This study was supported by the Higher Education Commission of Pakistan.

NOTE: The protocol here uses COMSOL, a multiphysics simulation software, to simulate the controlled sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using AC dielectrophoresis.
1. Chip design and parameter selection
<ol>
	<li>Open multiphysics software and select Blank Model. Right-click on the Global Definitions and select Parameters. Import the parameters given in Table 1 into global definitions as a text file or enter the values individually.</li>
	<li>Select Add Component from the home tab and add a 2D Component. Right-click on geometry and import the model file by double-clicking on the file.</li>
	<li>Choose a blank material and use the material properties from Table 1.</li>
	<li>Select Add Physics from the home tab, and type AC/DC. Under the AC/DC node, choose electric currents as Physics under the sub-node of electric fields and currents.</li>
	<li>Right-click on the Electric Current and choose the Current Conservation, Insulate, and Electric Potential sub-nodes to insulate the channel walls to assign potential to the electrodes.</li>
	<li>Select Add Physics from the home tab, and under the Fluid Flow node, choose Creeping Flow Physics under the sub-node of Single-Phase Flow. Right-click on Single-Phase Flow and render the chip boundaries as walls by using the Wall sub-node.</li>
	<li>Right click on Single-Phase Flow and add two inlet sub-nodes and one outlet sub-node.</li>
	<li>Assign the inlets using the inlet sub-node and use normal in Flow Velocity as the Boundary Condition. Assign the outlet using the outlet sub-node.</li>
	<li>Select Add Physics from the home tab, and under the Fluid Flow node, choose Particle Tracing Flow Physics under the sub node of Particle Tracing.</li>
	<li>Right-click on the Particle Tracing node and add the sub-nodes wall, two-particle property sub-nodes, two inlet sub-nodes, one outlet sub-node, two dielectrophoresis force sub-nodes, and one drag force sub-node.
	<ol>
		<li>Set Particle Properties for both MCF-7 and MCF-10A cells using the Particle Properties sub-node. Choose the particle properties from parameters under the Global Definition section.</li>
		<li>Add the Drag Force sub-node to assign the dielectrophoretic force to both types of cells.</li>
		<li>Add Particle Properties in this case from the parameter section. Add the Shell sub-node to model mammalian cells.</li>
	</ol>
	</li>
	<li>From the home tab, choose Add Mesh and select Fine Mesh. Choose Build Mesh from the home tab to build a mesh.</li>
	<li>From the home tab, click on Add Study to add three study steps. Study Step 1 is for simulating a frequency response; use a Frequency Domain sub-node.
	<ol>
		<li>To simulate creeping flow, choose a Stationary Study node. Add two time-dependent steps to simulate conditions with dielectrophoretic force and without dielectrophoretic force.</li>
		<li>For the no dielectrophoretic condition, choose Physics and Variables Selection, check the box titled Modify Model Configuration for the study step, and disable the Dielectrophoretic Step. For dielectrophoretic conditions, do not disable. Save the file and press Compute for the simulation to run. 
		NOTE: The microfluidic chip designed for the sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) has two separate inlets for cell mixture flow and for hydrodynamic flow focusing, respectively, with widths of 20 &#181;m and 40 &#181;m, respectively, as shown in Supplementary Figure 1 and Supplementary Figure 2.</li>
