Cell culture arrays using magnetic force-based cell patterning for dynamic single cell analysis

In order to understand the behavior of individual cells, single cell analyses have attracted attention since most cell-based assays provide data with values averaged across a large number of cells. Techniques for the manipulation and analysis of single cells are crucial for understanding the behavior of individual cells. In the present study, we have developed single cell culture arrays using magnetic force and a pin holder, which enables the allocation of the magnetically labeled cells on arrays, and have analyzed their dynamics. The pin holder was made from magnetic soft iron and contained more than 6000 pillars on its surface. The pin holder was placed on a magnet to concentrate the magnetic flux density above the pillars. NIH/3T3 fibroblasts that were labeled with magnetite cationic liposomes (MCLs) were seeded into a culture dish, and the dish was placed over the pin holder with the magnet. The magnetically labeled cells were guided on the surface where the pillars were positioned and allocated on the arrays with a high resolution. Single-cell patterning was achieved by adjusting the number of cells seeded, and the target cell was collected by a micromanipulator after removing the pin holder with the magnet. Furthermore, change in the morphology of magnetically patterned cells was analyzed by microscopic observation, and cell spreading on the array was observed with time duration. Magnetic force-based cell patterning on cell culture arrays would be a suitable technique for the analysis of cell behavior in studies of cell-cell variation and cell-cell interactions.     

Ferromagnetic micropallets for magnetic capture of single adherent cells

We present a magnetic micropallet array and demonstrate its capacity to recover specific, individual adherent cells from large populations and deliver them for downstream single cell analysis. A ferromagnetic photopolymer was formulated, characterized, and used to fabricate magnetic micropallets, which are microscale pedestals that provide demarcated cell growth surfaces with preservation of biophysical properties including photopatternability, biocompatibility, and optical clarity. Each micropallet holds a single adherent cell in culture, and hundreds of thousands of micropallets comprise a single micropallet array. Any micropallet in the array can be recovered on demand, carrying the adhered cell with it. We used this platform to recover selectively single cells, which were subsequently analyzed using single-cell RT-qPCR.     

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.     

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronectin coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4-fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties.     

Spatial confinement of avidin domains in microwell arrays

Microwell arrays were chemically etched across the distal faces of coherent fiber-optic bundles. A typical 1.6 mm diameter array comprised approximately 3000 individual microwells that were approximately 1-14 microm deep and approximately 22 microm wide. A methodology involving organosilane functionalized microwell surfaces and site-selective photobiotin chemistry was developed to partially fill microwells with a thin avidin layer. Avidin microwell arrays were characterized using charge coupled device optical microscopy and scanning electron microscopy. The avidin microwell arrays had individual well volumes that were six orders of magnitude smaller and up to 30-fold more numerous than commercially available avidin-coated microtiter plates. Preliminary results indicated that individual avidin microwells were ideally suited to house single biological cells. Using standard epifluorescence microscope optics and a mercury-arc lamp, an individual 22 microm wide microwell could be optically addressed and selectively filled with avidin without the use of a photolithographic mask. The ability to control both the size and position of avidin domains on the microwell array surface demonstrates the utility of this methodology towards fabricating a single microwell array with multianalyte sensing capabilities.     

Selective trapping of single mammalian breast cancer cells by insulator-based dielectrophoresis

The trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment.

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.     

Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing.     

Programmed trapping of individual bacteria using micrometre-size sieves

Monitoring the real-time behavior of spatial arrays of single living bacteria cells is only achieved with much experimental difficulty due to the small size and mobility of the cells. To address this problem, we have designed and constructed a simple microfluidic device capable of trapping single bacteria cells in spatially well-defined locations without the use of chemical surface treatments. The device exploits hydrodynamics to slow down and trap cells flowing near a narrow aperture. We have modeled this system numerically by approximating the motion of Escherichia coli cells as rigid 3-D ellipsoids. The numerical predictions for the speed and efficiency of trapping were tested by fabricating the devices and imaging GFP expressing E. coli at a high spatio-temporal resolution. We find that our numerical simulations agree well with the actual cell flow for varying trap geometries. The trapped cells are optically accessible, and combined with our ability to predict their spatial location we demonstrate the ease of this method for monitoring multiple single cells over a time course. The simplicity of the design, inexpensive materials and straightforward fabrication make it an accessible tool for any systems biology laboratory.     

Polystyrene-coated micropallets for culture and separation of primary muscle cells

Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells.     

