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1.
Microfluidic diagnostic devices often require handling particles or cells with different sizes. In this investigation, a tunable hydrophoretic device was developed which consists of a polydimethylsiloxane (PDMS) slab with hydrophoretic channel, a PDMS diaphragm with pressure channel, and a glass slide. The height of the hydrophoretic channel can be tuned simply and reliably by deforming the elastomeric diaphragm with pressure applied on the pressure channel. This operation allows the device to have a large operating range where different particles and complex biological samples can be processed. The focusing performance of this device was tested using blood cells that varied in shape and size. The hydrophoretic channel had a large cross section which enabled a throughput capability for cell focusing of ∼15 000 cells s−1, which was more than the conventional hydrophoretic focusing and dielectrophoresis (DEP)-active hydrophoretic methods. This tunable hydrophoretic focuser can potentially be integrated into advanced lab-on-a-chip bioanalysis devices.  相似文献   

2.
Current microfluidic techniques for isolating circulating tumor cells (CTCs) from cancer patient blood are limited by low capture purity, and dielectrophoresis (DEP) has the potential to complement existing immunocapture techniques to improve capture performance. We present a hybrid DEP and immunocapture Hele-Shaw flow cell to characterize DEP''s effects on immunocapture of pancreatic cancer cells (Capan-1, PANC-1, and BxPC-3) and peripheral blood mononuclear cells (PBMCs) with an anti-EpCAM (epithelial cell adhesion molecule) antibody. By carefully specifying the applied electric field frequency, we demonstrate that pancreatic cancer cells are attracted to immunocapture surfaces by positive DEP whereas PBMCs are repelled by negative DEP. Using an exponential capture model to interpret our capture data, we show that immunocapture performance is dependent on the applied DEP force sign and magnitude, cell surface EpCAM expression level, and shear stress experienced by cells flowing in the capture device. Our work suggests that DEP can not only repel contaminating blood cells but also enhance capture of cancer cell populations that are less likely to be captured by traditional immunocapture methods. This combination of DEP and immunocapture techniques to potentially increase CTC capture purity can facilitate subsequent biological analyses of captured CTCs and research on cancer metastasis and drug therapies.  相似文献   

3.
The present work demonstrates the use of a dielectrophoretic lab-on-a-chip device in effectively separating different cancer cells of epithelial origin for application in circulating tumor cell (CTC) identification. This study uses dielectrophoresis (DEP) to distinguish and separate MCF-7 human breast cancer cells from HCT-116 colorectal cancer cells. The DEP responses for each cell type were measured against AC electrical frequency changes in solutions of varying conductivities. Increasing the conductivity of the suspension directly correlated with an increasing frequency value for the first cross-over (no DEP force) point in the DEP spectra. Differences in the cross-over frequency for each cell type were leveraged to determine a frequency at which the two types of cell could be separated through DEP forces. Under a particular medium conductivity, different types of cells could have different DEP behaviors in a very narrow AC frequency band, demonstrating a high specificity of DEP. Using a microfluidic DEP sorter with optically transparent electrodes, MCF-7 and HCT-116 cells were successfully separated from each other under a 3.2 MHz frequency in a 0.1X PBS solution. Further experiments were conducted to characterize the separation efficiency (enrichment factor) by changing experimental parameters (AC frequency, voltage, and flow rate). This work has shown the high specificity of the described DEP cell sorter for distinguishing cells with similar characteristics for potential diagnostic applications through CTC enrichment.  相似文献   

4.
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   

5.
Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.  相似文献   

6.
Human mesenchymal stem cells (hMSCs) have three key properties that make them desirable for stem cell therapeutics: differentiation capacity, trophic activity, and ability to self-renew. However, current separation techniques are inefficient, time consuming, expensive, and, in some cases, alter hMSCs cellular function and viability. Dielectrophoresis (DEP) is a technique that uses alternating current electric fields to spatially separate biological cells based on the dielectric properties of their membrane and cytoplasm. This work implements the first steps toward the development of a continuous cell sorting microfluidic device by characterizing native hMSCs dielectric signatures and comparing them to hMSCs morphologically standardized with a polymer. A quadrapole Ti-Au electrode microdevice was used to observe hMSC DEP behaviors, and quantify frequency spectra and cross-over frequency of hMSCs from 0.010–35 MHz in dextrose buffer solutions (0.030 S/m and 0.10 S/m). This combined approach included a systematic parametric study to fit a core-shell model to the DEP spectra over the entire tested frequency range, adding robustness to the analysis technique. The membrane capacitance and permittivity were found to be 2.2 pF and 2.0 in 0.030 S/m and 4.5 pF and 4.1 in 0.10 S/m, respectively. Elastin-like polypeptide (ELP-) polyethyleneimine (PEI) copolymer was used to control hMSCs morphology to spheroidal cells and aggregates. Results demonstrated that ELP-PEI treatment controlled hMSCs morphology, increased experiment reproducibility, and concurrently increased hMSCs membrane permittivity to shift the cross-over frequency above 35 MHz. Therefore, ELP-PEI treatment may serve as a tool for the eventual determination of biosurface marker-dependent DEP signatures and hMSCs purification.  相似文献   

