A hydrogel-based microfluidic device for the studies of directed cell migration

We have developed a hydrogel-based microfluidic device that is capable of generating a steady and long term linear chemical concentration gradient with no through flow in a microfluidic channel. Using this device, we successfully monitored the chemotactic responses of wildtype Escherichia coli (suspension cells) to alpha-methyl-DL-aspartate (attractant) and differentiated HL-60 cells (a human neutrophil-like cell line that is adherent) to formyl-Met-Leu-Phe (f-MLP, attractant). This device advances the current state of the art in microchemotaxis devices in that (1) it demonstrates the validity of using hydrogels as the building material for a microchemotaxis device; (2) it demonstrates the potential of the hydrogel based microfluidic device in biological experiments since most of the proteins and nutrients essential for cell survival are readily diffusible in hydrogel; (3) it is capable of applying chemical stimuli independently of mechanical stimuli; (4) it is straightforward to make, and requires very basic tools that are commonly available in biological labs. This device will also be useful in controlling the chemical and mechanical environment during the formation of tissue engineered constructs.     

A biological sensor platform using a pneumatic-valve controlled microfluidic device containing Tetrahymena pyriformis

In this study, we introduce a microfluidic device equipped with pneumatically actuated valves, generating a linear gradient of chemoeffectors to quantify the chemotactic response of Tetrahymena pyriformis, a freshwater ciliate. The microfluidic device was fabricated from an elastomer, poly(dimethylsiloxane) (PDMS), using multi-layer soft lithography. The components of the device include electronically controlled pneumatic microvalves, microchannels and microchambers. The linear gradient of the chemoeffectors was established by releasing a chemical from a ciliate-free microchamber into a microchamber containing the ciliate. The ciliate showed chemotactic behaviours by either swimming toward or avoiding the gradient. By counting the number of ciliates residing in each microchamber, we obtained a precise time-response curve. The ciliates in the microfluidic device were sensitive enough to be attracted to 10 pmol glycine-proline, which indicates a 10(5) increase in the ciliate's known sensitivity. With the use of blockers, such as DL-2-amino-5-phosphonopentanoic acid (APPA) or lanthanum chloride (LaCl3), we have demonstrated that the NMDA (N-methyl-d-aspartate) receptor plays a critical role in the perception of chemoeffectors, whereas the Ca2+ channel is related to the motility of the ciliate. These results demonstrate that our microfluidic chemotaxis assay system is useful not only for the study of ciliate chemotaxis but also for a better understanding of the signal transduction mechanism on their receptors.     

Microfluidic device for analyzing preferential chemotaxis and chemoreceptor sensitivity of bacterial cells toward carbon sources

We present a novel microfluidic device that enables high sensitive analyses of the chemotactic response of motile bacterial cells (Escherichia coli) that swim toward a preferred nutrient by sorting and concentrating them. The device consists of the Y-shaped microchannel that has been widely used in chemotaxis studies to attract cells toward a high concentration and a concentrator array integrated with arrowhead-shaped ratchet structures beside the main microchannel to trap and accumulate them. Since the number of accumulated cells in the concentrator array continuously increases with time, the device makes it possible to increase the sensitivity of detecting chemotactic responses of the cells about 10 times greater than Y-shaped channel devices in 60 min. In addition, the device can characterize the relative chemotactic sensitivity of chemoreceptors to chemoeffectors by comparing the number of cells in the concentrator array at different distances from the channel junction. Since the device allows the analysis of both the chemotactic responses and the sensitivity of chemoreceptors with high resolution, we believe that not only can the device be broadly used for various microbial chemotaxis assays but it also can further the advancement of microbiology and even synthetic biology.     

