Microwave irradiation device

Abstract

A microwave irradiation device (100) comprises: a first chamber (11) in which an object (OB) is accommodated; a second chamber (12) provided in a lower portion of the first chamber (11); partition parts (31, 32) spatially partitioning the first chamber (11) and the second chamber (12); and an irradiation antenna (21) provided in a lower portion of the second chamber (12) to irradiate microwaves into the second chamber (12). The partition parts (31, 32) at least partly allow the microwaves irradiated into the second chamber (12) by the irradiation antenna (21) to be transmitted into the first chamber (11).

Description

Microwave irradiation device

[0001] The present invention relates to a microwave irradiation device.

[0002] The microwave aging device described in Patent Document 1 includes a microwave oscillator that radiates microwaves to food. The microwave oscillator includes an irradiation port that irradiates microwaves into an aging chamber in which the food is accommodated. The irradiation port is provided on the top surface of the aging chamber.

[0003] Japanese Patent Application Publication No. 2020-181756 (JP 2020-181756 A)

[0004] Here, steam is generated from the object irradiated with microwaves. In the microwave aging device described in Patent Document 1, the rising of the generated steam may cause water droplets to adhere to the irradiation port provided on the top surface of the aging chamber. Furthermore, if the object is housed in a container, the water droplets may adhere to the top lid of the container. In this case, the microwaves irradiated from the irradiation port are absorbed not by the object but by the water droplets attached to the irradiation port or the top lid of the container. This may result in the microwaves not being properly irradiated to the object. Or, the microwaves may not be absorbed by the object as planned.

[0005] An object of the present invention is to provide a microwave irradiation device that prevents microwaves from being absorbed by moisture in the environment unplanned, and that can easily be irradiated and absorbed by an object as planned.

[0006] The microwave irradiation device of the present invention comprises a first chamber in which an object is accommodated, a second chamber provided below the first chamber, a partition that spatially separates the first chamber from the second chamber, and an irradiation antenna provided below the second chamber that irradiates microwaves toward the second chamber, and the partition allows microwaves irradiated from the irradiation antenna to pass through to the first chamber at least in part.

[0007] According to this configuration, the irradiation antenna irradiates microwaves onto the object from below the object, so that the microwaves irradiated from the irradiation antenna are less likely to be absorbed by water droplets due to steam or condensation generated from the object, and the microwaves are irradiated and absorbed by the object as planned.

[0008] Furthermore, by providing a second chamber through which the microwaves emitted from the radiating antenna pass, the distance from the radiating antenna to the first chamber can be secured. This allows the microwaves to be radiated to any desired location on the object by changing the directivity of the microwaves emitted by the radiating antenna. This makes it easier to control the microwave irradiation position.

[0009] In the present invention, it is preferable that the partition portion has a portion where the object is provided that is permeable to microwaves.

[0010] According to this configuration, microwaves irradiated to the portion of the partition where the object is to be placed reach the object without being blocked by the partition, thereby allowing the microwaves to be appropriately irradiated to the object.

[0011] In the present invention, it is preferable that a blocking member for blocking microwaves is provided, and that the blocking member is provided in a portion of the partition where the object is not provided.

[0012] According to this configuration, microwaves irradiated to a portion of the partition where no target object is provided are blocked by the blocking member, thereby making it possible to prevent excessive microwave irradiation into the first chamber.

[0013] In the present invention, it is preferable to include a temperature control device that controls the temperature of the first chamber.

[0014] If the temperature of the object rises due to microwave irradiation, it is preferable to set the temperature of the first chamber containing the object low in order to keep the temperature of the object constant. With this configuration, the temperature of the first chamber is controlled by the temperature control device, so the temperature of the object can be maintained at an appropriate temperature.

[0015] In the present invention, it is preferable that a sensor for detecting the temperature of the object is provided, and that the sensor is provided below the second chamber.

[0016] According to this configuration, the sensor for detecting the temperature of the object is disposed in a position close to the irradiating antenna, so that wiring and the like can be shortened.

[0017] Furthermore, if the sensor is placed above the object, it may detect the temperature of steam generated from the object or water droplets due to condensation. With this configuration, the sensor is placed below the object, making the sensor less susceptible to the effects of steam generated from the object or water droplets due to condensation.

[0018] In the present invention, it is preferable that the device further comprises a control unit that controls the irradiating antenna and a sensor that detects the temperature of the object, and the control unit controls the irradiating antenna in response to a signal output by the sensor.

[0019] According to this configuration, the control unit controls the irradiation antenna in accordance with the signal output from the sensor, thereby maintaining the temperature of the object at an appropriate temperature.

[0020] In the present invention, it is preferable that a plurality of the irradiating antennas are provided, and the control unit controls the plurality of irradiating antennas individually in accordance with the signal output from the sensor.

[0021] According to this configuration, the control unit controls the plurality of irradiation antennas individually in accordance with the arrangement of the irradiation antennas, thereby maintaining the temperature of the object at an appropriate temperature.

[0022] In the present invention, it is preferable that a sensor for detecting the temperature of the object is provided, the temperature control device is provided above the first chamber, and the irradiation antenna and the sensor are provided below the second chamber.

[0023] With this configuration, the sensor for detecting the temperature of the object is located close to the radiation antenna, which shortens the wiring etc. Also, the radiation antenna and the temperature control device can be located in a position with ample space.

[0024] In the present invention, it is preferable to provide a carbon dioxide concentration control device that controls the concentration of carbon dioxide in the first chamber.

[0025] According to this configuration, the carbon dioxide concentration in the first chamber is maintained at an appropriate concentration by the carbon dioxide concentration control device.

[0026] In the present invention, it is preferable that the object is a culture medium containing cells, and the first chamber is a cell culture chamber.

[0027] According to this configuration, the secretion of intracellular substances to the outside of the cells is promoted by irradiating the culture medium containing the target cells with microwaves, thereby improving the efficiency of secretion of intracellular substances.

[0028] 1 is a front view of a microwave irradiation device. FIG. 2 is a front longitudinal cross-sectional view of a microwave irradiation device. FIG. 3 is a block diagram of a microwave irradiation device. FIG. 4 is a block diagram of an irradiation unit. FIG. 5 is a bottom cross-sectional view of an example of control of an irradiation antenna. FIG. 6 is a bottom cross-sectional view of an example of control of an irradiation antenna. FIG. 7 is a flowchart showing an example of control of an irradiation antenna. FIG. 8 is a front longitudinal cross-sectional view of another example of a microwave irradiation device. FIG. 9 is a front longitudinal cross-sectional view of another example of a microwave irradiation device. FIG. 10 is a schematic diagram showing the mechanism of exosome secretion promotion in a method for promoting secretion of intracellular substances. FIG. 11 is a graph showing the results of Example 1, illustrating an increase in the amount of exosome secretion due to short-term microwave irradiation (1 to 3 hours). FIG. 12 is a graph showing the results of Example 2, illustrating an increase in the amount of exosome secretion due to long-term microwave irradiation (24 hours). FIG. 13 is a graph showing the results of Example 3, a reproduction experiment of Example 2, illustrating an increase in the amount of exosome secretion due to long-term microwave irradiation (24 hours). FIG. 14 is a graph showing the results of Example 4, illustrating an increase in the amount of exosome secretion due to microwave irradiation for various cell lines. 1 is a graph showing the results of Example 5, which shows an increase in the amount of protein secreted by microwave irradiation. 2 is a graph showing the results of Example 6, which shows an increase in the amount of exosome secreted by microwave irradiation of cells cultured in a serum-free medium.

[0029] A microwave irradiation device according to the present invention will be described below with reference to the drawings. As will be described in detail later, this microwave irradiation device 100 can be used, for example, to promote the secretion of intracellular substances outside the cells by irradiating a culture medium containing cells with microwaves. In the following description, the direction of arrow U in the drawings will be referred to as the "upper side," the direction of arrow D as the "lower side," the direction of arrow R as the "right side," and the direction of arrow L as the "left side."

[0030] 1 and 2, the microwave irradiation device 100 includes a box 1 and an irradiation unit 2 that irradiates microwaves. The interior of the box 1 is spatially divided into a first chamber 11 that accommodates an object OB to be irradiated with microwaves, and a second chamber 12 that is provided below the first chamber 11, by a plate-like partition plate 31 (corresponding to the partition portion of the present invention).

[0031] The irradiation unit 2 has a plurality of irradiation antennas 21 that irradiate microwaves. The plurality of irradiation antennas 21 are provided below the second chamber 12. The irradiation antennas 21 are directed toward the second chamber 12. In this embodiment, the irradiation antennas 21 are directed toward the objects OB contained in the second chamber 12 and the first chamber 11. Specifically, the irradiation antennas 21 are directed upward.

[0032] The microwaves irradiated from the radiation antenna 21 to the second chamber 12 pass through the partition plate 31 and are irradiated onto the object OB contained in the first chamber 11. In other words, at least a portion of the partition plate 31 is capable of transmitting microwaves. The microwaves may be reflected by the inner surface of the box body 1 and then irradiated onto the object OB.

[0033] In this embodiment, the object OB is a culture medium containing cells. Details of the object OB will be described later. The object OB is accommodated in a container 32 for accommodating the object OB. The first chamber 11 is a cell culture chamber for culturing cells.

[0034] The box 1 has an interior member on its inner surface that absorbs or reflects microwaves. The interior member is made of, for example, metal such as aluminum or stainless steel, or a material such as ceramic or conductive rubber. The box 1 also has a heat insulating member between its inner and outer surfaces. The heat insulating member is made of, for example, hard urethane foam or glass wool.

[0035] As shown in FIGS. 1 and 2, the box 1 includes a plate-shaped top plate 13, a plate-shaped bottom plate 14 located below the top plate 13, and a door 15 fixed to the box 1 and capable of being opened and closed.

[0036] An upper space 17 is provided between the ceiling 11a of the first chamber 11 and the top plate 13. A lower space 18 is provided between the bottom 12a of the second chamber 12 and the bottom plate 14.

[0037] The door 15 has an optically transparent window portion. The window portion allows the object OB contained in the first chamber 11 to be observed. The window portion is formed of a material such as glass or plastic. The door 15 is fixed to the box body 1 via a hinge. The door 15 is rotatable around a vertical axis.

[0038] When the door 15 is closed, the first chamber 11 is sealed from the outside. This makes it possible to suppress the influence of the room temperature outside the box body 1 while the door 15 is closed, and to appropriately control the room temperature inside the first chamber 11.

[0039] As shown in Figures 1 and 2, the microwave irradiation device 100 includes a temperature control device 5 that controls the temperature of the first chamber 11, a carbon dioxide concentration control device 6 that controls the concentration of carbon dioxide in the first chamber 11, and an operation panel 10.

