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

Yuuwa Takahashi 1 and Shogo Miyata 2,*

1 Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan; [email protected] 2 Department of Mechanical Engineering, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan * Correspondence: [email protected]; Tel.: +81-45-566-1827

 Received: 16 June 2020; Accepted: 26 July 2020; Published: 29 July 2020 

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 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.

Keywords: cell sorting; dielectrophoresis; fluid-induced shear force; pluripotent stem cell; feeder cell

1. Introduction Regenerative medicine is a remarkable new approach that restores damaged tissues or organs in the human body by constructing three-dimensional tissues with cells and scaffold material. Pluripotent stem cells (PSCs) are considered as a promising cell source for regenerative medicine because of their high proliferation rate and pluripotency [1,2]. Although PSCs show the potential to differentiate multiple kinds of cells to regenerate biological tissues and organs, it is difficult to establish a PSC culture so as to maintain their pluripotency because it is easily lost by disturbing the cell passage procedure and changing the physical culture conditions (vibration, temperature, etc.). Usually, PSCs have been cultured on feeder cells [2], matrix-coated substrates [3–5], or surface-modified substrates [6–8] to maintain their pluripotency. Recently, cell-adhesive matrix coating has become a major approach to culture PSCs: gelatin or Matrigel for mouse embryonic stem cells (mESCs) and induced pluripotent stem cells (iPSCs), and Matrigel or laminin fragments (LN-511, LN-511-E8, etc.) for human iPSCs [9–12]. However, in the conventional method, the PSC culture is still effective on the feeder cell layer because this method is superior in terms of the proliferation rate and pluripotency stability of the culture. Nevertheless, in the PSC culture on feeder layers, the contamination of feeder cells is not negligible

Micromachines 2020, 11, 734; doi:10.3390/mi11080734 www.mdpi.com/journal/micromachines Micromachines 2020, 11, 734 2 of 13 when the PSCs are corrected for clinical or experimental use, such as cell sampling (gene and protein assay, histological analysis, etc.) and cell passage. Therefore, a simple and rapid method to eliminate feeder cells from the PSC culture in the cell collection process is required. Moreover, it is preferable to perform this method under damage-less and label-free conditions. A major method for cell sorting is fluorescence-activated cell sorting (FACS) [13,14]. For the FACS method, the cells are labeled with fluorescent dyes to distinguish the type and function of cells. The FACS system detects the fluorescence of labeled cells one-by-one; therefore, the sorting accuracy is high. Another method is magnetic-activated cell sorting (MACS) [15]. The MACS system distinguishes cells using a magnetically labeled antibody. These conventional methods are superior in terms of accuracy and efficacy; however, immunological or fluorescent staining is required to label the cells [16–18]. From this perspective, we focused on dielectrophoresis, which handles the movement of cells by electrical force only. Dielectrophoresis (DEP) is one of the most promising approaches for manipulating and separating cells. 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 manipulated by DEP forces to make it move toward high or low electric field regions, depending on the relative electric property of the cells, which is related to the cell type and function. Therefore, the cell type and function can be distinguished based only on electrical properties, without any fluorescent staining or magnetic antibodies. Many studies reported cell sorting devices using DEP phenomena to distinguish and manipulate various cells (stem cell, blood cell, cancer cell, etc.) [19–36]. These studies manipulate or separate the cells based only on the difference in DEP properties of cells, without any fluorescent staining and magnetic antibodies like FACS or MACS systems. Single cell manipulation could be performed using dielectrophoresis [19,20]. As for the cell sorting technology, living cells and other particles could be separated using dielectrophoresis [21–23]. The DEP sorting of living cells according to cell properties (function, kind, size, etc.) has also been performed; however, these approaches required a three-dimensional electrodes array or microfluidic flow control with a numerical simulation and validation [24–34]. In addition, some researchers reported accurate cell sorting methods for blood cells or oocytes [35,36]. However, the continuity of the sorting process was not sufficient. In our previous study, a novel simplified cell manipulating and sorting device using DEP and fluid-induced shear force was developed [37–39]. It consisted of a transparent parallel-lined interdigitated-electrode array on an indium-tin-oxide (ITO)-coated slide glass. The accuracy and simplicity of our sorting device was sufficient; however, the continuity of the sorting was not. The aim of this study is to develop a simple and continuous cell sorting system to discriminate cells based on the difference in DEP properties using a liquid flow control system.

