KR20060068979A - Systems of separating cells using ultrasound field and travelling wave dielectrophoresis - Google Patents

Abstract

The present invention relates to a cell separation system capable of efficiently separating cells without damage, and more particularly, to an ultra-fine cell separation system for performing selection and separation of cells using an ultrasonic field and traveling wave electrophoresis.

According to the cell separation system using the ultrasonic field and traveling wave genetic electrophoresis according to the present invention, since the cells are transported in a plane without using a separate channel, the interference of the formation of the ultrasonic field due to the channel can be eliminated, It doesn't happen at all.

In addition, since only the cells move without moving the fluid, the efficiency of cell transfer and separation is high. In addition, while observing the cell separation process through a CCD camera or the like, it is possible to perform cell separation more effectively by performing PID control.

In addition, the present invention can effectively separate cells from contaminants and the like or create a new Lab On a Chip system.

Description

System of Separating Cells Using Ultrasound Field and Traveling Wave Dielectrophoresis

Figure 1 shows the principle of traveling wave electrophoresis used in the present invention.

Figure 2a is a conceptual diagram showing the cell separation device of the cell separation system according to an embodiment of the present invention, Figure 2b is a side view of the cell separation device, Figure 2c is a cell separation system according to an embodiment of the present invention The force vector distribution in the fluid is shown to explain the cell arrangement using the ultrasonic field in the cell separation apparatus.

3 is a plan view showing a cell separation device of a cell separation system according to an embodiment of the present invention in order to show the movement of particles such as cells in more detail.

Figure 4 shows a cell separation system according to an embodiment of the present invention.

The present invention relates to a cell separation system capable of efficiently separating cells without damage, and more particularly, to an ultra-fine cell separation system for performing selection and separation of cells using an ultrasonic field and traveling wave electrophoresis.

Cell separation using genophoresis may be performed by using only positive / positive or traveling wave genophoresis, or by using electrophoretic migration or genophoretic retention that combines genetic and microfluidics. have. The electrophoretic migration or the electrophoretic retention has the disadvantage that the difference in the genetic characteristics between the cells must be somewhat large.

In addition, another cell separation method is a long-flow fraction, which is a method for measuring the separation and size distribution of colloids, particulate matter, and polymers, and is a fast and selective method for separating polymers and fine colloidal particles. Necessity and liquid chromatography have been developed to minimize the adsorption or shear degradation of sample materials induced in the stationary phase. The field-flow fraction may be divided into various types according to the external field or the driving force, sedimentation-flow fraction using centrifugal force, flow-flow fraction using secondary fluid flow, and temperature. The difference is the thermal-flow fraction using thermal diffusion and the electric-flow fraction using electric field, and the physical properties of the sample material, such as molecular weight, density, electrical properties, thermal diffusion coefficient, Stokes radius, etc. Used as Until now, the sediment-flow fraction is the most sensitive technique for separating cells, and there have been cases of neural stem cell separation using the sediment-flow fraction, but it is more suitable for the macro system than the micro system.

Alternatively, there is a microfluidic system using centrifugal force, which has the same shape as a compact disc, and is mainly used for transporting and mixing microfluids for diagnosis, and using a flow switch using a coriolis force generated in a rotating body. There is also a way to apply.

There are many cell separation methods and systems as described above, but using the conventional methods and systems alone has the disadvantages of being expensive, damaging to the cells, or not suitable for micro systems.

Therefore, there is a need for the development of a cell separation system capable of precisely and efficiently performing cell migration and separation without damaging the cells.

The present invention for solving the above problems, is connected to both ends of the upper glass substrate, piezoelectric transducer (transducer) for converting the electrical input from the outside into mechanical vibration applied to the upper glass substrate; And N electrodes arranged on a lower substrate parallel to the upper glass substrate, wherein a gap between the upper glass substrate and the lower substrate is filled with a fluid mixed with cells, and each electrode has a length of the piezoelectric transducer. The N electrodes are arranged in a direction perpendicular to a direction, and the N electrodes include cell separation devices arranged at regular intervals along the longitudinal direction of the piezoelectric transducer. System.

Preferably, the vibration applied to the upper glass substrate by the piezoelectric transducer generates a surface wave propagating through the upper glass substrate and collides with another surface wave propagating in a direction opposite to the traveling direction of the surface wave, thereby causing a cell in the fluid. Particles, such as or the like, generate standing waves that are perpendicular to the longitudinal direction of the piezoelectric transducer and move in a direction perpendicular to the upper glass substrate.

