Trapping single human osteoblast-like cells from a heterogeneous population using a dielectrophoretic microfluidic device

We describe a system for the isolation, concentration, separation, and recovery of human osteoblast-like cells from a heterogeneous population using dielectrophoretic ring traps. Cells flowing in a microfluidic channel are immobilized inside an electric field cage using negative dielectrophoresis. A planar ring electrode creates a closed trap while repelling surrounding cells. Target cells are identified by fluorescent labeling, and are trapped as they pass across a ring electrode by an automated system. We demonstrate recovery of small populations of human osteoblast-like cells with a purity of 100%, which in turn demonstrates the potential of such a device for cell selection from a heterogeneous population.     

Release monitoring of single cells on a microfluidic device coupled with fluorescence microscopy and electrochemistry

A method for monitoring the biological exocytotic phenomena on a microfluidic system was proposed. A microfluidic device coupled with functionalities of fluorescence imaging and amperometric detection has been developed to enable the real-time monitoring of the exocytotic events. Exocytotic release of single SH-SY5Y neuroblastoma cells was studied. By staining the cells located on integrated microelectrodes with naphthalene-2,3-dicarboxaldehyde, punctuate fluorescence consistent with localization of neurotransmitters stored in vesicles was obtained. The stimulated exocytotic release was successfully observed at the surface of SH-SY5Y cells without refitting the commercial inverted fluorescence microscope. Spatially and temporally resolved exocytotic events from single cells on a microfluidic device were visualized in real time using fluorescence microscopy and were amperometrically recorded by the electrochemical system simultaneously. This coupled technique is simple and is hoped to provide new insights into the mechanisms responsible for the kinetics of exocytosis.     

A microfluidic device for simultaneous electrical and mechanical measurements on single cells

This paper presents a microfluidic device for simultaneous mechanical and electrical characterization of single cells. The device performs two types of cellular characterization (impedance spectroscopy and micropipette aspiration) on a single chip to enable cell electrical and mechanical characterization. To investigate the performance of the device design, electrical and mechanical properties of MC-3T3 osteoblast cells were measured. Based on electrical models, membrane capacitance of MC-3T3 cells was determined to be 3.39±1.23 and 2.99±0.82 pF at the aspiration pressure of 50 and 100 Pa, respectively. Cytoplasm resistance values were 110.1±37.7 kΩ (50 Pa) and 145.2±44.3 kΩ (100 Pa). Aspiration length of cells was found to be 0.813±0.351 μm at 50 Pa and 1.771±0.623 μm at 100 Pa. Quantified Young's modulus values were 377±189 Pa at 50 Pa and 344±156 Pa at 100 Pa. Experimental results demonstrate the device's capability for characterizing both electrical and mechanical properties of single cells.     

Continuous removal of glycerol from frozen-thawed red blood cells in a microfluidic membrane device

Cryopreservation of human red blood cells (RBCs) in the presence of 40% glycerol allows a shelf-life of 10 years, as opposed to only 6 weeks for refrigerated RBCs. Nonetheless, cryopreserved blood is rarely used in clinical therapy, in part because of the requirement for a time-consuming (∼1 h) post-thaw wash process to remove glycerol before the product can be used for transfusion. The current deglycerolization process involves a series of saline washes in an automated centrifuge, which gradually removes glycerol from the cells in order to prevent osmotic damage. We recently demonstrated that glycerol can be extracted in as little as 3 min without excessive osmotic damage if the composition of the extracellular solution is precisely controlled. Here, we explore the potential for carrying out rapid glycerol extraction using a membrane-based microfluidic device, with the ultimate goal of enabling inline washing of cryopreserved blood. To assist in experimental design and device optimization, we developed a mass transfer model that allows prediction of glycerol removal, as well as the resulting cell volume changes. Experimental measurements of solution composition and hemolysis at the device outlet are in reasonable agreement with model predictions, and our results demonstrate that it is possible to reduce the glycerol concentration by more than 50% in a single device without excessive hemolysis. Based on these promising results, we present a design for a multistage process that is predicted to safely remove glycerol from cryopreserved blood in less than 3 min.     

