JP7202639B2 - Apparatus and method for separating and collecting cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to an apparatus and method for separating and collecting cells with high accuracy.

Techniques for selecting and isolating specific cells are indispensable in fields such as biotechnology and medicine. Flow cytometry using fluorescent labeling and magnetic cell separation are widely used, but they are very expensive and pose a serious obstacle to cells. In contrast, an inexpensive and non-invasive separation method combining dielectrophoresis, which is an alternating electrokinetic phenomenon, with microchannels has been devised.

A system that uses an insulating film with multiple pores for efficient separation processing, forms a non-uniform electric field in the pores and around the entrance, and separates a large number of cells at once by the difference in the dielectric properties of the cells. It has been known. However, since such techniques give priority to large-scale processing, the pore diameters are often extremely large compared to the cell size, conversely causing a decrease in separation accuracy.

Specifically, a cell separation system using an insulating membrane with pores having a diameter of 100 to 200 μm, which is considerably larger than the general size of cells to be separated (minimum diameter is several μm), is known (for example, , Patent Document 1, Non-Patent Documents 1 and 2). In this system, a non-uniform electric field is formed around the hole and a large number of cells can be separated at once. In particular, it separates those that do not enter the holes due to the negative dielectrophoretic force due to the application of alternating current and those that pass through the holes due to the electrophoretic force due to the direct current component. However, there is a problem that the separation accuracy is very poor.

A technique is also known in which cells are introduced and held in an array of pores with a diameter of 30 μm larger than the cells with a positive dielectrophoretic force, and the cells are fixed by another method, sampled and analyzed (for example, patent See Document 2, Non-Patent Document 3).

Another known technique is to use an insulating film with ultrafine pores with a diameter of 130 nm that cells cannot pass through, and trap cells near the film surface above the pores where the electric field strength is strong due to positive dielectrophoretic force ( See, for example, Non-Patent Document 4). However, in this technique, cells are adsorbed only by the dielectrophoretic force, so it is difficult to retain cells on the membrane surface due to the weak force of cell capture.

A technique is also known in which an insulating film with an array of pores with a diameter of 100 μm, which is considerably larger than the cells, is used, and the cells are adsorbed and concentrated on the walls of the pores or around the pore entrance where the electric field strength is strong due to the positive dielectrophoretic force. (See, for example, Non-Patent Document 5). However, even with this technique, the trapping power is weak and cells are difficult to retain.

By penetrating pores with a diameter of 400 μm perpendicularly to the multilayer electrode layer, multiple places with strong electric field strength are generated on the wall surface of the pores, and live cells are adsorbed by positive dielectrophoretic force, and dead cells are negative. There is also known a technique of passing through the pores without being adsorbed by the dielectrophoretic force (see, for example, Non-Patent Document 6). Although this technique can process large amounts, the separation accuracy is still low. In addition, since the adsorbed live cells come into contact with the metal electrode, there is concern about damage to the cells.

JP-A-2009-65967 Japanese Patent Application Laid-Open No. 2016-174596

Journal of the Institute of Electrostatics, 30, 140-145, 2006 Biotechnol Prog, 32, 1292-1300, 2016 PLoSOne, 2015Jun24;10(6):e0130418 Anal Chem, 80, 657-664, 2008 Electrophoresis, 30, 3153-3159, 2009 Proc Natl Acad Sci USA, 114, 4591-4596, 2017

The present invention has been made in view of the above problems. That is, it is an object of the present invention to provide an apparatus and a method capable of separating live cells and dead cells with high precision and firmly trapping them, without affecting or damaging cells due to Joule heat.

According to one embodiment, the present invention is as follows [1] to [13].
[1] A device for separating and collecting cells, comprising: a cell comprising two conductive layers provided facing each other; and one or more microporous thin films provided between the two conductive layers; (a) the micropores are cylindrical, and the diameter of the micropores is larger than the minimum diameter of the cells to be separated and 2.5 times or less the minimum diameter is, or
(b) the micropore has a non-cylindrical shape selected from a truncated cone shape, a staircase shape, an hourglass shape, a pincushion shape, or a pot shape, and the diameter of the inlet of the micropore is the minimum diameter of the cells to be separated; Larger, no more than 2.5 times the smallest diameter.
[2] In the separation and recovery apparatus described in [1] above, the conductive layers provided facing each other are electrodes forming the bottom surface and the top surface of the cell, and the microporous thin film is at least one of the It is preferable that the microporous insulating film is provided between the electrodes so as to be separated from the electrodes.
[3] In the separation/recovery apparatus described in [1] above, it is preferable that at least one of the conductive layers provided facing each other is a conductive coating layer that covers the surface of the microporous insulating film.
[4] In the separation and recovery apparatus described in [1] above, one of the conductive layers provided to face each other is an electrode constituting the bottom surface or top surface of the cell, and the conductive layer provided to face each other The other of the layers and the microporous membrane are preferably conductive nets or sieves spaced apart from the electrodes and covered with an insulating coating layer.
[5] In the separation and recovery apparatus described in [1] above, at least one of the conductive layers provided facing each other is a conductive mesh or a sieve, and the microporous thin film is the conductive mesh or sieve. It is preferably provided at a distance from or in contact with the .
[6] In the separation and recovery apparatus according to any one of [1] to [5] above, the microporous thin film is a track etched membrane having a thickness of 7 to 30 μm, a UV laser processed film, or a photolithography processed film. It is preferably a membrane.
[7] In the separation and recovery apparatus described in [2] above, it is preferable that the microporous insulating film is provided so as to be in contact with one of the electrodes and cover the electrode.
[8] In the separation and recovery device described in [2] above, the microporous insulating film is separated from both the electrode forming the bottom surface and the electrode forming the upper surface to separate the cells. is preferably provided in
[9] In the separation/collection device described in [2] above, it is preferable that the cell further includes an inlet for a sample suspension containing cells to be separated, and an outlet.
[10] In the separation and recovery apparatus described in [2] above, the one or more microporous thin films provided between the two conductive layers are two or more microporous insulating films spaced apart from each other. Preferably, the two or more microporous insulating films have different micropore diameters, and the micropore diameters decrease sequentially from the microporous insulating film near the top surface to the microporous insulating film near the bottom surface.
[11] In the separation/recovery apparatus described in any one of [3], [4] or [5] above, it is preferable that the upper surface is an open system.
[12] A method for separating and collecting cells, wherein a cell to be separated is provided in a cell comprising two conductive layers provided facing each other and one or more microporous thin films provided between the two conductive layers. and applying, to the pair of conductive layers, a frequency that selectively aspirates or ejects dead cells or live cells into the micropores.
[13] In the method for separating and collecting cells according to [12] above, it is preferable to generate a non-uniform electric field in the micropores of the microporous thin film by applying a frequency.

According to the apparatus and method for separating and collecting cells according to the present invention, cells having specific dielectric properties are introduced or not introduced into the micropores of the microporous insulating film by positive or negative dielectrophoretic force due to frequency application. By using micropores with a slightly larger diameter than the cells, the cells can be firmly captured to such an extent that they are not easily peeled off by water flow or the like. In addition, in the case of non-spherical cells in particular, only cells having specific dielectric properties can be sucked into the micropores even if the pores are difficult to pass through by filtration, so that the separation accuracy can be improved. Further, when the fine pore diameter of the fine pore insulating film is reduced, a strong electric field strength is obtained in the fine pores, so that a dielectrophoretic force is generated even at a low applied voltage. Therefore, the generation of heat is suppressed and damage to cells is reduced. Furthermore, not only the selective separation of live cells and dead cells by dielectric properties, but also the separation by cell size can be performed at the same time by using an appropriate micropore size. Furthermore, depending on the shape of the micropores, a non-uniform electric field is generated within the micropores, and a frequency that causes specific dielectrophoresis in living or dead cells is applied, and by continuously switching this, the micropores of the cells It is possible to repeat the suction to the inside and the discharge from the inside of the micropores. This makes it possible to pass only specific cells continuously through the micropores and separate them from one layer separated by a membrane or the like in the cells into another layer. Furthermore, by using a mesh-shaped conductor or a conductor film coated with a microporous insulating film as an electrode for applying a frequency, a cell with an open top surface is formed so that the cell suspension can be easily injected by dropping. becomes possible.

FIG. 1 is a diagram showing the configuration of a cell separation/collection device according to an embodiment of the present invention. FIG. 2 is a diagram showing the configuration of a cell separation/collection device according to another embodiment of the present invention. FIG. 3 is a photomicrograph at 100× before aspiration of cells into micropores with a pore size of 5 μm by dielectrophoresis. FIG. 4 is a photomicrograph at 100× magnification after cell aspiration into micropores with a pore size of 5 μm by dielectrophoresis. FIG. 5 is a 100-fold photomicrograph showing the state of cell aggregation in micropores with a pore diameter of 20 μm by dielectrophoresis. FIG. 6 is a graph showing the ratio of live cells and dead cells sucked into micropores for each external conductivity when the frequency is swept from the low frequency side. FIG. 7 is a graph showing the ratio of live cells and dead cells sucked into micropores for each external conductivity when the frequency is swept from the high frequency side. FIG. 8 shows micrographs of fluorescently stained live-dead mixed cell suspension before and after application of 20 MHz, where (A) is before application and (B) is after application. In (A) and (B), the left panel is a photograph of a bright field image, and the right panel is a photograph of a fluorescence image. FIG. 9 shows micrographs of a fluorescently stained live-dead mixed cell suspension before and after application of 50 kHz, where (A) is before application and (B) is after application. In (A) and (B), the left panel is a photograph of a bright field image, and the right panel is a photograph of a fluorescence image. FIG. 10 is a conceptual diagram showing an example of a microporous insulating film having cylindrical micropores. FIG. 11 is a graph showing the electric field intensity on the center line of the cylindrical pores of the example. FIG. 12 is a conceptual diagram showing an example of a microporous insulating film having truncated conical micropores. FIG. 13 is a graph showing the electric field intensity on the center line of the truncated conical micropores of the example. FIG. 14 is a conceptual diagram showing an example of a microporous insulating film having two steps of micropores. FIG. 15 is a conceptual diagram showing an example of a microporous insulating film having three steps of micropores. FIG. 16 is a conceptual diagram showing an example of a microporous insulating film having hourglass-shaped micropores. FIG. 17 is a conceptual diagram showing an example of a microporous insulating film having pincushion-shaped micropores. FIG. 18 is a conceptual diagram showing an example of a microporous insulating film having pot-shaped micropores. FIG. 19 is a conceptual diagram showing an example of a cell structure provided with a microporous insulating film whose upper surface is covered with a conductive coating layer. FIG. 20 is a conceptual diagram showing an example of a cell structure provided with a microporous insulating film whose upper surface and pore inner walls are coated with a conductive coating layer. FIG. 21 is a conceptual diagram showing an example of a cell structure provided with a microporous insulating film whose lower surface is covered with a conductive coating layer. FIG. 22 is a conceptual diagram showing an example of a cell structure provided with a microporous insulating film whose upper and lower surfaces are coated with conductive coating layers. FIG. 23 is a conceptual diagram showing an example of a cell configuration comprising a conductive mesh or sieve coated with an insulating coating layer. FIG. 24 is a conceptual diagram showing an example of a cell configuration in which a conductive mesh or sieve is provided as an electrode instead of the upper electrode. FIG. 25 is a conceptual diagram showing an example of a cell configuration in which conductive nets or sieves are provided as electrodes instead of top and bottom electrodes. FIG. 26 is a conceptual diagram showing an example of a cell configuration using a laminated membrane filter with channels using an ultra-thin film. FIG. 27 is a graph showing the relationship between frequency and the real part of the Clausius-Mossotti factor (Re CMf) versus the conductivity of an external solution in which live or dead cells are suspended. FIG. 28 is a graph showing the suction ratio and ejection ratio with respect to the frequency of applying viable cells to the truncated cone-shaped micropores in the medium solution. FIG. 29 is a graph showing the electric field intensity on the center line of the staircase (1-step and 2-step), hourglass, pincushion, and pot-shaped pores of the example. FIG. 30 is a graph showing the electric field intensity on the center line of the micropores in an embodiment in which a conductor film is attached to the microporous insulating film of the example and in an aspect in which an insulating film is attached to the conductor film composed of a conductive net or sieve. is. FIG. 31 shows a cell according to a modification of the second embodiment, which enables selective separation according to the cell diameter using a microporous insulating film having micropores with different pore sizes in the horizontal direction. It is a conceptual diagram. FIG. 32 is a conceptual diagram showing a cell provided with a conductive mesh or sieve as an electrode, with a funnel-shaped cell concentration area below the conductive mesh or sieve on the bottom side and an additional electrode.

