Microfluidics studies of the regulation of myoblast migration

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Rovei Miab, Ziba
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Cell migration is an essential process in which cells move from one location to another using different mechanisms. Migration occurs during development, and in the maintenance of multicellular organisms during wound healing, tissue regeneration, and immune and pathophysiological responses. In skeletal muscle, satellite cells with dual roles as muscle precursors and self-renewing unipotent adult stem cells, are resident on muscle fibers and normally mitotically inactive. Their activation and subsequent migration critically mediate muscle repair. While early literature suggested satellite cells could demonstrate multipotency (giving rise to bone or adipose cells, this has not held up to scrutiny1. In this three-part thesis research, C2C12 mouse myoblast morphology and migration were first studied using a microfluidic platform under a range of chemo- and hapto-taxis conditions. The haptotaxis substrate was found to modify myoblast chemotaxis. Since cultured cells and tissues produce a natural extracellular matrix (ECM) which is pivotal in cell migration behavior, a second set of experiments was designed to study the haptotaxis effect of a natural substrate produced by one set of differentiated myoblasts on the migration behavior of a second set of myoblasts. Cell behavior was examined in four microfluidic devices with pillars in the migration channel. Results showed that more myotubes would align and form in the device (device 1) with offset rows of pillars. Differences in flow rate and velocity significantly affected migration patterns. Fibronectin (FN) in an applied substrate also shortened the time to confluency. Considering the effects of ECM and FN on myoblast behavior, a final set of experiments explored the interaction between FN and integrin, the FN receptors. Four inhibitors were applied to interrupt this interaction in device 1. Anti-Integrin antibody had the strongest inhibitory effect, followed by in order by focal adhesion kinase, CS1, and RGD peptides. The capability of microfluidic devices is advantageous for controlling cellular microenvironments, and thus offers a valuable approach for quantitative studies of cell migration in vitro. Devices can be designed to incorporate conditions that mimic what is known of normal physiology and control microenvironmental changes that model particular situations of disease or tissue injury. Further research to identify a device that promotes muscle fiber growth and enable longer-term studies of myoblast behavior toward muscle tissue engineering and regeneration, can now utilize the results of these novel experiments showing the potent impact on myoblast migration and alignment of combined haptotaxis and chemotaxis stimuli, microfluidic device design, and the different interaction of the receptor, integrin with an applied FN substrate vs. a natural ECM.
Muscle cell migration, Microfluidics
1. Roveimiab, Z., Lin, F., & Anderson, J. E. (2020). Traction and attraction: haptotaxis substrates collagen and fibronectin interact with chemotaxis by HGF to regulate myoblast migration in a microfluidic device. American Journal of Physiology-Cell Physiology, 319(1), C75-C92.
2. Roveimiab, Z., Lin, F., & Anderson, J. Microfluidics Studies of the Regulation of Myoblast Migration and Differentiation Behaviour–Possible Application in Wound Healing. The FASEB Journal, 35.