Probing the mechanical properties of brain cancer cells using a microfluidic cell squeezer device

Despite being invasive within surrounding brain tissues and the central nervous system, little is known about the mechanical properties of brain tumor cells in comparison with benign cells. Here, we present the first measurements of the peak pressure drop due to the passage of benign and cancerous brain cells through confined microchannels in a “microfluidic cell squeezer” device, as well as the elongation, speed, and entry time of the cells in confined channels. We find that cancerous and benign brain cells cannot be differentiated based on speeds or elongation. We have found that the entry time into a narrow constriction is a more sensitive indicator of the differences between malignant and healthy glial cells than pressure drops. Importantly, we also find that brain tumor cells take a longer time to squeeze through a constriction and migrate more slowly than benign cells in two dimensional wound healing assays. Based on these observations, we arrive at the surprising conclusion that the prevailing notion of extraneural cancer cells being more mechanically compliant than benign cells may not apply to brain cancer cells.     

Modulating chemotaxis of lung cancer cells by using electric fields in a microfluidic device

We employed direct-current electric fields (dcEFs) to modulate the chemotaxis of lung cancer cells in a microfluidic cell culture device that incorporates both stable concentration gradients and dcEFs. We found that the chemotaxis induced by a 0.5 μM/mm concentration gradient of epidermal growth factor can be nearly compensated by a 360 mV/mm dcEF. When the effect of chemical stimulation was balanced by the electrical drive, the cells migrated randomly, and the path lengths were largely reduced. We also demonstrated electrically modulated chemotaxis of two types of lung cancer cells with opposite directions of electrotaxis in this device.     

Quantifying the volume of single cells continuously using a microfluidic pressure-driven trap with media exchange