		<li>Assign frequency (f0) under the Frequency Domain sub-node and voltage using the Electric Potential sub-node to the planer electrodes (295 &#181;m in width) placed along the top side wall of the sorting chamber. At the outlet, use the &#34;freeze&#34; wall condition to visualize the sorted particles.</li>
	</ol>
	</li>
</ol>
2. Mathematical model and computational analysis
<ol>
	<li>Verify the operating parameters for separating non-metastatic breast cancer cells and non-tumor breast epithelial cells inside the microfluidic device by setting up a computational fluid dynamic (CFD) study. 
	NOTE: Multiphysics software (AC/DC, Microfluidics, and Particle Tracking modules) was used for this purpose. The governing equations and theoretical background are given in detail in Supplementary File 1. The model was tested by using the dielectric properties of non-metastatic breast cancer cells (MCF7) and non-tumor breast epithelial cells (MCF-10A) reported in literature31,32, which are summarized in Table 1.</li>
	<li>Perform the CFD simulations by introducing non-metastatic breast cancer (MCF7) and non-tumor breast epithelial (MCF-10A) cell lines with a ratio of 1:1 at the cell mixture inlet.
	<ol>
		<li>Initially, perform a mesh independence study to optimize the mesh size for the simulations33. 
		NOTE: A mesh independence study was performed to find the best solution for the operating parameters. A set of five different mesh sizes was chosen to quantify the best possible element size for the convergence of the solution. It was observed that, when the total number of elements defining a mesh was 635 (coarser mesh), as shown in Supplementary Figure 3A, the sorting efficiency was at its lowest, with some of the MCF7 cells moving to the bottom outlet, as depicted in Supplementary Figure 3B. When the mesh size was increased to fine, the number of elements defining the mesh also increased to 2,288. The sorting efficiency was at its maximum in this case, with both MCF7 and MCF-10A cells moving toward their respective outlets. The finer mesh was also simulated, with the number of elements defining the mesh increasing to 3,188. The sorting efficiency remained unaffected beyond this point. Hence, we can safely say that fine mesh size works the best in our case.</li>
		<li>Solve two sets of CFD studies.</li>
		<li>For the first set, right -click on Study 1 and add the Parametric Sweep sub-node. Press the + sign to add fluid medium conductivity &#34;&#963;m&#34;&#160;as the sweep variable. Perform a parametric sweep study for the fluid medium conductivity &#963;m ranging from 0.01 S/m to 2.5 S/m, keeping the applied frequency, f (Hz), constant at a value of 800 kHz.</li>
		<li>For the second set, conduct a Parametric Sweep study by varying the applied AC frequency from 100 kHz to 100 MHz while keeping the conductivity of the fluid medium, &#963;m, fixed at 0.4 S/m for each case. This &#963;m value was chosen based on the results of the first CFD study as a maximum separation between MCF-7 and MCF-10A was observed at this value.</li>
		<li>The strength of the dielectrophoresis (DEP) force, FDEP (-), exerted on a dielectric spherical particle in a conductive medium is given by Equation 1T34: 
		FDEP&#160;<img alt="Equation 1" src="/files/ftp_upload/63850/63850eq01.jpg" style="margin: auto;" />&#160; [1] 
		Use Equation 1 under the dielectrophoretic force sub-node. In equation 1, r shows the radius of the particle on which FDEP is applied; K (-) is known as the Clausius-Mossotti factor; &#949;m (-) shows the dielectric permittivity of the medium; and E(V/m) is the root mean square value of the electric field.</li>