2. Materials and Methods

2.1. Dielectrophoresis For a spherical particle in a non-uniform electric field, the time-averaged DEP force is generated on the particle as: 3 2 F = 2πr ε εmRe[ fcm(ω)] E (1) DEP 0 ∇ rms where r is the radius of the microparticles, ε0 is the vacuum permittivity, εm is the relative permittivity of the surrounding medium, Re[f cm (ω)] is the real part of the Clausius–Mossoti (CM) factor, and Erms is the root mean square value of the imposed electric field [40]. The CM factor is related to the magnitude and direction of the DEP force. If the CM factor is positive, the DEP force caused on the particle is directed toward the region of high electric field intensity (positive-DEP). Conversely, if the CM factor is negative, the DEP force is directed toward the region of low electric field intensity (negative-DEP). Micromachines 2020, 11, 734 3 of 13

The CM factor is expressed as: εp∗ εm∗ fcm(ω) = − (2) εp∗ + 2εm∗ where εp∗ and εm∗ are the complex permittivities of the microparticles and the suspended medium, respectively. Each complex permittivity is defined as follows:

jσ ε∗ = ε ε (3) 0 − ω where ε is the relative permittivity of the particle or surrounding medium, σ is the electrical conductivity, and ω is the angular frequency of the applied AC electric field. This equation shows the dependency of the CM factor on not only the electric properties of the particle and surrounding medium but also on the frequency of the applied AC electric field. The frequency where the direction of the DEP force changes from n-DEP to p-DEP is called the crossover frequency. Our previous study reported that living cells and polystyrene beads could be separated based on DEP properties [19]. Therefore, cells could be distinguished based on differences in dielectrophoresis phenomena.

2.2. Cell Culture In this study, mouse embryonic stem cells (mESCs) and mouse embryonic fibroblast (MEF) cells were used for the DEP cell sorting experiments. The mouse embryonic cell line, ES-B3, was obtained from Riken Bioresource center (Tsukuba, Japan), and the mitomycin C-treated MEF cells were from ReproCELL Inc. (Yokohama, Japan). The ES-B3 cells were cultured in 75-cm2 flasks in Glasgow Modified Essential Medium (GMEM) supplemented with 10% fetal bovine serum (FBS), antimycotics-antibiotics, and 1000 U/mL leukemia inhibitory factor (LIF). The MEFs were cultured in 75-cm2 flasks in GMEM supplemented with 10% FBS and antimycotics-antibiotics. Both cells were incubated in 5% CO2 and 95% humidity at 37 ◦C. Before the DEP experiments, the ES-B3 cells were passaged twice and MEFs were passaged once. Prior to the experiments, the cells were detached from the flasks using 0.05% trypsin and suspended in a low-conductivity buffer (LCB; 10 mM HEPES, 0.1 mM CaCl2, and 59 mM D-glucose in sucrose solution) [37–39]. The concentration of each cell suspension for DEP characterization was 5.0 106 cells/mL, and the mixed ratio of ES-B3 and MEF × cells for the DEP cell-sorting experiment was set at 4:6, according to a conventional on-feeder culture.

2.3. DEP Characterization of ES-B3 and MEF Cells To sort ES-B3 and MEF cells from the mixed cell suspension, the DEP characteristics of ES-B3 and MEF cells were evaluated. To determine the crossover frequency between negative- and positive-DEP, the behavior of each cell was evaluated under various AC voltage frequencies. To cause the DEP phenomenon, a non-equal electric field was generated using transparent conductive glass (Figure1)[37,39] . This chamber consisted of a transparent parallel-line electrode array on a glass substrate, ITO-coated glass, and a silicone rubber gasket. The parallel-line electrode array was fabricated using ITO-coated glass (Geomatec Co., Ltd., Yokohama, Japan) as a conductive substrate. The thickness of the ITO layer was 1500 Å, and the resistance was 5 Ω/sq. The parallel-line electrode was patterned using laser etching techniques. The electrode array was designed to generate a highly non-uniform electric field [37,39]. The width of each electrode line was 20 µm, and the spaces between each electrode were 80 µm (Figure1a). The flow channel was made from a silicon rubber gasket to make a rectangular volume. The DEP chamber was formed by sandwiching the silicon rubber gasket between the parallel-line electrode array and a bare ITO-coated slide glass drilled with holes for the fluidic inlet and outlet. The thickness of the silicon rubber gasket was 500 µm. The cells were moved toward the electrodes by p-DEP and between electrodes by n-DEP in the DEP chamber (Figure1b). The AC electric field was applied between the parallel-line electrode array and bare ITO-coated glass, using a function generator (WF1974, NF Corp., Yokohama, Japan) and amplifier (BA4850, NF Corp., Micromachines 2020, 11, x 4 of 13

