JP2010227041A - Cell photoresponse control substrate, cell photoresponse control device, cell photoresponse detecting device, cell photoresponse control method and cell photoresponse detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell photoresponse control substrate having high time resolution and spacial resolution and capable of being miniaturized-highly integrated, a cell photoresponse control device using the substrate, a cell photoresponse control detecting device and the like. <P>SOLUTION: There are provided a photoresponse detecting method of cells, and the like, which method comprises, in a photoresponse detecting device, setting a photosensitive cell at an optically open part of a substrate, irradiating light from the open part side for a light source to the photosensitive cell, and detecting the signal generated by the photosensitive cell. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell light response control substrate, a cell light response control device, a cell light response detection device, a cell light response control method, and a cell light response detection method.

  More specifically, the present invention enables high temporal resolution and spatial resolution, enables downsizing of the apparatus, enables high integration, and controls and detects long-time / repetitive cellular photoresponse. The present invention relates to a response control substrate, a cell light response control device, a cell light response detection device, a cell light response control method, and a cell light response detection method.

  It has been clarified that cells transmit signals within cells and between cells, and examples of such signals include hormones and electrical stimulation.

  In signal transmission of nerve cells, action potentials and neurotransmitters that are electrical signals are signals. The ability to control the action potential of a nerve cell from the outside is extremely useful for studying a network of nerve cells.

  Conventionally, as an external control method of a nerve cell, for example, a method of inserting an electrode into a nerve cell and exciting it with an electrical signal (Patent Document 1 below) and a method of irradiating light from above (Patent Document 2) are known. . However, there has been no external control method for neuronal action potential that can be highly integrated and has high time and high spatial resolution. In addition, for cells having photosensitivity, there has been no method and apparatus for controlling and detecting the photoresponse of cells that have high temporal resolution and spatial resolution and can be miniaturized and highly integrated.

JP, 2004-206830, A The method of patent documents 1 inserts an electrode to a nerve cell, and had a problem in time resolution and spatial resolution.

JP, 2006-217866, A Since the method of patent documents 2 uses an upright epi-illumination fluorescence microscope, light is irradiated from the side of a cell put on a substrate. At this time, a plurality of lenses are required to converge the light applied to the cells, and there is a problem that the apparatus becomes large. In order to maintain the cells for a long time, it is necessary to immerse the cells in a cell maintenance solution such as physiological saline. In this case, in order for light to reach the cells from the light source, the lens, the upper surface of the case holding the cell maintenance solution, and the cell maintenance solution must pass through. Therefore, in order to measure long-time and repeated cell responses, it is necessary to further increase the size of the apparatus, and there is a problem in high integration.

  A first object of the present invention is to provide a cell photoresponse control substrate that has high time resolution and spatial resolution and can be miniaturized and highly integrated, which is used for cell control and photoresponse detection having photosensitivity. It is.

  A second object of the present invention is to provide a cell light response control device and a cell light response detection device that have high time resolution and spatial resolution and can be miniaturized and highly integrated using the substrate.

  A third object of the present invention is to provide a cell light response control method and a cell light response detection method using the substrate, the apparatus.

(First invention)
The structure of the first invention of the present application for solving the above-described problems is that a light transmission path that is a through hole is provided in a single layer structure substrate made of a light impermeable material, or a light transmissive material is provided in the through hole. It is a cell light response control substrate provided with a light transmission path filled.

  In the first invention described above, “filling through holes” with a light-transmitting material means filling with no gaps (airtight) into the through holes. This also applies to the following inventions.

(Second invention)
The structure of the second invention of the present application for solving the above-described problem is that at least a substrate first layer having the following first layer light transmission path bonded to each other, and a substrate second layer having a second layer light transmission path, at least: The cell light response control substrate has a multilayer structure and includes a single light transmission path formed by the first layer light transmission path and the second layer light transmission path.

  (1) A substrate first layer made of a light-transmitting material and having a portion corresponding to the following second-layer light transmission path defined as a first-layer light transmission path.

  (2) A second layer light transmission path which is a through hole is provided on a substrate made of a light impermeable material, or a second layer light transmission path which is formed by filling the through hole with a light transmissive material. Substrate second layer.

  In the second invention described above, the “part corresponding to the second layer light transmission path” refers to a part corresponding to an extension of the second layer light transmission path. This also applies to the following inventions. Moreover, regarding the first layer and the second layer of the substrate of the second invention, the “surface” in the following invention refers to the surface that is not the mutual bonding surface in the first layer or the second layer.

