Pressure application method and pressure application device

Abstract

The present invention addresses the problem of providing a method and a device for generating a periodically fluctuating pressure in a sealed container. The present invention relates to a pressure application method and a pressure application device, said pressure application method including generating a periodically fluctuating pressure in a sealed container by alternately repeating the following steps (a) and (b), wherein a biological sample is accommodated in the sealed container. (a) A step wherein the pressure in the sealed container is adjusted by a first pressure adjustment mechanism. (b) A step wherein the pressure in the sealed container is adjusted by a second pressure adjustment mechanism.

Description

Pressure application method and pressure application device

[0001] The present invention relates to a pressure application method and a pressure application device, and more particularly to a pressure application method and a pressure application device for generating a periodically fluctuating pressure in a sealed container.

[0002] Organs and cells in the body are exposed to various mechanical stresses (tension, shear stress, hydrostatic pressure, etc.). In particular, circulatory system organs such as the heart and blood vessels sense these mechanical stresses in response to cardiac pumping. Mechanical stress is known to be involved in cell differentiation, proliferation, and migration, but the cellular response to hydrostatic pressure, for example, remains largely unknown.

[0003] To study cellular responses to hydrostatic pressure in vivo, methods for applying pressure to static water in vitro have been investigated. Conventional methods for applying pressure to static water in vitro include the syringe method and liquid perfusion method. For example, Patent Document 1 proposes a method of applying pressure by sealing a system containing a cell suspension and changing the volume of the sealed space using a cylinder or the like. Patent Document 2 also discloses a method for producing three-dimensional cell aggregates, including a pressure application step in which a cell suspension is placed in a cell-containing container and pressure is applied to the cells in the container. Patent Document 3 discloses a perfusion culture device capable of culturing cells in a multilayer structure, configured to create a pressure difference by applying pressure from outside to the inside of at least one of the storage container and the culture container.

[0004] JP 2021-126051 A International Publication No. 2016 / 052472 JP 2018-64519 A

[0005] The syringe method described above allows for pressure increase and decrease by increasing or decreasing the volume of the sealed space, but it is not possible to apply a steep pressure like that of pulsation. Furthermore, while conventional liquid perfusion methods are said to be capable of generating periodic pressure fluctuations, it has been difficult to apply pressure while generating a minute pressure difference of approximately several tens of kPa in a sealed space with a period equivalent to that of pulsation, simulating the pulsation in vivo.

[0006] In other words, in the conventional technology, it was possible to apply extreme pressures such as low pressures or high pressures for a relatively long period of time or to apply pressures at a relatively long cycle (1 to 3 times / minute), but it was difficult to generate pressures such as high pressures or low pressures at a relatively short cycle. Therefore, in order to solve such problems of the conventional technology, the inventors have conducted research with the aim of providing a method and apparatus for generating cyclically fluctuating pressures at a relatively short cycle in a sealed container.

[0007] As a result of extensive research to solve the above problems, the inventors discovered that by alternately repeating a process of regulating the pressure inside the sealed container using a first pressure regulation mechanism and a process of regulating the pressure inside the sealed container using a second pressure regulation mechanism, it is possible to generate a periodically fluctuating pressure inside the sealed container with a relatively short period, and thus completed the present invention. Specifically, the present invention has the following features.

[0008] [1] A pressure application method comprising generating a periodically fluctuating pressure in a sealed container by alternately repeating the following steps (a) and (b): (a) adjusting the pressure in the sealed container by a first pressure adjustment mechanism; and (b) adjusting the pressure in the sealed container by a second pressure adjustment mechanism. [2] The pressure application method according to [1], wherein the first pressure adjustment mechanism comprises a first regulator and / or a first control valve, and the second pressure adjustment mechanism comprises a second regulator and / or a second control valve. [3] The pressure application method according to [1] or [2], comprising repeating 10 to 300 cycles per minute, where one cycle is defined as performing step (a) followed by step (b). [4] The pressure application method according to any of [1] to [3], wherein step (a) is a step of pressurizing the sealed container by the first pressure adjustment mechanism. [5] The pressure application method according to any one of [1] to [4], wherein step (a) is a step of adjusting the pressure inside the sealed container so that a pressure of less than 200 kPa is applied inside the sealed container. [6] The pressure application method according to any one of [1] to [5], wherein step (b) is a step of reducing the pressure inside the sealed container in a second pressure adjustment mechanism. [7] The pressure application method according to any one of [1] to [6], wherein step (b) is a step of adjusting the pressure inside the sealed container so that the pressure is reduced by 1 kPa or more from the pressure reached in step (a). [8] The pressure application method according to any one of [1] to [7], wherein the sealed container is equipped with a pressure sensor that can measure the pressure inside the sealed container in real time, and wherein the first pressure adjustment mechanism and the second pressure adjustment mechanism are each controlled according to the pressure value detected by the pressure sensor. [9] A pressure application device connected to an airflow supply source and a sealed container for accommodating a biological sample, the pressure application device comprising a first pressure adjustment mechanism for adjusting the pressure inside the sealed container and a second pressure adjustment mechanism for adjusting the pressure inside the sealed container, the first pressure adjustment mechanism and the second pressure adjustment mechanism operating alternately to generate a periodically fluctuating pressure inside the sealed container.

[10] The pressure application device according to [9], wherein the first pressure adjustment mechanism comprises a first regulator and / or a first control valve, and the second pressure adjustment mechanism comprises a second regulator and / or a second control valve.

[11] The pressure application device according to [9] or

[10] , wherein the first pressure adjustment mechanism and the second pressure adjustment mechanism operate so as to repeat 10 to 300 cycles per minute, where one cycle is a process of adjusting pressure by the first pressure adjustment mechanism followed by adjusting pressure by the second pressure adjustment mechanism.

[12] The pressure application device according to any of [9] to

[11] , wherein the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in series or in parallel.

[13] The pressure application device according to any of [9] to

[12] , wherein the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel.

[14] The pressure application device according to any of [9] to

[13] , wherein the first pressure adjustment mechanism includes a first control valve downstream of the first regulator, the second pressure adjustment mechanism includes a second control valve downstream of the second regulator, and the first control valve and the second control valve switch between connecting the sealed container to the first regulator and connecting the sealed container to the second regulator.

[15] The pressure application device according to any one of [9] to

[14] , wherein a sealed container is connected downstream of the first pressure adjustment mechanism and a supply source is connected upstream thereof, and wherein a sealed container is connected downstream of the second pressure adjustment mechanism and a supply source or an exhaust mechanism is connected upstream thereof.

[16] The pressure application device according to any one of [9] to

[15] , wherein the first pressure adjustment mechanism pressurizes the sealed container.

[17] The pressure application device according to any one of [9] to

[16] , wherein the first pressure adjustment mechanism adjusts the pressure inside the sealed container so that a pressure of less than 200 kPa is applied inside the sealed container.

[18] The pressure application device according to any one of [9] to

[17] , wherein the second pressure adjustment mechanism reduces the pressure inside the sealed container.

[19] The pressure application device according to any one of [9] to

[18] , wherein the second pressure adjustment mechanism adjusts the pressure inside the sealed container so that the pressure is reduced by 1 kPa or more from the pressure applied by the first pressure adjustment mechanism.

