ItemThe Effect of Microfluidic Geometry on Myoblast Migration(MDPI AG) Atmaramani, Rahul; Black, Brian J.; Lam, Kevin H.; Sheth, Vinit M.; Pancrazio, Joseph J.; Schmidtke, David W.; Alsmadi, Nesreen Zoghoul; 0000-0002-9325-547X (Atmaramani, R); 0000-0001-8276-3690 (Pancrazio, JJ); 0000-0001-6404-318X (Schmidtke, DW); Atmaramani, Rahul; Black, Brian J.; Lam, Kevin H.; Sheth, Vinit M.; Pancrazio, Joseph J.; Schmidtke, David W.; Alsmadi, Nesreen ZoghoulIn vitro systems comprised of wells interconnected by microchannels have emerged as a platform for the study of cell migration or multicellular models. In the present study, we systematically evaluated the effect of microchannel width on spontaneous myoblast migration across these microchannels-from the proximal to the distal chamber. Myoblast migration was examined in microfluidic devices with varying microchannel widths of 1.5-20 µm, and in chips with uniform microchannel widths over time spans that are relevant for myoblast-to-myofiber differentiation in vitro. We found that the likelihood of spontaneous myoblast migration was microchannel width dependent and that a width of 3 µm was necessary to limit spontaneous migration below 5% of cells in the seeded well after 48 h. These results inform the future design of Polydimethylsiloxane (PDMS) microchannel-based co-culture platforms as well as future in vitro studies of myoblast migration. © 2019 by the authors. ItemFunctional Cargo Delivery into Mouse and Human Fibroblasts Using a Versatile Microfluidic Device(Springer New York LLC) Lam, Kevin H.; Fernandez-Perez, A.; Schmidtke, David W.; Munshi, N. V.; 0000-0001-6404-318X (Schmidtke, DW); Lam, Kevin H.; Schmidtke, David W.Efficient intracellular cargo delivery is a key hurdle for the translation of many emerging stem cell and cellular reprogramming therapies. Recently, a microfluidic-based device constructed from silicon was shown to transduce macromolecules into cells via shear-induced formation of plasma membrane pores. However, the scalability and widespread application of the current platform is limited since physical deformation-mediated delivery must be optimized for each therapeutic application. Therefore, we sought to create a low-cost, versatile device that could facilitate rapid prototyping and application-specific optimization in most academic research labs. Here we describe the design and implementation of a microfluidic device constructed from Polydimethylsiloxane (PDMS) that we call Cyto-PDMS (Cytoplasmic PDMS-based Delivery and Modification System). Using a systematic Cyto-PDMS workflow, we demonstrate intracellular cargo delivery with minimal effects on cellular viability. We identify specific flow rates at which a wide range of cargo sizes (1–70 kDa) can be delivered to the cell interior. As a proof-of-principle for the biological utility of Cyto-PDMS, we show (i) F-actin labeling in live human fibroblasts and (ii) intracellular delivery of recombinant Cre protein with appropriate genomic recombination in recipient fibroblasts. Taken together, our results demonstrate that Cyto-PDMS can deliver small-molecules to the cytoplasm and biologically active cargo to the nucleus without major effects on viability. We anticipate that the cost and versatility of PDMS can be leveraged to optimize delivery to a broad array of possible cell types and thus expand the potential impact of cellular therapies. © 2018, Springer Science+Business Media, LLC, part of Springer Nature. ItemConstricted Microfluidic Devices to Study the Effects of Transient High Shear Exposure on Platelets(Amer Inst Physics, 2018-10-01) Alsmadi, Nesreen Z.; Shapiro, Sarah J.; Lewis, Christopher S.; Sheth, Vinit M.; Snyder, Trevor A.; Schmidtke, David W.; 0000-0001-6404-318X (Schmidtke, DW); Snyder, Trevor A.; Schmidtke, David W.Due to the critical roles that platelets play in thrombosis during many biological and pathological events, altered platelet function may be a key contributor to altered hemostasis, leading to both thrombotic and hemorrhagic complications. Platelet adhesion at arterial shear rates occurs through binding to von Willebrand Factor via the glycoprotein (GP) GPIb receptor. GPIb binding can induce platelet activation distinguishable by P-selectin (CD62P) surface expression and α(IIb)β₃ activation, resulting in platelet aggregation and formation of the primary hemostatic plug to stop bleeding. Previous studies have used cone and plate viscometers to examine pathologic blood flow conditions, applied shear rates that are relatively low, and examined exposure times that are orders of magnitude longer compared to conditions present in ventricular assist devices, mechanical heart valves, or pathologic states such as stenotic arteries. Here, we evaluate the effect of short exposure to high shear on granule release and receptor shedding utilizing a constricted microfluidic device in conjunction with flow cytometry and enzyme-linked immunosorbent assay. In this study, platelets were first perfused through microfluidic channels capable of producing shear rates of 80 000-100 000 s⁻¹ for exposure times of 0-73 ms. We investigated platelet activation by measuring the expression level of CD62P (soluble and surface expressed), platelet factor 4 (PF4), and beta-thromboglobulin (βTG). In addition, we measured potential platelet receptor shedding of GPVI and GPIb using flow cytometry. The results showed that a single pass to high shear with short exposure times (milliseconds) had no effect on the levels of CD62P, GPVI and GPIb, or on the release of alpha granule content (PF4, βTG, and sP-selectin). Published by AIP Publishing.