(Third invention)
The configuration of the third invention of the present application for solving the above problem is a cell light response control substrate, wherein the substrate according to the first invention or the second invention comprises any one of the following configurations.

  (1) The substrate having a single-layer structure described in the first invention includes an extracellular matrix-forming substance at the opening on the first surface side or the peripheral edge thereof in the light transmission path.

  (2) The multi-layer structure substrate described in the second invention comprises an extracellular matrix forming substance at the opening on the surface side of the first layer light transmission path, which is the first surface side, or at the peripheral edge thereof. Yes.

  In the above third invention, the “opening” of the light transmission path is a physical opening in the light transmission path which is a through hole, or a light transmission path formed by filling a light transmitting material into the through hole. The optical aperture in

(Fourth invention)
The structure of the fourth invention of the present application for solving the above problem is that the cell light response control substrate defined in (1) of the third invention is on the side opposite to the first surface side in the light transmission path. The second layer light in which the portion near the end on the second surface side is enlarged, or the cell light response control substrate defined in (2) of the third invention is the second surface side This is a cell light response control substrate in which a portion near the end portion on the surface side of the transmission path is enlarged.

(Fifth invention)
The structure of the fifth invention of the present application for solving the above-described problems is that the cell light response control substrate according to any one of the first invention to the fourth invention is a light transmission path or a second invention respectively defined in the first invention. A cell light response control substrate having a plurality of light transmission paths defined in the above.

(Sixth invention)
The structure of the sixth invention of the present application for solving the above problems is the cell photoresponsive control substrate according to any one of the first invention to the fifth invention, and the light transmission path of the cell photoresponsive control substrate. And a light source located on the second surface side.

(Seventh invention)
The structure of the seventh invention of the present application for solving the above problems is the cell light response control device according to the sixth invention, wherein the cell light is provided with a liquid reservoir capable of introducing a solution on the first surface side of the light transmission path. It is a response control device.

(Eighth invention)
The structure of the eighth invention of the present application for solving the above-described problems uses the cell light response control device described in the sixth invention or the seventh invention, and provides light sensitivity to the opening on the first surface side of the light transmission path. A cell light response control method of locating cells having light and irradiating the light sensitive cells with light from the second surface side of the light transmission path.

  In addition, regarding the above 8th invention, the cell which has photosensitivity is well-known, The preparation method is described in the said patent document 2 etc., for example.

(Ninth Invention)
The configuration of the ninth invention of the present application for solving the above problem is that, in the cell light response control method according to the eighth invention, a cell network is connected by connecting a cell having a signal transmission function to the cell having photosensitivity. This is a cell light response control method provided.

(Tenth invention)
The configuration of the tenth invention of the present application for solving the above-described problem is that the cell photoresponse detection device located in contact with or in the vicinity of the cell is further provided for the cell photoresponse control device described in the sixth invention or the seventh invention. It is a cell light response detection apparatus provided with a vessel.

(Eleventh invention)
The structure of the eleventh invention of the present application for solving the above-described problems is the use of the cell photoresponse detection device described in the tenth invention, wherein a cell having photosensitivity is provided at the opening on the first surface side of the light transmission path. It is a cell photoresponse detection method of locating and irradiating light having sensitivity to a cell from the second surface side of the light transmission path.

(Twelfth invention)
The structure of the twelfth aspect of the present invention for solving the above problem is that, in the cell photoresponse detection method according to the eleventh aspect of the present invention, a cell having a signal transduction function is connected to a cell having photosensitivity. Is a cell photoresponse detection method.

  The cell light response control substrate according to the present invention does not transmit light at portions other than the light transmission path. Then, light sensitive cells are positioned at the opening (physical opening or optical opening) on the first surface side of the light transmission path, and the light sensitive cells are stimulated by light. can do. If the opening is made as large as a cell body to be irradiated with light, only target cells can be stimulated independently. Furthermore, if the opening is made smaller than the cell body, high spatial resolution can be realized. A high temporal resolution can be realized by making light irradiation of the light source a short pulse irradiation by an arbitrary means.

  In addition, the cell light response control substrate of the present invention fulfills both functions of fixing cells and adjusting irradiation light. In the light irradiation method described in Patent Document 2 described above, the lens, the upper surface of the case for holding the cell maintenance solution, and the cell maintenance solution must pass through before the light reaches the cells from the light source. For example, since the cell light response control substrate adjusts the irradiation light, light reaches the cell from the light source without passing through the lens. Therefore, it is possible to reduce the size of the device that handles the light response of cells, and to achieve high integration.

