Latest Developments in Microfluidic Cell Biology ... - ACS Publications

Anal. Chem. 2010, 82, 4848–4864

Latest Developments in Microfluidic Cell Biology and Analysis Systems Georgette B. Salieb-Beugelaar,†,‡ Giuseppina Simone,† Arun Arora,† Anja Philippi,† and Andreas Manz*,†,§,| KIST Europe, Korea Institute of Science and Technology, Campus E71, 66123 Saarbru¨cken, Germany, MESA+ Institute for Nanotechnology/BIOS/Lab-on-a-Chip Group, Twente University, Building Carre´, 7500 AE, Enschede, The Netherlands, FRIAS, Freiburg Institute for Advanced Studies, Albert-Ludwig-Universita¨t Freiburg, Albertstrasse 19, 79104 Freiburg, Germany, and IMTEK, Institute for Microsystem Technology, University of Freiburg, Georges-Ko¨hler-Allee 103, 79110 Freiburg, Germany Review Contents Technology Designs and Microfabrication Surface Modification Optical Integration Flow Control Standard Operations Cell Cultivation Reactors and Mixers Manipulation of Cells Cell Counting and Flow Cytometry Separation, Sorting, and Trapping of Cells Detection Other Applications Clinical Diagnostics Cancer Research Drug Discovery and Screening Stem Cell Research Assays Single Cell Applications Neuroscience Microbiology Secretion/Release Studies Migration and Chemotaxis Intra- and Intercellular Signaling Cell Mechanics Tissue Models Assisted Reproduction Other Applications Literature Cited

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The multidisciplinary field of micro total analysis systems (µTAS) also called “lab-on-a-chip” has enormously expanded in the last 2 years. By cross referencing online keyword searches in citation and journal databases, several thousands of excellent publications were found for the period between March 2008 and February 2010. The publications are spread among a wide variety of journals but are most frequently found in journals with high impact factors such as Nature, Science, PNAS, Lab on a Chip, and Analytical Chemistry. The annual µTAS conference also served as a great source of information. Among the collected publications, * To whom correspondence should be addressed. E-mail: manz@ kist-europe.de. † KIST Europe, Korea Institute of Science and Technology. ‡ Twente University. § FRIAS Freiburg. | IMTEK Freiburg.

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we noticed the rapidly developing field of microfluidic systems for support in cell biology research. Since cell biology and tissue engineering are based mostly on molecular phenomena, we believe that papers targeting cell biology, in the sense of obtaining information, are related to chemistry and, therefore, should be covered in our review. For that reason, the initial plan to follow the earlier reviews (1) was adapted, and the reader can now find the corresponding references split into two papers; part one (2) is dealing with technology and more classical applications in analytical chemistry, whereas this second part is dealing with cell biology applications. We intend to provide an overview of the novel and significant developments and to assist the newcomers and more experienced with an update of the novel achievements. In this review, we have focused on research into fluidic microsystems for cell applications. For this reason, related research in the “nofluidics” micro cell plates, Petri dish-like cultures, “classic” microwell arrays, non-integrated sensors, theory, and molecular simulations as well as reviews and trend articles have been omitted. The section of technology in this review is limited and mainly is focused on the design and on-chip used technologies specific for cell applications. Furthermore, the rapidly growing field of droplet-based technologies is featured throughout the Review. Even though this review focuses on microfluidic systems for cells, it remains a multidisciplinary field, which sometimes makes it difficult to draw boundaries between the different presented activities. TECHNOLOGY Designs and Microfabrication. Continuous microfabrication developments have resulted in many innovations for cells-on-chip systems. Huang and co-workers (3) fabricated transparent silicon dioxide microtube arrays in which cells can be cultivated in a 2D confined environment. The fabrication technology consists of conventional photolithography, electron beam evaporation deposition, and chemical etching. By altering the geometry of the photoresist patterns and the deposition parameters, the size of the microtubes can be tuned. The manufacturing of cell adhesive “Janus” polyurethane microfibers were described by Jung et al. (4). The use of spontaneous carbon dioxide bubble formation during the synthesis in multiple laminar streams resulted in an asymmetrically porous polyurethane microfiber. The porous site of the fiber can be used for cultivating cells. Thermoplastic and 10.1021/ac1009707  2010 American Chemical Society Published on Web 05/12/2010

elastomeric polymers were used by Mehta and co-workers (5) to produce hard top, soft bottom microfluidic devices. Channel features were manufactured on rigid polymers such as polyethylene terephtalate glycol (PETG), cyclic olefin copolymer (COC), and polystyrene (PS) by hot embossing. These rigid structures were bonded onto elastomeric polymers such as polyurethane (PU) or polydimethylsiloxane (PDMS)-parylene C-PDMS. The devices were used for cell cultivation by employing the deformation of the device to perfuse and recirculate media. Hsieh and co-workers (6) presented the development of a microculture platform, capable of automatic cell plating, medium supply, waste removal, and real time monitoring of cellular responses. The platform was successfully tested with OC-2 cancer cells. Kim and co-workers (7) presented a microfluidic system that integrates pulsatile cell sorting, delivering, and cell cultivating functions on a single platform. The devices consisted of two reservoirs, a pulsatile pumping system containing two soft check valves, six switch valves, and three cultivation chambers integrated together. Damage-free transport of human breast adenocarcinoma cells (MCF-7) and mesenchymal stem cells (hMSCs) was demonstrated by the pumping system. Meyvantsson et al. (8) developed a device for the cultivation of cells that uses surface tension driven passive pumping with traditional fluid handling tools. The tubeless microfluidics presented here provides compartmentalization without sacrificing microfluidic manipulation and without the need for elastomers that adsorb hydrophobic compounds. HMT-3522 S1 and Hs578 S1 cells were patterned and cultivated for up to 4 days in the device. Surface Modification. Westcott and co-workers (9) developed a method to fabricate a variety of different topologies and surface chemistries on gold surfaces. A self-assembled monolayer (SAM) was formed on the gold by immersing the surface in an ethanolic solution containing tetra (ethylene glycol) undecane thiol. Adhesion of fibroblasts to gold surfaces with a SAM was successful after the addition of a hydrophobic hexadecanethiol to the gold surfaces in the microchannel. Goto et al. (10) generated a microand nanometer scale gold stripe pattern on quartz glass substrate by electron-beam lithography and metal sputtering. The surface was modified with alkanethiol resulting in control of both the topology and the chemical properties. Mouse fibroblast cells recognized the chemical properties and were cultivated. Optical Integration. Gottschamel et al. (11) developed a disposable microfluidic biofilm chip. In this device, the incorporation of high density interdigitated capacitors, which are isolated by a 700 nm SU-8 layer, enables contactless bioimpedance spectroscopy of cultivated cells. Additionally, respiration of the biofilm is provided by measuring the oxidized redox-mediators at band electrodes that are located in the microchannels. The disposable biofilm analysis platform is used to continuously monitor the dynamic responses of C. albicans to different glucose and galactose concentrations. To detect the small changes of oxygen encountered in the cellular environment, Nock et al. (12) developed polymer optical oxygen sensors. The sensors are manufactured from the oxygen-sensitive platinum(II) octaethylporphyrin ketone fluorescent dye which is dissolved in polystyrene and spin coated onto glass substrates. The sensor patterns were manufactured by soft lithography with PDMS stamps and reactive ion etching and subsequently integrated into a PDMS

