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 <stron...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

This protocol allows the separation of cancer and healthy cells in a controlled way by keeping the conductivity constant and changing the applied frequency. This protocol simulates the controlled sorting of non-metastatic breast cancer cells and non-tumor breast epithelial cells using AC dielectrophoresis. This technique is the first simulation-based example of inline separation of non-metastatic breast cancer cells and non-tumor breast epithelial cells based on their dielectric properties.To begin, open the Multiphysics software, select the blank model and right-click on the global definitions. Select parameters and import the parameters given in table one into global definitions as a text file or enter the values individually. Select the 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. Choose a blank material and use the material properties from table one. Go to the home tab, select Add Physics and type AC/DC.Then go to the AC/DC node under the subnode of electric fields and currents and choose electric currents as physics. Insulate the channel walls to assign potential to the electrodes by right-clicking on the electric current and choosing the current conservation, insulate, and electric potential subnodes. Next, select Add Physics from the home tab and under the fluid flow node, go to the subnode single phase flow and choose creeping flow physics.Right-click on the single phase flow and render the chip boundaries as walls using the wall subnode. Right-click on the single phase flow and add two inlet subnodes and one outlet subnode. Assign the inlets using the inlet subnode and use normal and flow velocity as the boundary condition.Assign the outlet using the outlet subnode. Then select Add Physics from the home tab and under the fluid flow node, go to the subnode of particle tracing and choose particle tracing flow physics. Right-click on the particle tracing node and check the settings.Set the particle properties for both MCF-10A and MCF-7 cells using the particle properties subnode. Choose the particle properties from the parameters under the global definition section. Add the drag force subnode to assign the dielectrophoretic force to both types of cells.In this case, add the particle properties from the parameter section. Now choose Add Mesh and select Fine Mesh from the home tab. For building a mesh, select Build Mesh and click on Add Study to add three study steps.Study step one is for simulating a frequency response and using a frequency domain subnode. To simulate creeping flow, choose a stationary study node. Add two time-dependent steps to simulate conditions with dielectrophoretic force and without dielectrophoretic force.For the node dielectrophoretic condition, choose the physics and variable selection, check the modify model configuration box for the study setup and disable the dielectrophoretic step. For dielectrophoretic conditions, do not disable. Run the simulation after saving the file.After performing the CFD simulations by introducing non-metastatic breast cancer and non-tumor breast epithelial cell lines, solve two sets of CFD studies. For the first set, right-click on study one and add the parametric sweep subnode. Press the plus sign to add fluid medium conductivity sigma_m as the sweep variable.Perform a parametric sweep study for the fluid medium conductivity sigma_m ranging from 0.01 to 2.5 Siemens per meter, keeping the applied frequency constant at 800 kilohertz. For the second set, conduct a parametric sweep study by varying the applied AC frequency from 100 kilohertz to 100 megahertz, keeping the conductivity of the fluid medium sigma_m fixed at 0.4 Siemens per meter. Calculate the strength of the dielectrophoresis force exerted on a dielectric spherical particle in a conductive medium using this equation under the dielectrophoretic force subnode.Use this equation for a spherical particle under the dielectrophoretic force subnode. For a spherical particle under the dielectrophoretic force subnode, use this equation. Use a modified form of the previous equation to model biological cells such as mammalian cells which are more complex and have a multi-layered structure.Then solve complex permeability using this equation. Then plot REK as a function of the applied electric field for cancer and healthy cells. Right-click on the results node.Add the particle evaluation subnode. And in the expression section, type fpt.deff1. K to plot the CM factor for particle one and fpt.deff2.K for particle two. Under the fluid medium conductivity of 0.01 Siemens per meter and AC frequency of 100 kilohertz, the MCF-10A and MCF-7 cells experience positive dielectrophoresis with a REK value of 0.82 and 0.76. At the conductivity of 0.4 Siemens per meter, MCF-10A and MCF-7 showed negative dielectrophoretic behavior with REK values of minus 0.46 and minus 0.31 respectively.When the conductivity was increased to 1.2 Siemens per meter, the cell lines experienced negative dielectrophoresis at 100 kilohertz with REK values minus 0.49 and minus 0.43. Under the conductivity of 0.01 Siemens per meter, both cell types experienced positive dielectrophoresis, moved toward the region of high electric field strength and moved out from the top outlet. MCF-10A cells moved to the top outlet, while MCF-7 cells moved to the bottom outlet when the conductivity was increased to 0.4 Siemens per meter with the applied frequency fixed at 0.8 megahertz.As the medium conductivity was increased to 1.2 Siemens per meter, the cell lines moved away from the regions of high electric field. At 100 kilohertz frequency, both the cell lines experienced negative dielectrophoresis and moved toward the bottom outlet. The behavior of both cell lines remained unchanged until 0.8 megahertz.Beyond that, MCF-10A changed their dielectrophoretic behavior and crossed over to the positive dielectrophoretic region. At 100 megahertz, both the cell lines experienced positive dielectrophoretic and moved toward the top outlet. These techniques will open new venues for researchers who want to separate viable and non-viable cells and sort different types of cancer cells if the dielectric properties are not the same.Also, sorting based on different sizes can be achieved using the same method.

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

Abstract