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

Summary

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.

Abstract

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.

Introduction

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'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 cells^7,8,9,10. 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 properties^11,12.

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 reactions^21,22. 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 channel^25,26. 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 V_pp. 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 chip^28,29. 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.

Protocol

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 Open multiphysics software and select Blank Model. Right-click on the Global Definitions and select Parameters. Import the parameters given in <stron…

Representative Results

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. …

Discussion

Microfluidic devices have been reported previously for cell culture, trapping, and sorting^47,52,53. 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…

Materials

COMSOL

COMSOL

multiphysics simulation software