The integration of semiporous membranes into poly(dimethylsiloxane) (PDMS) microfluidic devices is useful for mass transport control. Several methods such as plasma oxidation and manual application of PDMS prepolymer exist to sandwich such membranes into simple channel structures, but these methods are difficult to implement with reliable sealing and no leakage or clogging for devices with intricate channel features. This paper describes a simple but robust strategy to bond semiporous polyester and polycarbonate membranes between layers of PDMS microchannel structures effectively without channel clogging. A thin layer of PDMS prepolymer, spin-coated on a glass slide, is transferred to PDMS substrates with channel features as well as to the edges of the semiporous membrane by stamping. This thin PDMS prepolymer serves as "mortar" to strongly bond the two PDMS layers and seal off the crevices generated from the thickness of the membranes. This bonding method enabled the fabrication of an 8x12 criss-crossing microfluidic channel array with 96 combinations of fluid interactions. The capability of this device for bioanalysis was demonstrated by measuring responses of cells to different color fluorescent reagents.

The formation of spherical aggregates of cells called embryoid bodies (EBs) is an indispensable step in many protocols in which embryonic stem (ES) cells are differentiated to other cell types. Appropriate morphology and embryo size are critical for the sequential developmental stages of naturally conceived embryos. Likewise, regulating the size of EBs and the timing of their formation is crucial for controlling the differentiation of ES cells within the EB. Existing methods of formation of EBs, however, are tedious or provide heterogeneously-sized EBs. Here we describe a microfluidic system for straightforward synchronized formation of uniform-sized EBs, the size of which can be controlled by changing the cross-sectional size of microchannels in the microfluidic device. The device consists of two microchannels separated by a semi-porous polycarbonate membrane treated to be resistant to cell adhesion. ES cells introduced into the upper channel self-aggregate to form uniformly-sized EBs. The semi-porous membrane also allows subsequent treatment of the non-attached EBs with different reagents from the lower channel without the need for wash out because of the compartmentalization afforded by the membrane. This method provides a simple yet robust means to control the formation of EBs and the subsequent differentiation of ES cells in a format compatible for ES cell processing on a chip.

This paper describes the use of arrays of horizontally-oriented reservoirs to deliver liquids through microchannels at a constant flow rate over extended periods of time (hours to days). The horizontal orientation maintains a constant hydraulic pressure drop across microfluidic channels even as the volumes of liquids within the reservoirs change over time. For a given channel-reservoir system, the magnitude of the flow velocity depends linearly on the height difference between reservoirs. The simple structure and operation mechanism make this pumping system versatile. A one-inlet-one-outlet system was used to continuously deliver media for perfusion cell culture, and an array of inlet reservoirs coupled to an outlet reservoir via microchannels was used to drive flows of multiple laminar streams. The parallel pumping scheme conveniently generated various smooth and step concentration gradients, and allowed evaluation of the effect of colchicine on myoblasts. Since the reservoir arrays are configured to be compatible with commercialized multichannel pipettors designed for 96 well plate handling, this simple pumping scheme is envisioned to be broadly useful for medium to high throughput microfluidic perfusion cell culture assays, cell migration assays, multiple laminar flow drug tests, and any other applications needing multiple microfluidic streams.

This paper describes a simple reversible hydrogel patterning method for 3D cell culture. Alginate gel is formed in select regions of a microfluidic device through light-triggered release of caged calcium. In the pre-gelled alginate solution, calcium is chelated by DM-nitrophen (DM-n) to prevent cross-linking of alginate. After sufficient UV exposure the caged calcium is released from DM-n causing alginate to cross-link. The effect of using different concentrations of calcium and chelating agents as well as the duration of UV exposure is described. Since the cross-linking is based on calcium concentration, the cross-linked alginate can easily be dissolved by EDTA. We also demonstrate application of this capability to patterned microscale 3D co-culture using endothelial cells and osteoblastic cells in a microchannel.

Currently, average roughness (Ra), is the parameter specified for controlling surface quality. The influence of surface averageThe increased functionality of today's advanced electronics, such roughness (Ra) does not correlate well to peel strength between as multi-function mobile equipment, is driven by semiconductor epoxy substrates and plated copper. In this article, we will use 3-D technology development. The added functionality of semiconductor roughness parameters to provide a better insight into the relationship and passive devices increase circuit density and decrease the between peel strength and surface roughness. available assembly space in the device. Packaging substrates are controlled by semiconductor trends. As semiconductor chip EXPERIMENT performance improves, the substrates and packaging technologies must keep pace.

We present a microsystem capable of electrochemically patterning the dissolved oxygen gradients present in cell culture. Multiple microelectrodes in an array are separated from the cells by an oxygen permeable membrane. Each electrode generates distinct amounts of dissolved oxygen via electrolysis; these different sources superimpose to generate one-and two-dimensional microgradient profiles not possible with other methods. We believe this is the first technology that enables researchers to pattern localized oxygen doses and program arbitrary oxygen gradients with microscale resolution during cell culture. We present fluorescent and colorimetric data on the construction of two-dimensional oxygen microgradients in aqueous solution and preliminary cell culture results with C2C12 rat myoblasts.

Many biological studies, drug screening methods, and cellular therapies require culture and manipulation of living cells outside of their natural environment in the body. The gap between the cellular microenvironment in vivo and in vitro, however, poses challenges for obtaining physiologically relevant responses from cells used in basic biological studies or drug screens and for drawing out the maximum functional potential from cells used therapeutically. One of the reasons for this gap is because the fluidic environment of mammalian cells in vivo is microscale and dynamic whereas typical in vitro cultures are macroscopic and static. This presentation will give an overview of efforts in our laboratory to develop programmable microfluidic systems that enable spatio-temporal control of both the chemical and fluid mechanical environment of cells. The technologies and methods close the physiology gap to provide biological information otherwise unobtainable and to enhance cellular performance in therapeutic applications. Specific biomedical topics that will be discussed include subcellular signalling in normal and cancer cells, in vitro fertilization on a chip, studies of the effect of physiological and pathological fluid mechanical stresses on endothelial and epithelial cells, and microfluidic stem cell engineering. In the nanoscale regime, tunable nanochannels that can manipulate single DNA molecules will be discussed.