Below, the invention will be described in detail with reference to the drawings. The following description does not limit the invention

A device for separating and collecting cells according to the present invention comprises: a cell comprising two conductive layers provided facing each other; one or more microporous thin films provided between the two conductive layers; and an AC oscillator that applies a frequency within a predetermined range to the. In such a separation and recovery device, (a) the micropores are cylindrical, and the diameter of the micropores is larger than the minimum diameter of the cells to be separated and 2.5 times or less than the minimum diameter, or (b) ) The micropore has a non-cylindrical shape selected from a truncated cone shape, a staircase shape, an hourglass shape, a pincushion shape, or a pot shape, and the diameter of the inlet of the micropore is larger than the minimum diameter of the cells to be separated. , is less than or equal to 2.5 times the minimum diameter. The separating and collecting device according to the present invention forms an electric field stronger inside the micropores than outside the micropores, thereby attracting and holding predetermined cells in the micropores from one region separated by the microporous thin film. or, optionally, to be sucked into the micropores and ejected to the other area. Each embodiment will be described below.

[First embodiment]
The cell separation and recovery device according to the first embodiment of the present invention is characterized in that, in the separation and recovery device of the present invention, the two conductive layers provided facing each other form an electrode forming a bottom surface of the cell and an upper surface. It is a constituent electrode, and the microporous thin film is a microporous insulating film provided between at least one of the electrodes and separated from the electrodes. FIG. 1 shows a cell separating and collecting apparatus according to this embodiment. The cell separation/collection device according to the first embodiment is mainly composed of a cell 10 and an AC oscillator 20 .

The cell 10 and an AC oscillator 20 are connected, and the AC oscillator 20 is configured to apply a frequency within a predetermined range to the cell suspension 5 in the cell 10 . The cell 10 has a pair of electrodes 2a and 2b, and is arranged so that one electrode 2b is located on the bottom surface and the other electrode 2a is located on the top surface. FIG. 1 is a conceptual diagram of a cross section of a cell viewed from a direction perpendicular to the electric field direction. The electrode 2a forming the top surface and the electrode 2b forming the bottom surface are formed on glass substrates 1a and 1b, respectively. Suspended liquid 5 is sandwiched. A microporous insulating film 3 is provided on the liquid contact surface of the electrode 2b forming the bottom surface. Electrodes 2a and 2b are each connected to an AC oscillator 20 .

Each part constituting the cell separating and collecting apparatus will be further described. The cell 10 is a container for injecting a cell suspension containing cells to be separated and for applying a frequency within a predetermined range to the cell suspension. Such a cell 10 can be produced by sandwiching a spacer 4 of desired thickness between two opposing electrodes and injecting a cell suspension 5 . The cell 10 is arranged so that one of the two electrodes is on the bottom surface and the other is on the top surface.

As electrodes, conductive and optionally transparent electrodes can be used. In particular, from the viewpoint of making it possible to observe the process of separating and collecting cells, a transparent electrode can be used, and a conductive thin film such as ITO or FTO provided on a glass substrate can be used, but these are not limited. not. As an example, a transparent electrode or the like for a liquid crystal display can be used. As another example, a glass substrate or a plastic substrate obtained by vapor-depositing a thin film of gold or platinum with a thickness of about 10 to 100 nm has conductivity and transparency and can be used. When using a transparent electrode made of ITO on a glass substrate, the thickness of the glass substrate can be 0.12 to 1.5 mm, but is not limited to this range. Also, the thickness of the ITO film can be about 50 nm to 400 nm, but is not limited to this range. The opaque electrode is not particularly limited, and any general-purpose electrode can be used.

In the present embodiment, the microporous insulating film 3 is provided on the surface inside the cell of the electrode 2b, which is the bottom surface, that is, the liquid contact surface. The microporous insulating film 3 does not need to cover the entire surface of the electrode 2b inside the cell, but preferably covers at least the surface of the electrode 2b that is in contact with the cell suspension 5 . The microporous insulating film 3 is an insulating film having micropores 31 . The microporous insulating film 3 preferably has strength and flexibility, a smooth surface, low protein adsorption, and heat resistance. These characteristics can bring about the effects that the membrane is not broken in the separation operation, a high electric field can be easily formed in 31 parts of the micropores, the cell recovery rate can be increased, and autoclave sterilization is possible. Furthermore, from the viewpoint of observation with a microscope or the like, it is preferable that the material also has transparency. The microporous insulating film 3 that satisfies these properties may be made of plastic, particularly polycarbonate, polyethylene terephthalate, polyimide, polydimethylsiloxane (PDMS), epoxy resin photoresist, such as product name SU-8. (manufactured by Kayaku Microchem) can be used, but is not limited to these. The thickness of the microporous insulating film 3 is preferably about 7 to 30 μm. If it is too thin, the cells may not be accommodated in the pores, and the membrane may be easily broken. be. The microporous insulating film 3 is typically one continuous film. , may be connected in the horizontal direction.

The diameter or minimum diameter of the micropores 31 provided in the microporous insulating film 3 should be larger than the minimum diameter of the cells to be separated and 2.5 times or less the minimum diameter of the cells. Note that the pore diameter of the micropores is a cylindrical shape in which the pore diameter of each micropore is substantially uniform in the thickness direction of the membrane according to each aspect described in detail in the second embodiment. shall be expressed in terms of diameter. If the shape of the micropore is a non-cylindrical shape in which the diameter of one micropore varies along the thickness direction of the membrane, the minimum diameter shall be used. In the present invention, the shape of the cells to be separated is not particularly limited, so the diameter or minimum diameter of the micropores 31 is determined based on the minimum diameter of the cells. The minimum diameter of a cell is, for example, the diameter for a spherical cell, the minor axis diameter for a cylindrical or oval cell (the oval is a cross-section cut horizontally with the pointed end facing up or down). It is defined by the short axis diameter of the larger sphere in the case of a snowball-shaped (a shape in which two spheres are connected) cells. Typically, the diameter or minimum diameter of micropores 31 may be about 1 to 10 μm, but is not limited to a specific range.

It is preferable that the fine holes 31 have a substantially uniform hole diameter and have a uniform cylindrical shape (hole shape) that is steeply provided from the surface of the fine hole insulating film 3 . The fine holes 31 having such a shape are suitable for generating a non-uniform electric field necessary for dielectrophoresis of cells around the outside of the fine holes. Even when the diameter of the fine holes 31 is somewhat non-uniform, the diameter of the fine holes 31 refers to the minimum diameter. The fine pores 31 may or may not penetrate the membrane, but preferably have a depth greater than at least the maximum diameter of the cells to be separated. Furthermore, the depth direction of the micropores 31 is preferably substantially perpendicular to the surface (principal surface) of the film, but the depth direction is inclined at most about 40° with respect to the surface of the film. good too. Alternatively, the pores may be non-cylindrical pores, as detailed in the second embodiment.

The density of the fine holes 31 provided in the fine hole insulating film 3 is preferably about 100,000/cm ² or more and about 400,000/cm ² or less. not. Since such a microporous insulating film 3 has a small micropore diameter, it is easy to increase the micropore density, and by increasing the number of micropores, mass processing becomes possible. Also, by increasing the area of the film, mass processing becomes possible. A membrane filter having a non-smooth surface in which fibers are woven is not preferable as the microporous insulating film in the present invention because a non-uniform electric field is not formed and the electric field is made uniform. In addition, the hole diameters may be the same or different among the plurality of fine holes 31 provided in one fine hole insulating film 3 . By providing micropores with different diameters in one microporous insulating film 3, cells can be separated and sorted according to their diameters. A detailed aspect will be described later with reference to FIG.

A track-etched membrane can be used as an example of a preferable microporous insulating film 3 . A track-etched membrane is a microporous membrane formed by a track-etching method, and is particularly preferable from the viewpoint of uniformity of pore size and pore shape. According to the track etching method, a track-etched membrane having a desired pore size, pore shape, thickness and fine pore density can be appropriately manufactured. Moreover, track-etched membranes that are commercially available as filtration membrane filters can be used, and in this case, they are particularly advantageous in that they can be mass-produced, sterile, and disposable. Alternatively, the microporous insulating film 3 may be a film processed by UV laser or a film processed by photolithography.

