Thermal responsiveness evaluation device

Abstract

Provided is a thermal responsiveness evaluation device comprising: a culture chamber having a culture section for culturing nerve cells; and a measurement substrate for supporting the culture chamber. The culture chamber has: the culture section penetrating in a thickness direction and surrounded by a closed ring in a plan view; and a groove part provided on a rear surface of the culture chamber and extending from the culture section to a side surface of the culture chamber. The measurement substrate has: a base material; a thermal stimulation element provided on an upper surface of the base material; and a measurement part provided on the upper surface. The measurement part has: a measurement electrode; and a wire connected to the measurement electrode. A space surrounded by the upper surface of the base material and the groove part is a minute passage into which an axon extending from each nerve cell is introduced. At least a part of each of the thermal stimulation element and the measurement electrode is exposed on the minute passage.

Description

Thermal response evaluation device

[0001] The present invention relates to a thermal response evaluation device.

[0002] Traditionally, effective treatment methods for sensory disorders and pain have been investigated by using experimental models to evaluate how living organisms respond to stimuli such as sensation, pain, and inflammation. In recent years, from the perspective of reducing the number of experimental animals and concerns about species differences with humans, experimental models derived from stem cells, including human iPS cells (culture experimental models), have been attracting attention as an alternative to the traditional experimental models using small animals.

[0003] In the culture experimental models described above, neurons with preserved responsiveness to stimuli are often used. For example, an experimental model has been proposed in which sensory neurons are cultured on a substrate that allows measurement of the electrical activity of the neurons, and the responsiveness to thermal stimuli is evaluated by applying stimuli by heating the area around the substrate using a heater (see, for example, Non-Patent Document 1).

[0004] A. Odawara et al., Toxicological Sciences 188.1 (2022): 131-141.

[0005] In the technology described in Non-Patent Document 1, the periphery of the culture substrate is heated with a heater to apply thermal stimulation, so the entire neurons used receive the thermal stimulation. This makes it difficult to apply local stimulation to neurons, and it is not possible to determine where in the neurons the action potential is occurring.

[0006] The present invention has been made in view of the above circumstances, and aims to provide a thermal response evaluation device that can apply a localized thermal stimulus to cultured nerve cells and evaluate their response.

[0007] In order to solve the above-mentioned problems, one aspect of the present invention provides a thermal responsiveness evaluation device comprising: a culture chamber having a culture compartment for culturing neurons; and a measurement substrate supporting the culture chamber; the culture chamber having the culture compartment penetrating through the thickness direction and surrounded by a closed ring shape in a plan view; and a groove provided on the back surface of the culture chamber and extending from the culture compartment to a side surface of the culture chamber; the measurement substrate having a base material, a thermal stimulation element provided on an upper surface of the base material, and a measurement unit provided on the upper surface; the measurement unit having a measurement electrode and wiring connected to the measurement electrode; the space surrounded by the upper surface of the base material and the groove is a microchannel into which axons extending from the neurons are introduced; and at least a portion of the thermal stimulation element and the measurement electrode are exposed to the microchannel.

[0008] According to the present invention, a thermal response evaluation device can be provided that can apply a localized thermal stimulus to cultured nerve cells and evaluate the response.

[0009] FIG. 1 is a schematic perspective view of a thermal responsiveness evaluation device 1 according to an embodiment. FIG. 2 is a schematic perspective view showing a culture chamber 10. FIG. 3 is a plan view showing a measurement substrate 20. FIG. 4 is a block diagram illustrating a control unit 50. FIG. 5 is an explanatory diagram illustrating the operation of the thermal responsiveness evaluation device 1. FIG. 6 is a phase-contrast microscope image of the fabricated thermal responsiveness evaluation device. FIG. 7 is an explanatory diagram showing the response of a typical neuron.

[0010] The thermal response evaluation device according to this embodiment will be described below with reference to Figures 1 to 5. In all of the following figures, the dimensions and proportions of the components have been appropriately changed to make the drawings easier to understand.

[0011] In the following explanation, an xyz Cartesian coordinate system is set, and the positional relationships of the components are explained with reference to this xyz Cartesian coordinate system. Here, a predetermined direction in a horizontal plane is defined as the x-axis direction, a direction perpendicular to the x-axis direction in the horizontal plane is defined as the y-axis direction, and a direction perpendicular to both the x-axis and y-axis directions (i.e., the vertical direction) is defined as the z-axis direction.

