We describe a microfabricated airway system integrated with computerized air-liquid two-phase microfluidics that enables onchip engineering of human airway epithelia and precise reproduction of physiologic or pathologic liquid plug flows found in the respiratory system. Using this device, we demonstrate cellularlevel lung injury under flow conditions that cause symptoms characteristic of a wide range of pulmonary diseases. Specifically, propagation and rupture of liquid plugs that simulate surfactantdeficient reopening of closed airways lead to significant injury of small airway epithelial cells by generating deleterious fluid mechanical stresses. We also show that the explosive pressure waves produced by plug rupture enable detection of the mechanical cellular injury as crackling sounds.airway reopening ͉ small airway epithelial cells ͉ mechanical forces ͉ microfluidic cell culture

Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined. However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves. This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber. The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture. The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion. These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serumcontaining media. This technology will allow future microscale tissue or cell studies to be more accessible, especially for highthroughput, complex, and long-term experiments. The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use.pump ͉ valve ͉ bioreactor ͉ mixer ͉ perfusion A dvanced microfluidic cellular assays (1-6) and microscale tissue engineering studies (7-10) would benefit from robust and convenient methods to computer-control accurate spatiotemporal patterns of microfluidic flows in arrays of fluidic networks. In the past, fluidic control included syringe pumps (11), hydrogel valves (12), gravity-driven pumps (13), evaporation-based pumps (14, 15), acoustic pumps (16), gas-generationbased pumps (17, 18), and centrifugal force in CD chips (19). Electrokinetic flow (20-22), thermopneumatic (23, 24), pneumatic͞hydraulic (25), or mechanical (26, 27) valves and pumps have a high degree of control, but require external connection lines to larger equipment for actuation. A few fully integrated and self-packaged systems (23, 28) have been developed, but lack the reconfigurability inherent with numerous active valves and pumps.Here, we report a method to precisely control fluid flow inside elastomeric capillary networks by using multiple (tens to hundreds) computer-controlled, piezoelectric, movable pins. These pins are positioned as a grid on a refreshable Braille display (e.g., F. H. Papenmeier, Schwerte, Germany), which is a tactile device used by the visually impaired to read computer text. Each pin can act as a valve and be shifted upward to push against channels contained in silicone rubber and completely shut the channel. Three sequential valves of this...

Endothelial cells (ECs) lining the inner lumen of blood vessels are continuously subjected to hemodynamic shear stress, which is known to modify EC morphology and biological activity. This paper describes a self-contained microcirculatory EC culture system that efficiently studies such effects of shear stress on EC alignment and elongation in vitro. The culture system is composed of elastomeric microfluidic cell shearing chambers interfaced with computer-controlled movement of piezoelectric pins on a refreshable Braille display. The flow rate is varied by design of channels that allow for movement of different volumes of fluid per variable-speed pump stroke. The integrated microfluidic valving and pumping system allowed primary EC seeding and differential shearing in multiple compartments to be performed on a single chip. The microfluidic flows caused ECs to align and elongate significantly in the direction of flow according to their exposed levels of shear stress. This microfluidic system overcomes the small flow rates and the inefficiencies of previously described microfluidic and macroscopic systems respectively to conveniently perform parallel studies of EC response to shear stress.

Evaporation is a critical problem when handling submicroliter volumes of fluids. This paper characterizes this problem as it applies to microfluidic cell culture in poly(dimethylsiloxane) (PDMS) devices and provides a practical solution. Evaporation-mediated osmolality shifts through PDMS membranes with varying thicknesses (10, 1, 0.2, or 0.1 mm) were measured over 96 h. Even in humidified cell culture incubators, evaporation through PDMS and associated shifts in the osmolality of culture media was significant and prevented mouse embryo and human endothelial cell growth and development. A simple diffusion model, where the measured diffusion coefficient for PDMS matches reported values of approximately 10-9 m2/s, accounts for these evaporation and osmolality shifts. To overcome this problem, a PDMS-parylene-PDMS hybrid membrane was developed that greatly suppresses evaporation and osmolality shifts, yet possesses thinness and the flexibility necessary to interface with deformation-based microfluidic actuation systems, maintains the clarity for optical microscopy, and enables the successful development of single-cell mouse embryos into blastocysts under static conditions and culture of human endothelial cells under dynamic recirculation of submicroliter volumes of media. These insights and methods demonstrated specifically for embryo and endothelial cell studies will be generally useful for understanding and overcoming evaporation-associated effects in microfluidic cell cultures.

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.