In the present embodiment, the cell 10 is arranged so that the surface on which the microporous insulating film 3 is provided is the bottom surface when the cells are separated and collected. This is because predetermined cells are attracted to the microporous insulating film 3 by dielectrophoretic force and gravity and separated. In the illustrated embodiment, the liquid contact surface of the upper electrode is not provided with a coating film, but may be provided with an insulating coating layer of about 10 nm to 5 μm, for example. This is to prevent electrolysis and cell adsorption and improve the cell recovery rate. For the same purpose, in all the embodiments described later, the layer corresponding to the "two conductive layers provided facing each other" of the present invention, that is, the conductive layer to which the frequency is applied, the surface in contact with the cell suspension It can be coated with an insulating thin film.

The spacer 4 may be any material as long as it is insulating and inert to the cell suspension 5. For example, polydimethylsiloxane, silicon rubber, hard rubber, polyimide, resin such as SU-8 or plastic is used. can be, but are not limited to. The thickness of the spacer 4 is preferably 1-300 μm, more preferably 10-100 μm. The thickness of spacer 4 determines the distance between the electrodes and thus the electric field strength. However, the above values are typical thicknesses, and the thickness of the spacer 4 is preferably determined in consideration of the size (maximum diameter) of cells to be separated. That is, the thickness of the spacer 4 can be about 1 to 30 times the size of the cell, but is not limited to this range.

The cell 10 may simply sandwich the cell suspension 5 from the top and bottom, or may be a closed system. Alternatively, although not shown in FIG. 1, the flow cell may be provided with an inlet and an outlet for the cell suspension so that the cell suspension can flow. In this case, the cell may be provided with a pump, a reservoir, and the like for sending and draining the liquid. The size of the cell 10 is not particularly limited as long as the required amount of cells can be separated and collected.

The AC oscillator 20 may be one that can apply frequencies within a predetermined range. Specifically, a commercially available AC oscillator may be used as long as it can apply a frequency in the range of about 1 kHz to 100 MHz. However, it is preferable to have an internal resistance of 50Ω to 75Ω and an output of 10Vp-p. An amplifier can also be connected to the AC oscillator 20 . With such a configuration, the voltage can also be amplified. The output waveform of the AC oscillator 20 is not limited to a sine wave, and may be a square wave, a triangular wave, or the like. A high-frequency DC pulse can also be used.

Next, a method for separating and collecting cells according to one embodiment of the present invention will be described. The cell separation and recovery method according to the present embodiment can be performed using the cell separation and recovery device described above. The separation and recovery method according to the present embodiment is a cell composed of electrodes whose bottom faces and top faces face each other, and at least the liquid contact surface of the electrode constituting the bottom face is larger than the minimum diameter of a desired cell and A step of injecting a cell suspension containing cells to be separated into a cell coated with a microporous insulating film having a micropore diameter of 2.5 times or less, and applying dead or live cells to the pair of electrodes and applying a frequency that selectively attracts to the micropores.

A cell suspension containing cells to be separated is prepared as a step prior to each step of the separation and recovery method. Cells to be separated are not particularly limited, and any cells existing as dielectric particles covered with an insulating film can be separated. Therefore, the shape is also not limited, and examples include microbial cells and animal cells of arbitrary shapes such as spherical, cylindrical, oval, and snowball-shaped. Specific examples of separation targets include Schizosaccharomyces yeast such as cylindrical fission yeast (Schizosaccharomyces pombe), Saccharomyces yeast such as egg-shaped and snowball-shaped Saccharomyces cerevisiae, Candida yeast, Pichia yeast, Rhodotorula yeast, Platelets, algae, microalgae, unicellular organisms (euglena, paramecium, etc.), Escherichia coli, lactobacilli, salmonella, spherical yeast protoplasts with cell wall digestion, rat adrenal pheochromocytoma PC12, spheroids (cell clumps), cocci, streptococci Examples include, but are not limited to, cocci.

The cells may be of any origin, but may be, for example, recombinant proteins, antibiotics, vaccines, biofuels, or microbial cells used in food manufacturing processes. Cells can then be harvested by sampling from badges or lines in the manufacturing process. As an example, when used to remove dead cells after microbial fermentation, microbial cells can be collected and separated by continuously circulating to the outside through a connected tube or the like. Alternatively, the non-microbial cells may be blood-derived cells from humans or other mammals, or bacteria that cause sepsis.

The harvested cells can be pretreated to remove contaminants in advance using, for example, a centrifuge, a filter, or the like. This is because the pretreatment can improve the separation accuracy. Pretreated cells are used to prepare cell suspensions in the case of yeast. A cell suspension can be prepared by directly diluting a culture medium with ultrapure water, distilled water, ion-exchanged water, or a pH buffer. In addition, sorbitol solution, mannitol solution, glycerol solution, sucrose solution and the like can be used as non-electrolyte osmotic pressure adjusting agents, but are not limited to these. According to this aspect, pretreatment such as washing the cells with water, a low-conductivity solution, a non-electrolyte osmotic pressure adjusting solution, or the like can be omitted, and the labor and time required for washing can be reduced. Therefore, it becomes possible to directly supply the cell suspension to the cell by feeding the cell suspension from the culture vessel or the like, and rapid and continuous separation can be performed.

In the cell suspension, the cell concentration is preferably about 1×10 ⁶ to 1×10 ⁸ cells/mL, more preferably about 5×10 ⁶ to 5×10 ⁷ cells/mL. In addition, the electrical conductivity (also referred to as external electrical conductivity) of the cell suspension is preferably adjusted to 0.01 to 100 mS/m when collecting only dead cells by aspiration. More preferably, it is 1 to 50 mS/m. On the other hand, the electrical conductivity of the cell suspension is preferably 100 to 1000 mS/m when only viable cells are collected by aspiration. Within such a conductivity range, a positive dielectrophoretic force is generated and attracted in the frequency range where the Clausius-Mossotti factor of living cells is positive. Note that the Clausius-Mossotti factor is a factor that serves as an indicator of the state of polarization in an electric field, regarding cells as dielectric particles. Therefore, when the value of the dielectrophoretic force is positive, positive dielectrophoresis occurs, in which the cells migrate to the region where the electric field strength is strong. In order to achieve a conductivity of about 100 mS/m, it is possible to use a culture medium with almost the same conductivity (95 mS/m in the case of YE medium) as it is, which is advantageous. Under such conditions, it is preferable to apply a cell adsorption inhibitor, such as prevelex LS1004 manufactured by Nissan Chemical Industries, over the entire channel including pores and electrodes.

The cell suspension prepared as described above is injected into the cell 10 of the separation and recovery apparatus described above. About 0.1 to 10 μl of the sample solution can be collected and injected into the cell so that the diameter of the spot is as small as possible relative to the size of the cell. More preferably, the sample volume is 0.5 to 3 μl. In addition to the separation of live and dead cells, such a small amount of sample enables accurate determination of life and death.

Next, a step of applying a predetermined frequency to a pair of electrodes to selectively attract dead cells or live cells into the micropores is performed. Such frequencies are also referred to herein as "attraction frequencies". The attraction frequency means that only one of dead cells and live cells performs positive or negative dielectrophoresis and is attracted to the micropores, and the other of dead cells and live cells performs reverse dielectrophoresis and is attracted to the micropores by repulsive force. is the frequency repelled from It is known that living cells and dead cells can induce opposite dielectrophoresis by appropriately setting the frequency and external conductivity conditions. Based on this principle, the present invention selectively generates positive dielectrophoresis of only one of dead cells and living cells, guides them to the micropores 31 of the microporous insulating film 3, attracts and holds them in the micropores 31, Conversely, the cells are separated by causing the other cells to undergo negative dielectrophoresis so that they are repelled from the micropores 31 . In this specification, the frequency at which dead cells or living cells that are sucked and are present in the fine holes are selectively ejected from the fine holes is also referred to as "ejection frequency". The ejection frequency will be described in detail in the second embodiment.

The suction frequency and external conductivity described above can be obtained by preliminary experiments. Alternatively, it can be obtained by theoretical calculation of the relationship between the Clausius-Mossotti factor and frequency. A suitable frequency for a particular external conductivity is preferably obtained by preliminary experiments or theoretical calculations. For example, when the cells to be separated are fission yeast or budding yeast, and the external conductivity is 20 mS/m, a suitable suction frequency capable of selectively guiding the living cells 7 to the micropores 31 is 15 to 50 MHz. A suitable suction frequency that can selectively guide the dead cells 6 to the micropores 31 can be 10 to 60 kHz. Similarly, for an external conductivity of 50 mS/m, the preferred suction frequency for live cells may be 20-50 MHz and the preferred suction frequency for dead cells may be 50-250 kHz. When the external conductivity is 100 to 1000 mS/m, a suitable suction frequency for living cells can be about 0.5 to 300 MHz, more preferably 0.5 to 50 MHz. On the other hand, dead cells are not aspirated at all frequencies at this time.

After applying the suction frequency, by maintaining the frequency, the cells guided to the micropores 31 can be held in the micropores 31 . The present invention is advantageous over the prior art in that the cells sucked into the micropores can be firmly retained by using the predetermined microporous insulating film 3 . Also, by switching the frequency or changing the external conductivity while the cells are sucked into the micropores 31, the cells can be ejected from the micropores 31. FIG. Therefore, for example, when it is desired to separate and collect the living cells 7, positive dielectrophoresis is generated to suck the living cells 7 into the micropores 31, and then the cell suspension is replaced while the suction state is maintained. By generating negative dielectrophoresis, the live cells 7 can be ejected into the replaced liquid and only the live cells 7 can be selectively collected. Specifically, when the external conductivity is 20 mS/m, 20 MHz is applied, when the external conductivity is 100 mS/m, 1 MHz is applied, and when the external conductivity is 1000 mS/m, 10 MHz is applied. is sucked into the micropores 31, the liquid having the same external conductivity is replaced in that state, and a voltage of 50 kHz is applied, the living cells 7 can be ejected. Alternatively, if the same frequency is applied and the liquid is replaced with a liquid having an external conductivity of 1.5 mS/m or more, the living cells 7 can be ejected. On the other hand, with an external conductivity of 20 mS/m, dead cells 6 can be aspirated at 50 kHz and expelled at 50 MHz. In another embodiment, it is also possible to perform an operation of aspirating unnecessary cells first, and then collect the cells floating in the upper layer. In this case, although not shown, the cell can be provided with an inlet and an outlet to form a flow cell, thereby increasing throughput. According to these embodiments, selective separation of cells can be performed.