Preferably, the surface waves generate acoustic waves that propagate from the upper glass substrate to the lower substrate in the fluid, and the acoustic waves are reflected from the lower substrate to the upper glass substrate to generate an ultrasonic field in the fluid. .

Preferably, the electrode generates an electric field at predetermined time intervals sequentially from the first electrode to the Nth electrode. Assuming that there are N electrodes, if the first electrode is the electrode disposed on the leftmost side, the second electrode is an electrode disposed on the right side of the first electrode, and the third electrode is disposed on the right side of the second electrode. The Nth electrode will be the rightmost electrode. Of course, on the contrary, when the first electrode is the electrode disposed at the rightmost side, the Nth electrode will be the electrode disposed at the leftmost side.

Also preferably, the cells in the fluid are arranged in M rows parallel to the piezoelectric transducer by acoustic forces by the standing wave. That is, the M arrays are arranged parallel to the longitudinal direction in which the piezoelectric transducers are disposed.

In addition, the cells arranged in the M rows are moved in the direction of the N-th electrode by the electric field generated at regular intervals of time on the electrode. That is, the cells are generated by the electric field sequentially generated from the first electrode to the N-th electrode to move in the direction of the N-th electrode.

In addition, the acoustic force may be changed depending on at least one of the volume, density, and properties of the fluid in which the cells are mixed.

Further, preferably, the acoustic force may be adjusted according to the magnitude of the voltage applied to the piezoelectric transducer.

In addition, preferably, the cell separation system using the ultrasonic field and traveling wave genetic electrophoresis according to the present invention, observe the cell separation process in the cell separation device, the position of the cell separation device when the desired cells are not completely separated By moving, the cell separation control further comprises a PID control for the cell separation process.

Preferably, the cell separation control unit may change the frequency supplied to the cell separation device.

Preferably, the cell separation controller is connected to an illuminator located at the top of the cell separation device, a microscope located at the bottom of the cell separation device, the microscope so that the inside of the cell separation device can be observed. A CCD camera connected to the CCD camera, a video recorder for recording an image in the cell separation device obtained from the camera, and a vertical separation operation in the cell separation device from the recorded image while being connected to the video recorder. It may be configured to include an analyzer and the like to analyze the critical parameters.

Hereinafter, specific embodiments of the present invention will be described with reference to the accompanying drawings.

Figure 1 shows the principle of traveling wave electrophoresis used in the present invention.

Dielectrophoresis works on any material that can be neutral or polarized, unlike electrophoresis, which acts only on materials charged with either an anode or a cathode. In addition, the cells can be transported, separated, fixed, and rotated according to the electrode design, placement, and waveform of applied voltage, and the cells can be manipulated in a relatively simple and inexpensive process. In addition, it has the advantage of having flexibility, controllability and automation for cell subjects, and the specific immunity recognition by antigen-antibody reactions to isolate specific cells. As shown in FIG. 1, when there is a non-uniform electric field and there are polarized particles in the electric field, the dipoles induced in the non-uniform electric field generate a force on the particles. The force generated by the particles allows the particles to move in the electric field. When the particles (eg, cells) polarize more than the surrounding medium (eg, culture), the dipoles of the particles The particles are arranged in a denser region of the electric field, and the particles move to the denser region of the electric field. This phenomenon is called positive dielectrophoresis, and it can be seen that the particles on the left side of FIG. 1 are moved toward the higher density of the electric field by the positive dielectrophoresis.

On the other hand, when the particles are less polarized than the surrounding medium, the dipoles are arranged in areas of low density of the electric field, and the particles are also forced to move to areas of low density of the electric field, which is negative. This is referred to as dielectrophoresis, and indicates that the particle on the right side of FIG. 1 is moving toward the lower side of the electric field by the negative dielectrophoresis.

The movement of the particles in accordance with the density of the electric field is due to the dipoles induced by the force in the electrophoresis, the direction of which is independent of the electric field and only depends on the field gradient of the electric field.

When the particles have a homogeneous sphere shape, the electrophoretic force acting on the particles is determined as follows.

