Abstract:
Disclosed is a cell culture vessel of the present invention, which is composed of a space enclosed by a housing that supports a medium and a cell attachment part, disposed on the bottom surface of the space, that attaches and supports the cells, the cell attachment part having a cell immobilizing mechanism that guides the cells in the space to the cell attachment part to immobilize there and a cell detachment mechanism that detaches the cells attached in the cell attachment part, the cell immobilizing mechanism having a step of applying a voltage to an electrode disposed in the cell attachment part to generate a heterogeneous electric field in the space, and the cell detachment mechanism having a step of applying a voltage to an electrode disposed in the cell attachment part to induce electrolyte in the space.

Description:
TECHNICAL FIELD 
     The present invention relates to a cell culture vessel and a culture device. In particular, the present invention relates to a method for improving incubation efficiency. 
     BACKGROUND ART 
     Regenerative medicine has gotten a lot of attention as an innovative medical treatment, which enables basic remedy for damaged and/or defective cells, tissues, and organs. The regenerating tissue used for regenerative medicine, which is produced through the steps of collecting cells from the body of a patient or the other person; separating and purifying the collected cells in vitro, and growing and organizing the cells into tissue, is transplanted into the body of the patient. Tissue engineering, making advances yearly, has enabled the methods for forming one kind of cells into a sheet and for arranging several kinds of cells sterically to assemble an organ by artificial means to be developed. 
     To amplify therapeutic cells, in particular adherent cells in large quantities, an incubator large in area is useful. It is because adherent cells are amplified while expanding in the planar direction. On the other hand, it has a problem that as the area of an incubator becomes larger, its culture surface increasingly deforms; thereby, cells aggregate in a lower area, leading to deteriorated usage efficiency. As an effective technique for manipulating cells, electrophoresis has gotten attention. The systematic study and theoretical analysis of electrophoresis were set out by Pohl in 1970s (see Nonpatent Literature 1). Micro biological substances, such as bacteria and cells, have been already employed as a principal target to be manipulated since the initial study; accordingly, biotechnology is one of key applications of electrophoresis. 
     A dielectrophoretic force F DEP  exerted on dielectric particles is given by the following equation 1 (see Nonpatent Literature 1). In the following paragraph, how to calculate is explained taking an example of dielectric particles being cells.
 
[Mathematical formula 1]
 
 F   DEP =2πα 3 ∈ 0 ∈ m   Re[K]∇E   2   (Formula 1)
 
     Where a is the radius of a cell approximated to a spherical shape, ∈ 0 : electric permittivity in vacuum, ∈ m : specific electric permittivity in medium, E: electric field intensity, and ∇ is an operator representing a gradient. In this case, ∇E 2 , which is the gradient for the square of an electric field intensity (E 2 ), indicates how degree E 2  inclines at that point, namely, how suddenly the electric field spatially changes. K is called a Claudius-Mossotti number and is represented by an equation 2. Herein, assuming that ∈ b * and ∈ m * be complex dielectric constants for cells and a medium, respectively, and Re [K] be the real part of the Claudius-Mossotti number, Re [K]&gt;0 represents positive electrophoresis and the cells migrate in the same direction as that of the electric field gradient, namely toward an electric field concentration part. Re [K]&lt;0 represents negative electrophoresis and cells migrate in the direction apart from the electric field concentration part, namely toward a weak electric field part. 
     
       
         
           
             
               
                 
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     Formula 3 generally represents complex dielectric constant ∈ r     *   . 
     
       
         
           
             
               
                 
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     Where, ∈ r  is the specific electric permittivity for a cell or medium, σ is the electric conductivity of a cell or medium, and ω is the angular frequency of an applied electric field. As known from Formulae 1, 2, and 3, a dielectrophoretic force depends on the radius of a cell, the real part of a Claudius-Mossotti number, and an electric field intensity. Moreover, it is known that the real part of the Claudius-Mossotti number varies depending on the complex electric permittivity and electric field frequency of a cell and medium. 
     The DEPIM method, combining dielectrophoresis and impedance measurement, has been proposed as a method for measuring microbial counts using dielectrophoresis. The DEPIM method is characterized in that these parameters are appropriately selected and a positive dielectrophoretic force exerted on microorganisms is sufficiently increased to collect the microorganisms into an electrode gap, making electrical measurement to determine a microbial count in the sample solution (see Nonpatent Literature 2). 
     In addition, a culture device, which eliminates unnecessary cells from a cell suspension using negative dielectrophoresis to culture necessary cells at high concentrations, is disclosed (see Patent Literature 1 and Patent Literature 3). 
     Moreover, a method and apparatus, for collecting cells efficiently in a target area without losing the activity of functional cells using positive dielectrophoresis, is disclosed (see Patent Literature 2). 
     CITATION LIST 
     Patent Literature 
     
         
         Patent Literature 1: Japanese Unexamined Patent Application Publication No. 2009-291097 
         Patent Literature 2: Japanese Unexamined Patent Application Publication No. 2008-54511 
         Patent Literature 3: US Patent Application Publication No. 2008/0057505, Specification 
       
    
     Nonpatent Literature 
     
         
         Nonpatent Literature 1: H. Pohl: Dielectrophoresis, Cambridge University Press, Cambridge (1978) 
         Nonpatent Literature 2: J. Suchiro, R. Yatsunami, R. Hamada, M. Hara, J. Phys. D: Appl. Phys. 32 (1999) 2814-2820 
       