We demonstrate a microfluidic device capable of tracking the volume of individual cells by integrating an on-chip volume sensor with pressure-activated cell trapping capabilities. The device creates a dynamic trap by operating in feedback; a cell is periodically redirected back and forth through a microfluidic volume sensor (Coulter principle). Sieve valves are positioned on both ends of the sensing channel, creating a physical barrier which enables media to be quickly exchanged while keeping a cell firmly in place. The volume of individual Saccharomyces cerevisiae cells was tracked over entire growth cycles, and the ability to quickly exchange media was demonstrated.Measuring cell growth is of primary interest to researchers who seek to study the effects of drugs, nutrients, disease, and environmental stress. This has traditionally been accomplished by monitoring the optical transmittance of large ensembles of cells and applying the Beer-Lambert Law.^1,2 Such population-scale measurements provide important culture statistics, but averaging obscures the behaviour of individual cells. In addition, these techniques often require cell synchronicity in order to correlate growth with specific points in the cell cycle, but synchronicity typically decays rapidly in many cell lines including Saccharomyces cerevisiae (yeast) cultures.³ Researchers have thus adopted methods that study the growth of individual cells. Quantifying cellular growth is especially challenging since proliferating cells such as yeast or Escherichia coli are irregularly shaped, and will only increase in size by a factor of two.⁴ Growth will affect the mass, volume, and density of the cell; having access to each of these characteristics is important in obtaining a complete picture of this process. Time-lapse fluorescence microscopy can provide valuable information as to the cell cycle progression of individual cells,⁵ but 2D optics requires geometric assumptions, and, thus, can provide an incomplete picture of growth.^6,7Microfluidic lab-on-chip devices with integrated sensors can provide high-resolution growth tracking of individual cells, either through mass, volume, or density monitoring.^4,7,8 Recently, a microfluidic mass sensor was used to track the buoyant mass of individual cells using a suspended microchannel resonator (SMR).^4,9 Monitoring growth can also be accomplished by tracking volume using microfluidic volume sensors⁷ operating on the Coulter principle.¹⁰ Trapping can be achieved by either (1) cycling the target back and forth through the sensor (pressure-driven⁴ and electrokinetic⁷) or (2) holding a cell in place (posts,¹¹ chevron structure,¹² and E-Field¹³). The former, dynamic approach, allows a single cell to be sampled periodically by reversing flow directions after a cell is detected. Simple in its implementation, this technique also has the ability to compensate for a drifting baseline current resulting from parasitic ionic changes within the sensing channel or other sources of noise. On the other hand, static traps allow cells to be held in place while the buffer is rapidly exchanged.¹² The ability to dynamically change cellular growth conditions during an experiment can lead to significant insight into the behaviour of cells in environments of varying salinity,¹⁴ oxidative,^15,16 or osmotic conditions,¹⁷ as well as the effect of nutrients¹⁸ and drugs.¹⁹In this work, we propose a device capable of tracking growth using high-resolution volume measurements, combining the best attributes of both types of measurement systems; continuous baseline correction and the ability to rapidly exchange cell media. This is accomplished by using a pressure-driven, feedback-based dynamic trap, whereby a cell is cycled back and forth through the sensor within a microfluidic channel. On-chip sieve valves positioned at both ends of the sensing channel are able to selectively capture a cell while the solution is being replaced. As proof of principle, the volume of several individual yeast cells was monitored over the course of their respective growth cycles, and the ability to quantify growth response to media exchange was demonstrated.Devices were fabricated using multilayered soft lithography with polydimethylsiloxane (PDMS) molding.²⁰ The completed device is pictured in Figure 1(a); full fabrication protocols are presented as supplementary material.²¹ To maximize measurement sensitivity, it is optimal to choose a channel width and height slightly larger than the dimensions of the target cell.²² However, yeast cells are asymmetrically shaped and tend to tumble as they traverse the sensor. Preliminary testing suggested this effect could be mitigated by having cells flow along trajectories far from the electrodes (through buoyancy), where electric field is more uniform. Thus, a channel height of 20 μm was chosen as a compromise. Channel height increases to 28 μm in the wider part of the central and bypass channels, a result of using a mold made out of reflowed photoresist.²³ Channel width was set at 25 μm through the sensor, and widens to 80 μm at the sieve valves to facilitate valve actuation, which requires a high width to height ratio.²⁰ The fluidic layer is integrated in a 35 μm thick PDMS spin-coated layer, above which sits a 50 μm tall valve channel in a 4 mm PDMS layer. Tubing connects I1 and I2 to a common inlet vial, V1 and V2 to vials filled with deionised water and O1 and O2 connect to empty vials (not pictured). Inlet pressures I1 and I2, and valve pressures V1 and V2 are controlled with manual regulators (SMC IR2000-N02-R and SMC IR2010-N02-R); outlet pressures are computer-controlled (SMC ITV-1011). This pressure scheme is detailed elsewhere.²⁴ Current pulses caused by transiting particles/cells (Figure 1(d)) were acquired by applying a 50 kHz, 220 mV AC voltage between a pair of electrodes and measuring the drawn current. This frequency is sufficiently elevated to avoid the electrical double layer capacitance at the electrode-electrolyte interface,²⁵ but low enough to avoid sensitivity to cell impedance or substrate.²⁶ The electrical setup used for these experiments has been described previously.^24,27 A temperature controller maintains the device at 30 °C.Open in a separate windowFIG. 1.