A gradient-generating microfluidic device for cell biology

The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described. A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients. Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles. Here, we developed simple gradient-generating microfluidic devices with three separate inlets. Three microchannels combined into one microchannel to generate concentration gradients. The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF). Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations. The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis. Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.     

Migration of connective tissue-derived cells is mediated by ultra-low concentration gradient fields of EGF

The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.     

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.     

A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods

Background

Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories.

Results

We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient.

Conclusion

This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols.     

A Portable Chemotaxis Platform for Short and Long Term Analysis

Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours) and long-term (72 hours) concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.     

Microfluidic‐Assisted Blade Coating of Compositional Libraries for Combinatorial Applications: The Case of Organic Photovoltaics

Microfluidic technologies are highly adept at generating controllable compositional gradients in fluids, a feature that has accelerated the understanding of the importance of chemical gradients in biological processes. That said, the development of versatile methods to generate controllable compositional gradients in the solid‐state has been far more elusive. The ability to produce such gradients would provide access to extensive compositional libraries, thus enabling the high‐throughput exploration of the parametric landscape of functional solids and devices in a resource‐, time‐, and cost‐efficient manner. Herein, the synergic integration of microfluidic technologies is reported with blade coating to enable the controlled formation of compositional lateral gradients in solution. Subsequently, the transformation of liquid‐based compositional gradients into solid‐state thin films using this method is demonstrated. To demonstrate efficacy of the approach, microfluidic‐assisted blade coating is used to optimize blending ratios in organic solar cells. Importantly, this novel technology can be easily extended to other solution processable systems that require the formation of solid‐state compositional lateral gradients.     

Deep wells integrated with microfluidic valves for stable docking and storage of cells

In this paper, we describe a microfluidic mechanism that combines microfluidic valves and deep wells for cell localization and storage. Cells are first introduced into the device via externally controlled flow. Activating on-chip valves was used to interrupt the flow and to sediment the cells floating above the wells. Thus, valves could be used to localize the cells in the desired locations. We quantified the effect of valves in the cell storage process by comparing the total number of cells stored with and without valve activation. We hypothesized that in deep wells external flows generate low shear stress regions that enable stable, long-term docking of cells. To assess this hypothesis we conducted numerical calculations to understand the influence of well depth on the forces acting on cells. We verified those predictions experimentally by comparing the fraction of stored cells as a function of the well depth and input flow rate upon activation of the valves. As expected, upon reintroduction of the flow the cells in the deep wells were not moved whereas those in shallow wells were washed away. Taken together, our paper demonstrates that deep wells and valves can be combined to enable a broad range of cell studies.     

Experimental verification of the behavioral foundation of bacterial transport parameters using microfluidics

We present novel microfluidic experiments to quantify population-scale transport parameters (chemotactic sensitivity χ₀ and random motility μ) of a population of bacteria. Previously, transport parameters have been derived theoretically from single-cell swimming behavior using probabilistic models, yet the mechanistic foundations of this upscaling process have not been verified experimentally. We designed a microfluidic capillary assay to generate and accurately measure gradients of chemoattractant (α-methylaspartate) while simultaneously capturing the swimming trajectories of individual Escherichia coli bacteria using videomicroscopy and cell tracking. By measuring swimming speed and bias in the swimming direction of single cells for a range of chemoattractant concentrations and concentration gradients, we directly computed the chemotactic velocity V_C and the associated chemotactic sensitivity χ₀. We then show how μ can also be readily determined using microfluidics but that a population-scale microfluidic approach is experimentally more convenient than a single-cell analysis in this case. Measured values of both χ₀ [(12.4 ± 2.0) × 10⁻⁴ cm² s⁻¹] and μ [(3.3 ± 0.8) × 10⁻⁶ cm² s⁻¹] are comparable to literature results. This microscale approach to bacterial chemotaxis lends experimental support to theoretical derivations of population-scale transport parameters from single-cell behavior. Furthermore, this study shows that microfluidic platforms can go beyond traditional chemotaxis assays and enable the quantification of bacterial transport parameters.     

Density-dependent adaptive resistance allows swimming bacteria to colonize an antibiotic gradient

During antibiotic treatment, antibiotic concentration gradients develop. Little is know regarding the effects of antibiotic gradients on populations of nonresistant bacteria. Using a microfluidic device, we show that high-density motile Escherichia coli populations composed of nonresistant bacteria can, unexpectedly, colonize environments where a lethal concentration of the antibiotic kanamycin is present. Colonizing bacteria establish an adaptively resistant population, which remains viable for over 24 h while exposed to the antibiotic. Quantitative analysis of multiple colonization events shows that collectively swimming bacteria need to exceed a critical population density in order to successfully colonize the antibiotic landscape. After colonization, bacteria are not dormant but show both growth and swimming motility under antibiotic stress. Our results highlight the importance of motility and population density in facilitating adaptive resistance, and indicate that adaptive resistance may be a first step to the emergence of genetically encoded resistance in landscapes of antibiotic gradients.     

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping,Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.     

