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1.
Embryonic stem cells (ESCs) are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body (EB) formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method. 相似文献
2.
This paper reports a two-layered polydimethylsiloxane microfluidic device—Flip channel, capable of forming uniform-sized embryoid bodies (EBs) and performing stem cell differentiation within the same device after flipping the microfluidic channel. The size of EBs can be well controlled by designing the device geometries, and EBs with multiple sizes can be formed within a single device to study EB size-dependent stem cell differentiation. During operation of the device, cells are positioned in the designed positions. As a result, observation and monitoring specific population of cells can be achieved for further analysis. In addition, after flipping the microfluidic channel, stem cell differentiation from the EBs can be performed on an unconfined flat surface that is desired for various differentiation processes. In the experiments, murine embryonic stem cells (ES-D3) are cultured and formed EBs inside the developed device. The size of EBs is well controlled inside the device, and the neural differentiation is performed on the formed EBs after flipping the channel. The EB size-dependent stem cell differentiation is studied using the device to demonstrate its functions. The device provides a useful tool to study stem cell differentiation without complicated device fabrication and tedious cell handling under better-controlled microenvironments. 相似文献
3.
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
4.
Here, we report a novel method for the fabrication of polydimethylsiloxane microdevices with complicated 3-D structures, such as concave and crater shapes, using an easily machined polymethyl methacrylate mold combined with a one-step molding process. The procedure presented here enables rapid preparation of complex 3-D microstructures varying in shape and dimensions. To regulate embryoid body (EB) formation, we fabricated a microfluidic device with an array of concave microwells and found that EBs growing in microwells maintained their shape, viability, and a high degree of homogeneity. We believe that this novel method provides an alternative for rapid prototyping, especially in fabricating devices with curved 3-D microstructures. 相似文献
5.
A microfluidic device with planar square electrodes is developed for capturing particles from high conductivity media using negative dielectrophoresis (n-DEP). Specifically, Bacillus subtilis and Clostridium sporogenes spores, and polystyrene particles are tested in NaCl solution (0.05 and 0.225 S∕m), apple juice (0.225 S∕m), and milk (0.525 S∕m). Depending on the conductivity of the medium, the Joule heating produces electrothermal flow (ETF), which continuously circulates and transports the particles to the DEP capture sites. Combination of the ETF and n-DEP results in different particle capture efficiencies as a function of the conductivity. Utilizing 20 μm height DEP chambers, “almost complete” and rapid particle capture from lower conductivity (0.05 S∕m) medium is observed. Using DEP chambers above 150 μm in height, the onset of a global fluid motion for high conductivity media is observed. This motion enhances particle capture on the electrodes at the center of the DEP chamber. The n-DEP electrodes are designed to have well defined electric field minima, enabling sample concentration at 1000 distinct locations within the chip. The electrode design also facilitates integration of immunoassay and other surface sensors onto the particle capture sites for rapid detection of target micro-organisms in the future. 相似文献
6.
The recent development of microfluidic “lab on a chip” devices requires the need to continuously separate submicron particles. Here, we present a PDMS microfluidic device with sidewall conducting PDMS (AgPDMS) composite electrodes capable of separating submicron particles in hydrodynamic flow. In particular, the device can service dual functions. First, the AgPDMS composite electrodes embedded in a sidewall of the device channel allow for performing AC-dielectrophoretic (DEP) characterization through direct microscopic observation of particle behavior. Characterization experiments are carried out for numerous parameters including particle size, medium conductivity, and AC field frequency to reveal important dielectrophoresis DEP information in terms of the crossover frequency and positive/negative DEP behavior under specific frequencies. Second, the device offers an advantage that sidewall AgPDMS composite electrodes can produce strong DEP effects throughout the entire channel height, and thus the robustness of the on-chip particle separation is demonstrated for continuous separation in a flowing mixture of 0.5 and 5 μm particles with 100% separation efficiency. 相似文献
7.
