Efficient Dielectrophoretic Patterning of Embryonic Stem Cells in Energy Landscapes Defined by Hydrogel Geometries

In this study, we have developed an integrated microfluidic platform for actively patterning mammalian cells, where poly(ethylene glycol) (PEG) hydrogels play two important roles as a non-fouling layer and a dielectric structure. The developed system has an embedded array of PEG microwells fabricated on a planar indium tin oxide (ITO) electrode. Due to its dielectric properties, the PEG microwells define electrical energy landscapes, effectively forming positive dielectrophoresis (DEP) traps in a low-conductivity environment. Distribution of DEP forces on a model cell was first estimated by computationally solving quasi-electrostatic Maxwell’s equations, followed by an experimental demonstration of cell and particle patterning without an external flow. Furthermore, efficient patterning of mouse embryonic stem (mES) cells was successfully achieved in combination with an external flow. With a seeding density of 107 cells/mL and a flow rate of 3 μL/min, trapping of cells in the microwells was completed in tens of seconds after initiation of the DEP operation. Captured cells subsequently formed viable and homogeneous monolayer patterns. This simple approach could provide an efficient strategy for fabricating various cell microarrays for applications such as cell-based biosensors, drug discovery, and cell microenvironment studies

Culturing Aerobic and Anaerobic Bacteria and Mammalian Cells with a Microfluidic Differential Oxygenator

In this manuscript, we report on the culture of anaerobic and aerobic species within a disposable multilayer polydimethylsiloxane (PDMS) microfluidic device with an integrated differential oxygenator. A gas-filled microchannel network functioning as an oxygen−nitrogen mixer generates differential oxygen concentration. By controlling the relative flow rate of the oxygen and nitrogen input gases, the dissolved oxygen (DO) concentration in proximal microchannels filled with culture media are precisely regulated by molecular diffusion. Sensors consisting of an oxygen-sensitive dye embedded in the fluid channels permit dynamic fluorescence-based monitoring of the DO concentration using low-cost light-emitting diodes. To demonstrate the general utility of the platform for both aerobic and anaerobic culture, three bacteria with differential oxygen requirements (E. coli, A. viscosus, and F. nucleatum), as well as a model mammalian cell line (murine embryonic fibroblast cells (3T3)), were cultured. Growth characteristics of the selected species were analyzed as a function of eight discrete DO concentrations, ranging from 0 ppm (anaerobic) to 42 ppm (fully saturated)

Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells

Background Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands. Methodology/Principal Findings We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs. Conclusions/Significance Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.National Institutes of Health (U.S.) (NIH grant No. EB007278)Swiss National Science FoundationSwiss National Science Foundatio

From Cleanroom to Desktop: Emerging Micro-Nanofabrication Technology for Biomedical Applications

This review is motivated by the growing demand for low-cost, easy-to-use, compact-size yet powerful micro-nanofabrication technology to address emerging challenges of fundamental biology and translational medicine in regular laboratory settings. Recent advancements in the field benefit considerably from rapidly expanding material selections, ranging from inorganics to organics and from nanoparticles to self-assembled molecules. Meanwhile a great number of novel methodologies, employing off-the-shelf consumer electronics, intriguing interfacial phenomena, bottom-up self-assembly principles, etc., have been implemented to transit micro-nanofabrication from a cleanroom environment to a desktop setup. Furthermore, the latest application of micro-nanofabrication to emerging biomedical research will be presented in detail, which includes point-of-care diagnostics, on-chip cell culture as well as bio-manipulation. While significant progresses have been made in the rapidly growing field, both apparent and unrevealed roadblocks will need to be addressed in the future. We conclude this review by offering our perspectives on the current technical challenges and future research opportunities