Generation of Gradients on a Microfluidic Device: Toward a High-Throughput Investigation of Spermatozoa Chemotaxis

Various research tools have been used for in vitro detection of sperm chemotaxis. However, they are typically poor in maintenance of gradient stability, not to mention their low efficiency. Microfluidic device offers a new experimental platform for better control over chemical concentration gradient than traditional ones. In the present study, an easy-handle diffusion-based microfluidic chip was established. This device allowed for conduction of three parallel experiments on the same chip, and improved the performance of sperm chemotaxis research. In such a chip, there were six channels surrounding a hexagonal pool. The channels are connected to the hexagon by microchannels. Firstly, the fluid flow in the system was characterized; secondly, fluorescein solution was used to calibrate gradient profiles formed in the central hexagon; thirdly, sperm behavior was observed under two concentration gradients of progesterone (100 pM and 1 mM, respectively) as a validation of the device. Significant differences in chemotactic parameters were recognized between experimental and control groups (p < 0.05). Compared with control group, sperm motility was greatly enhanced in 1 mM group (p < 0.05), but no significant difference was found in 100 pM group. In conclusion, we proposed a microfluidic device for the study of sperm chemotaxis that was capable of generating multi-channel gradients on a chip and would help reduce experimental errors and save time in experiment.     

Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable,Competing Gradients

Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but was repelled from the stationary-phase signal indole. Cells also migrated toward higher concentrations of isatin (indole-2,3-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between attractant and repellent signals may be important determinants of bacterial colonization of the gut.Bacteria sense chemoeffectors using cell surface receptors (13, 29). Cells constantly monitor the concentration of specific molecules, comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement (6, 22). Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Natural habitats for Escherichia coli, such as the gastrointestinal (GI) tract, are typically heterogeneous and contain multiple chemoeffectors with potentially opposing effects. The integrated chemotactic response in such environments is thus likely to be an important factor in bacterial colonization.Conventional approaches for investigating bacterial chemotaxis, such as the swim plate and capillary (1) assays, are not ideal for quantifying bacterial migration. Chemotactic-ring formation in semisolid agar requires metabolizable attractants and is subject to multiple factors, and both it and the traditional capillary assay are poorly designed to investigate repellent taxis. Mao et al. (23) were the first to investigate bacterial taxis in a microfluidic flow cell. In their device, a concentration gradient is formed by the diffusive mixing of two inlet streams. However, the exposure to a fully developed gradient in this device is limited because it takes time for the gradient to develop.Variations of this technique, such as three-channel microfluidic devices (7, 8) in which a linear gradient is generated in the absence of flow or a T-channel device that monitors chemotaxis perpendicular to the direction of fluid flow (18), were developed subsequently. The T-channel system has many of the limitations of the device developed by Mao et al. (23), and nonflow systems, like the capillary assay (1), suffer from a lack of temporal stability of the gradients.Here, we report a flow-based microfluidic chemotaxis device that is coupled to a gradient generator. Bacteria are exposed to precise and temporally stable concentration gradients of chemoeffectors over the length of the microfluidic channel. This device was used to quantify E. coli chemotaxis in response to the canonical chemoeffectors l-aspartate and Ni²⁺. The device was also used to investigate chemotaxis toward cell-cell communication signals such as autoinducer-2 (AI-2), indole, and isatin that are likely to be present in the in vivo microenvironment in which E. coli is present (e.g., the human GI tract). The data obtained reinforce the idea that concentration-dependent interactions between different chemical signals could be important determinants of bacterial colonization in natural environments.     

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15–1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.     

Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates

In vitro recording of neuronal electrical activity is a widely used technique to understand brain functions and to study the effect of drugs on the central nervous system. The integration of microfluidic devices with microelectrode arrays (MEAs) enables the recording of networks activity in a controlled microenvironment. In this work, an integrated microfluidic system for neuronal cultures was developed, reversibly coupling a PDMS microfluidic device with a commercial flat MEA through magnetic forces. Neurons from mouse embryos were cultured in a 100 μm channel and their activity was followed up to 18 days in vitro. The maturation of the networks and their morphological and functional characteristics were comparable with those of networks cultured in macro-environments and described in literature. In this work, we successfully demonstrated the ability of long-term culturing of primary neuronal cells in a reversible bonded microfluidic device (based on magnetism) that will be fundamental for neuropharmacological studies.     

Microbial response to environmental gradients in a ceramic-based diffusion system

A solid, porous matrix was used to establish steady-state concentration profiles upon which microbial responses to concentration gradients of nutrients or antimicrobial agents could be quantified. This technique relies on the development of spatially defined concentration gradients across a ceramic plate resulting from the diffusion of solutes through the porous ceramic matrix. A two-dimensional, finite-element numerical transport model was used to predict the establishment of concentration profiles, after which concentration profiles of conservative tracers were quantified fluorometrically and chemically at the solid-liquid interface to verify the simulated profiles. Microbial growth responses to nutrient, hypochloride, and antimicrobial concentration gradients were then quantified using epifluorescent or scanning confocal laser microscopy. The observed microbial response verified the establishment and maintenance of stable concentration gradients along the solid-liquid interface. These results indicate the ceramic diffusion system has potential for the isolation of heterogeneous microbial communities as well as for testing the efficacy of antimicrobial agents. In addition, the durability of the solid matrix allowed long-term investigations, making this approach preferable to conventional gel-stabilized systems that are impeded by erosion as well as expansion or shrinkage of the gel.     