(a) Micrograph of the microfluidic device. Two parallel bypass channels are connected by a sensing channel with sensing electrodes. Pressure is applied at inlets (I1, I2) and outlets (O1, O2) to control flow conditions. Valves (V1, V2) are positioned over each end of the sensing channel. Food coloring is used to highlight the valve (red) and fluidic layers (blue). (b) Flow mode: valves are unpressurized, and cells flow freely through the device. (c) Trapping mode: valves are pressurized to capture a cell within the central channel. Pressure-driven flow cycles the cell back and forth across the sensor. (d)Typical current pulses measured for a yeast cell.The cell capture, media exchange, and detection process occurs as follows. A cell suspension is loaded into the bypass channel and made to flow through the central sensing channel by imposing a pressure gradient (Figure 1(b)). Cells flowing through the sensor are observed optically; once a cell of interest is observed (a cell without a bud), valves are sealed (V1 = V2 = 35 psi). This stops all flow through the sensor, and enables bypass channels to be flushed and replaced with fresh media. After 2 min, valve channels are pressurized to 24 psi where they compress the channel to a sufficient height to physically restrict the passage of yeast cells, while allowing the media to flow through the central channel (Figure 1(c)). The pressure gradient between bypasses causes the media in the central channel to be flushed out, while the target cell is physically trapped. Replacing the media in the central channel takes 2 min. At this stage, a pressure-driven feedback-based dynamic trap can be initiated. In this dynamic trap mode, the pressure settings at O1 and O2 are adjusted to redirect the cell back and forth through the sensor, based on current pulses measured from cells transiting through the sensor. Through custom LabView® software, these outlet pressure settings are feedback-adjusted to maintain a speed of 250 μm/s in both directions at a detection frequency of 30 cells/min (Figure 1(d)). To minimize the effects of channel stretching/shrinking, the sum of pressures at O1 and O2 is held constant. This precaution was taken since the sensing channel structured within the flexible PDMS polymer will alter its geometry based on internal pressure.²⁸ The short central channel ensures steady nutrient replenishment from the bypasses. For example, a glucose molecule takes ∼4 min to diffuse from the bypass to the electrodes. In practice, Taylor-Aris dispersion will reduce this replenishment time considerably. Based on video analysis, 25% of the central channel''s media is replenished every pressure reversal (video presented as supplementary material²¹). Polystyrene microspheres of 3.9 ± 0.3 μm, 5.6 ± 0.2 μm, and 8.3 ± 0.7 μm (NIST size standards) were used to calibrate the sensor, and obtain the current pulse-to-volume calibration for every solution (supplementary material²¹). The validity of this calibration method is discussed elsewhere.²⁹ Care was taken to limit trajectory-based variations in signal: the device is positioned with electrodes at the top of the sensing channel, and with the negatively buoyant cells/particles flowing along the bottom. Based on previous experimental and theory work, we found that signal amplitude can vary as much as 3.5 fold for different heights.²⁷ The effect of trajectory on current pulse amplitude has also been reported elsewhere.^30,31 In this work, buoyancy is used to ensure that the cell flows along a trajectory at the same distance from the electrodes for every measurement.Saccharomyces cerevisiae (BY4743 Mat a/alpha, genotype: his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0 ade2::LEU2/ade2::URA3) was cultured to exponential phase at 30 °C in an incubator/shaker in yeast bacto-peptone (YPD) with 2% w/v glucose, supplemented with 0.2 M NaCl, 0.05% bovine serum albumin (BSA) and 42 mg/l adenine. Sodium chloride was added to enable the current pulse measurement, at a concentration where cells are viable;³² BSA was used to prevent cell agglomeration; adenine was supplemented since this particular yeast mutant does not produce its own supply. A cell suspension was introduced into the device, from which a cell at the early stages of its cell cycle was captured, and dynamically trapped for 100 min. Three typical cell growth results are shown in Figure 2(a). Since the culture was not synchronized, this leads to variability between “initial” cell volumes: there is a 27% difference in initial volume between the cells identified by red squares and green triangles. This is caused by (1) optical limits, whereby cells chosen for study are not all at the exact same cell cycle stage and (2) differences in the age of the mother cell: the more buds a mother cell has produced, the larger it becomes.³³ On average, captured yeast cell demonstrated a doubling time consistent with growth rates under ideal incubator/shaker conditions; nutrient depletion, electric field, and shear stresses are not affecting growth. Optical inspection of budding cells confirms that most growth is occurring at the daughter cell, as expected.³³ An elevated signal-to-noise ratio allows for high resolution volumetric measurements (4 μm³); cell asymmetry⁷ and trajectory variability^27,30,31 lead to a relative standard deviation of 6% for cells and 4% for microspheres of similar size. While mass or protein synthesis methods have indicated linear³⁴ or exponential^4,6,35,36 growth curves, volume-based methods have suggested sigmoidal patterns.^7,37 Prior to daughter cell emergence, and later in the cycle as the daughter cell emerges, volumetric growth rate declines.³⁸ In this work, it is difficult to ascertain with mathematical rigor the shape of the growth profile; however, for each cell, volume increases steadily throughout the growth cycle before declining near the end of the cycle.Open in a separate windowFIG. 2.(a) Growth curves for 3 cells trapped in succession. Simultaneous optical and electrical measurements allow cell cycle stage to be correlated with volume. Pictures of cell corresponding to the red squares are presented in 15 min increments. A cell is cycled through the sensor every 2 s. For clarity, each data point for yeast volume represents the average of data points over a period of 5 min, with standard deviation. (b) Demonstration of an interrupted growth cycle, where YPD + 0.2 M NaCl was replaced with 0.2 M NaCl at 40 min, and then again returned to YPD + 0.2 M NaCl at 80 min. The media exchange process takes 4 min.To demonstrate our ability to easily exchange media while maintaining a trap, the solution was exchanged 40 min into a yeast growth cycle; culture media was replaced with a pure saline solution 0.2 M NaCl + 0.05% BSA, and then replaced again with culture media at 80 min (Figure 2(b)). Cell growth is halted temporarily while in saline solution, before resuming normal growth thereafter. The cell cycle time is extended by this period. The cell volume drifts downward after the initial solution change at 40 min. Though this drift lies within our uncertainty bounds, cellular responses to osmotic shock on similar timescales have been documented elsewhere.³⁹ This result demonstrates an ability to quickly exchange cell media, and observe cellular response.In conclusion, we have demonstrated a microfluidic device capable of maintaining a dynamic, pressure-driven cell trap, which can monitor cellular volume over the cell cycle. Concurrent optical microscopy allows for real-time visual inspection of the cells. In addition, sieve valve integration provides for the exchange of media or the addition of drugs. Such a platform could also be key in cancer cell cytotoxicity assays,⁴⁰ where growth response to anticancer drugs could be monitored.     

