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1.
Cellular transplantation is a promising technology with great clinical potential in regenerative medicine and disease management. However, effective control over patient immunological response is essential. The encapsulation of cells within hydrogel microspheres is an increasingly prevalent method for the protection of cellular grafts from immune rejection. Microfluidic “chip” reactors present elegant solutions to several capsule generation issues, including the requirement for intercapsule uniformity, high reproducibility, and sterile, good manufacturing practice compliance. This study presents a novel method for the on-chip production of stable, highly monodisperse alginate microspheres and demonstrates its utility in the encapsulation of an immortalized human-derived cell line. Four populations of immortalized human embryonic kidney cells (HEK293) were encapsulated on chip within monodisperse alginate capsules. Cell viability measurements were recorded for each of the four encapsulated populations for 90 days.  相似文献   

2.
Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.  相似文献   

3.
In this work, we demonstrate a robust and reliable approach to fabricate multi-compartment particles for cell co-culture studies. By taking advantage of the laminar flow within our microfluidic nozzle, multiple parallel streams of liquids flow towards the nozzle without significant mixing. Afterwards, the multiple parallel streams merge into a single stream, which is sprayed into air, forming monodisperse droplets under an electric field with a high field strength. The resultant multi-compartment droplets are subsequently cross-linked in a calcium chloride solution to form calcium alginate micro-particles with multiple compartments. Each compartment of the particles can be used for encapsulating different types of cells or biological cell factors. These hydrogel particles with cross-linked alginate chains show similarity in the physical and mechanical environment as the extracellular matrix of biological cells. Thus, the multi-compartment particles provide a promising platform for cell studies and co-culture of different cells. In our study, cells are encapsulated in the multi-compartment particles and the viability of cells is quantified using a fluorescence microscope after the cells are stained for a live/dead assay. The high cell viability after encapsulation indicates the cytocompatibility and feasibility of our technique. Our multi-compartment particles have great potential as a platform for studying cell-cell interactions as well as interactions of cells with extracellular factors.  相似文献   

4.
Cell encapsulation technology is a promising strategy applicable to tissue engineering and cell therapy. Many advanced microencapsulation chips that function via multiple syringe pumps have been developed to generate mono-disperse hydrogel beads encapsulating cells. However, their operation is difficult and only trained microfluidic engineers can use them with dexterity. Hence, we propose a microfluidic manifold system, driven by a single syringe pump, which can enable the setup of automated flow sequences and generate highly mono-disperse alginate beads by minimizing disturbances to the pump pressure. The encapsulation of P19 mouse embryonic carcinoma cells and embryonic body formation are demonstrated to prove the efficiency of the proposed system.  相似文献   

5.
Recent years have witnessed a strong trend towards analysis of single-cells. To access and handle single-cells, many new tools are needed and have partly been developed. Here, we present an improved version of a single-cell printer which is able to deliver individual single cells and beads encapsulated in free-flying picoliter droplets at a single-bead efficiency of 96% and with a throughput of more than 10 beads per minute. By integration of acoustophoretic focusing, the cells could be focused in x and y direction. This way, the cells were lined-up in front of a 40 μm nozzle, where they were analyzed individually by an optical system prior to printing. In agreement with acoustic simulations, the focusing of 10 μm beads and Raji cells has been achieved with an efficiency of 99% (beads) and 86% (Raji cells) to a 40 μm wide center region in the 1 mm wide microfluidic channel. This enabled improved optical analysis and reduced bead losses. The loss of beads that ended up in the waste (because printing them as single beads arrangements could not be ensured) was reduced from 52% ± 6% to 28% ± 1%. The piezoelectric transducer employed for cell focusing could be positioned on an outer part of the device, which proves the acoustophoretic focusing to be versatile and adaptable.  相似文献   

