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1.
Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.  相似文献   

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

3.
Microfluidic devices have emerged as important tools for experimental physiology. They allow to study the effects of hydrodynamic flow on physiological and pathophysiological processes, e.g., in the circulatory system of the body. Such dynamic in vitro test systems are essential in order to address fundamental problems in drug delivery and targeted imaging, such as the binding of particles to cells under flow. In the present work an acoustically driven microfluidic platform is presented in which four miniature flow channels can be operated in parallel at distinct flow velocities with only slight inter-experimental variations. The device can accommodate various channel architectures and is fully compatible with cell culture as well as microscopy. Moreover, the flow channels can be readily separated from the surface acoustic wave pumps and subsequently channel-associated luminescence, absorbance, and/or fluorescence can be determined with a standard microplate reader. In order to create artificial blood vessels, different coatings were evaluated for the cultivation of endothelial cells in the microchannels. It was found that 0.01% fibronectin is the most suitable coating for growth of endothelial monolayers. Finally, the microfluidic system was used to study the binding of 1 μm polystyrene microspheres to three different types of endothelial cell monolayers (HUVEC, HUVECtert, HMEC-1) at different average shear rates. It demonstrated that average shear rates between 0.5 s−1 and 2.25 s−1 exert no significant effect on cytoadhesion of particles to all three types of endothelial monolayers. In conclusion, the multichannel microfluidic platform is a promising device to study the impact of hydrodynamic forces on cell physiology and binding of drug carriers to endothelium.  相似文献   

4.
Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.  相似文献   

5.
Fluid shear stress (FSS) plays a critical role in regulating endothelium function and maintaining vascular homeostasis. Current microfluidic devices for studying FSS effects on cells either separate high shear stress zone and low shear stress zone into different culturing chambers, or arranging the zones serially along the flow direction, which complicates subsequent data interpretation. In this paper, we report a diamond shaped microfluidic shear device where the high shear stress zone and the low shear stress zone are arranged in parallel within one culturing chamber. Since the zones with different shear stress magnitudes are aligned normal to the flow direction, the cells in one stress group are not substantially affected by the flow-induced cytokine/chemokine releases by cells in the other group. Cell loading experiments using human umbilical vein endothelial cells show that the device is able to reveal stress magnitude-dependent and loading duration-dependent cell responses. The co-existence of shear stress zones with varied magnitudes within the same culturing chamber not only ensures that all the cells are subject to the identical culturing conditions, but also allows the resemblance of the differential shear stress pattern in natural arterial conditions. The device is expected to provide a new solution for studying the effects of heterogeneous hemodynamic patterns in the onset and progression of various vascular diseases.  相似文献   

6.
Adler M  Groisman A 《Biomicrofluidics》2012,6(2):24109-2410916
Mixing of liquids to produce solutions with different concentrations is one of the basic functionalities of microfluidic devices. Generation of specific temporal patterns of concentration in microfluidic devices is an important technique to study responses of cells and model organisms to variations in the chemical composition of their environment. Here, we present a simple microfluidic network that linearly converts pressure at an inlet into concentration of a soluble reagent in an observation region and also enables independent concurrent linear control of concentrations of two reagents. The microfluidic device has an integrated mixer channel with chaotic three-dimensional flow that facilitates rapid switching of concentrations in a continuous range. A simple pneumatic setup generating linear ramps of pressure is used to produce smooth linear ramps and triangular waves of concentration with different slopes. The use of chaotic vs. laminar mixers is discussed in the context of microfluidic devices providing rapid switching and generating temporal waves of concentration.  相似文献   

7.
Control of the 3D microenvironment for cultured cells is essential for understanding the complex relationships that biomolecular concentration gradients have on cellular growth, regeneration, and differentiation. This paper reports a microfluidic device for delivering gradients of soluble molecules to cells in an open reservoir without exposing the cells to flow. The cells are cultured on a polyester membrane that shields them from the flow that delivers the gradient. A novel "lid" design is implemented which prevents leakage from around the membrane without requiring sealing agents or adhesives. Once layers are molded, device fabrication can be performed within minutes while at room temperature. Surface gradients were characterized with epifluorescence microscopy; image analysis verified that sharp gradients (～33 μm wide) can be reproducibly generated. We show that heterogeneous laminar flow patterns of Orange and Green Cell Tracker (CT) applied beneath the membrane can be localized to cells cultured on the other side; concentration profile scans show the extent of CT diffusion parallel to the membrane's surface to be 10-20 μm. Our device is ideal for conventional cell culture because the cell culture surface is readily accessible to physical manipulation (e.g., micropipette access), the cell culture medium is in direct contact with the incubator atmosphere (i.e., no special protocols for ensuring proper equilibration of gas concentrations are required), and the cells are not subjected to flow-induced shear forces, which are advantageous attributes not commonly found in closed-channel microfluidic designs.  相似文献   

