Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays

We present a soft lithographic method to fabricate multiphenotype cell arrays by capturing cells within an array of reversibly sealed microfluidic channels. The technique uses reversible sealing of elastomeric polydimethylsiloxane (PDMS) molds on surfaces to sequentially deliver various fluids or cells onto specific locations on a substrate. Microwells on the substrate were used to capture and immobilize cells within low shear stress regions inside channels. By using an array of channels it was possible to deposit multiple cell types, such as hepatocytes, fibroblasts, and embryonic stem cells, on the substrates. Upon formation of the cell arrays on the substrate, the PDMS mold could be removed, generating a multiphenotype array of cells. In addition, the orthogonal alignment and subsequent attachment of a secondary array of channels on the patterned substrates could be used to deliver fluids to the patterned cells. The ability to position many cell types on particular regions within a two dimensional substrate could potentially lead to improved high-throughput methods applicable to drug screening and tissue engineering.     

A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array

We present a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 microm in height surrounded by multiple narrow perfusion channels 2 microm in height. The high aspect ratio (approximately 20) between the microchamber and the perfusion channels offers advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C and was grown to confluency. The microfluidic cell culture array could potentially offer an affordable platform for a wide range of applications in high throughput cell-based screening, bioinformatics, synthetic biology, quantitative cell biology, and systems biology.     

An integrated microfluidic culture device to regulate endothelial cell differentiation from embryonic stem cells

We developed an integrated microfluidic culture device to regulate embryonic stem (ES) cell fate. The integrated microfluidic culture device consists of an air control channel and a fluidic channel with 4×4 micropillar arrays. We hypothesized that the microscale posts within the micropillar arrays would enable the control of uniform cell docking and shear stress profiles. We demonstrated that ES cells cultured for 6 days in the integrated microfluidic culture device differentiated into endothelial cells. Therefore, our integrated microfluidic culture device is a potentially powerful tool for directing ES cell fate.     

Molded polyethylene glycol microstructures for capturing cells within microfluidic channels

The ability to control the deposition and location of adherent and non-adherent cells within microfluidic devices is beneficial for the development of micro-scale bioanalytical tools and high-throughput screening systems. Here, we introduce a simple technique to fabricate poly(ethylene glycol)(PEG) microstructures within microfluidic channels that can be used to dock cells within pre-defined locations. Microstructures of various shapes were used to capture and shear-protect cells despite medium flow in the channel. Using this approach, PEG microwells were fabricated either with exposed or non-exposed substrates. Proteins and cells adhered within microwells with exposed substrates, while non-exposed substrates prevented protein and cell adhesion (although the cells were captured inside the features). Furthermore, immobilized cells remained viable and were stained for cell surface receptors by sequential flow of antibodies and secondary fluorescent probes. With its unique strengths in utility and control, this approach is potentially beneficial for the development of cell-based analytical devices and microreactors that enable the capture and real-time analysis of cells within microchannels, irrespective of cell anchorage properties.     

Dual‐micropillar‐based microfluidic platform for single embryonic stem cell‐derived neuronal differentiation

We developed the dual‐micropillar‐based microfluidic platform to direct embryonic stem (ES) cell fate. 4 × 4 dual‐micropillar‐based microfluidic platform consisted of 16 circular‐shaped outer micropillars and 8 saddle‐shaped inner micropillars in which single ES cells were cultured. We hypothesized that dual‐micropillar arrays would play an important role in controlling the shear stress and cell docking. Circular‐shaped outer micropillars minimized the shear stress, whereas saddle‐shaped inner micropillars allowed for docking of individual ES cells. We observed the effect of saddle‐shaped inner micropillars on cell docking in response to hydrodynamic resistance. We also demonstrated that ES cells cultured for 6 days within the dual‐micropillar‐based microfluidic platform differentiated into neural‐like cells. Therefore, this dual‐micropillar‐based microfluidic platform could be a potentially powerful method for screening of lineage commitments of single ES cells.     

Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip

A microfluidic system was developed for the analysis of single biological cells, with functional integration of cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection in microfabricated channels of a single glass chip. Channels were 12 microm deep and 48 microm wide, with a simple crossed-channel design. The effective separation channel length was 35 mm. During sampling with a cell suspension (cell population 1.2 x 10(5) cells per mL in physiological salt solution), differential hydrostatic pressure (created by adjusting liquid levels in the four reservoirs) was used to control cell flow exclusively through the channel crossing. Single cell loading into the separation channel was achieved by electrophoretic means by applying a set of potentials at the four reservoirs, counteracting the hydrostatic flow. A special docking (adhering) procedure for the loaded cell was applied before lysis by repeatedly connecting and disconnecting a set of low potentials, allowing precise positioning of the cell within the separation channel. Cell lysis was then effected within 40 ms under an applied CE separation voltage of 1.4 kV (280 V cm(-1)) within the working electrolyte (pH 9.2 borate buffer) without additional lysates. The docked lysing approach reduced dispersion of released intracellular constituents, and significantly improved the reproducibility of CE separations. Glutathione (GSH) was used as a model intracellular component in single human erythrocyte cells. NDA derivatized GSH was detected using LIF. A throughput of 15 samples h(-1), a retention time precision of 2.4% RSD was obtained for 14 consecutively injected cells. The average cellular concentration of GSH in human erythrocytes was found to be 7.2 [times] 10(-4)+/- 3.3 x 10(-4) M (63 +/- 29 amol per cell). The average separation efficiency for GSH in lysed cells was 2.13 x 10(6)+/- 0.4 x 10(6) plates per m, and was about a factor of 5 higher than those obtained with GSH standards using pinched injection.     

Recent advances in the development of single cell analysis—A review

Development of techniques for the analysis of the content of individual cells represents an important direction in modern bioanalytical chemistry. While the analysis of chromosomes, organelles, or location of selected proteins has been traditionally the domain of microscopic techniques, the advances in miniaturized analytical systems bring new possibilities for separations and detections of molecules inside the individual cells including smaller molecules such as hormones or metabolites. It should be stressed that the field of single cell analysis is very broad, covering advanced optical, electrochemical and mass spectrometry instrumentation, sensor technology and separation techniques. The number of papers published on single cell analysis has reached several hundred in recent years. Thus a complete literature coverage is beyond the limits of a journal article. The following text provides a critical overview of some of the latest developments with the main focus on mass spectrometry, microseparation methods, electrophoresis in capillaries and microfluidic devices and respective detection techniques for performing single cell analyses.     

Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels

We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively.     

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

Nonlithographic fabrication of microfluidic devices

A facile nonlithographic method for expedient fabrication of microfluidic devices of poly(dimethylsiloxane) is described. Positive-relief masters for the molds are directly printed on smooth substrates. For the formation of connecting channels and chambers inside the polymer components of the microfluidic devices, cavity-forming elements are adhered to the surfaces of the masters. Using this nonlithographic approach, we fabricated microfluidic devices for detection of bacterial spores on the basis of enhancement of the emission of terbium (III) ions.     

Patterned cell culture inside microfluidic devices

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.     

Separation of two phenotypically similar cell types via a single common marker in microfluidic channels

To isolate clinically and biologically relevant cell types from a heterogeneous population, fluorescent or magnetic tagging together with knowledge of surface biomarker profiles represents the state of the art. To date, it remains exceedingly difficult to separate phenotypically and physically similar cell types from a mixed population. We report a microfluidic platform engineered to separate two highly similar cell types using a single antibody by taking advantage of subtle variations in surface receptor density and cell size. This platform utilizes antibody-conjugated surfaces in microfluidic channels together with precise modulation of fluid shear stresses to accomplish selective fractionation in a continuous flow process. Antibody conjugation density variation on the adhesive surfaces is achieved by covalently immobilizing an antibody in the presence of poly(ethylene glycol). This platform is used to demonstrate separation of two CD31 positive cell types, human umbilical vein endothelial cells and human micro vascular endothelial cells.     

