"Microfluidics Studies of the Regulation of Myoblast Migration and Differentiation Behaviour – Possible Application in Wound Healing".

R3710 --> Natural extracellular matrices made from cultured cells or tissues, are tissue and cell specific, and critical in tissue engineering and cellular applications. This study focused on the effects of a more naturally produced substrate, made by myotubes differentiated within a device channel, on migration and proliferation (haptotaxis) of a second set of cells (set2) loaded into the device. First, four different microfluidic devices were designed with pillars in either an offset pattern or aligned rows along the channel to investigate the myotube formation. C2C12 cells preloaded with Hoechst stain to label DNA (set1 cells), were loaded into device channels in medium with 2% serum to induce differentiation. Results showed that in devices with aligned rows, cells were more concentrated toward pillars, and their distance from a pillar was smaller than for cells in devices with offset rows of pillars. An average of 20‐30 set2 cells was tracked over 10 hours, by image capture every 2 hours in the 3 or 4 channels of each device. Results were compiled in Excel and analyzed by multi‐way ANOVAs using Jamovi software. Minimum distance to a pillar occurred rapidly, at day 0 for all four devices and increased over time. After allowing set1 cells to differentiate for 5 days, prestained set2 cells were loaded. The nucleus position of set2 cells was categorized as located nearest to one of four places in the migration channel: the nucleus of a set1 cell, an extension of a set1 cell, a pillar, or the device‐channel wall. Results showed that a 3‐channel device with offset rows of pillars was best able to lead set1 cells to form and align myotubes in the channel and then attract the most set2 cells to nuclei of those set1 cells. Since cellular proximity is critical to myotube formation and set 2 cells were closer to the set1 nuclei than to device pillars over 10 hours imaging. Differences in flow rate among the four devices suggest that the pillars' orientation, channel dimensions, and initial velocity are factors that influenced behavioural variations among set1 cells from the time of loading to the end of the 5‐day differentiation period. Experiments in devices that were precoated with a fibronectin substrate showed that fibronectin shortened the time to confluency of set1 cells by about 2 days. Perfusion of set1 cells after 5 days of differentiation with an RGD inhibitor ((Arg‐Gly‐Asp) peptide) of fibronectin binding to integrins, induced a significant shift in the behavior of set2 cells toward proximity to set1 nuclei, whereas without RGD, set2 cells moved toward a device pillar. Additional experiments targeting processes during the fibronectin signaling in migration are in progress to further explore mechanism targeted by variations in device design that affect cell behavior or movement. Further study of cellular taxis should provide clues to identifying a device that would best create a muscle by promoting muscle fiber growth (by fusion of set2 to set1 cells) or in the longer term, induce set2 cells to become quiescent satellite (stem) cells. [ABSTRACT FROM AUTHOR]