T cell chemotaxis in a simple microfluidic device

This paper describes the use of a simple microfluidic device for studying T cell chemotaxis. The microfluidic device is fabricated in poly(dimethylsiloxane) (PDMS) using soft-lithography and consists of a "Y" type fluidic channel. Solutions are infused into the device by syringe pumps and generate a concentration gradient in the channel by diffusion. We show that the experimentally measured gradient profiles agree nicely with theoretical predictions and the gradient is stable in the observation region for cell migration. Using this device, we demonstrate robust chemotaxis of human T cells in response to single and competing gradients of chemokine CCL19 and CXCL12. Because of the simplicity of the device, it can flexibly control gradient generation in space and time, and would allow generation of multiple gradient conditions in a single chip for highly parallel chemotaxis experimentation. Visualization of T cell chemotaxis has previously been limited to studies in 3D matrices or under agarose assays, which do not allow precise control or variation in conditions. Acknowledging the importance of lymphocyte homing in the adaptive immune response, the ability to study T cell chemotaxis in microfluidic devices offers a new approach for investigating lymphocyte migration and chemotaxis in vitro.     

Characterization of a membrane-based gradient generator for use in cell-signaling studies

This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel. The high fluidic resistance of the membrane limits fluid flow caused by pressure differences in the system, but allows diffusive transport of a chemical species through the membrane and into the channel. The large volume sink region at the end of the microfluidic channel helps to maintain spatial and temporal stability of the gradient. The chemical gradient in a 0.5 mm region near the sink region experiences a maximum of 10 percent change between the 6 and 24 h data points. We present the theory, design, and characterization of this device and provide an example of neutrophil chemotaxis as proof of concept for future quantitative cell-signaling applications.     

Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices

Immune cell migration is a fundamental process that enables immunosurveillance and immune responses. Understanding the mechanism of immune cell migration is not only of importance to the biology of cells, but also has high relevance to cell trafficking mediated physiological processes and diseases such as embryogenesis, wound healing, autoimmune diseases and cancers. In addition to the well-known chemical concentration gradient based guiding mechanism (i.e. chemotaxis), recent studies have shown that lymphocytes can respond to applied physiologically relevant direct current (DC) electric fields by migrating toward the cathode of the fields (i.e. electrotaxis) in both in vitro and in vivo settings. In the present study, we employed two microfluidic devices allowing controlled application of electric fields inside the microfluidic channel for quantitative studies of lymphocyte electrotaxis in vitro at the single cell level. The first device is fabricated by soft-lithography and the second device is made in glass with integrated on-chip electrodes. Using both devices, we for the first time showed that anti-CD3/CD28 antibodies activated human blood T cells migrate to the cathode of the applied DC electric field. This finding is consistent with previous electrotaxis studies on other lymphocyte subsets suggesting electrotaxis is a novel guiding mechanism for immune cell migration. Furthermore, the characteristics of electrotaxis and chemotaxis of activated T cells in PDMS microfluidic devices are compared.     

A parallel diffusion-based microfluidic device for bacterial chemotaxis analysis

We developed a multiple-channel microfluidic device for bacterial chemotaxis detection. Some characteristics such as easy operation, parallel sample adding design and fast result readout make this device convenient for most biology labs. The characteristic feature of the design is the agarose gel channels, which serve as a semi-permeable membrane. They can stop the fluid flow and prevent bacteria getting across, but permit the diffusion of small molecules. In the device fabrication process a novel thermal-based method was used to control the shape of agarose gel in the microfluidic channel. The chemical gradient is established by diffusion which can be precisely controlled and measured. Combined with an 8-channel pipette, different attractants, repellent chemicals or different bacteria were analyzed by a two step operation with a readout time of one hour. This device may be useful in the high throughput detection of chemotaxis related molecules and genes.     