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
8.
Vickers DA Kulik M Hincapie M Hancock WS Dalton S Murthy SK 《Biomicrofluidics》2012,6(2):24122-2412210
Embryonic stem (ES) cells are capable of proliferating and differentiating to form cells of the three embryonic germ layers, namely, endoderm, mesoderm, and ectoderm. The utilization of human ES cell derivatives requires the ability to direct differentiation to specific lineages in defined, efficient, and scalable systems. Better markers are needed to identify early differentiation. Lectins have been reported as an attractive alternative to the common stem cell markers. They have been used to identify, characterize, and isolate various cell subpopulations on the basis of the presentation of specific carbohydrate groups on the cell surface. This article demonstrates how simple adhesion assays in lectin-coated microfluidic channels can provide key information on the interaction of lectins with ES and definitive endoderm cells and thereby track early differentiation. The microfluidic approach incorporates both binding strength and cell surface receptor density, whereas traditional flow cytometry only incorporates the latter. Both approaches are examined and shown to be complementary with the microfluidic approach providing more biologically relevant information. 相似文献
9.
This paper presents a mathematical model for laser-induced rapid electro-kinetic patterning (REP) to elucidate the mechanism for concentrating particles in a microchannel non-destructively and non-invasively. COMSOL®(v4.2a) multiphysics software was used to examine the effect of a variety of parameters on the focusing performance of the REP. A mathematical model of the REP was developed based on the AC electrothermal flow (ACET) equations, the dielectrophoresis (DEP) equation, the energy balance equation, the Navier-Stokes equation, and the concentration-distribution equation. The medium was assumed to be a diluted solute, and different electric potentials and laser illumination were applied to the desired place. Gold (Au) electrodes were used at the top and bottom of a microchannel. For model validation, the simulation results were compared with the experimental data. The results revealed the formation of a toroidal microvortex via the ACET effect, which was generated due to laser illumination and joule-heating in the area of interest. In addition, under some conditions, such as the frequency of AC, the DEP velocity, and the particle size, the ACET force enhances and compresses resulting in the concentration of particles. The conditions of the DEP velocity and the ACET velocity are presented in detail with a comparison of the experimental results. 相似文献
10.
Mohammed Alshareef Nicholas Metrakos Eva Juarez Perez Fadi Azer Fang Yang Xiaoming Yang Guiren Wang 《Biomicrofluidics》2013,7(1)
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
11.
Melinda G. Simon Ying Li Janahan Arulmoli Lisa P. McDonnell Adnan Akil Jamison L. Nourse Abraham P. Lee Lisa A. Flanagan 《Biomicrofluidics》2014,8(6)
Dielectrophoresis (DEP) has proven an invaluable tool for the enrichment of populations of stem and progenitor cells owing to its ability to sort cells in a label-free manner and its biological safety. However, DEP separation devices have suffered from a low throughput preventing researchers from undertaking studies requiring large numbers of cells, such as needed for cell transplantation. We developed a microfluidic device designed for the enrichment of stem and progenitor cell populations that sorts cells at a rate of 150,000 cells/h, corresponding to an improvement in the throughput achieved with our previous device designs by over an order of magnitude. This advancement, coupled with data showing the DEP-sorted cells retain their enrichment and differentiation capacity when expanded in culture for periods of up to 2 weeks, provides sufficient throughput and cell numbers to enable a wider variety of experiments with enriched stem and progenitor cell populations. Furthermore, the sorting devices presented here provide ease of setup and operation, a simple fabrication process, and a low associated cost to use that makes them more amenable for use in common biological research laboratories. To our knowledge, this work represents the first to enrich stem cells and expand them in culture to generate transplantation-scale numbers of differentiation-competent cells using DEP. 相似文献
12.
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
13.
