Practical fabrication of microfluidic platforms for live-cell microscopy

Abstract

We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities.

Vibration of osteoblastic cells using a novel motion‐control platform does not acutely alter cytosolic calcium, but desensitizes subsequent responses to extracellular ATP

“…Having validated that we can precisely apply accelerations at specific frequencies with our platform, we examined the effects of LMHF vibration on live cells. Based on previous reports that other forms of mechanical stimuli (e.g., fluid shear) induce calcium signaling in osteoblastic cells (Donahue, Jacobs, & Donahue, ; Lorusso et al, ), we anticipated that vibration might also induce acute elevation of [Ca 2+ ] i . To investigate this possibility, we first loaded UMR‐106 rat osteoblastic cells with fura‐2, placed them in the vibration device, and monitored [Ca 2+ ] i levels using microspectrofluorometry (Figure ).…”

“…We first used the custom motion‐control platform to investigate whether vibration induces transient changes in [Ca 2+ ] i similar to those induced by other mechanical stimuli such as fluid shear, membrane stretch and deformation (Adachi et al, ; Donahue et al, ; Hung, Pollack, Reilly, & Brighton, ; Lorusso et al, ; Mikolajewicz et al, ; Nishitani, Saif, & Wang, ; Thompson et al, ; Wu, Wong, Glogauer, Ellen, & McCulloch, ). Under the conditions tested, changes in [Ca 2+ ] i were not observed in response to mechanical vibration of UMR‐106 cells, MC3T3‐E1 cells, or primary rat osteoclasts.…”

“…Thus, the fluid shear stress at the dish surface can be solved using the Navier–Stokes equation. Based on the conditions present in Figures , the maximal shear stress at the wall was calculated to be ~0.03 Pa, approximately an order of magnitude less than that used to stimulate [Ca 2+ ] i transients in osteoblasts and other cell types (Hung, Allen, Pollack, & Brighton, ; Kamioka et al, ; Lorusso et al, ; Lorusso et al, ; Masyuk et al, ). Indeed, in the present study, we observed that the resultant fluid shear due to vibration of the dish is insufficient to generate calcium transients in osteoblastic cells (Figures ).…”

“…Again, cell samples were excited with alternating wavelengths of 345 and 380 nm using a DeltaRam X illuminator and the emission signal was monitored at 510 ± 20 nm, as described previously (Wheal, Beach, Tanabe, Dixon, & Sims, ). Ratio images were acquired at 1 s intervals with a sCMOS camera (pco.edge 4.2 LT, 2048 × 2048 pixels; PCO AG, Kelheim, Germany) and EasyRatioPro 2.3 Software (Horiba‐ Photon Technology International) (Lorusso et al, ). The ratio of fluorescence intensities at 345/380 nm gives an index of [Ca 2+ ] i independent of cell thickness or dye leakage.…”

“…The biological mechanisms underlying mechanotransduction are areas of intense study (Wall et al, 2017). In cells of the osteoblast lineage, early signaling events activated by mechanical stimuli such as fluid flow and membrane stretch include transient elevation in the concentration of cytosolic free calcium ([Ca 2+ ] i ) (Jing et al, 2014;Lorusso et al, 2016;Mikolajewicz, Zimmermann, Willie, & Komarova, 2018;Thi, Suadicani, Schaffler, Weinbaum, & Spray, 2013). Furthermore, such responses have been shown to be dependent on the release of nucleotides, such as ATP, that act in an autocrine/ paracrine manner through Ca 2+ -mobilizing P2 receptors on the cell surface (Riddle, Taylor, Rogers, & Donahue, 2007).…”