    
     SUMMARY OF INVENTION 
     Technical Problem 
     However, it is difficult to manipulate cells and microorganisms directly in an ion-rich culture solution (namely, high electric conductivity) using dielectrophoresis described in BACKGROUND. For this reason, generally, the target cells are moved in an ion-poor buffer solution, manipulated, and then returned back in the original culture solution. As a result, a cell manipulation process is complicated, causing a problem of increased stress on the cells due to a change in culture environment. In addition, this method has another problem that generally, an enzyme is used to detach the cells grown during surface culture from the surface of culture medium, increasing load on the cells. 
     An object of the present invention is to simplify the cell manipulation process to reduce the stress on the cells, as well as the load on the grown cells exerted when detached from the surface of the culture medium in order to solve these problems. This makes possible to improve culture efficiency of a cell culture vessel and determine cell distribution and growth via electric signals. 
     Solution to Problem 
     To address the aforementioned problems, the key characteristics of the cell culture vessel of the present invention are as described below. 
     A cell culture vessel for supporting and culturing cells is composed of a space enclosed by a housing for supporting a medium and a cell attachment part disposed on the bottom surface of the space for attaching and supporting the cells therein. The cell attachment part has a cell immobilizing mechanism for guiding the cells to the cell attachment part from the cell space and immobilizing them therein, and a cell detachment mechanism for detaching the cells attached in the cell attachment part. The cell immobilizing mechanism includes a step of applying voltage in an electrode to generate an inhomogeneous electric field in the space, and the cell detachment mechanism includes a step of applying voltage in an electrode disposed in the cell attachment part to induce electrolysis in the space. 
     A cell culture device of the present invention is mainly characterized as described below. The cell culture device equipped with the cell culture vessel for supporting and culturing the cells therein is composed of a feeding/discharging part for feeding/discharging the medium into/from the cell culture vessel, and a power source for applying voltage to an electrode disposed in the cell culture vessel. A cell culture vessel is composed of a space enclosed by a housing for supporting a medium and a cell attachment part disposed on the bottom surface of the space for attaching and supporting the cells therein. The cell attachment part has a cell immobilizing mechanism for guiding the cells into the cell attachment part from the cell space and immobilizing them therein, and a cell detachment mechanism for detaching the cells attached in the cell attachment part. The cell immobilizing mechanism includes a step of applying voltage in an electrode to generate an inhomogeneous electric field in the space, and the cell detachment mechanism includes a step of applying voltage in an electrode disposed in the cell attachment part to induce electrolysis in the space. 
     It has been known that in the ion-rich environment with the electric conductivity of the medium equal to or less than 1000 mS/m, dielectrophoresis becomes negative always at the frequency equal to or less than 10 9  Hz. Taking advantage of cell migration in the direction apart from the electric field concentration, namely toward the weak electric field by negative electrophoresis, the present invention enables the cells to be immobilized in a desired location. 
     The cells may be detached from the surface of the culture medium by applying a direct current (DC) field; thereby, the need for the use of any enzyme (e.g., trypsin) in cell detachment, as with traditional apparatuses, is eliminated. 
     Advantageous Effects of Invention 
     The present invention enables the culture efficiency of the cell culture vessel to be improved and the cell distribution and growth to be determined via electric signals. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a diagram showing one configuration of a cell culture vessel of the present invention. 
         FIG. 2  is a diagram showing one configuration of an electrode disposed in a cell immobilizing mechanism of the present invention. 
         FIG. 3  is a diagram illustrating the relationship between the frequency of an alternate current (AC) field and the real part Re [K] of a Claudius-Mossotti number. 
         FIG. 4  is a diagram illustrating the use of circular electrodes as cell immobilizing electrodes. 
         FIG. 5  is a diagram illustrating the use of comb-shaped electrodes as the cell immobilizing electrodes. 
         FIG. 6  is a diagram illustrating the use of castle-wall electrodes as the cell immobilizing electrodes. 
         FIG. 7  is a diagram illustrating how to immobilize the cells by the cell immobilizing electrodes of the present invention. 
         FIG. 8  is a diagram illustrating how to grow the cells in the cell culture vessel of the present invention. 
         FIG. 9  is a view showing the state of the cell culture vessel before the cells are detached by the electrodes disposed in the cell detachment mechanism of the present invention. 
         FIG. 10  is a view the state of the cell culture vessel after the cells are detached by the electrodes disposed in the cell detachment mechanism of the present invention. 
         FIG. 11  is a view illustrating an equivalent circuit for the cells in the gap formed between the electrodes. 
         FIG. 12  is a diagram showing another configuration of the electrodes disposed in the cell detachment mechanism of the present invention. 
         FIG. 13  is a diagram showing the other configuration of the electrodes disposed in the cell detachment mechanism of the present invention. 
         FIG. 14  is a view showing the state of the cell culture vessel after the cells have been seeded according to an example 1. 
         FIG. 15  is a view showing the state of the cell culture vessel after the cells have been grown according to the example 1. 
         FIG. 16  is a view exemplifying the influence of an applied frequency on impedance between the electrodes of the present invention. 
         FIG. 17  is a view exemplifying a time-course change in impedance between the electrodes of the present invention. 
         FIG. 18  is a view showing a cell concentration apparatus according to an example 3 of the present invention. 
         FIG. 19  is a view illustrating the relationship between the frequency of an AC electric field and the real part RE [K] of a Claudius-Mossotti number. 
         FIG. 20  is a view illustrating the principle of concentration of the cell by dielectrophoresis of the present invention. 
         FIG. 21A  is a view illustrating a flow A of cell concentration by the cell concentration apparatus according to the example 3 of the present invention. 
         FIG. 21B  is a view illustrating a flow B of cell concentration by the cell concentration apparatus according to the example 3 of the present invention. 
         FIG. 21C  is a view illustrating a flow C of cell concentration by the cell concentration apparatus according to the example 3 of the present invention. 
         FIG. 21D  is a view illustrating a flow D of cell concentration by the cell concentration apparatus according to the example 3 of the present invention. 
         FIG. 22  is a view showing a cell concentration apparatus according to a fourth embodiment of the present invention. 
         FIG. 23A  is a view showing a flow A of concentration measurement at the cell concentration apparatus according to an example 4 of the present invention. 
         FIG. 23B  is a view showing a flow B of concentration measurement at the cell concentration apparatus according to the example 4 of the present invention. 
         FIG. 23C  is a view showing a flow C of concentration measurement at the cell concentration apparatus according to the example 4 of the present invention. 
         FIG. 23D  is a view showing a flow D of concentration measurement at the cell concentration apparatus according to the example 4 of the present invention. 
         FIG. 24  is a view illustrating an equivalent circuit for a cell disposed between the electrodes. 
         FIG. 25  is a view showing a cell concentration system of an example 5 of the present invention. 
         FIG. 26  is a flow sheet illustrating the step of controlling the cell concentration system according to the example 5 of the present invention. 
         FIG. 27A  is a view showing a cell concentration apparatus having a multilayer electrode structure according to an example 6 of the present invention. 
         FIG. 27B  is a view showing a cell concentration apparatus having a multilayer electrode structure according to the example 6 of the present invention. 