[20] The pressure application device according to any one of [9] to

[19] , wherein the sealed container is provided with a pressure sensor that can measure the pressure inside the sealed container in real time, and the operation of the first pressure adjustment mechanism and the second pressure adjustment mechanism is controlled according to the pressure value detected by the pressure sensor.

[0009] The present invention provides a method and apparatus for generating a periodically fluctuating pressure in a sealed container with a relatively short period. By using the method and apparatus of the present invention, it is possible to generate pressure in still water that corresponds to the pressure generated in response to pulsation in a living body, and it is expected that research into the cellular response to hydrostatic pressure generated in a living body will progress dramatically.

[0010] FIG. 1 is a diagram showing a pressure waveform when pressure fluctuations are generated in a sealed container using the pressure application device of this embodiment. FIG. 2 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 3 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 4 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 5 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 6 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 7 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 8 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 9 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 10 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 11 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 12 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 13 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 14 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. FIG. 15 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. Figure 16 is a graph showing the expression level of EGR1 in human aortic smooth muscle cells after cyclic hypertension. Figure 17 is a graph showing the expression level of EGR1 in human umbilical artery vascular smooth muscle cells after cyclic hypertension.

[0011] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0012] (Pressure application device / pressure application method) A first aspect of the present invention relates to a pressure application device connected to an airflow supply source and a sealed container that contains a biological sample. The pressure application device includes a first pressure adjustment mechanism for adjusting the pressure inside the sealed container and a second pressure adjustment mechanism for adjusting the pressure inside the sealed container, and the first pressure adjustment mechanism and the second pressure adjustment mechanism operate alternately to generate a periodically fluctuating pressure inside the sealed container. In the pressure application device, the first pressure adjustment mechanism and the second pressure adjustment mechanism operate alternately at an arbitrary set pressure and for an arbitrary set time.

[0013] A second aspect of the present invention relates to a pressure application method for a biological sample contained in a sealed container, the method comprising alternately repeating the following steps (a) and (b): (a) adjusting the pressure in the sealed container by a first pressure adjustment mechanism; and (b) adjusting the pressure in the sealed container by a second pressure adjustment mechanism. The second aspect of the present invention can be implemented, for example, by using the pressure application device described above.

[0014] The pressure application device of the first aspect has a first pressure adjustment mechanism and a second pressure adjustment mechanism, and therefore can generate a cyclically fluctuating pressure with a relatively short period in a sealed container containing a biological sample. Furthermore, the pressure application method of the second aspect can generate a cyclically fluctuating pressure with a relatively short period in a sealed container containing a biological sample by alternately repeating the above steps (a) and (b). Furthermore, when a liquid such as a culture medium is contained in the sealed container, the cyclically fluctuating pressure can be applied to the liquid (static water), and the hydrostatic pressure is transmitted to the biological sample.

[0015] If one cycle is defined as a process in which pressure is adjusted by the first pressure adjustment mechanism followed by pressure adjustment by the second pressure adjustment mechanism, the first pressure adjustment mechanism and the second pressure adjustment mechanism preferably operate so as to repeat 10 to 300 cycles per minute. That is, if one cycle of the pressure application method is defined as a process in which step (a) is followed by step (b), the cycle is preferably repeated 10 to 300 cycles per minute. The number of cycles per minute is preferably 20 or more, more preferably 25 or more, and even more preferably 30 or more. The number of cycles per minute is preferably 250 or less, more preferably 200 or less, even more preferably 180 or less, and particularly preferably 150 or less. In this way, in this embodiment, a periodically fluctuating pressure can be generated in the sealed container at a relatively short period.

[0016] Furthermore, in this embodiment, a pressure resembling in vivo pulsatile pressure can be applied within a sealed container, simulating in vivo pulsation. In this specification, "in vivo pulsatile pressure" refers to pressure that fluctuates in association with blood flow in a living body due to cardiac pulsation, and is also referred to as physiological pressure or physiological pulsatile pressure. "In vivo pulsatile pressure-like pressure" refers to pressure that fluctuates to mimic the periodic pressure fluctuations associated with blood flow in a living body due to cardiac pulsation. In this case, the pressure fluctuation period is approximately 10 to 300 times per minute, resulting in short periodic pressure fluctuations. Furthermore, while pressure values ​​measured in a living body vary from place to place, for example, when "in vivo pulsatile pressure-like pressure" is generated, pressure increases and decreases alternately within a range of 1 to 100 kPa (gauge pressure). When "in vivo pulsatile pressure-like pressure" is generated, the difference between the peak pressure during pressure increase and the peak pressure during pressure decrease is repeated to produce a minute pressure difference of several kPa to several tens of kPa. In this manner, when a "pressure similar to in vivo pulsatile pressure" is generated in this embodiment, it is possible to alternately generate a state in which the pressure in the sealed container is increased to 1 to 100 kPa (gauge pressure) at a cycle of, for example, 10 to 300 times / minute, preferably 30 to 250 times / minute, and a state in which the pressure in the sealed container is further decreased by several kPa to several tens of kPa from the peak pressure at the time of the increased pressure.

[0017] In general, the pressure waveform of mammalian blood pressure is observed to increase from low pressure to high pressure, followed by a two-stage decrease in pressure. In this embodiment, the above-described configuration has succeeded in reproducing such a two-stage decrease in pressure waveform. Specifically, by using the device having the above configuration, the pressure inside a sealed container can be increased from low pressure to high pressure in one go, and then decreased in two stages.

[0018] For example, Fig. 1 is a graph showing an example of a pressure waveform when pressure fluctuations are generated in a sealed container using the pressure application device or pressure application method of this embodiment. In the example shown in Fig. 1, the minimum pressure (low-pressure side pressure) in the pressure fluctuation is 17 kPa, the maximum pressure (high-pressure side pressure) is 27 kPa, the pressure rise time is 0.5 seconds, the pressure drop time is 0.5 seconds, and the time required for one cycle (one period) is 1 second. Furthermore, the pressure waveform shown in Fig. 1 is a waveform in which the pressure rises from a low pressure to a high pressure and then drops in two stages. For example, in Fig. 1, the time required for the first stage of pressure drop is t seconds, and the time required for the second stage of pressure drop is m seconds. In this way, this embodiment can generate a pressure similar to in vivo pulsatile pressure in a sealed container.

[0019] 2 is a schematic diagram showing the mechanism of the pressure application device of this embodiment. As shown in FIG. 2, the pressure application device 100 of this embodiment is connected to an airflow supply source 50 and a sealed container 5 that contains a biological sample, and the pressure application device 100 includes a first pressure adjustment mechanism 10 and a second pressure adjustment mechanism 20. In the pressure application device 100, the first pressure adjustment mechanism 10 and the second pressure adjustment mechanism 20 operate alternately to generate a periodically fluctuating pressure in the sealed container 5. By including multiple pressure adjustment mechanisms, such as the first pressure adjustment mechanism 10 and the second pressure adjustment mechanism 20, the pressure application device of this embodiment can generate a periodically fluctuating pressure in the sealed container 5.

[0020] In this embodiment, the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in series or in parallel. It is particularly preferable that the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel. When the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel, the pressure application device 100 has a first flow path 11 and a second flow path 21. For example, as shown in FIG. 2 , the first pressure adjustment mechanism 10 is provided in the first flow path 11, and the second pressure adjustment mechanism 20 is provided in the second flow path 21.