[0012] Furthermore, "up" refers to the +z direction, which is vertically upward, and "down" refers to the -z direction, which is vertically downward.

[0013] In the following description, "planar view" refers to viewing an object from vertically above (+z side) downward (-z direction).

[0014] 1 is a schematic perspective view of a thermal responsive evaluation device 1 of this embodiment. The thermal responsive evaluation device 1 has a culture chamber 10 and a measurement substrate 20. The thermal responsive evaluation device 1 may further have a culture ring 30, a light source 40, and a control unit 50.

[0015] The thermal responsiveness evaluation device 1 is used to evaluate the thermal responsiveness of nerve cells. That is, the thermal responsiveness evaluation device 1 can culture nerve cells in a culture chamber 10, apply a thermal stimulus to the axons of the cultured nerve cells, and evaluate the response to the applied thermal stimulus based on the action potentials emitted by the cells.

[0016] [Culture Chamber] Fig. 2 is a schematic perspective view showing the culture chamber 10. As shown in Figs. 1 and 2, the culture chamber 10 is a member that is rectangular in plan view, and has culture compartments 11 and a plurality of grooves 12. The culture chamber 10 is disposed on the upper surface 21a of the measurement substrate 20.

[0017] The culture compartment 11 is a through-hole that penetrates the culture chamber 10 in the thickness direction (Z direction) from the upper surface 10a to the lower surface (back surface) 10b and is surrounded by a closed ring shape in a plan view. The internal space of the culture compartment 11 is used as a space for culturing neurons.

[0018] 1 and 2, the culture compartment 11 is shown as a rectangle having sides parallel to the X and Y directions in plan view. Each side of the culture compartment 11 is preferably 5 mm to 20 mm in plan view.

[0019] The groove 12 is provided on the rear surface 10b of the culture chamber 10 and extends from the culture compartment 11 to the side surface 10x of the culture chamber. One end of the groove 12 reaches the side surface 10x, and the other end reaches the inner surface 11x of the culture compartment 11.

[0020] The grooves 12 extend linearly in the X direction in plan view, and multiple grooves 12 (four grooves in the figure) are provided in parallel in the Y direction. The grooves 12 may be partially curved. Alternatively, the culture chamber 10 may have only one groove 12.

[0021] The height H and width W1 of groove 12 are preferably 1 μm or more and 10 μm or less. Axons of nerve cells cultured in culture compartment 11 are introduced into groove 12. When the dimensions of groove 12 are within the above ranges, nerve cells cannot pass through groove 12, but axons can be introduced.

[0022] The length L of the groove 12 is preferably 500 μm or more and 5000 μm or less, so that the axons can sufficiently extend inside the groove 12.

[0023] Although there are no particular limitations on the material of the culture chamber 10, a resin material is preferable because it is easy to form, and a curable resin is more preferable. A curable silicone resin (silicone rubber) is preferable as the material of the culture chamber 10. By making the culture chamber 10 from silicone resin, it is easy to adhere the culture chamber 10 to the upper surface 21 a of the measurement substrate 20 and fix it.

[0024] The separation distance W2 between adjacent grooves 12 is preferably 100 μm or more, which ensures a sufficient contact area between the culture chamber 10 and the measurement substrate 20, making it easy to closely fix the two together.

[0025] The culture chamber 10 can be manufactured by forming a mold for the culture chamber 10 using photolithography technology, and then transferring the mold shape using a curable resin.

[0026] [Measurement Substrate] Fig. 3 is a plan view showing the measurement substrate 20. As shown in Figs. 1 and 3, the measurement substrate 20 has a base material 21, a thermal stimulation element 22, and a measurement unit 23. The thermal stimulation element 22 and the measurement unit 23 are provided on an upper surface 21a of the base material 21. The upper surface 21a of the base material 21 is the upper surface of the measurement substrate 20.

[0027] The substrate 21 (measurement substrate 20) supports the culture chamber 10 on its upper surface 21a from the rear surface 10b of the culture chamber 10. The space surrounded by the upper surface 21a of the substrate 21 and the grooves 12 is a microchannel MP into which axons extending from nerve cells are introduced. Each of the multiple grooves 12 forms a microchannel MP.

