A critical need for enhancing usability and capabilities of microfluidic technologies is the development of standardized, scalable, and versatile control systems1,2. Electronically controlled valves and pumps typically used for dynamic flow regulation, although useful, can limit convenience, scalability, and robustness3–5. This shortcoming has motivated development of device-embedded non-electrical flow-control systems. Existing approaches to regulate operation timing on-chip, however, still require external signals such as timed generation of fluid flow, bubbles, liquid plugs or droplets, or an alteration of chemical compositions or temperature6–16. Here, we describe a strategy to provide device-embedded flow switching and clocking functions. Physical gaps and cavities interconnected by holes are fabricated into a three-layer elastomer structure to form networks of fluidic gates that can spontaneously generate cascading and oscillatory flow output using only a constant flow of Newtonian fluids as the device input. The resulting microfluidic substrate architecture is simple, scalable, and should be applicable to various materials. This flow-powered fluidic gating scheme brings the autonomous signal processing ability of microelectronic circuits to microfluidics where there is the added diversity in current information of having distinct chemical or particulate species and richness in current operation of having chemical reactions and physical interactions.

Microfluidic bioreactors fabricated from highly gas-permeable poly(dimethylsiloxane) (PDMS) materials have been observed, somewhat unexpectedly, to give rise to heterogeneous long term responses along the length of a perfused mammalian cell culture channel, reminiscent of physiologic tissue zonation that arises at least in part due to oxygen gradients. To develop a more quantitative understanding and enable better control of the physical-chemical mechanisms underlying cell biological events in such PDMS reactors, dissolved oxygen concentrations in the channel system were quantified in real time using fluorescence intensity and lifetime imaging of an oxygen sensitive dye, ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP). The data indicate that despite oxygen diffusion through PDMS, uptake of oxygen by cells inside the perfused PDMS microchannels induces an axial oxygen concentration gradient, with lower levels recorded in downstream regions. The oxygen concentration gradient generated by a balance of cellular uptake, convective transport by media flow, and permeation through PDMS in our devices ranged from 0.0003 (mg/l)/mm to 0.7 (mg/l)/mm. The existence of such steep gradients induced by cellular uptake can have important biological consequences. Results are consistent with our mathematical model and give insight into the conditions under which flux of oxygen through PDMS into the microchannels will or will not contribute significantly to oxygen delivery to cells and also provide a design tool to manipulate and control oxygen for cell culture and device engineering. The combination of computerized microfluidics, in situ oxygen sensing, and mathematical models opens new windows for microphysiologic studies utilizing oxygen gradients and low oxygen tensions.

Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however, these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12h. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch.

Chemokine CXCL12 promotes CXCR4-dependent chemotaxis of cancer cells to characteristic organs and tissues, leading to metastatic disease. This study was designed to investigate how cells expressing CXCR7 regulate chemotaxis of a separate population of CXCR4 cells under physiologic conditions in which cells are exposed to gradients of CXCL12. We recapitulated a cancer-stroma microenvironment by patterning CXCR4-expressing cancer cells in microchannels at spatially defined positions relative to CXCL12-producing cells and CXCR7-expressing cells. CXCR7 scavenges and degrades CXCL12, which has been proposed to facilitate CXCR4-dependent chemotaxis through a source-sink model. Using the microchannel device, we demonstrated that chemotaxis of CXCR4 cells depended critically on the presence and location of CXCR7 cells (sink) relative to chemokine secreting cells (source). Furthermore, inhibiting CXCR4 on migrating cells or CXCR7 on sink cells blocked CXCR4-dependent chemotaxis toward CXCL12, showing that the device can identify new therapeutic agents that block migration by targeting chemoattractant scavenging receptors. Our system enables efficient chemotaxis under much shallower yet more physiological chemoattractant gradients by generating an in vitro microenvironment where combinations of cellular products may be secreted along with formation of a chemoattractant gradient. In addition to elucidating mechanisms of CXCL-12 mediated chemotaxis, this simple and robust method can be broadly useful for engineering multiple microenvironments to investigate intercellular communication.

This paper describes a cell-exclusion patterning method facilitated by a polymeric aqueous two-phase system. The immersion aqueous phase (polyethylene glycol) containing cells rehydrates a dried disk of the denser phase (dextran) on the substrate to form a dextran droplet. With the right properties of the phase-forming polymers, the rehydrating droplet remains immiscible with the immersion phase. Proper formulation of the two-phase system ensures that the interfacial tension between the rehydrating droplet and the surrounding aqueous phase prevents cells from crossing the interface so that cells only adhere to the regions of the substrate around the dextran phase droplet. Washing out the patterning two-phase reagents reveals a cell monolayer containing a well-defined circular gap that serves as the migration niche for cells of the monolayer. Migration of cells into the cell-excluded area is readily visualized and quantified over time. A 96-well plate format of this “gap healing” migration assay demonstrates the ability to detect inhibition of cell migration by known cytoskeleton targeting agents. This straightforward method, which only requires a conventional liquid handler and readily prepared polymer solutions, opens new opportunities for high throughput cell migration assays.

This mini-review provides a brief overview of recent devices that use networks of elastomeric valves to minimize or eliminate the need for interconnections between microfluidic chips and external instruction lines that send flow control signals. Conventional microfluidic control mechanisms convey instruction signals in a parallel manner such that the number of instruction lines must increase as the number of independently operated valves increases. The devices described here circumvent this "tyranny of microfluidic interconnects" by the serial encoding of information to enable instruction of an arbitrary number of independent valves with a set number of control lines, or by the microfluidic circuit-embedded encoding of instructions to eliminate control lines altogether. Because the parallel instruction chips are the most historical and straightforward to design, they are still the most commonly used approach today. As requirements for instruction complexity, chip-to-chip communication, and real-time on-chip feedback flow control arise, the next generation of integrated microfluidic circuits will need to incorporate these latest interconnect flow control approaches.

Generation of stable soluble-factor gradients in microfluidic devices enables studies of various cellular events such as chemotaxis and differentiation. However, many gradient devices directly expose cells to constant fluid flow and that can induce undesired responses from cells due to shear stress and/or wash out of cell-secreted molecules. Although there have been devices with flow-free gradients, they typically generate only a single condition and/or have a decaying gradient profile that does not accommodate long-term experiments. Here we describe a microdevice that generates several chemical gradient conditions on a single platform in flow-free microchambers which facilitates steady-state gradient profiles. The device contains embedded normally-closed valves that enable fast and uniform seeding of cells to all microchambers simultaneously. A network of microchannels distributes desired solutions from easy-access open reservoirs to a single output port, enabling a simple setup for inducing flow in the device. Embedded porous filters, sandwiched between the microchannel networks and cell microchambers, enable diffusion of biomolecules but inhibit any bulk flow over the cells.

The study of complex biological systems requires methods to perturb the system in complex yet controlled ways to elucidate mechanisms and dynamic interactions, and to recreate in vivo conditions in flexible in vitro set-ups. This paper reviews recent advances in the use of micro- and nanotechnologies in the study of complex biological systems and the advantages they provide in these two areas. Particularly useful for controlling the chemical and mechanical microenvironments of cells is a set of techniques called soft lithography, whereby elastomeric materials are used to transfer and generate micro- and nanoscale patterns. Examples of some of the capabilities of soft lithography include the use of elastomeric stamps to generate micropatterns of protein and the use of elastomeric channels to localize chemicals with subcellular spatial resolutions. These types of biological micro- and nanotechnologies combined with mathematical modeling will propel our understandings of cellular and subcellular physiology to new heights.