[0021] In this specification, "pressure adjustment" refers to pressurizing and / or depressurizing the inside of the sealed container 5. Note that, when the pressure inside the sealed container 5 is at a desired pressure value, maintaining the pressure inside the sealed container 5 without pressurizing or depressurizing it is also included in "pressure adjustment." However, it is preferable that the first pressure adjustment mechanism 10 is a mechanism that pressurizes and / or depressurizes the inside of the sealed container 5, and it is preferable that the second pressure adjustment mechanism 20 is a mechanism that pressurizes and / or depressurizes the inside of the sealed container 5.

[0022] In a particularly preferred embodiment of the present invention, the first pressure adjustment mechanism 10 is preferably a mechanism for pressurizing the inside of the sealed container 5 (high-pressure application mechanism), and the second pressure adjustment mechanism 20 is preferably a mechanism for depressurizing the inside of the sealed container 5 (low-pressure application mechanism). That is, in a preferred embodiment of the pressure application method, it is preferred that the step (a) is a step of pressurizing the inside of the sealed container 5 by the first pressure adjustment mechanism 10, and the step (b) is a step of depressurizing the inside of the sealed container 5 by the second pressure adjustment mechanism 20. It is particularly preferred that an airflow such as compressed gas is supplied into the sealed container 5 in the step (a).

[0023] The first pressure adjustment mechanism 10 is preferably a mechanism that adjusts (pressurizes) the pressure inside the sealed container 5 so that a pressure (gauge pressure) of less than 200 kPa is applied inside the sealed container 5. That is, step (a) in the pressure application method is preferably a step of adjusting the pressure inside the sealed container 5 so that a pressure (gauge pressure) of less than 200 kPa is applied inside the sealed container 5. The pressure applied by the first pressure adjustment mechanism 10 (the pressure value inside the sealed container 5 in step (a)) is more preferably 180 kPa or less, even more preferably 160 kPa or less, even more preferably 140 kPa or less, still more preferably 120 kPa or less, and particularly preferably 100 kPa or less. Furthermore, the pressure applied by the first pressure adjustment mechanism 10 (the pressure value inside the sealed container 5 in step (a)) is preferably 1 kPa or more, more preferably 5 kPa or more, and even more preferably 10 kPa or more.

[0024] The second pressure adjustment mechanism 20 is preferably a mechanism that adjusts (depressurizes) the pressure inside the sealed container 5 so that the pressure applied by the first pressure adjustment mechanism 10 is reduced by 1 kPa or more, and more preferably a mechanism that adjusts (depressurizes) the pressure inside the sealed container 5 so that the pressure is reduced by 1 kPa or more and 50 kPa or less. That is, step (b) is preferably a step of adjusting (depressurizing) the pressure inside the sealed container 5 so that the pressure is reduced by 1 kPa or more from the pressure reached in step (a), and more preferably a step of adjusting (depressurizing) the pressure inside the sealed container 5 so that the pressure is reduced by 1 kPa or more and 50 kPa or less. Thus, the fluctuation range of the pressure applied inside the sealed container 5 (maximum pressure - minimum pressure) is preferably 1 to 50 kPa. For example, when the pressure application device 100 is operated to simulate the blood pressure of an animal, it is preferable to set the pressure within a range of a maximum of 300 mmHg (approximately 40 kPa) and a minimum of 30 mmHg (approximately 4 kPa).

[0025] If the maximum pressure (absolute pressure) inside the sealed container 5 when the first pressure adjustment mechanism 10 adjusts the pressure inside the sealed container 5 is P, and the minimum pressure (absolute pressure) inside the sealed container 5 when the second pressure adjustment mechanism 20 adjusts the pressure inside the sealed container 5 is Q, the value of P / Q is preferably 1.000 or more, more preferably 1.001 or more, and even more preferably 1.010 or more. Furthermore, the value of P / Q is preferably 3 or less, more preferably 1.5 or less, even more preferably 1.35 or less, even more preferably 1.2 or less, particularly preferably 1.15 or less, and most preferably 1.1 or less.

[0026] In this embodiment, a biological sample is preferably contained in the sealed container 5. In this case, the biological sample may be placed directly in the sealed container 5, but it is preferable that a biological sample holding container 6 is contained in the sealed container 5, and the biological sample is preferably cultured in the biological sample holding container 6. Examples of the biological sample holding container 6 include a cell culture plate and a cell culture tank. It is preferable that the biological sample holding container 6 is filled with a medium for cell culture or the like. In this case, instead of applying pressure directly to the medium (liquid) containing the biological sample, pressure can be indirectly transmitted to the liquid by varying the pressure in the sealed container 5, thereby more faithfully reproducing physiological conditions.

[0027] Examples of biological samples include cells, tissues, blood, body fluids, DNA, RNA, proteins, etc. Among these, cells are preferred as biological samples. It can be said that tissues are aggregates of cells. Cells include all cells that make up a living organism, but also include unicellular organisms (amoeba, euglena, diatoms, etc.), multicellular organisms (daphnia, Spirogyra, etc.), and multicellular three-dimensional structures (organoids, spheroids, etc.).

[0028] Preferably, a culture medium containing a biological sample is contained within the sealed container 5. By applying cyclic pressure to the sealed container 5, cyclic pressure can be applied to the culture medium (static water), thereby applying mechanical stress to the biological sample. This allows the biological sample to be cultured under conditions closer to the in vivo environment, and also allows for research into cellular responses to mechanical stress. The sealed container 5 can also be attached to a microscope stage, in which case the biological sample can be observed while applying cyclic pressure.

[0029] The pressure application device 100 is connected to a supply source 50. The supply source 50 is an airflow supply source that supplies an airflow to the first pressure adjustment mechanism 10 and / or the second pressure adjustment mechanism 20. Preferably, the supply source 50 is a compressed gas supply source, and it is particularly preferable to supply compressed gas to the first pressure adjustment mechanism 10. Examples of the supply source 50 include a mechanism for supplying compressed gas, such as an air compressor or a gas cylinder. Compressed gas can be compressed from ordinary air, but any gas can also be used, such as carbon dioxide, inert gas, or a mixture of these. In this embodiment, compressed gas is introduced when applying pressure, thereby suppressing changes in the pH, carbon dioxide concentration, nitrogen gas concentration, and other parameters within the culture medium containing the biological sample. The discharge pressure of the supply source 50 is preferably less than 1000 kPa (gauge pressure), at which pneumatic equipment operates. The discharge pressure of the supply source 50 must be greater than the maximum pressure applied within the sealed container 5.

[0030] In this embodiment, when the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel, the first flow path 11 has the first pressure adjustment mechanism 10, and the sealed container 5 is connected downstream of the first pressure adjustment mechanism 10, and the supply source 50 is connected upstream of the first pressure adjustment mechanism 10. In this way, the first flow path 11 is a flow path connecting the upstream supply source 50 to the downstream sealed container 5. Furthermore, the second flow path 21 has the second pressure adjustment mechanism 20, and the sealed container 5 is connected downstream of the second pressure adjustment mechanism 20, and the supply source 50 or an exhaust mechanism (not shown) is connected upstream of the second pressure adjustment mechanism 20. In this way, the second flow path 21 is a flow path that has the second pressure adjustment mechanism 20 and connects to the downstream sealed container 5. The supply source 50 may be connected upstream of the second pressure adjustment mechanism 20, or an exhaust mechanism capable of exhausting may be provided.