observed using a phase-contrast microscope (Nikon Eclipse TE300, Nikon, Tokyo, Japan) with a digital video camera. For the DEP characterization, ES-B3 or MEF cell suspension in LCB was injected and subjected to an AC electric field 180 s after injection. The magnitude of the imposed AC voltage was 20 Vp-p, and the frequency was varied from 10 kHz to 1 MHz. The behavior of ES-B3 and MEF cells was observed by the digital camera on the microscope, and microphotographs were captured 180 s after each AC voltage frequency was imposed. The captured images were trimmed to 200 μm × 300 μm, and the number of cells on the electrodes (positive-DEP) and between the electrodes (negative-DEP) were counted. The ratio of cells indicating positive-DEP in the chamber was calculated to evaluate the crossover frequency. The dielectrophoretic property of a cell (indicating p-DEP or n-DEP) was assessed based on the region where the cell moved (Figure 1c). The frequency dependency of the DEP property was also evaluated as: Micromachines 2020, 11, 734 4 of 13 Ratio of cells indicating positive-DEP = NP/(NP + NN) (4)

where NP and NN are the number of cells indicating positive-DEP and negative-DEP, respectively. Yokohama, Japan). The applied voltage was monitored by an oscilloscope (TDS1001B, Tektronix, When the ratio of cells indicating positive-DEP is 50%, the frequency of the AC voltage is considered Beaverton, OR, USA) connected in parallel. The movements of the cells within the DEP chamber were a transition point from negative-DEP to positive-DEP. In this study, the crossover frequency of the observed using a phase-contrast microscope (Nikon Eclipse TE300, Nikon, Tokyo, Japan) with a digital DEP is defined as the frequency at which the ratio of cells indicating positive-DEP is 50%. video camera.

Figure 1. Dielectrophoretic characterization of living cells: (a) Schematic of dielectrophoresis (DEP) Figure 1. Dielectrophoretic characterization of living cells: (a) Schematic of dielectrophoresis (DEP) chamber;chamber; (b) Positive- (b) Positive- and negative- and negative- DEP of DEP living of cells;living (c cells;) Discrimination (c) Discrimination of positive- of positive- and negative-DEP. and negative- DEP. For the DEP characterization, ES-B3 or MEF cell suspension in LCB was injected and subjected to an AC electric field 180 s after injection. The magnitude of the imposed AC voltage was 20 Vp-p, and the frequency was varied from 10 kHz to 1 MHz. The behavior of ES-B3 and MEF cells was observed by the digital camera on the microscope, and microphotographs were captured 180 s after each AC voltage frequency was imposed. The captured images were trimmed to 200 µm 300 µm, × and the number of cells on the electrodes (positive-DEP) and between the electrodes (negative-DEP) were counted. The ratio of cells indicating positive-DEP in the chamber was calculated to evaluate the crossover frequency. The dielectrophoretic property of a cell (indicating p-DEP or n-DEP) was assessed based on the region where the cell moved (Figure1c). The frequency dependency of the DEP property was also evaluated as:

Ratio of cells indicating positive-DEP = NP/(NP + NN) (4) where NP and NN are the number of cells indicating positive-DEP and negative-DEP, respectively. When the ratio of cells indicating positive-DEP is 50%, the frequency of the AC voltage is considered a Micromachines 2020, 11, x 5 of 13