[0040] In the present embodiment, the radiation antenna 21 is provided in the lower space 18 with an radiation port through which microwaves are radiated facing the second chamber 12. However, the radiation antenna 21 may be provided on the inner surface of the second chamber 12 as long as it is located below the second chamber 12. Furthermore, the entire radiation antenna 21 may be provided in the lower space 18.

[0041] In the present embodiment, the irradiation unit 2 is provided in the lower space 18 of the box 1. However, the present invention is not limited to this, and as long as the irradiation antenna 21 is provided below the second chamber 12, the components of the irradiation unit 2 other than the irradiation antenna 21 may be provided at any position in the box 1.

[0042] The vertical distance L1 of the second chamber 12 can be, for example, about 50 to 200 mm. The distance L1 is the distance from the lower end of the partition plate 31 to the upper end of the bottom 12a of the second chamber 12.

[0043] [Partition Plate] In this embodiment, the object OB is placed on the partition plate 31. The partition plate 31 is fixed to the inner surface of the box body 1 by a fastener or the like. A seal member S1 is filled between the inner surface of the box body 1 and the partition plate 31 to prevent air from flowing from the first chamber 11 into the second chamber 12. This is not limiting, and any method for fixing the partition plate 31 to the box body 1 may be used. For example, the partition plate 31 may be welded to the inner surface of the box body 1.

[0044] The partition plate 31 has a transparent portion 31a that is transparent to microwaves. The transparent portion 31a is located at least where the object OB is provided. In this embodiment, the entire partition plate 31 is the transparent portion 31a. The transparent portion 31a is made of a material that is transparent to microwaves. The transparent portion 31a is made of, for example, glass such as quartz glass, sapphire glass, or heat-resistant glass, a resin material such as Teflon (registered trademark) or polypropylene, or a ceramic material such as high-purity alumina.

[0045] [Shielding Member] The microwave radiating device 100 includes a shielding member 4 that shields microwaves. The shielding member 4 is a sheet-like member. The shielding member 4 is made of a material that absorbs or reflects microwaves. The shielding member 4 is made of, for example, silicon carbide (SiC), titanium dioxide (TiO ２ They are made of semiconductor materials such as silicon dioxide, materials that absorb microwaves such as conductive rubber or ceramic, or materials that reflect microwaves such as aluminum or stainless steel.

[0046] The blocking member 4 is provided in a portion of the partition plate 31 where the object OB is not provided. In the present embodiment, the blocking member 4 has an opening at a location where a container 32 for containing the object OB is provided. As a result, when the entire partition plate 31 is made of a material that transmits microwaves, by arranging the blocking member 4 at any location on the partition plate 31, microwaves are absorbed or reflected at the location of the partition plate 31 where the blocking member 4 is provided. Therefore, the location of the partition plate 31 where microwaves are absorbed or reflected can be changed by the simple method of arranging the blocking member 4. This is not limited to this, and the microwave irradiation device 100 does not need to include the blocking member 4. In this case, the partition plate 31 may be formed of a material that transmits microwaves at the location where the object OB is provided, and may be formed of a material that absorbs or reflects microwaves at the location where the object OB is not provided. In other words, it is preferable that the partition plate 31 be capable of transmitting microwaves at the location where the object OB is provided.

[0047] [Temperature Control Device] The temperature control device 5 controls the room temperature of the first chamber 11 to a preset temperature. The temperature control device 5 has a Peltier element and a fan that blows air. Air from within the first chamber 11 is supplied by the fan to the heat-generating or cooling surface of the Peltier element. The supplied air is cooled by the temperature control device 5 before being supplied to the first chamber 11. Alternatively, the supplied air is heated by the temperature control device 5 before being supplied to the first chamber 11. In other words, the temperature control device 5 controls the temperature of the first chamber 11. In this embodiment, the temperature control device 5 can control the room temperature within the first chamber 11 within a range of 0°C to 40°C.

[0048] The temperature control device 5 is provided above the first chamber 11. Specifically, the temperature control device 5 is provided in the upper space portion 17.

[0049] The carbon dioxide concentration control device 6 supplies carbon dioxide gas to the first chamber 11 together with the air blown from the temperature control device 5. Specifically, the carbon dioxide concentration control device 6 supplies carbon dioxide gas into the first chamber 11 from a gas supply pipe connected to a gas supply source such as a gas cylinder. In this way, the carbon dioxide gas is supplied to the first chamber 11 together with the air cooled or heated by the temperature control device 5.

[0050] The operation panel 10 is provided at a position on the upper front side of the box body 1. The operation panel 10 is, for example, a touch panel. The operation panel 10 displays the set temperature of the object OB, the set room temperature of the first chamber 11, the microwave output of the irradiation unit 2, the reaction time, the airflow speed of the fan, the CO ２ Various setting values ​​such as concentration can be input. The input setting values ​​are output to the first control unit 27 or the second control unit 52. The operation panel 10 can also display the current values ​​for the above setting values. In addition, the operation panel 10 can also display graphs, alarms, etc.

[0051] 2 and 3 , the microwave irradiation device 100 includes a first control unit 27 (corresponding to a control unit) that controls the irradiation antenna 21, and a second control unit 52 that controls the temperature control device 5. The first control unit 27 is provided in the irradiation unit 2. The second control unit 52 is provided in the temperature control device 5.

[0052] The first control unit 27 and the second control unit 52 each have a memory unit and a CPU that executes a program. The memory unit is configured with, for example, an HDD, a ROM, or a non-volatile memory.

[0053] The first control unit 27 is capable of transmitting and receiving information to and from the second control unit 52 and the operation panel 10 via wired communication.

[0054] As shown in FIG. 3, the microwave irradiation device 100 includes a sensor 7 for detecting the temperature of the object OB and a CO 2 concentration sensor 102 for detecting the carbon dioxide concentration in the first chamber 11. ２ The device is equipped with a sensor 8 and a room temperature sensor 9 that detects the room temperature of the first chamber 11.

[0055] The sensor 7 is a non-contact sensor. Specifically, the sensor 7 is an infrared sensor that detects infrared rays emitted from the object OB. The sensor 7 is, for example, a thermopile infrared sensor. In this embodiment, the sensor 7 detects the temperature of the object OB by detecting the surface temperature of the bottom of the container 32 in which the object OB is stored. However, the sensor 7 may also directly detect the temperature of the object OB.

[0056] The sensor 7 is provided below the second chamber 12. The sensor 7 is provided in the lower space 18 with a portion of the sensor 7 facing the second chamber 12. The sensor 7 detects the temperature of the object OB placed on the partition plate 31 over time.

[0057] The first control unit 27 controls the irradiation unit 2 in response to the signal output from the sensor 7 so that the object OB reaches a preset target temperature V (see FIG. 7 ). In other words, the first control unit 27 controls the irradiation antenna 21 in response to the signal output from the sensor 7. The target temperature V of the object OB can be set by manually operating the operation panel 10.

[0058] The second control unit 52 controls the temperature control device 5 in response to a signal output from the room temperature sensor 9. In this embodiment, the second control unit 52 controls the temperature control device 5 so that the temperature in the first chamber 11 is 4°C. ２ The carbon dioxide concentration in the first chamber 11 is controlled in response to a signal output from the sensor 8. Specifically, the carbon dioxide concentration control device 6 controls the concentration of carbon dioxide in the first chamber 11 by supplying CO 2 from the gas supply pipe. ２ By supplying the gas, the concentration of carbon dioxide in the first chamber 11 is controlled.

[0059] With the above configuration, the blocking member 4 is disposed around the container 32, making it difficult for air from the first chamber 11 to enter between the bottom of the container 32 and the partition plate 31. As a result, water droplets due to condensation are less likely to adhere to the bottom of the container 32 than to the top of the container 32. This makes it possible to prevent the microwaves irradiated from the irradiation antenna 21 from being absorbed by water droplets adhering to the container 32 rather than by the object OB. It also makes it easier to maintain the object OB at an appropriate temperature.

[0060] [Irradiation Unit] Details of the irradiation unit 2 will be described with reference to Figures 2, 3, and 4. In this embodiment, the irradiation antenna 21 that irradiates microwaves is a circularly polarized patch antenna. However, the irradiation antenna 21 is not limited to this, and may be, for example, a dielectric antenna, a helical antenna, a coaxial antenna, or a waveguide aperture antenna. Furthermore, the irradiation antenna 21 may irradiate linearly polarized microwaves.

[0061] The irradiation unit 2 has four antennas as the plurality of irradiation antennas 21. In other words, the irradiation unit 2 has a first irradiation antenna 21a, a second irradiating antenna 21b, a third irradiating antenna 21c, and a fourth irradiating antenna 21d as the irradiation antennas 21. However, the number of the irradiation antennas 21 is not limited to this, and may be any number.

[0062] 4, the irradiation unit 2 includes an oscillator 22 that oscillates microwaves, a distributor 23 that distributes the oscillated microwaves, a phase shifter 24 that changes the directivity of the irradiation antenna 21, an amplifier 26 that amplifies the input microwaves to a desired output, and a circulator 25 that directs the direction of reflected power input from the irradiation antenna 21. The microwaves oscillated from the oscillator 22 are transmitted by a coaxial cable.

[0063] As shown in Fig. 4, the oscillator 22 is an electronic oscillator that controls the oscillation frequency in response to an input voltage (a DC voltage of approximately 1 V to 20 V). In other words, the oscillator 22 oscillates a microwave at a desired frequency in response to an input signal from the first control unit 27. In this embodiment, the oscillator 22 continuously changes the frequency of the oscillated microwave between approximately 2.4 GHz and 2.5 GHz. The power output from the oscillator 22 is input to the distributor 23.

[0064] The divider 23 divides the input power to each antenna. In this embodiment, the divider 23 divides the input power into four. In Fig. 4, the paths branched from the divider 23 are omitted except for the path connected to the first irradiating antenna 21a. The omitted paths have the same configuration as the path connected from the divider 23 to the first irradiating antenna 21a.

[0065] The power output from the distributor 23 is input to the phase shifter 24. The phase shifter 24 changes the phase difference between the antenna elements to change the directivity of the first irradiating antenna 21 a. Specifically, the phase shifter 24 changes the directivity in the vertical plane of the microwaves irradiated from the first irradiating antenna 21 a.

[0066] The amplifier 26 is a solid-state semiconductor amplifier having a semiconductor element, such as a GaAs, GaN, or LDMOS (Laterally Diffused MOS) transistor. The power output from the phase shifter 24 is amplified by the amplifier 26. This controls the output of the microwaves radiated from each radiation antenna 21.