[0031] The first flow path 11 is a flow path that mainly introduces compressed gas supplied from the supply source 50 into the sealed container 5, and the second flow path 21 is a flow path that mainly recovers gas from the sealed container 5. When the supply source 50 is connected to the second flow path 21 upstream of the second pressure adjustment mechanism 20, it is preferable that the recovered gas is exhausted via the second pressure adjustment mechanism 20. Note that, when the increased pressure value in the sealed container 5 is higher than the desired pressure value, the first flow path 11 can also serve as a flow path that recovers gas from the sealed container 5. In this case, the recovered gas is exhausted via the first pressure adjustment mechanism 10. On the other hand, when the decreased pressure value in the sealed container 5 is lower than the desired pressure value, the second flow path 21 can also serve as a flow path that introduces compressed gas supplied from the supply source 50 into the sealed container 5.

[0032] An exhaust valve may be provided in the sealed container 5. The pressure in the sealed container 5 may be reduced by rapidly exhausting air from the exhaust valve provided in the sealed container 5. It is also possible to simultaneously exhaust air via both the exhaust valve provided in the sealed container 5 and the second pressure adjustment mechanism 20. By appropriately controlling the pressure reduction method, it becomes easy to reduce the pressure in two stages, for example, and to easily control the pressure waveform.

[0033] The pressure application device 100 in this embodiment may have a first common flow path 30. In this case, as shown in FIG. 2 , the first flow path 11 and the second flow path 21 merge upstream to form the first common flow path 30. The first common flow path 30 is preferably connected to a supply source 50. The pressure application device 100 may also have a second common flow path 40. In this case, as shown in FIG. 2 , the first flow path 11 and the second flow path 21 merge downstream to form the second common flow path 40. The second common flow path 40 is preferably connected to the sealed container 5.

[0034] It is preferable that the first common flow path 30 and the second common flow path 40 each have a control valve at a point where they branch into the first flow path 11 and the second flow path 21. This control valve can, for example, block the airflow to one of the flow paths, thereby making it possible to supply compressed gas to only one of the flow paths (mainly the first flow path) or to discharge recovered gas to only one of the flow paths (mainly the second flow path).

[0035] 3 to 5 are schematic diagrams illustrating other aspects of the pressure application device 100 of this embodiment. As shown in FIG. 3, the pressure application device 100 may not have the second common flow path 40, and the first flow path 11 and the second flow path 21 may each be connected to the sealed container 5. In this case, the first flow path 11 serves as a supply flow path and the second flow path 21 serves as an exhaust flow path, which is expected to improve gas circulation. Also, as shown in FIG. 4, the pressure application device 100 may not have the first common flow path 30, and the first flow path 11 and the second flow path 21 may each be connected to a supply source 51 and a supply source 52. Furthermore, the second flow path 21 may be connected to an exhaust mechanism (open-to-atmosphere mechanism) without being connected to the supply source 52.

[0036] 5, the pressure application device 100 may not have the first common flow path 30 and the second common flow path 40, and the first flow path 11 and the second flow path 21 may be connected to the sealed container 5, and the first flow path 11 and the second flow path 21 may be connected to the supply source 51 and the supply source 52, respectively. Moreover, the second flow path 21 may be connected to an exhaust mechanism (open-to-atmosphere mechanism) without being connected to the supply source 52.

[0037] -Embodiment A- Figure 6 is a schematic diagram more specifically illustrating the first pressure regulating mechanism and the second pressure regulating mechanism shown in Figures 2 to 5. In embodiment A, it is preferable that the first pressure regulating mechanism includes a first regulator and / or a first control valve, and the second pressure regulating mechanism includes a second regulator and / or a second control valve, and it is more preferable that the first pressure regulating mechanism includes a first regulator and a first control valve, and the second pressure regulating mechanism includes a second regulator and a second control valve. As shown in Figure 6, in embodiment A, the first pressure regulating mechanism 10 includes a first regulator 12 and a first control valve 14, and the second pressure regulating mechanism 20 includes a second regulator 22 and a second control valve 24.

[0038] The control valve used in this embodiment is not particularly limited as long as it has the function of passing or blocking gas through the flow path. Examples of the control valve include a solenoid valve, a proportional control valve, an air-operated valve, a manual valve, and a mechanically operated valve. Among these, the control valve is preferably a solenoid valve, and the use of a solenoid valve makes it easier to achieve more regular cyclic pressurization.

[0039] In embodiment A, the first control valve 14 and the second control valve 24 are preferably two-port valves (on-off valves). In this embodiment A, the first regulator 12 and the second regulator 22 are connected in parallel, and the first regulator 12 is disposed upstream of the first control valve 14 in the first flow path. Furthermore, the second regulator is disposed upstream of the second control valve 24 in the second flow path.

[0040] The first regulator 12 and the second regulator 22 may each include a valve therein. The pressure at which the valve opens (operates) (the pressure at which compressed gas on the primary side of the regulator begins to be supplied to the secondary side) is adjusted as desired to determine the set pressure of the compressed gas introduced from the upstream (primary side) of the regulator to the downstream (secondary side). If the secondary side pressure of the regulator is lower than the set pressure, the regulator increases the secondary side pressure until it reaches the set pressure. If the secondary side pressure of the regulator is higher than the set pressure, the regulator releases excess pressure to the atmosphere via the regulator's relief mechanism, thereby reducing the secondary side pressure. In this way, the regulator increases or decreases the secondary side pressure based on the set pressure. The pressure at which the regulator's valve opens, i.e., the set pressure, may be adjusted manually or automatically.

[0041] The first control valve is provided downstream of the first regulator 12, and the second control valve 24 is provided downstream of the second regulator 22. The first control valve 14 and the second control valve 24 are downstream switching control valves that switch between the operation of the first regulator 12 and the operation of the second regulator 22. In other words, the first control valve 14 and the second control valve 24 serve to switch between the connection between the sealed container 5 and the first regulator 12 and the connection between the sealed container 5 and the second regulator 22.

[0042] For example, in this embodiment A, when the pressure application device 100 is operating, the first regulator 12 and the second regulator 22 are in an operating state and can apply and / or reduce pressure to reach a predetermined pressure. When applying pressure to the sealed container 5, the first control valve 14 opens for a predetermined time. Here, the predetermined time refers to the pressure increase time as shown in FIG. 1 , e.g., 0.5 seconds. The set pressure of the first regulator 12 is adjusted manually or automatically so that the pressure inside the sealed container 5 reaches a predetermined pressure value (maximum pressure value) within this predetermined time. When the pressure increase time is set to a short time, such as 0.5 seconds, if the set pressure value and the maximum pressure value are the same (set pressure value = maximum pressure value), the maximum pressure value may not be reached within the predetermined time. Therefore, it is preferable to set the set pressure value of the first regulator 12 higher than the maximum pressure value of the sealed container 5 (maximum pressure value < set pressure value). Note that if the "maximum pressure value < set pressure value" is set, the maximum pressure value inside the sealed container 5 may be reached earlier than the predetermined time. In this case, the amount of compressed gas supplied to the sealed container 5 in a predetermined time may be reduced by lowering the set pressure of the first regulator 12. In this way, it is preferable that the first regulator 12 is adjusted so that the pressure inside the sealed container 5 reaches the maximum value in a predetermined time.