		<li>Use Equation 2 for a spherical particle under the dielectrophoretic force sub-node. 
		<img alt="Equation 2" src="/files/ftp_upload/63850/63850eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;[2] 
		In Equation 2, <img alt="Equation 3" src="/files/ftp_upload/63850/63850eq03.jpg" style="margin: auto;" /> (-) shows the complex permittivity of the particle on which the DEP force is applied; <img alt="Equation 4" src="/files/ftp_upload/63850/63850eq04.jpg" style="margin: auto;" /> (-) shows the complex permittivity of the fluid surrounding the particle. The complex permittivity <img alt="Equation 3" src="/files/ftp_upload/63850/63850eq03.jpg" style="margin: auto;" /> and <img alt="Equation 4" src="/files/ftp_upload/63850/63850eq04.jpg" style="margin: auto;" /> are defined as follows35:</li>
		<li>Use Equation 3 for a spherical particle under the dielectrophoretic force sub-node: 
		<img alt="Equation 6" src="/files/ftp_upload/63850/63850eq06.jpg" style="margin: auto;" />&#160; &#160; &#160;[3] 
		In Equation 3, &#949;p (-) shows the real part of the complex permittivity of the particle; &#949;m (-) shows the&#160;real part of the complex permittivity of the fluid surrounding the particle; &#963;p (S/m) shows the particle conductivity; &#963;m (S/m) shows the conductivity of the medium surrounding the particle; and &#969; (Hz) is the frequency of the applied electric field. 
		NOTE: The sign of Re(K) determines the polarity of the FDEP. If the sign of Re(K) is negative, then the particle experiences a negative dielectrophoretic force (nDEP); contrary to this, if the sign of Re(K) is positive, it implies a positive dielectrophoretic force (pDEP). For the Clausius-Mossotti factor (K), the variation lies within the range of -1 to 1.</li>
	</ol>
	</li>
	<li>Use a modified form of Equation 3 to model biological cells such as mammalian cells, which are more complex and have a multilayered structure. 
	K (<img alt="Equation 7" src="/files/ftp_upload/63850/63850eq07.jpg" style="margin: auto;" />) = <img alt="Equation 8" src="/files/ftp_upload/63850/63850eq08.jpg" style="margin: auto;" />&#160; &#160; &#160;[4] 
	In Equation 4, <img alt="Equation 9" src="/files/ftp_upload/63850/63850eq09.jpg" style="margin: auto;" /> (-) incorporates both the complex permittivity of the cytoplasm, <img alt="Equation 10" src="/files/ftp_upload/63850/63850eq10.jpg" style="margin: auto;" /> (-), and the complex permittivity of the cell membrane, <img alt="Equation 11" src="/files/ftp_upload/63850/63850eq11.jpg" style="margin: auto;" /> (-), and is given as follows:36</li>
	<li>Use Equation 5 to solve &#34;<img alt="Equation 12" src="/files/ftp_upload/63850/63850eq12.jpg" style="margin: auto;" />&#34;: 
	<img alt="Equation 13" src="/files/ftp_upload/63850/63850eq13.jpg" style="margin: auto;" />&#160; &#160;&#160; [5] 
	In Equation 5, Rcyto (m) and Rmem (m) show the radius of the cell cytoplasm and cell membrane, respectively.</li>
	<li>Then, use Equation 4 to plot Re(K) as a function of the applied electric field for both cancer and healthy cells. Calculate the real part of the Clausius-Mossotti (CM) factor, Re(K), to quantify the dielectrophoretic force (DEP) that the particle experiences.</li>
	<li>Right-click on the Results node, add the Particle Evaluation sub-node, and in the expression section, type fpt.deff1.K to plot the CM factor for particle 1 and fpt.deff2.K for particle 2. 
	NOTE: All the protocol steps listed in the main text can be viewed in the protocol video (Video 1).</li>
</ol>