  In the cell light response control substrate according to the second invention, the shape of the light transmission path of the first layer (and thus the shape of the light transmission path opening on the first surface side of the substrate) depends on the light transmission path of the second layer. It is prescribed. That is, the “part corresponding to the extension of the light transmission path of the second layer” in the first layer becomes the light transmission path of the first layer.

  The cell light response control substrate according to the second invention is a multilayer in which a first layer made of a light-transmitting material and having no through-hole is joined to a second layer made of a light-impermeable material and having a through-hole. It is a structure. Therefore, even if the cell maintenance solution is dropped so as to cover the cells on the one surface side without causing liquid leakage from one surface of the substrate to the other surface while securing the light transmission path, the other surface side No liquid leaks.

  According to the cell light response control substrate according to the third invention, the cell body to be irradiated with light is autonomously guided to the opening of the light transmission path on the first surface side by the action of the extracellular matrix forming substance. And fix. Therefore, a skilled operation is not required for fixing the cells at a predetermined position, and it is not necessary to use means such as suction or to provide a cell fixing protrusion on the opening of the light transmission path. As a result, stress that weakens the photosensitive cell or shortens its life is not loaded. Therefore, since the favorable physiological state of the cell can be maintained for a time sufficient for performing control / detection by the light response of the cell, a reliable control / detection result can be obtained.

  When an extracellular matrix-forming substance having a size almost the same as that of a cell is provided, a single cell body to be irradiated with light can be brought into close contact with the substrate quickly, and high electrical seal resistance can be easily achieved. This reduces background noise in the detection of electrical signals.

  According to the fourth aspect of the present invention, the light irradiated to the cells can be adjusted by reducing the diameter by the diameter-enlarging shape of the second-layer light transmission path (the shape that reduces the diameter toward the first-layer side). Moreover, the light source for irradiation can also be incorporated in the diameter-expanded part of the second layer light transmission path. As a result, it is possible to further reduce the size and increase the integration of the device that handles the photoresponse of cells.

  According to the fifth invention, since a plurality of photoresponse control systems and photoresponse detection systems can be provided on a single cell photoresponse control substrate, the apparatus for handling the photoresponse of cells can be further reduced in size and heightened. Integration is possible. Furthermore, a plurality of independent optical response control systems and optical response detection systems of the same type or different types can be operated simultaneously on a single cell photoresponse control substrate.

  According to the sixth invention to the ninth invention, preferred cell light response control devices and cell light response control methods are provided. In addition, according to the tenth to twelfth inventions, preferred cell photoresponse detection devices and cell photoresponse detection methods are provided.

  Among these, when the cell light response control device or the cell light response detection device is provided with the liquid reservoir as in the seventh invention, the solution can be introduced to the first surface side of the substrate. If the introduced solution is a cell maintenance solution such as physiological saline, the physiological state of the cells to be controlled, detected, and measured can be maintained for a sufficient time for control, detection, and measurement by optical response. it can. If the introduced solution is a solution of a compound or the like that gives a stimulus other than light irradiation to cells, an apparatus for controlling, detecting and measuring the influence of the compound or the like on the light response is provided.

  Furthermore, according to the ninth and twelfth inventions, signal transmission in the network can be controlled. Signal transmission from photosensitive cells stimulated by light irradiation can be controlled.

  In particular, when the substrate has a plurality of light transmission paths, a network of a plurality of photosensitive cells (for example, a network of nerve cells) located at the opening of each light transmission path can be formed, and each light Sensitive cells can be controlled independently by each light source. Therefore, the signal transmission system can be controlled by simultaneously irradiating a plurality of cells with light. It is also possible to control the signal transmission system by irradiating light using a time difference.

  By irradiating light using the time difference, it is possible to detect or measure the influence of the time difference on the network. These patterns can be combined to detect or measure a preferred signal. Moreover, it becomes possible to detect or measure the interference between signals and the amplification effect. This makes it possible to detect or measure various patterns of network transmission depending on the timing of light irradiation.

  By combining these controls, it is possible to select a signal to be transmitted. As a result, the signal can be transmitted to the target to be transmitted. Thereby, various control patterns can be realized depending on the timing of light irradiation. If the detector is a measuring device, the transmitted signal can be measured. Further, when a plurality of detectors or measuring devices are provided from the upstream side to the downstream side of the network, it is possible to detect or measure the disappearance, reduction or amplification of the transmitted signal.