Microfluidic device for trapping and monitoring three dimensional multicell spheroids using electrical impedance spectroscopy

In this paper, we report the design, fabrication, and testing of a lab-on-a-chip based microfluidic device for application of trapping and measuring the dielectric properties of microtumors over time using electrical impedance spectroscopy (EIS). Microelectromechanical system (MEMS) techniques were used to embed opposing electrodes onto the top and bottom surfaces of a microfluidic channel fabricated using Pyrex substrate, chrome gold, SU-8, and polydimethylsiloxane. Differing concentrations of cell culture medium, differing sized polystyrene beads, and MCF-7 microtumor spheroids were used to validate the designs ability to detect background conductivity changes and dielectric particle diameter changes between electrodes. The observed changes in cell medium concentrations demonstrated a linear relation to extracted solution resistance (Rs), while polystyrene beads and multicell spheroids induced changes in magnitude consistent with diameter increase. This design permits optical correlation between electrical measurements and EIS spectra.     

Transport and shear in a microfluidic membrane bilayer device for cell culture

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.     

Gold nanoparticle-assisted single base-pair mismatch discrimination on a microfluidic microarray device

Two simple gold nanoparticle (GNP)-based DNA analysis methods using a microfluidic device are presented. In the first method, probe DNA molecules are immobilized on the surface of a self-assembled submonolayer of GNPs. The hybridization efficiency of the target oligonulceotides was improved due to nanoscale spacing between probe molecules. In the second method, target DNA molecules, oligonulceotides or polymerase chain reaction (PCR) amplicons, are first bound to GNPs and then hybridized to the immobilized probe DNA on a glass slide. With the aid of GNPs, we have successfully discriminated, at room temperature, between two PCR amplicons (derived from closely related fungal pathogens, Botrytis cinerea and Botrytis squamosa) with one base-pair difference. DNA analysis on the microfluidic chip avoids the use of large sample volumes, and only a small amount of oligonucelotides (8 fmol) or PCR products (3 ng), was needed in the experiment. The whole procedure was accomplished at room temperature in 1 h, and apparatus for high temperature stringency was not required.     

