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Separation of Viable and Nonviable Animal Cell Using Dielectrophoretic Filter Masaru Hakoda Dept. of Chemical and Environmental Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, Japan Yoshikazu Wakizaka Center for Advanced Science and Innovation, Osaka University, Osaka, Japan Yusuke Hirota Dept. Environment Management, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, Japan DOI 10.1002/btpr.394 Published online February 8, 2010 in Wiley Online Library (wileyonlinelibrary.com). Selective separation of cells using dielectrophoresis (DEP) has recently been studied and methods have been proposed. However, these methods are not applicable to large-scale separation because they cannot be performed efﬁciently. In DEP separation, the DEP force is effective only when it is applied close to the electrodes. Utilizing a DEP ﬁlter is a solution for large-scale separation. In this article, the separation efﬁciency for viable and nonviable cells in a DEP ﬁlter was examined. The effects of an applied AC electric ﬁeld frequency and the gradient of the squared electric ﬁeld intensity on a DEP velocity for the viable and nonviable animal cells (3-2H3 cell) were discussed. The frequency response of the DEP velocity differed between the viable and the nonviable cells. We deducted an empirical equation that can be used as guiding principle for the DEP separation. The results indicate that the viable and the nonviable cells were separated using the DEP ﬁlter, and the best operating condiC 2010 American Institions such as the applied voltage and the ﬂow rate were discussed. V tute of Chemical Engineers Biotechnol. Prog., 26: 1061–1067, 2010 Keywords: bioseparation, dielectrophoresis, animal cell, AC electric ﬁeld Introduction The dielectrophoresis (DEP) phenomenon was studied in detail by Pohl1 in 1960. DEP is the motion of dielectric particles caused by polarization effects in a nonuniform electric ﬁeld. This motion depends on the dielectric properties of the particle and medium, particle size, as well as the gradient of the squared electric ﬁeld intensity (!E2), which depends on the applied voltage and electrode geometry.1,2 Recently, the dielectric properties of bioparticles have been analyzed using the DEP phenomenon to identify methods of selective manipulation and separation.3 DEP has many useful biotechnological applications, for example, in the separation of viable and nonviable yeasts from their mixture, 4,5 in the separation of blood cells,6–8 and in the manipulation and the separation of submicron particles such as latex particles and viruses.9–11 For more details, see Refs. 12–15. However, the DEP force is effective only when it is close to the electrode because !E2 decreases rapidly away from the electrode. Therefore, these have not been applied to large-scale separations because they are the separation in the narrow domain on a micro device. However, the DEP ﬁlter has the large volume of separation products, and the possibility of continuous operation compared with the micro device. Few studies on separation devices using the DEP ﬁlter have been reported.16–21 Docoslis et al.16,17 used an etched Correspondence concerning this article should be addressed to M. Hakoda at [email protected] C 2010 American Institute of Chemical Engineers V silicon wafer between the electrode gaps to develop a ﬁlter, thereby resulting in continuous separation. Authors discussed the electrode geometries to improve the separation efﬁciency of the DEP ﬁlter.20,21 On the basis of the aforementioned paper, the electrodes of the DEP ﬁlter of present research were made, and the separation experiments were performed. This ﬁlter has a selective retention capability for viable cells, which is caused by a negative-DEP force that moves toward the low-electric ﬁeld region. It retains only the viable cells in mixture cells and discharges the nonviable cells. The purpose of this research was to examine the practicality of a cell separator that used the DEP ﬁlter. The effects of the experimental conditions on the separation efﬁciency were examined using a wire–wire type DEP ﬁlter. In addition, the electrical difference between viable and nonviable cells was clariﬁed by measuring the dielectric characteristic of animal cells. On the basis of those results, separation of the viable and nonviable cells was performed using the DEP ﬁlter. Moreover, the DEP separation by the difference in the activity of the cells was examined. Theory A time average dielectrophoresis force FDEP is applied in particles in the nonuniform AC electric ﬁeld and is theoretically shown by the following equation4: FDEP ¼ 2pr3 eM Re½KðxÞ rE2 (1) 1061 1062 Biotechnol. Prog., 2010, Vol. 26, No. 4 Figure 2. Conceptual diagram of viable and nonviable cell separation by dielectrophoretic ﬁlter. Materials