2.4. Continuous Cell Sorting Using DEP and Fluid Shear Forces The DEP cell-sorting system consisted of a custom-made syringe pump with a PC control system, two syringes filled with the cell-mixed suspension containing ES-B3 and MEF cells and LCB solution, and the DEP chamber described in Section 2.3, (Figure 2a). The outlet of the DEP chamber wasMicromachines connected2020 to, 11 two, 734 outlet ports, named port A (waste port) and port B (cell-collector port), through5 of 13 the switching channel. The DEP chamber was also connected to syringes containing cell suspension andtransition LCB solution. point from The negative-DEP custom syringe to positive-DEP.pump controll Ined this the study,flow rate the of crossover the cell frequencysuspension of and the LCB DEP solution.is defined The as thetwo frequency syringes were at which connected the ratio to ofeach cells other indicating by a silicone positive-DEP rubber istube 50%. (diameter: 1 mm) and introduced into the DEP chamber. The DEP chamber for cell sorting had the same transparent parallel-lined2.4. Continuous electrode Cell Sorting array Using on the DEP lower and Fluid surface Shear of Forcesthe chamber as described in Section 2.3. The flow channel of the DEP cell sorting chamber was made from a polydimethylsiloxane (PDMS) The DEP cell-sorting system consisted of a custom-made syringe pump with a PC control system, polymer sheet with a molded channel shape (Figure 2b). The DEP chamber was formed by a PDMS two syringes filled with the cell-mixed suspension containing ES-B3 and MEF cells and LCB solution, sheet sandwiched between the ITO glasses. The flow channel was designed to be 3 mm long from the and the DEP chamber described in Section 2.3 (Figure2a). The outlet of the DEP chamber was inlet to the electrode array, 5 mm wide, and 100 μm high. The length of the flow channel was designed connected to two outlet ports, named port A (waste port) and port B (cell-collector port), through to ensure that whole cells were dropped on the electrode array during the cell-sorting procedure [20]. the switching channel. The DEP chamber was also connected to syringes containing cell suspension DEP and fluid-flow-based cell sorting was performed as described below (Figure 3). First, the and LCB solution. The custom syringe pump controlled the flow rate of the cell suspension and LCB cell-mixed suspension was injected into the chamber at a flow rate of 0.024 mL/min, and an AC solution. The two syringes were connected to each other by a silicone rubber tube (diameter: 1 mm) electric field was imposed on the electrode array to cause p-DEP in MEF cells and n-DEP or neutral and introduced into the DEP chamber. The DEP chamber for cell sorting had the same transparent in ES-B3 cells (Step 1). In Step 1, the MEF cells were trapped on the electrodes, while the ES-B3 cells parallel-lined electrode array on the lower surface of the chamber as described in Section 2.3. The flow passed through the electrode array and were introduced into port B. After the line-electrode array channel of the DEP cell sorting chamber was made from a polydimethylsiloxane (PDMS) polymer was almost filled with the MEF cells, the AC voltage was switched off to remove the p-DEP force sheet with a molded channel shape (Figure2b). The DEP chamber was formed by a PDMS sheet (Step 2). Next, the bulk LCB buffer was injected at a flow rate of 2.4 mL/min to release the trapped sandwiched between the ITO glasses. The flow channel was designed to be 3 mm long from the inlet MEF cells from the electrode array, and they were introduced into port A (Step 3). To repeat this to the electrode array, 5 mm wide, and 100 µm high. The length of the flow channel was designed to sorting procedure from steps 1 to 3, the MEF cells could be continuously eliminated from the ES- ensure that whole cells were dropped on the electrode array during the cell-sorting procedure [20]. B3/MEF cell-mixed suspension.

(a)

(b)

FigureFigure 2. 2. (a(a) )Experimental Experimental setup setup for for the the DEP DEP cell-sorting cell-sorting system system combined combined with with the the fluid-control fluid-control system;system; ( (bb)) Flow Flow channel channel of of the the dielectropho dielectrophoreticretic chamber chamber and and electrode electrode array.

DEP and fluid-flow-based cell sorting was performed as described below (Figure3). First, the cell-mixed suspension was injected into the chamber at a flow rate of 0.024 mL/min, and an AC electric field was imposed on the electrode array to cause p-DEP in MEF cells and n-DEP or neutral in ES-B3 cells (Step 1). In Step 1, the MEF cells were trapped on the electrodes, while the Micromachines 2020, 11, 734 6 of 13

ES-B3 cells passed through the electrode array and were introduced into port B. After the line-electrode array was almost filled with the MEF cells, the AC voltage was switched off to remove the p-DEP force (Step 2). Next, the bulk LCB buffer was injected at a flow rate of 2.4 mL/min to release the trapped MEF cells from the electrode array, and they were introduced into port A (Step 3). To repeat this sorting procedure from steps 1 to 3, the MEF cells could be continuously eliminated from the ES-B3/MEF cell-mixedMicromachines suspension. 2020, 11, x 6 of 13

STEP 1 DEP ON Inject cell suspension LCB Trapping MEF using p-DEP force

Cell suspension

Port A Port B

STEP 2 Inject LCB LCB Trapping MEF using DEP Collecting all mESc in the DEP chamber to Port B

Cell suspension

Port A Port B

STEP 3 DEP OFF LCB Inject LCB Collecting MEF in Port A

Cell suspension

Port A Port B

FigureFigure 3. 3.Sorting Sorting procedure procedure using using positive positive dielectrophoresis dielectrophoresis and and fluid-flow fluid-flow control. control.