[0043] When the first control valve 14 is opened and the first regulator 12 is operated, compressed gas is discharged from the secondary side of the first regulator 12, and the inside of the sealed container 5 is pressurized. When the predetermined pressure inside the sealed container 5 becomes higher than the set pressure, the excess pressure may be released from the relief mechanism of the first regulator 12.

[0044] Next, simultaneously with the closing of the first control valve 14, the second control valve 24 opens for a predetermined time, and the second regulator 22 operates, thereby reaching a predetermined pressure value (minimum pressure value). The predetermined time is the pressure drop time shown in FIG. 1 , e.g., 0.5 seconds. The set pressure of the second regulator 22 is adjusted manually or automatically so that the pressure inside the sealed container 5 reaches the minimum pressure value within this predetermined time. When the pressure drop time is set to a short time, such as 0.5 seconds, if the set pressure value and the minimum pressure value are the same (set pressure value = minimum pressure value), the minimum pressure value may not be reached within the predetermined time. Therefore, the set pressure value of the second regulator 22 is set lower than the minimum pressure value of the sealed container 5 (minimum pressure value > set pressure value). Furthermore, if the "minimum pressure value > set pressure value" is set, the minimum pressure value inside the sealed container 5 may be reached earlier than the predetermined time. In this case, the set pressure of the second regulator 22 is increased to reduce the amount of compressed gas discharged from the sealed container 5 within the predetermined time. In this way, the second regulator 22 is adjusted so that the pressure inside the sealed container 5 reaches the minimum pressure value within a predetermined time.

[0045] When depressurizing the sealed container 5, the second regulator 22 typically recovers gas from the sealed container 5 and releases it through the relief mechanism of the second regulator 22. When the second control valve 24 opens, the internal volume expands from the sealed container 5 to the second regulator 22, causing the gas stored in the sealed container 5 to expand. This causes the pressure in the sealed container 5 to drop rapidly (t in FIG. 1). Immediately thereafter, the compressed gas is gradually released through the relief mechanism of the second regulator 22 (m in FIG. 1). Therefore, the waveform during pressure drop is two-stage, similar to pulsatile pressure. To ensure sufficient internal volume, a tank may be appropriately installed between the second control valve 24 and the second regulator 22, or the flow path between the second control valve 24 and the second regulator 22 may be lengthened. The gas released through the relief mechanism of the second regulator 22 may be released into the pressure application device 100; however, since the gas contains components different from those of the atmosphere, it may affect the human body. In this case, an exhaust port may be provided in the second flow path of the pressure application device 100, and piping may be provided between the second regulator 22 and the exhaust port, so that the gas can be exhausted to the outside of the pressure application device 100.

[0046] The pressure application device 100 may further include a tank 16 downstream of the first regulator 12. The tank 16 is preferably provided between the first regulator 12 and the first control valve 14. Depending on the insufficient supply pressure from the supply source 50 or the volume of the sealed container 5, the first regulator 12 may have difficulty increasing the pressure inside the sealed container 5 to a predetermined level within a predetermined time. In such cases, the shortage of the supply amount of compressed gas can be compensated for by appropriately installing the tank 16.

[0047] Furthermore, the sealed container 5 is preferably provided with a pressure sensor 7 that can measure the pressure inside the sealed container 5 in real time. In this embodiment, the pressure sensor 7 is preferably a gauge pressure sensor. The pressure sensor 7 is provided inside the sealed container 5 and measures the pressure applied inside the sealed container 5 in real time. Then, depending on the pressure value detected by the pressure sensor 7, the operation of the first pressure adjustment mechanism 10 (first regulator 12 and / or first control valve 14) and the second pressure adjustment mechanism 20 (second regulator 22 and / or second control valve 24) is controlled, respectively.

[0048] The pressure application device 100 preferably further includes a controller 60. The controller 60 can set the pressure application cycle, the maximum pressure value, the minimum pressure value, and the time and pressure value for two-stage pressure reduction. Time-dependent pressure value information detected by the pressure sensor 7 is provided to the controller 60, which feedback-controls the first pressure adjustment mechanism 10 and the second pressure adjustment mechanism 20 according to the pressure value detected by the pressure sensor 7 and the pressure and cycle set by the controller 60. The transmission of pressure value information detected by the pressure sensor 7 and the signal output from the controller 60 to each mechanism are indicated by dotted arrows in FIG. 6. The controller 60 performs feedback control by performing calculations in a calculation unit to match the pressure with a target value (set value) and outputting adjusted signals to each mechanism.

[0049] For example, when pressurization is performed by first pressure adjustment mechanism 10 (first regulator 12 and / or first control valve 14), if the output value from pressure sensor 7 does not reach a predetermined pressure value within the pressure increase time (pressure application period) set in controller 60, the output value from controller 60 to first pressure adjustment mechanism 10 is controlled, and first pressure adjustment mechanism 10 is adjusted so that it matches the predetermined pressure value. Alternatively, if the pressure increase time (pressure application period) when the output value from pressure sensor 7 reaches the maximum pressure value does not match the time set in controller 60, the output value from controller 60 to first pressure adjustment mechanism 10 is controlled, and first pressure adjustment mechanism 10 is adjusted so that it matches the set time.

[0050] When the pressure is reduced by the second pressure adjustment mechanism 20 (second regulator 22 and / or second control valve 24) and the output value from the pressure sensor 7 does not reach a predetermined pressure value within the pressure reduction time (pressure application period) set in the controller 60, the output value from the controller 60 to the second pressure adjustment mechanism 20 is controlled, and the second pressure adjustment mechanism 20 is adjusted so that it matches the predetermined pressure value. Alternatively, if the pressure reduction time (pressure application period) when the output value from the pressure sensor 7 reaches the minimum pressure value does not match the time set in the controller 60, the output value to the controller 60 is controlled, and the second pressure adjustment mechanism 20 is adjusted so that it matches the set time. For example, the first control valve 14 and the second control valve 24 are controlled by an output from the controller to each control valve in accordance with the pressure application period set in the controller 60.

[0051] In addition to the pressure control described above, the controller 60 can also output control signals for adjusting the temperature, humidity, gas composition, and the like within the sealed container 5. For example, in addition to the pressure sensor 7, the controller 60 can be equipped with a temperature sensor, a humidity sensor, a gas sensor, and the like, which can measure the pressure value within the sealed container 5 as well as the temperature, humidity, and gas composition within the sealed container 5 in real time. Information detected by these sensors is transmitted to the controller 60, allowing the controller 60 to output feedback control signals regarding the increase or decrease in temperature and humidity to the temperature control mechanism and humidity control mechanism within the sealed container 5. Examples of the temperature control mechanism include a heater, and examples of the humidity control mechanism include a humidifier. Furthermore, if a gas composition control mechanism such as a gas blender is connected to the supply source, the desired gas composition can be adjusted by outputting time-varying gas composition information or a control signal to the gas composition control mechanism. The controller 60 can also input pressure information, period information, temperature information, humidity information, gas composition information, and the like, and can store this data obtained from the sealed container 5 or output it to an external device.

[0052] The pressure application device 100 may be provided with a temperature and humidity control mechanism 8 inside or outside the sealed container 5. As the temperature and humidity control mechanism 8, for example, a heater is preferably provided below the biological sample container 6 inside the sealed container 5. This makes it possible to heat the culture medium inside the biological sample container 6 and keep it at a temperature range suitable for cell culture (for example, about 25 to 45°C).