Patterning cells and shear flow conditions: convenient observation of endothelial cell remoulding, enhanced production of angiogenesis factors and drug response

We present a method that allows patterning cells and shear flow conditions for endothelial cell based assays. This method is novel in combining (1) cell culture on the surface of a substrate both topographically and chemically patterned; (2) multi-shear flow assays after covering the cell substrate with a microfluidic cover plate containing microchannels of different channel widths, and (3) conventional immunostaining assays after removal of the cover plate. This method has the advantage of performing cell cultures and immunoassays in standard cell biology environments with open access, facilitating the formation of confluent cell layers and the observation of cell responses to shear-flow and drug stimulations. To obtain multi-shear stress conditions, a single channel with stepwise increasing channel widths was patterned on the surfaces of both the substrate and the microfluidic cover plate. As results, we observed excellent viability of endothelial cells in the whole range of applied shear stresses (0-25 dyn cm(-2)) and shear stress dependent cytoskeleton remoulding, activation of von Willebrand factor (vWF), and re-organisation of angiogenesis factors such as tetra peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) of endothelial cells. To validate this approach for drug analysis, we also studied drug effects under shear stress conditions. Our results indicate that the drug effect of combretastatin A-4, an anti-tumour vascular targeting drug, could be significantly enhanced under shear flow conditions.     

Transport, retention and fluorescent measurement of single biological cells studied in microfluidic chips

Cellular manipulation and fluorescent measurement were performed on two types of biological cells. First, transport and retention of yeast cells were demonstrated on a glass microfluidic chip, which consists of special U-shaped microstructures. These microstructures have the openings parallel to the liquid flow and weirs perpendicular to the flow. These allow the retention of yeast cells in the U-shaped pocket and drainage of liquid over the weirs. Thereafter, the same chip was used to carry out real-time fluorescent measurement for the cellular changes in single Jurkat T cells. In this case, the Jurkat cells were localized inside the straight portion of a microchannel. Fluorescent imaging on the same, single suspension cell was carried out to study two cellular processes occurring in viable cells, (1) the intracellular conversion of fluorescein diacetate (FDA) to fluorescein; (2) the degradation of an inhibitory protein, IkappaB, as involved in the NF-kappaB signalling pathway. In the former, the increase in fluorescent intensity of single Jurkat T cells (due to fluorescein formation) was measured; whereas in the latter, the decrease in the fluorescent intensity of a single transfected Jurkat cell (due to the degradation of the IkappaB-EGFP fusion protein) was monitored. In addition, we employed a Jurkat cell expressed with IkappaB-EGFP to probe any possible action of an herbal compound, isoliquiritigenin (IQ), on the degradation of IkappaB-EGFP. These examples have demonstrated that Jurkat cells remain viable within microfluidic channels for cellular studies and that the microfluidic chip can facilitate monitoring of cellular changes of biological cells at the single cell level and in the same cell.     

Peptide-mediated selective adhesion of smooth muscle and endothelial cells in microfluidic shear flow

Microfluidic devices have recently emerged as effective tools for cell separation compared to traditional techniques. These devices offer the advantages of small sample volumes, low cost, and high purity. Adhesion-based separation of cells from heterogeneous suspensions can be achieved by taking advantage of specific ligand-receptor interactions. The peptide sequences Arg-Glu-Asp-Val (REDV) and Val-Ala-Pro-Gly (VAPG) are known to bind preferentially to endothelial cells (ECs) and smooth muscle cells (SMCs), respectively. This article examines the roles of REDV and VAPG and fluid shear stress in achieving selective capture of ECs and SMCs in microfluidic devices. The adhesion of ECs in REDV-coated devices and SMCs in VAPG-coated devices increases significantly compared to that of the nontargeted cells with decreasing shear stress. Furthermore, the adhesion of these cells is shown to be independent of whether these cells flow through the devices as suspensions of only one cell type or as a heterogeneous suspension containing ECs, SMCs, and fibroblasts. Whereas the overall adhesion of cells in the devices is determined mainly by shear stress, the selectivity of adhesion depends on the type of peptide and on the device surface as well as on the shear stress.     

Monitoring spatial distribution of ethanol in microfluidic channels by using a thin layer of cholesteric liquid crystal

Monitoring spatial distribution of chemicals in microfluidic devices by using traditional sensors is a challenging task. In this paper, we report utilization of a thin layer of cholesteric liquid crystal for monitoring ethanol inside microfluidic channels. This thin layer can be either a polymer dispersed cholesteric liquid crystal (PDCLC) layer or a free cholesteric liquid crystal (CLC) layer separated from the microfluidic device by using a thin film of PDMS. They both show visible colorimetric responses to 4% of ethanol solution inside the microfluidic channels. Moreover, the spatial distribution of ethanol inside the microfluidic channel can be reflected as a color map on the CLC sensing layers. By using this device, we successfully detected ethanol produced from fermentation taking place inside the microfluidic channel. These microfluidic channels with embedded PDCLC or embedded CLC offer a new sensing solution for monitoring volatile organic compounds in microfluidic devices.     