According to the cell separation and recovery apparatus and method according to the present embodiment, by using a microporous insulating film having a micropore diameter of about the size of a cell, cells can be separated into micropores by the difference in positive or negative dielectrophoretic force acting on the cells. It can be sucked into the micropores or repulsed out of the micropores, and compared with the conventional technology, the separation accuracy is high, and the retention force of the cells in the micropores is also increased.

[Second embodiment]
FIG. 2 shows a cell separating and collecting apparatus according to a second embodiment of the present invention. The cell separation/collection device according to the second embodiment is mainly composed of a cell 30 and an AC oscillator 20 .

The cell separation and recovery apparatus according to the second embodiment has the same configuration of the glass substrates 1a and 1b, the electrodes 2a and 2b and the spacer 5 in the cell 30, and the AC oscillator 20 as in the first embodiment. The second embodiment differs from the first embodiment in that the upper layer region A in the cell 30 is provided with an inlet 8 for injecting the cell suspension 5 and an outlet 9, and in that the microporous insulating film 3 is installed. different.

In this embodiment, the microporous insulating film 3 is spaced apart from both electrodes 2a and 2b and divides the space inside the cell 30 into an upper layer region A in contact with the electrode 2a and a lower layer region B in contact with the electrode 2b. It is different in that it is provided as follows. The specifications of the fine hole insulating film 3 may be the same as those described in the first embodiment, but the fine holes 31 need to be provided so as to penetrate the fine hole insulating film 3 . Further, the upper layer region A and the lower layer region B are configured so that substances can flow only through the plurality of micropores 31 provided in the microporous insulating film 3 . The position where the microporous insulating film 3 is provided may be near the central portion as shown in the figure, or may be other locations.

The injection port 8 and the discharge port 9 are provided in such a manner that the cell suspension 5 can be injected into the upper layer region A. In the illustrated embodiment, the injection port 8 and the discharge port 9 are provided so as to pass through the glass substrate 1a and the electrode 2a, but they may be provided so as to pass through the spacer. An inlet 8 and an outlet 9 allow continuous injection and ejection of the cell suspension 5 into the cell 30, enabling high-volume processing. Although not shown, the lower region B may also be similarly provided with inlets and outlets in any form.

In the cell separating and collecting device according to the present embodiment, cells are intended to be sucked into the micropores 31 from one region separated by the microporous insulating film 3 and discharged from the micropores 31 to the other region. Therefore, the shape of the fine holes 31 can be varied. 10, 12, and 14 to 18 are schematic enlarged views of one fine hole 31 portion P in FIG. 2, and the mode of each fine hole will be described.

FIG. 10 shows an example of a microporous insulating film having cylindrical micropores according to the first embodiment. The cylindrical micropores are as described in detail in the first embodiment. The microporous insulating film 3a shown in FIG. 10 has micropores 31a with a hole diameter φ _i located on one surface serving as the suction port i and the other hole diameter φ _o serving as the discharge port o. A substantially uniform high electric field can be generated in such a cylindrical fine hole 31a.

In the second aspect, the shape of the fine holes 31 may be such that the hole diameter changes along the thickness direction of the fine hole insulating film 3 . As a mode in which the hole diameter changes along the thickness direction, for example, the hole diameter changes continuously from the suction port i located on one surface of the microporous insulating film 3 to the discharge port o located on the other surface. It may be of increasing frusto-conical shape. An example of a microporous insulating film having truncated conical micropores is shown in FIG. The microporous insulating film 3b shown in FIG. 12 has micropores 31b with a hole diameter φi located on one surface serving as the suction port _i smaller than the hole diameter φo on the other side serving as the discharge port _o . In such a truncated cone-shaped fine hole 31b, the larger the difference in hole diameter between the suction port i and the discharge port o, the greater the electric field strength near the suction port i with respect to the electric field strength at the discharge port o. A uniform electric field is formed. It should be noted that the electric field intensity anywhere inside the hole is greater than the electric field intensity outside the hole.

In the third embodiment, the shape of the micropores 31 is a step in which the diameter increases stepwise from the suction port i located on one surface of the micropore insulating film 3 to the discharge port o located on the other surface. can be of type In other words, the step-shaped micropores can also be said to have a shape in which a plurality of cylinders with different diameters are stacked in order of increasing or decreasing diameters. An example of a microporous insulating film having stepped micropores is shown in FIGS. 14 and 15. FIG. In the microporous insulating film 3c shown in FIG. 14, the hole diameter φ _i located on one surface that serves as the suction port i is smaller than the hole diameter φ _o that serves as the discharge port o, and the hole diameter changes in two stages. 31c. In the microporous insulating film 3d shown in FIG. 15, the hole diameter φi located on one surface serving as the suction port _i is smaller than the hole diameter φo on the other side serving as the discharge port _o , and the hole diameter is changed in three steps. 31d. 14 and 15, as the hole diameter increases, the electric field strength in the fine holes 31c and 31d decreases, and the electric field strength near the suction port i is larger than the electric field strength at the ejection port o. is formed. It should be noted that the electric field strength anywhere in the fine holes 31c and 31d is greater than the electric field strength outside the fine holes 31c and 31d. Note that the number of stages of the staircase type is not limited to a specific number.

In the fourth aspect, the shape of the fine holes 31 is such that the hole diameter continuously decreases from the suction port i located on one side of the fine hole insulating film 3 toward the central portion in the thickness direction of the film. It may be an hourglass shape in which the hole diameter continuously increases from the part toward the ejection port o located on the other surface of the microporous insulating film 3 . In other words, the hourglass-shaped micropore can be said to have a shape in which two truncated cones having the same upper surface diameter are stacked so that the upper surfaces are in contact with each other. An example of a microporous insulating film having hourglass-shaped micropores is shown in FIG. In the microporous insulating film 3e shown in FIG. 16, the hole diameter φi located on one surface serving as the suction port _i is the same as the hole diameter φo on the other side serving as the discharge port _o . It has a fine hole 31e having a portion with a minimum hole diameter. In the fine hole 31e shown in FIG. 16, the electric field intensity near the suction port i and the vicinity of the ejection port o are substantially the same, and a non-uniform electric field having the maximum electric field strength is formed at the portion where the hole diameter is minimized. It should be noted that the electric field strength at any location within the fine holes 31e is greater than the electric field strength outside the fine holes 31e.

In the fifth aspect, the shape of the fine holes 31 is such that the hole diameter gradually decreases from the suction port i located on one surface of the fine hole insulating film 3 toward the center in the thickness direction of the film. A pincushion shape may be used in which the hole diameter increases stepwise from the part toward the ejection port o located on the other surface of the microporous insulating film 3 . An example of a microporous insulating film having pincushion-shaped micropores is shown in FIG. In the microporous insulating film 3f shown in FIG. 17, the hole diameter φi located on one surface serving as the suction port _i and the hole diameter φo located on the other surface serving as the discharge port _o are the same, and the central portion in the film thickness direction is the same. 31 f of fine holes having a portion with a hole diameter smaller than these are provided. Such a non-uniform electric field in the fine holes 31f is substantially the same as in the fourth mode, the electric field intensity near the suction port i and the vicinity of the discharge port o being substantially the same, and the electric field intensity being maximum at the portion where the hole diameter is small. A non-uniform electric field is formed with It should be noted that the electric field strength at any location within the fine holes 31f is greater than the electric field strength outside the fine holes 31f.

In the sixth aspect, the shape of the micropores 31 is such that the pore diameter continuously increases from the suction port i located on one surface of the microporous insulating film 3 toward the central portion in the thickness direction of the film. It may be pot-shaped in which the hole diameter continuously decreases from the part toward the ejection port o located on the other surface of the fine hole insulating film 3 . In other words, the pot-shaped fine hole can be said to have a shape in which two truncated cones having the same diameter on the lower surface are stacked so that the lower surfaces are in contact with each other. An example of a microporous insulating film having pot-shaped micropores is shown in FIG. In the microporous insulating film 3g shown in FIG. 18, the hole diameter φi located on one surface serving as the suction port _i is the same as the hole diameter φo on the other side serving as the discharge port _o . 31 g of fine holes having a portion with a maximum hole diameter. In the fine holes 31g shown in FIG. 18, the suction port i and the discharge port o have substantially the same electric field strength, and a non-uniform electric field having the minimum electric field strength is formed at the portion where the hole diameter is minimized. It should be noted that the electric field strength at any location within the fine holes 31g is greater than the electric field strength outside the fine holes 31g.

In this way, a microporous insulating film having non-cylindrical micropores, in which a non-uniform electric field is likely to be formed in the micropores, applies a suction frequency to cells to cause positive dielectrophoresis, thereby generating an electric field It is possible to attract from the outside of the hole where the strength is weak into the hole where the electric field strength is strong. After the cells are sucked into the pores, they can be moved toward the ejection port o with a weaker electric field strength and ejected out of the pores by applying an ejection frequency and negative dielectrophoresis.

In one aspect, the cell separation and collection device according to the second embodiment can be produced using an insulating ultrathin film. An insulating ultra-thin film is a film having a very thin thickness, for example, about 10 μm or less. The composition is not particularly limited as long as it is insulating, but an example is polyimide having a thickness of about 5 μm. Using such an insulating ultra-thin film, a flow channel film having a flow channel having a thickness of about 20 μm and a width of about 200 μm, and a microporous insulating film (microporous film filter) A cell according to this embodiment can be formed. These can be laminated and adhered in order of insulating ultra-thin film/channel-forming film/microporous insulating film/channel-forming film/insulating ultra-thin film. A cell can be formed by contacting the top and bottom of this laminate (also referred to as a filter), that is, by sandwiching and adhering the top and bottom insulating ultra-thin films between two flat electrodes. At this time, the insulating ultra-thin film and the electrodes on the bottom and top surfaces must be brought into close contact with each other without gaps using a liquid having a high dielectric constant and viscosity, such as water or glycerol. This is for the purpose of eliminating an air layer and applying a voltage to the inside of the flow path. In the cell obtained by this mode, a high electric field can be formed in the micropores of the microporous insulating film in the laminate, and cells can be sucked and discharged. In addition, the use of the channel film has the advantage that the inlet and outlet of the cell suspension can be formed in the upper layer and the lower layer sandwiching the microporous insulating film. In this embodiment, since the cell suspension containing pathogens and the like does not come into direct contact with the electrode, the electrode portion can be reused and the filter can be used as is.

