Cell Pairing Using Microwell Array Electrodes Based on

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Cell pairing using microwell array electrodes based on dielectrophoresis Yuki Yoshimura, Masahiro Tomita, Fumio Mizutani, and Tomoyuki Yasukawa Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 20 Jun 2014 Downloaded from http://pubs.acs.org on June 27, 2014

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Analytical Chemistry

Cell pairing using microwell array electrodes based on dielectrophoresis

Yuki Yoshimura, a Masahiro Tomita, b Fumio Mizutani, a* Tomoyuki Yasukawa a*

a

Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori,

Ako, Hyogo 678-1297, Japan b

Division of Chemistry for Materials, Graduate School of Engineering, Mie University,

1577 Kurima-Machiya-cho, Tsu, Mie 514-8507, Japan

*CORRESPONDING AUTHOR: Tomoyuki Yasukawa Tel: +81-791-58-0171; Fax: +81-791-58-0493 E-mail: [email protected]

Fumio Mizutani E-mail: [email protected]

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Abstract We report a simple device with an array of 10,000 (100 × 100) microwells for producing vertical pairs of cells in individual microwells with a rapid manipulation based on positive dielectrophoresis (p-DEP). The areas encircled with micropoles which fabricated from an electrical insulating photosensitive polymer were used as microwells. The width (14 µm) and depth (25 µm) of the individual microwells restricted the size to two vertically aligned cells. The DEP device for the manipulation of cells consisted of a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower microwell array electrode fabricated on an ITO substrate. Mouse myeloma cells stained in green were trapped within 1 s in the microwells by p-DEP by applying an alternating current voltage between the upper ITO and the lower microwell array electrode. The cells were retained inside the wells even after switching off the voltage and washing with a fluidic flow. Other myeloma cells stained in blue were then trapped in the microwells occupied by the cells stained in green to form the vertical cell pairing in the microwells. Cells stained in different colors were paired within only 1 min and a pairing efficiency of over 50% was achieved.

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Introduction Precise pairing of two heterogeneous cells with a high efficiency is an important step to produce cell couplets by cell fusion. Chemical methods such as avidin-biotin recognition have frequently been used to produce pairs of two different types of cells,1, 2 but undesired pairing and multiple cell formation in suspension resulted in a low efficiency for the generation of reliable pairs. Alternating current (AC) electric fields have also been used to achieve cell alignment and contact before a direct current pulse was applied for cell fusion.3-5 The phenomenon of particle movement induced by the polarization in non-uniform AC electric fields is widely known as dielectrophoresis (DEP).6,

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The DEP technique has been widely applied in biological fields for the

detection of bacteria,8-12 trapping of biomacromolecules (DNA13-15 and proteins15,16), fractionation of blood cells,17-20 sorting of cells,21-25 and patterning with cells.26-29 Micro-orifice structures between two opposite electrodes in a microfluidic channel were developed for cell parting and fusion.30,

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The strong electric field

produced at the micro-orifice guided cells to small openings by positive-DEP (p-DEP). The throughput for pairing cells was dramatically improved on using a sheet with an array of micro-orifices,32 but a relatively long incubation time was required to remove

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couplets from the micro-orifices. The use of protruding microelectrodes incorporated into microchannels has also been reported for pairing cells.33-37 The application of an AC voltage to electrodes prepared on sidewalls of the channel directed cells towards the edges of microelectrodes by p-DEP to produce cell pairs. However, it is difficult to form pairs of two different cells at individual protruding electrodes. Recently, a p-DEP-based device was reported by Matsue’s group.38 Cells were captured on one side of gourd-shaped microwells on interdigitated band array (IDA) electrodes. The excess cells were removed, and the other types of cells were trapped on the adjacent microwells. However, it seems to be difficult to obtain the couples by applying an electric pulse voltage through the IDA electrodes because the electric field may not be concentrated on the positions in close contact with the cell pairs. Microwell structures fabricated on conductive materials were also used to direct cells to wells prior to implementing cell fusion.39, 40 Recently, we have also reported the formation of an array of cell pairs preliminarily.41 Both vertical and horizontal alignments were formed in the microwells due to the relative large size of the microwells. Moreover, the number of prepared microwells was only 400 (20 × 20). In the study described here, p-DEP was employed to guide cells to individual 10,000 (100 × 100) microwells with the width of

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single cell and depth of two single cells and the vertical alignment of two cells stained in different colors was achieved with high efficiency. Vertical alignment should be advantageous for the effective cell fusion due to the vertical electric field concentrated onto the close contact of vertically aligned pairs of cells.