         FIG. 27C  is a view showing a cell concentration apparatus having a multilayer electrode structure according to the example 6 of the present invention. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, by reference to the accompanying drawings, the embodiments of the present invention will be explained. It should be noted that the same signs are assigned to the same components in the drawings and the explanation of these components are omitted. 
     Hereinafter, by reference to the accompanying drawings, the embodiments will be explained. 
     First Embodiment 
     One example of the cell culture vessel of the present invention will be explained by reference to  FIG. 1 . 
     In  FIG. 1, 1  is the ceiling substrate of the cell culture vessel,  3  is an upper electrodes including an electrode couple, which is disposed on the ceiling substrate  1 .  2  is a bottom substrate of the cell culture vessel,  4  are lower electrodes for immobilizing the cells, which is disposed on the bottom substrate.  5  is an internal space of a cell culture vessel, and  5 A is a medium containing cells  5 B.  6  is a medium inlet, at which a valve  6 A is disposed,  7  is a medium outlet, at which a valve  7 A is disposed,  8  is a mixed gas inlet, at which a valve  8 A is disposed and  9  is a mixed gas outlet, at which a valve  9 A is disposed.  10  is an AC power source and  11  is an impedance measuring apparatus for measuring impedance between the electrode couple.  12  is a DC power source,  13 A is a switch for conducting electricity from the upper electrode  3  to the lower electrode  4  through the DC power source  12 , and  13 B is a switch for conducting electricity from the AC power source  10  to the lower electrode  4 . 
       FIG. 2  is a plan view showing one configuration of the electrodes disposed in the cell immobilizing mechanism.  4  is a thin film electrode disposed on the bottom surface and  14  is an on/off switch for supplying or interrupting current to/from the power source  10 .  15 A,  15 B, and  15 C are driving circuits for controlling the switch  14 . 
     The aforementioned the ceiling substrate  1  and the bottom substrate  2  may be formed using any of insulating materials, as their base material, such as glass, silicone, quartz plastics, polymers. Preferably, the ceiling substrate  1  and the bottom substrate  2  are formed using, as their material, any of materials with light transmittance to the degree that the cells are enabled to be observed under an optical microscope, and more preferably, for the surface of the bottom substrate  2 , a material is used, which may be modified through cleaning and preprocessing processes before the cells are attached thereon. 
     Generally, an ion-rich, highly-conducting medium (1000 mS/m) is used for cell culture, in particular for animal cell culture.  FIG. 3  is a diagram showing the relationship between the frequency of a DC electric field and the real part Re [K] of a Claudius-Mossotti number. As known from the figure, when the electric conductivity of the medium is equal to or higher than 1000 mS/m, dielectrophoresis is negative dielectrophoresis (negative DEP) in all cases at the frequency equal to or lower than 10 9  Hz. Specifically, the cells migrate in the direction away from the center of the electric field, namely toward the weak electric field. It should be noted that preferably, the applied frequency is equal to or lower than 10 7  Hz because the dielectrophoretic force is proportional to the amplitude of Re [K]. 
     As shown in  FIG. 4 , at the center of the four electrodes disposed in the cell immobilizing mechanism according to the first embodiment, a weak electric field is formed. This enables the cells in the highly-conducting medium to migrate into this weak electric field and be immobilized there. Moreover, individually controlling these four electrodes allows for control of cell distribution. It goes without saying that circular electrodes have been described in regard to the first embodiment, but rectangular or polygonal electrodes may be used. 
     The present invention is not limited to the electrodes according to the aforementioned first embodiment but may be the electrodes formed into the shape capable of generating the weak electric field shown in  FIG. 5  and  FIG. 6 ; it is because this type of electrodes enable the cells in the highly-conducting medium to migrate into the weak electric field and be immobilized there through a negative dielectrophoretic force. 
     In the case of the cells culture on the surface of the medium, it is desired that to grow the cells, a layer for facilitating cell attachment capacity, for example a polymeric membrane, is coated between the bottom surface of the incubator between the electrodes, as well as the surfaces of the electrodes. 
     Hereinafter, by reference to  FIGS. 7, 8, 9, and 10 , the flow of a process involving the steps of seeding cells homogenously, culturing the cells for growth, and detaching the cells is explained. 
     As shown in  FIG. 7 , cells  5 B seeded in five mediums  5 A in the cell culture vessel migrate into many weak electric fields of the lower electrode  4  and are immobilized there separately. Accordingly, it is possible to suppress the influence of the deformed bottom surface of the cell culture vessel, external vibrations during cell seeding, and medium vibration, achieving homogenous cell seeding over the whole surface of the cell culture vessel. Homogenous cell seeding improves the use efficiency of the cell culture vessel, increasing cell culture efficiency. Moreover, measuring changes in impedance in the gap between the lower electrodes  4  makes it possible to estimate the distribution of cells immobilized in the weak electric fields. Using this advantage, the cell distribution may be easily estimated using electric signals rather than an optical microscope. 
     During the step of culturing the cells (for example, animal cells) for growth, the cells are attached to the culture surface at 37° C. for growth. During the step of exchanging a mixed gas for culture (composed of air, 5% Co 2 , and 100% water), as shown in  FIG. 8 , the gas is introduced into the cell culture vessel from a mixed-gas inlet  8  thereof, and waste gas produced by culture is discharged from a mixed-gas outlet  9 . During the step of exchanging the medium, a new medium is introduced from a medium inlet  6  and waste medium is discharged from a medium outlet  7 . Since the culture surface is disposed directly above the lower electrodes  4 , measuring changes in impedance in the gap between the lower electrodes  4  allows for estimation of cell growth progress. This enables real-time measurement of the cell growth progress through the electric signals with no need for observation of the progress under an optical microscope. 
     To detach the grown cells from the culture surface, as shown in  FIG. 9 , first, the cell culture vessel is filled with the medium  5 A. Then, the switch  13 B is turned OFF and the switch  13 A is turned ON. This operation applies the DC electric field is applied between the lower electrode  4  and the upper electrode from the DC power source  12 . Applying an appropriate DC voltage enables the cells to be detached from the culture surface, as shown in  FIG. 10 , by the effect of electrolysis occurring on the surface of the lower electrode. In this case, the cells may be detached with no need for using the enzyme (for example, trypsin) as with conventional techniques. Accordingly, the cost of an enzyme may be saved. 
     The cells contained in the medium precipitate, when left as it is, spontaneously down toward the bottom of the cell culture vessel under its own weight. However, it takes long time, about several hours, for the cells to reach the bottom of the incubator and initiate their growth, especially for light cells; thereby they are likely to die before they initiate their growth. To solve this problem, it is required that an appropriate voltage is applied to facilitate cell immobilization. However, even though the voltage is applied, the precipitated cells are eccentrically deposited; thereby it is not expected that the cells grow homogeneously over a wide range. 
     According to the first embodiment of the present invention, it is expected that the death of the cells may be avoided. 
     Moreover, according to the first embodiment of the present invention, the cell culture vessel of the present invention enables the cells to be incubated more efficiently, the distribution and growth progress of the cells to be estimated, and the cells to be detached from the culture surface through electrophoresis. In other words, the cell culture vessel according to the first embodiment of the present invention has advantages of improving cell culture efficiency and reducing the running cost of the apparatus using the cell culture vessel. 
     Second Embodiment 
     With regard to the second embodiment of the present invention, explained is a method for estimating the distribution and growth progress of the cells by measuring the impedance between the lower electrodes of the present invention. 
     Hereinafter, assuming that the impedance between the lower electrodes be Z, capacitance be C, reactance be x, resistance be r, and resistor be R, the aforementioned method is explained using formulas 4 to 8 by reference to  FIG. 11 . 
     