In another aspect of the cell separation and collection device according to the second embodiment, the pore diameters of the micropores differ between the area where the micropore insulating film is continuous in the horizontal direction and the other area. The pore diameters of the micropores differ in the case of cylindrical micropores, and in the case of non-cylindrical micropores, it means that the diameters of the suction ports i differ. Such different micropore diameters can be appropriately determined by those skilled in the art according to the diameter of cells to be separated. As long as the pores have different diameters, the shape of the pores may be the same or different. Further, in the lower layer partitioned by the microporous insulating film, a plurality of regions with different pore diameters of the micropores are partitioned by walls that do not allow cells to migrate. FIG. 31 schematically shows such a cell. In the cell shown in FIG. 31, the microporous insulating film 3h is divided into a region having relatively large micropores _31h1 and a region having relatively small micropores _31h2 . _A spacer 4b is provided in the _lower layer of the cells vertically partitioned by the fine hole insulating film 3h. It is divided horizontally into a lower layer region B2 of the region having the In addition, discharge ports 9 are provided in the lower layer regions B1 and B2, respectively. The structure of the upper layer region A of the microporous insulating film 3h can be the same as that of the embodiment shown in FIG. In the cell according to this aspect, by providing regions with different pore diameters in the horizontal direction of the microporous insulating film, it is possible to separate cells specifically according to the size of the cells.

The separation and recovery method using the cell separation and recovery device according to the second embodiment can also be carried out in substantially the same manner as described in the first embodiment. Referring to FIG. 2, in the second embodiment, either the dead cells 6 or the live cells 7 are selectively moved to the lower layer region B through the micropores 31 by applying the suction frequency. to separate the cells. For example, a microporous insulating film 3 with a micropore diameter of 5 to 10 μm can be used to separate yeast cells. Then, the electrical conductivity of the cell suspension 5 can be set to 40-60 mS/m, and a suction frequency of 40-60 kHz can be applied. The dead cells 6 may be attracted to the micropores 31 by positive dielectrophoresis and fall to the lower layer region B. FIG. On the other hand, under the same conditions, the viable cells 7 are repelled from the micropores 31 by negative dielectrophoresis and remain in the upper region A. The dead cells 6 that have fallen to the lower layer region B do not return to the upper layer region A by passing through the micropores 31 again under the condition that the suction frequency is applied or under the condition that the frequency is not applied. It can be selectively separated into lower layer regions B. FIG. In particular, when a microporous insulating film 3 having a micropore diameter of about 10 μm is used, the cells are loosely held in the micropores, so that they are likely to freely fall to the lower layer region B depending on the flow velocity. In addition, by switching from the suction frequency to the ejection frequency of about 1 kHz, it is possible to generate negative dielectrophoresis in both live cells and dead cells, and the cells that are still retained by suction are actively moved to the lower layer region B. can be discharged. By repeating this process, clogging can be prevented, and target cells can be continuously sucked and ejected to move from the upper layer area A to the lower layer area B, thereby increasing the processing amount.

Alternatively, when live cells are sucked from the upper layer region A and ejected to the lower layer region B to be collected, the electrical conductivity of the cell suspension is preferably 70 to 1200 mS/m. Within such a conductivity range, a positive dielectrophoretic force is generated and attracted in the attraction frequency range (0.3 to 300 MHz) where the Clausius-Mossotti factor of living cells is positive. Then, by switching to the ejection frequency range in which the Clausius-Mossotti factor is negative, a negative dielectrophoretic force is generated and the living cells can be ejected out of the pores. In the cell suspension conductivity range of 70-1200 mS/m, dead cells have a negative Clausius-Mossotti factor over the entire frequency range. Therefore, negative dielectrophoresis occurs, and it is not attracted into the pores.

Furthermore, according to the aspect shown in FIG. 31, which is one aspect of the second embodiment, the suction frequency and the ejection frequency can be switched in the same manner as described above, but relatively large living cells 7a and relatively small living cells 7a can be switched. Cells 7b and 7b can be selectively separated from dead cells 6a and b, respectively, and recovered, enabling more accurate separation.

The cell separation and collection apparatus and method according to the second embodiment enable continuous cell separation by passing through the microporous insulating membrane, which is more advantageous in that mass processing becomes possible. In particular, by processing the shape inside the micropores, the electric field strength is changed from the suction port to the ejection port, and by switching the non-uniform electric field in the pores and the applied frequency, positive or negative dielectrophoresis can be achieved. Generating active passage of either live or dead cells through the micropores allows for cell separation.

[Third embodiment]
A device for separating and collecting cells according to a third embodiment of the present invention comprises: a cell comprising two conductive layers provided facing each other; and one or more microporous membranes provided between the two conductive layers; An AC oscillator that applies a frequency within a predetermined range between the two conductive layers, and at least one of the conductive layers provided facing each other is a conductive coating layer that covers the microporous insulating film.

In one aspect of the present embodiment, unlike the first and second embodiments, there is no upper surface electrode of the cell, and instead the upper surface of the microporous insulating film (the surface opposite to the surface facing the bottom surface) ) with a conductive coating layer. FIG. 19 is a partial conceptual diagram of a cell cross section according to this embodiment. In the cell shown in FIG. 19, a bottom electrode 2b is provided on the bottom surface, and a microporous insulating film 3b having truncated conical pores 31b is provided across a gap filled with a cell suspension. Also, the upper surface of the microporous insulating film 3b is covered with a conductive coating layer 11. As shown in FIG. AC oscillator 20 is arranged so that a frequency can be applied between bottom electrode 2b and conductive coating layer 11 . Above the conductive coating layer 11, a portion that fills the cell suspension may be provided, and above that, the top surface of the cell may be provided, or the cell may be an open cell with no top surface. . The upper surface of the cell may be made of glass or the like, but is not particularly limited. The shape of the pores of the microporous insulating film 3b is not limited to the illustrated mode, and may be any mode described in the second embodiment. Although not shown, the bottom electrode may not exist, the top electrode may exist, and only the bottom surface (the surface facing the bottom surface) of the microporous insulating film may be provided with the conductive coating layer. In this case, the cell may have a funnel-shaped cell concentration portion below the microporous insulating film. Then, cells can be moved to the site, and observation and collection of a very small number of cells can be facilitated.

The conductive coating layer 11 may be a metal layer or a conductive polymer layer. Although the thickness is not particularly limited, it may be about 0.01 to 5 μm. In addition, it is preferable not to provide a conductive coating layer on the inner walls of the micropores of the micropore insulating film 3b. This is because if a conductive coating layer is provided on the inner wall of the micropore as in the cell shown in FIG. 20, it is difficult to generate a non-uniform electric field inside the micropore, making it difficult to attract cells.

In another aspect of this embodiment, neither the top electrode nor the bottom electrode of the cell is present, and both of the conductive layers provided facing each other may be conductive coating layers that cover the microporous insulating film. . In the cell shown in FIG. 22, a microporous insulating film 3b having truncated cone-shaped pores 31b is provided. be done. An AC oscillator 20 is arranged such that a frequency can be applied between the conductive coating layers 11a,b. Above the conductive coating layer 11a, a portion that fills the cell suspension may be provided, and above that, the top surface of the cell may be provided, or the cell may be an open cell with no top surface. . Under the conductive coating layer 11b, a portion filled with the cell suspension may be provided between the bottom surface (not shown) and the bottom surface. Also in this aspect, the conductive coating layer is not provided on the inner walls of the micropores. Alternatively, a funnel-shaped cell concentration portion may be provided below the microporous insulating film.

An insulating coating layer can be formed on the surface of any of the conductive coating layers of the present embodiment that is in contact with the cell suspension. Even if the insulating coating layer is formed, an electric field can be formed in the micropores, and electrolysis of the cell suspension can be prevented, and cell adsorption to the conductive coating layer can be prevented. The specifications of the insulating coating layer can be the same as those of the insulating coating layer 3 of the fourth embodiment described later.

According to the present embodiment, without providing a top electrode and/or a bottom electrode, by applying a frequency to the conductive coating layer 11 coated on the microporous insulating film 3b and the bottom electrode or the top electrode, or the conductive coating layer A dielectrophoretic force can be applied to cells by applying a frequency between 11a and 11b. For this reason, the cell can be an open system in which the upper surface is not sealed, and injection of the cell suspension becomes easier.

[Fourth embodiment]
A cell separation and recovery device according to a fourth embodiment of the present invention comprises: a cell comprising two conductive layers provided facing each other; one or more microporous thin films provided between the two conductive layers; an alternating current oscillator that applies a frequency within a predetermined range between two conductive layers, one of the conductive layers provided facing each other is an electrode that constitutes the bottom surface or the top surface of the cell; The other of the conductive layers provided and the microporous membrane is a conductive net or sieve coated with an insulating coating layer, provided at a distance from the electrodes.

In this embodiment, unlike the first and second embodiments, either the top electrode or the bottom electrode of the cell does not exist. Also, the microporous membrane is a conductive mesh or sieve covered with an insulating coating layer over its entire surface. That is, the microporous thin film plays a role of allowing cells to pass through and separate them, and a role of an electrode for applying frequency. FIG. 23 is a partial conceptual diagram of a cell cross section according to this embodiment. In the cell shown in FIG. 23, an upper surface electrode 2a is provided on the upper surface of the cell, and a conductive mesh or sieve 12 is provided across a gap filled with cell suspension. The entire surface of the conductive mesh or sieve 12, that is, all surfaces that can come into contact with the cell suspension, is provided with an insulating coating layer 3a, which functions as a microporous insulating membrane. Although not shown, below the conductive mesh or sieve 12 is provided a cell bottom with a gap filled with the cell suspension. The bottom portion may be made of glass or the like, but is not particularly limited. An AC oscillator 20 is arranged such that a frequency can be applied between the top electrode 2a and the conductive mesh or sieve 12. FIG. Instead of the embodiment shown in FIG. 23, it is also possible to provide a bottom electrode without providing the top electrode, and apply a frequency between the bottom electrode and the conductive mesh or sieve.

The conductive mesh or sieve 12 may be a flat plate mesh or sieve made of metal or conductive polymer, and a microsieve with any mesh size on the order of micrometers can be used. is not limited. The mesh size of the mesh or sieve is such that the pores after forming the insulating coating layer 3a with a predetermined thickness on its surface have the preferred pore sizes described in the first and second embodiments. can be selected as appropriate. The insulating coating layer 3a provided on the surface of the conductive mesh or sieve may be made of polyimide, silicone resin, oxide thin film of aluminum or silicon, polydimethylsiloxane, or epoxy resin photoresist. The thickness of the insulating coating layer 3a may be, for example, about 0.1 to 5 μm, and may be provided with a uniform thickness over the entire surface of the conductive net or sieve, or may have a partially different thickness. may be By adopting the structure of the present embodiment, it is possible to manufacture fine pores with high accuracy by precision processing or the like, and there are advantages such as heat resistance, thinness, and mechanical strength being easily obtained.