은 주위 매질의 유전율,

은 델 벡터 연산자, E는 전기장의 크기, Re[K(ω)]는 클라우지우스-모소티(Clausius-Mossotti) 인자(factor)의 실수부를 뜻하며, 상기 클라우지우스-모소티 인자는 다음과 같이 정의된다. In Equation 1, r is the radius of the particles,

Is the permittivity of the surrounding medium,

Is the Dell vector operator, E is the magnitude of the electric field, Re [K (ω)] is the real part of the Clausius-Mossotti factor, and the Clausius-Mossottie factor is defined as do.

은 매질의 복소 유전율을,

은 입자의 복소 유전율을 뜻하며, 복소 유전율은 다음과 같이 정의된다. here,

The complex permittivity of the medium,

Is the complex permittivity of the particle, and the complex permittivity is defined as follows.

는 전도율,

은 유전율 및

는 입력 전기장의 주파수이다. At this time,

Is the conductivity,

Permittivity and

Is the frequency of the input electric field.

에 따라 변화하므로, 유전영동 힘의 세기 또한 주파수에 따라 변함을 알 수 있다. 또한, Re[K(ω)]의 크기는 입자가 주위 배양액보다 더 분극화가 잘 되는가에 따라서도 변하게 된다. 따라서, 입자가 주위 매질에 비해 분극화가 잘 되는 경우에는, Re[K(ω)]가 양의 값을 갖는데, 이 경우에 상기 입자는 전기장의 밀도가 높은 곳으로 이동하게 된다. 반대로, 입자가 주위 매질에 비해 분극화가 잘 안 되는 경우, 즉, Re[K(ω)]가 음의 값을 갖는 경우에는, 전기장의 밀도가 낮은 곳으로 상기 입자가 이동하게 된다. From Equation 1, Re [K (ω)] is the frequency of the input electric field.

Since it varies according to, the strength of the dielectrophoretic force also changes with frequency. The size of Re [K (ω)] also varies depending on whether the particles are more polarized than the surrounding culture. Thus, when the particles are more polarized relative to the surrounding medium, Re [K (ω)] has a positive value, in which case the particles move to a higher density of the electric field. In contrast, when the particles are less polarized compared to the surrounding medium, that is, when Re [K (ω)] has a negative value, the particles move to a place where the density of the electric field is low.

If the polarized particles are in a rotating electric field, the induced dipole rotates at the same time as the rotating electric field, and considering that it takes some time to form the dipole, if the rotational speed of the electric field is sufficiently high, Torque is generated in the particles with a delay compared to the change in the electric field. At this time, the direction of rotation of the particles is the direction of rotation of the electric field or vice versa, depending on whether the delay between the electric field and the dipole exceeds 180 degrees.

When the above phenomenon is applied to electrodes and particles arranged linearly on a plane, the particles may be moved. The electrodes may be arranged in parallel at regular intervals like a track of a train, and then the electrodes are arranged in the order in which they are arranged. In the case of generating an electric field in the electrode, if the wave of the electric field generating the electric field is fast enough, it takes time to form a dipole in the particle, so that torque is generated in the particle and the particle follows the electrode. You can move.

It is called traveling wave electrophoresis to allow the particles to travel along the electrodes arranged in a plane as described above, and the cell separation system according to the present invention uses the traveling wave dielectric electrophoresis as described above.

Hereinafter, with reference to Figures 2a and 2b showing a cell separation device of a cell separation system according to an embodiment of the present invention will be described in more detail.

Figure 2a conceptually shows a cell separation device of a cell separation system according to an embodiment of the present invention, Figure 2b is a side view of the cell separation device.

In the cell separation device of the present invention, as shown in FIG. 2B, a piezoelectric transducer 21 is connected to both ends of the upper glass substrate 23, and the piezoelectric transducer 21 is fixed to the clamper 22. The lower substrate 24 together with the upper glass substrate 23 constitutes a chamber, and contains a fluid in which particles such as cells to be separated are mixed. The lower substrate 24 does not necessarily need to be a glass substrate, and may be made of a material such as silicon. However, as shown in FIG. 2B, when the microscope for observing the fluid is installed below the lower substrate 24, the lower substrate 24 may be made of glass.