[0053] When the pressure application device 100 has the second common flow path 40, an inlet / outlet pipe for supplying compressed gas and recovering compressed gas is connected to the sealed container 5. In this case, the sealed container 5 may be provided with an inlet / outlet port 9. By connecting the second common flow path 40 to the inlet / outlet port 9, gas can be supplied to the sealed container 5 and the gas can be recovered. Furthermore, when cyclic pressurization is not performed, the inlet / outlet port 9 can be closed to maintain the sealed state inside the sealed container 5.

[0054] -Embodiment B- In embodiment B, as shown in FIG. 7, the second pressure regulating mechanism 20 has a second regulator 22, a second control valve 24a, and a second control valve 24b. In embodiment B, it is preferable that the second control valve 24a is a distribution valve (three-port valve), and the second control valve 24b is a flow control valve. In this case, it is preferable that the second regulator 22, the second control valve 24a, and the second control valve 24b are arranged in this order from the upstream side. In embodiment B, the second flow path 21 is branched via the second control valve 24a into a first exhaust flow path 21a including the second regulator 22 and a second exhaust flow path 26 (exhaust mechanism 26) that is directly exhausted from the second control valve 24a.

[0055] The second control valve 24a is an exhaust control valve that switches the connection of the exhaust airflow to either the first exhaust flow path 21a or the second exhaust flow path 26. The second control valve 24a has a first port P connected to the second control valve 24b, a second port Q connected to the first exhaust flow path 21a, and a third port R connected to the second exhaust flow path 26. The second control valve 24a switches the first port P to connect to either the second port Q or the third port R. When the pressure inside the sealed container 5 is to be rapidly reduced, the second control valve 24a connects the first port P to the third port R. The second flow path 21 is connected to the second exhaust flow path 26, and gas is exhausted. The third port R does not have to be connected to the second exhaust flow path 26; in that case, gas may be directly exhausted from the third port R. On the other hand, when the pressure inside the sealed container 5 is to be gradually reduced, the second control valve 24a connects the first port P to the second port Q. The second flow path 21 is then connected to the first exhaust flow path 21a, and the gas is exhausted via the relief mechanism of the second regulator 22. In this way, by appropriately changing the exhaust flow path, it is possible to obtain a two-stage pressure reduction waveform.

[0056] 8, the relief mechanism of the second regulator 22 may be connected to the second exhaust flow path 26, and the gas exhausted from the relief mechanism of the second regulator 22 may be discharged from the second exhaust flow path 26. In this case, the gas may be exhausted in a concentrated manner from one exhaust port via the second exhaust flow path 26. Alternatively, the housing of the pressure application device 100 may be provided with respective exhaust ports and piping, and the gas may be exhausted to the outside.

[0057] -Embodiment D- In the case where the pressure application device 100 has a second common flow path 40, a control valve 42 may be provided in the second common flow path 40 (FIG. 9). In this case, the control valve 42 corresponds to the first control valve 14 and the second control valve 24. The control valve 42 is a three-port valve. The control valve 42 is provided downstream of the first regulator 12 and the second regulator 22. The control valve 42 is a downstream switching control valve that switches between operation of the first regulator 12 and operation of the second regulator 22.

[0058] The control valve 42 has a first port P connected to the second common flow path 40, a second port Q connected to the first flow path 11, and a third port R connected to the second flow path 21. The control valve 42 switches between connecting the first port P to either the second port Q or the third port R. When the first port P and the second port Q are connected and the second common flow path 40 is connected to the first flow path 11, gas is supplied from the supply source 50 through the first regulator 12 to the sealed container 5. On the other hand, when the first port P and the third port R are connected and the second common flow path 40 is connected to the second flow path 21, the gas in the sealed container 5 is exhausted through the relief mechanism of the second regulator 22. To obtain a two-stage pressure-drop waveform, a long flow path between the control valve 42 and the second regulator 22 may be provided, or a tank may be provided between the control valve 42 and the second regulator 22. As shown in FIG. 9, by reducing the number of control valves to one, the pressure application device 100 can be made smaller, and the manufacturing costs and the like can be reduced.

[0059] -Embodiment E- When the pressure in the sealed container 5 is repeatedly increased and decreased at a relatively short cycle (e.g., a pulsatile pressure of 10 to 300 cycles per minute), as in this embodiment, excess pressure often does not occur. When excess pressure does not occur, the first pressure adjustment mechanism 10 does not need to be provided with an exhaust mechanism. Therefore, a flow control valve can be selected instead of the first regulator. Examples of flow control valves include a proportional control valve, a needle valve, a speed controller, and the throttle valve disclosed in Japanese Patent No. 6134287. In particular, when electrical control is performed, it is preferable to use a proportional control valve or the throttle valve disclosed in Japanese Patent No. 6134287. Similarly, a flow control valve can be selected instead of the second regulator. Examples of flow control valves include a proportional control valve, a needle valve, a speed controller, and the throttle valve disclosed in Japanese Patent No. 6134287. In particular, when electrical control is performed, it is preferable to use a proportional control valve or the throttle valve disclosed in Japanese Patent No. 6134287.

[0060] For example, in this embodiment E, as shown in FIG. 10 , the first pressure adjustment mechanism 10 includes a first control valve 14 and a needle valve 18, and the second pressure adjustment mechanism 20 includes a second control valve 24 and a needle valve 28. The needle valve 18 is connected to a supply source 50, and the needle valve 28 is connected to an exhaust flow path. When increasing the pressure, the interior of the sealed container 5 reaches a predetermined pressure within a predetermined time by adjusting the aperture of the needle valve 18. Similarly, when decreasing the pressure, the interior of the sealed container 5 reaches a predetermined pressure within a predetermined time by adjusting the aperture of the needle valve 28. At this time, the second flow path 21 is not connected to the supply source 50, and the gas filled in the sealed container 5 is exhausted. The exhausted gas may be exhausted into the pressure application device 100, or may be exhausted to the outside of the pressure application device 100 by providing an exhaust port in the housing of the pressure application device 100 and connecting the exhaust port to the needle valve 28 through piping.

[0061] -Embodiment F- In this embodiment F, the second regulator 22 in FIG. 7 of embodiment B can be replaced with a needle valve (FIG. 11). In this embodiment F, as shown in FIG. 11, the first exhaust flow path 21a provided with the needle valve does not need to be connected to a supply source. Gas flowing through the first exhaust flow path 21a during pressure reduction is exhausted via the needle valve. Note that, as shown in FIG. 8, the first exhaust flow path 21a may be connected to the second exhaust flow path 26, thereby concentrating exhaust at one location (concentrated exhaust).

[0062] 12, in this embodiment G, the first regulator 12 and the second regulator 22 can be replaced with needle valves 18 and 28, respectively. In this case, the upstream of the needle valve 28 is not connected to the supply source but is exhausted.