[0028] The base material 21 is a plate material that is rectangular in plan view. The base material 21 does not need to be optically transparent, but it is preferable that it is transparent to light of the wavelength of laser light described below. The top surface 21a of the base material 21 is insulating. It is also preferable that the top surface 21a of the base material 21 has low thermal conductivity.

[0029] As long as the effect of the invention is not impaired, organic or inorganic materials can be selected as the material for the substrate 21. Glass is preferred as an inorganic material.

[0030] Examples of organic materials that form the substrate 21 include polymeric materials and elastomers. Examples of polymeric materials include thermoplastic resins such as polyvinyl chloride, polystyrene, ABS resin, and polylactic acid, and thermosetting resins such as polyimide and phenolic resin. Examples of elastomers include polysilicone and synthetic rubber.

[0031] The thermal stimulation elements 22 have the property of absorbing light and generating heat. In Fig. 3, a plurality of (12 in the figure) thermal stimulation elements 22 each having a rectangular shape in plan view are shown.

[0032] The plurality of thermal stimulation elements 22 are arranged in a matrix of 3 rows x 4 columns by forming an element array 22A in which three thermal stimulation elements 22 are arranged in the X direction, and the four formed element arrays 22A are arranged in the Y direction. The plurality of thermal stimulation elements 22 are each independent on the upper surface 21 a.

[0033] The material of the thermal stimulation element 22 is not particularly limited as long as it absorbs light and generates heat and is not cytotoxic. The material of the thermal stimulation element 22 is not particularly limited as long as it exhibits the above properties, and examples thereof include graphene, gold nanoparticles, and black phosphorus, which are used as photothermal conversion materials.

[0034] The thermal stimulation elements 22 preferably have a side length in the range of 10 μm to 200 μm. There are no particular restrictions on the number of thermal stimulation elements 22 or the spacing between the thermal stimulation elements 22, and these can be set appropriately depending on the purpose of the invention.

[0035] The measurement unit 23 detects an action potential generated in an axon. The measurement unit 23 has a measurement electrode 231 and a wiring 232 connected to the measurement electrode 231. The measurement unit 23 also has an insulating layer 233 and a reference electrode 239.

[0036] 3 shows a plurality of (16 in the figure) measuring units 23. The plurality of measuring units 23 are independent from each other on the upper surface 21a.

[0037] The measurement electrodes 231 are rectangular in plan view and are arranged in the center of the substrate 21 in plan view. The size of each measurement electrode 231 is preferably 10 μm or more and 200 μm or less on a side. The multiple measurement electrodes 231 are arranged between the three thermal stimulation elements 22 included in the element array 22A and on both sides of the element array 22A in the X direction, and are arranged in a matrix of 4 rows x 4 columns.

[0038] That is, the measurement electrodes 231 and the thermal stimulation elements 22 are arranged alternately along the micropath.

[0039] The thermal stimulation elements 22 and the measurement electrodes 231 overlap the groove portions 12 in a plan view, and at least a portion of them is exposed to the micropassage MP. The thermal stimulation elements 22 and the measurement electrodes 231 that make up the element array 22A are arranged along the micropassage MP.

[0040] Furthermore, the plurality of thermal stimulation elements 22 and the plurality of measurement electrodes 231 are arranged independently for each micropassage MP without spanning adjacent micropassages MP.

[0041] One end of the wiring 232 is connected to the measurement electrode 231, and the other end extends to the end 21b in the +X direction of the upper surface 21a of the substrate 21. The other ends of the multiple wirings 232 are arranged at equal intervals.

[0042] Reference electrodes 239 are provided on both sides of the wiring 232 arranged at the end 21 b. A control unit 50, which will be described later, constantly measures the potential difference between the reference electrode 239 and the measurement electrode 231. The reference electrode 239 is used to detect a change in potential outside the cell when a nerve cell fires, from the potential difference between the reference electrode 239 and the measurement electrode 231.

[0043] The end of the reference electrode 239 can be set appropriately depending on the purpose, as long as it is possible to detect the potential difference between it and the measuring unit 23 .

