(501c) A Microfluidic Migration Assay for Single-Cell Tracking in Well-Controlled and Non-Flowing Gradient Fields

The investigation of cellular response to soluble gradients is important for understanding migration phenomena, such wound healing, embryonic development, and cancer metastasis. Traditional migration assays provide end-point (e.g. Boyden, transwell) or time-lapse (e.g. Dunn) information; however, these methods employ gradients with suboptimal control and stability. Microfluidic assays have emerged, due in large part to the stability offered by laminar flows. Most microfluidic methods require either direct flow over cells or diffusion in static fields, which could lead to undesired shear effects or lack of control, respectively. Such trade-offs pose challenges to achieving optimal microenvironments in migration assays.

We address the abovementioned challenges by developing a device that produces a soluble concentration gradient that is (i) fully stable, (ii) switchable, and (iii) generates zero net flow through a cell culture chamber. The device contains a novel microfluidic architecture, where a flowing sheet acts as a gradient "source." Gradient information from the source is translated into the chamber by diffusion with high fidelity. As it is sealed in all other directions, there is no net flow through the chamber, which minimizes the possibility of shear-induced cellular behavior. An upstream flow network selects streams for different stages of each experiment, such as cell loading/seeding, growth, serum deprivation, or gradient activation. Finite element models show a steady-state gradient with trace velocities, and the gradient is verified via intensity measurements of fluorescent markers. We validated the design by measuring responses of NIH/3T3 cells to growth factor gradients. Cells are seeded at low density in chambers (1000x300 μm) pre-incubated with extracellular matrix (i.e. fibronectin). Single cells are tracked by calculating centroid trajectories from traces of cell edges, yielding step-velocities and morphological readouts (e.g. aspect ratio, angular orientation). These data are compared by diverse graphical and statistical means, establishing the effectiveness of the device in performing high-resolution migrational studies.