and Methods Materials Figure 1. Schematic diagrams of experimental apparatus for dielectrophoretic velocity measurement and the electrode geometry. where r is the particle radius, eM is the real part of the medium permittivity, and E is the electric ﬁeld intensity. Re[K(x)] indicates the real part of the Clausius-Mossotti function and is given by the following equations: KðxÞ ¼ ep eM ep þ 2eM (2) where e* p and e* M are the complex permittivity of the particle and the medium, respectively. Re[K(x)] depends on the frequency of the applied electric ﬁeld and also on the dielectric property of the particles and medium. When Re[K(x)] [ 0, the particles move toward the high electric ﬁeld region, called the positive DEP. On the other hand, when Re[K(x)] \ 0, the particles move toward the low electric ﬁeld region, called the negative DEP. !E2 depends on the electric ﬁeld intensity, the electrode shape, and the shape of the device. Stokes law is shown by the following equation: Fdrag ¼ 6pg rv (3) where Fdrag is the drag force, g is the viscosity of the medium, and v is the particle velocity. For small particles and ignoring the Brownian motion and buoyancy force, it can be assumed that the DEP force and the drag force are equal. Therefore, the DEP velocity can be given by the following equation: vDEP ¼ r eM Re½KðxÞ rE2 3g 2 (4) As seen in Eqs. 1 and 4, the most important factors for determining the DEP force and the DEP velocity are Re[K(x)] and !E2. The !E2 is another factor that greatly depends on the applied voltage and the electrode shape. The DEP force is inﬂuenced by the electrode geometry and the geometry of the separation device. However, !E2 decreases abruptly with increase of the distance from the electrode. Mouse hybridoma 3-2H3 cells (RCB0867, Riken Gene Bank, Japan) of free-ﬂoating cells, HeLa cells (RCB0007, Riken Gene Bank, Japan), and MDCK cells (03-360, Dainippon Sumitomo Pharmaceutical Co., Ltd., Japan) of anchorage-dependent cells were used. The growth medium used was DMEM (D6429, Sigma Co., Ltd.) supplemented with 10% FCS, 100 mg/L streptomycin sulfate, and 100 U/mL crystalline potassium penicillin G. The 3-2H3 cells were cultured in a petri dish at 310 K in a humidiﬁed 5% CO2 incubator. The cells were suspended in isotonic solution consisting of 8.5 %(w/v) sucrose plus 0.3 %(w/v) dextrose buffer, and the cell concentrations were adjusted to about 106 cells/mL. The nonviable cells were obtained by an autoclave (SS-325, TOMY) at 80 C for 10 min. The cell concentration was measured using a hemocytometer. Measuring method for DEP velocity Schematic diagrams of the experimental apparatus for the DEP velocity measurement and the geometry of the electrodes are shown in Figure 1. The electrodes were made of nickel, and both the electrode wires were 50 lm in diameter. The distance between the wire-wire electrodes installed horizontally is 350 lm. To measure the DEP velocity of the cells, the position of individual cells was recorded as a function of time in an arbitrary area divided into 50 lm units. An ac voltage was applied to the electrodes using a function generator (Model 33250A, Agilent Technologies, USA). The movement of a single cell subjected to the DEP force was observed by a CCD camera system (CCD Micro Scope Inf500, Moritex Co., Japan and Trinitron, SONY Co., Japan). All experiments were carried out in a temperature controlled room at 298 1 K. DEP ﬁlter apparatus Conceptual diagram of the separation of viable and nonviable cell separation by the DEP ﬁlter (cross sectional view) is shown in Figure 2. This ﬁlter has a selective retention capability for viable cells that is caused by a negative-DEP force which moves toward the low electric ﬁeld region. On the other hand, since nonviable cell has small DEP force, it passes through between the DEP ﬁlters by the ﬂow of liquid. The cells are separated by this method close to the electrode which the DEP force is effectively acted. A schematic diagram of the cell separation apparatus with the DEP ﬁlter and a photograph of the ﬁlter electrode are Biotechnol. Prog., 2010, Vol. 26, No. 4 1063 Figure 3. Schematic diagrams of dielectrophoretic separation apparatus and photograph of ﬁlter electrode. (a) Schematic diagram of the experimental apparatus for cell separation. (b) Side viewof DEP ﬁlter unit. (c) Photograph of wire electrode for DEP ﬁlter. shown in Figure 3. The upper part of the DEP ﬁlter unit consisted of a circulation liquid gateway, a permeation liquid exit, and the ﬁlter electrode part (Figure 3b). The electrodes were made of nickel, and both the electrode diameter and electrode gap were 200 lm (Figure 3c). Moreover, the diameter of the permeation part of the DEP ﬁlter was 10 mm. The DEP ﬁlter unit middle part and the lower part consisted of a silicon rubber spacer of 2 mm in thickness and an acrylic resin