[0044] The material of the measurement electrode 231 is not particularly limited as long as it is a conductor that is not cytotoxic, but from the viewpoint of cell observation, it is preferable that it be optically transparent. Examples of materials for the measurement unit 23 and the reference electrode 239 include graphene and indium tin oxide (ITO).

[0045] The materials of the wiring 232 and the reference electrode 239 are not particularly limited as long as they are conductors that are not cytotoxic, but from the viewpoint of cell observation, they are preferably optically transparent. The materials of the wiring 232 and the reference electrode 239 may be the same as or different from that of the measurement electrode 231.

[0046] The dimensions (width, thickness, length) of the measuring section 23 and the reference electrode 239 can be appropriately selected from the configurations normally adopted in order to improve detection accuracy in terms of the design concept of electrical circuits.

[0047] The insulating layer 233 is provided at the end 21b of the base material 21 between the wires 232 and between the wire 232 and the reference electrode 239. The insulating layer 233 extends from the end 21b in the −X direction.

[0048] The material of the insulating layer 233 may be a known material that is normally used as an insulating layer for an electric circuit and does not have cytotoxicity.

[0049] In the measurement substrate 20, a certain area where the insulating layer 233 is provided is a connection pad 235 for electrical connection with other devices.

[0050] Such a measuring section 23 can be formed appropriately using known photolithography techniques.

[0051] 1, the culture ring 30 is a cylindrical member provided on the upper surface 21a of the measurement substrate 20. In the thermal response evaluation device 1, the culture chamber 10 is disposed inside the culture ring 30.

[0052] A space 30S surrounded by the inside of the culture ring 30 and the upper surface 21a of the measurement substrate 20 functions as a reservoir for storing a culture solution when culturing nerve cells.

[0053] 1, the light source 40 is a laser light source that emits laser light LB. The light source 40 emits laser light LB with a spot diameter of, for example, 100 μm. It is preferable that the light source 40 irradiates the laser light LB onto the thermal stimulation element 22 from the lower surface (the surface on the −Z side) of the measurement substrate 20.

[0054] The laser light LB is light with a wavelength that can be absorbed by the thermal stimulation element 22. For example, the laser light LB can be red laser light with a wavelength of 785 nm. When the thermal stimulation element 22 is irradiated with the laser light LB, it absorbs the laser light LB and generates heat.

[0055] The light source 40 may have a position adjustment member that moves the light source 40 to adjust its position so that the laser light LB can be irradiated onto a specific thermal stimulation element 22 among the plurality of thermal stimulation elements 22. The light source 40 may also have an optical path changing member that changes the irradiation position of the laser light LB. A known mirror or light guiding optical system can be used as the optical path changing member.

[0056] 4 is a block diagram illustrating the control unit 50. The control unit 50 is connected to the measurement substrate 20 and the light source 40. The control unit 50 controls the operation of the light source 40. The control unit 50 also detects the thermal responsiveness of the nerve cells based on the action potential of the axon detected by the measurement unit 23.

[0057] The control unit 50 includes a calculation unit 51, a current pulse generation unit 52, a reception unit 53, and a storage unit 54. The control unit 50 may include an input device 59.

[0058] The calculation unit 51 sets the intensity and pulse width of the laser light LB to be emitted from the light source 40, and calculates and determines the irradiation conditions of the laser light LB. The calculation unit 51 also A / D converts the electrical signal ES detected via the measurement unit 23, and calculates and determines the action potential of the nerve cell based on the electrical signal ES.

[0059] The current pulse generating unit 52 generates a current pulse to be input to the light source 40 based on the irradiation conditions of the laser light LB generated by the calculating unit 51. The generated current pulse is input to the light source 40, and the laser light LB is emitted from the light source 40.

[0060] The receiving unit 53 is connected to the measuring unit 23 and receives the electrical signal transmitted via the measuring unit 23. The receiving unit 53 may amplify the received electrical signal, remove noise contained in the electrical signal, or perform other processing.