Before the cell-sorting experiment, the DEP chamber was degassed and sterilized. To sterilize the Before the cell-sorting experiment, the DEP chamber was degassed and sterilized. To sterilize chamber, the flow channel was filled with 70% ethanol for 5 min and washed twice with LCB solution. the chamber, the flow channel was filled with 70% ethanol for 5 min and washed twice with LCB The DEP chamber was also filled with 0.2% bovine serum albumin (BSA) solution for anti-cell adhesion solution. The DEP chamber was also filled with 0.2% bovine serum albumin (BSA) solution for anti- in the flow channel. Following the sterilization of the DEP chamber, the cell-mixed suspension was cell adhesion in the flow channel. Following the sterilization of the DEP chamber, the cell-mixed suspension was introduced into the chamber at a flow rate of 0.024 mL/min. In the sorting experiment, the AC electric field was set as 20 Vp-p at 110 kHz to cause p-DEP for MEF cells only. To evaluate the purification efficiency of the DEP cell-sorting system, the number of injected cells (Nin) and those of ports A and B (NA, NB) were counted (Figure 4). The number of injected cells containing both ES-B3 and MEF cells was evaluated in order to compute the average number of cells passing through the first line of the electrode-array for 10 s. The numbers of cells in ports A and B were counted using a hemocytometer. The ES-B3 and MEF cells were distinguished based on the differences in the cell diameter; the diameter of ES-B3 cells ranged from 5–8 μm, and that of MEF cells ranged from 10–20 μm. The purification ratio of DEP cell sorting was determined so as to measure

Micromachines 2020, 11, 734 7 of 13 introduced into the chamber at a flow rate of 0.024 mL/min. In the sorting experiment, the AC electric

fieldMicromachines was set as 2020 20 ,V 11p-p, x at 110 kHz to cause p-DEP for MEF cells only. 7 of 13 To evaluate the purification efficiency of the DEP cell-sorting system, the number of injected cellsthe (N existingin) and thoseratio of of ES-B3 ports cells A and in Bport (NA B, N(collectingB) were counted port). Moreover, (Figure4). the The continuity number of of injected cell sorting cells was containingevaluated both so as ES-B3 to measure and MEF the cells purification was evaluated ratio at in multiple order to cycles compute of our the DEP average cell-sorting number system. of cells passing through the first line of the electrode-array for 10 s. The numbers of cells in ports A and B were counted using a hemocytometer. The ES-B3 and MEF cells were distinguished based on the differences Port A in the cell diameter; the diameter of ES-B3 cells ranged from 5–8 µm, and that of MEF cells ranged Micromachines 2020, 11, x (N ) 7 of 13 Injected cell A Purity = NB/(NA + NB) from 10–20 µm. The purification ratioDEP ofchamber DEP cell sorting was determined so as to measure the existing ratio of ES-B3number cells in port B (collecting port). Moreover, the continuity of cell sorting was evaluated so the existing ratio of ES-B3 cells in(Cell port sorting) B (collecting port). Moreover, the continuityCell loss = of N cellin -( sortingNA + N Bwas) (Nin) asevaluated to measure so theas to purification measure the ratio purification at multiple ratio cycles at multiple of ourPort DEP cycles B cell-sorting of our DEP system. cell-sorting system. (NB)

Port A (N ) Injected cell Figure 4. Evaluation of the purity of mouse ESCsA and cell lossPurity of sorted = N cells.B/(NA + NB) DEP chamber number (Cell sorting) 3. Results(N ) and Discussion Cell loss = Nin -(NA + NB) in Port B 3.1. DEP Characterization of ES-B3 and MEF (NB)