High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species.     

Mapping of Enzyme Kinetics on a Microfluidic Device

A microfluidic platform or “microfluidic mapper” is demonstrated, which in a single experiment performs 36 parallel biochemical reactions with 36 different combinations of two reagents in stepwise concentration gradients. The volume used in each individual reaction was 36 nl. With the microfluidic mapper, we obtained a 3D enzyme reaction plot of horseradish peroxidase (HRP) with Amplex Red (AR) and hydrogen peroxide (H₂O₂), for concentration ranges of 11.7 μM to 100.0 μM and 11.1 μM to 66.7 μM for AR and H₂O₂, respectively. This system and methodology could be used as a fast analytical tool to evaluate various chemical and biochemical reactions especially where two or more reagents interact with each other. The generation of dual concentration gradients in the present format has many advantages such as parallelization of reactions in a nanoliter-scale volume and the real-time monitoring of processes leading to quick concentration gradients. The microfluidic mapper could be applied to various problems in analytical chemistry such as revealing of binding kinetics, and optimization of reaction kinetics.     

Synergistic NGF/B27 gradients position synapses heterogeneously in 3D micropatterned neural cultures

Native functional brain circuits show different numbers of synapses (synaptic densities) in the cerebral cortex. Until now, different synaptic densities could not be studied in vitro using current cell culture methods for primary neurons. Herein, we present a novel microfluidic based cell culture method that combines 3D micropatterning of hydrogel layers with linear chemical gradient formation. Micropatterned hydrogels were used to encapsulate dissociated cortical neurons in laminar cell layers and neurotrophic factors NGF and B27 were added to influence the formation of synapses. Neurotrophic gradients allowed for the positioning of distinguishable synaptic densities throughout a 3D micropatterned neural culture. NGF and B27 gradients were maintained in the microfluidic device for over two weeks without perfusion pumps by utilizing a refilling procedure. Spatial distribution of synapses was examined with a pre-synaptic marker to determine synaptic densities. From our experiments, we observed that (1) cortical neurons responded only to synergistic NGF/B27 gradients, (2) synaptic density increased proportionally to synergistic NGF/B27 gradients; (3) homogeneous distribution of B27 disturbed cortical neurons in sensing NGF gradients and (4) the cell layer position significantly impacted spatial distribution of synapses.     

Colorimetric device for measurement of transvascular fluid flux in blood-perfused organs

The aim of this study was to develop a device capable of measuring transvascular fluid flux in blood-perfused organs. For any given blood flow through the organ (QT), transvascular flux (QF) can be considered as the fraction of QT exchange. Presumably, QF would change the background concentration of an impermeable tracer residing in the perfusate. Thus QF could be calculated from the relative changes in tracer concentration for any given QT. We have used Blue Dextran (1 g/l of blood) as the reference tracer. Because the minimum molecular weight of Blue Dextran is 2 X 10(6), we anticipated it to behave as an impermeable tracer in most organs. QF was simulated with continuous infusions of plasma, normal saline solution, and a 50% mixture of both. Changes in Blue Dextran concentration were continuously followed colorimetrically by changes in transmission of specific light at a wavelength of 632 nm. Because 632-nm light is affected by hematocrit and O2 saturation changes, two additional wavelengths were used: 815-nm, which is not affected by saturation or Blue Dextran concentration changes, was used to account for changes in hematocrit, and 887-nm specific light, which is not affected by Blue Dextran, served to correct for saturation changes. Red cells could not be used as the reference tracer because of the possibility of hematocrit changes independent of fluid flux (Fahraeus effect). The device so constructed proved capable of measuring rates of fluid infusion in the order of 0.1% of QT with a variability of 10% around the mean.     

An integrated microfluidic platform for magnetic microbeads separation and confinement

An innovative microfluidic platform for magnetic beads manipulation is introduced, consisting of novel microfabricated 3D magnetic devices positioned in a microfluidic chamber. Each magnetic device comprises of an embedded actuation micro-coil in various design versions, a ferromagnetic pillar, a magnetic backside plate and a sensing micro-coil. The various designs of the micro-coils enable efficient magnetic beads trapping and concentration in different patterns. The finite element analysis (FEA) results show a significant increase of the developed force on suspended magnetic beads when the magnetic pillar and backside plate were integrated into the device structure. These simulation results were confirmed experimentally by measuring the magnetic beads trapping ratios for the different designs and structures of the devices under continuous flow conditions. The trapping ratios and profiles were studied using beads counting, measuring the change of inductance with the sensing micro-coil and by image processing. The devices have efficiently demonstrated a controlled and localized magnetic beads trapping and concentration at small spatial locations for the first time. The new results shown in this study demonstrate the feasibility of efficiently using these original devices as key elements in complex bio-analysis systems.