[0061] The storage unit 54 may include both a volatile memory that temporarily stores the results of calculations performed by the calculation unit 51 and a nonvolatile storage device that stores a plurality of irradiation conditions for the laser beam LB as specified values. The storage unit 54 may store information such as the spot diameter of the laser beam LB, the size of the thermal stimulation element 22, and the photothermal conversion efficiency of the thermal stimulation element 22 in a nonvolatile storage area. When calculating the irradiation conditions for the laser beam LB, the calculation unit 51 can retrieve this information from the storage unit 54 and calculate the irradiation conditions for the laser beam LB based on the set values ​​for the amount of heat generated by the thermal stimulation element 22 and the heat generation time.

[0062] The input device 59 is used to input information such as the spot diameter of the laser light LB, the size of the thermal stimulation element 22, and the photothermal conversion efficiency of the thermal stimulation element 22 to the control unit 50. As the input device 59, an input device such as a keyboard or a mouse can be used.

[0063] [Explanation of Operation] Figure 5 is an explanatory diagram illustrating the operation of the thermal responsiveness evaluation device 1. As shown in Figure 5, nerve cells C are cultured in the culture compartment 11, and axons A extend into the microchannel MP. With the thermal responsiveness evaluation device 1 in this state, laser light LB is irradiated onto the thermal stimulation element 22 from a light source (not shown).

[0064] In the thermal stimulation element 22, the laser light LB is converted into heat according to the properties of the material, and heats a part of the axon A that overlaps with the thermal stimulation element 22 (indicated by the symbol α in FIG. 5).

[0065] The amount of heat generated by the thermal stimulation element 22 can be roughly calculated depending on conditions such as the laser intensity (pulse amplitude) of the laser light LB, the pulse width, the spot diameter, the size (volume) of the thermal stimulation element 22, and the photothermal conversion efficiency of the thermal stimulation element 22. Of these conditions, by setting the laser spot diameter, the size of the thermal stimulation element 22, and the photothermal conversion efficiency to fixed values ​​(constants), the amount of heat generated can be controlled by the laser intensity and pulse width of the laser light LB.

[0066] In response to the thermal stimulus, an action potential is generated in a portion α of the axon A. The generated action potential is transmitted as an electrical signal ES through the axon A. When the electrical signal ES reaches the measurement electrode 231, the measurement unit 23 detects the electrical signal ES and transmits it to the control unit 50 via the wiring 232.

[0067] The control unit 50 evaluates the thermal response of the nerve cells based on the received electrical signal ES (action potential).

[0068] The control unit 50 synchronizes the time when the laser light LB is emitted from the light source 40 with the time when the electrical signal ES is detected, and calculates the elapsed time from the time when the laser light LB is emitted until the electrical signal ES is received. The response speed of the neuron C that has received a thermal stimulus can be obtained from the calculated elapsed time and the distance between the thermal stimulus element 22 and the measurement electrode 231.

[0069] Electrical signals ES are sequentially transmitted from a plurality of measurement electrodes 231 arranged along the micropath MP to the control unit 50. This allows the speed at which an action potential generated by a thermal stimulus propagates within the nerve cell C to be evaluated.

[0070] Furthermore, by setting the measurement electrodes 231 and the thermal stimulation elements 22 at equal intervals, it is easy to determine the speed at which the action potential is propagated within the axon A from the time at which the action potential is detected by each of the plurality of measurement electrodes 231. With this configuration, it is also easy to evaluate the relationship between the position of the axon A and the response speed.

[0071] Furthermore, by repeatedly irradiating the laser light LB, it becomes possible to evaluate desensitization when thermal stimulation is repeated multiple times.

[0072] In the thermal response evaluation device 1 described above, the thermal stimulation elements 22 are minute components with a side length of 10 μm to 200 μm and are formed discretely. When heated, the thermal stimulation elements 22 are irradiated with laser light LB to generate heat, thereby applying a μm-scale local stimulation to the axon A of the nerve cell C, and the responsiveness of the axon A to the thermal stimulation can be evaluated.

[0073] Furthermore, the thermal stimulation element 22 is a very small component, with each side measuring between 10 μm and 200 μm, and therefore does not accumulate heat easily. Therefore, it is believed that once irradiation with the laser light LB is stopped, the element quickly cools down and heating does not continue. This allows the thermal response evaluation device 1 to apply a short-term stimulus on the millisecond scale to the axon A of the nerve cell C and evaluate the responsiveness of the axon A to thermal stimuli.

