Efficient dielectrophoretic cell enrichment using a dielectrophoresis-well based system.
Ontology highlight
ABSTRACT:
SUBMITTER: Abdul Razak MA
PROVIDER: S-EPMC3869820 | biostudies-other | 2013
REPOSITORIES: biostudies-other
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Project description:Recent advancements in micro/nanofabrication techniques have led to the development of portable devices for high-throughput single-cell analysis through the isolation of individual target cells, which are then paired with functionalized microbeads. Compared with commercially available benchtop instruments, portable microfluidic devices can be more widely and cost-effectively adopted in single-cell transcriptome and proteome analysis. The sample utilization and cell pairing rate (∼33%) of current stochastic-based cell-bead pairing approaches are fundamentally limited by Poisson statistics. Despite versatile technologies having been proposed to reduce randomness during the cell-bead pairing process in order to statistically beat the Poisson limit, improvement of the overall pairing rate of a single cell to a single bead is typically based on increased operational complexity and extra instability. In this article, we present a dielectrophoresis (DEP)-assisted dual-nanowell array (ddNA) device, which employs an innovative microstructure design and operating process that decouples the bead- and cell-loading processes. Our ddNA design contains thousands of subnanoliter microwell pairs specifically tailored to fit both beads and cells. Interdigitated electrodes (IDEs) are placed below the microwell structure to introduce a DEP force on cells, yielding high single-cell capture and pairing rates. Experimental results with human embryonic kidney cells confirmed the suitability and reproducibility of our design. We achieved a single-bead capture rate of >97% and a cell-bead pairing rate of >75%. We anticipate that our device will enhance the application of single-cell analysis in practical clinical use and academic research.
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Project description:Human African trypanosomiasis (HAT), also known as sleeping sickness, is a vector-borne neglected tropical disease endemic to rural sub-Saharan Africa. Current methods of early detection in the affected rural communities generally begin with general screening using the card agglutination test for trypanosomiasis (CATT), a serological test. However, the gold standard for confirmation of trypanosomiasis remains the direct observation of the causative parasite, Trypanosoma brucei. Here, we present the use of dielectrophoresis (DEP) to enrich T. brucei parasites in specific locations to facilitate their identification in a future diagnostic assay. DEP refers to physical movement that can be selectively induced on the parasites when exposing them to electric field gradients of specific magnitude, phase and frequency. The long-term goal of our work is to use DEP to selectively trap and enrich T. brucei in specific locations while eluting all other cells in a sample. This would allow for a diagnostic test that enables the user to characterize the presence of parasites in specific locations determined a priori instead of relying on scanning a sample. In the work presented here, we report the characterization of the conditions that lead to high enrichment, 780% in 50 s, of the parasite in specific locations using an array of titanium microelectrodes.
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Project description:Blood vessel formation, during either normal vascular reconstruction or pathogenic tumour formation, relies upon highly organized cell-cell interactions. Isolating the function of any particular component of this cell-cell communication is often difficult, given the vast complexity of communication networks in multicellular systems. One way to address this problem is to analyse cell-cell communication on the most elementary scale--cell pairs. Here, we describe an integrated dielectrophoretic (DEP)-microfluidic device allowing for such analysis. Single cancer and endothelial cells (ECs) and cell pairs were patterned using DEP force and cultured within a minimally stressful microfluidic channel network. Controlling both the initial cell positions and extracellular environment, we investigated cell motility in homo- and heterotypic cell pairs under diverse conditions. We found that secreted collagen IV and soluble vascular endothelial growth factor have considerable guidance effect on ECs at the level of two interacting cells. Cell interaction rules extracted from the experiments of cell pairs were used to mathematically predict branching patterns characteristic of developing multicellular blood vessels. This integrative analysis method can be extended to other systems involving complex multicellular interactions.
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Project description:Cells mechanical property assessment has been a promising label-free method for cell differentiation. Several methods have been proposed for single-cell mechanical properties analysis. Dielectrophoresis (DEP) is one method used for single-cell mechanical property assessment, cell separation, and sorting. DEP method has overcome weaknesses of other techniques, including compatibility with microfluidics, high throughput assessment, and high accuracy. However, due to the lack of a general and explicit model for this method, it has not been known as an ideal cell mechanical property evaluation method. Here we present an explicit model using the most general electromagnetic equation (Maxwell Stress Tensor) for single-cell mechanical evaluation based on the DEP method. For proof of concept, we used the proposed model for differentiation between three different types of cells, namely erythrocytes, peripheral blood mononuclear cells (PBMC), and an epithelial breast cancer cells line (T-47D). The results show that, by a lumped parameter that depends on cells' mechanical and electrical properties, the proposed model can successfully distinguish between the mentioned cell types that can be in a single blood sample. The proposed model would open up the chance to use a mechanical assessment method for cell searching in parallel with other methods.
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Project description: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.
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Project description:The microfluidic dielectrophoretic (MF-DEP) chip is a new, economical and readily-available technology that might be used to enrich X-sperm for increasing female offspring in dairy farms. In this study, we sought to develop an MF-DEP chip to enrich X bovine sperm. The MF-DEP chip was composed of an electrode attached to a glass slide and a microchannel made from polydimethylsiloxane. Sex-sorted sperm from flow cytometry were used to identify optimal electric field conditions while unsorted sperm were later tested for sorting efficiency. The results show that during dielectrophoresis some sperm attached to the electrode (called positive DEP; pDEP) whereas other moved away from the electrode (called negative DEP; nDEP). X and Y-sperm responded to dielectrophoresis differently depending on various factors such as buffers, voltages, and frequencies. We found that the condition 4 V 1 MHz significantly reduced (P < 0.05) the percentage of Y-sperm to nearly 30 and therefore enriched X-sperm. The sorting efficiency was dependent on buffer, bull, sorting cycle, flow rate, electrical voltage, and frequency. Notably, the best sorting buffer found in this experiment was the conducting buffer, but this buffer significantly reduced sperm viability and motility. Other sperm-friendly buffers, TRIS and mHTF, were also used, but could not enrich X-sperm. In conclusion, this is a proof of concept that the MF-DEP chip can be effectively used to enrich bovine X-sperm. However, more research must be performed particularly to find the best sorting buffer to effectively sex-sort sperm while providing high motility and sperm viability.
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