microfluidic device. The sensors were tested by fluorescence microscopy detecting gaseous and dissolved oxygen. Lensless high-resolution on-chip optofluidic microscopes were developed by Cui and co-workers (13). Microfluidic flow is used to deliver specimens across array(s) of micrometer size apertures defined on a metal-coated complementary metal-oxide semiconductor (CMOS) sensor to generate direct projection images. Two different systems were developed. The first system utilizes a gravitydriven microfluidic flow for sample scanning and is suited for imaging elongate objects, such as C. elegans. The second system employs an electrokinetic drive for flow control and is suited for imaging cells and other spherical/ellipsoidal objects. Hajjoul et al. (14) developed a device for fast 3D particle tracking in living cells based on V-shaped micromirrors. These micromirrors are used to observe fluorescent specimen from multiple vantage points and provide a simultaneous stereo image, which can be recombined for 3D reconstruction. The functionality of the device was proven with the study of chromatin dynamics using yeast strains with one or two green fluorescent protein (GFP) tagged gene loci. Flow Control. 3D microfluidic networks for constructing a cell cultivation array were monolithically fabricated by Liu and co-workers (15). In this device, composed of parylene-C and PDMS layers, they integrated a combinatorial mixer with three inputs and one control channel, which can deliver simultaneously to the eight cultivation chambers of the device. Using the device, it was possible to cultivate rat neuroblastoma cells. Breuer (16) demonstrated that thousands of individual self-organizing bacterial cells can generate collective fluid motion that can pump fluid autonomously through a microfabricated channel for several hours at speeds of 25 µm s-1. By varying the geometry of the microchannel or the chemical stimuli in the buffer, the bacterial pump can be tuned. To simultaneously deliver many different profiles of stimuli to adherent cells using a single input control, King et al. (17) developed a flow-encoded switching design (FES). This design is based on laminar flow and diffusion limited mixing and is controlled by a single differential pressure. Wu and co-workers (18) presented a high throughput perfusion-based microbioreactor platform and highlighted their developed integrated serpentine-shaped pneumatic micropump. Three rectangular pneumatic chambers are connected by pneumatic microchannels with U-shaped corners. The design is such that the S-shaped micropump can be fine-tuned using the fluidic resistance of the system. The functionality of this system was tested by cultivating oral cancer cells for 48 h and subsequent testing of their viability. Park and co-workers (19) presented a microfluidic living cell array that consists of eight cultivation chambers. The flow streams vertically to pass over or under channels and on-chip valves. The design is such that the chambers are continuously perfused; no cross-chamber communication is possible, and the cells are protected from undesired shear stresses. The device was used to characterize different phenotypes of alveolar epithelial type II cells. Evander et al. (20) manufactured a glass device that uses acoustic forces. Inside a separation channel, with two buffer inlets, cells can be switched from one buffer to another without mechanically stressing the cells. Van der Steen et al. (21) presented an active mixing method for a microbioreactor. They generated an Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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oscillating fluid flow when varying the pressure to a microchannel looping tangentially into a cylindrical microreactor. STANDARD OPERATIONS Cell Cultivation. Mammalian Cells. Torisawa and co-workers (22) developed a method for the formation of cocultivated spheroids of various geometries and compositions in order to manipulate cell-cell interaction dynamics in 3D. The formation of “Janus” spheroids constructed of mouse embryonic cells (mES) and hepatocytes next to the regional differentiation of the mES was shown. Huang et al. (23) developed a device for the side-byside patterning of multiple gel types that allows the cultivation of different cell types and real time imaging of the interaction of cells to both autocrine and paracrine signaling. The capability of the device was tested by cultivating metastatic breast cancer cells next to macrophage cells. Wang et al. (24) reported a simple method for the cultivation and self-loading of mammalian cells in a microfluidic system with multiple chambers. This system is suitable for high throughput screening. Jang et al. (25) developed an osteoblast-based 3D continuous perfusion microfluidic system for drug screening. With the system, they were able to monitor the cells for 10 days and sampling of supernatant for an osteoblast differentiation assay was possible. Lii and co-workers (26) developed a 3D microenvironment for real-time monitoring of an array of mammalian cells. In this device, pneumatically actuated microvalves enable the selective delivery of reagents to individual 3D chambers as well as spatial control of diffusion pathways between the neighboring chambers. Yu et al. (27) reported an integrated microfluidic cell-cultivation platform for the parallel analysis of microenvironmental cues on different mammalian cells and their cellular responses to external stimuli. Several cell lines were cultivated and tested on drug-induced apoptotic responses; additionally, on-chip transfection of a reporter gene was performed. Hufnagel et al. (28) presented a device for the cultivation of mammalian cells and delivery into microfluidic microdroplets. Cell growth for up to 7 days was demonstrated for CHO-K1 cells, as well as successful transfection on-chip. Long and co-workers (29) developed a system for encapsulation and cultivation of neuron cells in microgel particles. Preliminary viability tests indicated the potential of the device for cell cultivation, growth analysis, and differentiation. Reusable chips made of optical-grade PDMS bonded to glass chips were reported by Wlodkowic et al. (30). The devices were tested for the cultivation of mammalian cells, adenoviral gene delivery to mammalian cells, and gravity enforced formation of multicellular tumor spheroids. Yang et al. (31) presented a multilayered device, manufactured from PDMS, for the separation of chemical stimulants over single living cells vertically through aqueous-phase separation by the use of laminar flow. NIH3T3 fibroblasts were successfully cultivated on top of single micrometer-scale channels inside a larger channel. Next to this, with the device, the labeling of the apical domain of the membrane of single cells through the main channel with simultaneous and distinct labeling of the basal domain via the lower microchannels was possible. Zhang et al. (32) reported a platform with eight microsieves in each cell cultivation chamber to enable continuous cell cultivation. With the sieves, uniform cell loading and distribution can be obtained. The utility of the device was demonstrated with the cultivation of the adherent cell line BALB/ 3T3. O’Neill and co-workers (33) developed a cell curtain which 4850


is defined as a PDMS wall that extends from the ceiling of a cell cultivation microchamber to within micrometers of the chamber floor. The cell curtain facilitates cellular assays and was demonstrated by observing a monolayer of human epidermal keratinocyte (HEK) colonies for 48 h longer than that observed with a non-curtained conventional microfluidic chamber. Villa-Diaz and co-workers (34) developed a device that allows the cultivation and experimentation on individual human embryonic stem cell (hESC) colonies in dynamic (flow applied) or static (without flow) conditions. Cardiomyocytes are known to dedifferentiate due to the lack of appropriate microenvironmental cues; therefore, Au et al. (35) designed a device on which simultaneous application of topographical and electrical cues are exposed to the cells. This device enables the cultivation of differentiated cardiomyocytes. Yamada and co-workers (36) developed a device for the millisecond treatment of cells via a two-step carrier-medium exchange. As a result of the rapid process, the medium can be hydrodynamically exchanged without significant diffusive mixing. Zhang and Roper (37) presented a perfusion system for the automatic delivery of temporal gradients to cells. The capability of the device was shown by the delivery of glucose gradients between 3 and 20 mM single islets of Langerhans, which resulted in clear and intense oscillations in the intracellular Ca2+ concentration. Embryo Cultivation System. Kim et al. (38) described a microfluidic in vitro cultivation system for the improvement of embryonic development within a constricted channel that mimics peristaltic muscle contraction. The fertilized bovine embryos were cultivated on the microfluidic in vitro cultivation system until the blastocyst, hatching, or hatched blastocyst stages. The reported results suggest that the effect of constriction is vital for the early development of bovine embryos in assisted-reproduction research. Kimura and co-workers presented (39) a device for the preparation of embryos, which can be used for assisted reproduction technology. The device was examined on the trapping, cultivation, and release of mouse embryos. Yeast Cells. Huang and co-workers (7) presented rolled-up transparent microtubes, which were used to study the mechanical interaction between the tubes and the 2D confined cultivated yeast cells. Kurth et al. (40) manufactured a bilayer microfluidic chip which allowed the separation of cell handling and cultivation from the supply of the chemicals of interest. The chip was tested with yeast cells. Rowat et al. (41) designed a microfluidic device that tracks the lineages of single cells in parallel by constraining them to grow in lines for as many as eight divisions. The functionality of the device was tested with S. cerevisiae. Luo et al. (42) described a high-throughput microfluidic system that was used for diffusionbased monolayer yeast cell culture monitoring. The system was demonstrated by the investigation of budding and fission yeast cultures for many cellular generations. Tissues. High-density arrays of hepatocytes in a tissue-like microarchitecture were presented by Zhang et al. (43). In the bioreactor, the environment mimics physiological liver mass transport that enables the cultivation of cells without nutrition limitation for over 1 week. Bruzewicz et al. (44) presented a device in which mammalian cells were cultivated in 3D using gels. The design is such that cells were able to proliferate to densities comparable to those of native tissues. The capability of the device was shown with the cultivation of 3T3 fibroblasts and HepG2 liver

cells. Zhang et al. (45) developed a device for the 3D cultivation of multiple cell types in compartmentalized microenvironments. Different cell types were cultivated, including liver, lung, kidney, and adipose tissue, and it was shown that controlled release of substitute growth factors inside such a compartment was possible by means of gelatin microspheres mixed with cells. Kimura et al. presented a device for the long-term perfusion cultivation and online monitoring of intestinal tissue (46) models. The perfusion and fluorescence measurements of the cultivation media for each channel can be conducted by the on-chip pumping system and optical fiber detection system. The utility of the device was demonstrated by the cultivation of Caco-2 cells for more than 2 weeks and the online fluorescent monitoring of Rhodamine 123. Various Cell Types. Wong et al. (47) presented a method for controlling the distribution of multiple cell types within a 3D hydrogel including the application of soluble factors such as cytokines. Vickerman and co-workers (48) designed a 3D cell cultivation device with the ability to generate gradients (nonreactive solute), surface shear, interstitial flow, and real time image cells in situ. Human adult dermal microvascular endothelial cells (HMVEC-ad) were cultivated for up to 7 days. Peng et al. (49) designed a device for the cultivation of many types of cells, including high motility and swimming cells. Yeast cells, red blood cells, rabbit bone marrow aspirate, and dinoflagellate swimming cells were successfully introduced into the device for multicell retention, multicell cultivation, and observation. Many cell-cell interactions were observed. For the cultivation of aerobic and anaerobic bacteria and mammalian cells, Lam et al. (50) developed a platform integrated with a differential oxygenator. Sensors consisting of an oxygen sensitive dye embedded in the fluidic channels permit the dynamic fluorescence-based monitoring of the dissolved oxygen concentration using light emitting diodes. The bacteria E. coli, A. viscosus, and F. nucleatum as well as the mammalian murine embryonic fibroblast cells 3T3 were cultivated. Kim et al. (51) presented a device for the cocultivation of epithelial cells and bacteria enabling the investigation for host-pathogen interactions. Commensal E. coli was cultivated next to HeLa epithelial cells. Reactors and Mixers. Titmarsh and Cooper-White (52) reported a scalable microbioreactor architecture that uses nested dilution structures to generate a full-factorial array of cell cultivation conditions. The device can be used for the analysis of provision of multiple soluble factors in cellular microenvironments. The validation of the device was demonstrated by the cultivation of the EGFP expressing murine microphage cell-line RAW264.7/ ELAM-eGFP. Protein expression levels correlated with the magnitude of the stimulus and, therefore, was used to demonstrate the utility of the device. Kortmann et al. (53) presented a bioreactor in which cells are trapped by contactless negative dielectrophoresis (nDEP) and cultivated in a constant medium flow. Joule heating by nDEP was used to control and maintain the cultivation temperature by a Peltier device. S. cerevisiae were successfully cultivated for up to four generations in the reactor. Manipulation of Cells. Cell Encapsulation. The rapidly developing field of droplet fluidics resulted in a variety of standard operations, for example, the encapsulation of cells into droplets, gels, and polymers. Having a single cell in a single droplet opens a world of possibilities for quantitative biomolecular studies. The