  In addition, when a solution reservoir is provided and a solution such as a compound that gives a stimulus other than light irradiation to a cell is introduced, the stimulus other than light irradiation is focused on a specific target such as a signal transducing cell or a light-sensitive cell. Can be given. As a result, the influence of stimuli other than light on the network can be examined. In other words, it is possible to screen for stimuli that have some influence on the network.

The substrate for cell light response control is shown. 1 shows a cell light response control substrate having a laminated structure. 1 shows a cell light response control substrate comprising an extracellular matrix-forming substance. The cell light response control board | substrate with which the opening part of the 2nd surface side of the board | substrate in a light transmission path was expanded is shown. The cell light response control apparatus is shown. FIG. 6A is a sectional view of the cell light response control substrate having a plurality of light transmission paths, and FIG. 6B is a view of the substrate as viewed from the first surface side. The cell light response control apparatus provided with the liquid reservoir part is shown. A state in which a plurality of cell bodies are positioned in the opening on the first surface side of a single light transmission path is shown. The Example of the photoresponse detection of a cell is shown. The output from the cell network is shown. An extracellular matrix-forming substance and a neuronal cell body printed in a lattice pattern are shown.

  Embodiments of the present invention will be described below with reference to the drawings. The technical scope of the present invention is not limited by these embodiments.

  One embodiment of a single layer structure cell light response control substrate according to the first invention is shown in FIG.

  The substrate P1 is made of a light-impermeable material and includes a light transmission path 1a that is a through hole. This through hole may be filled with a light transmissive material. Accordingly, the substrate P1 does not transmit light at portions other than the light transmission path 1a. Although the kind of the light-impermeable material which comprises the board | substrate P1 is not limited, For example, a silicon substrate, a colored glass board, a colored plastic board, a ceramic board etc. can be illustrated. The means for forming the light transmission path 1a which is a through hole is not limited, but various known processing techniques such as a combination of electron beam exposure and plasma etching, focused ion beam processing, laser processing, light exposure and plasma etching, and the like can be applied. The light transmission path 1a is used for light stimulation of cells having photosensitivity.

  One embodiment of a substrate for cell light response control having a multilayer structure according to the second invention is shown in FIG. The substrate P2 includes a substrate first layer 2 and a substrate second layer 3 bonded to each other. The substrate first layer 2 is made of a light-transmitting material, and examples of the constituent material thereof include transparent glass, plastic, silicon oxide, silicon nitride, and sapphire plate. The substrate second layer 3 is made of a light-impermeable material, and examples of the constituent material thereof include a silicon substrate, a colored glass plate, a colored plastic plate, and a ceramic plate.

  In addition to the above configuration, the substrate P2 transmits light further to the surface side of the substrate first layer 2 in addition to the silicon layer as the substrate second layer 3 and the silicon oxide layer as the substrate first layer 2. An SOI substrate having a silicon layer with such a very thin thickness can also be exemplified.

  The substrate P2 includes a light transmission path 1b including a second layer light transmission path 1c in the substrate second layer 3 and a first layer light transmission path 1d in the substrate first layer 2. The second layer light transmission path 1c is a through hole provided in the substrate second layer 3, but this through hole may be filled with a light transmissive material. The first-layer light transmission path 1d is a part indicated by a broken line in FIG. 2, but specifically, as a part corresponding to an extension of the second-layer light transmission path 1c without providing a through hole or the like. It is an optical light transmission path that is conceived.

  FIG. 3 shows a case where the substrate P2 having a multilayer structure is provided with the extracellular matrix-forming substance 6 at the opening 5 on the first surface side 4 and the peripheral edge thereof in the light transmission path 1b. In the above-described substrate P1, when the light transmission path 1a is a through hole, an extracellular matrix forming substance 6 may be provided on the periphery of the opening 5.

  Examples of the extracellular matrix-forming substance 6 include polylysine, collagen (type I, type II, type IV), fibronectin, laminin, proteoglycan (versican, decorin, etc.), proteoglycan (aggrecan), hyaluronic acid, link protein, laminin, enteractin, Chondroitin sulfate proteoglycan, tenascin, proteoglycan (chondroitin sulfate proteoglycan, heparan sulfate proteoglycan (such as perlecan), keratan sulfate proteoglycan, dermatan sulfate proteoglycan), hyaluronic acid (a type of glycosaminoglycan), tenascin, enteractin, elastin, etc. Is done.