       
         
           
             
               
                 
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     The formula 4 represents a synthetic impedance Z in a CR parallel equivalent circuit, the formula 5 represents a resistance r in the CR parallel equivalent circuit, the formula 6 represents a reactance x in the CR parallel equivalent circuit, the formula 7 represents a resistor R in the CR parallel equivalent circuit, and the formula 8 represents capacitance C in the parallel equivalent circuit. 
       FIG. 11  shows the electric state between the lower electrodes  16  of the cell culture vessel by means of the equivalent circuit. There exists the medium containing the cells between the electrodes  16 . The capacitance (C)  17  configured using the medium as an inter-electrode dielectric and the electric resistor (R)  18  connect in parallel between the electrodes  16  before the cells migrate into the gap between the electrodes. Specifically, the count of the cells, which are locally immobilized may be estimated based on the degree, to which the impedance between the lower electrodes of the cell culture vessel. Moreover, when the locally immobilized cells divide and grow, the impedance increases; this makes it possible to estimate the cell growth progress. Accordingly, electric signals may be used to assess the cell growth progress easily and rapidly with no observation under an optical microscope. 
     Third Embodiment 
     With respect to the third embodiment of the present invention, the gap distance between the electrodes of the cell immobilizing mechanism, applied voltage, and applied frequency are explained. 
     An electric field intensity E between the electrodes of the cell immobilizing mechanism may be represented by the formula 9. 
     
       
         
           
             
               
                 
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     Where, E is the electric field intensity, V is the applied voltage, and d is the gap distance. Water, which is a principal component of the medium for the cell culture, undergoes electrolysis theoretically at 1.23 V; thereby, the applied voltage V need to be set to 1.23 V and preferably, it is equal to and higher than 1 V. However, a lower applied voltage has a disadvantage that it induces only a weak dielectrophoretic force, taking long time for cell growth; accordingly, the lower limit of the applied voltage is preferably approx. 20 mV from the practical view. Moreover, when the cells are manipulated using the thin film electrodes, the electric field intensity E need to be equal to or higher than 1×10 4  V/m; thereby gap distance d between electrodes becomes equal to or lower than 123 μm. Furthermore, in the case of the cells, the average diameter of them is 10 μm; accordingly, the gap distance between the electrodes is preferably 20 to 30 μm. The formula 10 represents the amplitude of the impedance between the aforementioned electrodes. 
     
       
         
           
             
               
                 
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                     10 
                   
                   ) 
                 
               
             
           
         
       
     
