Microfluidic Studies Of Vascular Smooth Muscle Cell Migration

[Objective]Atherosclerosis and restenosis are major causes of morbidity and mortality. These processes develop secondary to endothelial injury and once this injury occurs, an essential element in the development of both these processes is the vascular smooth muscle cell (VSMC) migration. However, current studies of VSMC are based on conventional cell culture approaches (e.g., culture dish and flask), which allows no control over the spatial/temporal distribution of the cells and biomolecules and thus cannot recapitulate local in vivo microenvironments. Microfluidics is the science and technology of manipulating and detecting fluids in the microscale. Due to its dimensional comparison with biological cells and capabilities of defining local biophysical, biochemical and physiological cues, a microfluidic assay has been used to reconstruct more in vivo like cell culture models, cell migration models and high throughput quantify of different types of VSMC migration in the conditions with various concentrations of chemokines. Moreover, changes involved in VSMC proliferation and migration are explored within the microfluidic devices compared to traditional assays. In conclusion, our studies locate a novel platform in the development of atherosclerosis and restenosis, where chemokines could regulate VSMC proliferation and migration, leading to deepen our understanding of the pathophysiology function of VSMC and suggest perspective strategies in the prevention of restenosis.[Methods]1. A microfluidic device was presented for enabling culture of VSMCs where extracellular matrix coating (bare glass, poly-1-lysine, fibronectin, collagen), VSMC seeding, culture and immunostaining are demonstrated in a tubing-free manner.2. A microfluidic wound healing assay was presented to quantify the migration of five cell types of VSMC, where gravity was used to generate a laminar flow within microfluidic channels, enabling cell seeding, culture, and wound generation. The effects of channel geometries (channel height (100 μm vs.250 μm) and PDMS thickness (2 mm or 8 mm)), surface modifications (fibronectin vs. collagen) and chemokines (FBS free,10%FBS,10%FBS+PDGF-BB,10%FBS+TNF-a) on the migration of VSMCs were investigated and compared in a tubing-free manner.3. A 384-well microfluidic wound-healing assay was presented for enabling the wound generation in a high-throughput and well-controlled manner. The effects of different cell types of VSMC and various concentrations of chemokines (FBS, PDGF-BB, TNF-a, Ang Ⅱ) on the migration of VSMCs were investigated and compared.4. Each photo of VSMC was measured in the Image Pro Plus 6.0 software, two persons back to back, in average, with microsoft excel 2007 software input and statistical analysis. ANOVA was used for multiple-group comparisons where values of P<0.05 (*) and P<0.01 (**) were considered statistical significance and high statistical significance, respectively.[Results]1. This part developed a tubing-free microfluidic platform for VSMC culture where extracellular matrix coating, VSMC seeding, culture and observation, and immunostaining were demonstrated. Based on gravity and operation optimization, even distribution of cells within microfluidic channels (96.0±16.3/mm2) was realized. Compared to bare glass surface, extracellular matrix (collagen, poly-1-lysine and fibronectin) coated substrates lead to VSMC proliferations and phenotype variations in a time sequence.2. This part revealed that 1) height of the micro channels had a significant impact on cell migration 2) the surface coating of collagen induced more migration of VSMCs than fibronectin coated surfaces and 3) platelet derived growth factor BB resulted in maximal cell migration compared to tumor necrosis factor alpha and fetal bovine serum. Furthermore, migrations of five types of VSMCs (e.g., one human cell line, two primary cell types, and VSMCs isolated from two human samples) were quantified, finding that VSMCs from the human cell line and human samples demonstrated comparable migration distances, which were significantly lower than the migration distances of two primary cell types.3. This part found that 1) with increasing concentration of FBS, VSMC migration distance increased, confirmed the feasibility of the large sequence scratches migration technology 2) with increasing concentration of PDGF-BB, VSMC cell migration distance increased, but there was no saturation trend 3) with increasing concentration of PDGF-BB, the number of primary VSMC of atherosclerotic plaque migration increased, and in the position of 200 ng/ml arrived in peak, and then began to decline 4) with increasing concentration of TNF-a, the number of primary VSMC of atherosclerotic plaque migration had no obvious change 5) with increasing concentration of Ang Ⅱ, the number of primary VSMC of atherosclerotic plaque migration had a downward trend.[Conclusion]1. As a platform technology, this microfluidic device was confirmed to be capable of functioning as a new VSMC culture model for VSMC studies.2. As a platform technology, this tubing-free microfluidic wound healing assay may function as a new model to study migration of VSMCs within microfluidic environments.3. As a platform technology, this 384 cell migration assay may function as a high throughput, fully automatable, suitable for real-time viewing model.