encapsulation, incubation, and manipulation of individual cells in picoliter aqueous drops in a carrier fluid was described by Koster et al. (54). Oil drops can also be used for single-cell encapsulation. Edd et al. (55) described a method for injecting cells into monodisperse picoliter volume drops with the frequency of drop formation. The biopolymer alginate is also used to generate single cell containing droplets. This biopolymer is produced from a brown algae and in a liquid state called Na-alginate. The transition to a gel state of this biopolymer occurs when a solution with Ca2+ ions is introduced, which affects the viability of the cells. To enhance the viability, Kim et al. (56) proposed a rapid oil-exchange chip where the toxic calcified oleic acid, used to polymerize the droplets, is transformed to mineral oil. Morimoto et al. (57) developed a manufacturing method of semipermeable microcapsules composed of a alginate-poly-L-lysine membrane. The advantages of these microcages are that the cells are protected from mechanical stress and they have the possibility to move freely within their capsule. The captured cells can be observed continuously. Another extensively used inert biomaterial is polyethylene glycol (PEG). The fabrication of large numbers of cell-laden PEG microparticles using a continuous microfluidic process called stop-flow lithography was developed by Panda et al. (58). Here, photo-cross-linkable prepolymer containing cells flow through the microfluidic channels. After UV light exposure, the cells are permanently encapsulated. Perroud and co-workers (59) described isotropically etched radial micropores embedded in microfluidic channels. According to the dimensions of the pores manufactured, they were used to encapsulate individual E. coli cells. The trapping of macrophages in micropores was also described using other pore dimensions. Chabert and Viovy (60) presented a purely hydrodynamic method for on-chip cell encapsulation into picoliter drops followed by spontaneous self-sorting of these droplets. Encapsulation uses a cell-triggered RayleighPlateau instability in a flow-focusing geometry whereas the selfsorting is resulting from the lateral drift of deformable objects in a shear flow and sterically driven dispersion in a compressional flow. Electroporation of Cells. Zhan et al. (61) described the encapsulation of cells into aqueous droplets whereafter the cells are electroporated. Valley et al. (62) presented a microfluidic device for single cell light induced electroporation for the introduction of exogenous molecules across the impermeable cell membrane and dielectric manipulation of cells. Ionescu-Zanetti et al. (63) showed that electrophoresis can be used to assist loading by first preconcentrating molecules of interest at the cell-channel interface. Then, the electric field is used to drive these molecules into the cell postelectroporation. The anionic fluorescent molecules calcein and Oregon green dextran were successfully delivered in Hela cells with an enhanced delivery rate of more than an order of magnitude when compared to diffusion alone, subsequent to electroporesis. Valero et al. (64) presented a device capable of the electroporation of single cells for gene transfection and protein dynamic studies. They show the transport of propidium iodide into single mouse C2C12 cells and the nuclear translocation of green fluorescent erk1 producing C2C12 and human mesenchymal stem cells within 15 min after fibroblast growth factor stimulation. Olbrich et al. (65) presented an electroporation chip Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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for adherent cells on photochemically modified polymer surfaces. Electroporation was demonstrated by the transfection of human embryonic kidney cells with the enhanced green fluorescent protein. Wang and co-workers (66) developed a semicontinuous flow electroporation chip for the in vitro delivery of DNA. In this device, cells and plasmid DNA flowed continuously through a serpentine channel of which the walls were also serving as electrodes. K562 leukemia cells and mouse embryonic cells were successfully transfected. Focusing, Guiding, and Patterning of Cells. The use of a microvortex to focus, guide, and sort particles was reported by Hsu et al. (67). While investigating the possibilities for cells, they found that the microvortex can be used to focus cells into single or multiple streamlines following the surface patterns. Karnik and co-workers (68) reported the nanomechanical control of a HL-60 cell rolling in 2D in the direction of the flow by patterning surfaces with P-selectin receptors. Kim et al. (69) reported the investigation of T. pyriformis in a low Reynolds number fluidic environment. Electrical stimulation in the form of a direct current through the containing fluid causes a change in swimming direction toward the electrode. Photosimulation, by high broadband light, results in a rotational motion of the cells. Shi and co-workers (70) used standing surface acoustic waves to actively pattern cells. The use of the acoustic tweezers was verified with red blood cells and E. coli cells. Choi et al. (71) presented a device for the hydrophoretic focusing which utilizes transverse pressure gradients generated by a V-shaped obstacle array (VOA). The focusing pattern can be modulated by varying the gap height of the VOA. Red blood cells were used to study the hydrophoretic focusing pattern of biconcave disk shaped particles. Transport of Cells. Son and Garell (72) developed a digital microfluidic platform for the transport of live cells using droplets. This was demonstrated with the programmed transport of live yeast cells and a zebrafish embryo within the droplets. Manneberg and co-workers (73) reported a device for the flow-free transport and positioning of cells in microchannels by frequency-modulated ultrasound. Cell Lysis and Laser Surgery. Chung and Lu (74) developed a high-throughput cell microsurgery device that kills cells with a laser. This tool, for the study of cells, was demonstrated with the multiplex microfluidic manipulation of C. elegans, imaging processing, and automation, allowing for high-throughput cell ablations. Guo and co-workers (75) presented a femtosecond laser nanoaxotomy lab-on-a-chip for the in vivo study of the regeneration of nerve cells. A femtosecond laser was used on C. elegans while minimally affecting the worm. Quinto-Su et al. (76) developed a device for laser beam induced cell lysis. Non-adherent BAF-3 cells were studied using time-resolved fluorescent imaging and revealed cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics resulting from the laser pulse. Santillo and co-workers (77) described a device that was characterized for studying cell lysis of A. vulgaris over time. Cells were captured in the chambers and subsequently exposed to a constant flow of biocidal agents which causes the lysis. This device offers the possibility to study individual cell lysis. Bao et al. (78) examined the selective release of specific intracellular molecules calcein and a protein kinase (Syk), both EGFP tagged, from chicken B cells during electroporation at single cell 4852


level. In another investigation, Bao and Lu (79) reported the trapping and electrical lysis of bacteria cells. Here, a microscale bead array is used, and the optimal parameters for rapid electrical lysis for the release of intracellular materials of E. coli cells is presented. This has been done with a different device. Murakami et al. (80) developed a device for the sequential trapping, labeling, and content extraction of single cells. Non-fluorescent COS-1 cells were captured in single cell pockets; the nucleus was stained with a Hoechst dye, and finally, cell lysis was induced after exposure to a 10% sodium dodecyl sulfate (SDS) solution. They were able to collect the lysate of individual cells. Fusion. Skelley et al. (81) developed a device to trap and properly pair thousands of cells. Different cells types were paired including fibroblasts, mouse embryonic cells, and myeloma cells. With the device, both chemical and electrical fusion was demonstrated. Wu et al. (82) designed a device for the hydrodynamic trapping of cancer cells in controlled geometries followed by the formation of spheroids which is enhanced by the maintenance of compact groups of the trapped cells due to continuous perfusion. The size of the spheroids is uniformly increasing with increasing flow rate. Other. Adamo and Jensen (83) designed a device for the microinjection into single cells. The cells are moved onto a fixed needle by fluid streams. The device can handle only a few cells per single experimental session. Hela cells were used to investigate the capability of the device. Chokshi et al. (84) presented two microfluidic approaches for immobilizing C. elegans on-chip. The first method is the creation of a CO2 microenvironment, which can be used for long-term immobilization (1 to 2 h), and the second method is the restriction of the worm’s movement by a deformable PDMS membrane. Both methods can be used in the device. Klauke et al. (85) described the regional stimulation of single adult cardiac myocytes. In the device, flow rates of the laminar streams were adjusted such that the fluid interface between an injection flow and a perfusion flow was manipulated laterally to stimulate regions of the cell surface. Cell Counting and Flow Cytometry. A high-throughput microfluidic-based biophysical flow cytometer technique was presented by Rosenbluth and co-workers (86). They show that it is possible to measure single cell transit times of blood cell populations passing through in vitro capillary networks. The clinical relevance of the device was demonstrated with the characterization of mechanical properties of cells involved in two model diseases, sepsis, and leukostasis, respectively. Golden et al. (87) developed a multiwavelength microflow cytometer using grooved microfluidic channel generated sheath flow. E. coli assays were performed to demonstrate the capability of the device. Hur and co-workers (88) designed a high-throughput sheath-free positioning system that was tested with blood. Both red and white blood cells where automatically detected with high sensitivity and specificity for each cell type. Holmes et al. (89) developed a cytometer device based on impedance measurements of each single cell. Verification of cell dielectric parameters was performed by simultaneously measuring fluorescence from cluster of differentiation (CD) antibody-conjugated cells. This enabled direct correlation of impedance signals from individual cells with phenotype. Fu et al. (90) developed a cytometer device in which the particles are concentrated in the center of the sample stream

by the use of a 2D hydrodynamic focusing technique and a microweir structure. Cells or particles are detected and counted by laser induced fluorescence. Bao and co-workers (91) developed an electroporative flow cytometer for the study of single cell mechanics. The change in cytoskeleton dynamics associated with malignancy and metastasis was observed during the flow-through electroporation due to the altered deformability. Lee et al. (92) reported a micro flow cytometry device integrated with a tunable liquid filled microlens structure for optical detection. The functionality of the device using fluorescence detection and counting of circulating tumor cells (CTC) was reported. Tan and co-workers (93) designed a device which is capable of the enumeration and isolation of cancer cells of breast and colon origin in blood samples by utilizing the stiffer and larger size characteristics of cancer cells as compared to blood constituents. Thorslund and co-workers (94) designed a PDMS-based disposable microfluidic system for the on-chip counting of CD4+ lymphocytes. The micropillared sensor surface works by pure capillary action, and sample filling and rinsing are performed without external equipment. Wang et al. (95) designed a CD4+ counting device based on optical fluorescence detection with resistive pulse sensing enhanced by metal oxide semiconductor field effect transistor (MOSFET). The MOSFET signal indicates the total number of the cells passing through the detection channel, while the concurrent fluorescent signal records only the number of cells tagged with a specific fluorescent dye. A CD4+ counting platform, presented by Wang et al. (96) , enumerates the CD4+ lymphocytes from whole blood using chemiluminescence detection. Microfabricated traps in the device are coated with anti-CD4+ antibody to isolate the cells. On the basis of cell surface-bound CD3 antibodies conjugated with horseradish peroxidase, incubation with chemiluminescent substrate produced a current in the photodetector proportional to the number of captured cells. Cheng et al. (97) reported an improved design of a two stage microfluidic device to deplete monocytes from whole blood followed by immunoaffinity-based CD4+ capture. Kim and co-workers (98) designed a chip with polyelectrolytic gel electrodes (PGE) used to rapidly count the number of red blood cells in diluted whole blood based on the variance in impedance between two PGEs when a cell passes through it. Lee et al. (99) presented a method of measuring the cell concentration using two electrical cell counters across a fixed control volume. With this device, cells are counted at the inlet and outlet of a fixed control volume and the cell concentration is measured by calculating the number of cells in the fixed control volume. The device was demonstrated by counting of red blood cells. Separation, Sorting, and Trapping of Cells. Separation. A centrifugal microfluidic device for high throughput and continuous cell/plasma separation by the use of two-layer laminar flow was developed by Funamoto et al. (100). Human mesenchymal stem cells are very attractive for multiple research interests since they can differentiate in multiple lineages for cell therapy. Wu et al. (101) developed a device utilizing louver-array structures for the separation of amniotic fluid mesenchymal stem cells. The soft inertial force separation of bacteria from human blood cells based on size differences was presented by Wu and co-workers (102). Here, the use of a specific channel geometry together with acting and protecting sheath flow results in a soft inertial force effect on the cells. The inertial interactions inside the curved and focused