The authors declare no potential conflicts of interest.

Microfluidic devices have been reported previously for cell culture, trapping, and sorting47,52,53. The fabrication of these devices in the cleanroom is an expensive process, and it is imperative to quantify the output and efficiency of a proposed microfluidic device through CFD simulations. This study presents the design and simulations of an AC-dielectrophoretic microfluidic device for the continuous separation of non-metastat...

A malignant tumor that develops in and around the breast tissue is a frequent cause of breast cancer in women worldwide, causing a critical health problem1. Breast tumors before metastasis can be treated through surgery if detected at an early stage, but if ignored, they can have severe implications on the patient&#39;s life by spreading to their lungs, brain, and bones. The treatments offered at later stages, such as radiation and chemical-based therapies, have severe side effects2. Recent studies have reported that an early diagnosis of breast cancer reduces the mortality rate by 60%3. Hence, it is imperative to work toward personalized early detection methods. To this end, researchers working in different fields of science and technology have used microfluidics to develop devices for the early diagnosis of breast cancer4. These methods include cell affinity micro-chromatography, magnetic-activated micro-cell sorters, size-based cancer cell capture and separation, and on-chip dielectrophoresis (DEP)5,6. These microfluidic techniques reported in the literature enable precise cell manipulation, real-time monitoring, and sorting of well-defined samples, which serve as an intermediate step in many diagnostic and therapeutic applications5. The integration of these sorting mechanisms with microfluidics offers flexible and reliable manipulation of the target cells7,8,9,10. One of the main advantages of such an integration is the ability to work with fluid samples in nano to microliter volumes and also being able to manipulate the electrical properties of the sample fluid. By adjusting the conductivity of the suspending fluid inside microfluidic devices, the biological cells can be sorted based on their sizes and differences in their dielectric properties11,12.
Among these techniques, on-chip DEP is often preferred as it is a label-free cell sorting technique that exploits the electric properties of the biological samples. DEP has been reported to manipulate bio-samples such as DNA13, RNA14, proteins15, bacteria16, blood cells17, circulating tumor cells (CTCs)18, and stem cells19. Microfluidic devices that employ DEP for sorting biological samples have been reported extensively in literature20. Reservoir-based DEP microfluidic (rDEP) devices for sorting viable and non-viable yeast cells have been reported that protect the cells from the adverse effects of electrochemical reactions21,22. Piacentini et al. reported a castellated microfluidic cell sorter that separated red blood cells from platelets with an efficiency of 97%23. On-chip DEP devices with asymmetric orifices and embedded electrodes have also been reported to sort viable and non-viable cells24. Valero and Demierre et al. modified the castellated microfluidic cell sorter by introducing two arrays of microelectrodes on both sides of the channel25,26. This helped in focusing the cells in the center of the channel. Zeynep et al. presented a DEP-based microfluidic device to separate and concentrate MCF7 breast cancer cells from leukocytes27. They reported an efficiency of extracting MCF7 cells from leukocytes between 74%-98% with a frequency of 1 MHz and an applied voltage ranging from 10-12 Vpp. Supplementary Table 1 represents a qualitative and quantitative comparison between the DEP-based microfluidic sorting devices based on their design, electrode configuration, and operating parameters (applied frequency and voltage).
More recently, researchers have tried to measure the differences in the dielectric behavior of breast epithelial cells (MCF-10A) and non-metastatic breast cancer cells (MCF-7) inside a microfluidic chip28,29. Jithin et al. also characterized the dielectric responses of different cancer cell lines using an open-ended coaxial probe technique with frequencies between 200 MHz and 13.6 GHz30. These differences in the dielectric responses of MCF-7 and MCF-10A cell lines can be exploited to separate them in runtime and can lead to the development of personalized early-stage diagnosis devices.
In this article, we simulate the controlled sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using AC dielectrophoresis. The region of change in the electric field influences the sorting inside the microfluidic chip. The proposed technique is easy to implement and allows for the integration of the sorting technique into various microfluidic chip layouts. Computational fluid dynamics (CFD) simulations were carried out to study the separation of non-metastatic breast cancer cells and non-tumor breast epithelial cells by varying the conductivity of the fluid medium in which cells were suspended. In these simulations, it is shown that, by keeping the conductivity constant and by changing the applied frequency, the separation of cancer cells and healthy cells can be controlled.

<table><tbody><tr><td>COMSOL</td><td>COMSOL</td><td></td><td>multiphysics simulation software</td></tr></tbody></table>

microfluidic device for the separation of non-metastatic (mcf-7) and non-tumor (mcf-10a) breast cancer cells using ac dielectrophoresis