  The extracellular matrix-forming substance 6 is provided in a thickness that does not inhibit light transmission. Further, the setting range of the extracellular matrix-forming substance 6 is preferably set to the same level or not so different from the cell size, and more specifically, the area is 75% or more and 150% or less of the area of the cells to be fixed. Preferably there is. Usually, it is preferable to provide the extracellular matrix forming substance 6 in a circular or rectangular pattern with a size of 10 to 30 μm. In addition, it is preferable to set the extracellular matrix-forming substance 6 in a shape pattern that is preliminarily matched to the shape that is naturally taken at the time of cell colonization because stress on the cells can be further reduced. When the extracellular matrix-forming substance 6 is set to approximately the size of a cell body, a single cell body can be positioned in the opening 5 quickly and accurately.

  When the extracellular matrix-forming substance 6 is provided in a portion that is not the opening 5 on the first surface side 4 of the substrate P2, a signal transducing cell is located in this portion to form a substrate that can form a cell network. It is also possible. In this case, for example, the extracellular matrix forming substance 6 can be provided in a lattice shape, a spiral shape, or the like.

  The extracellular matrix-forming substance 6 can be provided on the substrate by, for example, dissolving it in a physiological saline buffered with phosphoric acid, applying it directly on the substrate, and drying it. Preferably, the extracellular matrix-forming substance is applied onto the substrate in a desired shape and amount using a printing method using the micro contact printing method described in Soft Matter 2007, 3, 168-177. Can be arranged. This method can be applied to the various substrates described above.

  FIG. 4 shows a case where the opening 8 on the second surface side opposite to the first surface side 4 in the light transmission path 1b of the substrate P2 is larger than the opening 5 on the first surface side.

  In FIG. 4, the portion of the substrate second layer 3 in the light transmission path 1 b is a through hole and has a shape that gradually increases in diameter toward the second surface side. In this case, the shape of the boundary portion 7 between the second layer light transmission path 1c of the substrate second layer 3 and the substrate first layer 2 defines the shape of the first layer light transmission path 1d in the substrate first layer 2, As a result, the shape of the opening 5 on the first surface side is defined.

As a method of forming such a pyramid-shaped or conical-shaped hole that gradually expands in diameter (a shape in which the hole gradually decreases in the depth direction), when the substrate second layer 3 is a silicon substrate, a diamond drill has a constant depth. After the vertical hole is formed, the required part can be processed by removing it with TMAH etching. In place of TMAH etching, etching using XeF ₂ gas can be performed in the same manner. In either case, etching can be performed at a rate of about 0.1 microns or more per minute. Even when a substrate other than the silicon substrate is used, the through holes can be provided by various known methods. For example, a method using a drill or normal electron beam exposure can be used, and further, through-holes may be formed by plasma etching after pattern formation by exposure lithography.

  When a through-hole having a gradually expanding diameter is formed, a device that handles the light response of cells can be miniaturized and highly integrated by incorporating a light source into the through-hole.

  A plurality of light transmission paths as described above may be provided on the substrate P1 or the substrate P2. When one cell is fixed with respect to one light transmission path and each cell is controlled independently, it is preferable to provide a distance of about 0.1 mm or more between each light transmission path. Further, when the extracellular matrix forming substance 6 is provided so as to connect the light transmission paths, a substrate capable of forming a network of photosensitive cells is obtained.

  FIG. 5 shows a specific embodiment of the cell light response control apparatus. A light source 9 is provided on the opening 8 side on the second surface side of the substrate P2. Although the lens and the optical fiber are not shown in the drawing, they are necessary or unnecessary depending on the type of the light source, and are collectively referred to as a light source. Irradiation light 10 (indicated by solid arrows) emitted from the light source 9 passes through the light transmission path 1b and is irradiated to the cell body 11 fixed by the extracellular matrix forming substance 6. The photoreceptor 12 of the cell body 11 causes a light response by the irradiation light 10.

  At this time, since an appropriate light wavelength or the like differs depending on the cell body 11 or the photoreceptor 12, various kinds of light can be appropriately selected and used according to the characteristics of the cell body 11 or the photoreceptor 12. For example, when the cell body having a blue light receptor is irradiated with light, the irradiation light 10 is preferably blue light. As for the light source, for example, an optical fiber output end as a light source can be embedded in the second layer light transmission path 1c. It is also possible to embed a light emitting diode with a lens in the second layer light transmission path 1c as a light source.