plate, respectively. A cell suspension in a reservoir was sent to the ﬁlter unit with a peristaltic pump, released at from Exit B and returned to the reservoir again (Figure 3a). The permeation ﬂowing rate to the DEP ﬁlter was kept constant with another pump connected to Exit A. The retention ratio of cells was deﬁned by the following equation: Retention ratio ½% ¼ XB 100 XA þ XB (5) where, XA and XB are the cell concentrations at Exit A and Exit B, respectively. Results and Discussions Effect of frequency on DEP velocity To examine the optimum frequency for the separation of viable and nonviable 3-2H3 cells, the DEP velocity was measured by using the apparatus shown in Figure 1. For the viable cells, cells that were in the logarithm growth period 1064 Biotechnol. Prog., 2010, Vol. 26, No. 4 Figure 4. Effect of applied electric ﬁeld frequency on dielectrophoretic velocity. Figure 5. Effect of !E2 on dielectrophoretic velocity for viable cells. of 3 days after seeding were used, and the nonviable ones were obtained by heating the viable cells at 356 K for 10 min. The effect of frequency on the DEP velocity of the 32H3 cells is shown in Figure 4. The nonviable cells displayed a weak positive-DEP at the frequency range of 1 kHz–1 MHz. On the other hand, the viable cells have shown a negative-DEP at 10 kHz or less and a positive-DEP over 100 kHz. This result indicates that viable and nonviable cells are separable under the experimental conditions for a frequency of 10 kHz or less using the DEP ﬁlter. Effect of !E2 on DEP velocity To examine the inﬂuence of !E2 on the DEP velocity for the viable cells, the DEP velocities of the cells that move between the wire electrodes were measured. The relation between the DEP velocity and !E2 in a certain position between the electrodes was measured. The factor !E2, which affects the DEP force, was analyzed using 2D ﬁnite element analysis software (Maxwell 2D, Ansoft Corp., UAS). Figure 5 shows the inﬂuence of the analytic value of !E2 on the DEP velocity measured under the conditions of the frequency of 1 kHz and applied voltage of 14 Vpp. Based on these experimental results, the empirical equation was obtained: Figure 6. Effect of frequency on retention ratio for viable cells. (Cultivated 3 days, viability: more than 90%). They found that the DEP velocity is equal to Eq.4. Therefore, the constant (3 1017) must be equal to (r2eMRe [K(x)])/3g in Eq.4. 2 mL/min, and permeation ﬂow rate of 0.4 mL/min. The experimental results are shown in Figure 6. The retention ratio decreased with the increase in frequency and in the case of 1 kHz was the highest. This result agrees with the measurement results of the DEP velocity shown in Figure 4. The aforementioned result shows that the optimum frequency for the retention of viable cells was 1 kHz. Effect of frequency on the retention of viable cells Effect of applied voltage on viable cell retention The retention ratios of viable 3-2H3 cells were measured using the DEP separation apparatus shown in Figure 3. Cells at 3 days after seeding are deﬁned as viable ones. The cell separation experiments were carried out under the conditions: frequency of 1, 5, and 10 kHz, circulation ﬂow rate of The effect of the applied voltage on the retention ratio of the 3-2H3 viable cells was examined under a frequency of 1 kHz. The experiment was carried out at 2 mL/min circulation ﬂow rate, 0.4 or 0.8 mL/min permeation ﬂow rate. The experimental results are shown in Figure 7. The retention vDEP ¼ 3 1017 rE2 (6) Biotechnol. Prog., 2010, Vol. 26, No. 4 1065 Figure 8. Effect of frequency on retention ratio. Figure 7. Effect of applied voltage on retention ratio for viable cells. (Mixture cell suspension of cultivated 3 days and 5 days cells, viability: 55%) (Cultivated 3 days, viability: more than 90%). ratios were over 50% regardless of the applied voltage. As deﬁned in Eq. 5, the retention ratio becomes 50% when the cells concentration at Exit A and that at Exit B are equal. At the permeation ﬂow rate of 0.4 mL/min, the retention ratio increased over the applied voltage of 20 Vpp and the retention ratio of 95% was obtained at 40 Vpp. At the permeation ﬂow rate of 0.8 mL/min, the retention ratio increased over 30 Vpp, and the retention ratio was 95% at 50 Vpp. In the cell separation using the DEP ﬁlter, it is thought that the minimum voltage holding the cells can be estimated by Eq. 6. As a result, the critical voltage necessary for retaining the viable cells was 20 Vpp under the condition of a frequency of 1 kHz and a permeate ﬂow rate of 0.4 mL/min, and it was 27 Vpp under the condition of a permeate ﬂow rate of 0.8 mL/min. The experimental results of the applied voltage on which the retention rate of the viable cells increased agreed well with the critical voltage obtained by Eq. 6. In the case of the cell separation using