Experimental Section Fabrication of a dielectrophoretic pairing device The dielectrophoretic patterning device consisted of an indium tin oxide (ITO) electrode and an ITO microwell array electrode that was fabricated by conventional photolithography. The mask plate for the micropole array with 10,404 (102 × 102) with clear square (length of sides 27 µm) and the microwell array with 10,000 (100 × 100) black circles (diameter 16 µm) bridged with black rectangles (length 16 µm) is designed (Figure 2A). The distance between centers of the neighboring circles was set at 32 µm. Micropole array was fabricated by negative photoresist (SU-8 3025, MicroChem Corp., Newton, MO) to used the areas enclosed with micropoles as microwells. A 30-µm thick polyester film (Nitto Denko, Osaka, Japan) was used as a spacer to fabricate the fluidic

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channel and this was sandwiched between the upper ITO electrode and the lower ITO electrode of the microwell array. Both the upper and lower electrodes were treated for 3 h with 2-methoxy(polyethyleneoxy)propyltrichlorosilane (Gelest, Inc., Morrisville, PA) to prevent the non-specific adsorption of cells.

Pairing of myeloma cells stained in two different colors in individual microwells Mouse myeloma cells (average diameter 12 µm) stained with 5-(and -6)-carboxyfluorescein diacetate, succinimidyl ester (CFDA SE, green fluorescence, Thermo Fisher Scientific K.K.) and cells stained with Hoechst 33342 (blue fluorescence, Sigma-Aldrich Japan) were prepared to demonstrate the cell pairing in microwells. A cross-sectional view of the DEP device and the method of cell pairing are schematically represented in Figure 1. A suspension (4.0 × 107 cells mL-1) of cells stained in green with CFDA SE in 250 mM sucrose solution (conductivity, 0.2 mS m–1) was introduced into the fluidic channel of the device (Figure 1A). An AC voltage (10 V peak-to-peak (10 Vpp)) in the p-DEP frequency region (1.0 MHz) was immediately applied between the upper and lower electrodes to provide the alternating electric field in the channel by the function generator (7075, Hioki E.E. Co., Ueda, Japan). A strong electric field was

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formed in the microwells due to the presence of micropoles caused by the insulating photoresist, which had a low dielectric constant (approximately 3).42 Under these conditions, cells were directed into the microwells by the attractive force of p-DEP (Figure 1B). Fresh 250 mM sucrose solution was injected into the channel to remove excess cells 10 s after the AC voltage was applied (Figure 1C). A suspension of cells (4.0 × 107 cells mL–1) stained in blue with Hoechst 33342 was subsequently introduced into the channel of the device (Figure 1D). The attractive force of p-DEP was again used to transport the cells into the microwells occupied with the cells stained in green (Figure 1E). The efficiency of the cell occupation in the microwells was defined as the ratio of the average number of microwells with cells to the total number of microwells. Manipulation of the cells by p-DEP was performed under an optical and fluorescent microscope (IX72, Olympus, Tokyo, Japan) equipped with a charge-coupled device camera (DP72, Olympus).

Results and Discussion Fabricated microwell array electrode

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Microscopy images of the fabricated micropoles and microwells are shown in Figures 2B and 2C. Microwells with a square shape (approximately 14 × 14 µm) were formed between the SU-8 micropoles, which had a circular shape (30 µm diameter). A cross-sectional image of the array electrode along the line in Figure 2C is shown in Figure 2D. Valley structures were formed between the SU-8 micropoles by removing the photoresist in the development step of the lithography process. These valleys correspond to the rectangular bridges in the photomask design. The depth of the microwells was estimated from the image of the valleys, and a value of 25 µm was obtained. The size of the microwells was slightly smaller than that in the mask design. The decrease in the valley shape and size may be due to diffraction of the UV light at small notches in the design edges. In contrast, incomplete wells, with a thickness of 25 µm, were fabricated on the SU-8 layer using the microwell array design (16 µm diameter) without the bridges. Thus, the introduction of bridges to fabricate micropoles allowed the fabrication of microwells with a high depth-to-diameter aspect ratio by conventional photolithography with a negative photoresist (SU-8 3025) due to the smooth supply of developer.