On the other hand, the cell separation device of the cell separation system according to the present invention, for example, the size of the upper glass substrate 23 is 20 millimeters (mm) * 20 millimeters (mm) * 1 millimeter (mm), the upper glass The distance between the substrate 23 and the lower substrate 24 may be configured to be 200 micrometers (μm), and when the frequency of the electrical input applied to the piezoelectric transducer is 1.2 megahertz (MHz), The length can be configured to be 1.7 millimeters. In this case, the volume of the fluid will be 20 millimeters (mm) * 20 millimeters (mm) * 200 micrometers (μm).

The piezoelectric transducer 21 converts an input electrical input into a mechanical output, that is, vibration, and transmits the electrical input to the connected upper glass substrate 23, wherein the upper glass substrate 23 is not a flexible material. In the case of vibrating at a frequency of sufficient magnitude (for example, 100 to 20 megahertz) despite being a substrate, as shown in FIG. 2A, the upper glass substrate 23 is composed of a flexible material. It has the same effect as it does.

More specifically, by applying an RF voltage to each piezoelectric transducer 21 connected to a function generator for supplying a sinusoidal wave, vibrations having a sufficient magnitude of frequency can be applied to the upper glass substrate. 23), resulting in a surface wave propagating the upper glass substrate surface in the x-axis direction. The surface wave generates sound waves in a nearby fluid, which is a fluid wave. The surface wave collides with another surface wave traveling in a direction opposite to the traveling direction of the surface wave while satisfying a predetermined condition, and changes into a standing wave that does not proceed any further. At this time, the other surface wave traveling in a direction opposite to the traveling direction of the surface wave is generated from the piezoelectric transducer located opposite to the piezoelectric transducer which generated the surface wave. As described above, the surface wave is changed to a standing wave so that particles such as cells in the fluid cannot move in the x-axis direction.

Meanwhile, the fluid wave generated in the fluid by the surface wave travels from the upper glass substrate to the lower substrate direction, that is, the -y axis direction, and the fluid wave reaching the lower substrate, that is, the acoustic wave An ultrasonic field is generated in the fluid as it is reflected back and proceeds toward the upper glass substrate. The arrangement of particles such as cells by the ultrasonic field will be described in more detail with reference to FIG. 2C.

On the other hand, the force generated from the standing wave and applied to particles such as cells in the fluid (hereinafter, 'acoustic force') can be obtained as follows.

는 상기 유체 내의 정재파의 파수(wave number),

는 세포 등의 입자의 반지름, x는 상기 정재파에 의한 입자의 변위, Φ는 속도 포텐셜(velocity potential)의 크기를 의미한다. At this time,

Is the wave number of standing waves in the fluid,

Is the radius of the particles such as cells, x is the displacement of the particles by the standing wave, Φ means the magnitude of the velocity potential (velocity potential).

In Formula 4, F _Y is a density-compressibility element, and can be expressed as follows.

는 상기 유체의 밀도,

는 상기 세포 등의 입자의 밀도,

는 유체 내 음속(speed of sound),

는 세포 등의 입자 내의 음속을 의미한다.

Is the density of the fluid,

Is the density of particles such as cells,

Is the speed of sound in the fluid,

Means the speed of sound in particles such as cells.

By the acoustic force represented by Equation 4, particles such as cells move to the position closest to the position with the minimum acoustic potential energy separated by half the amplitude of the acoustic wavelength. In the same principle as described above, particles such as cells in the fluid are arranged according to the wavelength of the acoustic wave generated in the fluid. As can be seen from Equation 4, the volume, density, and properties of the particles, such as cells, are shown. Since the magnitude of the acoustic force varies depending on such factors, it becomes possible to arrange cells or particles of the same type in each line.

At this time, when the cells need to be moved by a distance of more than half the size of the acoustic wavelength, they are moved through phase modulation or position control of the upper glass substrate. In addition, in order to keep the cells from moving further within the standing wave, the acoustic energy density must be greater than the critical energy density.

On the other hand, arranging the cells or particles of the same type in one line is caused by two-dimensional vibration, for which two piezoelectric transducers connected to the upper glass substrate operate. Alternatively, if one-dimensional vibration is desired, one piezoelectric transducer may be operated, and the three-dimensional vibration may cause particles to collect in an area other than a line.

To recap, the ultrasound field in the fluid generated by vibrating the upper glass substrate of the chamber containing the mixed fluid of the cells and the like at an appropriate frequency by two piezoelectric transducers connected to the upper glass substrate. ) Causes particles such as cells in the fluid to be arranged in a line as shown in FIG. 2A.