6.
Label-free isolation of single cells is essential for the growing field of single-cell analysis. Here, we present a device which prints single living cells encapsulated in free-flying picoliter droplets. It combines inkjet printing and impedance flow cytometry. Droplet volume can be controlled in the range of 500 pl–800 pl by piezo actuator displacement. Two sets of parallel facing electrodes in a 50 μm × 55 μm channel are applied to measure the presence and velocity of a single cell in real-time. Polystyrene beads with <5% variation in diameter generated signal variations of 12%–17% coefficients of variation. Single bead efficiency (i.e., printing events with single beads vs. total number of printing events) was 73% ± 11% at a throughput of approximately 9 events/min. Viability of printed HeLa cells and human primary fibroblasts was demonstrated by culturing cells for at least eight days.  相似文献   

7.
In this study, a microfluidic process is proposed for preparing monodisperse micrometer-sized hydrogel beads. This process utilizes non-equilibrium aqueous droplets formed in a polar organic solvent. The water-in-oil droplets of the hydrogel precursor rapidly shrunk owing to the dissolution of water molecules into the continuous phase. The shrunken and condensed droplets were then gelled, resulting in the formation of hydrogel microbeads with sizes significantly smaller than the initial droplet size. This study employed methyl acetate as the polar organic solvent, which can dissolve water at 8%. Two types of monodisperse hydrogel beads—Ca-alginate and chitosan—with sizes of 6–10 μm (coefficient of variation < 6%) were successfully produced. In addition, we obtained hydrogel beads with non-spherical morphologies by controlling the degree of droplet shrinkage at the time of gelation and by adjusting the concentration of the gelation agent. Furthermore, the encapsulation and concentration of DNA molecules within the hydrogel beads were demonstrated. The process presented in this study has great potential to produce small and highly concentrated hydrogel beads that are difficult to obtain by using conventional microfluidic processes.  相似文献   

8.
In this paper, we demonstrate for the first time the technique to using microfluidics to fabricate tissue engineering scaffolds with uniform pore sizes. We investigate both the bubble generation of the microfluidic device and the application of foam as a tissue engineering scaffold. Our microfluidic device consists of two concentric tapered channels, which are made by micropipettes. Nitrogen gas and aqueous alginate solution with Pluronic® F127 surfactant are pumped through the inner and the outer channels, respectively. We observe rich dynamic patterns of bubbles encapsulated in the liquid droplets. The size of the bubble depends linearly on the gas pressure and inversely on the liquid flow rate. In addition, monodisperse bubbles self-assemble into crystalline structures. The liquid crystalline foams are further processed into open-cell solid foams. The novel foam gel was used as a scaffold to culture chondrocytes.  相似文献   

9.
The successful encapsulation of human hepatocellular carcinoma (HepG2) cells would greatly assist a broad range of applications in tissue engineering. Due to the harsh conditions during standard chitosan fiber fabrication processes, encapsulation of HepG2 cells in chitosan fibers has been challenging. Here, we describe the successful wet-spinning of chitosan-alginate fibers using a coaxial flow microfluidic chip. We determined the optimal mixing conditions for generating chitosan-alginate fibers, including a 1:5 ratio of 2% (w∕w) water-soluble chitosan (WSC) solution to 2% (w∕w) alginate solution. Ratio including higher than 2% (w∕w) WSC solution increased aggregation throughout the mixture. By suspending cells in the WSC-alginate solution, we successfully fabricated HepG2 cell-laden fibers. The encapsulated HepG2 cells in the chitosan-alginate fibers were more viable than cells encapsulated in pure alginate fibers, suggesting that cross-linked chitosan provides a better environment for HepG2 cells than alginate alone. In addition, we found that the adhesion of HepG2 cells on the chitosan-alginate fiber is much better than that on the alginate fibers.  相似文献   