[0067] The power output from the amplifier 26 is input to the first irradiating antenna 21a via the circulator 25. The reflected power output from the first irradiating antenna 21a, reflected by the inner surface of the second chamber 12, and input to the first irradiating antenna 21a is directed by the circulator 25 toward the termination resistor RE. The level of the reflected power is input to the first control unit 27. The first control unit 27 controls the frequency, phase, or output of the microwaves output from each irradiating antenna 21. This adjusts the impedance with the object OB, thereby reducing the level of the reflected power.

[0068] [Regarding Control of Irradiation Antenna] Next, an example of control of the irradiating antenna 21 will be described with reference to the flowcharts shown in Figures 5, 6, and 7. Note that the order of the steps described below may be reversed, and multiple steps may be performed simultaneously, as long as no contradiction occurs.

[0069] In the example shown below, the partition plate 31 has an elongated shape in a plan view. Also, Figures 5 and 6 are cross-sectional bottom views of the object OB placed on the partition plate 31, as viewed from the irradiating antenna 21 side (below).

[0070] 5 and 6, the longitudinal direction of the partition plate 31 (left-right direction in the figures) is referred to as the x-direction, and the lateral direction of the partition plate 31 (up-down direction) in the figures is referred to as the y-direction. For the sake of explanation, the partition plate 31 is divided into four parts in the x- and y-directions, and these parts are referred to as a first region E1, a second region E2, a third region E3, and a fourth region E4 in a clockwise direction from the upper left in the figures.

[0071] 5, four objects OB are placed on the partition plate 31. Specifically, the objects OB are evenly arranged in the x direction and the y direction, with one object OB in each of the first area E1, the second area E2, the third area E3, and the fourth area E4.

[0072] 5, the plurality of objects OB are arranged at positions overlapping with the respective irradiating antennas 21 in a plan view. Specifically, the object OB1 arranged in the first region E1 is arranged at a position overlapping with the first irradiating antenna 21a in a bottom view. The object OB2 arranged in the second region E2 is arranged at a position overlapping with the second irradiating antenna 21b in a bottom view. The object OB3 arranged in the third region E3 is arranged at a position overlapping with the third irradiating antenna 21c in a bottom view. The object OB4 arranged in the fourth region E4 is arranged at a position overlapping with the fourth irradiating antenna 21d in a bottom view.

[0073] As shown in FIGS. 5 and 7 , a first state ST1 and a second state ST2 indicate the state of the temperature of the object OB detected by the sensor 7. Specifically, in the first state ST1, the temperature of the object OB is the target temperature V. In the second state ST2, the temperature of the object OB is not the target temperature V. In this embodiment, the second state ST2 is a state in which the temperature of the object OB is lower than the preset target temperature V. In this embodiment, the target temperature V is 37° C. However, the target temperature V is not limited to this and can be set arbitrarily.

[0074] A signal from the sensor 7 is input to the first control unit 27 (step S001). The first control unit 27 individually controls the multiple irradiation antennas 21 in response to the signal output from the sensor 7. Specifically, the first control unit 27 controls the irradiation antennas 21 so that the temperature of each object OB is maintained at 37° C. In this embodiment, the first control unit 27 changes the directivity of the irradiation antennas 21 so that the temperature of the object OB becomes the target temperature V.

[0075] A more detailed description will be given below. As shown in Fig. 5, the first control unit 27 recognizes that the object OB1 in the first region E1 is in the second state ST2 (step S002: No). In other words, the temperature of the object OB1 in the first region E1 is lower than the target temperature V. In this case, the first control unit 27 controls the irradiating antenna 21 so that the temperature of the object OB1 becomes the target temperature V (step S003). Specifically, the first control unit 27 changes the directivities of the first irradiating antenna 21a, the second irradiating antenna 21b, the third irradiating antenna 21c, and the fourth irradiating antenna 21d.

[0076] In the present embodiment, the first control unit 27 changes the directivity of the second irradiating antenna 21b toward the first region E1 without changing the directivity of the first irradiating antenna 21a. As a result, microwaves are irradiated onto the object OB1 from the first irradiating antenna 21a and the second irradiating antenna 21b, and the temperature of the object OB1 increases.

[0077] The signal from the sensor 7 is again input to the first control unit 27 (step S001), and the first control unit 27 recognizes that the temperature of the object OB1 in the first region E1 is in the first state ST1. In other words, the first control unit 27 recognizes that the temperature of the object OB1 is the target temperature V (step S002: Yes).

[0078] The first control unit 27 recognizes that the objects OB2, OB3, and OB4 in the second area E2, the third area E3, and the fourth area E4 are in the first state ST1 (step S004: Yes, step S007: Yes, step S0010: Yes).

[0079] 6, all of the objects OB1, OB2, OB3, and OB4 are in the first state ST1, and the work is completed. The above example is not limited to this, and the second state ST2 may be a state in which the temperature of the object OB is higher than a preset target temperature V. Furthermore, in the above example, the entire object OB1 in the first region E1 was in the second state ST2, but only a portion of the object OB1 may be in the second state ST2.

[0080] The microwave irradiation device 100 of this embodiment is a device for more efficiently promoting the secretion of intracellular substances from target cells. The microwave irradiation device 100 irradiates microwaves onto the target object OB to promote the secretion of intracellular substances outside the cells, thereby increasing the amount of intracellular substances secreted outside the cells. Microwaves promote the secretion of intracellular substances outside the cells by stimulating the intracellular substances themselves inside the cells, as well as membrane structures and organelles that encapsulate or attach the intracellular substances, and the cytoplasmic matrix that supports the intracellular substances. This increases the amount of intracellular substances secreted outside the cells. Furthermore, according to this embodiment, the secretion of intracellular substances can be promoted without the addition of special chemicals.

[0081] In this embodiment, the microwave frequency irradiated by the irradiating unit 2 is electromagnetic waves in the range of 300 MHz to 300 GHz. Preferably, the microwave frequency is 433.05 MHz to 434.79 MHz, 902 MHz to 928 MHz, 2.4 GHz to 2.5 GHz, 5.725 GHz to 5.875 GHz, 24 GHz to 24.25 GHz, 61 GHz to 61.5 GHz, 122 GHz to 123 GHz, or 244 GHz to 246 GHz (ISM band). In this embodiment, microwaves are irradiated onto the object OB at a microwave frequency of 2.40 GHz to 2.50 GHz. Irradiation of microwaves in this frequency range can improve the efficiency of promoting the secretion of intracellular substances without adversely affecting cell viability.

[0082] [Method for Promoting Secretion of Intracellular Substances] The inventors conducted the following test using the microwave irradiation device 100 described above to confirm the effect of promoting secretion of intracellular substances in a cell sample.

[0083] The method for promoting secretion of an intracellular substance according to this embodiment includes the following steps: (Step) Promoting secretion of an intracellular substance from cells by irradiating a sample containing cells with microwaves.

[0084] The method for promoting secretion of an intracellular substance according to this embodiment includes a step of irradiating a sample containing cells as an object OB with microwaves.

[0085] A cell is a living organism surrounded by a membrane structure that isolates it from the outside world. It contains genetic information and its expression mechanism, enabling self-renewal. In general, in eukaryotic organisms, cells consist of a membrane, nucleus, and cytoplasm. The membrane is composed of a lipid bilayer and proteins embedded in or bound to the lipid. The nucleus contains chromosomes and nucleoli. Chromosomes contain genetic information, while nucleoli synthesize ribosomal RNA, which is involved in protein synthesis, and assemble ribosomes. Cytoplasmic organelles, such as the endoplasmic reticulum, ribosomes, Golgi apparatus, mitochondria, and lysosomes, perform specific functions necessary for the activity of living organisms, such as synthesizing proteins based on genetic information, synthesizing ATP as an energy source, and breaking down substances using enzymes.

[0086] Cells contain substances derived from the nuclear and cytoplasmic organelles mentioned above, as well as substances that have been internalized via endocytosis from extracellular or cell membrane-resident materials. These intracellular substances include those that carry genetic information and those that play important roles in biological phenomena such as intercellular signaling, biological defense, and homeostasis. Examples of intracellular substances include polymers such as nucleic acids, proteins, peptides, and polysaccharides, as well as their component molecules, such as nucleotides, amino acids, and monosaccharides, as well as lipids, vitamins, and hormones. Cells are also known to secrete extracellular substances in capsules measuring tens to hundreds of nanometers in size and surrounded by a lipid bilayer membrane. These vesicles are called extracellular vesicles, and are classified into exosomes, microvesicles, apoptotic bodies, and other types based on their size and production mechanism. These extracellular vesicles can also be considered a type of intracellular substance.

[0087] These extracellular vesicles reflect the characteristics of the cells that secreted them (secreting cells). For example, extracellular vesicles secreted by cancer cells contain cancer-specific substances. Therefore, analyzing extracellular vesicles can provide information such as the genetic information of cells.

[0088] Furthermore, after being released from the extracellular space, extracellular vesicles are taken up by other cells (recipient cells). The intracellular substances contained within the extracellular vesicles are released into the recipient cells, resulting in functional and physiological changes in the recipient cells. In this way, extracellular vesicles are involved in intercellular molecular transport and function as a tool for intercellular signaling. It has been reported that the signaling mechanisms mediated by such extracellular vesicles may be involved in various physiological functions and the progression and onset of diseases such as cancer progression and metastasis, and viral infection. For example, extracellular vesicles secreted by cancer cells are thought to deliver substances related to angiogenesis, metastasis, and immune evasion to recipient cells, contributing to the creation of a microenvironment favorable to cancer survival.

[0089] In addition, extracellular vesicles are not only present between cells, but are also abundant in bodily fluids such as blood, tissue fluid, lymph, cerebrospinal fluid, and urine, and have been reported to circulate throughout the body. This allows extracellular vesicles to transmit information to specific cells in distant organs and tissues.

[0090] As described above, extracellular vesicles reflect the characteristics of secreting cells, and because they circulate in body fluids, they are expected to be used as disease biomarkers. For example, detecting disease-related extracellular vesicles in blood or urine can be used to test and diagnose diseases. In particular, substances that are not found in extracellular vesicles derived from normal cells but are specifically found in extracellular vesicles derived from cancer cells can be used as tumor markers, and are expected to be applied to cancer testing and diagnosis.

[0091] Furthermore, attempts are being made to develop a drug delivery system using extracellular vesicles as a carrier, taking advantage of the signal transduction mechanism of extracellular vesicles. For example, it has been reported in animal experiments that cancer shrinkage was confirmed when extracellular vesicles containing anti-cancer drugs were administered into the bloodstream. This indicates that extracellular vesicles can transport the drugs encapsulated in them to the recipient cancer cells.