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Cell trapping in microwells by p-DEP Dielectrophoretic trapping of mouse myeloma cells in the microwell array was studied using the DEP device. A suspension of myeloma cells stained in green was introduced into the device and an AC voltage was subsequently applied to guide the cells to the microwells by p-DEP. A fluorescence microscopy image before application of the voltage is shown in Figure 3A. Cells were randomly dispersed in the channel and were carried from the bottom to the top by the slight fluidic flow (Supporting Information, Movie). On applying the AC voltage (10 Vpp), the dispersed cells immediately, i.e., within 1 s, stayed at positions with microwells. The image obtained after the sucrose solution was injected into the channel is shown in Figure 3B. In the microwells trapped with two cells, almost all cells in the top component were removed by the fluidic flow, but cells in the bottom component were retained in the microwells because the fluidic flow could not remove cells from microwells with a depth of 25 µm, i.e., twice the cell diameter or more. An optical image of cells trapped in microwells by p-DEP is shown in Figure 3C. It can be seen that individual cells were trapped within the microwells enclosed with micropoles. These results clearly indicate that the cell array could be formed on the microwell array electrode easily and rapidly by p-DEP.

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In the investigation of the applied voltage for p-DEP manipulation of cells, the application of 10 Vpp was required to guide cells to the bottom of microwells. The application of low voltage resulted in the incomplete trapping, and hence a removal of the trapped cells from the microwells after the voltage was switched off. However, the application of voltage over 15 Vpp resulted in the destruction of cell membrane and elution of cytoplasm. Subsequently, we investigated the effect of the electric fields on cell viability by dynamic cell staining with 4', 6-Diamidino-2-phenylindole, dihydrochloride (DAPI). Myeloma cells suspended in 250 mM sucrose solution containing 1.5 µM DAPI for 20 min were trapped in microwells by p-DEP (10 Vpp) and subjected to the AC voltage during 20 s. The ratio of DAPI-positive (dead) cells which was stained in blue, was estimated immediately after the application of the AC voltage and 20 min after the AC voltage was switched off and found to be approximately 4% and 6%, respectively. The ratios evaluated were nearly the same as those of the stained cells in the original cell suspensions. The results suggested that cell viability is not adversely affected under the p-DEP manipulation in this work.

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The fluorescence intensity along the arrowed column in Figure 3B is shown in Figure 4A. There are twenty microwells in the column. Fluorescence signals were observed at identical intervals, and these correspond to stained cells trapped in microwells. Clear signals were not observed from the positions between the microwells. These results indicate that almost all of the excess cells were removed from the channel after flushing with the fluidic flow. The optical and fluorescence images for the ninth and seventh microwells from the left-hand side in Figure 4A, respectively, are shown in Figures 4B and C. A fluorescence signal was not observed from the ninth well because a cell was not trapped in this location (Figure 4B), whereas a signal was clearly obtained from the single cell trapped in the seventh well (Figure 4C). The signal obtained from the fourth well, in which two cells were trapped, had a slightly higher intensity than that obtained from wells in which single cells were trapped, because two cells were vertically trapped in a single well (Figure 4D). We investigated the relationship between the number of cells trapped in the single microwells and the relative fluorescence intensities obtained for the 520 individual microwells. It was found that relative fluorescence intensities were obtained in the range of 100–225 when single cells were trapped in the individual microwells. In this analysis, the average

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brightness obtained from a single pixel (0.64 × 0.64 µm) was used as a relative fluorescence intensity. The cell occupation efficiency of the microwells with single cells was estimated and found to be 65−85% from ten data values. The difference of the initial concentrations of cells presented at the area on the microwell array could be the major cause of the large difference of efficiencies. No trapping of cells was observed without the p-DEP attractive force in this experimental period and more, i.e., 30 min.

Pairing of cells stained in different colors in microwells by p-DEP Fluorescence microscopy images of paired cells trapped in microwells are shown in Figures 5A and 5B. Myeloma cells stained with different colors were successively guided to the microwells. Cells stained in blue were directed to microwells occupied with cells stained in green. Images were obtained after the removal of excess cells to downstream. During cell removal, the p-DEP attractive force was applied in order to retain the cells as these were the top component in each pair. Both types of cells, i.e., those stained in green and in blue, were trapped in the microwells. In this experiment, the cell occupation efficiencies in the microwells were 67% and 75% for green and blue cells, respectively. A combination of the images in Figures 5A and 5B is

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shown in Figure 5C. Green, blue, and light blue signals were observed from the microwells. A detail of the fluorescence image of single-cell pairs trapped in single microwells, representing the rectangular area in Figure 5C, is shown in Figure 5D. Clear light blue signals can be seen in the first and second microwells from the top, while green and blue signals can be seen from the third and fourth microwells, respectively. The rectangles shown in Figures 5A and 5B correspond to the microwells in Figure 5C. The green signals from the first, second, and third microwells (Figure 5A) can clearly be seen, as can the blue signals from the first, second, and fourth microwells (Figure 5B). The light blue color indicates that a green cell and a blue cell are vertically aligned in the microwell to form single-cell pairs. Cells stained in different colors can be paired within only 1 min with a pairing efficiency of 53%. Only two cell manipulation cycles of 10 s are required along with a step to remove the excess cells for 20 s. Thus, the use of the present device allows large numbers of cell pairs (over 5,000 pairs) to be easily and rapidly produced. The vertical alignment of cell pairs would be a distinct advantage for producing couplets by cell fusion using electric pulse fields generated between the upper electrode and the lower microwell array electrode, which were used for the cell pairing by p-DEP. Thus, we aim to optimize the scale of the microwells in order to

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significantly improve the efficiency of vertical cell pairing for applications in biological fields such as a reprogramming in hybrids and hybridoma engineering.