The ES-B3 cells showed n-DEP under 90 kHz of the AC electric field, whereas p-DEP was over 130 kHz (FigureFigureFigure 5). 4.The 4.Evaluation Evaluation MEF showed of of the the purity p-DEPpurity of of continuo mouse mouse ESCs ESCsusly and andfrom cell cell 10 loss loss kHz of of sorted sortedto 1 MHz cells. cells. of the AC electric field (Figure 6). The ratios of ES-B3 and MEF cells indicating p-DEP are shown in Figure 7. The 3. Results and Discussion 3.crossover Results and frequency Discussion of ES-B3 cells was considered approximately 110 kHz, whereas that of MEF cells 3.1.was DEP under Characterization 10 kHz. of ES-B3 and MEF 3.1. DEPThe Characterization dielectrophoretic of ES-B3 property and MEF of living cells depends mainly on the electric property and The ES-B3 cells showed n-DEP under 90 kHz of the AC electric field, whereas p-DEP was over diameterThe ES-B3 of the cells cells showed [40]. Theref n-DEPore, under it was 90 kHzconsidered of the ACthat electric ES-B3 field,and MEF whereas cells p-DEPshowed was different over 130 kHz (Figure5). The MEF showed p-DEP continuously from 10 kHz to 1 MHz of the AC electric field 130dielectrophoretic kHz (Figure 5). propertiesThe MEF showedbecause p-DEP of their continuo differencuslye in from cell 10size kHz and to functionality. 1 MHz of the Based AC electric on this (Figure6). The ratios of ES-B3 and MEF cells indicating p-DEP are shown in Figure7. The crossover fielddifference (Figure in 6). the The DEP ratios property, of ES-B3 the ES-B3 and MEF and MEFcells cellsindicating could bep-DEP distinguis are shownhed under in Figure an AC 7. voltage The frequency of ES-B3 cells was considered approximately 110 kHz, whereas that of MEF cells was under crossoverof 110 kHz, frequency where theof ES-B3 DEP force cells ofwas the considered ES-B3 cell approximatelywas negligible 110and kHz, the MEF whereas cells thatshowed of MEF a positive cells 10 kHz. wasDEP under and were10 kHz. trapped on the electrode array. The dielectrophoretic property of living cells depends mainly on the electric property and diameter0s of the cells [40]. Therefore, it was considered that ES-B3 and MEF cells showed different dielectrophoretic properties because of their difference in cell size and functionality. Based on this difference in the DEP property, the ES-B3 and MEF cells could be distinguished under an AC voltage of 110 kHz, where the DEP force of the ES-B3 cell was negligible and the MEF cells showed a positive DEP and were trapped on the electrode array.

0s 60 s

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10 kHz 110 kHz 500 kHz 60 s FigureFigure 5. Dielectrophoresis5. Dielectrophoresis of of ES-B3 ES-B3 cells cells using using a a parallel parallel line-electrode line-electrode arrayarray beforebefore (0(0 s) and after after (60 (60s) s) dielectrophoresis. dielectrophoresis.

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Figure 5. Dielectrophoresis of ES-B3 cells using a parallel line-electrode array before (0 s) and after (60 s) dielectrophoresis.

Micromachines 2020, 11, 734 8 of 13 MicromachinesMicromachines 2020 2020, 11, 11, x, x 8 8of of 13 13

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Figure 6. Dielectrophoresis of MEF cells using a parallel line-electrode array before (0 s) and after FigureFigure 6. 6. Dielectrophoresis Dielectrophoresis of of MEF MEF cells cells using using a aparallel parallel lin line-electrodee-electrode array array before before (0 (0 s) s) and and after after (60 (60 (60 s) dielectrophoresis. s)s) dielectrophoresis. dielectrophoresis.

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2020 RATIO OF CELLS INDICATING P-DEP RATIO OF CELLS INDICATING RATIO OF CELLS INDICATING P-DEP RATIO OF CELLS INDICATING 0 0 Ratio of cells indicationg p-DEP (%) Ratio of cells indicationg p-DEP (%) 1010 50 50 70 70 90 90 110 110 130 130 150 150 200 200 500 500 1000 1000 FrequencyFrequencyFREQUENCYFREQUENCY (kHz) (kHz) (KHZ) (KHZ)

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Micromachines 2020, 11, x 9 of 13