[0092] It has also been reported that extracellular vesicles secreted by mesenchymal stem cells (hereinafter sometimes abbreviated as "MSCs") have shown therapeutic effects against various diseases. MSCs are somatic stem cells present in adults and are known to be involved in tissue repair, and are expected to contribute to regenerative medicine and cell therapy. Recently, it has been reported that MSC-derived extracellular vesicles can have therapeutic effects similar to MSCs. For example, they are expected to be used not only in the treatment of various diseases such as kidney disease, myocardial disorders, and brain diseases, but also in the field of cosmetic surgery, such as for anti-aging effects through tissue repair and hair regeneration.

[0093] As such, intracellular substances such as extracellular vesicles are expected to be used in a variety of fields, including disease biomarkers (disease diagnosis), drug delivery, and treatment / regenerative medicine, and research on extracellular vesicles is rapidly expanding. As mentioned above, extracellular vesicles are secreted by almost all cells, but the extracellular vesicles secreted from each cell exhibit diversity, with various functions and properties depending on the cell type and condition. Therefore, in order to use extracellular vesicles for diagnosis and treatment, it is necessary to isolate and purify extracellular vesicles secreted from the target cells in sufficient quantity and quality.

[0094] To meet this need, techniques for promoting the secretion of extracellular vesicles and techniques for enhancing the therapeutic effects of extracellular vesicles have been reported. For example, it has been reported that 3D culture and a hypoxic environment promote the secretion of extracellular vesicles from MSCs, improving their functions in tissue repair, angiogenesis, and immunoregulation. It has also been reported that the therapeutic effects of extracellular vesicles are improved by chemical stimuli such as various cytokines, such as TNFα and interleukin-1β, EP4 antagonists, glycyrrhetinic acid, kartogenin, and thrombin, and physical stimuli, such as titanium surfaces and low-intensity pulsed ultrasound. Serum-free and animal-derived component-free media that enhance the secretion of extracellular vesicles have also been reported.

[0095] However, recovery of intracellular substances secreted outside the cells required a long time, and the recovery rate and purity were insufficient. In particular, recovery of extracellular vesicles remaining inside the cells was difficult, hindering efficient recovery of extracellular vesicles. As exemplified above, various techniques for recovering extracellular vesicles have been reported, but a standardized method for separating and recovering extracellular vesicles has yet to be established. Furthermore, promoting the extracellular secretion of intracellular substances can contribute to improving the utility of those substances and advancing functional research. Therefore, there was a need for a technology that promotes the extracellular secretion of intracellular substances, such as extracellular vesicles.

[0096] Details of the subject OB are described below. The cells contained in the subject OB according to this embodiment are not particularly limited, as long as they produce the desired intracellular substance, such as extracellular vesicles. For example, exosomes are known to be produced by cells, and exosomes reflect the characteristics of the producing cells and the production environment. Therefore, cells capable of producing exosomes with the desired characteristics can be appropriately selected and used as the subject of the method for promoting secretion of intracellular substances according to this embodiment.

[0097] Here, the intracellular substance targeted for secretion promotion by the method for promoting secretion of an intracellular substance according to this embodiment refers to substances generally present within or on the membrane structure of a cell, and does not include organelles such as the nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, ribosomes, lysosomes, centrosomes, cytoskeleton, cell membrane, chloroplasts, or cell wall. Examples of intracellular substances include, but are not limited to, nucleic acids, proteins, peptides, polysaccharides, oligosaccharides, and their constituent components such as nucleotides, amino acids, and monosaccharides, as well as lipids, vitamins, steroids, and other small molecule compounds. Intracellular substances also include substances bound to glycoproteins and glycolipids. Intracellular substances also include membrane vesicles encapsulating these substances. Examples of nucleic acids include, but are not limited to, DNA and RNAs such as mRNA, rRNA, tRNA, miRNA, dsRNA, and siRNA. Examples of proteins and peptides include, but are not limited to, enzymes, cytokines, receptors, immunoglobulins, hormones, transcription factors, chaperones, and transport proteins including transmembrane proteins. Examples of the sugars include, but are not limited to, sugar chains involved in glycosylation of proteins and lipids, etc. Examples of the lipids include, but are not limited to, fatty acids, triglycerides, sterols, phospholipids, etc.

[0098] The plasma membrane vesicles are vesicles composed of membrane structures such as lipid bilayers present within cells, including membrane vesicles involved in endocytosis and exocytosis. Specifically, they include vesicles involved in the transport and secretion of proteins and lipids between organelles or between the plasma membrane and organelles, such as transport vesicles and secretory granules. Proteins are synthesized within cells by ribosomes. Secretory proteins are synthesized by ribosomes bound to the endoplasmic reticulum and then transported to the Golgi apparatus as transport vesicles. After undergoing glycosylation and processing in the Golgi apparatus, they are enveloped in a membrane to form secretory granules. Secretory granules fuse with the plasma membrane as needed and release their contents extracellularly. Exosomes are also known as secretory granules secreted extracellularly. Exosomes, together with microvesicles and apoptotic bodies, are collectively referred to as extracellular vesicles. Extracellular vesicles are a collective term for vesicles with heterogeneous lipid bilayer structures secreted from cells. They are classified into exosomes, microvesicles, and apoptotic bodies based on their production mechanisms. In terms of size, exosomes are 30-150 nm in diameter, microvesicles are 100-1000 nm in diameter, and apoptotic bodies are 50-50,000 nm in diameter. The contents of these extracellular vesicles depend greatly on the type of cell that produces them. These extracellular vesicles can also be considered a type of intracellular substance.

[0099] Furthermore, the origin of the cells is not particularly limited, and cells capable of producing intracellular substances with desired characteristics can be appropriately selected from the nuclei of eukaryotic cells such as animal cells, plant cells, fungi, and protists, or prokaryotic cells such as bacteria. Therefore, the cells can be somatic cells, germ cells, induced pluripotent stem cells (iPS cells), tumor cells, etc. Furthermore, the origin of the cells is not particularly limited, and they can be naturally derived cells collected from animals, plants, bacteria, etc., cell lines or immortalized cells established from such naturally derived cells, or artificially recombinant cells. Therefore, cells into which exogenous genes, such as genes derived from viruses such as adenovirus, adeno-associated virus, and lentivirus, have been artificially introduced are also included. Immortalized cells can be produced, for example, by introducing a telomerase reverse transcriptase gene into primary culture cells. Furthermore, tissues, which are aggregates of cells, can also be used. The animal cells include, but are not limited to, cells derived from mammals such as humans, monkeys, horses, sheep, goats, mice, rats, hamsters, and guinea pigs, etc. Furthermore, the cells may be fetal or adult cells.

[0100] Somatic cells are broadly classified into differentiated cells and stem cells. Examples of differentiated cells include, but are not limited to, hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, epithelial cells, epidermal cells, endothelial cells, fibroblasts, kidney cells, lung cells, cardiac muscle cells, and blood cells such as lymphocytes, erythrocytes, leukocytes, monocytes, and macrophages. Stem cells are broadly classified into embryonic stem cells (ES cells) and adult stem cells. Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, and intestinal stem cells. Examples of germ cells include, but are not limited to, ova, egg cells, sperm, and sperm cells. Examples of the tumor cells include, but are not limited to, tumor cells derived from hematopoietic cells such as leukemia, malignant lymphoma, and myeloma; tumor cells derived from epithelial cells such as lung cancer, breast cancer, gastric cancer, colon cancer, uterine cancer, ovarian cancer, and head and neck cancer; and tumor cells derived from non-epithelial cells such as osteosarcoma, chondrosarcoma, rhabdomyosarcoma, fibrosarcoma, and leiomyosarcoma. Furthermore, the method for promoting secretion of an intracellular substance according to this embodiment can target not only one type of cell but also a combination of multiple types of cells as targets for promoting secretion of an intracellular substance.

[0101] For example, extracellular vesicles have been reported to be involved in cancer development, progression, and metastasis, cell differentiation and tissue regeneration, immune system regulation, and the like, and are known to be actively produced in undifferentiated cells. Therefore, the stem cells, tumor cells, fetal cells, and the like can preferably be used as targets for the method for promoting secretion of intracellular substances according to this embodiment. Preferably, HEK293-based cells, such as HEK293T cells established by introducing the E1 gene of human adenovirus type 5 into human fetal kidney cells, or ASC52telo cells established by immortalizing mesenchymal stem cells derived from human adipose tissue can be used.

[0102] Furthermore, the morphology or state of the cells that are the subject of the method for promoting secretion of intracellular substances according to this embodiment is not particularly limited, but cultured cells are preferred. When the cells are cultured cells, examples include a state in which the cells are suspended in a medium (suspension culture), a state in which the cells are adhered to a carrier surface (adhesion culture), or a state in which the cells are embedded within a carrier (embedded culture). The cells may be dispersed singly or aggregated to form cell clumps. Furthermore, the adhesion culture involves growing the target cells in a monolayer on a carrier surface, such as the culture surface of a culture vessel, but may also be grown using feeder cells or the like as a supplementary scaffold.

[0103] The cultured cells are cultured using a medium, additives, culture vessels, equipment, etc. required depending on the type of target cells, etc.

[0104] The medium is not particularly limited as long as it is capable of growing and maintaining the target cells, and any medium known in the art can be used. Liquid or solid media are acceptable, but liquid media are preferred. The medium generally contains a carbon source, a nitrogen source, inorganic salts, and the like. The carbon source includes an inorganic carbon source such as carbonate and an organic carbon source such as glucose, while the nitrogen source includes an inorganic nitrogen source such as ammonium salt or nitrate, and organic nitrogen sources such as various amino acids and peptone. Examples of inorganic salts include sodium, potassium, calcium, magnesium, phosphorus, sulfur, and iron. The medium may also be formulated to be free of serum or components derived from xenogeneic animals. The medium may be a natural or synthetic medium. Examples of synthetic media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 medium, DMEM / F12 medium, Glasgow's Minimum Essential Medium (IMDM), Iscove's Modified Dulbecco's Medium (IMDM), Minimum Essential Medium (MEM), Eagle's MEM medium, and RPMI-1640 medium. Furthermore, the medium may be a medium optimized for the growth and maintenance of a specific cell type, or a known medium optimized for the production of a desired intracellular substance. For example, when culturing human mesenchymal stem cells, MSH-BM (Shimadzu Diagnostics) or the like can be used.

[0105] Taking into consideration the nutritional requirements of the target cells, additives such as serum, various amino acids, vitamins, sugars, proteins, inorganic salts, organic salts, hormones, growth factors, cytokines, organic compounds, extracellular matrices, and antibiotics may be added to the medium as needed.