     Where S is the facing surface areas of the electrodes. As known from the formula 10, with d between the electrode gaps being constant, the larger the applied frequency f, the smaller the impedance. Specifically, applying high frequency decreases the resistance between the electrodes, causing a larger current to flow. This elevates the medium temperature, causing the environment appropriate for cell culture to be deteriorated or a current control system to be complicated. In addition, considering the technique for achieving a high frequency apparatus, to gain a practical dielectrophoretic force, the applied frequency is preferably equal to or lower than 10 MHz. However, with a lower applied frequency, electrolysis of water occurs readily; accordingly, the lower limit is preferably approx. 100 Hz. 
     Fourth Embodiment 
     With respect to the fourth embodiment, another cell culture vessel of the present invention is explained by reference to  FIGS. 12 and 13 . 
     In the fourth embodiment, an expansion mechanism  3 A shown in  FIG. 12  and a side electrode  3 B shown in  FIG. 13  are the same as those in the first embodiment. Hereinafter, the same signs are assigned to the same parts as those described with respect to the first embodiment to omit duplicated explanation and only different parts will be explained. 
     In the cell culture vessel configured as shown in  FIG. 12 , to detach the cultured and grown cell, the upper electrode  3  is caused to come into contact with the top surface of the medium  5 A by means of an expansion mechanism, and turns the switch  13 B OFF and the switch  13 A ON. This operation applies a DC electric field between the lower electrode  4  and the upper electrode  3  from the power source  12 . Applying an appropriate voltage enables the cells to be detached from the culture surface through the effect of electrolysis occurring on the surface of the lower electrode. 
     In the cell culture vessel shown in  FIG. 13 , the electrodes of the detachment mechanism are disposed on the side surface of the cell culture vessel. Moreover, the switch  13 B is turned OFF and the switch  13 A is turned ON. This operation applies a DC electric field between the lower electrode  4  and the upper electrode  3  from the power source  12 . Applying an appropriate voltage enables the cells to be detached from the culture surface through the effect of electrolysis occurring on the surface of the lower electrode. 
     Example 1 
     In the example 1, a castle-wall electrodes are used for cell immobilization, cell count measurement, and cell growth progress measurement.  FIG. 14  shows the state of the medium after cell seeding and  FIG. 15  shows the state of the medium after cell growth. In the example 1, 3T3 cells (cultivated strain of the fibroblast cells derived from mouse skin), and the DMEM medium with calf serum and an antibiotic substance added are used. Note that the average diameter of 3T3 cells is 10 μm and the electric conductivity of the medium is 1200 mS/m. 
       FIG. 16  shows the influence of the applied voltage of 0.1 V on changes in impedance between the electrodes with the gap distance between the electrodes of 20 m. As known from  FIG. 16 , the impedance of the medium containing the 3T3 cells is higher than that of the medium alone. It is because the 3T3 cells have been immobilized in the weak electric field by the negative dielectrophoretic force.  FIG. 17  shows a time-course change in impedance between the electrodes at the applied voltage of 0.1 V and the applied frequency of 1 KHz. The impedance between the electrodes clearly increased over time. This suggests that the cells in the medium are rapidly immobilized in the weak electric field between the electrodes. Moreover, comparison of this change in impedance with the result of microscopic observation gives the impedance count for each cell; accordingly, the count of the immobilized cells between the electrodes may be found based on the change in impedance. 
     With respect to the example 1, the result of the use of the 3T3 cells and the DMEM medium have been explained; however, the use of the cells derived from any other animal of comparable size and another medium with electric conductivity equivalent to that of the DMEM medium may give the same result. 
     As known from  FIG. 16 , after the cells are cultured for 24 hours at 37° C. while the mixed gas of air, 5% CO 2 , and 100% water is being flown into the cell culture vessel the impedance between the electrodes further increases. This increase in impedance between the electrodes may be associated with the increased count of the cells after cell growth. Taking advantage of this phenomenon, the cell growth progress may be easily determined using the electric signals. 
     Example 2 
     With respect to the example 2, the result of an experiment, in which the cultured cells are detached from the medium surface using the detachment mechanism of the present invention. Since the experimental conditions are the same as those of the example 1, the explanation of them is omitted. After 24-hour culture, 0.5 V of voltage was applied between the upper electrode  3  and lower electrode  4  of the cell culture vessel from the DC power source  12 . Two hours after applying the voltage, it was observed that the cells were gradually detached away from the medium surface. To facilitate this detachment step, the applied voltage may be increased; however, it is concerned about the possibility of damage to the cells due to strong electrolysis. Taking advantage of this phenomenon, the cells may be detached with no need for an enzyme for detachment as with conventional techniques, for example trypsin, reducing the running cost. 
     On the other hand, no technique method for concentrating the cells using dielectrophoresis has been reported. 
     With respect to the example 3 and its succeeding examples, an apparatus for concentrating the cells in the medium efficiently with less load on the cells using negative dielectrophoresis is explained. 
     Example 3 
     One configuration of the cell concentrating apparatus according to the example 3 of the present invention is explained by reference to  FIG. 18 . 
     In  FIG. 18, 101  is a piston-type incubator,  102  are concentrating electrodes containing electrode couples  102   a  and  102   b  disposed on the bottom surface of the piston-type incubator  101 .  103  is an AC power source,  104  is a switch between the concentrating electrode  102  and the AC power source  103 .  105  is a cell suspension vessel and  106  is the medium containing cells  107 .  108  is a driving mechanism equipped with a support mechanism  108 A.  109  is a discharge mechanism equipped with a discharge tube  110 . 
     The concentrating electrode  102  disposed on the bottom surface of the piston-type incubator  101  may be formed directly of, for example metal wire, or may be formed by evaporating or fixing a metal material on a solid insulating substrate made of any of materials such as glass, silicone, quartz, plastics, and polymers and then forming a through hole between the electrodes. Moreover, it is desired that any of materials capable of suppressing the chemical reaction with the medium and the influence on the cells is used for the aforementioned electrodes and the support member. The allowable materials for the electrodes include platinum, gold, chromium, palladium, silver, aluminum, tungsten, and ITO, or any combination of them. It goes without saying that the cross-sectional shape of the concentering electrode is preferably circular but may be other shapes such as rectangle and polygon. 
     Generally, ion-rich media with high conductivity (1000 mS/m or higher) is used for culturing the cells, especially animal cells.  FIG. 19  is a view showing the relationship between the frequency of the AC electric field and real part Re [K] of the Claudius-Mossotti number. In this figure, the results of dielectrophoresis are shown for each of media with electric conductivity of 0.1, 1, 10, 100, and 1000 mS/m. In the figure, the areas, where the dielectrophoretic force F DEP  is positive and negative, are also shown. As known from this figure, with the media with electric conductivity equal to or higher than 1000 mS/m, dielectrophoresis becomes negative at the frequency equal to or lower than 10 9  Hz in all cases. Specifically, the cells migrate in the direction away from the center of the electric field, namely toward the weak electric field. Note that since the dielectrophoretic force is proportional to the amplitude of Re [K], the applied frequency is preferably equal to or lower than 10 7  Hz. 