Mucin (MUC5AC) expression by lung epithelial cells cultured in a microfluidic gradient device

We have developed a microfluidic gradient device for controlling mucin gene expression of NCI-H292 epithelial cells derived from lung tissues. We hypothesized that gradient profiles would control mucin gene expression of lung epithelial cells. However, it was not possible to generate various stable gradient profiles using conventional culture methods. To address this limitation, we used a microfluidic gradient device to create various gradient profiles (i.e. non-linear, linear, and flat) in a temporal and spatial manner. NCI-H292 lung epithelial cells were exposed to concentration gradients of epidermal growth factor in a microfluidic gradient device with continuous medium perfusion. We demonstrated an effect of gradient profiles on mucin expression of lung epithelial cells cultured in the microfluidic gradient device. It was revealed that NCI-H292 lung epithelial cells exposed to the flat gradient profile of the epidermal growth factor exhibited high expression of mucin as compared with cells exposed to non-linear and linear gradient profiles. Therefore, this microfluidic gradient device could be a potentially useful tool for regulating the mucin expression of lung epithelial cells exposed to chemokine gradient profiles.     

Chemotaxis in microfluidic devices--a study of flow effects

The use of microfluidic devices has become increasingly popular in the study of chemotaxis due to the exceptional control of flow properties and concentration profiles on the length scale of individual cells. In these applications, it is often neglected that cells, attached to the inner surfaces of the microfluidic chamber, are three-dimensional objects that perturb and distort the flow field in their vicinity. Depending on the interplay of flow speed and geometry with the diffusive time scale of the chemoattractant in the flow, the concentration distribution across the cell membrane may differ strongly from the optimal gradient in a perfectly smooth channel. We analyze the underlying physics in a two-dimensional approximation and perform systematic numerical finite element simulations to characterize the three-dimensional case and to identify optimal flow conditions.     

Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients

Experimental systems that provide temporal and spatial control of chemical gradients are required for probing into the complex mechanisms of eukaryotic cell chemotaxis. However, no current technique can simultaneously generate stable chemical gradients and allow fast gradient changes. We developed a microfluidic system with microstructured membranes for exposing neutrophils to fast and precise changes between stable, linear gradients of the known chemoattractant Interleukin-8 (IL-8). We observed that rapidly lowering the average concentration of IL-8 within a gradient, while preserving the direction of the gradient, resulted in temporary neutrophil depolarization. Fast reversal of the gradient direction while increasing or decreasing the average concentration also resulted in temporary depolarization. Neutrophils adapted and maintained their directional motility, only when the average gradient concentration was increased and the direction of the gradient preserved. Based on these observations we propose a two-component temporal sensing mechanism that uses variations of chemokine concentration averaged over the entire cell surface and localized at the leading edge, respectively, and directs neutrophil responses to changes in their chemical microenvironment.     

Mimic Nature Using Chemotaxis of Ionic Liquid Microdroplets for Drug Delivery Purposes

Due to the growing prevalence of incurable diseases, such as cancer, worldwide, nowadays, the development of smart drug delivery systems is an inevitable necessity. Chemotaxis-driven movement of ionic liquid microdroplets containing therapeutic compounds is a well-known example of a smart drug delivery system. This review aims to classify, summarize, and compare ionic liquid-based chemotaxis systems in an easily understandable article. Chemotaxis is the basis of the movement of cells and microorganisms in biological environments, which is the cause of many vital biochemical and biological processes. This review attempts to summarize the available literature on single-component biomimetic and self-propelling microdroplet systems based on ionic liquids, which exhibit chemotaxis and spontaneously move in a determined direction by an external gradient, particularly a chemical change. It also aims to review artificial ionic liquid-based chemotaxis systems that can be used as drug carriers for medical purposes. The various ionic liquids used for this purpose are discussed, and different forms of chemical gradients and mechanisms that cause movement in microfluidic channels will be reviewed.     

Effects of flow and diffusion on chemotaxis studies in a microfabricated gradient generator

An understanding of chemotaxis at the level of cell-molecule interactions is important because of its relevance in cancer, immunology, and microbiology, just to name a few. This study quantifies the effects of flow on cell migration during chemotaxis in a microfluidic device. The chemotaxis gradient within the device was modeled and compared to experimental results. Chemotaxis experiments were performed using the chemokine CXCL8 under different flow rates with human HL60 promyelocytic leukemia cells expressing a transfected CXCR2 chemokine receptor. Cell trajectories were separated into x and y axis components. When the microchannel flow rates were increased, cell trajectories along the x axis were found to be significantly affected (p < 0.05). Total migration distances were not affected. These results should be considered when using similar microfluidic devices for chemotaxis studies so that flow bias can be minimized. It may be possible to use this effect to estimate the total tractile force exerted by a cell during chemotaxis, which would be particularly valuable for cells whose tractile forces are below the level of detection with standard techniques of traction-force microscopy.     

