JP7703310B2 - Cell culture method, culture medium production method, culture medium and culture device - Google Patents

Description

The present invention relates to a cell culture method, a culture medium production method, a culture medium, and a culture apparatus .

In the field of regenerative medicine, three-dimensional cells such as spheroids and organoids, which are produced by culturing human cells, are considered promising. The transplantation of three-dimensional cells into a designated site allows the cells to form a scaffold structure with each other, making apoptosis less likely to occur than with the transplantation of single cells or two-dimensional cells, and thus increasing the cell survival rate after transplantation.

In recent years, spheroids and organoids have been used in the process of culturing pluripotent ES cells and pluripotent stem cells such as ips cells in an undifferentiated state in three-dimensional suspension culture and inducing further differentiation. There is a demand for such spheroids and organoids to be produced reliably and in large quantities, with long-term stability and the desired size. Non-Patent Document 1 discloses the usefulness of cell spheroids and various methods for producing them.

Patent No. 6118544 Patent No. 4456176

"Development of cell spheroidization technology and its application to cell therapy" Drug Delivery System 28-1, P45-53, 2013

However, in the process of 3D culture, when 3D cells reach a certain size, there is a tendency for the cells inside to not receive enough oxygen, leading to necrosis. For this reason, although various culture methods have been devised, this necrosis has become a bottleneck, making it difficult to efficiently and mass-produce 3D cells. In addition, there has been a demand for more efficient and mass-produced 2D cells, in addition to 3D cells.

The present invention has been made to solve the above problems. Therefore, the purpose of the present invention is to provide a culture method, a method for producing a culture medium, a culture medium, and a culture device that can efficiently produce two-dimensional or three-dimensional cells in large quantities.

To this end, the present invention provides a culture method for culturing cells using a culture liquid containing ultra-fine bubbles generated by generating heat from a heating element to cause film boiling at the interface between the liquid and the heating element, wherein the liquid is sterilized purified water .

The present invention makes it possible to efficiently produce two-dimensional or three-dimensional cells in large quantities.

FIG. 1 illustrates an example of a UFB generating device. FIG. 2 is a schematic diagram of a pre-treatment unit. 2A and 2B are schematic diagrams of a dissolving unit and diagrams for explaining a dissolved state of a liquid. FIG. 2 is a schematic diagram of a T-UFB generation unit. FIG. 4 is a diagram for explaining details of a heating element. 1A and 1B are diagrams for explaining the state of film boiling in a heating element. FIG. 1 is a diagram showing how UFB is generated with the expansion of film boiling bubbles. FIG. 1 is a diagram showing how UFB is generated as film boiling bubbles shrink. FIG. 1 illustrates how reheating a liquid produces UFB. FIG. 1 is a diagram showing how UFB is generated by shock waves generated when bubbles generated by film boiling collapse. FIG. 2 is a diagram illustrating an example of the configuration of a post-processing unit. FIG. 1 is a diagram showing a first example of the first embodiment together with a comparative example. FIG. 2 is a schematic diagram for explaining the state of aggregation and dispersion of T-UFB. FIG. 1 is a schematic diagram for explaining the difference between a spheroid and an organoid. FIG. 11 is a diagram illustrating a second example of the first embodiment. FIG. 11 is a diagram illustrating a third example of the first embodiment. FIG. 13 is a schematic diagram of a culture device used in a second embodiment. FIG. 13 is a diagram showing a modified example of the culture device used in the second embodiment.

[First embodiment]
In recent years, technologies have been developed that utilize the properties of fine bubbles such as microbubbles with a diameter of micrometers and nanobubbles with a diameter of nanometers. In particular, the usefulness of ultra-fine bubbles (hereinafter, also referred to as "UFB") with a diameter of less than 1.0 μm has been confirmed in various fields.

Patent Document 1 discloses a microbubble generating device that generates microbubbles by ejecting a pressurized liquid in which gas is pressurized and dissolved from a pressure reducing nozzle. Patent Document 2 discloses a device that generates microbubbles by repeatedly splitting and merging a gas-mixed liquid using a mixing unit.