Francesco Piraino ?eila Selimovi? Marco Adamo Alessandro Pero Sam Manoucheri Sang Bok Kim Danilo Demarchi Ali Khademhosseini 《Biomicrofluidics》2012,6(4)
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
14.
Cell-secreted soluble factor signaling in a diffusion dominant microenvironment plays an important role on early stage differentiation of pluripotent stem cells invivo. In this study, we utilized a membrane-based two-chambered microbioreactor (MB) to differentiate mouse embryonic stem cells (mESCs) in a diffusion dominant microenvironment of the top chamber while providing enough nutrient through the bottom chamber. Speculating that accumulated FGF4 in the small top chamber will augment neuronal differentiation in the MB culture, we first differentiated mESCs for 8 days by using a chemically optimized culture medium for neuronal induction. However, comparison of cellular morphology and expression of neuronal markers in the MB with that in the 6-well plate (6WP) indicated relatively lower neuronal differentiation in the MB culture. Therefore, to investigate whether microenvironment in the MB facilitates non-neuronal differentiation, we differentiated mESCs for 8 days by using chemically defined basal medium. In this case, differentiated cell morphology differed markedly between the MB and 6WP cultures: epithelial sheet-like morphology in the MB, whereas rosette morphology in the 6WP. Expression of markers from the three germ layers indicated lower neuronal but higher meso- and endo-dermal differentiation of mESCs in the MB than the 6WP culture. Moreover, among various cell-secreted soluble factors, BMP4 expression was remarkably upregulated in the MB culture. Inhibition of BMP4 signaling demonstrated that enhanced effect of upregulated BMP4 was responsible for the prominent meso- and endo-dermal differentiation in the MB. However, in the 6WP, downregulated BMP4 had a minimal influence on the differentiation behavior. Our study demonstrated utilization of a microbioreactor to modulate the effect of cell-secreted soluble factors by autoregulation and thereby inducing alternative self-capability of mESCs. Understanding and implementation of autoregulation of soluble factors similar to this study will lead to the development of robust culture systems to control ESC behavior. 相似文献
15.
Ronald Pethig 《Biomicrofluidics》2010,4(2)
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
16.
A modified theory is proposed for extracting cell dielectric properties from the peak frequency measurement of electrorotation (ER) and the crossover frequency measurement of dielectrophoresis (DEP). Current theory in the literature is based on the low frequency (DC) approximations for the equivalent cell permittivity and conductivity, which are valid when the measurements are performed in a medium with conductivity less than 1 mS/m. The present theory extracts the cell properties through optimizing an expression for the medium conductivity in terms of the peak ER, or DEP crossover, frequency according to its definition using full expressions of equivalent cell permittivity and conductivity. Various levels of approximation of the theory are proposed and discussed through a scaling analysis. The present theory can extract both membrane and interior properties from the low and the high peak ER, or DEP crossover, frequencies for any medium conductivity provided the peak ER, or DEP crossover, frequency exists. It can be reduced to the linear theory for the low peak ER and DEP crossover frequencies in the literature when the medium conductivity is less than 10 mS/m. However, we can determine the membrane capacitance and conductance via the slope and intercept, respectively, of the straight line fitting of the ER peak and DEP frequency against medium conductivity data according to the linear theory only when the intercept dominates the experimental uncertainty, which occurs when the medium conductivity is less than 1 mS/m in practice. 相似文献
17.