Micro-/nanofluidic device for tunable generation of a concentration gradient: application to Caenorhabditis elegans chemotaxis

In this study, we propose a novel micro-/nanofluidic device that can generate a chemical concentration gradient using a parallel nanochannel as gradient generator. This device is easy to fabricate, showing high reproducibility. Its main feature is the multiple-nanochannel-based gradient generator, which permits the diffusion of small molecules and tunably generates concentration gradients. The nanopattern for the nanochannels can be rapidly and easily fabricated by wrinkling a diamond-like carbon thin film which is deposited on a polydimethylsiloxane substrate; the generation of the concentration gradient can be adjusted by controlling the dimensions of the nanochannels. The developed gradient generator is embedded into a microfluidic device to study chemotaxis in the nematode Caenorhabditis elegans, which has a highly developed chemosensory system and can detect a wide variety of chemical molecules. This device shows good performance for rapid analysis of C. elegans chemotaxis under sodium chloride stimuli.

Separation of progressive motile sperm from mouse semen using on-chip chemotaxis

We present a novel method for the separation of progressive motile sperm from non-progressive motile and immotile sperm. This separation was accomplished by inducing chemotaxis along a longitudinal chemical gradient in a microchip composed of a biocompatible polydimethysiloxane layer and a glass substrate. In a preliminary experiment using fluorescent rhodamine B as a marker, we verified that a chemical gradient was generated by diffusion within the microchannel. We used acetylcholine as a chemoattractant to evaluate the chemotactic response of sperm. We tested the response to a 1/2 to 1/64 dilution series of acetylcholine. The results of a mouse sperm chemotaxis assay showed that progressive motile sperm swam predominantly toward the outlet at an optimal chemical gradient of 0.625 (mg/ml)/mm of acetylcholine. This device provides a convenient, disposable, and high-throughput platform that could function as a progressive motile sperm sorter for potential use in intracytoplasmic sperm injection.     

Micellar affinity gradient focusing in a microfluidic chip with integrated bilinear temperature gradients

Micellar affinity gradient focusing (MAGF) is a microfluidic counterflow gradient focusing technique that combines the favorable features of MEKC and temperature gradient focusing. MAGF separates analytes on the basis of a combination of electrophoretic mobility and partitioning with the micellar phase. A temperature gradient is produced along the separation channel containing an analyte/micellar system to create a gradient in interaction strength (retention factor) between the analytes and micelles. Combined with a bulk counterflow, species concentrate at a unique point where their total velocity sums to zero. MAGF can be used in scanning mode by varying the bulk flow so that a large number of analytes can be sequentially focused and passed by a single detection point. In this work, we develop a bilinear temperature gradient along the separation channel that improves separation performance over the conventional linear designs. The temperature profile along the channel consists of a very sharp gradient used to preconcentrate the sample followed by a shallow gradient that increases resolution. We fabricated a hybrid PDMS/glass microfluidic chip with integrated micro heaters that generate the bilinear profile. Performance is characterized by separating several different samples including fluorescent dyes using SDS surfactant and pI markers using both SDS and poly-SUS surfactants as the micellar phase. The new design shows a nearly two times improvement in peak capacity and resolution in comparison to the standard linear temperature gradient.     