[Fifth embodiment]
A cell separation and recovery device according to a fifth embodiment of the present invention comprises: a cell comprising two conductive layers provided facing each other; one or more microporous thin films provided between the two conductive layers; an AC oscillator that applies a frequency within a predetermined range between two conductive layers, at least one of the conductive layers provided facing each other is a conductive mesh or a sieve, and the microporous thin film is the conductive layer. It is a microporous insulating membrane provided in spaced relation to or in contact with a mesh or sieve.

In this embodiment, unlike the first and second embodiments, there is no top electrode of the cell, and instead a conductive mesh or sieve is used. FIG. 24 is a partial conceptual diagram of a cross section of a cell according to this embodiment. In the cell shown in FIG. 24, a bottom electrode 2b is provided on the bottom, and a microporous insulating film 3b having truncated conical pores 31b is provided across a gap filled with cell suspension. A conductive mesh or sieve 12 is provided above the microporous insulating film 3b with a gap filled with the cell suspension. Above the conductive mesh or sieve 12 there may be a top surface of the cell, or it may be an open cell with no top surface. Use of this embodiment of the cell requires that the sieve electrode is completely immersed in the cell suspension. This is to allow cells to pass through the mesh of the sieve electrode. An AC oscillator 20 is arranged such that a frequency can be applied between the bottom electrode 2b and the conductive mesh or sieve 12 . The shape of the pores of the microporous insulating film 3b is not limited to the illustrated mode, and may be any mode described in the second embodiment. In the illustrated embodiment, the conductive mesh or sieve 12 is arranged apart from the microporous insulating film 3b, but the conductive mesh or sieve 12 is provided in contact with the microporous insulating film 3b. may have been When the conductive mesh or sieve 12 and the microporous insulating film 3b are in contact with each other, the electric field strength distribution is similar to that shown in FIG.

Further, the device according to this embodiment may have no top or bottom electrode, but rather a microporous membrane between two opposing conductive meshes or sieves. FIG. 25 is a partial conceptual diagram of a cell cross section according to this embodiment. In the cell shown in FIG. 25, conductive nets or sieves 12a,b are provided above and below the cell, and a gap filled with the cell suspension is separated by a truncated cone-shaped pore 31b between them. A microporous insulating film 3b is provided. Above the conductive mesh or sieve 12a, there may be a top surface of the cell, or it may be an open cell with no top surface. However, for the same reason as before, the use of this embodiment of the cell requires that the sieve electrode be completely immersed in the cell suspension. Also, although not shown, a bottom portion is provided below the conductive net or sieve 12b with a gap for filling the cell suspension. An AC oscillator 20 is arranged so that a frequency can be applied between the two conductive nets or sieves 12a,b. The shape of the pores of the microporous insulating film 3b is not limited to the illustrated mode, and may be any mode described in the second embodiment. Further, in the illustrated embodiment, the conductive meshes or sieves 12a,b are spaced apart from the microporous insulating film 3b, but one or both of the conductive meshes or sieves 12a,b Both may be provided in contact with the microporous insulating film 3b. It is theoretically assumed that when the microporous insulating film 3b is in contact with and sandwiched between the conductive nets or sieves 12a and 12b, the electric field intensity distribution will be similar to that shown in FIG. be done.

The conductive mesh or sieve 12 may be a flat plate mesh or sieve made of metal or conductive polymer, and a microsieve with any mesh size on the order of micrometers can be used. is not limited. The mesh size of the net or sieve may be a size that allows cells to pass through and excludes contaminants, and may be, for example, about 2 to 300 μm. The thickness of the mesh or sieve is not particularly limited, but may be about 10 to 100 μm. By using a conductive mesh or sieve instead of the top electrode, the cell suspension can be injected into the cell through the mesh of the mesh or sieve, and an open cell can be obtained. In addition, by using a conductive mesh or sieve instead of the bottom electrode, a funnel-shaped cell concentration portion is provided below the conductive mesh or sieve under the microporous insulating film, and cells are moved to this portion. This has the advantage of facilitating observation and recovery of a very small number of cells.

FIG. 32 is a conceptual schematic diagram showing a cell in which a funnel-shaped cell concentration portion is installed. The cell shown in FIG. 32 has a funnel-shaped cell concentration area below a conductive mesh or sieve 12b located below the cell. An electrode 2c is provided at the tip of the cell concentration portion, and a cell outlet 9 is provided near the tip. The material of the electrode 2c is not particularly limited, and any of the electrodes mentioned in the first embodiment can be used, but it is particularly preferable to use a transparent electrode such as ITO from the viewpoint of enabling observation. In the cell according to this aspect, the first AC oscillator 20a applies frequency between the two conductive nets or sieves 12a,b, the lower conductive net or sieve 12b and the second AC oscillator 20a applies frequency between the electrodes 2c. 2 AC oscillator 20b. In this embodiment, it is preferable to apply a frequency that causes positive dielectrophoresis to the second AC oscillator 20b for concentration recovery. In order to recover cells from the discharge port 9, it is preferable to apply a frequency that causes negative dielectrophoresis.

Any of the conductive nets or sieves of this embodiment can have an insulating coating layer formed on the surface in contact with the cell suspension. Even if an insulating coating layer is formed, an electric field can be formed in the micropores, and electrolysis of the cell suspension can be prevented, and cell adsorption to the conductive net or sieve can be prevented. The specifications of the insulating coating layer can be the same as those of the insulating coating layer 3 of the fourth embodiment.

[Sixth embodiment]
A cell separation and collection device according to a sixth embodiment of the present invention comprises: a cell comprising two conductive layers provided facing each other; one or more microporous thin films provided between the two conductive layers; an AC oscillator that applies a frequency within a predetermined range between two conductive layers, the conductive layers provided facing each other are electrodes forming the bottom surface and the top surface of the cell, and the microporous thin film comprises at least Two or more microporous insulating films provided between the electrodes separated from one of the electrodes, wherein one or more microporous thin films provided between the two conductive layers are provided apart from each other wherein the micropore diameters of the two or more microporous insulation films are different from each other, and the micropore diameters gradually decrease from the microporous insulation film near the top surface to the microporous insulation film near the bottom surface.

This embodiment is a variation of the second embodiment in which two or more microporous insulating electrodes are spaced between the opposed bottom and top electrodes, spaced apart from each of these electrodes and spaced from each other. A membrane is provided. Then, the pore diameters of the micropores provided in the microporous insulating film gradually decrease from the microporous insulating film closer to the upper electrode to the microporous insulating film closer to the lower electrode. The number of microporous insulating films to be provided may be two or more, and may be three, four, five, or six or more, and is not particularly limited. In the embodiment using two microporous insulating films, the cells are vertically divided into three-layer regions by the films, and in the embodiment using three microporous insulating films, the cells are vertically divided into four-layer regions by the films. be able to. Further, the shape of the holes may be any of the forms described in the second embodiment, and the hole shapes of the two or more microporous insulating films may be different. In any case, it is particularly preferable to provide truncated conical micropores. In the present embodiment, the progressively decreasing micropore diameter means that the diameter of the cylindrical micropores gradually decreases. On the other hand, in the case of non-cylindrical micropores, it means that the size of the suction port gradually decreases.

According to this embodiment, by providing two or more microporous insulating films, it is possible to separate cells of different sizes one after another, thereby enabling more accurate cell separation.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited by the following examples.

(1) Preparation of Cell Samples After culturing fission yeast (cylindrical) and budding yeast (snowball-shaped) in a natural medium to the logarithmic growth phase, they were washed with distilled water using a centrifuge to prepare samples. Dead cells were prepared by sterilization by heat treatment at 70° C. and washing with distilled water. The conductivity of the cell suspension was adjusted to 20-100 mS/m with potassium chloride. In order to distinguish live cells from dead cells, live cells were fluorescently stained with SYTO9 that emits green fluorescence, and dead cells were fluorescently stained with PI that emits red fluorescence.

(2) Experimental Apparatus and Experimental Operation In this example, a cell was assembled using ITO (indium-tin oxide) transparent electrodes of 25×20 mm so as to allow microscopic observation. First, an insulating spacer of 50 μm was used to secure the distance between the top and bottom electrodes. A 20 μm-thick polycarbonate microporous insulating film (track-etched membrane, manufactured by Merck) was laid in the gap so as to be in contact with the electrode surface on the bottom side to form the cell of this example. 1.5 μL of cell suspension was injected onto the microporous insulating membrane of this cell (FIG. 1). This was placed on an inverted microscope and observed while applying an AC voltage with an oscillator.

(3) Difference in cell attraction to micropores with respect to pore size When an AC voltage was applied, a phenomenon was observed in which fission yeast cells on the membrane surface were attracted into the micropores due to the dielectrophoretic force (Figs. 3 and 4). Also, this phenomenon was achievable at a minimum of 2V. This voltage is about 1/15 of the disclosure of Patent Document 1. However, when the pore diameter was considerably larger than the cells, a plurality of cells were adsorbed and aggregated on the inner surface of the pore (Fig. 5). When an insulating film with a pore size considerably larger than that of the cells was used, the cells sometimes passed through the pores, and the accuracy of separating live cells and dead cells was lowered. In experiments conducted using other microbial cells, the diameter of the micropores that were aspirated was about 1 to 2 times the size (minimum diameter) of the cells. Table 1 shows whether or not each microorganism species can be introduced into the micropores. In the table, + indicates that introduction was possible, and - indicates that introduction was not possible. +- indicates that those with a nearly spherical shape were introduced, and those with buds were caught in the entrance and could not be introduced.

(4) Difference in frequency characteristics between live cells and dead cells sucked into micropores When the fission yeast cell suspension has a conductivity of 20 mS/m and a micropore size of 5 μm, only viable cells are fine when 20 MHz is applied. It was found that only dead cells were sucked into the micropores (Fig. 7) when 50 kHz was applied (Fig. 6).

(5) Confirmation of separation of live and dead cells by fluorescence-stained cells Under the conditions of (4) above, only live cells can be aspirated from a cell suspension in which live and dead are mixed (Fig. 8), and only dead cells can be aspirated. It was confirmed by fluorescent staining that it was possible (FIG. 9).