Frequency discretization in dielectrophoretic assisted cell sorting arrays to isolate neural cells

We present an automated dielectrophoretic assisted cell sorting (DACS) device for dielectric characterization and isolation of neural cells. Dielectrophoretic (DEP) principles are often used to develop cell sorting techniques. Here we report the first statistically significant neuronal sorting using DACS to enrich neurons from a heterogeneous population of mouse derived neural stem/progenitor cells (NSPCs) and neurons. We also study the dielectric dispersions within a heterogeneous cell population using a Monte-Carlo (MC) simulation. This simulation model explains the trapping behavior of populations as a function of frequency and predicts sorting efficiencies. The platform consists of a DEP electrode array with three multiplexed trapping regions that can be independently activated at different frequencies. A novel microfluidic manifold enables cell sorting by trapping and collecting cells at discrete frequency bands rather than single frequencies. The device is used to first determine the percentage of cells trapped at these frequency bands. With this characterization and the MC simulation we choose the optimal parameters for neuronal sorting. Cell sorting experiments presented achieve a 1.4-fold neuronal enrichment as predicted by our model.     

Integrated microelectrode array and microfluidics for temperature clamp of sensory neurons in culture

A device for cell culture is presented that combines MEMS technology and liquid-phase photolithography to create a microfluidic chip that influences and records electrical cellular activity. A photopolymer channel network is formed on top of a multichannel microelectrode array. Preliminary results indicated successful local thermal control within microfluidic channels and control of lamina position over the electrode array. To demonstrate the biological application of such a device, adult dissociated dorsal root ganglion neurons with a subpopulation of thermally-sensitive cells are attached onto the electrode array. Using laminar flow, dynamic control of local temperature of the neural cells was achieved while maintaining a constant chemical culture medium. Recording the expected altered cellular activity confirms the success of the integrated device.     

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging, a powerful analytical method to study single-cell behavior, such as gene expression and protein profiling, provides an essential basis for modern medical diagnosis. The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years. Simultaneously, chip-based living single-cell imaging is also limited by complicated trapping steps, low cell utilization, and difficult high-resolution imaging. To solve these problems, an ultra-thin temperature-controllable microwell array chip (UTCMA chip) was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells. The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope (or confocal microscope) with a high magnification objective (100 × oil lens), and the temperature of the chip can be controlled by combining it with a home-made temperature control device. High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall. The cell utilization rate, single-cell capture rate, and microwell occupancy rate are all close to 100% in the microwell array. This method will be useful in rare single-cell research, extending its application in the biological and medical-related fields, such as early diagnosis of disease, personalized therapy, and research-based on single-cell analysis.     

Fast-lysis cell traps for chemical cytometry

Electrically addressable cell traps were integrated with capillary electrophoresis for the analysis of the contents of single adherent cells. Electrodes composed of indium tin oxide were patterned on a glass surface followed by formation of topographical cell traps using 1002F photoresist. Single cells trapped in the holes could be lysed in less than 66 ms by applying a brief electric field (10 ms) across the electrode beneath the cell and the ground electrode placed in the aqueous media above the cell traps. The gas formed during cell lysis remained localized within the cavity formed by the 1002F photoresist. The retention of the gas in the cell trap enabled the cell traps to be coupled to an overlying capillary without blockage of the capillary. Single cells cultured in the traps were loaded with fluorescein and Oregon Green and then electrically lysed. By simultaneous application of an electric field to the capillary, the cell's contents were loaded into the capillary and electrophoretically separated. Orgeon Green and fluorescein from a single cell were fully resolved in less than two minutes. The use of a single patterned electrode beneath the 1002F cell trap yielded a simple easily fabricated design that was robust when immersed in aqueous solutions. Moreover, the design can easily be scaled up to create arrays of adherent cells for serial analyses using a single capillary or for parallel analysis by mating to an array of capillaries. Enhancing the rate of analysis of single adherent cells would enable a greater understanding of cellular physiology.     

A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response

External forces are increasingly recognized as major regulators of cellular structure and function, yet the underlying mechanism by which cells sense forces and transduce them into intracellular biochemical signals and behavioral responses ('mechanotransduction') is largely undetermined. To aid in the mechanistic study of mechanotransduction, herein we devised a cell stretching device that allowed for quantitative control and real-time measurement of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached to the microposts. Using the mPAM, we studied the live-cell subcellular dynamic responses of contractile forces in vascular smooth muscle cells (VSMCs) to a sustained static equibiaxial cell stretch. Our data showed that in response to a sustained cell stretch, VSMCs regulated their cytoskeletal (CSK) contractility in a biphasic manner: they first acutely enhanced their contraction to resist rapid cell deformation ('stiffening') before they allowed slow adaptive inelastic CSK reorganization to release their contractility ('softening'). The contractile response across entire single VSMCs was spatially inhomogeneous and force-dependent. Our mPAM device and live-cell subcellular contractile measurements will help elucidate the mechanotransductive system in VSMCs and thus contribute to our understanding of pressure-induced vascular disease processes.     