  The illustrated cell body 11 is a light-responsive cell body that shows some response by light stimulation. There are various types of responses, such as gene expression, electrical signal transmission, or signal transduction substance secretion. As the cell body 11, various cell bodies such as animal cells, plant cells, and microbial cells can be considered. Among plant cells and microbial cells, cells having colored bodies such as chloroplasts, cells that measure day length by light irradiation, cells that generate signals by light irradiation, and the like are preferable. When a nervous system or a nerve cell network is to be controlled, a nerve cell to which photosensitivity is imparted by gene transfer is preferable.

  When the cell light response control device shown in FIG. 5 is further provided with a light response detector, the light response detection device can be obtained. If the optical response detector is an optical response measuring device, it becomes an optical response measuring device. The optical response detector is not particularly limited. When the cell body 11 is a nerve cell imparted with photosensitivity, a MOS diode using a MOS transistor can be exemplified. The MOS diode can be provided on the first surface side of the substrate P2 (or the substrate P1). You may provide a detector and a measuring device in required places, such as the middle and end point of signal transmission.

  As shown in FIG. 6A, when the substrate P2 has a plurality of light transmission paths 1b, the light response control device and the light response detection device include a plurality of light sources or a plurality of light response detectors, You may have a measuring device. In FIG. 6B, the opening 5 is shown and the extracellular matrix forming material 6 is not shown, but the plurality of openings 5 are formed in, for example, a lattice by the extracellular matrix forming material 6. If connected, a cell network can be formed.

  The optical response control device, the optical response detection device, and the optical response detection device may include a plurality of light sources, a plurality of optical response detectors, and an optical response measuring device. These numbers will vary depending on the purpose of the device. When it is desired to control a plurality of light responses and detect light responses independently with one apparatus, light sources and detectors are required as many as the number of control systems or detection systems. When signal transmission from the cell body 11 is controlled or detected, a light source corresponding to the light transmission path to be used as the start or intermediate light stimulus input unit may be provided. In addition, a detector or a measuring device may be provided at a necessary location such as an intermediate or end point of signal transmission.

  FIG. 7 shows a specific embodiment of a cell light response control device provided with a liquid reservoir. That is, the liquid reservoir 14 can be formed by providing the liquid reservoir partition wall 13 as a housing around the opening on the first surface side of the light transmission path in the substrate. Various solutions can be introduced into the liquid reservoir 14 in accordance with purposes such as cell control. If the solution to be introduced is a cell maintenance solution such as physiological saline, the good physiological state of the cells to be controlled / detected should be maintained for a sufficient time to perform control / detection / measurement by optical response. Can do. If the introduced solution is a solution of a compound or the like that gives a stimulus other than light irradiation to cells, an apparatus for controlling, detecting and measuring the influence of the compound or the like on the light response is provided.

  FIG. 8 shows a state in which a plurality of cell bodies 11 are positioned in the opening 5 on the first surface side of the substrate. In FIG. 8, for the convenience of illustration, the extracellular matrix forming substance 6 is omitted, and the light source provided on the second surface side of the substrate is not shown.

  In the case of FIG. 8, the opening 5 is larger than the cell body 11. When the plurality of cell bodies 11 are positioned in the single opening 5 using the extracellular matrix forming substance 6, the plurality of cell bodies can be stimulated simultaneously by one light irradiation. And it is possible to control each signal transmission started simultaneously.

  Examples of the present invention will be described below.

(Example 1: Photosensitive nerve cell)
The photosensitive neuron is prepared based on the description in Patent Document 2 described above, and is obtained by expressing channel rhodopsin 2 (ChR2), which is a photosensitive ion channel, in the neuron PC12.

  That is, a chop-2-Venus construct was introduced into PC12 cells to express protein molecules. Cells expressing channelrhodopsin 2 protein were identified by Venus fluorescence. When observed with a confocal microscope, channelrhodopsin 2 protein was localized on the surface of PC12 cells and was selectively expressed in the cell membrane.

It is known that PC12 cells are differentiated into nerve cells when neurogrose factor (NGF) is added. In this example, before adding NGF, the culture solution (DMEM (Dulbeco's Modified Eagles
Medium + 10% Horse serum + 5% FBS + 1% steptomycin-penicillin solution) Thus, PC12 expressing ChR2 almost uniformly was obtained. NGF was added and further cultured for 2-3 days to obtain ChR2 expressing neurons.