8.
In this article, we present a microfluidic platform for passive fluid pumping for pump-free perfusion cell culture, cell-based assay, and chemical applications. By adapting the passive membrane-controlled pumping principle from the previously developed perfusion microplate, which utilizes a combination of hydrostatic pressure generated by different liquid levels in the wells and fluid wicking through narrow strips of a porous membrane connecting the wells to generate fluid flow, a series of pump-free membrane-controlled perfusion microfluidic devices was developed and their use for pump-free perfusion cell culture and cell-based assays was demonstrated. Each pump-free membrane-controlled perfusion microfluidic device comprises at least three basic components: an open well for generating fluid flow, a micron-sized deep chamber/channel for cell culture or for fluid connection, and a wettable porous membrane for controlling the fluid flow. Each component is fluidically connected either by the porous membrane or by the micron-sized deep chamber/channel. By adapting and incorporating the passive membrane-controlled pumping principle into microfluidic devices, all the benefits of microfluidic technologies, such as small sample volumes, fast and efficient fluid exchanges, and fluid properties at the micro-scale, can be fully taken advantage of with this pump-free membrane-controlled perfusion microfluidic platform.  相似文献   

9.
Biomicrofluidics is an emerging field at the cross roads of microfluidics and life sciences which requires intensive research efforts in terms of introducing appropriate designs, production techniques, and analysis. The ultimate goal is to deliver innovative and cost-effective microfluidic devices to biotech, biomedical, and pharmaceutical industries. Therefore, creating an in-depth understanding of the transport phenomena of cells and biomolecules becomes vital and concurrently poses significant challenges. The present article outlines the recent advancements in diffusion phenomena of cells and biomolecules by highlighting transport principles from an engineering perspective, cell responses in microfluidic devices with emphases on diffusion- and flow-based microfluidic gradient platforms, macroscopic and microscopic approaches for investigating the diffusion phenomena of biomolecules, microfluidic platforms for the delivery of these molecules, as well as the state of the art in biological applications of mammalian cell responses and diffusion of biomolecules.  相似文献   

10.
The application of microfluidic technologies to stem cell research is of great interest to biologists and bioengineers. This is chiefly due to the intricate ability to control the cellular environment, the reduction of reagent volume, experimentation time and cost, and the high-throughput screening capabilities of microscale devices. Despite this importance, a simple-to-use microfluidic platform for studying the effects of growth factors on stem cell differentiation has not yet emerged. With this consideration, we have designed and characterized a microfluidic device that is easy to fabricate and operate, yet contains several functional elements. Our device is a simple polyester-based microfluidic chip capable of simultaneously screening multiple independent stem cell culture conditions. Generated by laser ablation and stacking of multiple layers of polyester film, this device integrates a 10 × 10 microwell array for cell culture with a continuous perfusion system and a non-linear concentration gradient generator. We performed numerical calculations to predict the gradient formation and calculate the shear stress acting on the cells inside the device. The device operation was validated by culturing murine embryonic stem cells inside the microwells for 5 days. Furthermore, we showed the ability to maintain the pluripotency of stem cell aggregates in response to concentrations of leukemia inhibitory factor ranging from 0 to ∼1000 U/ml. Given its simplicity, fast manufacturing method, scalability, and the cell-compatible nature of the device, it may be a useful platform for long-term stem cell culture and studies.  相似文献   

11.
In this article, we present a simple, rapid prototyped polystyrene-based microfluidic device with three-dimensional (3D) interconnected microporous walls for long term perfusion cell culture. Patterned 3D interconnected microporous structures were created by a chemical treatment together with a protective mask and the native hydrophobic nature of the microporous structures were selectively made hydrophilic using oxygen plasma treatment together with a protective mask. Using this polystyrene-based cell culture microfluidic device, we successfully demonstrated the support of four days perfusion cell culture of hepatocytes (C3A cells).  相似文献   

12.
We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data.  相似文献   

13.
Microfluidic technology has tremendously facilitated the development of in vitro cell cultures and studies. Conventionally, microfluidic devices are fabricated with extensive facilities by well-trained researchers, which hinder the widespread adoption of the technology for broader applications. Enlightened by the fact that low-cost microbore tubing is a natural microfluidic channel, we developed a series of adaptors in a toolkit that can twine, connect, organize, and configure the tubing to produce functional microfluidic units. Three subsets of the toolkit were thoroughly developed: the tubing and scoring tools, the flow adaptors, and the 3D cell culture suite. To demonstrate the usefulness and versatility of the toolkit, we assembled a microfluidic device and successfully applied it for 3D macrophage cultures, flow-based stimulation, and automated near real-time quantitation with new knowledge generated. Overall, we present a new technology that allows simple, fast, and robust assembly of customizable and scalable microfluidic devices with minimal facilities, which is broadly applicable to research that needs or could be enhanced by microfluidics.  相似文献   