sample flow result in deflection of larger cells. Di Carlo and coworkers (103) used asymmetric channel geometry for the use of differential inertial focusing. The device was tested with diluted whole blood and platelets, and the sample could be successfully enriched or depleted. Green et al. (104) separated large epithelial cells from the smaller fibroblast cells using deterministic lateral displacement. SooHoo and Walker (105) presented a two phase microfluidic system capable of controlling the location of cells and the position of the interface. Additionally, they showed that cells trapped at the interface could be collected. The functionality of the device was tested by separating leukocytes from whole blood. Dielectrophoresis (DEP) is a powerful tool for the separation and sorting of different types of cells based on their difference in the dielectric properties. Pommer et al. (106) presented the DEP separation of platelets from diluted whole blood in microfluidic channels, whereas Srivastava et al. (107) , presented the DEP characterization of erythrocytes. In the latter, positive blood types of the ABO typing system were investigated. A novel method for on-chip continuous separation of dividing and non-dividing cells based on their dielectric properties was presented by Demierre et al. (108). The principle of this separation is the use of two opposite DEP forces at multiple frequencies (Braschler et al. (109) ). The cells flow through the microfluidic device and are focused to different streamlines in the flow channel. Another related strategy is the lateral flow-through separation of cells, presented by Wang et al. (110). The lateral separation is enabled by the interdigitated electrodes which are used to generate the non-uniform electric fields and balanced DEP forces along the width of the microchannel. Choi et al. (111) presented the selection of porcine oocytes for in vitro fertilization based on differences in DEP responses of the cells. The selected group of cells that moved showed a better developmental potential with respect to the cells that stayed. Vahey and Voldman (112) introduced a new technique called isodielectric separation (IDS) for sorting cells based on their difference in electrical phenotypes. This technique is analogue to isoelectric focusing; however, here an electrical conductivity gradient is used instead of a pH gradient. Lu et al. (113) used DEP to pattern neurons and astrocytes separately in a microfluidic device where after a 3D matrix is introduced into the microchannel to polymerize around the cells. Vykoukal and co-workers (114) used dielectrophoretic field flow fractionation (DEP-FFF) for the enrichment of a putative stem cell population from an enzyme digested adipose tissue derived cell suspension. The capability of the device was demonstrated with independent runs for cells labeled with FITC-conjugated antibodies against two putative cell markers, NG2 and nestin, and yielded nearly identical elution profiles. Sankaran et al. (115) developed a method to capture mammalian cells by DEP using transparent indium tin oxide electrodes. The advantage of these transparent electrodes is that the cells can be monitored with conventional optical microscopy. Koo et al. (116) described the efficient capture of biotinylated heat shock protein 60 (Hsp60), a eukaryotic mitochondrial chaperon protein, as a capture molecule for living L. monocystogenes in a microfluidic environment. The addition of a DEP force increased the capture rate by 60%. Urdaneta and Smela (117) designed a method using multiple frequencies to counteract electric field distortions that interfere with the DEP manipulation of cells. This was shown using multiple frequency DEP to load Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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many cells into a cage in a matter of seconds in fluid flows of up to 300 µm s-1, which could not be done with single frequency DEP. A method for single cell immobilization using negative DEP was presented by Thomas and co-workers (118). This method enables the trapping of cells in high conductivity physiological media. The operation of the device was shown with HeLa cells against a moving fluid. Fan and co-workers (119) used dielectrophoresis (DEP) and electrowetting-ondielectric (EWOD) forces to demonstrate the cross-scale electric manipulations of cells and droplets. Cell Sorting. Johansson and co-workers (120) developed an acoustic microfluidic system for miniaturized fluorescence activated cell sorting. An acoustic wave is formed after the excitation of the miniaturized piezoelectric transducer at the bottom of the microchannel resulting in fluidic movement. They were capable of separating single cells from EGFP expressing β-cells. An optofluidic platform for cell sorting was presented by Lau et al. (121). Label-free cell identification and continuous cell sorting based on Raman spectroscopy was shown for two different leukemia cell lines. Another optical sorting device was presented by Perroud et al. (122) , who extended the principle of optical tweezers to actively sort cells. Hydrodynamically focused cells were sorted based on their fluorescence signals. The selectivity of the device was shown by the sorting of F. tularensis. Hulme and co-workers (123) used ratchets inside a microfluidic device to direct the motion of the swimming E. coli bacteria and three sorting junctions to isolate successively shorter populations of bacteria. A multimagnet activated cell sorter was developed by Adams et al. (124). Cells were labeled via target specific affinity reagent and with two different magnetic tags with distinct saturation magnetization and size. Simultaneous spatially addressable sorting of multiple target cell types in a continuous flow manner was shown. Kose et al. (125) presented the use of biocompatible ferrofluids for the label-free cellular manipulation and sorting via biocompatible ferrofluids. This low-cost platform exploits differences in particle size, shape, and elasticity to achieve rapid and efficient separation. The shape-based separation of live blood cells from sickle cells and bacteria demonstrated the proof of principle. The on-chip sorting of C. elegans based on morphological and intensity features was developed by Chung et al. (126). This device is capable of self-regulated sample loading and automatic sample positioning and can be combined with any microscope setup. Cell Trapping. Kovarik and Jacobson (127) presented an integrated nanopore/microchannel device for AC electrokinetic trapping. High field strengths and gradients, generated in the vicinity of the nanopores, resulted in a combination of both electrophoretic and dielectrophoretic trapping. C. crescentus bacteria were successfully trapped. Hunt et al. (128) reported an integrated circuit/microfluidic chip to use a program to trap and move cells and droplets with dielectrophoresis. The device is capable of simultaneously and independently controlling the location of thousands of dielectric objects. Additionally, it can trap and move a picoliter of water in oil droplets, split them into two, and mix two droplets into one. Ku¨hn et al. (129) presented a trap that relies on propagation loss in confined modes in liquid-core optical waveguides to trap cells. E. coli was trapped to demonstrate the capability of the device. In another investigation (130), they 4854


presented the electro-optical trapping with optical excitation power levels that are 5 orders of magnitude lower than in conventional force traps. Here, the device was demonstrated by the photobleaching dynamics of stained DNA in E. coli cells. Lin and coworkers (131) presented trapping using two counter-rotating microvortices which trap the cells within the confinement. Trapping and releasing of human embryonic kidney (HEK) cells, red blood cells, and IgG antibodies were demonstrated. Manneberg et al. (132) presented a 3D ultrasonic cage for both trapping and characterization of individual cells. In this device, the cage was simultaneously actuated at two frequencies, and therefore, it was demonstrated that HEK cells could be trapped as a 2D monolayer or as a 3D aggregate. Lay and co-workers (133) designed microfilters featuring a raindrop bypass filtering architecture, which significantly reduces the likelihood of clogging at the cost of limited cell lost. This architecture has a substantially low pressure drop. The protozoan cells C. parvum and G. lamblia were successfully trapped. E.coli bacteria could be trapped after modifying dimensions of the filters. Selection by Specific Adhesion. Plouffe et al. (134) produced coated microfluidic channels with tetrapeptides and captured endothelial cells, smooth muscle cells, and fibroblasts from heterogeneous suspensions. In another investigation (135), they captured cardiac fibroblast by the use of a thin hydrogel layer in a microfluidic channel which was enriched with specific peptides. After capturing of the cardiac cells to the specific peptide, the gel was dissolved by ethylene diamine tetraacetic acid (EDTA), resulting in a release of the captured cells. Phillips and co-workers (136) developed a device modified with aptamers that captures rare (cancer) cells to achieve a rapid assay without pretreatment of cells. In this way, rare cells can be enriched and captured prior to detection. Xu and co-workers (137) also designed a device for the enrichment, sorting, and detection of multiple cancer cells by the use of aptamers. The device employs cell-affinity chromatography based on the selective cell-capture of immobilized DNA aptamers and yields a 135-fold enrichment of rare cells in a single run. The authors also introduced an enrichment factor that enables an independent measure of the enrichment capability of a given device. Dharmasiri et al. (138) developed a device for the specific adhesion of rare circulating prostate tumor cells resident in a peripheral blood matrix using anti-PSMA aptamers which were immobilized on the surface. Cells were intact released by a trypsin containing fluid and counted using a contact conductivity sensor. Detection. Segerink and co-workers (139) developed a device for the on-chip concentration and detection of spermatozoa in semen using electrical impedance measurements. They demonstrated that a change in the electrical impedance signal is related to the size of a passing cell and demonstrated the capability to distinguish between spermatozoa and HL-60 cells suspended in washing medium. Shabani et al. (140) presented a method for the specific and direct detection of bacteria, using T4 bacteriophages as recognition receptors which are covalently immobilized onto functionalized screen-printed carbon electrode microarrays. The functionality was tested by impedance measurements of bound E.coli. Cho and co-workers (141) proposed a platform which utilized aptamers and surface plasmonics for a long-term and real-time assessment and label-free detection of secreted growth factors under the spatial and temporal control of a

