259604 Elucidation of Mast Cell Localization Using a Microfluidic Device That Generates a Controllable Diffusion-Driven SCF Gradient

Thursday, November 1, 2012: 3:51 PM
Somerset West (Westin )
Meghaan M. Smith, Amir Shamloo, Maheswaran Mani, Milan Manchandia, Kenneth Weinberg and Sarah C. Heilshorn, Stanford University, Stanford, CA

Mast cells, granular cells that reside in tissue, have become a well-known modulator of wound healing. Investigators have proven that the absence of mast cells greatly impairs healing of skin wounds created in mast cell-deficient murine models. Subsequent reconstitution with bone marrow-derived mast cells (BMMCs) reversed this effect. These studies also demonstrated that the extent of mast cell degranulation, which promotes healing by increasing vascular permeability, neutrophil recruitment, and wound closure, correlates inversely with distance from the lesion. Thus, regulation of mast cell migration and localization is critical for wound healing.

In addition to its chemotactic properties, stem cell factor (SCF, aka kit ligand) is required for mast cell growth, differentiation, and survival; thus, SCF is key in regulating mast cell localization. To shed light on the mechanism behind SCF-dependent localization, we developed a gradient-generating microfluidic device, which offers several advantages over traditional chemotaxis assays, including establishment of a temporally stable gradient, minimal use of expensive reagents and cells, and the ability to directly visualize the migration of single cells. The devices employed for these studies generated a stable linear SCF gradient across a central channel containing BMMCs without exposing the cells to convective flow.The SCF concentration profile is dictated by Fickian diffusion across the cell culture chamber, which enables direct control over the gradient steepness and average concentration. A concentration gradient of 8 ng/ml/mm was determined to be sufficient to induce BMMC chemotaxis. Analysis of the time-lapse microscopic videos enabled tracking of the migratory paths of hundreds of individual cells, which were then quantified to determine speed, persistence time, and chemotactic index (CI) as a function of SCF gradient and concentration. Interestingly, we found that within the same 8 ng/ml/mm gradient, BMMCs exposed to the highest average SCF concentration (10 ng/ml) displayed greater migration efficiency (CI = 0.32), than cells exposed to lower concentrations (6 ng/ml, CI = 0.10, p<0.05), while cell speed was largely unaffected. As expected, BMMCs not subjected to an SCF gradient displayed random migration within the devices. These data reinforce the concept that chemokinesis, i.e. the effect of a chemokine on random cell motility and speed, is a distinct process from chemotaxis, which requires asymmetric reorganization of the cytoskeleton. These results further suggest that efficient cell polarization requires both a minimum gradient steepness as well as a minimum SCF concentration. Further studies of BMMC response to SCF may help elucidate the mechanism behind mast cell migration and localization in response to a lesion. The knowledge acquired through these studies will aid in the development of chemokine-delivery therapies for dermal wound healing of chronic lesions.

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