Such UFBs are expected to be used in the field of regenerative medicine. However, according to the inventors' investigations, it has been confirmed that UFBs produced by the methods disclosed in Patent Documents 1 and 2 are difficult to store for long periods of time.

<<Configuration of UFB generating device>>
1 is a diagram showing an example of a UFB generator applicable to this embodiment. The UFB generator 1 of this embodiment includes a pre-treatment unit 100, a dissolving unit 200, a T-UFB generation unit 300, a post-treatment unit 400, and a recovery unit 500. A liquid W such as tap water supplied to the pre-treatment unit 100 is subjected to a process specific to each unit in the above order, and is recovered as a T-UFB-containing liquid in the recovery unit 500. The function and configuration of each unit will be described below.

Figure 2 is a schematic diagram of the pretreatment unit 100. The pretreatment unit 100 of this embodiment performs degassing treatment on the supplied liquid W. The pretreatment unit 100 mainly includes a degassing container 101, a shower head 102, a vacuum pump 103, a liquid inlet 104, a liquid circulation path 105, and a liquid outlet 106. Liquid W, such as tap water, is supplied to the degassing container 101 from the liquid inlet 104 via a valve 109. At this time, the shower head 102 provided on the degassing container 101 atomizes the liquid W and sprays it into the degassing container 101. The shower head 102 is for promoting the vaporization of the liquid W, but a centrifuge or the like can also be used as a mechanism for producing the vaporization promotion effect.

After a certain amount of liquid W has been stored in the degassing container 101, the decompression pump 103 is operated with all valves closed to discharge the gas components that have already been vaporized and also promote the vaporization and discharge of the gas components dissolved in the liquid W. At this time, the internal pressure of the degassing container 101 may be reduced to approximately several hundred to several thousand Pa (1.0 Torr to 10.0 Torr) while checking the pressure gauge 108. Gases that are degassed by the degassing unit 100 include, for example, nitrogen, oxygen, argon, and carbon dioxide.

The above-described degassing process can be repeatedly performed on the same liquid W by using the liquid circulation path 105. Specifically, the valve 109 of the liquid inlet path 104 and the valve 110 of the liquid outlet path 106 are closed, and the valve 107 of the liquid circulation path 105 is opened, and the shower head 102 is operated. As a result, the liquid W stored in the degassing container 101 and degassed once is sprayed again into the degassing container 101 via the shower head 102. Furthermore, by operating the decompression pump 103, the vaporization process by the shower head 102 and the degassing process by the decompression pump 103 are repeatedly performed on the same liquid W. Then, each time the above-described repeated process is performed using the liquid circulation path 105, the gas components contained in the liquid W can be gradually reduced. When the liquid W degassed to the desired purity is obtained, the valve 110 is opened, and the liquid W is sent to the dissolving unit 200 via the liquid outlet path 106.

In FIG. 2, the degassing unit 100 is shown, which vaporizes the dissolved material by reducing the pressure of the gas section, but the method of degassing the dissolved liquid is not limited to this. For example, a heat-boiling method may be adopted in which the liquid W is boiled to vaporize the dissolved material, or a membrane degassing method may be adopted in which hollow fibers are used to increase the interface between the liquid and the gas. As a degassing module using hollow fibers, the SEPAREL series (manufactured by Dainippon Ink Co., Ltd.) is commercially available. This uses poly-4-methylpentene-1 (PMP) as the raw material for the hollow fiber membrane, and is mainly used for the purpose of degassing air bubbles from inks supplied to piezoelectric heads. Furthermore, two or more of the vacuum degassing method, the heat-boiling method, and the membrane degassing method may be used in combination.

By performing the above-described degassing process as a pretreatment, the purity and solubility of the desired gas in the liquid W can be increased in the dissolution process described below. Furthermore, the purity of the desired UFB contained in the liquid W can be increased in the T-UFB production unit described below. In other words, by providing the pretreatment unit 100 before the dissolution unit 200 and the T-UFB production unit 300, it becomes possible to efficiently produce a highly pure UFB-containing liquid.