Uniform‐sized neurosphere‐mediated motoneuron differentiation in microwell arrays

We developed the photocrosslinkable hydrogel microwell arrays for uniform‐sized neurosphere‐mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform‐sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform‐sized neurospheres harvested from microwell arrays were replated into laminin‐coated substrate. In parallel, uniform‐sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three‐dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150 μm in diameter) on motoneuron differentiation, showing that the largest uniform‐sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform‐sized neurosphere‐mediated motoneuron differentiation.     

Dynamic trapping and high-throughput patterning of cells using pneumatic microstructures in an integrated microfluidic device

Microfluidic trapping methods create significant opportunities to establish highly controlled cell positioning and arrangement for the microscale study of numerous cellular physiological and pathological activities. However, a simple, straightforward, dynamic, and high-throughput method for cell trapping is not yet well established. In the present paper, we report a direct active trapping method using an integrated microfluidic device with pneumatic microstructures (PμSs) for both operationally and quantitatively dynamic localization of cells, as well as for high-throughput cell patterning. We designed and fabricated U-shape PμS arrays to replace the conventional fixed microstructures for reversible trapping. Multidimensional dynamics and spatial consistency of the PμSs were optically characterized and quantitatively demonstrated. Furthermore, we performed a systematic trapping investigation of the PμSs actuated at a pressure range of 0 psi to 20 psi using three types of popularly applied mammalian cells, namely, human lung adenocarcinoma A549 cells, human hepatocellular liver carcinoma HepG2 cells, and human breast adenocarcinoma MCF-7 cells. The cells were quantitatively trapped and controlled by the U-shape PμSs in a programmatic and parallel manner, and could be opportunely released. The trapped cells with high viability were hydrodynamically protected by the real-time actuation of specifically designed umbrella-like PμSs. We demonstrate that PμSs can be applied as an active microfluidic component for large-scale cell patterning and manipulation, which could be useful in many cell-based tissue organization, immunosensor, and high-throughput imaging and screening.     

A microfluidic concentrator array for quantitative predation assays of predatory microbes

We present a microfabricated concentrator array device that makes it possible to quantify the predation rate of Bdellovibrio bacteriovorus, a predatory microbe, toward its prey, Escherichia coli str. MG1655. The device can accumulate both prey and predator microbes sequentially within a series of concentrator arrays using the motility of the microbes and microfabricated arrowhead-shaped ratchet structures. Since the device can constrain both prey and predator cells within 200 pL chambers at a desired range of cell densities, it was demonstrated that the device cannot only enhance the possibility of studying predation processes/cycles directly at a single cell level but can also quantify the predation rates indirectly by measuring the time-dependent fluorescent intensity signals from the prey. Furthermore, the device can produce a wide range of initial prey to predator density ratios within various concentrator arrays through the use of microfluidic mixer structures on a single array chip, which allows us to study many different conditions with a single set of cultures, and quantitatively characterize the predation behaviour/rate. Lastly, we note that this novel concentrator array device can be a very powerful tool facilitating studies of microbial predations and microbe-microbe interaction and may be broadly used in other microbial biotechnological applications.     

Microfluidic arrays for logarithmically perfused embryonic stem cell culture

We present a microfluidic device for culturing adherent cells over a logarithmic range of flow rates. The device sets flow rates through four separate cell-culture chambers using syringe-driven flow and a network of fluidic resistances. The design is easy to fabricate with no on-chip valves and is scalable both in the number of culture chambers as well as in the range of applied flow rates. Using particle velocimetry, we have characterized the flow-rate range. We have also demonstrated an extension of the design that combines the logarithmic flow-rate functionality with a logarithmic concentration gradient across the array. Using fluorescence measurements we have verified that a logarithmic concentration gradient was established in the extended device. Compared with static cell culture, both devices enable greater control over the soluble microenvironment by controlling the transport of molecules to and away from the cells. This approach is particularly relevant for cell types such as embryonic stem cells (ESCs) which are especially sensitive to the microenvironment. We have demonstrated for the first time culture of murine ESCs (mESCs) in continuous, logarithmically scaled perfusion for 4 days, with flow rates varying >300x across the array. Cells grown in the slowest flow rate did not proliferate, while colonies grown in higher flow rates exhibited healthy round morphology. We have also demonstrated logarithmically scaled continuous perfusion culture of 3T3 fibroblasts for 3 days, with proliferation at all flow rates except the slowest rate.     

Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.