(6) Simulation of electric field strength inside micropore We simulated the electric field intensity of The conditions for the simulation were as shown in Table 2 below. Under any simulation conditions, the liquid gap between the microporous insulating film and the top electrode was 25 μm, the liquid gap between the microporous insulating film and the bottom electrode was 25 μm, and the AC voltage was 10 MHz and 2V. In Table 2, the notation of the hole diameter φ is the diameter for the cylindrical type, the diameter φ i of the suction port _i / the diameter φ _o of the discharge port o for the truncated cone type, and the suction port i for the stepped type. Diameter φ _i of /Intermediate Part Diameter (in the case of a three-step stepped type with an intermediate part)/Diameter φ _o of the outlet o, for the hourglass shape, the diameter φ _i of the inlet i/diameter of the minimum part/ For the pincushion type, the diameter φ _i of the suction port _i /the diameter of the intermediate portion/diameter φ _o of the discharge port _o . The diameter of the part/diameter φ _o of the outlet o shall be stated.

Cylindrical pores are shown schematically in FIG. 10 and have a shape corresponding to commercially available track edged membranes. In the cylindrical pores of simulation numbers 1 to 3, a substantially uniform electric field is generated in each of the pores, and the electric field strength is the liquid portion between the micropore insulating film outside the pores and the upper electrode. , or stronger than the liquid portion between the microporous insulating film and the bottom electrode. From this, it was found that cells can be sucked from the liquid part in cylindrical pores, but discharge can occur in both vertical directions. FIG. 11 is a graph showing the electric field intensity on the center line of the cylindrical micropores of simulation numbers 1-3. A center line shall mean the central axis of a micropore (cylinder). In the graph, the distance (μm) means the distance from the bottom electrode. From FIG. 11, it was found that the smaller the micropore diameter, the more uniform the inside of the micropore and the stronger the electric field strength. Also, although not shown, it was found that the larger the micropore diameter, the more concentrated the electric field at the ends of the suction port i and the discharge port o.

The truncated conical micropores are as schematically shown in FIG. In the truncated conical micropores of simulation numbers 4 to 6, a non-uniform electric field was formed in which the electric field strength gradually weakened from the suction port i toward the discharge port o. The electric field strength inside the micropores was stronger than that of the liquid portion between the microporous insulating film and the top electrode, or between the microporous insulating film and the bottom electrode, which is outside the micropores. FIG. 13 is a graph showing the electric field intensity on the centerline of the truncated conical micropores of simulation numbers 4-6. The larger the hole diameter difference between the hole diameter φ _i of the suction port i and the hole diameter φ _o of the discharge port o, the greater the electric field strength difference. It was presumed by simulations that a microporous insulating film provided with truncated conical micropores enables suction and ejection by forming a non-uniform electric field.

In addition, an experiment to verify the suction and discharge of cells using truncated cone-shaped micropores was performed as follows. The truncated conical micropores are processed by dry etching one side of a commercially available polycarbonate isopore (manufactured by Merck) with a hole diameter of 5 μm for 15 minutes with a high-frequency oxygen plasma device (plasma reactor PR-301, Yamato Scientific). A microporous insulating film having a hole diameter _φi of 5 μm and a hole diameter φo of the ejection port _o of 7 μm was obtained. Specifically, the conditions disclosed in Reference Anal Chem, 76, 2025-2030, 2004 were used.

A cell was assembled using two 25×20 mm ITO transparent electrodes according to the first embodiment (FIG. 1). First, an insulating spacer of 100 μm was used to secure the distance between the top and bottom electrodes. A truncated cone-shaped microporous insulating film having a thickness of 23 μm was laid so as to be in contact with the electrode surface on the bottom side of the gap to form the cell of this example. 1 μL of the cell suspension was injected onto the microporous insulating membrane of this cell. This was placed on an inverted microscope and observed while applying a voltage of 4 V from 10 kHz to 10 MHz with an AC oscillator. A cell suspension was prepared by suspending fission yeast cells in YE medium (95 mS/m) to a concentration of 1×10 ⁷ cells/mL.

In the case of the suction verification experiment, the suction port i was placed on the upper side and the rate of cells being sucked was measured. bottom. FIG. 28 shows the suction rate and ejection rate for the applied frequency of living cells. It was attracted by positive dielectrophoresis at 500 kHz or higher, and the highest attraction rate was at 1 MHz. On the other hand, the aspirated cells were ejected by negative dielectrophoresis at 500 kHz or lower, and the ejection rate was the highest at 50 kHz. In addition, dead cells did not cause absorption into the pores at all frequencies. It was confirmed that these experimental results approximately agree with the positive/negative of the real part of the Clausius-Mossotti factor shown in FIG.

The stepped micropores are as shown in schematic diagrams in FIGS. In simulation numbers 7 and 8, a non-uniform electric field was formed in which the electric field strength gradually weakened from the suction port i toward the discharge port o. A substantially uniform electric field was generated in the portion having the same micropore diameter. In addition, the electric field intensity inside the micropores was stronger than the liquid portion between the micropore insulating film and the top electrode, or the liquid portion between the micropore insulating film and the bottom electrode, which is outside the micropores. FIG. 29 is a graph showing the electric field intensity on the center line of the step-shaped, hourglass-shaped, pincushion-shaped, and pot-shaped micropores of simulation numbers 7 to 11. FIG. The definitions of the center line and the distance (μm) are the same as in FIG. 11 above. Since a non-uniform electric field could be formed in the stepped micropores as well, it is presumed that the liquid is sucked and ejected in the same manner as in the truncated conical micropores.

The hourglass-shaped micropores are as shown in the schematic diagram of FIG. 16 . Referring to FIG. 29, in the hourglass-shaped fine hole of simulation number 9, the electric field strength increases from the inlet i toward the portion where the hole diameter is minimal (referred to as the hole diameter minimum portion), and the discharge from the hole diameter minimum portion. A non-uniform electric field is formed in which the electric field intensity weakens toward the exit o. In hourglass-shaped micropores, the electric field intensity is strong at the minimum diameter portion in the central portion of the micropore insulating film. However, it is presumed that the cells that are sucked into the micropore and reach the minimum pore diameter are then affected by an electric field similar to the truncated cone-shaped micropore and are sucked and ejected in the same way as the truncated cone-shaped micropore. be.

The pincushion micropores are as shown in the schematic diagram of FIG. Referring to FIG. 29, in the pincushion micropores of simulation No. 10, the electric field strength in the portion where the diameter is small in the intermediate portion was stronger than the electric field strength in the vicinity of the suction port i and the discharge port o. The electric field intensity at any portion inside the micropore was stronger than the liquid portion outside the micropore between the microporous insulating film and the top electrode or between the microporous insulating film and the bottom electrode. The distribution tendency of the electric field intensity in the pincushion-shaped micropore conforms to that of the hourglass-shaped micropore, and it is presumed that cells can be sucked and discharged in the same way as in the hourglass-shaped micropore.

The pot-shaped pores are as shown in the schematic diagram of FIG. 18 . Referring to FIG. 29, in the pot-shaped pore of simulation number 11, the electric field strength in the vicinity of the suction port i and the discharge port o was stronger than the electric field strength in the portion where the diameter of the intermediate portion was maximized. Therefore, it is presumed that by applying a predetermined frequency, cells are sucked from the suction port i, move downward, and are ejected from the ejection port o.

In simulation numbers 12 to 14, the influence of the film thickness of the micropore insulating film was examined for truncated cone-shaped micropores. As a result, it was shown that the thicker the insulating film, the lower the electric field strength in the micropores, but the non-uniform electric field is formed in the micropores in the truncated conical shape. However, if the film thickness of the microporous insulating film is close to the cell diameter, it is presumed that the discharge by negative dielectrophoresis is rapid. In addition, if the movement distance of the cells in the micropores is long, that is, if the film thickness of the micropore insulating film is too thick, the cells are again attracted to the inlet i side by positive dielectrophoresis before being ejected from the ejection port o. It is expected that there will be a risk that

(7) Conductive film coating on the surface of the microporous insulating film A microporous insulating film having a thickness of 25 µm and having truncated conical pores with a hole diameter φi of 5 µm at the inlet _i and a hole diameter φo at the outlet _o of 10 µm. A thin film coating of gold was applied to the surface on which the suction port i of was located. The thickness of the gold thin film was about 100 nm. A schematic diagram is shown in FIG. The cell configuration was such that an AC oscillator 20 was connected between the thin gold film 11 functioning as a top electrode and the flat plate bottom electrode 2b without providing a top electrode so that an AC voltage could be applied. When a cell suspension is injected from the top of this cell and an alternating current of 10 MHz and 2 V is applied, the electric field strength near the suction port i is strong in the micropores, and the electric field strength is weak toward the discharge port o. A non-uniform electric field was formed. In this embodiment, by providing a conductive film on the upper surface of the microporous insulating film instead of the upper electrode, it was possible to inject the cell suspension as an open system without sealing the upper portion. However, in this embodiment, the electric field near the upper portion of the suction port i was weak, so the suction ability was lowered. FIG. 30 is a graph showing the result of simulating the electric field intensity on the center line of the micropores of the mode shown in FIGS. 19 to 23 under the same simulation conditions as in the previous example. In FIG. 30, "upper surface conductor coating" is a graph showing the electric field intensity on the center line of the truncated cone-shaped micropores of the example shown in FIG. The definitions of the center line and the distance (μm) are the same as in FIG. 11 above.

A fine-pore insulating film having a thickness of 25 μm and having similar truncated conical pores was coated with a thin film of gold on the surface where the suction port i was located and the inner wall surface of the fine pores. A schematic diagram is shown in FIG. Similarly, the surface of the 25 μm-thick microporous insulating film having truncated conical pores on which the outlets o are located was coated with gold as a thin film. A schematic diagram is shown in FIG. 20 and 21, the coating thickness of the gold thin film was the same as in the embodiment shown in FIG. 19 above. In the embodiments of FIGS. 20 and 21, an AC oscillator 20 is connected between the gold thin film 11 functioning as the upper electrode and the flat plate bottom electrode 2b without providing the upper electrode, and an AC voltage can be applied. assembled the cell. However, in these cell configurations, even if an AC voltage was applied, a strong electric field capable of attracting was not formed near the inlet i. In FIG. 30, "upper surface conductor coating" is a graph showing the electric field intensity on the center line of the truncated conical micropore of the example shown in FIG. 20, and "lower surface conductor coating" is the example shown in FIG.