10.
Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels.  相似文献   

11.
This paper presents the design, fabrication, and testing of a magnetophoretic bioseparation chip for the rapid isolation and concentration of CD4 + T cells from the peripheral blood. In a departure from conventional magnetic separation techniques, this microfluidic-based bioseperation device has several unique features, including locally engineered magnetic field gradients and a continuous flow with a buffer switching scheme to improve the performance of the separation process. Additionally, the chip is capable of processing significantly smaller sample volumes than conventional methods and sample losses are eliminated due to decreased handling. Furthermore, the possibility of sample-to-sample contamination is reduced with the disposable format. The overall dimensions of the device were 22 mm by 60 mm by 1 mm, approximately the size of a standard microscope slide. The results indicate a cell purity of greater than 95% at a sample flow rate of 50 ml/h and a cell recovery of 81% at a sample flow rate of 10 ml/h. The cell purity was found to increase with increasing the sample flow rate. However, the cell recovery decreases with an increase in the flow rate. A parametric study was also performed to investigate the effects of channel height, substrate thickness, magnetic bead size, and number of beads per cell on the cell separation performance.  相似文献   

12.
Herein, we present a large-area 3D hemispherical perforated microwell structure for a bead based bioassay. Such a unique microstructure enables us to perform the rapid and stable localization of the beads at the single bead level and the facile manipulation of the bead capture and retrieval with high speed and efficiency. The fabrication process mainly consisted of three steps: the convex micropatterned nickel (Ni) mold production from the concave micropatterned silicon (Si) wafer, hot embossing on the polymer matrix to generate the concave micropattened acrylate sheet, and reactive ion etching to make the bottom holes. The large-area hemispherical perforated micropatterned acrylate sheet was sandwiched between two polydimethylsiloxane (PDMS) microchannel layers. The bead solution was injected and recovered in the top PDMS microchannel, while the bottom PDMS microchannel was connected with control lines to exert the hydrodynamic force in order to alter the flow direction of the bead solution for the bead capture and release operation. The streptavidin-coated microbead capture was achieved with almost 100% yield within 1 min, and all the beads were retrieved in 10 s. Lysozyme or thrombin binding aptamer labelled microbeads were trapped on the proposed bead microarray, and the in situ fluorescence signal of the bead array was monitored after aptamer-target protein interaction. The protein-aptamer conjugated microbeads were recovered, and the aptamer was isolated for matrix assisted laser desorption/ionization time-of-flight mass spectrometry analysis to confirm the identity of the aptamer.  相似文献   

13.
Manipulation of magnetic beads plays an increasingly important role in molecular diagnostics. Magnetophoresis is a promising technique for selective transportation of magnetic beads in lab-on-a-chip systems. We investigate periodic arrays of exchange-biased permalloy microstripes fabricated using a single lithography step. Magnetic beads can be continuously moved across such arrays by combining the spatially periodic magnetic field from microstripes with a rotating external magnetic field. By measuring and modeling the magnetophoresis properties of thirteen different stripe designs, we study the effect of the stripe geometry on the magnetophoretic transport properties of the magnetic microbeads between the stripes. We show that a symmetric geometry with equal width of and spacing between the microstripes facilitates faster transportation and that the optimal period of the periodic stripe array is approximately three times the height of the bead center over the microstripes.  相似文献   

14.
We present a method capable of rapidly (∼20 s) determining the density and mass of a single leukemic cell using an optically induced electrokinetics (OEK) platform. Our team had reported recently on a technique that combines sedimentation theory, computer vision, and micro particle manipulation techniques on an OEK microfluidic platform to determine the mass and density of micron-scale entities in a fluidic medium; the mass and density of yeast cells were accurately determined in that prior work. In the work reported in this paper, we further refined the technique by performing significantly more experiments to determine a universal correction factor to Stokes'' equation in expressing the drag force on a microparticle as it falls towards an infinite plane. Specifically, a theoretical model for micron-sized spheres settling towards an infinite plane in a microfluidic environment is presented, and which was validated experimentally using five different sizes of micro polystyrene beads. The same sedimentation process was applied to two kinds of leukemic cancer cells with similar sizes in an OEK platform, and their density and mass were determined accordingly. Our tests on mouse lymphocytic leukemia cells (L1210) and human leukemic cells (HL-60) have verified the practical viability of this method. Potentially, this new method provides a new way of measuring the volume, density, and mass of a single cell in an accurate, selective, and repeatable manner.  相似文献   