chemically simulated tumor environment. The device was tested with the detection of vascular endothelial growth factor of human breast cancer cells upon a continuous stimulation with estradiol for 37 h. Lin et al. (142) integrated optical microsensors into a cell cultivation microchannel device. The sensors are integrated along the microchannel and enable the study of the concentration gradients of various metabolites as well as the cell uptake and perfusion rate of growth medium. The capability of the device was shown by glucose and oxygen concentration in the microenvironment of the cultivated mammalian cells. The audio frequency electrical impedance method on-chip was used by Hua and Pennell (143) for the single-cell real-time measuring of volume changes independent of cell shape. The capability of the device was tested by Madin-Darby canine kidney (MDCK) epithelial cells. Neurotransmitters and hormones are secreted in discrete packets in a process called quantal exocytosis. Gao et al. (144) developed a device for the amperometric measurement of quantal exocytosis of catecholamines from on-chip trapped bovine chromaffin cells. Zhao et al. (145) described a device that enables the chemiluminescent detection for single cells after intracellular labeling. Human blood cells were assayed to determine intracellular content of glutathione (GSH). Individual cells were labeled, injected, inline lysed, and on-chip separated. Chemiluminescent detection was based on the on-chip oxidation reaction of luminol labeled GSH with NaBrO. Ferrier et al. (146) presented a microwave interferometric system which is capable of detecting capacitance changes and actuation of single cells. Zheng et al. (147) reported a new approach to overcome the double-layer capacitance in micro impedance sensing for particles in fluidic flow. They introduced a parallel inductor to induce system resonance. The system is capable of sensing the differences in between erythrocytes and leukocytes. Mellors and co-workers (148) developed a platform for the rapid lysis of free single cells in a free solution electrophoresis channel. The cellular constituents were separated. An integrated electrospray emitter was used for ionization of the separated components, which enables the direct coupling of a mass spectrometer without the use of external pressure sources. Other. Sechan and co-workers (149) developed a cell deformability monitoring chip to measure the cell lysis rate that is dependent on the areal strain of the cell membrane. Courtois and co-workers (150) presented a method to control the retention of small molecules in emulsion microdroplets for the use in cellbased assays. The leaking of content into the oil phase is strongly dependent on the surfactant concentration. The addition of bovine serum albumin (BSA) suppresses this most likely through its capability to bind and adsorb on oil-water interfaces. The utility of BSA was demonstrated by following the alkaline phosphatase activity expressed by E. coli cells. Clark et al. (151) developed a dual-chip microfluidic platform that coupled perfusion of cultivated adipocytes with an online continuous flow fluorescent enzyme assay for monitoring the glycerol secretion. Cell characterization by the use of a protein-functionalized pore was presented by Carbonaro and co-workers (152). Specific interactions between the functionalized proteins and a cell surface marker retard the cell while passing through the pore, thus leading to an increased pulse duration that indicates the presence of that specific biomarker. Mukundan and Pruitt (153) developed a device to measure the stiffness of cells cultivated on suspended structures. In this

device, an actuator is integrated into a planar force sensing system. The system was used to measure the mechanics of live MadinDarby canine kidney cells in media. APPLICATIONS Clinical Diagnostics. Cell Studies. Liu et al. (154) designed a device for the rapid determination of superoxide-free radical in hepatocellular carcinoma cells. The device uses on-chip capillary electrophoresis and laser interference fluorescence and was successfully tested with phorbol 12-myristate 13-acetate stimulated RAW264.7 macrophages. Mycoplasma pneumonia can cause severe infections in patients who received transplants. Kim and co-workers (155) designed a device for the rapid detection of M. pneumonia using latex immunoagglutination and static light scattering. The latex immunoagglutination was performed with serially diluted M. pneumonia solutions using highly carboxylated polystyrene particles conjugated with monoclonal anti-M. pneumonia. Optical fibers located around the viewing cell of the device were used to measure the increase in light scattering of the aggregated particles. Mohammed and co-workers (156) developed and characterized a device to perfuse pancreatic islets while simultaneously characterizing the functionality of the islets. This was done using fluorescence imaging of the mitochondrial membrane potential and intercellular calcium and in addition to the enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin. This was demonstrated on both mouse and human islets. The device could be useful for testing islet functionality prior to transplantation into recipients. Inglis and coworkers (157) developed a method to measure the hydrodynamic size of platelets, a parameter associated with morphological change and activation of platelets. The device may be useful to provide rapid diagnostic information about the hemostatic condition of a blood sample without the use of an activation specific label or marker. Gutierrez et al. (158) developed two devices for the studies of shear-dependent adhesion. One device has an array of eight flow chambers with shear stress varying by a factor of 1.93 between adjacent chambers. The other device allows simultaneous high resolution fluorescence imaging of platelets from two different blood samples. The devices were subsequently used to study the roles of extra- and intracellular domains of RIIbβ3, a platelet receptor which is a central mediator of platelet aggregation and thrombus formation. Tovar-Lopez and co-workers (159) developed a platform for measuring platelet function and aggregation based on localized strain rate microgradients. The incorporated contraction-expansion geometries inside the device generate strain rate conditions mimicking the effects of pathological changes in blood vessel geometry. Abbyad et al. (160) developed a droplet-based microfluidic device in which the red blood cells sickling process can be studied. Low oxygen partial pressure is one of the triggers of the sickling of red blood cells and can be controlled by the oxygen partial pressure of the incoming oil and aqueous phase. In this way, a microdroplet is subjected to a rapid change in oxygen concentration. Lee et al. (161) developed a diagnostic magnetic resonance sensor that combines a miniaturized NMR probe with targeted magnetic nanoparticles for detection and molecular profiling of cancer cells. The clinical utility of the device was evaluated using fine needle aspirates from a panel of xenograft tumor models and showed that the detection Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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sensitivities were as good as those achieved with clinical methods (e.g., flow cytometry and Western blot analysis) in less than 15 min. Bacteria. Boedicker et al. (162) developed a plug-based device which enables rapid detection and antibiotic susceptibility screening of bacteria in samples, including complex biological matrixes without preincubation. With this device, the authors were able to make a chart of antibiotic sensitivity of methilicillin resistant S. aureus (MRSA) and the minimal inhibitory concentration of the drug cefoxitin. Kastrup and co-workers (163) demonstrated, using a microfluidic device and patterning, that the spatial localization of bacteria substantially affects coagulation of human blood and plasma. B. cereus and B. anthracis, the anthrax causing pathogen, directly initiated coagulation of blood in minutes when bacteria cells were clustered. B. anthracis, required secreted zinc metalloprotease InhA1, which activated prothrombin and factor x directly, and this mechanism was referred to as “quorum acting” which does not require a change in gene expression. This mechanism can be rapid and independent of bacterium to bacterium communication. Sepsis caused by gram positive and gram negative bacteria is the leading cause of death in intensive care units. Mahalanabis et al. (164) have developed a detection system for gram positive and gram negative bacteria from whole blood on a disposable microfluidic chip. Viruses. Even though viruses are not cells, we decided to include them here since they infect cells and do play an important role in various fields related to human health, such as clinical diagnostics. Reichmuth et al. developed an electrophoretic immunoassay for viral particles using open channel for the detection of swine influenza virus (165). Huh and co-workers have developed a fully integrated microfluidic system for sensing infectious viral disease (166). Birnbaumer et al. have reported molecular imprinted polymers integrated on a microfluidic biochip for detection of viruses using contactless dielectric microsensors (167). Cancer Research. Cheung and co-workers (168) used a microchannel device functionalized by N-cadherin antibodies to study the capturing and detachment behavior of a homogeneous suspension of N-cadherin expressing PC3N prostate and MDAMB-231-N breast cancer cells subjected to hydrodynamic flow. For both cell types, capturing was most successful when no flow was present while the required flow rate for cell detachment is random in nature. Song et al. (169) presented a microfluidic vasculature system to model interaction between circulating cancer cells with microvascular endothelium at potential sites of metastasis. CXCL12, a chemokine strongly implicated in metastasis, acts through the receptor CXCR4 on endothelium to promote the adhesion of circulating breast cancer cells which suggests that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers. The effects of electrochemotherapy on human breast cancer cells was studied by Choi et al. (170). The device mimics a clinical electroporator with a circular needle array and maintains a similar electric field strength distribution. It was tested in two- and six-electrode modes using propidium iodide and bleomycin followed by the characterization of the electroporation. The microfluidic electroporation of circulating tumor cells and blood cells was investigated by Bao and coworkers (171). They demonstrated the selective electroporation 4856