3(a) and (b) are schematic diagrams of the dissolving unit 200 and diagrams for explaining the dissolved state of the liquid. The dissolving unit 200 is a unit that dissolves a desired gas in the liquid W supplied from the pre-processing unit 100. The dissolving unit 200 of this embodiment mainly includes a dissolving container 201, a rotating shaft 203 to which a rotating plate 202 is attached, a liquid inlet path 204, a gas inlet path 205, a liquid outlet path 206, and a pressure pump 207.

The liquid W supplied from the pretreatment unit 100 is supplied to the dissolution container 201 through the liquid inlet 204 and stored there. On the other hand, the gas G is supplied to the dissolution container 201 through the gas inlet 205.

When a predetermined amount of liquid W and gas G are stored in the dissolution vessel 201, the pressure pump 207 is operated to raise the internal pressure of the dissolution vessel 201 to about 0.5 MPa. A safety valve 208 is provided between the pressure pump 207 and the dissolution vessel 201. In addition, the gas G supplied to the dissolution vessel 201 is turned into bubbles by rotating the rotating plate 202 in the liquid via the rotating shaft 203, thereby increasing the contact area with the liquid W and promoting dissolution in the liquid W. This operation is continued until the solubility of the gas G reaches almost the maximum saturated solubility. At this time, a means for lowering the temperature of the liquid may be provided in order to dissolve as much gas as possible. In addition, in the case of a gas that is difficult to dissolve, it is also possible to raise the internal pressure of the dissolution vessel 201 to 0.5 MPa or more. In that case, it is necessary to optimize the material of the vessel for safety reasons.

When liquid W is obtained in which the components of gas G are dissolved at the desired concentration, the liquid W is discharged via the liquid outlet path 206 and supplied to the T-UFB generation unit 300. At this time, the back pressure valve 209 adjusts the flow pressure of the liquid W so that the pressure during supply does not become higher than necessary.

Figure 3(b) is a schematic diagram showing how the gas G mixed in the dissolution container 201 dissolves. The bubbles 2 containing the components of the gas G mixed in the liquid W dissolve from the part in contact with the liquid W. As a result, the bubbles 2 gradually shrink, and the gas-dissolved liquid 3 is present around the bubbles 2. Because of the buoyancy acting on the bubbles 2, the bubbles 2 move to a position away from the center of the gas-dissolved liquid 3, or separate from the gas-dissolved liquid 3 to become residual bubbles 4. In other words, the liquid W supplied to the T-UFB generation unit 300 via the liquid outlet 206 contains a mixture of the gas-dissolved liquid 3 surrounding the bubbles 2 and the gas-dissolved liquid 3 and the gas-dissolved liquid 3 separated from the bubbles 2.

In the figure, the gas-dissolved liquid 3 means "a region in the liquid W where the dissolved concentration of the mixed gas G is relatively high." The gas components actually dissolved in the liquid W have the highest concentration around the bubbles 2 or at the center of the region even when separated from the bubbles 2, and the concentration of the gas components decreases continuously the further away from that position. That is, in FIG. 3(b), the region of the gas-dissolved liquid 3 is surrounded by a dashed line for the purpose of explanation, but in reality, no such clear boundary exists. In addition, in this embodiment, it is acceptable for gas that is not completely dissolved to exist in the liquid in the form of bubbles.

Figure 4 is a schematic diagram of the T-UFB generation unit 300. The T-UFB generation unit 300 mainly comprises a chamber 301, a liquid inlet passage 302, and a liquid outlet passage 303, and the flow from the liquid inlet passage 302 through the chamber 301 toward the liquid outlet passage 303 is formed by a flow pump (not shown). As the flow pump, various pumps such as a diaphragm pump, a gear pump, and a screw pump can be used. The liquid W introduced from the liquid inlet passage 302 contains a gas-dissolved liquid 3 of the gas G mixed in by the dissolving unit 200.

An element substrate 12 with a heating element 10 is disposed on the bottom surface of the chamber 301. When a predetermined voltage pulse is applied to the heating element 10, bubbles 13 (hereinafter also referred to as film boiling bubbles 13) are generated in the area in contact with the heating element 10 due to film boiling. Then, as the film boiling bubbles 13 expand and contract, ultra-fine bubbles (UFB11) containing gas G are generated. As a result, a UFB-containing liquid W containing a large number of UFB11 is discharged from the liquid discharge path 303.