[0106] Examples of the serum include, but are not limited to, bovine serum such as fetal bovine serum (FBS), horse serum, human serum, and serum albumin. Examples of the various amino acids include, but are not limited to, essential and non-essential amino acids such as arginine, glutamine, serine, and cysteine. Examples of the vitamins include, but are not limited to, vitamin A (retinol), vitamin B1 (thiamine), vitamin B2 (riboflavin), pantothenic acid, inositol, and vitamin E (tocopherol). Examples of the sugars include, but are not limited to, glucose, galactose, maltose, and fructose. Examples of the proteins include, but are not limited to, transferrin and lactoferrin. Examples of the inorganic salts include, but are not limited to, sodium, potassium, calcium, magnesium, phosphorus, sulfur, iron, chlorine, sodium selenite, and sodium bicarbonate. Examples of the organic acid salts include, but are not limited to, sodium and potassium salts of citric acid, isocitric acid, succinic acid, pyruvic acid, fumaric acid, oxaloacetic acid, malic acid, and lactic acid. Examples of the hormones include, but are not limited to, insulin, dexamethasone, hydrocortisone, progesterone, estradiol, melatonin, prostaglandins, prolactin, glucagon, leptin, and thyroxine. Examples of the growth factors include, but are not limited to, TGF-α, TGF-β, FGF, EGF, BDNF, and VEGF. Examples of the cytokines include, but are not limited to, interleukins such as IL-2, interferons such as IFN-α, IFN-β, and IFN-γ, G-CSF, and M-CSF. Examples of the organic compounds include, but are not limited to, mercaptoethanol, ethanolamine, polyethylene glycol, and cholesterol.Examples of the extracellular matrix include, but are not limited to, collagen, proteoglycan, fibronectin, elastin, laminin, hyaluronic acid, tenascin, vitronectin, thrombospondin, osteopontin, etc. In addition, the above-mentioned additives may be added in combination.

[0107] The culture vessel used for culturing can be any of various culture vessels known in the art, and the shape, size, material, etc. are not particularly limited. Examples of the culture vessel include, but are not limited to, dishes, flasks, plates such as multi-well plates, bottles, bags, and tubes. Preferably, the culture surface is the bottom surface of the culture vessel, and the bottom surface is flat. Examples of materials include, but are not limited to, glass (e.g., quartz glass, sapphire glass, etc.), polystyrene, polypropylene, polyvinyl chloride, polyethylene, polycarbonate, and polyethylene terephthalate. In addition, the culture vessel can be provided with a separate lid, which may be made of the same material as the culture vessel or a different material.

[0108] Furthermore, the culture vessel may be coated with a coating agent on the portion that comes into contact with cells, such as the culture surface. Coating the culture surface can suppress changes in cell morphology, such as suppressing cell detachment and aggregation, and can improve the efficiency of promoting secretion of intracellular substances from cells. As the coating agent, a cell adhesive substance such as a natural polymer, such as an extracellular matrix, or an artificial polymer can be used.

[0109] Suitable natural coating agents include the extracellular matrix and cell adhesive proteins that interact with the extracellular matrix. Examples include, but are not limited to, collagen, proteoglycan, fibronectin, elastin, laminin, hyaluronic acid, tenascin, vitronectin, thrombospondin, osteopontin, cellulose chitin, chitosan, cadherin, integrin, selectin, dextran sulfate, and heparan sulfate. Furthermore, natural coating agents modified by genetic engineering or other techniques can also be used. Examples of artificial coating agents include, but are not limited to, amino acid polymers such as poly-L-lysine, poly-D-lysine, and poly-L-ornithine; polysaccharides such as agarose and alginate gel; polylactic acid, polyglycolic acid, polycaprolactone, and polyethyleneimine. Furthermore, a combination of these coating agents may be used for coating.

[0110] The temperature and pH of the culture medium for the cultured cells, the carbon dioxide concentration in the culture vessel, and other conditions can be appropriately set depending on the type of cultured cells and the culture medium. Culture can be performed using an incubator known in the art. The microwave irradiation device 100 of this embodiment may be configured to be used without microwave irradiation. For example, the first chamber 11 of the microwave irradiation device 100 of this embodiment can be used as a cell culture chamber. The temperature of the culture medium is not particularly limited, but can be between 4°C and 65°C, preferably between 30°C and 40°C, and particularly preferably 37°C. Because microwave irradiation increases the temperature of the culture medium, it is preferable to appropriately cool the culture environment so that the culture medium can be maintained at a suitable temperature. Furthermore, the pH of the culture medium can be set to 5 to 9, preferably between 6 and 8, and particularly preferably 7.4. The carbon dioxide concentration in the culture vessel is also not particularly limited, but can be set to 4 to 10% by volume, e.g., approximately 5% by volume, relative to the air.

[0111] In the method for promoting secretion of intracellular substances according to this embodiment, the sample is not particularly limited as long as it contains cells targeted for promoting secretion of intracellular substances. Therefore, in addition to cell cultures, samples can also include those obtained by dispersing the cells in a buffer solution such as phosphate-buffered saline (PBS), or biological samples containing the cells, such as body fluids and tissues, including blood, cerebrospinal fluid, urine, saliva, amniotic fluid, and ascites. A culture medium containing the target cells is particularly preferred, and microwave irradiation of the culture medium can promote secretion of intracellular substances from the target cells. Furthermore, in the case of cell cultures, the medium composition during cell growth and microwave irradiation may be the same or different. For example, a medium supplemented with xenogeneic animal components such as FBS may be used during growth, and a medium free of such components may be used during microwave irradiation. Alternatively, a medium free of xenogeneic animal components such as FBS may be used during growth, and a medium containing such components may be used during microwave irradiation.

[0112] The cell culture is preferably cultured to 70-100% confluency, preferably 70-80% subconfluent. Cells at such confluency are suitable as target cells for promoting secretion of intracellular substances due to their favorable cell proliferation rate and nutrient supply, and can improve the efficiency of promoting secretion of intracellular substances without adversely affecting cell viability. Furthermore, the cell may be in an overconfluent state, where cell proliferation has progressed further from confluency. Here, confluency refers to the percentage of cells covering the culture surface of a culture vessel. For example, in the case of adherent cells, 50% confluency refers to a state in which approximately half of the culture surface is covered with cells. Therefore, in the case of adherent cells, confluency (100% confluency) refers to a state in which cells are in close contact with each other and cover the culture surface without any gaps, while overconfluency refers to a state in which cells are partially overlapping each other in addition to being confluent. Confluency can be measured, for example, by observing the state of the culture vessel using a microscope.

[0113] In the method for promoting secretion of intracellular substances according to this embodiment, the sample (OB) is irradiated with microwaves. The microwave irradiation can be performed using a microwave oscillator known in the art. For example, a solid-state semiconductor oscillator using semiconductor elements can be used. The output value of the microwave oscillator is not limited, and can be any output value of, for example, 100 W or less, preferably 50 W or less. The microwave irradiation can preferably be performed using the microwave irradiation device 100 according to this embodiment described above, but is not limited thereto. As long as the microwave can be irradiated to the object OB, any device known in the art can be used.

[0114] The microwave irradiation to the object can be transient, continuous, or intermittent. The transient irradiation means that the microwave is irradiated for a short period of time (for example, seconds). The continuous irradiation means that the microwave is irradiated for a fixed period of time, and the frequency and output value may be constant or variable. The intermittent irradiation means that the irradiation is repeated and stopped, and the interval between the irradiation and the stop may be constant or variable, and the frequency and output value during irradiation may be constant or variable. Specifically, the microwave is preferably irradiated to the object continuously for 30 minutes to 72 hours, and particularly preferably for 1 hour to 48 hours or 3 hours to 24 hours.

[0115] Because microwave irradiation raises the temperature of the sample (OB) to be irradiated with microwaves, it is preferable to set the temperature of the first chamber 11, which contains the object OB and is irradiated with microwaves, low in order to maintain a temperature suitable for cell growth and maintenance. For example, by setting the temperature of the first chamber 11 to 0-10°C, preferably 4°C, the temperature of the object OB can be controlled to a temperature suitable for cell growth and maintenance, such as 37°C. Such temperature control can be performed by a temperature control device 5 that measures the temperature of the object OB using a sensor 7 and controls the temperature of the first chamber 11 based on the measurement results, or a first control unit 27 that controls the microwave irradiation in response to a signal output by the sensor 7.

[0116] For example, when microwaves are continuously irradiated, the output can be varied from 0 to 50 W, the temperature of the first chamber 11 can be set to 4°C, and the microwave irradiation intensity can be controlled so that the temperature of the object OB is 37°C. Alternatively, the output can be set to 30 W and continuous irradiation can be performed. Furthermore, when microwaves are intermittently irradiated, the cycle can be set to 1 to 60 seconds, for example, 1, 5, 10, 30, or 60 seconds, and the microwaves can be irradiated intermittently. Preferably, the output can be fixed at 50 W, and a pattern of 5 seconds of irradiation, 5 seconds of pause, 7 seconds of irradiation, 3 seconds of pause, 3 seconds of irradiation, and 7 seconds of irradiation can be repeated. Furthermore, a pattern of 7 seconds of irradiation and 3 seconds of pause can be repeated.

[0117] The microwave irradiation time can be appropriately set depending on the cell growth rate, the type of intracellular substance to be secreted, etc., and irradiation can be carried out for, for example, several hours to several days.

[0118] In this case, the microwave frequency is preferably swept at 2.40 to 2.50 GHz for 20 seconds, as shown in Figures 3 and 8 of Japanese Patent Laid-Open No. 2024-033865.

[0119] Specifically, in the example of Figure 3 of the above-mentioned prior patent document, in the first cycle, the oscillation frequency is changed in a time-increasing direction at a 250 millisecond period from 2421 MHz to 2430 MHz, 2439 MHz, 2448 MHz, 2457 MHz, 2466 MHz, and 2475 MHz by a change amount of 9 MHz. At this time, the upper limit frequency value is set to 2480 MHz, and the frequency value obtained by adding the change amount (4 MHz) exceeding 2480 MHz to the start frequency value of the first cycle is set as the start frequency value of the second cycle. In the second cycle, starting from the start frequency of 2425 MHz, the oscillation frequency is changed in a time-increasing direction at a sweep period of 250 milliseconds and a change amount of 9 MHz. If the upper limit frequency value of 2480 MHz is exceeded, the start frequency of the third cycle of sweep irradiation is set in a similar manner, and the third cycle of sweep irradiation is also performed in the same manner as the first and second cycles. In this way, the start frequency of the first cycle is set to 2421 MHz, the start frequency of the second cycle is set to 2425 MHz, the start frequency of the third cycle is set to 2429 MHz, the start frequency of the fourth cycle is set to 2424 MHz, the start frequency of the fifth cycle is set to 2428 MHz, the start frequency of the sixth cycle is set to 2423 MHz, the start frequency of the seventh cycle is set to 2427 MHz, the start frequency of the eighth cycle is set to 2422 MHz, and the start frequency of the ninth cycle is set to 2426 MHz. In this example, it is the tenth cycle that the start frequency becomes the same value, and from the tenth cycle onwards, the oscillation frequency is repeatedly swept in the same way as the first to ninth cycles.