the DEP ﬁlter, Eq. 6 was effective for deciding the permeation ﬂow rates and the applied voltage, etc. Figure 9. Effect of frequency on retention ratio. Separation of viable and nonviable cells using the DEP ﬁlter The viable cells and the nonviable cells were separated using the DEP ﬁlter. Authors reported that there are relationship between the dielectric characteristic and the growth activity in the cultivation process of the 3-2H3 cells.22 Cells cultured for 3 days after seeding were chosen as the viable ones and cells cultured for 5 days after seeding were chosen as the nonviable cells. In the separation experiment of the viable and nonviable cells, both of the cells were adjusted to the same concentration. The circulation ﬂow rate was 2 mL/min, and the permeation ﬂow rate was 0.4 mL/min. The experimental results are shown in Figure 8. Since the retention ratios of the nonviable cells were 50–60%, it is concluded that the nonviable cells permeated the DEP ﬁlter without the action of the DEP (Cultivated 5 days, viability: 20%). force. On the other hand, the retention ratio of the viable cells was around 80%, so the viable and the nonviable cells were separated by using the DEP ﬁlter. The mixture suspension of cells cultured for 3 days and for 5 days was separated using the DEP ﬁlter. However, the retention ratios for the viable cells in the experiment using the mixture suspension shown in Figure 8 were lower than those in the experiments using only the cells cultured for 3 days in Figure 6. To examine this phenomenon, the cells cultured for 5 days were separated under the same conditions, and the experimental results are shown in Figure 9. The retention ratios of the nonviable cells were about 50–60%, which agrees well with the results of the mixture suspension shown in Figure 8, 1066 Biotechnol. Prog., 2010, Vol. 26, No. 4 Figure 10. Dielectrophoretic behaviors of various species of viable and nonviable cells. and the nonviable cells were not retained with the DEP ﬁlter. On the other hand, the retention ratios of the viable cells cultured for 5 days were about 60%, and the viable cells were not almost retained either. The retention ratios of the viable cells of the mixture cells suspension cultured for 3 days and 5 days were lower than that of the cells cultured for 3 days. This is because the viable cells cultured for 5 days permeated the DEP ﬁlter. As a result of the microscopy, no difference in size could be observed in viable cells cultured for 3 days and the viable cells cultured for 5 days. From the results, it found that there are ‘‘dielectric differences’’ between the viable cells cultured for 3 days and that for 5 days. Moreover, the results shown in Figure 8 show that there was no inﬂuence of cell size on the retention ratios of the viable and nonviable cells since the retention ratios of both the viable cells and the nonviable cells were not inﬂuenced by the applied voltage. The aforementioned results suggest that the differences of the DEP force between the viable and the nonviable cells are not the difference in the cell size but the differences in the electrical characteristics of the cells. Moreover, it was proved that the electrical characteristics of the cells are inﬂuenced by the cultivation days of the cells. The DEP characteristic of various viable and nonviable cells To examine the possibility of separating cells of a different species, the direction of the DEP was observed using the apparatus in Figure 1. The effects of the frequency on the DEP characteristics of the 3-2H3 cells, the MDCK cells, and the HeLa cells are shown in Figure 10. Consequently, in the three species of the cells, the DEP characteristic of the viable cells and the nonviable cells differed. Furthermore, a speciﬁc frequency that gives negative-DEP force only to the viable cells existed. It has to be proven that the viable cells and the nonviable cells can be separated using the DEP ﬁlter even in cells other than the 3-2H3 cells. Moreover, it is con- sidered that separation by cell species is possible, since the DEP characteristics differed according to the cells species. Conclusions This article shows that separation by retaining only the viable cells by negative DEP and removing the nonviable cells is possible using the DEP ﬁlter. The results of the measurement of the DEP velocity using a wires-type electrode unit indicate that the optimum frequency for separating the viable and the nonviable 3-2H3 cells is 1 kHz. Furthermore, an empirical equation for separation using the proposed DEP ﬁlter was obtained. In the separation experiment using the DEP ﬁlter, the retention ratio of the viable cells was maximum at the frequency of 1 kHz. The empirical equation was an indicator for determining the permeation ﬂux and the applied voltage. 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