[0063] -Embodiment H- In this embodiment H, as shown in FIG. 13 , a control valve 24d, which is a two-port valve, may be further provided on the second flow path 21. The installation of the control valve 24d facilitates two-stage pressure reduction. The control valve 24d is a two-port valve and is provided between the needle valve 28 and the control valve 42. When reducing the pressure, the control valve 42 first switches to the second flow path 21, connecting the sealed container 5 to the second flow path 21. This causes the gas stored in the sealed container 5 to expand, resulting in a sudden drop in pressure. When the control valve 24d subsequently opens, the gas after the sudden drop in pressure is discharged. At this time, the needle valve 28 throttles the exhaust flow rate, easing the pressure drop. Thus, the control valve 24d is an exhaust control valve for obtaining a two-stage pressure reduction waveform. To ensure sufficient internal volume, a tank may be appropriately provided between the control valve 42 and the control valve 24d, or the flow path between the control valve 42 and the control valve 24d may be lengthened. The control valve 42 corresponds to the first control valve 14 and the second control valve 24. The first pressure regulating mechanism 10 includes a first regulator 12.

[0064] -Embodiment I- In this embodiment I, as shown in Figure 14, a control valve 32, which is a three-port valve, may be installed in the first common flow path 30, and a control valve 42 may be installed in the second common flow path 40. In this case, for example, the second pressure adjustment mechanism 20 may include a check valve 25a as the second control valve. A needle valve 28 and a tank 29 may be installed in the second flow path. In this case, the check valve only needs to be located upstream of the tank 29, and may also be located upstream of the needle valve 28. The control valve 32 has a first port P connected to the first common flow path 30, a second port Q connected to the supply source, and a third port R connected to the exhaust flow path. The control valve 42 connects the first port P to either the second port Q or the third port R. The control valve 32 is an upstream switching control valve that is provided upstream of the first pressure regulating mechanism 10 and the second pressure regulating mechanism 20, for example, in the first common flow path 30, and switches the first pressure regulating mechanism 10 and the second pressure regulating mechanism 20 so that they are connected to either the supply source or the exhaust flow path.

[0065] During pressure increase, the first port P and the second port Q of the control valve 32 are connected, connecting the first common flow path 30 to the supply source. Gas is then supplied to the sealed container 5 via the first flow path 11. At the same time, gas supplied from the supply source also flows into the second flow path 21, but is prevented from flowing downstream by the check valve 25a, which is the second control valve. The check valve 25a prevents gas from being supplied to the tank 29, achieving a rapid increase in pressure within the sealed container 5 within a predetermined time. During pressure decrease, the first port P and the third port R of the control valve 42 are connected, connecting the second common flow path 40 to the second flow path 21. This causes the gas filling the sealed container 5 to expand, resulting in a rapid drop in pressure. At the same time, the control valve 32 is switched, connecting the first port P and the third port R, thereby connecting the first common flow path 30 to the exhaust flow path, resulting in a gradual drop in pressure. Instead of providing the tank 29, the flow path between the check valve 25a and the control valve 42 may be lengthened. Moreover, the needle valve 28 may be provided in the exhaust flow path of the control valve 32 instead of in the second flow path 21. The control valve 42 corresponds to the first control valve 14 and the second control valve 24. The first pressure regulating mechanism 10 includes a first regulator 12.

[0066] -Embodiment J- In this embodiment J, the first pressure adjustment mechanism 10 and the second pressure adjustment mechanism 10 may be connected in series as shown in Fig. 15. In this manner, when the flow path formed by connecting the first pressure adjustment mechanism 10 and the second pressure adjustment mechanism 10 in series is defined as the third flow path 70, the first pressure adjustment mechanism 10 may include a speed controller 14c as the first control valve 14, and the second pressure adjustment mechanism 20 may include a speed controller 24c as the second control valve 24. It is preferable that the third flow path 70 is connected to the control valve 32 upstream and to the sealed container 5 downstream. The control valve 32 has a second port Q connected to the supply source 50, a first port P connected to the third flow path 70, and a third port R connected to the exhaust flow path.

[0067] The speed controller 14c includes a check valve 15a that blocks flow from upstream to downstream and a needle valve 15b that throttles the flow rate of the flow path. The speed controller 24c includes a check valve 25a that blocks flow from downstream to upstream and a needle valve 25b that throttles the flow rate of the flow path. The speed controller 14c and the speed controller 24c may be arranged in any order, upstream or downstream.

[0068] During pressure increase, the first port P and the second port Q of the control valve 32 are connected, and gas is supplied from the supply source to the third flow path 70. A portion of the gas supplied is controlled by the check valve 15a of the speed controller 14c, and the flow rate is further controlled by the needle valve 15b. Since the check valve 25a of the speed controller 24c does not obstruct the flow from upstream, gas adjusted by the speed controller 14c is supplied to the sealed container 5. In this way, the speed controller 14c adjusts the pressure during pressure increase so that the pressure inside the sealed container 5 reaches a predetermined value within a predetermined time. During pressure decrease, the first port P and the third port R of the control valve 32 are connected, and the gas inside the sealed container 5 is exhausted. When the control valve 32 is switched, the gas filled inside the sealed container 5 expands, causing a rapid reduction in pressure. Immediately thereafter, the pressure gradually decreases via the speed controller 24c. The check valve 25a of the speed controller 24c controls a portion of the gas flowing in from the sealed container 5, and the needle valve 25b controls the flow rate of gas exhausted upstream from the sealed container 5. In this way, the speed controller 24c adjusts the pressure during pressure reduction so that the pressure inside the sealed container 5 reaches a predetermined pressure value within a predetermined time.

[0069] (Applications) In this embodiment, periodic pressure fluctuations can be generated within a sealed container. In this embodiment, a minute pressure differential of approximately several kPa to several tens of kPa can be generated in a sealed space at a period equivalent to the pulsation, simulating the pulsation in a living body. This allows a pressure similar to the pulsatile pressure in a living body to be applied within the sealed space. Such periodic pressure fluctuations are also propagated to a biological sample container housed within the sealed container, and can also impose pressure fluctuations on the liquid (e.g., culture medium) within the biological sample container. In other words, since this embodiment also allows the application of periodic hydrostatic pressure, for example, by applying periodic hydrostatic pressure while culturing cells in a liquid (e.g., culture medium) within the biological sample container, research on cellular responses to hydrostatic pressure in a living body can be conducted.

[0070] The pressure application device and pressure application method of the present invention can generate a pressure similar to in vivo pulsatile pressure in vitro, and can also apply a pressure similar to in vivo pulsatile pressure to a liquid (hydrostatic liquid). This makes it possible to reproduce physiological conditions in vitro, and is expected to be applied to research on cellular responses to mechanical stress, for example.

[0071] Furthermore, by using the pressure application device and pressure application method of the present invention, it is possible to artificially create an environment in which blood pressure is low or high, which makes it possible to conduct research on cellular responses caused by blood pressure reduction or increase, and to evaluate the efficacy of drugs such as antihypertensives.

[0072] Furthermore, by using the pressure application device and pressure application method of the present invention, it is possible to study the responses of tissues and cells to pressure in tissues other than the circulatory system that are periodically subjected to pressure (such as masticatory muscles and joints), and it can also be used to study the mechanisms of adaptation to abnormal environments such as high pressure.

[0073] The features of the present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0074] (Preparation of Biological Samples) Commercially available human aortic smooth muscle cells and human umbilical artery vascular smooth muscle cells were each placed in a 12-well plate at 15 × 10 ４ The cells were seeded at 10 cells / well and cultured for 24 hours in Dulbecco's Modified Eagle Medium (hereinafter referred to as DMEM) containing 10% bovine serum (in an incubator at 37°C and 5% CO ２ ).