Nikolic-Jaric M Romanuik SF Ferrier GA Cabel T Salimi E Levin DB Bridges GE Thomson DJ 《Biomicrofluidics》2012,6(2):24117-2411715
Dielectric particles flowing through a microfluidic channel over a set of coplanar electrodes can be simultaneously capacitively detected and dielectrophoretically (DEP) actuated when the high (1.45 GHz) and low (100 kHz–20 MHz) frequency electromagnetic fields are concurrently applied through the same set of electrodes. Assuming a simple model in which the only forces acting upon the particles are apparent gravity, hydrodynamic lift, DEP force, and fluid drag, actuated particle trajectories can be obtained as numerical solutions of the equations of motion. Numerically calculated changes of particle elevations resulting from the actuation simulated in this way agree with the corresponding elevation changes estimated from the electronic signatures generated by the experimentally actuated particles. This verifies the model and confirms the correlation between the DEP force and the electronic signature profile. It follows that the electronic signatures can be used to quantify the actuation that the dielectric particle experiences as it traverses the electrode region. Using this principle, particles with different dielectric properties can be effectively identified based exclusively on their signature profile. This approach was used to differentiate viable from non-viable yeast cells (Saccharomyces cerevisiae). 相似文献
18.
An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting 总被引：1，自引：0，他引：1
Multi-target pathogen detection using heterogeneous medical samples require continuous filtering, sorting, and trapping of debris, bioparticles, and immunocolloids within a diagnostic chip. We present an integrated AC dielectrophoretic (DEP) microfluidic platform based on planar electrodes that form three-dimensional (3D) DEP gates. This platform can continuously perform these tasks with a throughput of 3 μL∕min. Mixtures of latex particles, Escherichia coli Nissle, Lactobacillus, and Candida albicans are sorted and concentrated by these 3D DEP gates. Surface enhanced Raman scattering is used as an on-chip detection method on the concentrated bacteria. A processing rate of 500 bacteria was estimated when 100 μl of a heterogeneous colony of 107 colony forming units ∕ml was processed in a single pass within 30 min. 相似文献
19.
Dielectrophoretic capture voltage spectrum for measurement of dielectric properties and separation of cancer cells 总被引：1，自引：0，他引：1
In this paper, a new dielectrophoresis (DEP) method based on capture voltage spectrum is proposed for measuring dielectric properties of biological cells. The capture voltage spectrum can be obtained from the balance of dielectrophoretic force and Stokes drag force acting on the cell in a microfluidic device with fluid flow and strip electrodes. The method was demonstrated with the measurement of dielectric properties of human colon cancer cells (HT-29 cells). From the capture voltage spectrum, the real part of Clausius-Mossotti factor of HT-29 cells for different frequencies of applied electric field was obtained. The dielectric properties of cell interior and plasma membrane were then estimated by using single-shell dielectric model. The cell interior permittivity and conductivity were found to be insensitive to changes in the conductivity of the medium in which the cells are suspended, but the measured permittivity and conductivity of cell membrane were found to increase with the increase of medium conductivity. In addition, the measurement of capture voltage spectrum was found to be useful in providing the optimum operating conditions for separating HT-29 cells from other cells (such as red blood cells) using dielectrophoresis. 相似文献
20.
Dielectrophoresis (DEP) of biomolecules has large potential to serve as a novel selectivity parameter for bioanalytical methods such as (pre)concentration, fractionation, and separation. However, in contrast to well-characterized biological cells and (nano)particles, the mechanism of protein DEP is poorly understood, limiting bioanalytical applications for proteins. Here, we demonstrate a detailed investigation of factors influencing DEP of diagnostically relevant immunoglobulin G (IgG) molecules using insulator-based DEP (iDEP) under DC conditions. We found that the pH range in which concentration of IgG due to streaming iDEP occurs without aggregate formation matches the pH range suitable for immunoreactions. Numerical simulations of the electrokinetic factors pertaining to DEP streaming in this range further suggested that the protein charge and electroosmotic flow significantly influence iDEP streaming. These predictions are in accordance with the experimentally observed pH-dependent iDEP streaming profiles as well as the determined IgG molecular properties. Moreover, we observed a transition in the streaming behavior caused by a change from positive to negative DEP induced through micelle formation for the first time experimentally, which is in excellent qualitative agreement with numerical simulations. Our study thus relates molecular immunoglobulin properties to observed iDEP, which will be useful for the future development of protein (pre)concentration or separation methods based on DEP. 相似文献