     By reference to  FIG. 20 , the principle of cell concentration will be explained. A gravity force G, buoyant force F B , and viscous force F DRAG  are exerted on the cells  107  seeded on the medium  106 . In this case, when the concentrating electrode  102 , to which the AC voltage is applied, approaches the cells  107 , the dielectrophoretic force F DEP  is further exerted thereon. When the concentrating electrode  102  is inserted down into the cell suspension vessel  105  vertically from the top side, the cells  107  contained in the medium move toward the bottom of the cell suspension vessel  105  together with the concentrating electrode  102 , provided that the condition F DEP +G&gt;F B  is met. At the same time, the medium passes through the gap between the concentrating electrodes  102  to the piston-type incubator  101 , in which the medium is discharged. Thus, cell concentration may be achieved. 
     With respect to the example 3, the technique for inserting the concentrating electrode  102  down into the cell suspension vessel  105  vertically from the top side has been described; however, it goes without saying that the concentrating electrode  102  may be inserted from the bottom side or the lateral side. Insertion of the concentrating electrode  102  into the cell suspension vessel  105  from the top side is preferable because it improves cell concentration efficiency by exerting the gravity force G and the dielectrophoretic force from their individual directions. 
     By reference to  FIGS. 21A to 21D , the flow of the process of concentrating the cells according to the example 3 will be explained. First, as shown in  FIG. 21A , the switch  104  is closed and an AC voltage is applied to the concentrating electrode  102 . Second, as shown in  FIG. 21B , the driving mechanism  108  moves the piston-type incubator  101  into the cell suspension vessel  105 . The cells  107  are pressed down against the bottom surface of the cell suspension vessel  105  and coagulate thereon, while the medium  106  passes through the through-hole of the concentrating electrode  102  and moves upward. Third, shown in  FIG. 21C , the medium  106 , which moved upward, is discharged outside from the cell suspension vessel  105  by means of the discharge mechanism  109  and the discharge tube  110 . Finally, as shown in  FIG. 21D , the switch  104  is opened to return the piston-type incubator  101  back to its original position shown in  FIG. 21A  by means of the driving mechanism  108 . This operation allows for concentrating the cell suspension in the cell suspension vessel  5 . 
     Herein, the gap distance between the concentrating electrodes  102 , and the applied voltage and applied frequency are explained. 
     The electric field intensity E between cell concentrating electrodes is represented by the above formula 9. 
     Water, which is a principal component of the medium for cell culture, undergoes electrolysis theoretically at 1.23 V; thereby, the applied voltage need be set to 1.23 V or lower. Moreover, as shown in the formula 1, since the dielectrophoretic force is proportional to the applied voltage. At the applied voltage lower than 20 mV, the dielectrophoretic force becomes smaller, namely the force for driving the cells is decreased; accordingly, the applied voltage is preferably equal to or higher than 20 mV. 
     To manipulate the cells, the electric field intensity E equal to or higher than 1×10 4  V/m is required, resulting in the gap distance d between the electrodes being equal to or lower than 123 μm. Furthermore, for the animal cells, of which average diameter is 10 μm, the gap distance between the electrodes is preferably within the range from 20 to 30 μm. 
     The above formula 10 represents the amplitude of the impedance between the aforementioned electrodes. 
     In the formula, S is the area between the opposing electrodes. As known from the formula 10, assuming that the gap distance d between the electrodes is constant, the larger the applied frequency f, the smaller the impedance. Specifically, when high frequency is applied, the resistance between the electrodes, increasing flowing current. This elevates the medium temperature, causing the environment appropriate for cell culture to be deteriorated or a current control system to be complicated. In addition, considering the technique for achieving a high frequency apparatus, the applied frequency is preferably equal to or lower than 10 MHz. However, with higher electric conductivity of the medium, electrolysis of water occurs even when low-frequency AC voltage is applied; accordingly, the applied voltage is preferably equal to or higher than 100 MHz. 
     According to the example 3 of the present invention, the cell concentrating apparatus of the present invention is capable of concentrating the cells easily and efficiently by means of dielectrophoretic force, rather than the conventional membrane separation process, centrifuge separation method, and precipitation separation method. 
     Example 4 
     The example 4 of the present invention measures the impedance between the concentrating electrode and the bottom surface electrode of the present invention to determine the cell concentration. The example 4 is explained by reference to  FIG. 22 . Hereinafter, the same signs are assigned to the same parts as those described in the example 3 to omit their explanation and only the different parts are explained. 
       113  is a bottom surface electrode disposed on the bottom surface of the cell suspension vessel,  111  is an impedance measuring apparatus electrically connected to the bottom surface electrode  113 , and  112  is a switch connecting the concentrating electrode  102  and the impedance measuring apparatus.  114  is a position sensor of the concentrating electrode, and  115  is magnetic sheet for the position sensor. 
     The flow of a process of measuring the cell concentration according to the example 4 of the present invention is explained by reference to  FIGS. 23A to 23D . First, as shown in  FIG. 23A , the switch  104  is closed and the switch  112  is opened. Second, as shown in  FIG. 23B , the driving mechanism  108  moves the piston-type incubator  101  toward the cell suspension vessel  105 . The cells  107  are coagulated on the bottom surface of the cell suspension vessel  105  by means of dielectrophoresis, while the medium  106  passes through the through-hole of the concentrating electrode  102  and moves upward. Third, as shown in  FIG. 23C , the medium  106 , which moved upward, is discharged outside from the cell suspension vessel  105  by means of the discharge mechanism  109  and the discharge tube  110 . At this point, the switch  104  is opened and the switch  112  is closed. The impedance measuring apparatus  111  measures the impedance between the concentrating electrode  102  and the bottom surface electrode  113  to estimate the count of the cells contained in the medium. Moreover, the position sensor  114  measures the volume of the cell suspension. This allows for measurement of the concentration of the concentrated cells. Finally, as shown in  FIG. 23D , the switch  112  is opened to return the piston-type incubator  101  back to its original position shown in  FIG. 23A  by means of the driving mechanism  108 . This operation allows for concentrating the cell suspension in the cell suspension vessel  105  and measuring the concentration of the concentrated cells. 
     Herein, the method for determining the cell concentration by measuring the impedance between the concentrating electrode  102  and the bottom surface electrode  113  is explained. 