In this case, the particles such as cells in the fluid are arranged in a line by the vibration by the ultrasonic field means a line in a state in which the cell separation device according to an embodiment of the present invention in a plan view, and the three-dimensional observation of the fluid In this case, not all cells, etc. are arranged in a line. The fluid of the cell separation device according to the embodiment of the present invention will be described below with reference to FIG. 2C. However, specific formulas relating to the relationship between the wavelength of the ultrasonic wave and the position of the particles to be described by FIG. 2C will be omitted herein.

In FIG. 2C, the y-axis represents the direction in which acoustic waves are generated due to vibration, that is, the direction perpendicular to the upper glass substrate, and the x-axis represents the direction in which particles in the fluid move.

As mentioned above, when a vibration having a sufficient magnitude of frequency is applied to the upper glass substrate (y = 0), a surface wave moving in the x-axis direction is generated on the upper glass substrate, which is in contact with the upper glass substrate. Acoustic waves traveling in the -y axis direction occur in the fluid to generate an ultrasound field in the fluid. The acoustic wave travels in the fluid from the upper glass substrate side toward the lower substrate (y = -0.75), where the reflected wave is generated by the acoustic wave. At this time, the acoustic wave generated by the vibration of the upper glass substrate and the reflected wave reflected from the lower substrate overlap in the fluid. At this time, a force vector capable of moving particles such as cells in the fluid is generated in the fluid, and has a distribution as shown in FIG. 2C, so that particles such as cells in the fluid are collected at the point where the force vectors are collected. The force potential is arranged at a low point (marked with x). Without consideration of the force vector, when viewed in a direction perpendicular to the upper glass substrate and the lower substrate at a point of y> 0, particles such as cells in the fluid are arranged parallel to the z axis at every x value by standing waves. Will be achieved. In this case, the array of particles such as cells arranged parallel to the z-axis does not have only one array for each constant x value, but in reality, the array of particles such as cells for each of the constant x values has a plurality of arrays parallel to the z-axis. This is due to the distribution of the force vectors mentioned above.

For reference, the circular solid line in FIG. 2C is a line connecting points having the same force potential.

3 is a plan view showing a cell separation device of a cell separation system according to an embodiment of the present invention in order to show the movement of particles such as cells in more detail.

FIG. 3 shows a collection port by traveling wave dielectric electrophoresis in a state where cells and the like are already arranged in a line by an ultrasonic field generated by the piezoelectric transducer 31 vibrating the upper glass substrate 33 at an appropriate frequency. collection well) (37).

It can be seen that the seven electrodes 38 are arranged on the lower substrate of the cell separation device, but the number of electrodes 38 according to the present invention is not limited by FIG. 3. In addition, the electrode has a width of, for example, 20 micrometers, and may be configured in the form of a layer of gold (Au) having a thickness of 2000 Angstroms stacked on a layer of 300 Angstroms thick chromium (Cr). The chromium layer may be used to enhance adhesion between the gold layer and the glass substrate.

When the rightmost of the electrodes is called the first electrode and the leftmost electrode is called the seventh electrode, particles such as cells move from the first electrode toward the seventh electrode in the direction of the arrow. The movement of particles, such as, is accomplished by traveling wave dielectrophoresis.

That is, when the electric field is sequentially generated from the first electrode to the seventh electrode, and the speed of electric field generation is sufficiently high, torque is generated on the particles such as the cells, and the particles move toward the seventh electrode. The collected particles are separated through the collection well 37.

Figure 4 shows a cell separation system according to an embodiment of the present invention.

Cell separation system according to an embodiment of the present invention is the amplifier 41, the sine wave generator 42, the illuminator (illuminator) 43, the cell separation device 44, XYZ drive stage 45, the microscope 46 ), A CCD camera 47, a video recorder 48, and an analyzer 49.

The cell separation device 44 is a cell separation device that has already been described in FIGS. 2 (a), 2 (b) and 3, and the electrical input from the amplifier 41 and the sine wave generator 42 By being applied to the piezoelectric transducer of the cell separation device, the arrangement of particles such as cells by the ultrasonic field in the cell separation device is achieved.