7.
Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O2 plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force (FDEP) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 μl/h and 78.7% at 80 μl/h, for 100 μm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10).  相似文献   

8.
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   

9.
Cell fusion consists of inducing the formation of a hybridoma cell containing the genetic properties of the progenitor cells. Such an operation is usually performed chemically or electrically. The latter method, named electrofusion, is considered as having a strong potential, due to its efficiency and non-toxicity, but deserves further investigations prior to being applicable for key applications like antibody production and cancer immunotherapy. Indeed, to envision such applications, a high amount of hybrid cells is needed. In this context, we present in this paper a device for massive cell pairing and electrofusion, using a microarray of non-connected conductive pads. The electrofusion chamber––or channel––exposes cells to an inhomogeneous electric field, caused by the pads array, enabling the trapping and pairing of cells with dielectrophoresis (DEP) forces prior to electrofusion. Compared to a mechanical trapping, such electric trapping is fully reversible (on/off handling). The DEP force is contactless and thus eases the release of the produced hybridoma. Moreover, the absence of wire connections on the pads permits the high density trapping and electrofusion of cells. In this paper, the electric field mapping, the effect of metallic pads thickness, and the transmembrane potential of cells are studied based on a numerical model to optimize the device. Electric calculations and experiments were conducted to evaluate the trapping force. The structure was finally validated for cell pairing and electrofusion of arrays of cells. We believe that our approach of fully electric trapping with a simple structure is a promising method for massive production of electrofused hybridoma.  相似文献   

10.
A review is presented of the present status of the theory, the developed technology and the current applications of dielectrophoresis (DEP). Over the past 10 years around 2000 publications have addressed these three aspects, and current trends suggest that the theory and technology have matured sufficiently for most effort to now be directed towards applying DEP to unmet needs in such areas as biosensors, cell therapeutics, drug discovery, medical diagnostics, microfluidics, nanoassembly, and particle filtration. The dipole approximation to describe the DEP force acting on a particle subjected to a nonuniform electric field has evolved to include multipole contributions, the perturbing effects arising from interactions with other cells and boundary surfaces, and the influence of electrical double-layer polarizations that must be considered for nanoparticles. Theoretical modelling of the electric field gradients generated by different electrode designs has also reached an advanced state. Advances in the technology include the development of sophisticated electrode designs, along with the introduction of new materials (e.g., silicone polymers, dry film resist) and methods for fabricating the electrodes and microfluidics of DEP devices (photo and electron beam lithography, laser ablation, thin film techniques, CMOS technology). Around three-quarters of the 300 or so scientific publications now being published each year on DEP are directed towards practical applications, and this is matched with an increasing number of patent applications. A summary of the US patents granted since January 2005 is given, along with an outline of the small number of perceived industrial applications (e.g., mineral separation, micropolishing, manipulation and dispensing of fluid droplets, manipulation and assembly of micro components). The technology has also advanced sufficiently for DEP to be used as a tool to manipulate nanoparticles (e.g., carbon nanotubes, nano wires, gold and metal oxide nanoparticles) for the fabrication of devices and sensors. Most efforts are now being directed towards biomedical applications, such as the spatial manipulation and selective separation∕enrichment of target cells or bacteria, high-throughput molecular screening, biosensors, immunoassays, and the artificial engineering of three-dimensional cell constructs. DEP is able to manipulate and sort cells without the need for biochemical labels or other bioengineered tags, and without contact to any surfaces. This opens up potentially important applications of DEP as a tool to address an unmet need in stem cell research and therapy.  相似文献   

11.
This paper presents a field-flow method for separating particle populations in a dielectrophoretic (DEP) chip with asymmetric electrodes under continuous flow. The structure of the DEP device (with one thick electrode that defines the walls of the microfluidic channel and one thin electrode), as well as the fabrication and characterization of the device, was previously described. A characteristic of this structure is that it generates an increased gradient of electric field in the vertical plane that can levitate the particles experiencing negative DEP. The separation method consists of trapping one population to the bottom of the microfluidic channel using positive DEP, while the other population that exhibits negative DEP is levitated and flowed out. Viable and nonviable yeast cells were used for testing of the separation method.  相似文献   