Homogeneous reversed-phase agarose thermogels for electrochromatography

A method for the derivatization of agarose by covalent attachment of hydrophobic ligands for reversed-phase (RP) chromatographic separation and ionic groups for generation of electroosmosis under electrochromatographic conditions in the capillaries or microfluidic channels filled with the thermogel of this agarose derivative is described. The product renders a capability of reversible thermogelation. The thermogels formed provide sufficient hydrophobicity and electroosmosis for the separations of the analytes under RP mobile-phase conditions and electric field applied. The gels may be used repeatedly without loss of resolution. They are thermally replaceable and UV transparent (providing possibility in column/in-gel detection), require no covalent attachment to the capillary inner wall (or microchip channel), and are suitable for isocratic or gradient operation in the aqueous-organic mobile phases.     

Generation of concentration gradient by controlled flow distribution and diffusive mixing in a microfluidic chip

We have developed a simple method to generate a concentration gradient in a microfluidic device. This method is based on the combination of controlled fluid distribution at each intersection of a microfluidic network by liquid pressure and subsequent diffusion between laminas in the downstream microchannel. A fluid dynamic model taking into account the diffusion coefficient was established to simulate the on-chip flow distribution and diffusion. Concentration gradients along a distance of a few hundred micrometers were generated in a series of microchannels. The gradients could be varied by carefully regulating the liquid pressure applied to the sample injection vials. The observed concentration gradients of fluorescent dyes generated on the microfluidic channel are consistent with the theoretically predicted results. The microfluidic design described in this study may provide a new tool for applications based on concentration gradients, including many biological and chemical analyses such as cellular reaction monitoring and drug screening.     

A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment

While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g. pipettes), that provides robust soluble factor control within a three-dimensional biological matrix. We demonstrate long-lasting linear and non-linear concentration profiles that were maintained for up to ten days using 34.5 muL solute volume. We also demonstrate the ability to superimpose local soluble factor pulses onto existing gradients via defined dosing windows. The combination of long-term and transient gradient characteristics within a three-dimensional environment opens the door for signaling studies that investigate the migratory behavior of cells within a biologically representative matrix. To this end, we apply temporally evolving and long-lasting gradients to study the chemotactic responses of human neutrophils and the invasion of metastatic rat mammary adenocarcinoma cells (MtLN3) within three-dimensional collagen matrices.     

Gradient generation by an osmotic pump and the behavior of human mesenchymal stem cells under the fetal bovine serum concentration gradient

This paper describes a method to generate a concentration gradient using an osmosis-driven pump, without the need for bulky peripheral devices, such as an electric syringe pump or a pneumatic pump. By the osmosis, the flow in the microfluidic channel can be controlled even to a very slow speed (nanolitre scale), which enables its application to generate the stable and wide (width = 4 mm) concentration gradient profile, even within a short flow path. A computational simulation was also performed to predict the local distribution of the solute concentration and velocity-pressure profile in the microfluidic chip. The performance of the osmosis-driven pump was evaluated by culturing human mesenchymal stem cells within the concentration gradient of fetal bovine serum. The effects of the gradient on attachment, viability and morphology of the cells were analyzed and quantified. The cell density in a higher serum concentration region was twice greater than that in the pure culture media. The compact, cost-effective, self-powered and osmosis-based gradient generation system can be useful for biomedical and chemical applications.     

In vitro 3D collective sprouting angiogenesis under orchestrated ANG-1 and VEGF gradients

Sprouting angiogenesis requires a coordinated guidance from a variety of angiogenic factors. Here, we have developed a unique hydrogel incorporating microfluidic platform which mimics the physiological microenvironment in 3D under a precisely orchestrated gradient of soluble angiogenic factors, VEGF and ANG-1. The system enables the quantified investigation in chemotactic response of endothelial cells during the collective angiogenic sprouting process. While the presence of a VEGF gradient alone was sufficient in inducing a greater number of tip cells, addition of ANG-1 to the VEGF gradient enhanced the number of tip cells that are attached to collectively migrated stalk cells. The chemotactic response of tip cells attracted by the VEGF gradient and the stabilizing role of ANG-1 were morphologically investigated, elucidating the 3D co-operative migration of tip and stalk cells as well as their structures. We found that ANG-1 enhanced the connection of the stalk cells with the tip cells, and then the direct connection regulated the morphogenesis and/or life cycle of stalk cells.