Patterning, integration and characterisation of polymer optical oxygen sensors for microfluidic devices

This paper describes a process for the layer-by-layer fabrication and integration of luminescent dye-based optical oxygen sensors into microfluidic devices. Application of oxygen-sensitive platinum(ii) octaethylporphyrin ketone fluorescent dye dissolved in polystyrene onto glass substrates by spin-coating was studied. Soft lithography with polydimethylsiloxane (PDMS) stamps and reactive ion etching in oxygen plasma were used to produce sensor patterns with a minimum feature size of 25 microm. Sensors patterns were integrated into a PDMS microfluidic device by plasma bonding. No degradation of the sensor response as a result of the lithography and pattern-transfer processes was detected. Gaseous and dissolved oxygen (DO) detection was characterised using fluorescence microscopy. The intensity signal ratio of the sensor films was found to increase almost two-fold from 3.6 to 6.8 by reducing film thickness from 1.3 microm to 0.6 microm. Calibration of DO measurement showed linear Stern-Volmer behaviour that was constant for flow rates from 0.5 to 2 mL min(-1). The calibrated sensors were subsequently used to demonstrate laterally resolved detection of oxygen inside a microfluidic channel. The fabrication process provides a novel, easy to use method for the repeatable integration of optical oxygen sensors into cell-culture and lab-on-a-chip devices.     

Retention forces of a liquid slug in a rough capillary tube with symmetric or asymmetric features

On surfaces with asymmetric "sawtooth" features, liquid slugs or drops tend to move preferentially in one direction. In this theoretical study, the imbalance of capillary forces that leads to directionally biased wetting is examined. Capillary tubes with symmetric and asymmetric sawtooth features were used to estimate the ratios of retention forces in opposing directions. Our analysis suggests that the difference between the retention force in one direction versus the other can be maximized by increasing feature asymmetry and minimizing inherent hysteresis of the materials of construction. This work has implications for small channels or surfaces of fluid-handling components found in microfluidic devices and fuel cells.     

Patterned self-assembled beads in silicon channels.

A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 microm wide and 50 microm deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 microm, has also been generated on the bottom of a 500 microm wide and 50 microm deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.     

Photocured biodegradable polymer substrates of varying stiffness and microgroove dimensions for promoting nerve cell guidance and differentiation

Photocross-linkable and biodegradable polymers have great promise in fabricating nerve conduits for guiding axonal growth in peripheral nerve regeneration. Here, we photocross-linked two poly(ε-caprolactone) triacrylates (PCLTAs) with number-average molecular weights of ～7000 and ～10?000 g mol(-1) into substrates with parallel microgrooves. Cross-linked PCLTA7k was amorphous and soft, while cross-linked PCLTA10k was semicrystalline with a stiffer surface. We employed different dimensions of interests for the parallel microgrooves, that is, groove widths of 5, 15, 45, and 90 μm and groove depths of 0.4, 1, 5, and 12 μm. The behaviors of rat Schwann cell precursor line (SpL201) cells with the glial nature and pheochromocytoma (PC12) cells with the neuronal nature were studied on these microgrooved substrates, showing distinct preference to the substrates with different mechanical properties. We found different threshold sensitivities of the two nerve cell types to topographical features when their cytoskeleton and nuclei were altered by varying the groove depth and width. Almost all of the cells were aligned in the narrowest and deepest microgrooves or around the edge of microgrooves. Oriented SpL201 cell movement had a higher motility as compared to unaligned ones. After forskolin treatment, SpL201 cells demonstrated significantly upregulated S-100 and O4 on stiffer substrates or narrower microgrooves, suggesting more differentiation toward early Schwann cells (SCs). PC12 neurites were oriented with enhanced extension in narrower microgrooves. The present results not only improve our fundamental understanding on nerve cell-substrate interactions, but also offer useful conduit materials and appropriate feature dimensions to foster guidance for axonal growth in peripheral nerve regeneration.