Designing tools for quantitative, single-cell analysis of mammalian cell migration [electronic resource]

Mammalian cell migration guides many important processes within the human body, from physiological activities such as embryonic development, immune cell trafficking, and wound healing to pathological conditions like cancer metastasis, multiple sclerosis, and rheumatoid arthritis. Cell migration is a highly complex, tightly regulated biological process that is dependent on the cellular microenvironment: composition, rigidity, and topography of the extracellular matrix, as well as chemical cues, can modulate migration by altering cell speed, direction, and persistence. Conventional techniques for studying cell migration in response to chemical cues (e.g., scratch, micropipette, and Transwell assays) are limited in their ability to 1) maintain a stable concentration gradient and 2) allow longitudinal, quantitative, single-cell analyses of cell migration. The first limitation makes these techniques inappropriate for long-term studies of chemotaxis, which require a stable concentration gradient. The second limitation can cause data to be misinterpreted, as increases in proliferation (chemoproliferation) and speed (chemokinesis) in response to soluble cues can be misconstrued as directed migration (chemotaxis). To address these limitations, we developed a shear-free, microfluidic gradient generator to study cell migration. We used our microfluidic device to study the impact of soluble and insoluble cues on mammalian cell migration in two distinct applications: 1) chemotaxis of murine mast cells to kit ligand (KL) and 2) chemotaxis and chemokinesis of primary human myoblasts to basic fibroblast growth factor (bFGF). These applications are important for understanding the complex responses required for effective wound healing and muscle regeneration, respectively. In the first application, we found that KL caused a complex chemotactic response in mast cells, invoking chemoattraction at high concentrations and chemorepulsion at low concentrations. While the ability for a single cytokine to induce both attraction and repulsion is not unknown, this is the first reported observation of a bimodal chemotactic response mediated by a receptor tyrosine kinase (RTK). In the second application, we studied the impact of the underlying extracellular matrix (ECM) on the bFGF-stimulated responses (i.e., chemoproliferation, chemokinesis, and chemotaxis) of primary human myoblasts. We revealed that while the underlying ECM substrate did affect chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. Although laminin promoted significantly faster migration speeds than fibronectin or collagen without bFGF stimulation, the addition of bFGF increased migration speed by equal amounts on all substrates, indicating an additive, not synergistic effect. In contrast to the robust chemokinesis observed, our studies revealed weak chemoattraction in myoblasts exposed to a bFGF gradient. Together, these data indicate that changes in the muscle ECM, which can occur as a result of aging or disease, may impact muscle regeneration by altering proliferation and migration. The ECM substrates used in the microfluidic studies described above are all naturally derived materials (e.g., laminin). Naturally derived biomaterials exhibit high batch-to-batch variation and material properties (e.g., stiffness and ligand density) cannot be independently tuned. To study the effects of substrate stiffness and topography on cell behavior, without altering the density of cell-adhesive ligands, we used an engineered extracellular matrix (eECM) comprised of elastin-like proteins (ELP) and cell-adhesive domains containing the RGD motif found in fibronectin. Soft lithography techniques were used to pattern the ELP substrates to create a grooved topography. We found that patterned substrates induced cell alignment and increased migration speed. Altering the ratio of ELP and crosslinker increased substrate stiffness and, consequently, resulted in faster cell migration rates. Myoblast migration is a key step in muscle regeneration, and culturing cells for 10 days on ELP substrates resulted in the formation of multi-nucleated myotubes that expressed [alpha]-actinin (a marker of mature muscle). Many processes essential for human health are governed by a cell's ability to efficiently migrate within the body, making mammalian cell migration an essential topic for scientific investigation. We have developed two novel in vitro platforms that enable longitudinal, quantitative, single-cell analyses of mammalian cell migration in response to the microenvironment. These tools allowed us to deconstruct complex migratory behaviors in response to soluble (i.e, chemical) and insoluble (i.e., substrate composition, topography, and stiffness) cues that have important repercussions for wound healing and muscle regeneration. Understanding the mechanisms used to regulate mammalian cell migration holds the promise of new therapeutic approaches for disease treatments, cell transplantation therapies, and the development of artificial tissues.

Keywords: Chemical Engineering.
Advisor: Heilshorn Sarah primary advisor. Thesis advisor
Wang Clifford (Clifford Lee) primary advisor. Thesis advisor
Fuller Gerald G.