14.
This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes.  相似文献   

15.
Spatially and temporally resolved delivery of soluble factors is a key feature for pharmacological applications. In this framework, microfluidics coupled to multisite electrophysiology offers great advantages in neuropharmacology and toxicology. In this work, a microfluidic device for biochemical stimulation of neuronal networks was developed. A micro-chamber for cell culturing, previously developed and tested for long term neuronal growth by our group, was provided with a thin wall, which partially divided the cell culture region in two sub-compartments. The device was reversibly coupled to a flat micro electrode array and used to culture primary neurons in the same microenvironment. We demonstrated that the two fluidically connected compartments were able to originate two parallel neuronal networks with similar electrophysiological activity but functionally independent. Furthermore, the device allowed to connect the outlet port to a syringe pump and to transform the static culture chamber in a perfused one. At 14 days invitro, sub-networks were independently stimulated with a test molecule, tetrodotoxin, a neurotoxin known to block action potentials, by means of continuous delivery. Electrical activity recordings proved the ability of the device configuration to selectively stimulate each neuronal network individually. The proposed microfluidic approach represents an innovative methodology to perform biological, pharmacological, and electrophysiological experiments on neuronal networks. Indeed, it allows for controlled delivery of substances to cells, and it overcomes the limitations due to standard drug stimulation techniques. Finally, the twin network configuration reduces biological variability, which has important outcomes on pharmacological and drug screening.  相似文献   

16.
There is great interest in highly sensitive separation methods capable of quickly isolating a particular cell type within a single manipulation step prior to their analysis. We present a cell sorting device based on the opposition of dielectrophoretic forces that discriminates between cell types according to their dielectric properties, such as the membrane permittivity and the cytoplasm conductivity. The forces are generated by an array of electrodes located in both sidewalls of a main flow channel. Cells with different dielectric responses perceive different force magnitudes and are, therefore, continuously focused to different equilibrium positions in the flow channel, thus avoiding the need of a specific cell labeling as discriminating factor. We relate the cells’ dielectric response to their output position in the downstream channel. Using this microfluidic platform that integrates a method of continuous-flow cell separation based on multiple frequency dielectrophoresis, we succeeded in sorting viable from nonviable yeast with nearly 100% purity. The method also allowed to increase the infection rate of a cell culture up to 50% of parasitemia percentage, which facilitates the study of the parasite cycle. Finally, we prove the versatility of our device by synchronizing a yeast cell culture at a particular phase of the cell cycle avoiding the use of metabolic agents interfering with the cells’ physiology.  相似文献   

17.
We have investigated the bonding stability of various silane treatments for the integration of track-etched membranes with poly(dimethylsiloxane) (PDMS) microfluidic devices. We compare various treatments using trialkoxysilanes or dipodal silanes to determine the effect of the organofunctional group, cross-link density, reaction solvent, and catalyst on the bond stability. We find that devices made using existing silane methods delaminated after one day when immersed in cell culture medium at 37 °C. In contrast, the dipodal silane, bis[3-(trimethoxysilyl)propyl]amine, is shown to yield stable and functional integration of membranes with PDMS that is suitable for long-term cell culture. To demonstrate application of the technique, we fabricated an open-surface device in which cells cultured on a track-etched membrane can be stimulated at their basal side via embedded microfluidic channels. C2C12 mouse myoblasts were differentiated into myotubes over the course of two weeks on these devices to demonstrate biocompatibility. Finally, devices were imaged during the basal-side delivery of a fluorescent stain to validate the membrane operation and long-term stability of the bonding technique.  相似文献   

18.
Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell–cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.  相似文献   

19.
We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells.  相似文献   

20.
We present a low cost microfluidic chip integrating 3D micro-chambers for the capture and the analysis of cells. This device has a simple design and a small footprint. It allows the implementation of standard biological protocols in a chip format with low volume consumption. The manufacturing process relies on hot-embossing of cyclo olefin copolymer, allowing the development of a low cost and robust device. A 3D design of microchannels was used to induce high flow velocity contrasts in the device and provide a selective immobilization. In narrow distribution channels, the liquid velocity induces a shear stress that overcomes adhesion forces and prevents cell immobilization or clogging. In large 3D chambers, the liquid velocity drops down below the threshold for cell attachment. The devices can be operated in a large range of input pressures and can even be handled manually using simple syringe or micropipette. Even at high flow injection rates, the 3D structures protect the captured cell from shear stress. To validate the performances of our device, we implemented immuno-fluorescence labeling and Fluorescence in Situ Hybridization (FISH) analysis on cancer cell lines and on a patient pleural effusion sample. FISH is a Food and Drug Administration approved cancer diagnostic technique that provides quantitative information about gene and chromosome aberration at the single cell level. It is usually considered as a long and fastidious test in medical diagnosis. This process can be easily implanted in our platform, and high resolution fluorescence imaging can be performed with reduced time and computer intensiveness. These results demonstrate the potential of this chip as a low cost, robust, and versatile tool adapted to complex and demanding protocols for medical diagnosis.  相似文献   

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