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using the principle of the polarization of the cancer cells in the sample volume by applying an external electric field. This article demonstrates how a microfluidic platform can be utilized for high-throughput continuous sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using hydrodynamic dielectrophoresis (HDEP) from the cell mixture. By generating an electric field between two electrodes placed side-by-side with a micron-sized gap between them in an HDEP microfluidic chip, non-tumor breast epithelial cells (MCF-10A) can be pushed away, exhibiting negative DEP inside the main channel, while the non-metastatic breast cancer cells follow their course unaffected when suspended in cell medium due to having conductivity higher than the membrane conductivity. To demonstrate this concept, simulations were performed for different values of medium conductivity, and the sorting of cells was studied. A parametric study was carried out, and a suitable cell mixture conductivity was found to be 0.4 S/m. By keeping the medium conductivity fixed, an adequate AC frequency of 0.8 MHz was established, giving maximum sorting efficiency, by varying the electric field frequency. Using the demonstrated method, after choosing the appropriate cell mixture suspension medium conductivity and frequency of the applied AC, maximum sorting efficiency can be achieved.

Investigating the optimal operational parameters for effective DEP-based sorting of non-metastatic breast cancer (MCF-7) and non-tumor breast epithelial (MCF-10A) cells 
To achieve a successful separation of non-metastatic breast cancer (MCF-7) and non-tumor breast epithelial (MCF-10A) cells with divergent dielectric properties when undergoing dielectrophoresis, their K factors should be distinct by keeping the applied frequency fixed37,38. ...

Watch this Scientific Journal Video about Microfluidic Device for the Separation of Non-Metastatic MCF-7 and Non-Tumor MCF-10A Breast Cancer Cells Using AC Dielectrophoresis at JoVE.com

Microfluidic Device for the Separation of Non-Metastatic MCF-7 and Non-Tumor MCF-10A Breast Cancer Cells Using AC Dielectrophoresis

Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells.

Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using ...

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2.3K Views.  Foundation University Islamabad. Dit protocol maakt de scheiding van kanker en gezonde cellen op een gecontroleerde manier mogelijk door de geleidbaarheid constant te houden en de toegepaste frequentie te veranderen. Dit protocol simuleert de gecontroleerde sortering van niet-gemetastaseerde borstkankercellen en niet-tumor borstepitheelcellen met behulp van AC-dielectroforese. Deze techniek is het eerste simulatie-gebaseerde voorbeeld van inline scheiding van niet-gemetastaseerde borstkankercellen en niet-tumor borstepitheel...

Video: Microfluidic Device for the Separation of Non-Metastatic MCF-7 and Non-Tumor MCF-10A Breast Cancer Cells Using AC Dielectrophoresis

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Bioengineering

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

Medicine

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Cancer Research

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) offer a patient-derived cell source for conducting mechanistic studies of diseases or for several therapeutic applications. Understanding hMSC properties, such as their electrical behavior at various maturation stages, has become more important in recent years. Dielectrophoresis (DEP) is a method that can manipulate cells in a nonuniform electric field, through which information can be obtained about the electrical properties of the cells, such as the cell membrane capacitance and permittivity. Traditional modes of DEP use metal electrodes, such as three-dimensional electrodes, to characterize the response of cells to DEP. In this paper, we present a microfluidic device built with a photoconductive layer capable of manipulating cells through light projections that act as in situ virtual electrodes with readily conformable geometries. A protocol is presented here that demonstrates this phenomenon, called light-induced DEP (LiDEP), for characterizing hMSCs. We show that LiDEP-induced cell responses, measured as cell velocities, can be optimized by varying parameters such as the input voltage, the wavelength ranges of the light projections, and the intensity of the light source. In the future, we envision that this platform could pave the way for technologies that are label-free and perform real-time characterization of heterogeneous populations of hMSCs or other stem cell lines.

Here, we present light-induced dielectrophoresis as a label-free approach for characterizing heterogeneous cell lines, specifically human mesenchymal stem cells (hMSCs). This paper describes a protocol for using and optimizing a microfluidic device with a photoconductive layer to characterize the electrical behavior of hMSCs without altering their native state.

Human mesenchymal stem cells (hMSCs) offer a patient-derived cell source for conducting mechanistic studies of diseases or for several therapeutic ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis

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