     Hereinafter, the impedance Z between the concentrating electrode and the bottom surface electrode is explained by reference to  FIG. 24  and the above formulas 4 to 8. 
     In the formulas, the capacitance is represented by C, the reactance is represented by x, the resistance is represented by r, and the resistor is R. 
     The formula 4 represents the synthetic impedance Z in the CR parallel equivalent circuit, the formula 5 represents the resistance r in the CR parallel equivalent circuit, the formula 6 represents the reactance x in the CR parallel equivalent circuit, the formula 7 represents the resistor R in the CR parallel equivalent circuit, and the formula 8 represents the capacitance C in the CR parallel equivalent circuit. 
       FIG. 24  shows the electric state between the lower electrodes  116  of the cell culture vessel by means of an equivalent circuit. There exists the medium containing the cells between the electrodes  116 . The capacitance (C)  17  configured using the medium as an inter-electrode dielectric and the electroconductive resistor (R)  18  connect in parallel between the electrodes  116  before the cells migrate into the gap between the electrodes. 
     The medium is homogenous liquid. In contrast, the cell is enclosed with an almost insulating cell membrane and therefore, large differences in capacitance and resistance are observed between the cell and the medium. Specifically, the capacitance and resistance of the medium have been measured in advance and when the cells are seeded in the medium, the cell count is determined based on the changes in capacitance and resistance. Since the impedance may be assessed based on the capacitance and resistance, the cell count may be estimated based on the impedance by assessing, in advance, the relationship between the cell count and the impedance. In other words, the cell count may be estimated based on the impedance between the concentrating electrode and the bottom surface electrode. 
     Example 5 
     By reference to  FIG. 25 , the cell concentration system according to the example 5 of the present invention. 
     In  FIG. 25 , the parts excluding a control processor  119  and a monitor  120  are the same as those explained with respect to the example 4. Note that the broken line in  FIG. 25  is an electric signal line connecting the control processor  119  to individual electric control parts. 
     The cell concentration system shown in  FIG. 25  is capable of controlling and monitoring the steps of concentrating the cells and measuring the cell concentration explained above with respect to the example 4. First, the switch  104  is closed and the switch  112  is opened. Second, the driving mechanism  108  moves the piston-type incubator  101  to the cell suspension vessel  105 . Third, the medium  106  is discharged outside of the cell suspension vessel  105  by means of the discharge mechanism  109  and the discharge tube  110 . At this point, the switch  104  is opened and the switch  112  is closed. The impedance measuring apparatus  111  measures the impedance between the concentrating electrode  102  and the bottom surface electrode  113  to estimate the count of the cells contained in the medium. Moreover, the position sensor  114  measures the volume of the cell suspension. This allows for measurement of concentration of the concentrated cells. Finally, the switch  112  is opened to return the piston-type incubator  101  to its original position by means of the driving mechanism  108 . This operation enables the cell suspension contained in the cell suspension vessel  105  to be concentrated and the concentration of the concentrated cells to be measured. At this point, the driving speed of the driving mechanism  108 , and the position of the concentrating electrode, the volume of the cell suspension, and cell concentration may be monitored. 
     Taking advantage of the above functions, the cells may be concentrated to the target level. The flow sheet of controlling the process is shown in  FIG. 26 . First, in the first step ST 1 , it is determined whether or not the cell concentration is the target one. If it is target one, the process goes to the step ST 2  for opening the switch  104  and the switch  112 . When the step ST 2  is finished, control is transferred to the main routine in the step ST 5 . On the other hand, if the cell concentration is not target one, the process goes to the step ST 3  of concentrating the cells contained in the medium by opening the switch  104 , turning the AC power source  103  ON, and moving the driving mechanism  108  down. Moreover, the process goes to the step ST 4  of opening the switch  104  and closing the switch  112 , turning the AC power source OFF and the impedance measuring apparatus  111  ON to measure the impedance and the position. The step ST 3  for concentrating the cells to the target cell concentration and the step ST 4  of measuring the impedance and the position are repeated until the condition set in the step ST 1  is met. 
     Example 6 
     A cell concentration apparatus according to the example 6 of the present invention is explained by reference to  FIGS. 27A to 27C . Herein after, the same signs are assigned to the same parts as those explained with regard to the examples 3 and 4 to omit the explanation of them and only different parts are explained. 
     According to the example 6, the concentrating electrode  102  is disposed on the wall of the cell suspension vessel  105  in the form of multi-layer electrodes  102 A to  102 C. The corresponding one of switches  104 A to  104 C is disposed between each of the concentrating electrode  102 A to  102 C and the AC power source  103 . 
     The flow of the process for concentrating the cells according to the example 6 is explained by reference to  FIGS. 27A to 27C . First, as shown in  FIG. 27A , the switch  104 A is closed, the switch  104 B is opened, the switch  104 C is opened, and an AC voltage is applied to the concentrating electrode  102 A to coagulate the cells at the bottom of the concentrating electrode  102 A. Second, the switch  104 A is opened, the switch  104 B is closed, the switch  104 C is opened, and an AC voltage is applied to the concentrating electrode  102 B to coagulate the cells at the bottom of the concentrating electrode  102 B. Third, as shown in  FIG. 27B , the switch  104 A is opened, the switch  104 B is opened, the switch  104 C is closed, and an AC voltage is applied to the concentrating electrode  102 C to coagulate the cells at the bottom of the concentrating electrode  102 C. Finally, as shown in  FIG. 27C , the medium  106  on the concentering electrode  102 C may be discharged outside of the cell suspension vessel  105  by means of the discharge mechanism  109  and the discharge tube  110 . It goes without saying that three-layer concentrating electrode has been explained with regard to the example 6, but a two-layer or four layer or more concentrating electrode may be used. 
     Focusing on cell concentration, the present invention has been explained. The principle of the apparatus and the system of the present invention may be inversely used to dilute the cell suspension to the target concentration. 
     As long as the features of the present invention are not lost, the present invention is not limited to the aforementioned embodiments and examples and includes other embodiments, which may be considered within the scope of the technical idea of the present invention. 
     Hereinafter, the embodiments of the present invention are summarized and recited. 
     (1) A cell concentration apparatus for concentrating cells contained in a medium, which includes a cell suspension vessel that supports the media containing the cells, a piston-type incubator moving toward the cell suspension vessel, an electrode disposed on the bottom surface of the piston-type vessel, a through-hole, which pierces through the bottom surface of the piston-type incubator disposed between the electrodes, a power source that applies an AC voltage to the electrode, a driving mechanism that moves the piston-type incubator up from and down to the cell suspension vessel, and a discharge mechanism that discharges the medium, which enters the piston-type vessel through the through-hole.
 