Figures 5(a) and (b) are diagrams showing the detailed structure of the heating element 10. Figure 5(a) shows a cross-sectional view of the vicinity of the heating element 10, and figure 5(b) shows a cross-sectional view of the element substrate 12 of a wider area including the heating element 10.

As shown in Fig. 5A, the element substrate 12 of this embodiment has a thermal oxide film 305 as a heat storage layer and an interlayer film 306 that also serves as a heat storage layer laminated on the surface of a silicon substrate 304. A SiO2 film or a SiN film can be used as the interlayer film 306. A resistive layer 307 is formed on the surface of the interlayer film 306, and wiring 308 is partially formed on the surface of the resistive layer 307. The wiring 308 can be an Al alloy wiring such as Al, Al-Si, or Al-Cu. A protective layer 309 made of a _SiO2 film or _{a Si3N4} film is formed on the surfaces of the wiring 308, the resistive layer 307, and the interlayer film 306.

On the surface of the protective layer 309, a cavitation-resistant film 310 is formed in the area corresponding to the heat application portion 311 that will eventually become the heating element 10, and around the area, to protect the protective layer 309 from chemical and physical shocks associated with the heat generation of the resistance layer 307. On the surface of the resistance layer 307, the area where the wiring 308 is not formed is the heat application portion 311 where the resistance layer 307 generates heat. The heat-generating portion of the resistance layer 307 where the wiring 308 is not formed functions as the heating element (heater) 10. In this way, the layers in the element substrate 12 are formed sequentially on the surface of the silicon substrate 304 by semiconductor manufacturing technology, and the silicon substrate 304 is provided with the heat application portion 311.

The configuration shown in the figure is an example, and various other configurations are applicable. For example, a configuration in which the stacking order of the resistive layer 307 and the wiring 308 is reversed, and a configuration in which an electrode is connected to the underside of the resistive layer 307 (a so-called plug electrode configuration) are applicable. In other words, as described below, any configuration is acceptable as long as it can heat the liquid with the thermal action portion 311 and cause film boiling in the liquid.

Figure 5 (b) is an example of a cross-sectional view of a region including a circuit connected to wiring 308 in the element substrate 12. An N-type well region 322 and a P-type well region 323 are partially provided on the surface layer of the silicon substrate 304, which is a P-type conductor. A P-MOS 320 is formed in the N-type well region 322 and an N-MOS 321 is formed in the P-type well region 323 by the introduction and diffusion of impurities such as ion implantation by a general MOS process.

The P-MOS 320 is composed of a source region 325 and a drain region 326 formed by partially doping N-type or P-type impurities into the surface layer of the N-type well region 322, and a gate wiring 335. The gate wiring 335 is deposited on the surface of the N-type well region 322, except for the source region 325 and the drain region 326, via a gate insulating film 328 several hundred angstroms thick.

The N-MOS 321 is composed of a source region 325 and a drain region 326 formed by partially doping N-type or P-type impurities into the surface layer of the P-type well region 323, and a gate wiring 335. The gate wiring 335 is deposited on the surface of the part of the P-type well region 323 excluding the source region 325 and the drain region 326, via a gate insulating film 328 having a thickness of several hundred Å. The gate wiring 335 is made of polysilicon having a thickness of 3000 Å to 5000 Å deposited by the CVD method. The P-MOS 320 and N-MOS 321 form the C-MOS logic.

In the P-type well region 323, an N-MOS transistor 330 for driving an electrothermal conversion element (heat generating resistor element) is formed in a portion different from the N-MOS 321. The N-MOS transistor 330 is composed of a source region 332 and a drain region 331 partially formed on the surface layer of the P-type well region 323 by processes such as the introduction and diffusion of impurities, and a gate wiring 333. The gate wiring 333 is deposited on the surface of the P-type well region 323 in the portion excluding the source region 332 and the drain region 331 via a gate insulating film 328.