(Example 2: Substrate and apparatus)
FIG. 9 is a cross-sectional view of a cell light response detection apparatus using an SOI substrate 23 composed of a second silicon layer 20 as a second substrate, a silicon oxide layer 21 as a first layer, and a first silicon layer 22 as a substrate. The figure is shown.

  The light transmission path 1 b of the SOI substrate 23 includes a second layer light transmission path 1 c provided in the second silicon layer 20 (a through hole having a shape that gradually increases in diameter toward the second surface side) and a silicon oxide layer 21. It consists of the first layer light transmission path 1d which is a corresponding part. The SOI substrate 23 is provided with a plurality of light openings 5.

  An extracellular matrix forming material 6 is provided in a plurality of light openings 5 so as to have a circular shape with a diameter of 20 to 30 μm, and a lattice pattern is formed between them using the extracellular matrix forming material 6 as shown in FIG. Connected. Laminin or collagen I was used as the extracellular matrix-forming substance 6, and the microcontact printing method in which the extracellular matrix-forming substance 6 was provided by printing was used to form the above pattern.

  Although not shown, a liquid reservoir 14 as shown in FIG. 7 is provided on the first surface side of the SOI substrate 23, a culture solution containing PC12 cells 19 expressing ChR2 is introduced, and NGF is added. Culture was performed for 4 days. PC12 cells 19 migrated in the culture medium and settled on extracellular matrix-forming substance 6. The PC12 cells 19 which are neurons grew along the pattern of the extracellular matrix-forming substance 6 at the intersections of the lattice pattern as shown in FIG. 11 so that the axon came on the lattice line.

  Since the fluorescent dye FITC was previously labeled on the extracellular matrix-forming substance 6 laminin or collagen I, the printed pattern of the extracellular matrix could be observed with a fluorescence microscope.

(Example 3: Light irradiation to cell body, detection of light response)
As shown in FIG. 9, when the PC12 cell 19 was irradiated with a light source 24 of a laser diode (LD) having a wavelength of 475 nm, an output current as shown in FIG. 10 could be observed.

  In the current observation of this example, a hole having a diameter of about 1 micron is made in the first layer light transmission path 1d, and nystatin is administered to the cell membrane in that part to make a fine hole, and the inside of the cell and the lower part of the substrate. Were measured at the same potential, and the channel current was measured in a so-called whole cell mode in which electrodes were arranged above and below the substrate.

  In FIG. 10, the place where the current is lowered indicates the time of light irradiation. In this case, the intensity of the irradiation light is 3.3 mW, the diameter of the light flux in the irradiation area of the irradiation light is about 30 μm, which is almost the same as the cell size, and it is possible to excite only one cell. Met. Although not shown in FIG. 9, a liquid reservoir is provided on the first surface side of the SOI substrate 23, and physiological saline is introduced into the liquid reservoir as a cell maintenance solution.

The threshold value of the light intensity necessary for generating the action potential of the nerve cell depends on the expression level of the photosensitive protein, but is usually about 0.5 W / cm ² according to the following literature.

Literature: T. Ishizuka et al, "Kinetic evaluation of
photosensitivity in genetically engineered neurons expressing green algae
light-gated channels "Neuroscience Research, 54 (2006) 85-94).

In the case of FIG. 9, a laser beam having an output of 6 mW and a transmission wavelength of 475 nm is condensed to 30 μm by a lens, and silicon oxide (not shown) on the first surface side of the SOI substrate 23, the first silicon layer 22, When the silicon oxide layer 21 is 1 μm, 1 μm, and 3 μm, respectively, the intensity of light irradiated to the cell is 700 W / cm ² , and an intensity sufficient to generate an action potential is realized. In the case of laser light, since there is a margin of light intensity in this way, it is possible to generate an action potential sufficiently even when the output end of the optical fiber is brought close to the opening of the light transmission path. Since the diameter of the opening of the light transmission path can be freely selected within a range of 10 to 200 microns, nerve cells can be arranged with a high degree of integration and can be photoexcited independently or simultaneously. Moreover, as reported in the following literature, neurons expressing ChR2 generate action potentials by light stimulation and transmit this electrical signal in the axon direction.

Literature: Georg Nagel et al, "Channelrhodopsin-2, a
directly light-gated cation-selective membrane channel ", Proceedings of
the National Academy of Sciences of the United States of America, 100 (2003)
13940-13945).