(2) The cell concentrating apparatus described in (1) which is characterized in that the electrode disposed on the bottom surface of the piston-type incubator presses the cells in a cell suspension against the bottom of the cell suspension vessel by means of a diectrophoretic force.
 
(3) The cell concentrating apparatus described in (1) which further includes an impedance measuring apparatus that measures the impedance between the electrode disposed on the bottom surface of the piston-type incubator and the electrode disposed on the bottom surface of the cell suspension vessel to estimate the count of the cells in the medium based on the measured impedance.
 
(4) The cell concentrating apparatus described in (3), which further includes a position sensor that measures the position of the electrode, in which it finds the volume of the cell suspension based on the measured electrode position to determine the cell concentration.
 
(5) The cell concentration apparatus for concentrating the cells contained in the medium, characterized by including a cell suspension vessel that supports the medium contained the cells, a plurality of electrodes disposed on the inner wall of the cell suspension vessel in the multi-layer form, a through-holes formed between the electrodes, a power source that applies an AC voltage to the electrode, a switch that switches among the plurality of electrodes to which the AC voltage is applied, and a discharge mechanism that discharges the medium which enters the cell suspension vessel.
 
(6) The cell concentrating apparatus described in (5) which is characterized in that the plurality of electrodes disposed on the inner wall of the cell suspension vessel in the multi-layer form presses the cells in the cell suspension against the bottom of the cell suspension vessel by means of a negative dielectrophoretic force.
 
(7) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the voltage applied for generating an electric field between the electrodes is within the range from 20 mV to 1.23 V.
 
(8) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the frequency applied for generating an electric field between the electrodes is within the range from 100 Hz to 10 MHz.
 
(9) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the gap distance between the electrodes is equal to or less than 123 μm.
 
(10) The cell concentrating apparatus described in any one of (1) to (6) which is characterized in that the electrodes are made of any one of platinum, gold, chrome, palladium, rhodium, silver, aluminum, tungsten, and ITO, or any combination of them.
 
(11) A cell concentrating system composed of a cell concentrating apparatus described in any one of (1) to (6); and a control processor that controls the individual parts of the cell concentrating apparatus.
 
(12) A method for concentrating cells using a cell concentrating apparatus described in one of (1) and (2), which includes: supplying a medium containing cells in the cell suspension vessel; moving the piston-type incubator downward while applying an AC voltage to the electrode; and discharging the medium, which enters the piston-type incubator through the through-holes.
 
(13) A method for concentrating the cells using the cell concentrating apparatus described in (4), which includes: supplying the medium containing the cells in the cell suspension vessel; moving the piston-type incubator downward while applying an AC voltage to the electrode; and discharging the medium, which enters the piston-type incubator through the through-holes; measuring the impedance between the electrode disposed on the bottom surface of the piston-type incubator and the electrode disposed on the bottom surface of the cell suspension vessel using the impedance gauge and finding the volume of the cell suspension using the position sensor; finding the cell concentration based on the measured impedance and volume of the cell suspension; and ending the cell concentration process if the found cell concentration has reached the target level, and going to the step of moving the piston-type incubator downward if it is lower than the target level.
 
(14) A method for concentrating the cells using the cell concentrating apparatus described in one of (5) and (6), which includes: supplying the medium containing the cells in the cell suspension vessel; switching among the electrodes arranged in the multi-layer form to apply the AC voltage to them sequentially from one end; and discharging the medium, which enters the cell suspension vessel through the through-holes.
 
     Note that according to the above-recited embodiments of the present invention, the cells contained in the medium may be concentrated efficiently with less load on them. The cell concentration may be measured by means of electric signals. 
     REFERENCE SIGNS LIST 
       1  . . . Incubator ceiling substrate,  2  . . . Incubator bottom substrate,  3  . . . Upper electrode,  3 A . . . Expansion mechanism,  3 B . . . Side electrode,  4  . . . Lower electrode,  5  . . . Inside of the incubator,  5 A . . . Culture medium,  5 B . . . Cell,  6  . . . Medium inlet,  6 A . . . Medium inlet valve,  7  . . . Medium outlet,  7 A . . . Medium outlet valve,  8  . . . Mixed-gas inlet,  8 A . . . Mixed-gas inlet valve,  9  . . . Mixed-gas outlet,  9 A . . . Mixed-gas outlet valve,  10  . . . AC power source,  11  . . . Impedance measuring apparatus,  12  . . . DC power source,  13 A . . . Switch,  13 B . . . Switch,  14  . . . Switching element,  15 A . . . Driving circuit,  15 B . . . Driving circuit,  15 C . . . Driving circuit,  16  . . . Electrode,  17  . . . Capacitance C,  18  . . . Resistor R,  101  . . . Piston-type incubator,  102  . . . Concentrating electrode,  103  . . . AC power source,  104  . . . Switch,  105  . . . Cell suspension vessel,  106 . Medium,  107  . . . Cell,  108  . . . Driving mechanism,  108 A . . . Support mechanism,  109  . . . Discharge mechanism,  110  . . . Discharge tube,  111  . . . Impedance measuring apparatus,  112  . . . Switch,  113  . . . Bottom surface electrode,  114  . . . Position sensor,  115  . . . Magnetic sheet,  116  . . . Electrode, C 17  . . . Capacitance C, R 18 . Resistor R,  119  . . . Control processor,  120  . . . Monitor.