of tumor cells among blood cells. Using coherent anti-Stokes Raman scattering (CARS) and fluorescence microscopy tools, a dramatic increase in the size of the nucleus of a tumor cell in response to the applied field was shown. Drug Discovery and Screening. Brouzes et al. (172) developed a droplet-based microfluidic technology that enables high throughput screening of single mammalian cells. The validation of the full droplet screening workflow was done by screening of an optically coded droplet-based drug library for its cytotoxic effects against human monocytic U937 cells. An osteoblast-based 3D continuous perfusion microfluidic system for drug screening was developed by Jang and co-workers (25). With this system, they were able to monitor cells for 10 days and take samples of the supernatant system for the osteoblast differentiation assay. Wlodkowic et al. (173) developed a microfluidic array for the realtime screening of anticancer drugs against arrays of single cells. Human HL60 cells were used to quantify on-chip anticancer drug induced apoptosis. Ainslie and co-workers (174) developed a device for enhanced bioadhesive drug delivery. The system is capable of both targeting and asymmetric release of small molecules in flow through a cell monolayer. Shi et al. (175) designed an array device capable of the encapsulation of C. elegans into droplets and enabling the characterization of the worm behavior in response to neurotoxin at single-animal resolution. Li et al. (255) developed a microfluidic system for the real-time detection of drug effects, based on the quantitative measurement of calibrated cytosolic calcium on single cancer cells. This method is rapid in detecting early event cytotoxicity of drug candidates on cancer cells; results can be obtained in hours instead of waiting for a couple of days. The effect of isoliquiritigenin (IQ) was studied on leukemia cells, and it was found that a concentration of 50 µM causes a sustained elevation of the cytosolic calcium concentration and cytotoxic effects on the cells. The effect of cytostatic drugs on-chip was also studied by Komen et al. (177). They present the development of two devices to validate the cell cultivation properties and analysis of the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine. The viability of the cells was assessed using the life stain Calcein-AM and the death dye propidium iodide (PI). Chung et al. (178) presented a clonal cultivation and chemodrug assay of heterogeneous cells (PC3 prostate carcinoma cells) using microfluidic single cell array chips. Here, single cells can be automatically loaded into the microwell array and cultivated or assayed using the continuous fluid flow without cross migration of cells between the neighboring microwells. The analysis of nonadherent apoptotic cells by a quantum dots probe in a microfluidic device for drug screening was presented by Zhao and co-workers (179). In this investigation, leukemic HL-60 cells were immobilized and cultivated in the device. Annexin V conjugated quantum dots were used to distinguish apoptotic from unaffected cells with a single cell resolution. The diffusion time of the quantum dots was reduced to 5 min before imaging. Yeon and Park (180) presented a drug permeability assay to investigate the delivery path of drugs in humans. On this device, human colon adenocarcinoma endothelial cells (Caco-2) were trapped into microholes without damage and the permeability of 10 drugs was measured. Multidrug resistance (MDR) is a major cause of failure in cancer therapy. Li and co-workers (176) reported an approach combined with the

same single cell analysis to investigate the modulation of MDR. The device is capable of selecting and retaining single MDR cancer cells and was used to investigate drug efflux inhibition in leukemia cell lines. For the 3D hydrogel cell cultivation and testing the cytotoxicity of anticancer drugs while reproducing multiorgan interaction, Sung and Shuler (181) developed a microfluidic device. Separate cultivation chambers representing the liver, tumor, and marrow are connected by channels mimicking blood flow. Colon cancer cells (HCT-116) and hepatoma cells (HepG2/ C3a) were encapsulated in Matrigel and cultivated in the tumor and liver chamber, respectively. Myeloblasts (Kasumi-1) were encapsulated in alginate in the marrow chamber. The effect of the prodrug Tegafur was successfully investigated in this device showing the death of the liver cells after the metabolism of Tegafur into 5-fluorouracil in the liver cells. Ku et al. (182) developed a method to monitor platelet adhesion to immobilized endothelium in the presence of a known platelet activator and antiadhesion drug. Dworak and Wheeler (183) presented a multielectrode-array platform for the long-term and highly selective recordings of axonal signals in microtunnels. The influence of the drug medivavaine was successfully identified in the significant change in the mean spiking rate and conduction velocity. Fillafer et al. (184) developed an acoustically driven biochip to investigate the impact of flow on the cell association of targeted drug carriers. This device can accurately mimic a wide range of physiological flow conditions and was used to study the interaction characteristics of protein coated particles with cells. Stem Cell Research. For the investigation of the influence of spatial restriction and adhesive interactions on hematopoietic stem and progenitor cell (HSCs) fate decisions, Kurth et al. (185) developed a device containing a set of fibronectin-coated micrometer sized cavities. Human CD133+ HSCs were isolated after being cultivated on these surfaces and analyzed. Surprisingly, it was found that the proliferation and differentiation of the HSCs were decreased, suggesting that with these cavities the cells could be kept in a quiescent and immature state for a period. Fung and co-workers (186) developed a platform for controlling the differentiation of embryoid bodies (EB). Using the laminar characteristics at low Reynolds number and high Peclet numbers, they induced cell differentiation on half of the EB while maintaining the other half in uninduced stages and proved the potential of the use of microfluidic technology for manipulation of EBs and embryonic stem cells in tissue engineering. Wu et al. (101) developed a device for the cultivation and differentiation of human mesenchymal stem cells (MSC). Human amniotic fluid-derived MSCs were successfully cultivated and several methods, such as immunofluorescence staining, were used to asses the differentiation of the MSCs. An integrated microfluidic device for the reproducible and quantitative cultivation and analysis of human embryonic stem cells was presented by Kamei and co-workers (27). Knock-in cell lines with EGFP driven by the endogenous OCT4 promotor were constructed, and on-chip immunoassays of several pluripotency markers were carried out to confirm the maintenance of their pluripotency. Assays. Huebner and co-workers (187) reported a device in which microdroplets in an array format are entirely controlled by liquid flow. Such an array-based approach was used to characterize droplet shrinkage, aggregation of encapsulated E. coli cells, and

enzymatic reactions. Clausell-Tormos et al. (188) described a droplet-based platform in which cells are grown in aqueous microcompartiments separated by an inert perfluorocarbon carrier oil. Human cells and C. elegans survived and proliferated within the microcompartiments for several days. To prove the utility of this device, an automated fluorescence-based analysis of single cells in individual compartments after 16 h of incubation was demonstrated. Courtois et al. (150) described a device for droplet formation, incubation, and screening. Monitoring time-dependent in vitro expression from single genes of cells in picoliter droplets proved the functionality of the device. Barbulovic-Nad et al. (189) presented a digital microfluidic method for cell-based assays. The automated manipulation of multiple reagents in addition to reduced reagent use and analysis time is advantageous for cellbased assays. The applicability of the device was performed with a cytotoxicity assay using Jurkat cells. Lee and co-workers (190) developed a miniaturized diagnostic magnetic resonance (DMR) system. The device is capable of detecting bacteria with high sensitivity and identification of a small number of cells. In addition, real-time molecular parallel analysis of protein biomarkers was demonstrated. Single Cell Applications. Liu et al. (191) designed a permalloy-based magnetic single cell array. Immunomagnetic labeled cells will interact with the magnetic fields and can be captured at the magnetic trap sites. This device was tested using fixed and live Jurkat cells. Further development by Sakaki et al. (192) resulted in an automated single cell arraying and analysis instrument, which was tested with human T-lymphocytes and by analyzing their uptake dynamics of calcein acetoxymethylester. Yu and co-workers (193) developed a platform for the chemical analysis of single cell lysates. Single K562 cells were lysed, and the analysis of glutathione and Rhodamine 123 was performed by laser interference fluorescence. Schumann et al. (194) presented a device for the concomitant detection of the CYP1A1 enzymatic activity and CYP1A1 protein in individual human urothelial cells using a bilayer microfluidic device. Neuroscience. A compartmentalized cocultivation device which can be used for the research of central nervous axon myelination was presented by Park and co-workers (195). In this work, they showed isolated neuronal cell bodies and dendrites from axons growing through an array of axon guiding microchannels into a compartment in which myelin producing glia, oligodendrocytes (OLs), were placed. OL progenitors cocultivated with axons differentiated into mature OLs. The analysis of neuronal growth cone responses to simple or composite gradients of precisely generated and aligned surface bound and diffusible cues were possible with a platform fabricated by Wang et al. (196). They investigated on-chip how the polarity of growth of Xenopus embryonic spinal neurons is regulated by gradients of surface bound laminin and diffusible brain-derived neurotrophic factor gradients. A platform for the study of single mammalian axonal injury and subsequent regeneration has been developed by Kim et al. (197). Dorsal root ganglion and cortical neurons of embryonic rats were used. The device was also capable of the creation of in vitro neuronal circuit models and to study the disruption of these circuits after spatially selective induced axonal injuries. Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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Microbiology. Liu and co-workers (198) designed a dropletbased device for the isolation, incubation, and parallel functional testing and identification by fluorescence in situ hybridization (FISH) of rare microbial single-copy cells from multispecies mixtures using the combination of chemistrode and stochastic confinement. Volfson et al. (199) analyzed the spatial organization of E. coli cells growing in a microfluidic chemostat and showed that purely biomechanical short-range interactions can lead to highly ordered structures in a growing bacterial population. Min and co-workers (200) present a single-cell motility assay using optical tweezers that allows the quantification of bacterial swimming in a well controlled environment. The utility was shown by the quantification of higher order features of bacterial swimming. Bacteria were detected by light and fluorescence microscopy. Ma¨nnik et al. (201) studied peritrichously flagellated E. coli and B. subtilis in microchannels where the width of the channels exceeds their diameters and showed that these bacteria are still motile. For smaller widths, the motility vanishes but bacteria can still pass through these channels by growth and division. Kim et al. (202) presented the construction of a community of three different species of wild type soil bacteria using a microfluidic device to control spatial structure and chemical communication. Using this system, it was found that the defined microscale spatial structure is both necessary for the stable coexistence of interacting bacterial species in the synthetic community. Stocker et al. (203) used microfluidic devices to create nutrient patches with environmentally realistic dimensions and dynamics for the study of the chemotactic response of the marine bacteria P. haloplanktis and found that this response is more than ten times faster than the classic chemotaxic model of E.coli. Additionally they demonstrated that such a rapid response allows P. haloplanktis to colonize nutrient plumes for realistic particle sinking speeds, with up to 4-fold nutrient exposure compared with non-motile cells. Lee et al. (204) demonstrated a chemotaxic assay using capillary filling and direct fluidic contacting on the developed chip. Clear migration of E. coli cells toward the chemo effector was observed. Krommenhoek and co-workers (205) showed the development of a microchip with integrated electrochemical sensors for the measurements of pH, temperature, dissolved oxygen, and viable biomass concentration under yeast cultivation conditions. The biomass sensor was based on impedance spectroscopy. Bryan et al. (206) developed a device for the study of cell growth. A suspended microchannel resonator with a Coulter counter is used to measure the mass, volume, and density of budding yeast cells through the cell cycle. The device is also suitable for other nonadherent cells. Secretion/Release Studies. Wan and co-workers (207) studied the shear-induced ATP release from red blood cells. Measurements were performed in microfluidic channels that include a narrow constriction. The higher shear stress in the constrictions served as the mechanical stimulus on the cells. The magnitude and duration were varied using different designs by changing the width and the length of the constrictions. Dittami and Rabbit (208) developed a device that is capable of resolving single vesicle quantal release, in the zeptomolar range, as well as the kinetics associated with the vesicle fusion process. The capability was demonstrated by the electrically depolarization of rat pheochromocytoma (PC12) cells and the simultaneously 4858