[0074] Furthermore, if a thermal stimulus is applied to the axon A by using, for example, resistance heating caused by passing electricity through electrical wiring, the wiring on the measurement substrate will become congested, making the configuration complex. In a thermal response evaluation device with such a configuration, the wiring will be close to each other, making leakage current more likely to occur and difficult to control. In contrast, the thermal response evaluation device 1 described above uses the thermal stimulus element 22 for heating, reducing the number of wirings on the measurement substrate and simplifying the configuration.

[0075] If a method of directly heating the axon A with laser light is considered, it would be difficult to provide appropriate heating within a short period of time because the laser light would be absorbed and attenuated by the culture solution surrounding the axon A. In contrast, the thermal response evaluation device 1 described above can provide short-term stimulation by using the thermal stimulation element 22 to provide heating.

[0076] As a result, the thermal response evaluation device 1 configured as described above can provide a thermal response evaluation device that can evaluate the response of cultured neurons by applying localized thermal stimuli to them. This makes it possible to reproduce the signal transmission process from thermoreception in a culture experimental system similar to that in the body, enabling research on thermoreception at the cellular level.

[0077] For example, it will be possible to visualize at the cellular level 1) the response time delay from heat stimulation to the generation of an action potential, 2) the relationship between the propagation characteristics and the response threshold of heat stimulation, and 3) the desensitization phenomenon to high-frequency stimulation. Furthermore, by analyzing these phenomena in conjunction with cellular information such as protein and gene expression, our understanding of thermoreception will advance dramatically.

[0078] In this embodiment, the light source 40 may have a position adjustment member or an optical path change member to control the irradiation position of the laser light LB, but this is not limiting. For example, the position of the light source 40 may be fixed, and an XY stage may be used as a mounting table for placing the measurement substrate 20 equipped with the culture chamber 10, so that the relative positions of the light source 40 and the thermal stimulation element 22 on the measurement substrate 20 can be changed.

[0079] While the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to these examples. The shapes and combinations of the components shown in the above examples are merely examples, and various modifications can be made based on the design, specifications, etc., without departing from the spirit of the present invention.

[0080] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0081] [Fabrication of a device for evaluating thermal response] The culture chamber was fabricated using polydimethylsiloxane (PDMS), a silicone rubber widely used in fluidic devices. The desired structure of the culture chamber was obtained by pouring uncrosslinked PDMS into a mold for the microchannel and culture compartment fabricated by photolithography and then thermally curing it.

[0082] The measurement substrate was fabricated by etching the ITO film on a glass substrate with an ITO film using photolithography to form wiring and a reference electrode. After applying a graphene dispersion for inkjet printing, the substrate was etched with oxygen plasma via a protective film fabricated by photolithography to form measurement electrodes and thermal stimulation elements.

[0083] Furthermore, poly(chloro-p-xylylene) (Parylene C) was vapor-deposited, and then the Parylene C was removed from the areas corresponding to the measurement electrodes, wiring, and thermal stimulation elements using oxygen plasma treatment to form an insulating layer.

[0084] To improve cell adhesion, the surface was treated with polyethyleneimine to obtain a measurement substrate.

[0085] The position was confirmed under a microscope, and the culture chamber was placed on the measurement substrate so that the measurement electrodes and the thermal stimulation elements were aligned with the microchannels, thereby obtaining a device for evaluating thermal response.

[0086] 6 shows a phase-contrast microscope image of the fabricated thermal response evaluation device. The height of the microchannel MP was set to 10 μm. It was confirmed that the measurement electrodes 231 and the thermal stimulation elements 22 were arranged along the microchannel MP.

[0087] The measurement electrode 231 was a square with sides of 50 μm, and the interval between adjacent measurement electrodes 231 along the micropassage MP was 450 μm. The measurement electrode 231 was disposed in the center between adjacent measurement electrodes 231, and the interval between the measurement electrode 231 and the thermal stimulation element 22 was set to be equal.

[0088] The thermal stimulation element 22 was a square with each side being 100 μm.

[0089] [Evaluation of thermal stimulus responsiveness] When sensory neurons collected from the dorsal root ganglia of a rat fetus were cultured in the culture compartment of the fabricated thermal stimulus responsive device, axonal extension into the microchannel MP was confirmed within about one week. An experiment to evaluate thermal responsiveness was conducted after 2 to 3 weeks of culture.