High-throughput sorting of drops in microfluidic chips using electric capacitance

We analyze a recently introduced approach for the sorting of aqueous drops with biological content immersed in oil, using a microfluidic chip that combines the functionality of electrowetting with the high throughput of two-phase flow microfluidics. In this electrostatic sorter, three co-planar electrodes covered by a thin dielectric layer are placed directly below the fluidic channel. Switching the potential of the central electrode creates an electrical guide that leads the drop to the desired outlet. The generated force, which deflects the drop, can be tuned via the voltage. The working principle is based on a contrast in conductivity between the drop and the continuous phase, which ensures successful operation even for drops of highly conductive biological media like phosphate buffered saline. Moreover, since the electric field does not penetrate the drop, its content is protected from electrical currents and Joule heating. A simple capacitive model allows quantitative prediction of the electrostatic forces exerted on drops. The maximum achievable sorting rate is determined by a competition between electrostatic and hydrodynamic forces. Sorting speeds up to 1200 per second are demonstrated for conductive drops of 160 pl in low viscosity oil.     

Discrimination between the human prostate normal cell and cancer cell by using a novel electrical impedance spectroscopy controlling the cross-sectional area of a microfluidic channel

The prostate biopsy method shows a high false negative result because the suspicious tissue considered as cancer is not confirmed during tissue sampling. Thus, repeated biopsy procedures and diagnostic errors in relation to prostate cancer frequently occur. The purpose of this research is to enhance the prostate cancer detection rate by using microfluidic electrical impedance spectroscopy (μEIS), which allows real-time measurement of the electrical impedance of a single human prostate normal cell and cancer cell. The μEIS was equipped with a movable flexible membrane, which is operated by pneumatic pressure to capture the single cell on the surface of sensing electrodes. The forced tight contact between the cell and electrodes makes it possible to measure the electrical characteristics of the cell with a high sensitivity. The μEIS discriminates well between normal human prostate cells (RWPE-1) and cancer cells (PC-3) at 8.7 kHz based on the electrical signal responses of the cells. The average difference rates of admittance magnitude and susceptance are 54.55% and 54.59%, respectively. The developed μEIS also shows high repeatability, which was verified by a deionized water test conducted before and after each cell assay; the maximum variance of both the impedance and admittance at 8.7 kHz was as small as 9.48%.     

On demand nanoliter-scale microfluidic droplet generation,injection, and mixing using a passive microfluidic device

We here present and characterize a programmable nanoliter scale droplet-on-demand device that can be used separately or readily integrated into low cost single layer rapid prototyping microfluidic systems for a wide range of user applications. The passive microfluidic device allows external (off-the-shelf) electronically controlled pinch valves to program the delivery of nanoliter scale aqueous droplets from up to 9 different inputs to a central outlet channel. The inputs can be either continuous aqueous fluid streams or microliter scale aqueous plugs embedded in a carrier fluid, in which case the number of effective input solutions that can be employed in an experiment is no longer strongly constrained (100 s–1000 s). Both nanoliter droplet sequencing output and nanoliter-scale droplet mixing are reported with this device. Optimization of the geometry and pressure relationships in the device was achieved in several hardware iterations with the support of open source microfluidic simulation software and equivalent circuit models. The requisite modular control of pressure relationships within the device is accomplished using hydrodynamic barriers and matched resistance channels with three different channel heights, custom parallel reversible microfluidic I/O connections, low dead-volume pinch valves, and a simply adjustable array of external screw valves. Programmable sequences of droplet mixes or chains of droplets can be achieved with the device at low Hz frequencies, limited by device elasticity, and could be further enhanced by valve integration. The chip has already found use in the characterization of droplet bunching during export and the synthesis of a DNA library.     