3.2. Purification Efficacy of DEP Cell-Sorting Device Micromachines 2020, 11, 734 9 of 13 In this study, a cell-sorting experiment on mESCs and feeder cells was performed using a DEP device combined with a fluid-flow control system, and the purification efficacy of mESCs was The dielectrophoretic property of living cells depends mainly on the electric property and evaluated. During one cycle of cell sorting, the ES-B3 cells passed through the line-electrode array diameter of the cells [40]. Therefore, it was considered that ES-B3 and MEF cells showed different into port B; meanwhile, the MEF cells were trapped on the electrode array by p-DEP force and the dielectrophoretic properties because of their difference in cell size and functionality. Based on this trapped MEF cells were collected into port A (Video S1). Figure 8 shows the photomicrograph at each difference in the DEP property, the ES-B3 and MEF cells could be distinguished under an AC voltage step of the DEP cell-sorting process. In step 1, the ES-B3/MEF-mixed cell suspension was injected into of 110 kHz, where the DEP force of the ES-B3 cell was negligible and the MEF cells showed a positive the DEP chamber (Figure 8a). Following step 1, the ES-B3 cells passed the line-electrode array, DEP and were trapped on the electrode array. whereas the MEF cells were trapped on the electrode array by the p-DEP force against the fluid- 3.2.induced Purification shear force Efficacy (step of DEP 2; Figure Cell-Sorting 8b). After Device 80% of the electrode array was covered by the trapped cells (Figure 8c), the p-DEP force was turned off in order to release and collect the MEF cells into port A (stepIn this 3; Figure study, 8d). a cell-sorting Figure 8 shows experiment the purification on mESCs ratio and feederof ES-B3 cells cells was in performedport B for multiple using a DEP cell- devicesorting combined cycles. The with measured a fluid-flow purification control system, ratio of and ES-B3 the purification cells was about efficacy 59% of before mESCs cell was sorting evaluated. and Duringincreased one to cycle about of 94% cell sorting,in port B the after ES-B3 the cellsfirst cycl passede of throughcell sorting. the line-electrodeThe purification array ratio into decreased port B; meanwhile,to approximately the MEF 90% cells during were five trapped cycles on (Figure the electrode 9). The array decrease by p-DEP in the force purity and of the ES-B3 trapped cells MEF was cellsderived were from collected the residual into port cells A in (Video the corners S1). Figure of the8 showschamber the or photomicrograph the connective regions at each of stepthe silicone of the DEPrubber cell-sorting tubes. However, process. the In steppurity 1, of the ES-B3 ES-B3 cells/MEF-mixed reached a cell plateau suspension at approximately was injected 90% into and the did DEP not chamberchange after (Figure 10 cycles8a). Following of cell sorting step 1,(data the not ES-B3 show cellsn). passedTherefore, the line-electrodethe continuity array,and efficacy whereas of theDEP MEFcell-sorting cells were can trapped be maintained on the electrode if a large array amount by theof cell p-DEP sorting force is against required. the fluid-induced shear force (step 2;There Figure are8 b).superior After 80%conventional of the electrode cell-sorting array me wasthods, covered such by as theFACS trapped and MACS, cells (Figure for evaluating8c), the p-DEPthe cell force type was or turnedcell function off in orderquickly to releaseand accurately. and collect Although the MEF these cells intomethods port A are (step effective 3; Figure for8 d).cell Figuresorting,8 showsthe labeling the purification of cells by ratioantibody of ES-B3 or fluorescent cells in port dye B is for required multiple [4,5]. cell-sorting Our newly cycles. developed The measuredDEP cell-sorting purification system ratio does of not ES-B3 require cells antibodies was about, fluorescence 59% before cell dyes, sorting magnetic and beads, increased or expensive to about 94%equipment. in port B In after addition, the first the cycle process of cell of sorting. our DEP The ce purificationll-sorting system ratio decreased was simple to approximatelywhen compared 90% to duringother DEP five cell-sorting cycles (Figure systems9). The [41–46], decrease with in theno puritycomplicated of ES-B3 processes cells was and derived with a fromhigh theextensibility. residual cellsFurthermore, in the corners our ofDEP the cell-sorting chamber or system the connective does not regions require of a thenumerical silicone analysis rubber tubes. of the However, fluid-flow the in puritythe DEP of ES-B3chamber. cells By reached using a plateaumultiplex at DEP-chamber approximately or 90% by andextending did not the change width after of the 10 cycleschamber, of cell the sortingthroughput (data notof the shown). DEP cell-sorting Therefore, thesystem continuity could andbe larger. efficacy Therefore, of DEP cell-sorting our DEP-cell can besorting maintained system ifwould a large be amount applicable of cell for sorting research is required.and clinical uses requiring a large number of purified cells.

a

b Feeder cells (trapped)

mES cells (through)

Figure 8. Cont.

MicromachinesMicromachines2020 2020, 11, 11, 734, x 1010 of of 13 13 Micromachines 2020, 11, x 10 of 13 c c Feeder cells (trapped)Feeder cells (trapped)

Feeder cells d (DEPFeeder off, cells collected(DEP off, into d wastecollected port) into waste port)