  The action potential can be detected by a MOS transistor as shown in the following document.

Literature: Peter Fromherz, Physica E, "Semiconductor
chips with ion channels, nerve cells and brain ", 16 (2003) 24-34, etc.) The potential change is detected with high sensitivity by placing the cell part where the action potential is to be detected at the gate of the MOS transistor. be able to.

  The present embodiment can be used as a transmitting element that transmits such an action potential. In the prior art, such transmission is configured to irradiate light from above the cells while observing with a microscope or as described in the above paper, so it was difficult to increase the degree of integration of the elements. . Such an element of the present embodiment is useful for elucidating the cause of neurodegenerative diseases such as Alzheimer and for drug development, in addition to functional analysis of the neural cell network itself.

  According to the present invention, there is provided an advantageous photoresponse control substrate used for control of cells having photosensitivity and detection of photoresponse. In addition, cell photoresponse control device, cell photoresponse detection device, cell photoresponse control using these substrates and devices, which have high time resolution and spatial resolution and can be miniaturized and highly integrated using this substrate. Alternatively, a method for detecting cell light response is provided.

P1, P2 Substrate 1 Light transmission path 2 Substrate first layer 3 Substrate second layer 4 First surface side of substrate 5 Opening 9, 24 Light source 11 Cell body 14 Liquid reservoir 15 Synapse 16, 17, 18 MOS transistor 23 SOI substrate

Claims (12)

For cell light response control in which a light transmission path that is a through hole is provided on a substrate having a single layer structure made of a light impermeable material, or a light transmission path that is formed by filling the through hole with a light transmission material substrate. The first layer light transmission path has a multilayer structure including at least a substrate first layer having the following first layer light transmission path bonded to each other and a substrate second layer having a second layer light transmission path. And a second layer light transmission path, a cell light response control substrate constituting a single light transmission path.
(1) A substrate first layer made of a light-transmitting material and having a portion corresponding to the following second-layer light transmission path defined as a first-layer light transmission path.
(2) A second layer light transmission path which is a through hole is provided on a substrate made of a light impermeable material, or a second layer light transmission path which is formed by filling the through hole with a light transmissive material. Substrate second layer. The cell light response control substrate according to claim 1 or 2, wherein the substrate has any of the following configurations.
(1) The substrate having a single layer structure described in claim 1 is provided with an extracellular matrix forming substance at the opening on the first surface side in the light transmission path or at the peripheral edge thereof.
(2) The substrate having a multilayer structure according to claim 2 is provided with an extracellular matrix forming substance in the opening on the surface side of the first layer light transmission path, which is the first surface side, or in the peripheral portion thereof. . The cell light response control substrate defined in (1) has an enlarged diameter in the vicinity of the end portion of the second surface side opposite to the first surface side in the light transmission path, or 4. The cell according to claim 3, wherein the cell light response control substrate defined in (2) has a diameter enlarged in the vicinity of the end portion on the surface side of the second layer light transmission path which is the second surface side. Optical response control board. The cell light response control substrate according to any one of claims 1 to 4, wherein each of the cell light response control substrates has a plurality of light transmission paths defined in claim 1 or a plurality of light transmission paths defined in claim 2. substrate. Cell light comprising: the cell photoresponsive control substrate according to any one of claims 1 to 5; and a light source positioned on the second surface side of the light transmission path of the cell photoresponsive control substrate. Response control device. The cell light response control device according to claim 6, wherein a liquid reservoir capable of introducing a solution is provided on the first surface side of the light transmission path in the cell light response control device. Using the cell light response control device according to claim 6 or 7, a cell having photosensitivity is positioned in the opening on the first surface side of the light transmission path, and the second surface of the light transmission path A cell photoresponse control method for irradiating a cell having photosensitivity from the side. The cell light response control method according to claim 8, wherein in the cell light response control method, a cell network is provided by connecting a cell having a signal transmission function to a cell having photosensitivity. 8. A cell photoresponse detection device according to claim 6 or 7, further comprising a cell photoresponse detector in contact with or in the vicinity of the cell. Using the cell light response detecting device according to claim 10, cells having photosensitivity are positioned in the opening on the first surface side of the light transmission path, and the light is transmitted from the second surface side of the light transmission path. A cell photoresponse detection method for irradiating sensitive cells with light. 12. The cell photoresponse detection method according to claim 11, wherein a cell network is provided by linking cells having a signal transmission function to cells having photosensitivity.