15.
Detection of individual target cells among a large amount of blood cells is a major challenge in clinical diagnosis and laboratory protocols. Many researches show that two dimensional cells array technology can be incorporated into routine laboratory procedures for continuously and quantitatively measuring the dynamic behaviours of large number of living cells in parallel, while allowing other manipulations such as staining, rinsing, and even retrieval of targeted cells. In this study, we present a high-density cell self-assembly technology capable of quickly spreading over 300 000 cells to form a dense mono- to triple-layer cell arrangement in 5 min with minimal stacking of cells by the gentle incorporation of gravity and peripheral micro flow. With this self-assembled cell arrangement (SACA) chip technology, common fluorescent microscopy and immunofluorescence can be utilized for detecting and analyzing target cells after immuno-staining. Validated by experiments with real human peripheral blood samples, the SACA chip is suitable for detecting rare cells in blood samples with a ratio lower than 1/100 000. The identified cells can be isolated and further cultured in-situ on a chip for follow-on research and analysis. Furthermore, this technology does not require external mechanical devices, such as pump and valves, which simplifies operation and reduces system complexity and cost. The SACA chip offers a high-efficient, economical, yet simple scheme for identification and analysis of rare cells. Therefore, potentially SACA chip may provide a feasible and economical platform for rare cell detection in the clinic.  相似文献   

16.
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   

17.
We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-μl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, “amplicon-in-answer-out” operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis.  相似文献   

18.
We present a 91 MHz surface acoustic wave resonator with integrated microfluidics that includes a flow focus, an expansion region, and a binning region in order to manipulate particle trajectories. We demonstrate the ability to change the position of the acoustic nodes by varying the electronic phase of one of the transducers relative to the other in a pseudo-static manner. The measurements were performed at room temperature with 3 μm diameter latex beads dispersed in a water-based solution. We demonstrate the dependence of nodal position on pseudo-static phase and show simultaneous control of 9 bead streams with spatial control of −0.058 μm/deg ± 0.001 μm/deg. As a consequence of changing the position of bead streams perpendicular to their flow direction, we also show that the integrated acoustic-microfluidic device can be used to change the trajectory of a bead stream towards a selected bin with an angular control of 0.008 deg/deg ± 0.000(2) deg/deg.  相似文献   

19.
Herein, we demonstrate a novel method for the generation of monodisperse cell-like structures containing a biocompatible hydrogel matrix surrounded by a membrane responsive to chemical cues. Specifically, we employ droplet-based microfluidics to generate PEG-PLA polymersomes encapsulating alginate in liquid form. We investigate alginate core gelation by creating an osmotic pressure gradient across the polymeric membrane that, through expansion, allows the passage of calcium ions. The effects of calcium concentration on the core gelation are explored.  相似文献   

20.
Droplet microfluidics is a powerful method used to characterize chemical reactions at high throughput. Often detection is performed via in-line optical readout, which puts high demands on the detection system or makes detection of low concentration substrates challenging. Here, we have developed a droplet acoustofluidic chip for time-controlled reactions that can be combined with off-line optical readout. The principle of the platform is demonstrated by the enzymatic conversion of fluorescein diphosphate to fluorescein by alkaline phosphatase. The novelty of this work is that the time of the enzymatic reaction is controlled by physically removing the enzymes from the droplets instead of using chemical inhibitors. This is advantageous as inhibitors could potentially interact with the readout. Droplets containing substrate were generated on the chip, and enzyme-coupled microbeads were added into the droplets via pico-injection. The reaction starts as soon as the enzyme/bead complexes are added, and the reaction is stopped when the microbeads are removed from the droplets at a channel bifurcation. The encapsulated microbeads were focused in the droplets by acoustophoresis during the split, leaving the product in the side daughter droplet to be collected for the analysis (without beads). The time of the reaction was controlled by using different outlets, positioned at different lengths from the pico-injector. The enzymatic conversion could be measured with fluorescence readout in a separate PDMS based assay chip. We show the ability to perform time-controlled enzymatic assays in droplet microfluidics coupled to an off-line optical readout, without the need of enzyme inhibitors.  相似文献   

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