200 ∝m 200 ∝m

Figure 8. Purification of mESCs (ES-B3) from the ES/feeder cell-mixed suspension using DEP and Figure 8. Purification of mESCs (ES-B3) from the ES/feeder cell-mixed suspension using DEP and fluid-flowFigure 8. Purificationcontrol. (a) Injectionof mESCs of (ES-B3)mixed-cell from suspension; the ES/feeder (b) Trapping cell-mixed of suspensionMEF cells by using positive-DEP DEP and fluid-flow control. (a) Injection of mixed-cell suspension; (b) Trapping of MEF cells by positive-DEP forcefluid-flow and purification control. (a) ofInjection ES-B3 cells of mixed-cell from mixed-cell suspension; suspension; (b) Trapping (c) Covering of MEF of cells 80% byof thepositive-DEP electrode force and purification of ES-B3 cells from mixed-cell suspension; (c) Covering of 80% of the electrode arrayforce byand MEF purification cells; (d) ofRelease ES-B3 ofcells trapped from mixed-cellMEF cells. suspension; (c) Covering of 80% of the electrode array by MEF cells; (d) Release of trapped MEF cells. array by MEF cells; (d) Release of trapped MEF cells. 1.2 injected cell number MEF 1.2 1 injected cell number ES-B3MEF 93% ES-B3 1 94% 0.8 93% 88% 89% 94% 87% 0.8 88% 89% 87% 0.6 (cells) 0.6

(cells) 0.4 0.4 0.2 0.2 Cell number portCell of sorted B cells in 0 Cell number portCell of sorted B cells in 0 12345 12345Sort cycle (-) Sort cycle (-) Figure 9. Purification ratio of ES-B3 cells in the sorted-cell suspension and total cell loss compared to theFigure injected 9. Purification cell number. ratio of ES-B3 cells in the sorted-cell suspension and total cell loss compared to theFigure injected 9. Purification cell number. ratio of ES-B3 cells in the sorted-cell suspension and total cell loss compared to There are superior conventional cell-sorting methods, such as FACS and MACS, for evaluating the injected cell number. the4. Conclusions cell type or cell function quickly and accurately. Although these methods are effective for cell sorting, the labeling of cells by antibody or fluorescent dye is required [4,5]. Our newly developed 4. Conclusions DEP cell-sortingIn this study, system a novel does DEP not requirecell-sorting antibodies, device fluorescenceusing dielectrophoresis dyes, magnetic and beads,fluid-induced or expensive shear equipment.force Inwas this developed Instudy, addition, a novelin order the DEP process to cell-sortingsort of mouse our DEP embryonicdevice cell-sorting using cells dielectrophoresissystem and feeder was cells. simple and As whenfluid-induced a result, compared the feeder shear to othercellsforce were DEP was cell-sorting eliminateddeveloped fromin systems order the ES/feeder [to41 sort–46], mouse with mixed-cell no embryonic complicated suspension cells processes and by feederDEPand combined cells. with As a with higha result, fluid-induced extensibility. the feeder Furthermore,shearcells were force. eliminated Our our device DEP from cell-sorting could the alsoES/feeder system increase mixed-cell does the notpurity require suspension of the anumerical mixed by DEP cell combined analysis suspension of with the in fluid-flowfluid-induceda continuous in mannershear force. in order Our todevice process could a larg alsoe amount increase of the cell pu suspension.rity of the mixed cell suspension in a continuous manner in order to process a large amount of cell suspension.

Micromachines 2020, 11, 734 11 of 13 the DEP chamber. By using a multiplex DEP-chamber or by extending the width of the chamber, the throughput of the DEP cell-sorting system could be larger. Therefore, our DEP-cell sorting system would be applicable for research and clinical uses requiring a large number of purified cells.

4. Conclusions In this study, a novel DEP cell-sorting device using dielectrophoresis and fluid-induced shear force was developed in order to sort mouse embryonic cells and feeder cells. As a result, the feeder cells were eliminated from the ES/feeder mixed-cell suspension by DEP combined with fluid-induced shear force. Our device could also increase the purity of the mixed cell suspension in a continuous manner in order to process a large amount of cell suspension.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-666X/11/8/734/s1, Video S1: Whole process of DEP cell sorting, from step 1 to step 3. Author Contributions: Conceptualization, S.M.; methodology, S.M. and Y.T.; investigation, Y.T.; resources, S.M.; data curation, Y.T.; writing—original draft preparation, Y.T.; writing—review and editing, S.M.; visualization, S.M.; supervision, S.M.; project administration, S.M.; funding acquisition, S.M. Both authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a research grant (Adaptable and Seamless Technology Transfer Program through Target-driven R&D) from JST. Acknowledgments: The authors are grateful to Shinya Takeuchi of Nissha Co., Ltd. for fabricating the micro transparent electrodes. Conflicts of Interest: The authors declare no conflicts of interest.

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