Microfluidic perfusion systems, characterized by deterministic flow, low reagent consumption, small dead volumes, large integration in small footprints, high-throughput operation, and low-cost fabrication, are being increasingly used for cell culture studies in applications such as basic cell biology, molecular biological assays, tissue engineering, and systems biology. We report a multipurpose, pressure-driven and computer-controlled microfluidic perfusion device containing sixteen inlets and a large cell culture chamber. The user can choose, with sub-second temporal resolution, (a) to feed the chamber with one of 16 inlets, all 16 inlets, or one of 64 combinations of 2, 4, or 8 inlets using a binary multiplexer; (b) to introduce into the chamber a heterogeneous laminar flow of the inlets, a smoothened gradient, or a fully homogenized mixture; (c) to bypass the chamber in order to purge the inlet lines so as to minimize the dead volume; (d) to generate asymmetrical and curvilinear flow patterns within the chamber by opening side outlets; and (e) to slow down the flow by combinatorially adding segments of high fluid resistance (sixteen different levels of flow rates are possible using only four valves). All functionalities are combined to create complex gradient patterns and sequential perfusions within the central chamber.

Gradients of biochemical molecules play a key role in many physiological processes such as axon growth, tissue morphogenesis, and trans-epithelium nutrient transport, as well as in pathophysiological phenomena such as wound healing, immune response, bacterial invasion, and cancer metastasis. In this paper, we report a microfluidic transwell insert for generating quantifiable concentration gradients in a user-friendly and modular format that is compatible with conventional cell cultures and with tissue explant cultures. The device is simply inserted into a standard 6-well plate, where it hangs self-supported at a distance of ~250 μm above the cell culture surface. The gradient is created by small microflows from the device, through an integrated track-etched porous membrane, into the cell culture well. The microfluidic transwell can deliver stable, quantifiable gradients over a large area with extremely low fluid shear stress to dissociated cells or tissue explants cultured independently on the surface of a 6-well plate. We used finite-element modeling to describe the porous membrane flow and molecular transport and to predict gradients generated by the device. Using the device, we applied a gradient of the chemotactic peptide N-Formyl-Met-Leu-Phe (fMLP) to a large population of HL-60 cells (a neutrophil cell line) and directly observed the migration with time-lapse microscopy. On quantification of the chemotactic response with an automated tracking algorithm, we found 74% of the cells moving towards the gradient. Additionally, the modular design and low fluid shear stress made it possible to apply gradients of growth factors and second messengers to mouse retinal explant cultures. With a simplified interface and well-defined gradients, the microfluidic transwell device has potential for broad applications to gradient-sensing biology.

Control of the 3D microenvironment for cultured cells is essential for understanding the complex relationships that biomolecular concentration gradients have on cellular growth, regeneration, and differentiation. This paper reports a microfluidic device for delivering gradients of soluble molecules to cells in an open reservoir without exposing the cells to flow. The cells are cultured on a polyester membrane that shields them from the flow that delivers the gradient. A novel "lid" design is implemented which prevents leakage from around the membrane without requiring sealing agents or adhesives. Once layers are molded, device fabrication can be performed within minutes while at room temperature. Surface gradients were characterized with epifluorescence microscopy; image analysis verified that sharp gradients (∼33 μm wide) can be reproducibly generated. We show that heterogeneous laminar flow patterns of Orange and Green Cell Tracker (CT) applied beneath the membrane can be localized to cells cultured on the other side; concentration profile scans show the extent of CT diffusion parallel to the membrane's surface to be 10-20 μm. Our device is ideal for conventional cell culture because the cell culture surface is readily accessible to physical manipulation (e.g., micropipette access), the cell culture medium is in direct contact with the incubator atmosphere (i.e., no special protocols for ensuring proper equilibration of gas concentrations are required), and the cells are not subjected to flow-induced shear forces, which are advantageous attributes not commonly found in closed-channel microfluidic designs.

We have investigated the bonding stability of various silane treatments for the integration of track-etched membranes with poly(dimethylsiloxane) (PDMS) microfluidic devices. We compare various treatments using trialkoxysilanes or dipodal silanes to determine the effect of the organofunctional group, cross-link density, reaction solvent, and catalyst on the bond stability. We find that devices made using existing silane methods delaminated after one day when immersed in cell culture medium at 37 C. In contrast, the dipodal silane, bis [3-(trimethoxysilyl)propyl]amine, is shown to yield stable and functional integration of membranes with PDMS that is suitable for long-term cell culture. To demonstrate application of the technique, we fabricated an open-surface device in which cells cultured on a track-etched membrane can be stimulated at their basal side via embedded microfluidic channels. C2C12 mouse myoblasts were differentiated into myotubes over the course of two weeks on these devices to demonstrate biocompatibility. Finally, devices were imaged during the basalside delivery of a fluorescent stain to validate the membrane operation and longterm stability of the bonding technique. V C 2014 AIP Publishing LLC.

Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Wellcharacterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to preestablished cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.