[0090] A laser light having a wavelength of 785 nm and a spot diameter of 100 μm was emitted from the laser light source and irradiated onto the thermal stimulation element 22. The laser light had an output of 200 mW and a pulse width of 300 μm.

[0091] FIG. 7 is an explanatory diagram showing the response of a typical nerve cell, and is a diagram showing the detection result of an action potential.

[0092] When laser light was irradiated onto the thermal stimulation element 22 closest to the cell body of nerve cell C (T0), a peak indicating an action potential was measured approximately 1 ms later (T1 = 1 ms) in the signals SA and SB detected by the measurement electrodes 231A and 231B on both sides of the thermal stimulation element 22. In Figure 7, "*" indicates a peak indicating an action potential. The results showed that an action potential was generated in axon A within 1 ms after applying a thermal stimulus.

[0093] Furthermore, the propagation of action potentials was measured from electrodes arranged on a 100 μm scale, confirming that it is possible to evaluate the stimulus response associated with local stimulation.

[0094] Furthermore, in the multiple measurement electrodes 231 arranged along the microchannel, action potentials were measured with a delay in the order of signals SC to SH detected by measurement electrodes 231C, 231D, 231E, 231F, 231G, and 231H as they moved away from the thermal stimulation element 22.

[0095] These results confirmed that the fabricated thermal response evaluation device can evaluate the response of sensory neurons to localized thermal stimuli on the micrometer scale and short-term thermal stimuli on the millisecond scale, demonstrating the usefulness of the present invention.

[0096] 1...thermal response evaluation device, 2...culture, 10...culture chamber, 10a, 21a...upper surface, 10b...lower surface (rear surface), 10b...rear surface, 10x...side surface, 11...culture compartment, 12...groove portion, 20...measurement substrate, 21...base material, 22...thermal stimulation element, 23...measurement unit, 30S...space, 40...light source, 50...control unit, 231, 231A, 231C...measurement electrode, 232...wiring, A...axon, C...nerve cell, LB...laser light, MP...microchannel

Claims

1. A thermal responsive evaluation device comprising: a culture chamber having a culture compartment for culturing neurons; and a measurement substrate supporting the culture chamber, wherein the culture chamber has the culture compartment penetrating through the thickness direction and surrounded by a closed ring in a plan view; and a groove provided on the back surface of the culture chamber and extending from the culture compartment to a side surface of the culture chamber, the measurement substrate having a base material, a thermal stimulation element provided on the upper surface of the base material, and a measurement unit provided on the upper surface, the measurement unit having a measurement electrode and wiring connected to the measurement electrode, the space surrounded by the upper surface of the base material and the groove is a microchannel into which axons extending from the neurons are introduced, and at least a portion of the thermal stimulation element and the measurement electrode are exposed to the microchannel.

2. A thermal response evaluation device as described in claim 1, wherein the measurement substrate has a plurality of the thermal stimulation elements and a plurality of the measurement sections, and the plurality of the thermal stimulation elements and the plurality of the measurement electrodes are arranged along the micropath.

3. A thermal response evaluation device as described in claim 2, wherein the culture chamber has a plurality of groove portions, each of the plurality of groove portions forms a micropassage between itself and the upper surface of the base material, and the plurality of thermal stimulation elements and the plurality of measurement electrodes are arranged independently for each micropassage.

4. A thermal response evaluation device according to claim 2 or 3, wherein the plurality of thermal stimulation elements and the plurality of measurement units are alternately arranged at equal intervals along the micropath.

5. A thermal response evaluation device according to any one of claims 1 to 4, comprising a light source for irradiating the thermal stimulus element with laser light.

6. A thermal response evaluation device as described in claim 5, which has a control unit connected to the measurement substrate and the light source, and the control unit controls the light source and evaluates and detects the thermal response of the nerve cell based on the action potential transmitted via the measurement unit.

7. A thermal response evaluation device as described in claim 6, wherein the control unit measures the elapsed time from the time when the laser light is irradiated to the time when the action potential is detected, and evaluates the response speed of the nerve cell to thermal stimulation.

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