[0075] (Cultivation under pressure application conditions) The 12-well plate containing the cultured cells was placed in a sealed container (pressure-resistant chamber) of a pressure application device, and the sealed container (pressure-resistant chamber) was placed in an incubator and subjected to cyclic pressure application at 1 Hz (repetition rate of once per second) for 1 hour. The cyclic pressure application was performed under conditions that mimicked in vivo pulsation, with the pressure inside the sealed container adjusted to 17 kPa to 28 kPa, with one cycle of 17 kPa / 28 kPa.

[0076] (Analysis) Each cell was removed from the sealed container (pressure-resistant chamber), total RNA was extracted from the cells, and cDNA was prepared. The expression level of EGR1 (Early Growth Response 1), a gene known to respond to mechanical stress, was measured by quantitative PCR. RNA extraction and purification were performed using a commercially available RNA extraction reagent, and cDNA was prepared using a commercially available kit containing reverse transcriptase. The reaction time and reaction temperature during reverse transcription were as recommended by the manufacturer. Using this cDNA as a template, human EGR1 (Early Growth Response 1) gene-specific primers and commercially available real-time PCR reagents, EGR1 gene expression levels were measured by quantitative PCR. Quantitative PCR was performed in two steps, with the annealing and extension temperature set to 60°C. Quantitative PCR analysis was performed using the ΔΔCt method, and the expression level of 18s rRNA was used as an endogenous control. 16 and 17 show the expression level of EGR1 in each cell type. The control (expression level 1) was cells cultured for 1 hour in an incubator under atmospheric pressure without cyclic pressurization.

[0077] 16 and 17, a significant increase in the expression level of EGR1 was observed in cells cultured in a device that generated cyclic pressure that mimicked in vivo pulsation. It was confirmed that the pressure application device of the present invention generates cyclic pressure similar to in vivo pulsation pressure within the culture device, and that cyclic pressure mimicking in vivo pulsation (cyclic hydrostatic pressure application) is applied to the cells in the culture medium.

[0078] 5 Sealed container 6 Biological sample container 7 Pressure sensor 8 Temperature and humidity control mechanism 9 Inlet / outlet port 10 First pressure adjustment mechanism 11 First flow path 12 First regulator 14 First control valve 14c Speed ​​controller 15a Check valve 15b Needle valve 16 Tank 18 Needle valve 20 Second pressure adjustment mechanism 21 Second flow path 21a First exhaust flow path 22 Second regulator 24, 24a, 24b, 24d Second control valve 24c Speed ​​controller 25a Check valve 25b Needle valve 26 Second exhaust flow path (exhaust mechanism) 28 Needle valve 29 Tank 30 First common flow path 32 Control valve 40 Second common flow path 42 Control valves 50, 51, 52 Supply source 60 Controller 70 Third flow path 100 Pressure application device P First port Q Second port R Third port

Claims

1. A pressure application method comprising generating a periodically fluctuating pressure in a sealed container by alternately repeating the following steps (a) and (b), wherein a biological sample is contained in the sealed container: (a) a step of adjusting the pressure in the sealed container by a first pressure adjustment mechanism; and (b) a step of adjusting the pressure in the sealed container by a second pressure adjustment mechanism.

2. The pressure application method according to claim 1, wherein the first pressure adjustment mechanism comprises a first regulator and / or a first control valve, and the second pressure adjustment mechanism comprises a second regulator and / or a second control valve.

3. The pressure application method according to claim 1, wherein one cycle is defined as step (a) followed by step (b), and the method comprises repeating 10 to 300 cycles per minute.

4. The pressure application method according to any one of claims 1 to 3, wherein step (a) is a step of pressurizing the inside of the sealed container in a first pressure adjustment mechanism.

5. The pressure application method according to claim 4, wherein the step (a) is a step of adjusting the pressure inside the sealed container so that a pressure of less than 200 kPa is applied inside the sealed container.

6. A pressure application method according to any one of claims 1 to 3, wherein step (b) is a step of reducing the pressure inside the sealed container in a second pressure adjustment mechanism.

7. The pressure application method according to claim 6, wherein the step (b) is a step of adjusting the pressure inside the sealed container so that the pressure reached in the step (a) is reduced by 1 kPa or more.

8. A pressure application method according to any one of claims 1 to 3, wherein the sealed container is equipped with a pressure sensor capable of measuring the pressure inside the sealed container in real time, and the first pressure adjustment mechanism and the second pressure adjustment mechanism are each controlled according to the pressure value detected by the pressure sensor.

9. A pressure application device connected to an airflow supply source and a sealed container that contains a biological sample, the pressure application device comprising a first pressure adjustment mechanism for adjusting the pressure inside the sealed container and a second pressure adjustment mechanism for adjusting the pressure inside the sealed container, the first pressure adjustment mechanism and the second pressure adjustment mechanism operating alternately to generate a periodically fluctuating pressure inside the sealed container.

10. The pressure application device according to claim 9, wherein the first pressure adjustment mechanism comprises a first regulator and / or a first control valve, and the second pressure adjustment mechanism comprises a second regulator and / or a second control valve.

11. A pressure application device as described in claim 9, wherein the first pressure adjustment mechanism and the second pressure adjustment mechanism operate in such a manner that 10 to 300 cycles are repeated per minute, where the process of adjusting pressure by the first pressure adjustment mechanism followed by adjusting pressure by the second pressure adjustment mechanism is defined as one cycle.

12. A pressure application device according to any one of claims 9 to 11, wherein the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in series or in parallel.

13. A pressure application device according to any one of claims 9 to 11, wherein the first pressure adjustment mechanism and the second pressure adjustment mechanism are connected in parallel.

14. The pressure application device according to claim 10, wherein the first pressure adjustment mechanism comprises a first control valve downstream of a first regulator, the second pressure adjustment mechanism comprises a second control valve downstream of a second regulator, and the first control valve and the second control valve switch between the connection between the sealed container and the first regulator and the connection between the sealed container and the second regulator.

15. A pressure application device according to claim 13, wherein the sealed container is connected downstream of the first pressure adjustment mechanism and the supply source is connected upstream of the first pressure adjustment mechanism, and the sealed container is connected downstream of the second pressure adjustment mechanism and the supply source or an exhaust mechanism is connected upstream of the second pressure adjustment mechanism.

16. A pressure application device according to any one of claims 9 to 11, wherein the first pressure adjustment mechanism applies pressure to the inside of the sealed container.

17. The pressure application device according to claim 16, wherein the first pressure adjustment mechanism adjusts the pressure inside the sealed container so that a pressure of less than 200 kPa is applied inside the sealed container.

18. A pressure application device according to any one of claims 9 to 11, wherein the second pressure adjustment mechanism reduces the pressure inside the sealed container.

19. A pressure application device according to claim 18, wherein the second pressure adjustment mechanism adjusts the pressure inside the sealed container so that the pressure applied by the first pressure adjustment mechanism is reduced by 1 kPa or more.

20. A pressure application device according to any one of claims 9 to 11, wherein the sealed container is equipped with a pressure sensor capable of measuring the pressure inside the sealed container in real time, and the operation of the first pressure adjustment mechanism and the second pressure adjustment mechanism are each controlled according to the pressure value detected by the pressure sensor.

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