Abstract
Gradients of soluble molecules coordinate cellular communication in a diverse range of multicellular systems. Chemokine-driven chemotaxis is a key orchestrator of cell movement during organ development, immune response and cancer progression. Chemotaxis assays capable of examining cell responses to different chemokines in the context of various extracellular matrices will be crucial to characterize directed cell motion in conditions which mimic whole tissue conditions. Here, a microfluidic device which can generate different chemokine patterns in flow-free gradient chambers while controlling surface extracellular matrix (ECM) to study chemotaxis either at the population level or at the single cell level with high resolution imaging is presented. The device is produced by combining additive manufacturing (AM) and soft lithography. Generation of concentration gradients in the device were simulated and experimentally validated. Then, stable gradients were applied to modulate chemotaxis and chemokinetic response of Jurkat cells as a model for T lymphocyte motility. Live imaging of the gradient chambers allowed to track and quantify Jurkat cell migration patterns. Using this system, it has been found that the strength of the chemotactic response of Jurkat cells to CXCL12 gradient was reduced by increasing surface fibronectin in a dose-dependent manner. The chemotaxis of the Jurkat cells was also found to be governed not only by the CXCL12 gradient but also by the average CXCL12 concentration. Distinct migratory behaviors in response to chemokine gradients in different contexts may be physiologically relevant for shaping the host immune response and may serve to optimize the targeting and accumulation of immune cells to the inflammation site. Our approach demonstrates the feasibility of using a flow-free gradient chamber for evaluating cross-regulation of cell motility by multiple factors in different biologic processes.
Highlights
In nature, gradients of soluble molecules guide cellular responses in biologic activities ranging from embryogenesis [1], tissue regeneration [2] and axon growth in neural development [3] to immune cell activation and migration [4]
We have shown that the fibronectin concentration can regulate the extent of random motility of the Jurkat cells, we did not see a significant difference in the percentage of motile cells or their average velocities with 100-nM CXCL12 gradient in chambers coated with low (25 μg/mL) or high fibronectin concentrations (1000 μg/mL) (Figure 4a,b vs. Figure 4e)
We developed a flow-free gradient chamber within a microfluidic system that enables the generation and maintenance of single or combinatorial concentration gradients
Summary
Gradients of soluble molecules guide cellular responses in biologic activities ranging from embryogenesis [1], tissue regeneration [2] and axon growth in neural development [3] to immune cell activation and migration [4]. Cells initially seeded in the upper chamber sense the gradient and migrate into the lower chamber. The major limitation of Boyden chambers and other similar systems lies in their inability to track spatial and temporal parameters of cell migration. Since these systems allow only limited control over the microenvironment, cells cannot be challenged by sequential events, such as the ability on one chemokine to sensitize cells to exposure by subsequent factors
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