amperometric detection of exocytotic catecholamine release. Chen et al. (209) developed a microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in cultivation. RNA of high quality can be extracted from these microvesicles in a single wash-through procedure providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer. Dishinger et al. (210) developed a device for high-throughput automated and online monitoring of insulin secretion from individual islets of Langerhans. Utilizing serial immunoassays in 15 channels simultaneously every 10 s, the device was capable of performing 5400 immunoassays per hour, which results in insulin release profiles that captured single islet secretion. Another device using droplet-based microfluidics was developed by Easley et al. (211). With this device, the zinc secretion from pancreatic islets can be quantitatively measured with a high temporal resolution using a fluorescent indicator. Real time storage of secretions into droplets effectively preserves the temporal chemical information and allowed reconstruction of the secretory time record. Ammonia metabolism is an important marker and a critical function in cell sources employed in cell therapy for liver failure. Satoh and coworkers (212) developed a device for the on-chip cultivation and monitoring of the ammonia metabolism of hepatocytes. To evaluate the metabolism, ammonium is first transformed into a gaseous ammonia by raising the pH of the cell cultivation medium, which then dissipates into an air gap in the microfluidic channel, which dissolves into the electrolyte solution. The concentration is finally determined by an integrated ammonia sensor. Kortmann et al. (213) used single cell analysis platform with a confocal microscope and demonstrated the real time monitoring of the scFv-EGFP fusion protein secretion rates by S. pombe directly downstream of a cell. The “chemistrode” was introduced by Chen and co-workers (214). This droplet-based device enables stimulation, recording, and analysis of molecular signals with high spatial and temporal resolution. They successfully showed the measurements of insulin secretion of a single islet of Langerhans at a frequency of 0.67 Hz. Migration and Chemotaxis. Receptor desensitization plays an important role in migration efficiency and is most commonly studied using bath application of chemotactic factor solutions instead of presenting cells with gradients analogous to those they would experience in vivo. Therefore, Keenan and coworkers (215) presented a method for the gradient induced neutrophil desensitization based on an open microfluidic chamber. Chung et al. (216) developed a platform which has the capability to control the biochemical and biomechanical forces within a 3D scaffold coupled with accessible image acquisition. The platform was used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. The device has also been evaluated on endothelial cell response when cocultivated with physiologically relevant cell types. Irimia and Toner (217) designed a series of microfluidic devices that mechanically constrain migrating cancer cells inside microchannels with cross-section comparable to cell size. They observed unexpectedly fast and persistent movement of cancer cells of different types in one direction for several hours. This motility occurs spontaneously

in the absence of external gradients and suggests the presence of intrinsic mechanisms driving cancer cell motility that are induced in conditions of mechanical confinement. The exposure to drugs targeting the microtubules lead to a small number of cancer cells whose motility remained comparable to the untreated cells. Doran and co-workers (218) presented a device, which was used to assess rates of mouse fibroblasts (NIH 3T3) and human oseosarcoma (SaOS2) cell migration on surfaces functionalized with various extracellular matrix proteins as a demonstration that confining cell migration within a microchannel produces consistent and robust data. Lautenschla¨ger and co-workers (219) developed a device for the investigation of the regulatory role of cell mechanics for the migration of differentiating myeloid cells. They used a non-contact microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia cells, which occurs during differentiation therapy with retinoic acid. Agrawal et al. (220) designed a device for the on-chip neutrophil isolation of whole blood flowed by chemotaxis studies. The functionality of the device was tested with three different cell adhesion molecules for isolation (P-selectin, E-selectin, and fibronectin). Subsequent analysis of neutrophil migration in chemoattractant gradients of formyl-methyl-leucyl-phenylalanine (fMLP) and interleukin 8 (IL-8) shows higher average velocities over E-selectin compared to P-selectin. Shamloo and co-workers (221) describe a device capable of generating stable concentration gradients of biomolecules in a cell cultivation chamber while minimizing the fluid shear stress experienced by the cells. The polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. Results suggest that the absolute concentration of VEGF enhances the total filopodia extended while the gradient steepness induces the total number of filopodia, cell polarization, and subsequent directed migration. Abhyankar et al. (222) developed a platform which provides a robust soluble factor control within a 3D biological matrix. With this device, they studied the migration behavior of human neutrophils within a 3D collagen matrix in response to a temporally evolving fMLP gradient and the invasion and migration of metastatic rat mammary adenocarcinoma cells in response to a stable gradient of epidermal growth factor (EGF). An agarose-based 3D microfluidic chemotaxis device was produced by Haessler et al. (223). Here, fluid flow and chemical concentration gradients are decoupled from each other by the use of an agarose gel, which is a physical barrier for convective fluid flow, however, allowing protein diffusion. With this device, they successfully quantified the chemotactic response of murine dendritic cells to a gradient of the lymphoid chemokine CCL19. A static microfluidic gradient generator for chemotaxis studies was described by Kim and co-workers (224). In this device, the media or the chemoattractant are supplied by diffusion. The capability of the device to generate concentration gradients was confirmed by the chemotaxis of neutrophils to IL-8. Intra- and Intercellular Signaling. Hirsch and co-workers (225) presented a two-module microfluidic platform for simultaneous multi-time point stimulation and lysis of T-cells. This

platform enables the study of early time point signaling activation with a resolution down to 20 s, while using only a small amount of cells and reagents. The capability was presented by the activation of six important proteins in the signaling cascade and was quantified upon stimulation with a soluble form of R-CD3. Domenech et al. (226) presented the use of microchannels to study soluble factor signaling, providing an improved sensitivity as well as the ability to move beyond existing cocultivation and conditioned medium paradigms. Even so, the presented data suggest that microcultivation can be used to unmask effects of population demographics. Taylor and coworkers (227) developed a high-throughput microfluidic imaging platform for single cell studies of a network under hundreds of combined genetic perturbations and time-varying stimulant sequences. In the presented work, they successfully investigated the MAPK signaling of S. cerevisiae. Measurements of the pathway that responds to high osmolarity of S. cerevisiae were reported by Hersen et al. (228). This HOG-MAP kinase pathway is shown to act as a low-pass filter, integrating the signal when it changes rapidly and following it faithfully when it changes more slowly. Previously unknown bounds on all of the in vivo reaction rates acting in this pathway were measured, and it was found that the two component Ssk1 branch of this pathway was capable of fast signal integration, whereas the kinase Ste11 branch was not. Faley et al. (229) investigated both normal and chronic myeloid leukemia (CML) stem cell responses to the tyrosine kinase inhibitor dasatinib, a drug approved for the treatment of CML. Dynamic, on-chip three color viability assays revealed that differences in responses of normal and CML stem/progenitor cells to dasatinib were observed. In another investigation, they designed a platform for the real-time analysis of multiple T-cells in parallel. With this platform, it is possible to study both cell-cell interactions and intercellular signaling events of nonadherent cells. This was demonstrated by probing the intercellular communication between trapped dendritic and T-cells. Andrew and co-workers (230) developed a platform for probing singular networks by combining a programmable system for generating arbitrarily complex temporal input stimulant concentration profiles with real time monitoring of cell physiological response. This was presented by monitoring the response of individual cells on histamine concentration changes. Moraes et al. (231) presented an array platform for high-throughput screening of cellular response to cyclic substrate deformation. They demonstrated the effects of mechanical stimulation on the differential accumulation of β-catenin in the nuclei of mesenchymal progenitor cells. Srivastava and co-workers (232) developed a chip that relies on monolithic microfluidic technology to rapidly conduct signaling studies. This phosphoflow chip (pFC) allows hostpathogen phosphoprofiling in 30 min. The platform integrates cell stimulation and preparation, microscopy, and subsequent flow cytometry. The validation of the device, mitogen activated protein kinases ERK1/2 and p38 in response to E. coli lipopolysaccharide (LPS) stimulation of murine macrophage cells (RAW 264.7), was monitored. Cell Mechanics. To study the influence of shear stress on cells, Chau et al. (233) developed a device for the simultaneous evaluation of 10 different shear stresses ranging over 2 orders of Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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magnitude (0.07-13 Pa). To demonstrate the utility of the device, human umbilical vein endothelial cells (HUVECs) were exposed to the shear stress profiles for over a 20 h perfusion period and the von Willebrand factor (vWF) was investigated. Rossi et al. (234) reported a device for the investigation of biochemical and mechanical response of individual endothelial cells to different fluid dynamical conditions. The tapered geometry of the flow chamber creates a predefined homogeneous shear gradient on the cell. The promotor activity of the shear responsive transcription factor KLF2 with the driving gene for the green fluorescent protein (GFP) demonstrated the functionality of the device. Rydholm et al. (235) developed a device for the studies of primary cilium mediated cellular response to dynamic flow in asymmetric microfluidic environments. The primary cilium functions as a mechanosensor in renal tubular epithelium, sensing the extracellular flow. Shao and co-workers (236) developed an integrated chip for the cultivation and analysis of endothelial cells exposed to a pulsatile and oscillatory shear stress. Since endothelial cells play a role in the protein transport from blood to tissue, the transport of FITC labeled albumin was monitored in a static condition or after exposure to shear stress produced by the pulsatile and oscillatory flow. Tissue Models. The in vitro analysis of human hepatoma cells and primary rat hepatocytes was possible in a device with intrinsic microvascular-based channels developed by Carraro and co-workers (237). By the continuous in vitro perfusion of medium, the cells proliferated and maintained their hepatic functions. A multiwell plate for 3D liver tissue engineering was presented by Domansky et al. (238). Their developed bioreactor for the maintenance of 3D tissue cultures under constant perfusion is integrated into an array. Each bioreactor in the array is fluidically isolated from and contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The functionality of the new hepatic tissue was shown by immunostaining for hepatocyte and liver sinusoidal endothelial cells. Lee et al. (239) presented a device for the synthesis of cell-laden alginate hollow fibers using microvascularized tissue engineering applications. The feasibility of constructing 3D microvascularized structures were investigated. HIVE-78 cell laden fibers were fabricated and embedded into an agar gelatin fibronectin hydrogel were smooth muscle cells (HIVS-125) were cultivated. The cells were cultivated for 7 days, and using confocal laser microscopy, and it was shown that the fibers maintained their hollow structure. Another method was described by Mirsaidov et al. (240) , the live cell lithography to create tissues. This method uses multiple laminar flow and optical tweezers to organize cells into a complex array. The cells are then encapsulated in a 3D photo polymerizable hydrogel that mimics an extra cellular matrix. A heterogeneous array of E.coli genetically engineered with a lac switch was formed and evaluated on viability and metabolic activity. Puleo et al. (241) described a device containing collagen vitrigel which is used as a functional and sacrificial growth substrate for the development of corneal microtissue patches. Evaluation of the device was done by the miniaturization of the standard transepithelial permeability assay. In vivo tissue studies can be carried out under in vivo conditions using the chip fabricated by Hattersley et al. (242). Liver tissue has been kept functional 4860