In this example, an N-MOS transistor 330 is used as the driving transistor for the electrothermal conversion element. However, the driving transistor is not limited to the N-MOS transistor 430, and may be any transistor capable of driving multiple electrothermal conversion elements individually and capable of obtaining the fine structure described above. Also, in this example, the electrothermal conversion element and its driving transistor are formed on the same substrate, but they may be formed on separate substrates.

Between each element, such as between P-MOS 320 and N-MOS 321, and between N-MOS 321 and N-MOS transistor 330, oxide film isolation regions 324 are formed by field oxidation with a thickness of 5000 Å to 10000 Å. Each element is isolated by this oxide film isolation region 324. In the oxide film isolation region 324, the part corresponding to the thermal action portion 311 functions as the first heat storage layer 334 on the silicon substrate 304.

An interlayer insulating film 336 made of a PSG film or BPSG film having a thickness of about 7000 Å is formed on the surface of each element of the P-MOS 320, N-MOS 321, and N-MOS transistor 330 by the CVD method. After the interlayer insulating film 336 is flattened by heat treatment, an Al electrode 337, which becomes the first wiring layer, is formed through a contact hole penetrating the interlayer insulating film 336 and the gate insulating film 328. An interlayer insulating film 338 made of a SiO2 film having a thickness of 10000 Å to 15000 Å is formed on the surface of the interlayer insulating film 336 and the Al electrode 337 by the plasma CVD method. On the surface of the interlayer insulating film 338, a resistive layer 307 made of a TaSiN film having a thickness of about 500 Å is formed by the co-sputtering method in the portion corresponding to the heat application portion 311 and the N-MOS transistor 330. The resistive layer 307 is electrically connected to the Al electrode 337 near the drain region 331 through a through hole formed in the interlayer insulating film 338. On the surface of the resistive layer 307, an Al wiring 308 is formed as a second wiring layer that serves as wiring to each electrothermal conversion element. The wiring 308, the resistive layer 307, and the protective layer 309 on the surface of the interlayer insulating film 338 are made of a SiN film with a thickness of 3000 Å formed by a plasma CVD method. The cavitation-resistant film 310 deposited on the surface of the protective layer 309 is made of at least one metal selected from Ta, Fe, Ni, Cr, Ge, Ru, Zr, Ir, etc., and is made of a thin film with a thickness of about 2000 Å. As the resistive layer 307, various materials can be applied as long as they can cause film boiling in liquid, such as TaN0.8, CrSiN, TaAl, WSiN, etc. other than the above-mentioned TaSiN.

Figures 6(a) and (b) are diagrams showing the state of film boiling when a predetermined voltage pulse is applied to the heating element 10. Here, the case where film boiling is caused under atmospheric pressure is shown. In Figure 6(a), the horizontal axis indicates time. The vertical axis of the lower graph indicates the voltage applied to the heating element 10, and the vertical axis of the upper graph indicates the volume and internal pressure of the film boiling bubbles 13 generated by film boiling. Meanwhile, Figure 6(b) shows the state of the film boiling bubbles 13 in correspondence with timings 1 to 3 shown in Figure 6(a). Each state will be explained below along the time axis.

Before a voltage is applied to the heating element 10, the pressure inside the chamber 301 is kept at approximately atmospheric pressure. When a voltage is applied to the heating element 10, film boiling occurs in the liquid in contact with the heating element 10, and the generated bubbles (hereinafter referred to as film boiling bubbles 13) expand due to the high pressure acting from the inside (timing 1). The bubbling pressure at this time is considered to be approximately 8 to 10 MPa, which is close to the saturated vapor pressure of water.

The voltage application time (pulse width) is approximately 0.5 to 10.0 usec, but even after the voltage is no longer applied, the film boiling bubble 13 continues to expand due to the inertia of the pressure obtained at timing 1. However, inside the film boiling bubble 13, the negative pressure generated by the expansion gradually increases, acting in a direction that causes the film boiling bubble 13 to contract. Eventually, at timing 2 when the force of inertia and the negative pressure are balanced, the volume of the film boiling bubble 13 reaches a maximum, after which it rapidly contracts due to the negative pressure.