[0120] In the example shown in Figure 8 of the aforementioned prior patent document, in the first cycle, the oscillation frequency is changed in a time-increasing direction at a 250 millisecond period from 2421 MHz to 2430 MHz, 2439 MHz, 2448 MHz, 2457 MHz, 2466 MHz, and 2475 MHz by a change of 9 MHz. The upper limit frequency is set to 2480 MHz, and the frequency value obtained by subtracting the change amount exceeding 2480 MHz from the upper limit frequency value of 2480 MHz is set as the start frequency value for the second cycle. In the second cycle, the oscillation frequency is changed in a decreasing direction starting from the start frequency of 2476 MHz at a sweep period of 250 milliseconds and a change amount of 9 MHz. If the frequency becomes smaller than the lower limit frequency value of 2421 MHz, the frequency obtained by adding the change amount (8 MHz) that makes the frequency smaller than the lower limit frequency value is set as the start frequency for the third cycle. In the third cycle, the oscillation frequency is changed over time in an increasing direction from a starting frequency of 2429 MHz by an increment of 8 MHz. In this way, sweep irradiation is performed at different starting frequencies up to the ninth cycle. In the tenth cycle, the oscillation frequency may be returned to the first cycle, or the oscillation frequency may be swept in the opposite direction from the first cycle to the ninth cycle, with 2480 MHz (the final frequency of the ninth cycle, 2480 MHz, plus the frequency exceeding the upper limit frequency value in the ninth cycle, 0 MHz) as the starting frequency for the tenth cycle.

[0121] In the above-described steps of the method for promoting secretion of intracellular substances according to this embodiment, irradiating the sample with microwaves promotes secretion of intracellular substances from cells contained in the sample, thereby increasing the amount of secreted intracellular substances. Here, "secretion" refers to the release of substances within or on the membrane structure of a cell to the outside of the membrane structure of the cell. For example, it includes the extracellular release of substances formed by intracellular synthesis or metabolism. In particular, it includes the case where a membrane structure containing an intracellular substance as an inclusion fuses with the membrane structure of the cell and releases the inclusion to the outside of the membrane structure (so-called exocytosis), and the release of intracellular substances to the outside of the membrane structure via permeation through the membrane structure or via a pump on the membrane structure without changing the membrane structure of the cell (so-called transudation secretion). Here, preferably, "secretion" refers to the release of intracellular substances to the outside of the membrane structure of the cell without destroying the cell membrane, but does not include the release of intracellular organelles to the outside of the membrane structure. For example, when the sample is a cell culture, it refers to the release of intracellular substances into the culture medium.

[0122] The secretion-promoting effect of the method for promoting secretion of intracellular substances according to this embodiment will be explained using extracellular vesicles as an example of intracellular substances. Extracellular vesicles are variously named based on their particle size, constituent components, and production mechanism, but are broadly classified into three types based on their production mechanism: exosomes, microvesicles, and apoptotic bodies. Exosomes are produced by early endosomes formed intracellularly by endocytosis, which then transition to late endosomes, and then budding from these late exosomes. Endosomes containing numerous exosomes are called multivesicular bodies (hereinafter sometimes abbreviated as "MVLs"). These MVLs fuse with the cell membrane, and exosomes are secreted extracellularly by exocytosis. As shown in Figure 10 , microwaves stimulate exosomes present inside cells, stimulate and destroy the endosomes and MVBs containing exosomes, and stimulate fusion of MBVs with the cell membrane, thereby promoting the secretion of exosomes to the extracellular space. This increases the extracellular secretion of exosomes. Microvesicles are produced by directly budding from the cell membrane and released extracellularly. Apoptotic bodies are formed in the late stages of apoptosis. During the apoptosis process, cells undergo cell shrinkage and nuclear condensation and fragmentation, resulting in the production of apoptotic bodies, which are fragmented cells wrapped in the cell membrane. In the examples below, the method for secreting intracellular substances according to this embodiment was confirmed to increase the amount of exosomes, which are extracellular vesicles, over time upon microwave irradiation, suggesting that the method may not only be a transient stimulus but also activate the production and secretion pathways of extracellular vesicles described above.

[0123] The method for promoting secretion of an intracellular substance according to this embodiment may include a step of separating and recovering the intracellular substance, the secretion of which has been promoted from the microwave-treated cells in the above step, from a sample containing the cells. The method for promoting secretion of an intracellular substance according to this embodiment releases the intracellular substance outside the cells, making it possible to recover the intracellular substance, thereby enabling efficient recovery of the intracellular substance.

[0124] The step of separating and recovering intracellular substances whose secretion has been promoted from cells from a cell-containing sample can be performed using separation and purification methods known in the art, depending on the intracellular substance to be separated, and the method is not particularly limited. Here, "separation and recovery" refers to distinguishing and enriching intracellular substances from other components in the sample, and includes selectively collecting or concentrating the intracellular substances. In the method for promoting secretion of intracellular substances according to this embodiment, the intracellular substances are produced in large quantities by the microwave-irradiated cells and secreted extracellularly. Therefore, the microwave-irradiated sample can be subjected to, for example, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, limiting membrane filtration, polyethylene glycol precipitation, or affinity methods such as antibody or magnetic beads using markers specific to the intracellular substances, to separate and purify the intracellular substances from the sample, but these methods are not limited thereto. When the sample is a cell culture, the cell culture medium, i.e., the culture supernatant, is subjected to the above-mentioned method. Markers specific to intracellular substances, such as exosomes, can be used, for example, as exosome markers, such as surface proteins or proteins concentrated inside exosomes, including, but not limited to, tetraspanin proteins such as CD9, CD63, and CD81, heat shock proteins such as Hsp60, Hsp70, and Hsp90, flotillin, TSG101, ALIX, and phosphatidylserine.

[0125] The separated and purified intracellular substances can be confirmed using techniques known in the art as methods for detecting and analyzing the intracellular substances, depending on the intracellular substance to be isolated. For example, in the case of exosomes, they can be detected and analyzed by methods such as confirming lipid bilayer membrane microparticles through morphological observation, or by immunoassays using the above-mentioned exosome markers as indicators. For example, electron microscopy, Western blotting, flow cytometry, and ELISA can be used. Sandwich ELISA is particularly preferred. As described in the Examples below, Tim4-CD63 sandwich ELISA can be used. Tim4 specifically binds to phosphatidylserine, a phospholipid present on the membrane surface of exosomes, allowing for highly sensitive capture of exosomes. For example, after reacting a sample with a Tim4-immobilized plate to immobilize exosomes, exosomes can be detected with high sensitivity by using a biotin-labeled antibody against CD63 for primary detection and horseradish peroxidase (HRP)-labeled streptavidin for secondary detection.

[0126] In the method for promoting secretion of intracellular substances according to this embodiment, the secretion of intracellular substances from the cells to the outside of the cells is promoted by irradiating the cells with microwaves, thereby increasing the amount of secreted intracellular substances. In the examples below, it has been confirmed that microwave irradiation promotes the secretion of exosomes and intracellular proteins. Therefore, the method for promoting secretion of intracellular substances according to this embodiment may be applicable to efficiently promoting the secretion of various intracellular substances, including exosomes and proteins.

[0127] The present invention will be described in more detail below based on examples. However, these examples are provided to more specifically illustrate the present invention, and the present invention is not limited to these examples.

[0128] (Example 1) Examination of the effect of short-term microwave irradiation on exosome secretion

[0129] [Experimental Materials] The following cells, culture and microwave irradiation containers, culture media, and microwave irradiation equipment were used. Cells: HEK293T cells (human embryonic kidney cells: SV40 Large T antigen expression) (adherent cells) Culture and microwave irradiation containers: 6-well plates (uncoated) (TPP, 92406) Culture media: Growth medium: DMEM containing 10% FBS Recovery medium: DMEM (-NaHCO ３ + HEPES: FBS not included) Microwave irradiation device: Aging Booster (registered trademark) (manufactured by Shikoku Keisoku Kogyo Co., Ltd.) (frequency continuously changed from 2.4 GHz to 2.5 GHz, output 0 to 50 W)

[0130] [Experimental Method] HEK293T cells were seeded in a 6-well plate (N=1) and incubated in growth medium at 37°C in 5% CO until they reached confluence. ２ The cells were cultured under atmospheric conditions. Subsequently, the medium was replaced with 4 mL of recovery medium. After the medium replacement, HEK293T cells were irradiated with microwaves under atmospheric conditions using a microwave irradiation device (microwave irradiation group: MW). Microwave irradiation was performed for 1, 2, and 3 hours. During microwave irradiation, the microwave irradiation device was swept at 2.40 GHz to 2.50 GHz / 20 seconds, and the output was changed from 0 to 50 W, and irradiation was performed continuously. At this time, the temperature inside the microwave irradiation device was set to 4°C, and the microwave irradiation intensity (output) was controlled so that the medium temperature was 37°C. After microwave irradiation, the culture supernatant was collected, and the amount of exosomes in the culture supernatant was measured using Tim4-CD63 sandwich ELISA (Fujifilm Wako, 298-80601). The cells were detached using TrypLE® Select (Gibco, 12604013) and cell viability was measured by trypan blue staining. The control group (Con) (N=1) was incubated in a 37°C incubator for the same time as the microwave irradiation without microwave irradiation.

[0131] [Results] (Cell viability and total viable cell count) The cell viability was nearly 100% in both the microwave-irradiated and control groups, and no effect of microwave irradiation was observed. In addition, the number of viable cells recovered was 0.8 to 1.1 × 10 in both groups.６ The number was around 100, and no significant fluctuations were observed.

[0132] (Exosome secretion amount) The exosome secretion amount is shown in Figure 11. The vertical axis of Figure 11 represents the absorbance of the enzyme activity derived from the enzyme-labeled secondary antibody that captured exosomes in the Tim4-CD63 sandwich ELISA, measured at 450 nm (subwavelength 620 nm), and indicates the amount of secreted exosomes. In the microwave-irradiated group, the amount of exosome secretion increased over time with microwave irradiation time, and it was confirmed that microwave irradiation increases the amount of exosome secretion.

[0133] From the above, it was confirmed that microwave irradiation promotes exosome secretion from cells and increases the amount of exosome secretion. Since this promotion of secretion increases over time with microwave irradiation time, it is thought that this is not a transient promotion of exosome secretion due to microwave stimulation of the cells, but rather an activation of the exosome production and secretion pathway in the cells. Furthermore, short-term microwave irradiation of approximately 1 to 3 hours did not induce cell death or growth inhibition and did not have any adverse effects on the cells.