A microfluidic membrane device to mimic critical components of the vascular microenvironment

Vascular function, homeostasis, and pathological development are regulated by the endothelial cells that line blood vessels. Endothelial function is influenced by the integrated effects of multiple factors, including hemodynamic conditions, soluble and insoluble biochemical signals, and interactions with other cell types. Here, we present a membrane microfluidic device that recapitulates key components of the vascular microenvironment, including hemodynamic shear stress, circulating cytokines, extracellular matrix proteins, and multiple interacting cells. The utility of the device was demonstrated by measuring monocyte adhesion to and transmigration through a porcine aortic endothelial cell monolayer. Endothelial cells grown in the membrane microchannels and subjected to 20 dynes∕cm(2) shear stress remained viable, attached, and confluent for several days. Consistent with the data from macroscale systems, 25 ng∕ml tumor necrosis factor (TNF)-α significantly increased RAW264.7 monocyte adhesion. Preconditioning endothelial cells for 24 h under static or 20 dynes∕cm(2) shear stress conditions did not influence TNF-α-induced monocyte attachment. In contrast, simultaneous application of TNF-α and 20 dynes∕cm(2) shear stress caused increased monocyte adhesion compared with endothelial cells treated with TNF-α under static conditions. THP-1 monocytic cells migrated across an activated endothelium, with increased diapedesis in response to monocyte chemoattractant protein (MCP)-1 in the lower channel of the device. This microfluidic platform can be used to study complex cell-matrix and cell-cell interactions in environments that mimic those in native and tissue engineered blood vessels, and offers the potential for parallelization and increased throughput over conventional macroscale systems.     

Rapid separation of bacteriorhodopsin using a laminar-flow extraction system in a microfluidic device

A protein separation technology using the microfluidic device was developed for the more rapid and effective analysis of target protein. This microfluidic separation system was carried out using the aqueous two-phase system (ATPS) and the ionic liquid two-phase system (ILTPS) for purification method of the protein sample, and the three-flow desalting system was used for the removal of salts from the sucrose-rich sample. Partitioning of the protein sample was observed in ATPS or ILTPS with the various pHs. The microdialysis system was applied to remove small molecules, such as sucrose and salts in the microfluidic channel with the different flow rates of buffer phase. A complex purification method, which combines microdialysis and ATPS or ILTPS, was carried out for the effective purification of bacteriorhodopsin (BR) from the purple membrane of Halobacterium salinarium, which was then analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and matrix-assisted laser desorption∕ionization time-of-flight. Furthermore, we were able to make a stable three-phase flow controlling the flow rate in the microfluidic channel. Our complex purification methods were successful in purifying and recovering the BR to its required value.     

Efficient capture of circulating tumor cells with a novel immunocytochemical microfluidic device

Ability to perform cytogenetic interrogations on circulating tumor cells (CTCs) from the blood of cancer patients is vital for progressing toward targeted, individualized treatments. CTCs are rare compared to normal (bystander) blood cells, found in ratios as low as 1:10⁹. The most successful isolation techniques have been immunocytochemical technologies that label CTCs for separation based on unique surface antigens that distinguish them from normal bystander cells. The method discussed here utilizes biotin-tagged antibodies that bind selectively to CTCs. The antibodies are introduced into a suspension of blood cells intending that only CTCs will display surface biotin molecules. Next, the cell suspension is passed through a microfluidic channel that contains about 9000 transverse, streptavidin coated posts. A CTC making contact with a post has the opportunity to engage in a biotin-streptavidin reaction that immobilizes the cell. Bystander blood cells remain in suspension and pass through the channel. The goal of the present study is to establish the technical performance of these channels as a function of antigen density and operating conditions, especially flow rate. At 18 μL/min, over 70% of cells are captured at antigen densities greater than 30 000 sites/cell while 50% of cells are captured at antigen densities greater than 10 000. It is found that lower flow rates lead to decreasing cell capture probabilities, indicating that some streamlines develop which are never close enough to a post to allow cell-post contact. Future modeling and streamline studies using computational fluid dynamics software could aid in optimization of channel performance for capture of rare cells.     

