated PDMS-based μ-fluidic devices that consisted of an array of parallel μ-channels with cross-sectional areas that varied from 100 μm2 ((W)idth x (H)eight = 10 x 10 μm2, partial confining) to 30 μm2 (W x H = 3 x 10 μm2, confining). Next, we employed photopatterning to exclusively deposit collagen-type I to microchannel walls (Fig. 1 A). Aortic SMCs were introduced into the microchannels via pressure-driven flow and their behavior was examined over a period of three days (Fig. 1B). Using this assay, we investigated how long-term 3D confinement affected cell proliferation and survival.
Results, Conclusions, and Discussions:: Results: Our innovative technology allowed us to successfully entrap SMCs with high efficiency in both confining and partially confining μ-channels (Fig. 1B). Our results also showed that prolonged cell culture in confining μ-channels reduced cell division (Fig. 1C) and triggered higher cell death rates ( ~30%) in SMCs compared to less restrictive microenvironments (W x H =10 x 10 μm2) or 2D-like environments (W x H =400 x 50 μm2) (Fig. 1D). This outcome was not due to nutrient deprivation since cell access to nutrients was confirmed by monitoring the diffusion of a fluorescent dye in these channels. Conclusion: These findings suggest that confinement might play a crucial role in regulating SMC behavior, which could have important implications for tissue engineering applications. Further studies are required to identify the underlying mechanisms governing how smooth muscle cell behavior is influenced by microenvironments and to enhance the development of these environments to produce functional smooth muscle tissues. With the aid of such technology, previously unidentified molecular mechanisms governing cell function in (patho) physiologically significant microenvironments will be uncovered, paving the way for the creation of novel interventions intended to slow the progression of pathologic conditions.