Similarly, the surface of the 25 μm-thick microporous insulating film having truncated conical pores on which the suction port i is located and the surface on which the discharge port o is located were coated with a thin film of gold. FIG. 22 shows a schematic diagram. Also in the embodiment of FIG. 22, the thickness of the gold thin film was the same as that of the embodiment shown in FIG. In the embodiment of FIG. 22, neither a top electrode nor a bottom electrode is provided. An AC oscillator 20 was connected between the provided gold thin films 11b to assemble a cell in which an AC voltage could be applied. In this cell configuration, a non-uniform electric field was formed in the micropores that enabled suction and ejection. In FIG. 30, "double-sided conductive film" is a graph showing the electric field intensity on the center line of the truncated cone-shaped micropores of the example shown in FIG.

(8) Insulating film coating on conductive membrane filter surface The entire surface of a 23 μm thick metal sieve plate (micro sieve) having cylindrical micropores with a pore diameter of 5 μm was coated with an electrically insulating silicone resin. . The coating thickness of the silicone resin was about 1 μm. A schematic diagram is shown in FIG. The cell configuration was such that an AC oscillator 20 was connected between the electrode 2a arranged on the upper surface and the metal sieve plate 12 without providing a bottom electrode so that an AC voltage could be applied. In this embodiment, the insulating film 3a coated on the metal sieve plate 12 functions as a microporous insulating film. When a cell suspension is injected from the top of this cell and an alternating current of 10 MHz and 2 V is applied, the electric field strength near the inlet i is strong in the cylindrical fine hole 31a, and the electric field strength is weak toward the outlet o. A non-uniform electric field was formed. In FIG. 30, "conductor insulating film" is a graph showing the electric field intensity on the center line of the cylindrical micropores of the example shown in FIG.

(9) Use of metal flat plate sieve as electrode An example using a metal flat plate sieve (metal micro sieve) having micro-size gaps without using an upper electrode was examined. A schematic diagram is shown in FIG. An AC oscillator 20 was connected between the metal plate sieve 12 and the bottom electrode 2b made of ITO to prepare a cell in which an AC voltage could be applied. In this cell, a microporous insulating film having a thickness of 25 μm and having truncated conical micropores 31b with a hole diameter φ _i of the inlet i of 5 μm and a hole diameter φ _o of the outlet o of 10 μm is used as a flat metal sieve. provided between the bottom electrodes. When a cell suspension is injected from the top of this cell and an alternating current of 10 MHz and 2 V is applied, the electric field intensity is strong near the inlet i and weak toward the outlet o in the truncated cone-shaped micropores. A non-uniform electric field capable of suction and ejection was formed. It is important to inject the cell suspension so that the position of the sieve 12 is lower than the liquid surface. If the cells are at the same position as the liquid surface, they will not fall because they will stay in the gaps of the sieve due to surface tension. Also, no significant non-uniform electric field was formed in the gaps and the periphery of the flat metal sieve 12 . This indicates that the cells can pass through the gaps of the sieve 12 and fall down, since the cells are not captured by dielectrophoresis in the gaps of the sieve 12 .

An embodiment was studied in which neither the top electrode nor the bottom electrode were used, but instead two metal flat plate sieves with micro-sized gaps were used. FIG. 25 shows a schematic diagram. An AC oscillator 20 is connected between the flat metal sieve 12a provided above the cell that functions as a top electrode and the flat metal sieve 12b provided below the cell that functions in the same manner as the bottom electrode, and an alternating voltage can be applied. An embodiment cell was fabricated. In this cell, a fine hole insulating film 3b having a thickness of 25 µm and having a truncated conical fine hole 31b with a hole diameter φi of 5 µm at the inlet _i and a hole diameter φo at the outlet _o of 10 µm is formed by two sheets of metal. It was provided between the flat sieves 12a and 12b. When a cell suspension is injected from the top of this cell and an alternating current of 10 MHz and 2 V is applied, the electric field strength near the inlet i is strong in the truncated cone-shaped fine hole 31b, and the electric field strength is increased toward the outlet o. A weakening non-uniform electric field was formed. Similar to the embodiment shown in FIG. 24, it is necessary to inject the cell suspension so that the position of the sieve 12 is lower than the liquid surface. Also, no significant non-uniform electric field was formed in the gaps between and around the metal flat plate sieves 12a and 12b. This indicates that cells can pass through the gaps between the sieves 12a and 12b, since cells are not captured by dielectrophoresis in the gaps between the sieves 12a and b.

In the example shown in Figures 24 and 25, the cell suspension can be injected from the top (open system) and the cells pass through the interstices without being caught in the metal part of the flat metal sieve and through the microporous insulation. It easily reached the membrane and was sucked into the pores. In particular, when Tokyo Process Service Micro Sieve (mesh line number: 114 L/inch, mesh pitch: 223 μm, opening width: 196 μm, thickness: 17 μm, made of nickel) is used as a metal flat plate sieve, the light transmittance is high, so it can be used with a microscope. Observation was also possible. According to these embodiments, there is no need for precise flow rate control using an expensive syringe pump, and by changing the gap size of the metal plate sieve (micro sieve), it is possible to pretreat more cells than the target cells. It was also possible to remove large-sized impurities and the like.

(10) Laminated Membrane Filter with Channel Using Ultrathin Film A cell was produced using a laminated membrane filter with channels using an ultrathin film. A schematic diagram of the cross section of this filter is shown in FIG. The produced filter was provided with an insulating ultra-thin film 13 made of polyimide on the top and bottom surfaces of the cells, and a microporous insulating film 3b was provided via a gap filled with the cell suspension. Such a filter includes a polyimide insulating ultra-thin film 13 (Kapton 20EN, Toray Dupont) with a thickness of 5 μm, a channel film with a thickness of 20 μm (Hybrid adhesive sheet SJ41, Tomoegawa Paper Works, a channel with a width of 200 μm). grooves), the microporous insulating film 3b, the flow path film, and the insulating ultra-thin film 13 are laminated and bonded in this order. The laminated flow path filter was sandwiched between the top electrode 2a and the bottom electrode 2b, each of which was made of a flat plate ITO, and the electrodes were brought into tight contact with water or the like to form a cell. Also in the cell of this example, a high electric field was formed in the pores and the cells could be attracted. In this embodiment, since the cell suspension containing pathogens and the like does not come into direct contact with the electrodes, the electrode portions 2a and 2b can be reused and the filter can be used as is.

(11) Suction and discharge of living cells by using a high-conductivity suspension solution Living cells or dead cells of yeast are used as models of dielectric particles, and the conductivity of the external solution in which each model cell is suspended is 20 to 1000 mS /. m and varying the frequency, the relationship between the frequency and the real part of the Clausius-Mossotti factor (Re CMf) was calculated. The real part of the Clausius-Mossotti factor was calculated based on the following equation. where ε _p ^* and ε _s ^* represent the complex dielectric constants of the particles and solvent, respectively.

The results are shown in FIG. If the conductivity of the external solution is about 100 mS / m, a positive dielectrophoretic force is generated in the frequency range where the Clausius-Mossotti factor of the living cell is positive, and the pores described in each embodiment of the present invention Live cells are aspirated. Then, by switching the frequency to a range in which the Clausius-Mossotti factor of living cells is negative, a negative dielectrophoretic force is generated in the living cells and can be ejected from the pores. This almost coincided with the experimental result of the suction rate/discharge rate shown in FIG.

The device and method of the present invention are useful not only in cell culture and cell assays, but also in medical applications such as the separation and detection of infectious bacteria and tumor cells in blood and urine.

10, 30 cell, 20, 20a, 20b AC oscillator 1a glass substrate, 1b glass substrate,
2a top electrode, 2b bottom electrode, 2c electrode 3 microporous insulating film, 31 micropore 4 spacer, 5 cell suspension,
6 dead cells, 7 live cells, 8 inlet, 9 outlet, 11 conductive coating layer, 12 conductive mesh or sieve, 13 insulating ultra-thin film.

Claims (13)

a cell comprising two conductive layers provided facing each other and one or more microporous membranes provided between the two conductive layers;
an AC oscillator that applies a frequency within a predetermined range between the two conductive layers; .5 times or less, or
(b) the micropore has a non-cylindrical shape selected from a truncated cone shape, a staircase shape, an hourglass shape, a pincushion shape, or a pot shape, and the diameter of the inlet of the micropore is the minimum diameter of the cells to be separated; larger and no greater than 2.5 times the smallest diameter,
Cell separation and recovery device. the conductive layers provided facing each other are electrodes forming the bottom surface and the top surface of the cell;
wherein the microporous thin film is a microporous insulating film provided between the electrodes and separated from at least one of the electrodes;
The separation and recovery device according to claim 1. 2. The separation and recovery device according to claim 1, wherein at least one of the conductive layers provided opposite to each other is a conductive coating layer that covers the surface of the microporous insulating film. one of the conductive layers provided to face each other is an electrode constituting the bottom surface or the top surface of the cell;
The other of the conductive layers provided opposite to each other and the microporous thin film are conductive nets or sieves provided apart from the electrodes and covered with an insulating coating layer,
The separation and recovery device according to claim 1. 2. The method according to claim 1, wherein at least one of the conductive layers provided opposite to each other is a conductive mesh or sieve, and the microporous thin film is provided apart from or in contact with the conductive mesh or sieve. separation and recovery equipment. The device according to any one of claims 1 to 5, wherein the microporous thin film is a track-etched membrane, a UV laser-processed film, or a photolithographically-processed film with a thickness of 7-30 µm. 3. The device of claim 2, wherein the microporous insulating membrane is provided in contact with and covering one of the electrodes. 3. The device according to claim 2, wherein the microporous insulating film is provided so as to separate the cells from both the electrodes forming the bottom surface and the electrodes forming the top surface. 3. The apparatus of claim 2, wherein the cell further comprises an inlet for a sample suspension containing cells to be separated and an outlet. The one or more microporous thin films provided between the two conductive layers are two or more microporous insulating films spaced apart from each other, and the two or more microporous insulating films have different micropore diameters. 3. The device according to claim 2, wherein the micropore diameter decreases sequentially from the microporous insulating film near the top surface to the microporous insulating film near the bottom surface. 6. A device according to any one of claims 3, 4 or 5, wherein the upper surface is an open system. injecting a cell suspension containing cells to be separated into a cell comprising two conductive layers provided facing each other and one or more microporous membranes provided between the two conductive layers;
The pair of conductive layers apply a positive dielectrophoretic force to the dead or living cells to attract them into the micropores, and a negative dielectrophoretic force to repel or eject them from the micropores. A method for separating and collecting cells, comprising the step of switching and applying frequency . 13. The method of claim 12, wherein the step of applying a frequency generates a non-uniform electric field within the pores of the microporous membrane.

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