Controlled electroporation of the plasma membrane in microfluidic devices for single cell analysis

Chemical cytometry on a single cell level is of interest to various biological fields ranging from cancer to stem cell research. The impact chemical cytometry can exert in these fields depends on the dimensionality of the retrievable analytes content. To this point, the number of different analytes identifiable and additionally their subcellular localization is of interest. To address this, we present an electroporation based approach for selective lysis of only the plasma membrane, which permits analysis of the dissolved cytoplasm, while reducing contributions from the nucleus and membrane bound fractions of the cell analytes. The use of 100 μs long pulse and a well defined DC electric field gradient of ∼4.5 kV·cm⁻¹ generated by 3D electrodes initiates release of a cytoplasm marker in ≪1 s, while retaining nuclear fluorescence markers.     

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.     

A microfluidic device for simultaneous measurement of viscosity and flow rate of blood in a complex fluidic network

Blood viscosity has been considered as one of important biophysical parameters for effectively monitoring variations in physiological and pathological conditions of circulatory disorders. Standard previous methods make it difficult to evaluate variations of blood viscosity under cardiopulmonary bypass procedures or hemodialysis. In this study, we proposed a unique microfluidic device for simultaneously measuring viscosity and flow rate of whole blood circulating in a complex fluidic network including a rat, a reservoir, a pinch valve, and a peristaltic pump. To demonstrate the proposed method, a twin-shaped microfluidic device, which is composed of two half-circular chambers, two side channels with multiple indicating channels, and one bridge channel, was carefully designed. Based on the microfluidic device, three sequential flow controls were applied to identify viscosity and flow rate of blood, with label-free and sensorless detection. The half-circular chamber was employed to achieve mechanical membrane compliance for flow stabilization in the microfluidic device. To quantify the effect of flow stabilization on flow fluctuations, a formula of pulsation index (PI) was analytically derived using a discrete fluidic circuit model. Using the PI formula, the time constant contributed by the half-circular chamber is estimated to be 8 s. Furthermore, flow fluctuations resulting from the peristaltic pumps are completely removed, especially under periodic flow conditions within short periods (T < 10 s). For performance demonstrations, the proposed method was applied to evaluate blood viscosity with respect to varying flow rate conditions [(a) known blood flow rate via a syringe pump, (b) unknown blood flow rate via a peristaltic pump]. As a result, the flow rate and viscosity of blood can be simultaneously measured with satisfactory accuracy. In addition, the proposed method was successfully applied to identify the viscosity of rat blood, which circulates in a complex fluidic network. These observations confirm that the proposed method can be used for simultaneous measurement of viscosity and flow rate of whole blood circulating in the complex fluid network, with sensorless and label-free detection. Furthermore, the proposed method will be used in evaluating variations in the viscosity of human blood during cardiopulmonary bypass procedures or hemodialysis.     

Dielectrophoretic microfluidic device for the continuous sorting of Escherichia coli from blood cells

Microfluidic diagnostic devices promise faster disease identification by purifying and concentrating low-abundance analytes from a flowing sample. The diagnosis of sepsis, a whole body inflammatory response often caused by microbial infections of the blood, is a model system for pursuing the advantages of microfluidic devices over traditional diagnostic protocols. Traditional sepsis diagnoses require large blood samples and several days to culture and identify the low concentration microbial agent. During these long delays while culturing, the physician has little or no actionable information to treat this acute illness. We designed a microfluidic chip using dielectrophoresis to sort and concentrate the target microbe from a flowing blood sample. This design was optimized using the applicable electrokinetic and hydrodynamic theories. We quantify the sorting efficiency of this device using growth-based assays which show 30% of injected microbes are recovered viable, consistent with the electroporation of target cells by the dielectrophoretic cell sorters. Finally, the results illustrate the device is capable of a five-fold larger microbe concentration in the target analyte stream compared to the waste stream at a continuous sample flow rate of 35 μl∕h.