[0134] (Example 2) Study 1 on the effect of long-term microwave irradiation on exosome secretion

[0135] [Experimental Materials] The following cells, culture and microwave irradiation container, medium, and microwave irradiation device were used, as in Example 1. Cells: HEK293T cells Culture and microwave irradiation container: 6-well plate (uncoated) Medium: Growth medium: DMEM containing 10% FBS Recovery medium: DMEM (-NaHCO3 + HEPES: FBS-free) Microwave irradiation device: Aging Booster (registered trademark)

[0136] [Experimental Method] HEK293T cells were seeded in a 6-well plate (N=1) and cultured in growth medium until subconfluent. Subsequently, the medium was replaced with 4 mL of recovery medium. After the medium replacement, the HEK293T cells were irradiated with microwaves using a microwave irradiation device (microwave irradiation group: MV). Microwave irradiation was carried out for 24 hours. During microwave irradiation, the microwave irradiation device was swept at 2.40 GHz to 2.50 GHz / 20 seconds, and the output was changed from 0 to 50 W, and irradiation was carried out continuously. The temperature inside the microwave irradiation device was set to 4°C, and the microwave irradiation intensity (output) was controlled so that the medium temperature was 37°C. After microwave irradiation, the culture supernatant was collected, and the amount of exosomes in the culture supernatant was measured using Tim4-CD63 sandwich ELISA. After washing with PBS, the cells were detached using TrypLE® Select (Gibco, 12604013), and cell viability was measured by trypan blue staining. The control group (Con) (N=1) was incubated in a 37°C incubator for the same time as the microwave irradiation, but without microwave irradiation.

[0137] [Results] (Cell viability and total viable cell count) The cell viability was nearly 100% in both the microwave-irradiated and control groups, and no effect of microwave irradiation was observed. The total number of viable cells recovered was also 3.0 × 10 ６ The number was around 100, and no significant fluctuations were observed.

[0138] (Amount of exosome secretion) The amount of exosome secretion is shown in Figure 12. As in Figure 11, the vertical axis of Figure 12(a) is absorbance measured at 450 nm (subwavelength 620 nm), and indicates the amount of secreted exosomes. Figure 12(b) shows the absorbance per unit cell, converted to the amount of exosome secretion per unit cell. The amount of exosome secretion increased in the microwave-irradiated group compared to the control group, demonstrating that microwave irradiation increases the amount of exosome secretion. The amount of exosomes per unit cell increased 1.69-fold compared to the control group.

[0139] From the above, it was confirmed that 24 hours of microwave irradiation promoted exosome secretion from cells and increased the amount of exosome secretion, just like short-term irradiation. Furthermore, even a relatively long period of microwave irradiation, such as 24 hours, did not induce cell death or growth inhibition, and did not have any adverse effects on cells.

[0140] (Example 3) Study on the effect of long-term microwave irradiation on exosome secretion 2

[0141] [Experimental Materials and Methods] A reproduction experiment was carried out in Example 2. The experimental materials and method were the same as those in Example 2, except that the control group was carried out in 3 wells (N=3).

[0142] [Results] (Cell viability and total viable cell count) The cell viability was nearly 100% in both the microwave-irradiated and control groups. The total viable cell count was also 0.6-1.1 x 10 in the control group. ６ and in the microwave irradiation group, it was around 0.9 × 10 ６ The values ​​were around 100,000, and no significant fluctuations were observed in either case.

[0143] (Exosome secretion amount) The exosome secretion amount is shown in Figure 13. As in Figures 11 and 12 above, the vertical axis of Figure 13(a) is absorbance measured at 450 nm (subwavelength 620 nm), indicating the amount of secreted exosomes. Figure 13(b) shows the absorbance per unit cell, converted to the amount of exosome secretion per unit cell. The amount of exosome secretion increased in the microwave-irradiated group compared to the control group, demonstrating that microwave irradiation increases the amount of exosome secretion. The amount of exosomes per unit cell was 1.37 times that of the control group. The results of this example reproduced the results of Example 2.

[0144] From the above, it can be seen that microwave irradiation promotes exosome secretion from cells, increases the amount of exosome secretion, and efficiently promotes exosome secretion from cells in a reproducible manner.

[0145] (Example 4) Examination of the effect of microwave irradiation on exosome secretion in various cell lines

[0146] [Experimental Materials] The following cells, culture and microwave irradiation containers, media, and microwave irradiation equipment were used.

[0147] Experimental system using HEK293T cells, similar to Examples 1 to 3. Cells: HEK293T cells (adherent cells). Culture and microwave irradiation container: 6-well plate (coating: poly-L-lysine (PLL: Sigma-Aldrich P5899)). Culture medium: Growth medium: DMEM containing 10% FBS. Recovery medium: DMEM (-NaHCO ３ + HEPES: FBS not included) Microwave irradiation device: Aging Booster (registered trademark)

[0148] Experimental system using ASC52telo cells Cells: ASC52telo cells (immortalized adipose-derived human mesenchymal stem cells: ATCC, SCRC-4000) (adherent cells) Culture and microwave irradiation container: 6-well plate (uncoated) Culture medium: Growth medium: MesenPRO RS Medium (Gibco, 12746012) Recovery medium: DMEM (-NaHCO ３ + HEPES: FBS not included) Microwave irradiation device: Aging Booster (registered trademark)

[0149] In an experimental system using ASC52telo cells, coated plates were used for the culture vessel and microwave irradiation vessel to prevent cell detachment from the culture vessel. The cationic polymer poly-L-lysine was used as the coating agent. It was found that the use of the coating agent significantly improved cell detachment and aggregation from the plate.

[0150] HEK293T cells or ASC52telo cells were seeded in a 6-well plate (N=1) and cultured in growth medium until subconfluent. The medium was then replaced with 4 mL of recovery medium. After the medium replacement, the HEK293T cells or ASC52telo cells were irradiated with microwaves using a microwave irradiation device (microwave irradiation group: MW). Microwave irradiation was performed for 24 and 48 hours. During microwave irradiation, the microwave irradiation device was swept at 2.40 GHz to 2.50 GHz / 20 seconds, and the output was varied from 0 to 50 W, resulting in continuous irradiation. The internal temperature of the microwave irradiation device was set to 4°C, and the microwave irradiation intensity (output) was controlled so that the medium temperature was 37°C. After microwave irradiation, the culture supernatant was collected, and the amount of exosomes in the culture supernatant was measured using Tim4-CD63 sandwich ELISA. After washing with PBS, the cells were detached using TrypLE® Select, and cell viability was measured by trypan blue staining. A control group (Con) (N=3) was prepared by incubating the cells in a 37°C incubator for the same time as the microwave irradiation without irradiating them.

[0151] [Results] (Cell viability and total viable cell count) The cell viability was nearly 100% for both HEK293T cells and ASC52telo cells in both the microwave-irradiated and control groups. Although a decrease in cell count was observed after 48 hours of irradiation, the cell count increased between 24 and 48 hours of irradiation, possibly highlighting the cell proliferation inhibitory effect of long-term culture.

[0152] (Exosome Secretion Amount) Figure 14 shows the amount of exosome secretion from cells irradiated with microwaves for 48 hours. Figure 14 shows the relative ratio of exosome secretion amount converted per unit cell from absorbance measured at 450 nm (subwavelength 620 nm). Figure 14(a) shows the results for HEK293T cells, and Figure 14(b) shows the results for ASC52telo. In both cell types, the amount of exosome secretion per unit cell increased in the microwave-irradiated group compared to the control group, demonstrating that microwave irradiation promotes exosome secretion and increases the amount of exosome secretion. The exosome amount ratio per unit cell was 1.50-fold higher in HEK293T cells and 1.60-fold higher in ASC52telo cells compared to the control group.

[0153] As described above, although a slight decrease in cell number was observed with long-term microwave irradiation, the exosome secretion rate per unit cell increased, demonstrating that microwave irradiation can efficiently promote exosome secretion even with long-term irradiation such as 48 hours. Furthermore, regardless of the cell line, microwave irradiation was found to have the effect of increasing the amount of exosome secretion.

[0154] (Example 5) Examination of the effect of microwave irradiation on protein secretion

[0155] [Experimental Materials] The following cells, culture and microwave irradiation container, medium, and microwave irradiation device were used, as in Examples 2 and 3. Cells: HEK293T cells (adherent cells) Culture and microwave irradiation container: 6-well plate (uncoated) Medium: Growth medium: DMEM containing 10% FBS Recovery medium: DMEM (-NaHCO ３ + HEPES: FBS not included) Microwave irradiation device: Aging Booster (registered trademark)

[0156] HEK293T cells were seeded in a 6-well plate (N=1) and cultured in growth medium until subconfluent. The medium was then replaced with 4 mL of the above-mentioned recovery medium. After the medium replacement, the HEK293T cells were irradiated with microwaves using a microwave irradiation device (microwave irradiation group: MW). Microwave irradiation was carried out for 24 hours. During microwave irradiation, the microwave irradiation device was swept at 2.40 GHz to 2.50 GHz for 20 seconds, and the output was varied from 0 to 50 W, resulting in continuous irradiation. The internal temperature of the microwave irradiation device was set to 4°C, and the microwave irradiation intensity (output) was controlled so that the medium temperature reached 37°C. After microwave irradiation, the culture supernatant was collected, and the total protein content in the culture supernatant was measured using the microBCA method. Specifically, the culture supernatant was subjected to ultrafiltration (Amicon Ultra 10k device) to concentrate the protein (2 ml → 500 μL), while the buffer was exchanged with ultrapure water to remove phenol red from the medium. Subsequently, the total protein content was measured using the microBCA method. The control group (Con) was a group of cells that were incubated in a 37°C incubator for the same time as the microwave irradiation but without microwave irradiation, and then the same treatment was performed (N = 1 or N = 3).

[0157] [Results] The results are shown in Figure 15. Figures 15(a) and (b) show the results of an experiment conducted on the microwave-irradiated group and the control group of Example 2 above. The vertical axis of Figure 15(a) shows the amount of protein secretion (µg / mL), and the vertical axis of Figure 15(b) shows the relative ratio of the amount of protein secretion per unit cell, with the control group shown as 1. Figures 14(c) and (d) show the results of an experiment conducted on the microwave-irradiated group and the control group of Example 3 above. The vertical axis of Figure 15(c) shows the amount of protein secretion (µg / mL), and the vertical axis of Figure 15(d) shows the relative ratio of the amount of protein secretion per unit cell, with the control group shown as 1. A significant increase in protein secretion was observed in both cases, with the protein secretion ratio per unit cell increasing 2.88-fold in the microwave-irradiated group of Example 2 compared to the control group, and increasing 1.94-fold in the microwave-irradiated group of Example 3 compared to the control group.

