Analysis of U87 glioma cells by dielectrophoresis.

Glioblastoma multiforme is the most aggressive and invasive brain cancer consisting of genetically and phenotypically altering glial cells. It has massive heterogeneity due to its highly complex and dynamic microenvironment. Here, electrophysiological properties of U87 human glioma cell line were measured based on a dielectrophoresis phenomenon to quantify the population heterogeneity of glioma cells. Dielectrophoretic forces were generated using a gold-microelectrode array within a microfluidic channel when 3 Vpp and 100, 200, 300, 400, 500 kHz, 1, 2, 5, and 10 MHz frequencies were applied. We analyzed the dielectrophoretic behavior of 500 glioma cells, and revealed that the crossover frequency of glioma cells was around 140 kHz. A quantifying dielectrophoretic movement of the glioma cells exhibited three distinct glioma subpopulations: 50% of the glioma cells experienced strong, 30% of the cells were spread in the microchannel by moderate, and the rest of the cells experienced very weak positive dielectrophoretic forces. Our results demonstrated the dielectrophoretic spectra of U87 glioma cell line. Dielectrophoretic responses of glioma cells linked population heterogeneity to membrane properties of glioma cells rather than their size distribution in the population.

Long-term migratory velocity measurements of single glioma cells using microfluidics.

Microfluidic platforms enabling single-cell measurements notably contribute to the identification and observation of rare cancer cells that are involved in tumor invasion. Most aggressive, invasive, and heterogeneous glioblastoma cells cause incurable primary brain tumors. Infiltrating gliomas of a brain tumor microenvironment have been intensively studied using conventional assays. Still, quantitative, simple, and precise tools are required for long-term, steady-state migratory-velocity measurements of single glioma cells. To measure long-term velocity changes and investigate the heterogeneity of glioma cells under different growth conditions, we developed a microfluidic platform. We cultured U87 glioma cells in the microfluidic device using either regular growth medium or conditional medium composed of 50% basal medium and 50% macrophage-depleted medium. We microscopically monitored the behavior of 40 glioma cells for 5 days. Using acquired images, we calculated cellular circularity and determined the migratory velocities of glioma cells from 60 h to 120 h. The mean migratory velocity values of the glioma cells were 1.513 µm h-1 in the basal medium and 3.246 µm h-1 in the conditional medium. The circularity values of the glioma cells decreased from 0.20-0.25 to 0.15-0.20 when cultured in the conditional medium. Here, we clearly showed that the glioma cells lost their circularity and increased their steady-state velocities; in other words, they adopted an invasive glioma phenotype in the presence of macrophage-depleted medium. Besides, the heterogeneity of the circularity and the velocity of glioma cells were enhanced in the conditional medium.

Quantifying Deformation and Migration Properties of U87 Glioma Cells Using Dielectrophoretic Forces.

Glioblastoma multiforme is one of the most aggressive malignant primary brain tumors. To design effective treatment strategies, we need to better understand the behavior of glioma cells while maintaining their genetic and phenotypic stability. Here, we investigated the deformation and migration profile of U87 Glioma cells under the influence of dielectrophoretic forces. We fabricated a gold microelectrode array within a microfluidic channel and applied sinusoidal wave AC potential at 3 Vpp, ranging from 30 kHz to 10 MHz frequencies, to generate DEP forces. We followed the dielectrophoretic movement and deformation changes of 100 glioma cells at each frequency. We observed that the mean dielectrophoretic displacements of glioma cells were significantly different at varying frequencies with the maximum and minimum traveling distances of 13.22 µm and 1.37 µm, respectively. The dielectrophoretic deformation indexes of U87 glioma cells altered between 0.027-0.040. It was 0.036 in the absence of dielectrophoretic forces. This approach presents a rapid, robust, and sensitive characterization method for quantifying membrane deformation of glioma cells to determine the state of the cells or efficacy of administrated drugs.

Single-Cell Mechanophenotyping in Microfluidics to Evaluate Behavior of U87 Glioma Cells.

Integration of microfabricated, single-cell resolution and traditional, population-level biological assays will be the future of modern techniques in biology that will enroll in the evolution of biology into a precision scientific discipline. In this study, we developed a microfabricated cell culture platform to investigate the indirect influence of macrophages on glioma cell behavior. We quantified proliferation, morphology, motility, migration, and deformation properties of glioma cells at single-cell level and compared these results with population-level data. Our results showed that glioma cells obtained slightly slower proliferation, higher motility, and extremely significant deformation capability when cultured with 50% regular growth medium and 50% macrophage-depleted medium. When the expression levels of E-cadherin and Vimentin proteins were measured, it was verified that observed mechanophenotypic alterations in glioma cells were not due to epithelium to mesenchymal transition. Our results were consistent with previously reported enormous heterogeneity of U87 glioma cell line. Herein, for the first time, we quantified the change of deformation indexes of U87 glioma cells using microfluidic devices for single-cells analysis.

Quantifying Heterogeneity According to Deformation of the U937 Monocytes and U937-Differentiated Macrophages Using 3D Carbon Dielectrophoresis in Microfluidics.

A variety of force fields have thus far been demonstrated to investigate electromechanical properties of cells in a microfluidic platform which, however, are mostly based on fluid shear stress and may potentially cause irreversible cell damage. This work presents dielectric movement and deformation measurements of U937 monocytes and U937-differentiated macrophages in a low conductive medium inside a 3D carbon electrode array. Here, monocytes exhibited a crossover frequency around 150 kHz and presented maximum deformation index at 400 kHz and minimum deformation index at 1 MHz frequencies at 20 Vpeak-peak. Although macrophages were differentiated from monocytes, their crossover frequency was lower than 50 kHz at 10 Vpeak-peak. The change of the deformation index for macrophages was more constant and lower than the monocyte cells. Both dielectric mobility and deformation spectra revealed significant differences between the dielectric responses of U937 monocytes and U937-differentiated macrophages, which share the same origin. This method can be used for label-free, specific, and sensitive single-cell characterization. Besides, damage of the cells by aggressive shear forces can, hence, be eliminated and cells can be used for downstream analysis. Our results showed that dielectric mobility and deformation have a great potential as an electromechanical biomarker to reliably characterize and distinguish differentiated cell populations from their progenitors.

Single Cell Level Dielectrophoretic Responses & Dielectrophoretic Deformations of Monocytes to Quantify Population Heterogeneity.

Single-cell dielectrophoretic movement and dielectrophoretic deformation of monocyte cells were interrogated applying 20 Vpp, 50 kHz to 1 MHz signal in the 3D carbon electrode array. Heterogeneity of the monocyte population is shown in terms of the crossover frequencies, translational movement, and deformation index of the cells. The results presented that crossover range for monocytes was 100 kHz - 200 kHz, the translational movement of the cells was rapidly altered when the initial positions of the cells were in the negative dielectrophoretic region. Finally, the deformation index of the monocyte population varied from 0.5 to 1.5.