Continuous ES/Feeder Cell-Sorting Device Using Dielectrophoresis and Controlled Fluid Flow

Abstract

Pluripotent stem cells (PSCs) are considered as being an important cell source for regenerative medicine. The culture of PSCs usually requires a feeder cell layer or cell adhesive matrix coating such as Matrigel, laminin, and gelatin. Although a feeder-free culture using a matrix coating has been popular, the on-feeder culture is still an effective method for the fundamental study of regenerative medicine and stem cell biology. To culture PSCs on feeder cell layers, the elimination of feeder cells is required for biological or gene analysis and for cell passage. Therefore, a simple and cost-effective cell sorting technology is required. There are several commercialized cell-sorting methods, such as FACS or MACS. However, these methods require cell labeling by fluorescent dye or magnetic antibodies with complicated processes. To resolve these problems, we focused on dielectrophoresis (DEP) phenomena for cell separation because these do not require any fluorescent or magnetic dyes or antibodies. DEP imposes an electric force on living cells under a non-uniform AC electric field. The direction and magnitude of the DEP force depend on the electric property and size of the cell. Therefore, DEP is considered as a promising approach for sorting PSCs from feeder cells. In this study, we developed a simple continuous cell-sorting device using the DEP force and fluid-induced shear force. As a result, mouse embryonic stem cells (mESCs) were purified from a mixed-cell suspension containing mESCs and mouse embryonic fibroblasts (MEFs) using our DEP cell-sorting device.

Dielectrophoretic Micro-Organization of Chondrocytes to Regenerate Mechanically Anisotropic Cartilaginous Tissue

“…The cells were isolated from the digest by centrifugation for 5 min and rinsed twice with phosphate-buffered saline (PBS) containing antibiotics–antimycotics. Finally, after centrifugation for 5 min, the cells were resuspended in a low-conductivity osmotically balanced buffer (LC buffer: 10 mM HEPES, 0.1 mM CaCl 2 , and 59 mM D-glucose in sucrose solution [ 28 , 29 , 30 , 31 ]) for dielectrophoresis experiments. The total cell number was counted using a hemocytometer to adjust the cell concentration prior to the experiments.…”

“…DEP is a phenomenon that occurs under an applied non-uniform electric field, inducing dipoles within a polarized cell in a buffer solution. The cell, in a non-uniform electric field, can be moved by DEP forces toward high or low electric field regions, depending on the relative electric property of cells, which is related to the cell type and function [ 21 , 28 ]. A high electric field is localized at the line-shaped electrodes, and cells are localized to the high or low electric field region due to positive- or negative-dielectric forces depending on their electric properties ( Figure 2 ).…”

“…Albrecht et al reported the encapsulation of living cell arrays using dielectrophoresis and photo-cross-linked hydrogels [ 23 , 24 , 25 ]. In our previous studies, we reported cell manipulation and separation systems [ 28 , 29 , 30 ] and a cell-patterning technology in hydrogels using dielectrophoresis [ 31 ]. However, there have not been any studies on remodeling the mechanical anisotropic tissue based on controlled cellular organization by dielectrophoresis.…”

Dielectrophoresis applications in biomedical field and future perspectives in biomedical technology

“…However, sorting and separation of accurate stem cell phenotype for stem cell transplantation still need to be performed. Recently, the use of DEP for the sorting of differentiated and undifferentiated progeny [95,96], discrimination of stem cell at different developmental stage [95], sorting of stem cell subpopulation from cells [42,97], separation and enrichment of bone marrow‐derived mesenchymal stem cells (BMSCs) from cells [32], separation of target stem cell from contaminant cell (feeder cell) has been studied [29], and separation of viable and nonviable stem cells [98].…”

“…Hence, the DEP parameter can be controlled to prevent the electric field from affecting cell viability. The polarization of cells in a nonuniform electric field is performed to achieve cell separation [27,28], sorting [29,30], and enrichment [31,32].…”