Michael B. Sano

Contactless dielectrophoresis: a new technique for cell manipulation.

Dielectrophoresis (DEP) has become a promising technique to separate and identify cells and microparticles suspended in a medium based on their size or electrical properties. Presented herein is a new technique to provide the non-uniform electric field required for DEP that does not require electrodes to contact the sample fluid. In our method, electrodes are capacitively-coupled to a fluidic channel through dielectric barriers; the application of a high-frequency electric field to these electrodes then induces an electric field in the channel. This technique combines the cell manipulation abilities of traditional DEP with the ease of fabrication found in insulator-based technologies. A microfluidic device was fabricated based on this principle to determine the feasibility of cell manipulations through contactless DEP (cDEP). We were able to demonstrate cell responses unique to the DEP effect in three separate cell lines. These results illustrate the potential for this technique to identify cells through their electrical properties without fear of contamination from electrodes.

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

BackgroundTherapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.MethodsA combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.ResultsNo visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.ConclusionsH-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.

Modeling and development of a low frequency contactless dielectrophoresis (cDEP) platform to sort cancer cells from dilute whole blood samples.

Contactless dielectrophoresis (cDEP) devices are a new adaptation of dielectrophoresis in which fluid electrodes, isolated from the main microfluidic channel by a thin membrane, provide the electric field gradients necessary to manipulate cells. This work presents a continuous sorting device which is the first cDEP design capable of exploiting the Clausius-Mossotti factor at frequencies where it is both positive and negative for mammalian cells. Experimental devices are fabricated using a cost effective technique which can achieve 50 μm feature sizes and does not require the use of a cleanroom or specialized equipment. An analytical model is developed to evaluate cDEP devices as a network of parallel resistor-capacitor pairs. Two theoretical devices are presented and evaluated using finite element methods to demonstrate the effect of geometry on the development of electric field gradients across a wide frequency spectrum. Finally, we present an experimental device capable of continuously sorting human leukemia cells from dilute blood samples. This is the first cDEP device designed to operate below 100 kHz resulting in successful manipulation of human leukemia cells, while in the background red blood cells are unaffected.

Selective concentration of human cancer cells using contactless dielectrophoresis

This work is the first to demonstrate the ability of contactless dielectrophoresis (cDEP) to isolate target cell species from a heterogeneous sample of live cells. Since all cell types have a unique molecular composition, it is expected that their dielectrophoretic (DEP) properties are also unique. cDEP is a technique developed to improve upon traditional and insulator‐based DEP devices by replacing embedded metal electrodes with fluid electrode channels positioned alongside desired trapping locations. Through the placement of the fluid electrode channels and the removal of contact between the electrodes and the sample fluid, cDEP mitigates issues associated with sample/electrode contact. MCF10A, MCF7, and MDA‐MB‐231 human breast cells were used to represent early, intermediate, and late‐staged breast cancer, respectively. Trapping frequency responses of each cell type were distinct, with the largest difference between the cells found at 20 and 30 V. MDA‐MB‐231 cells were successfully isolated from a population containing MCF10A and MCF7 cells at 30 V and 164 kHz. The ability to selectively concentrate cells is the key to development of biological applications using DEP. The isolation of these cells could provide a workbench for clinicians to detect transformed cells at their earliest stage, screen drug therapies prior to patient treatment, increasing the probability of success, and eliminate unsuccessful treatment options.

Towards the creation of decellularized organ constructs using irreversible electroporation and active mechanical perfusion

BackgroundDespite advances in transplant surgery and general medicine, the number of patients awaiting transplant organs continues to grow, while the supply of organs does not. This work outlines a method of organ decellularization using non-thermal irreversible electroporation (N-TIRE) which, in combination with reseeding, may help supplement the supply of organs for transplant.MethodsIn our study, brief but intense electric pulses were applied to porcine livers while under active low temperature cardio-emulation perfusion. Histological analysis and lesion measurements were used to determine the effects of the pulses in decellularizing the livers as a first step towards the development of extracellular scaffolds that may be used with stem cell reseeding. A dynamic conductivity numerical model was developed to simulate the treatment parameters used and determine an irreversible electroporation threshold.ResultsNinety-nine individual 1000 V/cm 100-μs square pulses with repetition rates between 0.25 and 4 Hz were found to produce a lesion within 24 hours post-treatment. The livers maintained intact bile ducts and vascular structures while demonstrating hepatocytic cord disruption and cell delamination from cord basal laminae after 24 hours of perfusion. A numerical model found an electric field threshold of 423 V/cm under specific experimental conditions, which may be used in the future to plan treatments for the decellularization of entire organs. Analysis of the pulse repetition rate shows that the largest treated area and the lowest interstitial density score was achieved for a pulse frequency of 1 Hz. After 24 hours of perfusion, a maximum density score reduction of 58.5 percent had been achieved.ConclusionsThis method is the first effort towards creating decellularized tissue scaffolds that could be used for organ transplantation using N-TIRE. In addition, it provides a versatile platform to study the effects of pulse parameters such as pulse length, repetition rate, and field strength on whole organ structures.