12.
Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.  相似文献   

13.
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   

14.
The dielectric properties of tumour cells are known to differ from normal blood cells, and this difference can be exploited for label-free separation of cells. Conventional measurement techniques are slow and cannot identify rare circulating tumour cells (CTCs) in a realistic timeframe. We use high throughput single cell microfluidic impedance cytometry to measure the dielectric properties of the MCF7 tumour cell line (representative of CTCs), both as pure populations and mixed with whole blood. The data show that the MCF7 cells have a large membrane capacitance and size, enabling clear discrimination from all other leukocytes. Impedance analysis is used to follow changes in cell viability when cells are kept in suspension, a process which can be understood from modelling time-dependent changes in the dielectric properties (predominantly membrane conductivity) of the cells. Impedance cytometry is used to enumerate low numbers of MCF7 cells spiked into whole blood. Chemical lysis is commonly used to remove the abundant erythrocytes, and it is shown that this process does not alter the MCF7 cell count or change their dielectric properties. Combining impedance cytometry with magnetic bead based antibody enrichment enables MCF7 cells to be detected down to 100 MCF7 cells in 1 ml whole blood, a log 3.5 enrichment and a mean recovery of 92%. Microfluidic impedance cytometry could be easily integrated within complex cell separation systems for identification and enumeration of specific cell types, providing a fast in-line single cell characterisation method.  相似文献   

15.
High-throughput size-based rare cell enrichment using microscale vortices   总被引：2，自引：0，他引：2  
Cell isolation in designated regions or from heterogeneous samples is often required for many microfluidic cell-based assays. However, current techniques have either limited throughput or are incapable of viable off-chip collection. We present an innovative approach, allowing high-throughput and label-free cell isolation and enrichment from heterogeneous solution using cell size as a biomarker. The approach utilizes the irreversible migration of particles into microscale vortices, developed in parallel expansion-contraction trapping reservoirs, as the cell isolation mechanism. We empirically determined the critical particle∕cell diameter D(crt) and the operational flow rate above which trapping of cells∕particles in microvortices is initiated. Using this approach we successfully separated larger cancer cells spiked in blood from the smaller blood cells with processing rates as high as 7.5×10(6) cells∕s. Viable long-term culture was established using cells collected off-chip, suggesting that the proposed technique would be useful for clinical and research applications in which in vitro culture is often desired. The presented technology improves on current technology by enriching cells based on size without clogging mechanical filters, employing only a simple single-layered microfluidic device and processing cell solutions at the ml∕min scale.  相似文献   

16.
Blood cell sorting is critical to sample preparation for both clinical diagnosis and therapeutic research. The spiral inertial microfluidic devices can achieve label-free, continuous separation of cell mixtures with high throughput and efficiency. The devices utilize hydrodynamic forces acting on cells within laminar flow, coupled with rotational Dean drag due to curvilinear microchannel geometry. Here, we report on optimized Archimedean spiral devices to achieve cell separation in less than 8 cm of downstream focusing length. These improved devices are small in size (<1 in.2), exhibit high separation efficiency (∼95%), and high throughput with rates up to 1 × 106 cells per minute. These device concepts offer a path towards possible development of a lab-on-chip for point-of-care blood analysis with high efficiency, low cost, and reduced analysis time.  相似文献   

17.
We report the development and results of a two-step method for sorting cells and small particles in a microfluidic device. This approach uses a single microfluidic channel that has (1) a microfabricated sieve which efficiently focuses particles into a thin stream, followed by (2) a dielectrophoresis (DEP) section consisting of electrodes along the channel walls for efficient continuous sorting based on dielectric properties of the particles. For our demonstration, the device was constructed of polydimethylsiloxane, bonded to a glass surface, and conductive agarose gel electrodes. Gold traces were used to make electrical connections to the conductive gel. The device had several novel features that aided performance of the sorting. These included a sieving structure that performed continuous displacement of particles into a single stream within the microfluidic channel (improving the performance of downstream DEP, and avoiding the need for additional focusing flow inlets), and DEP electrodes that were the full height of the microfluidic walls (“vertical electrodes”), allowing for improved formation and control of electric field gradients in the microfluidic device. The device was used to sort polymer particles and HeLa cells, demonstrating that this unique combination provides improved capability for continuous DEP sorting of particles in a microfluidic device.  相似文献   

18.
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19.
Chen H  Li J  Zhang H  Li M  Rosengarten G  Nordon RE 《Biomicrofluidics》2011,5(4):44117-4411713
Continuous cell tracking by time-lapse microscopy has led to detailed study of cell differentiation pathways using single cell fate maps. There are a multitude of cell fate outcomes, so hundreds of clonal division histories are required to measure these stochastic branching processes. This study examines the principle of condensing cell imaging information into a relatively small region to maximize live cell imaging throughput. High throughput clonal analysis of non-adherent cells by continuous live cell tracking was possible using a microwell perfusion array with an internal volume of 16 μl and 600 microwells at the base. This study includes examination of biocompatibility of buffer systems, connecting tubing, cell culture substrates, and media degradation. An intermittent perfusion protocol was selected for long-term time-lapse imaging of KG1a cells in the microwell array; 1500 clones were simultaneously cultured and scanned every 3 min at 100 × magnifications for 6 days. The advantages of perfusion microwell culture are continuous long-term cell tracking, higher cell imaging throughput, and greater control over cell microenvironment. Microwell devices facilitate high throughput analysis of cell lineage development and measurement of the probability distribution for cell life events such as mitosis.  相似文献   

20.
Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.  相似文献   

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