The cell separation system according to the present invention uses the illuminator 43, the microscope 46, the CCD camera 47, the video recorder 48 and the analyzer 49 to observe the inside of the cell separation device. When the cell separation process is not performed smoothly, that is, when the cell separation in the cell separation device is not completely performed, the desired cell is separated more completely by using a feedback method to increase the separation efficiency for the cells to be separated. do. In this case, the lower substrate of the cell separation device 44 is preferably a transparent glass substrate.

In addition, the XYZ driving stage 45 is connected to the cell separation device, more specifically, the lower substrate of the cell separation device, so that the cell separation device can move in the X-axis, Y-axis and Z-axis. . On the other hand, the analyzer 49 serves to obtain the critical parameters required for cell manipulation in the ultrasonic field, such as the operation speed of other cells, from the image data recorded in the video recorder.

As described above, the present invention described in the present specification is only an example, and the present invention is not limited by the above description, and simple changes, substitutions, and modifications of the embodiments are not allowed to depart from the scope of the present invention. It will be apparent to those of ordinary skill in the field.

According to the cell separation system using the ultrasonic field and traveling wave genetic electrophoresis according to the present invention, since the cells are transported in a plane without using a separate channel, the interference of the formation of the ultrasonic field due to the channel can be eliminated, It does not occur at all.

In addition, since only the cells move without moving the fluid, the efficiency of cell transfer and separation is high. In addition, while observing the cell separation process through a CCD camera or the like, it is possible to perform cell separation more effectively by performing PID control.

In addition, the present invention can effectively separate cells from contaminants and the like or create a new Lab On a Chip system.

Claims (10)

A piezoelectric transducer connected to both ends of the upper glass substrate, the piezoelectric transducer converting an electrical input from the outside into mechanical vibration and applying it to the upper glass substrate; And N electrodes arranged on a lower substrate parallel to the upper glass substrate, Between the upper glass substrate and the lower substrate is filled with a fluid mixed with cells, each electrode is placed in a direction perpendicular to the longitudinal direction of the piezoelectric transducer, the N electrodes are the longitudinal direction of the piezoelectric transducer Cell separation system using an ultrasonic field and traveling wave electrophoresis, characterized in that it comprises a cell separation device arranged along a predetermined interval along. According to claim 1, The vibration applied to the upper glass substrate by the piezoelectric transducer generates surface waves traveling through the upper glass substrate, and collides with other surface waves traveling in a direction opposite to the traveling direction of the surface waves, thereby causing particles such as cells in the fluid. And a standing wave moving perpendicularly to the longitudinal direction of the piezoelectric transducer and moving in a direction perpendicular to the upper glass substrate. The method of claim 2, The surface wave generates an acoustic wave propagating from the upper glass substrate to the lower substrate in the fluid, and the acoustic wave is reflected from the lower substrate to the upper glass substrate to generate an ultrasonic field in the fluid. Cell Separation System Using Ultrasonic Field and Wave Propagation. According to claim 1, The electrode is a cell separation system using an ultrasonic field and traveling wave dielectric electrophoresis, characterized in that for generating an electric field at a predetermined time interval sequentially from the first electrode to the N-th electrode. The method of claim 3, wherein Cells in the fluid is a cell separation system using an ultrasonic field and traveling wave electrophoresis, characterized in that arranged in M rows parallel to the piezoelectric transducer by the acoustic force by the standing wave. The method of claim 5, The cells arranged in the M columns are moved in the direction of the N-electrode by the electric field generated by a predetermined time interval sequentially on the electrode, the cell separation system using the ultrasonic wave and traveling wave electrophoresis. The method of claim 5, The acoustic force is a cell separation system using an ultrasonic field and traveling wave electrophoresis, characterized in that the value is changed by at least one of the volume, density, and properties of the fluid in which the cells are mixed. The method of claim 5, The acoustic force may be adjusted according to the magnitude of the voltage applied to the piezoelectric transducer. The method according to any one of claims 1 to 8, And a cell separation control unit for observing the cell separation process in the cell separation device and PID control of the cell separation process by moving the position of the cell separation device when the desired cell is not completely separated. Cell Separation System Using Ultrasonic Field and Wave Propagation. The method of claim 9, The cell separation control unit is a cell separation system using an ultrasonic field and traveling wave genetic electrophoresis, characterized in that for controlling the cell separation process by changing the frequency of the voltage supplied to the cell separation device.

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