Conclusions A device with an array of 10,000 microwells (100 × 100) was fabricated to form vertically aligned pairs of cells by p-DEP. The use of the design with an array of micropoles allowed the fabrication of microwells between micropoles with the appropriate width and depth for the vertical alignment of two cells. The formation of a relatively strong electric field from the microwells to the main channel resulted in the rapid (within 1 s) navigation of cells from the channel to the microwells. Vertically aligned cell pairings can be achieved by the trapping of two cells stained in different colors in microwells. Two cells can be paired with a pairing efficiency of approximately 50%. The time required for the formation of the array of cell pairs is as short as 1 min. Therefore, large numbers of cell pairs (over 5,000 pairs) can be prepared rapidly. The vertical electric field generated by the upper and lower electrodes can be concentrated onto the close contact of vertically aligned pairs of cells with a spherical shape. As a

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consequence, efficient cell fusion to produce cell couplets is feasible using this device. The simple structure of the device described here is also advantageous in that the numbers of microwells could be increased for high-throughput operation.

Acknowledgements This work was partly supported by a Grant-in-Aid for Scientific Research (No. 24360339) and also by a Grant-in-Aid for Scientific Research Innovative Areas “Bio Assembler” of The Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Supporting information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure captions Figure 1. Cross-section of the dielectrophoresis (DEP) device and the cell pairing method using positive-DEP (p-DEP) manipulation. (A) Injection of the suspension of cells stained in green into the device. (B) Trapping of cells by the p-DEP attractive force. (C) Removal of excess cells. (D) Injection of other cells stained in blue into the device. (E) Trapping of cells in microwells already occupied by cells trapped in the first step with p-DEP.

Figure 2. (A) The photomask and (B) a fabricated microwell array electrode. (C) Enlarged image of Figure 2B. (D) Cross-section of the microwell array along the solid line in Figure 2C.

Figure 3. Fluorescence microscopy images (A) before the application of the voltage and (B) after the sucrose solution was injected into the channel to remove excess cells. (C) Cells trapped in microwells by p-DEP.

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Figure 4. (A) Fluorescence intensity along the arrowed column in Figure 3B. Optical and fluorescence images for (B) the ninth well (without a cell), (C) seventh well (with a single cell), and (D) fourth well (with two cells).

Figure 5. Fluorescence microscopy images of the paired myeloma cells stained (A) in green and (B) in blue. (C) Combined image from Figures 5A and 5B. (D) Enlargement of the rectangular area in Figure 5C.

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Figure 1. Cross-section of the dielectrophoresis (DEP) device and the cell pairing method using positive-DEP (p-DEP) manipulation. (A) Injection of the suspension of cells stained in green into the device. (B) Trapping of cells by the p-DEP attractive force. (C) Removal of excess cells. (D) Injection of other cells stained in blue into the device. (E) Trapping of cells in microwells already occupied by cells trapped in the first step with pDEP. 83x74mm (300 x 300 DPI)



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Figure 2. (A) The photomask and (B) a fabricated microwell array electrode. (C) Enlarged image of Figure 2B. (D) Cross-section of the microwell array along the solid line in Figure 2C. 91x99mm (300 x 300 DPI)


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Figure 3. Fluorescence microscopy images (A) before the application of the voltage and (B) after the sucrose solution was injected into the channel to remove excess cells. (C) Cells trapped in microwells by p-DEP. 28x9mm (300 x 300 DPI)



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Figure 4. (A) Fluorescence intensity along the arrowed column in Figure 3B. Optical and fluorescence images for (B) the ninth well (without a cell), (C) seventh well (with a single cell), and (D) fourth well (with two cells). 83x49mm (300 x 300 DPI)


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Figure 5. Fluorescence microscopy images of the paired myeloma cells stained (A) in green and (B) in blue. (C) Combined image from Figures 5A and 5B. (D) Enlargement of the rectangular area in Figure 5C. 83x39mm (300 x 300 DPI)



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82x38mm (300 x 300 DPI)


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Cell Pairing Using Microwell Array Electrodes Based on

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