[0158] (Example 6) Examination of the effect of microwave irradiation on exosome secretion in cells cultured in serum-free medium

[0159] [Experimental Materials] The following cells, culture and microwave irradiation container, medium, and microwave irradiation device were used. Cells: ASC52telo cells Culture and microwave irradiation container: 6-well plate (uncoated) Medium: Growth medium: MesenPRO RS Medium (Gibco, 12746012) Recovery medium: MSH-BM / Supplement A (serum-free medium) (Shimadzu Diagnostics Co., Ltd., 66213 (MSH-BM), 66214 (Supplement A)) Microwave irradiation device: Aging Booster (registered trademark)

[0160] ASC52telo cells were seeded in a 6-well plate (N=1) and cultured in growth medium until subconfluent or overconfluent. The medium was then replaced with 4 mL of recovery medium. After the medium replacement, the ASC52telo cells were irradiated with microwaves using a microwave irradiation device (microwave irradiation group: MW). Microwave irradiation was carried out for 24 hours. During microwave irradiation, the microwave irradiation device was swept at 2.40 GHz to 2.50 GHz / 20 seconds, and the output was varied from 0 to 50 W, resulting in continuous irradiation. The internal temperature of the microwave irradiation device was set to 4°C, and the microwave irradiation intensity (output) was controlled so that the medium temperature reached 37°C. After microwave irradiation, the culture supernatant was collected, and the amount of exosomes in the culture supernatant was measured using Tim4-CD63 sandwich ELISA. After washing with PBS, the cells were detached using TrypLE® Select, and cell viability was measured by trypan blue staining. A control group (Con) (N=3) was prepared by incubating the cells in a 37°C incubator for the same time as the microwave irradiation without irradiating them.

[0161] [Results] (Cell viability and total viable cell count) The cell viability of the microwave-irradiated group was reduced compared to the control group, but was 95% in the subconfluent case and 100% in the overconfluent case, indicating that microwave irradiation did not have a significant effect on cell viability. The total viable cell count of the control group was 2 x 10 in the subconfluent case. ５ In the microwave irradiation group, the mean age was 1.5 × 10 ５ Although a decrease in the total viable cell count was confirmed, no significant effect was observed. On the other hand, in the case of overconfluence, no significant difference in the total viable cell count was observed compared to the control group.

[0162] (Cell Morphology) Cell morphology was also observed for the microwave-irradiated group and the control group, which used DMEM (FBS-free) as the recovery medium. When DMEM was used as the recovery medium, changes in cell morphology were observed in both the microwave-irradiated group and the control group. On the other hand, in this example, when MSH-BM was used as the recovery medium, cell weakening was confirmed in both the microwave-irradiated group and the control group in the case of subconfluence, and such weakening was more observed in the microwave-irradiated group. However, when the cells were overconfluent, there was no change in cell morphology in either the microwave-irradiated group or the control group.

[0163] (Exosome Secretion Amount) The exosome secretion amount is shown in Figure 16. Figure 16 shows the exosome amount calculated from the absorbance measured at 450 nm (subwavelength 620 nm). Figures 16(a) and (b) show the relative ratio of the secreted exosome amount and the relative ratio of the exosome amount per unit cell in the case of subconfluent cultures, respectively. Figures 16(c) and (d) show the relative ratio of the secreted exosome amount and the relative ratio of the exosome amount per unit cell in the case of overconfluent cultures, respectively. In both the subconfluent and overconfluent cultures, the exosome secretion amount increased compared to the control group, and it was confirmed that microwave irradiation increases the exosome secretion amount, as in the above examples. Specifically, the exosome amount ratio was confirmed to increase by 1.25-fold in the subconfluent cultures and 1.46-fold in the overconfluent cultures compared to the control group. Furthermore, the ratio of exosome quantity per unit cell was confirmed to be 1.59-fold higher in the subconfluent group and 1.70-fold higher in the overconfluent group compared to the control group.

[0164] As described above, although a slight decrease in cell number and changes in cell morphology were observed in the case of subconfluent cultures, the amount of exosomes secreted and the amount of exosomes secreted per cell increased. In the case of overconfluent cultures, the amount of exosomes secreted and the amount of exosomes secreted per cell increased without any significant effect on cell survival or proliferation. Therefore, it can be seen that exosome secretion can be efficiently promoted under all conditions, regardless of the confluency of the cells during culture or the type of medium.

[0165] [Other Embodiments] The present invention is not limited to the above-described embodiment. For example, the present invention may be configured as in the following other embodiments. In the other embodiments described below, the same components as those in the above-described embodiment are assigned the same numbers and symbols as those in the above-described embodiment.

[0166] [1] In the above-described embodiment, the irradiating unit 2 has a plurality of irradiating antennas 21. However, the present invention is not limited to this, and the irradiating unit 2 may have a single irradiating antenna 21.

[0167] [2] In the above-described embodiment, a seal member S1 is filled between the inner surface of the box body 1 and the partition plate 31 to prevent air from the first chamber 11 from flowing into the second chamber 12. However, this is not limiting, and the seal member S1 does not have to be filled between the inner surface of the box body 1 and the partition plate 31. Furthermore, the partition plate 31 may be detachable from the box body 1, and air from the first chamber 11 may flow into the second chamber 12.

[0168] [3] The microwave irradiation device does not need to include a partition plate 31. In this case, as shown in Fig. 8, the interior of the box 1 may be spatially divided into a first chamber 11 and a second chamber 12 by a container 32 (corresponding to a partition in the present invention) in which the object OB is accommodated. Specifically, left and right protrusions 16 are provided on the inner surface of the box 1. A plurality of left and right protrusions 16 are arranged vertically with a gap between them. The container 32 is held in the box 1 by being inserted between the upper and lower protrusions 16.

[0169] [4] As shown in Fig. 9, the interior of the box 1 may be spatially divided into a first chamber 11 and a second chamber 12 by a container 32 and a partition plate 31. Specifically, the partition plate 31 has an opening where the container 32 is placed. The container 32 has a flange-shaped portion that protrudes toward the outside of the container 32. In this case, the container 32 may be fitted into the opening of the partition plate 31 from above, and the flange-shaped portion may be held on the upper surface of the blocking member 4.

[0170] [5] In the above-described embodiment, the object OB is a culture medium containing cells. However, the object OB may be, for example, a food product or other solid or liquid. In this case, the partition plate 31 may be made of a punched metal or a mesh-like metal member having openings.

[0171] [6] In the above-described embodiment, the temperature control device 5 is an air-cooled device using a Peltier element. However, the temperature control device 5 may be a heat pump device using a compressor. Alternatively, the temperature control device 5 may have a heater and control the temperature of the first chamber 11 by heating with the heater.

[0172] [7] In the above-described embodiment, the temperature control device 5 is provided in the upper space 17. However, the present invention is not limited to this, and the temperature control device 5 may be provided in other parts of the box 1. For example, the temperature control device 5 may be provided on the rear surface of the box 1. Furthermore, the temperature control device 5 may be fixed to the outer surface of the box 1 and attached externally.

[0173] [8] In the above-described embodiment, the sensor 7 is a non-contact sensor. However, the sensor 7 is not limited to this, and may be a contact sensor provided on the bottom surface of the container 32.

[0174] [9] In the above-described embodiment, the first control unit 27 is provided in the irradiation unit 2. However, the present invention is not limited to this, and the first control unit 27 may be provided outside the irradiation unit 2.

[0175]

[10] In the above-described embodiment, the target temperature V of the object OB can be set by manually operating the operation panel 10. However, the first control unit 27 may be capable of wired or wireless communication with an external CPU, and the first control unit 27 may set the target temperature V of the object OB in response to a signal input from the external CPU.

[0176]

[11] The first control unit 27 may control the strength of the microwaves emitted by each irradiation antenna 21 by changing the power output by the amplifier 26 so that the temperature of the object OB becomes the target temperature V.

[0177]

[12] In the above-described embodiment, the microwave radiating device 100 is configured by one box body 1. However, the microwave radiating device 100 may be configured by a plurality of boxes. For example, the microwave radiating device 100 may be configured by combining a first box body that configures the first chamber 11 and a second box body that configures the second chamber 12.

[0178]

[13] The configurations disclosed in the above-described embodiments (including other embodiments, the same applies below) can be applied in combination with configurations disclosed in other embodiments, unless a contradiction arises. Furthermore, the embodiments disclosed in this specification are merely examples, and the embodiments of the present invention are not limited thereto and can be appropriately modified within the scope of the purpose of the present invention.

[0179] The present invention can be applied to a microwave irradiation device.

[0180] 4: Shielding member 5: Temperature control device 6: Carbon dioxide concentration control device 7: Sensor 11: First chamber 12: Second chamber 21: Irradiation antenna 27: First control unit (control unit) 31: Partition plate (partition unit) 32: Container (partition unit) 100: Microwave irradiation device OB: Object

Claims

1. A microwave irradiation device comprising: a first chamber in which an object is stored; a second chamber provided below the first chamber; a partition that spatially separates the first chamber from the second chamber; and an irradiation antenna provided below the second chamber that irradiates microwaves toward the second chamber, wherein at least a portion of the partition allows microwaves irradiated from the irradiation antenna to pass through to the first chamber.

2. The microwave irradiation device according to claim 1, wherein the partition section is capable of transmitting microwaves at the location where the object is placed.

3. A microwave irradiation device as described in claim 2, further comprising a blocking member for blocking microwaves, the blocking member being provided in a location in the partition where the object is not provided.

4. The microwave irradiation device according to claim 1, further comprising a temperature control device for controlling the temperature of the first chamber.

5. The microwave irradiation device according to claim 1, further comprising a sensor for detecting the temperature of the object, the sensor being provided below the second chamber.

6. The microwave irradiation device according to claim 1, further comprising: a control unit that controls the irradiation antenna; and a sensor that detects the temperature of the object, wherein the control unit controls the irradiation antenna in response to a signal output by the sensor.

7. The microwave irradiation device according to claim 6, further comprising a plurality of said irradiation antennas, wherein said control unit controls said plurality of irradiation antennas individually in accordance with the signal output from said sensor.

8. A microwave irradiation device according to claim 4, further comprising a sensor for detecting the temperature of the object, wherein the temperature control device is provided above the first chamber, and the irradiation antenna and the sensor are provided below the second chamber.

9. The microwave irradiation device according to claim 1, further comprising a carbon dioxide concentration control device for controlling the concentration of carbon dioxide in the first chamber.

10. The microwave irradiation device according to any one of claims 1 to 9, wherein the object is a culture medium containing cells, and the first chamber is a cell culture chamber.

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