Theoretical Considerations of Tissue Electroporation With High-Frequency Bipolar Pulses

This study introduces the use of high-frequency pulsed electric fields for tissue electroporation. Through the development of finite element models and the use of analytical techniques, electroporation with rectangular, bipolar pulses is investigated. The electric field and temperature distribution along with the associated transmembrane potential development are considered in a heterogeneous skin fold geometry. Results indicate that switching polarity on the nanosecond scale near the charging time of plasma membranes can greatly improve treatment outcomes in heterogeneous tissues. Specifically, high-frequency fields ranging from 500 kHz to 1 MHz are best suited to penetrate epithelial layers without inducing significant Joule heating, and cause electroporation in underlying cells.

Contactless dielectrophoretic spectroscopy: Examination of the dielectric properties of cells found in blood†

The use of non‐invasive methods to detect and enrich circulating tumor cells (CTCs) independent of their genotype is critical for early diagnostic and treatment purposes. The key to using CTCs as predictive clinical biomarkers is their separation and enrichment. This work presents the use of a contactless dielectrophoresis (cDEP) device to investigate the frequency response of cells and calculate their area‐specific membrane capacitance. This is the first demonstration of a cDEP device which is capable of operating between 10 and 100 kHz. Positive and negative dielectrophoretic responses were observed in red blood cells, macrophages, breast cancer, and leukemia cells. The area‐specific membrane capacitances of MDA‐MB231, THP‐1 and PC1 cells were determined to be 0.01518 ± 0.0013, 0.01719 ± 0.0020, 0.01275 ± 0.0018 (F/m2), respectively. By first establishing the dielectrophoretic responses of cancerous cells within this cDEP device, conditions to detect and enrich tumor cells from mixtures with non‐transformed cells can be determined providing further information to develop methods to isolate these rare cells.

Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells

In this study, the electrical properties of four different stages of mouse ovarian surface epithelial (MOSE) cells were investigated using contactless dielectrophoresis (cDEP). This study expands the work from our previous report describing for the first time the crossover frequency and cell specific membrane capacitance of different stages of cancer cells that are derived from the same cell line. The specific membrane capacitance increased as the stage of malignancy advanced from 15.39 ± 1.54 mF m(-2) for a non-malignant benign stage to 26.42 ± 1.22 mF m(-2) for the most aggressive stage. These differences could be the result of morphological variations due to changes in the cytoskeleton structure, specifically the decrease of the level of actin filaments in the cytoskeleton structure of the transformed MOSE cells. Studying the electrical properties of MOSE cells provides important information as a first step to develop cancer-treatment techniques which could partially reverse the cytoskeleton disorganization of malignant cells to a morphology more similar to that of benign cells.

Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis

Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool.

Joule heating effects on particle immobilization in insulator‐based dielectrophoretic devices

In this work, the temperature effects due to Joule heating obtained by application of a direct current electric potential were investigated for a microchannel with cylindrical insulating posts employed for insulator‐based dielectrophoresis. The conductivity of the suspending medium, the local electric field, and the gradient of the squared electric field, which directly affect the magnitude of the dielectrophoretic force exerted on particles, were computationally simulated employing COMSOL Multiphysics. It was observed that a temperature gradient is formed along the microchannel, which redistributes the conductivity of the suspending medium leading to an increase of the dielectrophoretic force toward the inlet of the channel while decreasing toward the outlet. Experimental results are in good agreement with simulations on the particle‐trapping zones anticipated. This study demonstrates the importance of considering Joule heating effects when designing insulator‐based dielectrophoresis systems.