and viable for over 70 h. Samples were also disaggregated in situ on-chip into individual primary cells, using a collagenase digestion procedure, enabling further cell analysis to be carried out off-line. A lung organomimetic microdevice was developed by Huh and Ingber (243), which enables the generation of functional alveolar epithelial tissues and reproduction of their dynamic mechanical microenvironment in vitro. Assisted Reproduction. To assess the potential of on-chip fertilization, sperm was studied by Lopez-Garcia and co-workers (244). The fluid motion of the sperm, the interaction with the flow, and spermocyte attachment were investigated. For this study, bovine sperm was used. Another clinically relevant application would be to determine the metabolic activities of embryos produced through assisted reproduction since there is increasing evidence that embryos with greater developmental capacity have distinct metabolic profiles. Urbanski et al. (245) presented an automated system which assesses the embryonic metabolism noninvasively. This was done by combining automated measurements of previously characterized enzyme-linked assays. Other Applications. Cartwright et al. (246) developed a device to immobilize, orient, and image muscle formation in Drosphila embryos without affecting the development of the organism. The Drosphila embryos expressed the green fluorescent protein in the membranes of muscle precursor cells. After imaging, the embryos were flushed away of the device and the wild type embryos developed normally and emerged as adult flies within 12 days. Glawdel et al. (247) presented a microfluidic device with integrated electroosmotic pumps to control the flow, a concentration gradient generator that produced a stable linear gradient in a cell chamber for a fish cell line (RTgill-W1). This cell line is extremely suitable for potential portable water quality testing. To investigate the plasma membranes of cells, Lanigan and co-workers (248) developed a microfluidic platform which uses optically trapped lipid coated oil droplets (smart droplet microtools, SDMs). With this device, it was shown that fusion of the SDMs with the cell plasma membrane was possible and that material plasma from the plasma membrane to the droplet via the tether was seen to occur. On-chip separation of the target cells from the bulk SDM population and from downstream analysis modules was also performed. Shah et al. (249) reported a EWOD driven droplet device integrated with optoelectronic tweezers as an automated platform for cellular isolation and analysis. The applicability of the device was shown by the use of HeLa cells over a relatively large area combined with reprogrammable microfluidic operations of independent droplets. Zeng et al. (250) developed a method for non-invasive and high-throughput on-chip immobilization of physiologically active C. elegans without the use of anaesthesia or cooling. The capability of the device was demonstrated by the observation and manipulation of subcellular features in immobilized animals using twophoton microscopy and femtosecond-laser microsurgery. Ino et al. (251) developed a microfluidic device with an interdigitated array of electrodes for the detection of hormone active chemicals using genetically engineerd yeast cells. The yeast cells were successfully used as biosensors since they produce β-galactosidase in the presence of the hormone active chemical, 17β-estradiol. Lee and co-workers (252) reported the application

of on-chip optofluidic microscopy for the imaging of G. lamblia trophozoites and cysts. The system performs imaging by flowing or scanning the target cells or objects across a slanted hole array. The rendered images are at a resolution comparable to the hole size. Subcellular content and the trophozoites’ flagella were clearly imaged with the device at an achieved focal plane resolution of 800 nm. Jang and Suh (253) presented the development of a simple multilayered device to cultivate and analyze renal tubular cells. The functionality of the device was shown by cultivating primary rat inner medullary collecting duct cells and verifying the enhanced cell polarization, cytoskeletal reorganization, and molecular transport by hormonal stimulations. Cleaning blood with a micromagneticmicrofluidic device was developed by Yung et al. (254). The device generates magnetic field gradients across vertically stacked channels, which enables the continuous and high throughput immunomagnetic microbead-based separation of fungi from whole blood. ACKNOWLEDGMENT G.B.S.-B., G.S., and A.A. contributed equally to this work. The authors thank Prof. Edwin Carlen (Twente University, EWI/BIOS Lab-on-a-chip Group, The Netherlands) for proofreading of the manuscript. Gabrielle Sprave is thanked for her excellent assistance in many different ways during the making of the two reviews. Georgette B. Salieb-Beugelaar received her M.Sc. in Chemistry in 2003. After working for almost 10 years at the Clinical Genetic Department of the Academic Medical Centre of Amsterdam, where she was involved in epigenetic DNA studies, she decided to switch fields in 2005. In 2009, she obtained her Ph.D. in nanofluidics at the BIOS/Lab on a Chip group of Prof. Albert van den Berg, the MESA+ Institute, Twente University, The Netherlands. Her thesis presents the investigation of the electrokinetic transport of DNA molecules in nanoslits. Presently, she is working as a postdoc in both the Korea Institute of Science and Technology in Germany and at the University of Twente. Her research interests include micro- and nanofluidics for biomolecular separation, clinical diagnostics, cell studies, tissue engineering, DNA confinement studies, and archaeometry. Giuseppina Simone received her M.Sc. in Chemical Engineering from the University “Federico II” of Naples, Italy. She spent 3 years doing research on polymers at the University of Naples and at CNR in Milan before embarking on her Ph.D. at the University “La Sapienza” in Rome and at the Technical University of Denmark. She studied and characterized organic-inorganic hybrid composites, and she developed an optical sensor based on a nanocomposite for microfluidic devices. At Silicon Biosystems in Bologna, she worked on microfluidic and technologies for rare cell sorting, and she gained an extensive experience in microfluidics and cellomics. She was a Postdoctoral fellow at the National Nanotechnology Laboratory of CNR-INFM, at the Department of Medicine of Harvard Medical School and Harvard-MIT Health Sciences and Technology, and at the Danish Technological Institute working on microfluidics applied to early cancer diagnosis. Since November 2009, she has joined Prof. Andreas Manz’s group at Korea Institute of Science and Technology in Germany. Her research interests lie in microfluidics for clinical diagnostics and rare cell detection, in engineering surfaces for the life sciences, and in microfabrication and integration. Arun Arora obtained his PhD from Imperial College, London with Prof. Andreas Manz where he developed an online wireless electrochemilumenescence detection system for capillary electrophoresis on glass chip. He spent two more years in Imperial College working on electrochemical sensors for water quality monitoring with Prof Nigel Graham, Civil Engineering Department; enzyme electrodes for glucose with Dr Danny O’Hare, Biosensors group, Bioengineering Department; and solid state gold/ iridium oxide pH electrodes as a possible prime power source for a nonresonant electrostatic energy harvesting from a rolling mass with Prof Erik Yeatman, Optical and Semiconductor Devices Group, Department of Electrical and Electronic Engineering. Since November 2009, he has joined Prof. Andreas Manz’s microfluidic group at Korea Institutes of Technology, Saarbrucken, Germany. His interests include exploring and developing new

material for microfluidic chip fabrication for droplet-based applications and electrophoresis methods development by employing fluorescence, electrochemiluminescence, and electrochemical detection techniques.

Anja Philippi studied Biology at the University of Saarland (Saarbru ¨ cken, Germany) and obtained her Ph.D. from the University of Regensburg (Germany). In 2008, she joined the Korea Institute of Science and Technology (KIST) Europe in Saarbru ¨ cken, Germany. After an initial period as postdoctoral fellow, she became a team leader in the newly founded Interdisciplinary Human Biotechnology Group in April 2010. Her research interests include cellular cancer therapies and targeted drug delivery. Andreas Manz obtained his Ph.D. from the Swiss Federal Institute of Technology (ETH) Zurich, Switzerland, with Professor W. Simon. His thesis dealt with the use of microelectrodes as detectors for picoliter-size volumes. He spent 1 year at Hitachi Central Research Lab in Tokyo, Japan, as a postdoctoral fellow and produced liquid chromatography column on a chip. At Ciba-Geigy, Basel, Switzerland, he developed the concept of miniaturized total analysis systems and built a research team on-chip-based analytical instrumentation during 1988-1995. He was professor for analytical chemistry at Imperial College in London, 1995-2003. Subsequently, he was the head of ISAS in Dortmund, Germany, and a Professor for analytical chemistry at the University of Dortmund. Since October 2009, he has been the head of research at KIST, Saarbru ¨ cken. His research interests include microfluidics for chemical analysis, biomimetic microfabrication, nanomedicine, cancer research, and the “Human Document Project”.

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