When the film boiling bubbles 13 disappear, they do not disappear over the entire surface of the heating element 10, but in one or more extremely small areas. Therefore, in the heating element 10, a force greater than that generated when the film boiling bubbles 13 disappear is generated in the extremely small area where the film boiling bubbles 13 disappear (timing 3) than when the bubbles bubble at timing 1.

The generation, expansion, contraction and disappearance of the film boiling bubbles 13 as described above are repeated each time a voltage pulse is applied to the heating element 10, and each time a new UFB 11 is generated.

Next, we will explain in more detail how UFB11 is generated during the processes of generation, expansion, contraction, and disappearance of film boiling bubbles 13.

Figures 7(a) to (d) are diagrams showing how UFB11 is generated with the generation and expansion of film boiling bubbles 13. Figure 7(a) shows the state before a voltage pulse is applied to the heating element 10. Liquid W mixed with gas-dissolved liquid 3 flows inside the chamber 301.

Figure 7(b) shows how a voltage is applied to the heating element 10, and film boiling bubbles 13 are uniformly generated over almost the entire area of the heating element 10 in contact with the liquid W. When a voltage is applied, the surface temperature of the heating element 10 rises rapidly at a rate of 10°C/μsec or more, and when it reaches approximately 300°C, film boiling occurs and film boiling bubbles 13 are generated.

The surface temperature of the heating element 10 continues to rise to about 600-800°C while the pulses are being applied, and the liquid around the film boiling bubble 13 is also rapidly heated. In the figure, the region of liquid located around the film boiling bubble 13 and rapidly heated is shown as the unfoamed high temperature region 14. The gas-dissolved liquid 3 contained in the unfoamed high temperature region 14 exceeds the thermal solubility limit and precipitates to become UFB. The precipitated bubbles have a diameter of about 10 nm to 100 nm and have high gas-liquid interface energy. Therefore, they do not disappear in a short time, but float independently in the liquid W. In this embodiment, the bubbles generated by thermal action when the film boiling bubble 13 expands are called the first UFB 11A.

Figure 7 (c) shows the process of the film boiling bubble 13 expanding. Even after the application of the voltage pulse to the heating element 10 is terminated, the film boiling bubble 13 continues to expand due to the inertia of the force obtained when it was generated, and the unfoamed high-temperature region 14 also moves and diffuses due to inertia. In other words, in the process of the film boiling bubble 13 expanding, the gas-dissolved liquid 3 contained in the unfoamed high-temperature region 14 precipitates as new bubbles, becoming the first UFB 11A.

Figure 7 (d) shows the state when the film boiling bubble 13 has reached its maximum volume. The film boiling bubble 13 expands due to inertia, but as it expands, the negative pressure inside the film boiling bubble 13 gradually increases, acting as a negative pressure that tries to contract the film boiling bubble 13. Then, when this negative pressure balances with the force of inertia, the volume of the film boiling bubble 13 reaches its maximum, and then it begins to contract.

Figures 8(a) to (c) are diagrams showing how UFB 11 is generated as film boiling bubble 13 contracts. Figure 8(a) shows the state in which film boiling bubble 13 has started to contract. Even after film boiling bubble 13 starts to contract, the surrounding liquid W still has an inertial force in the direction of expansion. Therefore, the extreme periphery of film boiling bubble 13 is subjected to an inertial force acting in a direction away from heating element 10 and a force acting toward heating element 10 as film boiling bubble 13 contracts, resulting in a reduced pressure region. In the figure, such a region is shown as an unfoamed negative pressure region 15.

The gas-dissolved liquid 3 contained in the unfoamed negative pressure region 15 exceeds the pressure dissolution limit and precipitates as bubbles. The precipitated bubbles have a diameter of about 100 nm, and do not disappear within a short time thereafter, but float independently within the liquid W. In this embodiment, the bubbles precipitated in this way due to the pressure action when the film boiling bubbles 13 contract are referred to as second UFB11B.

Figure 8 (b) shows the process of the film boiling bubble 13 shrinking. The speed at which the film boiling bubble 13 shrinks is accelerated by the negative pressure, and the unfoamed negative pressure region 15 also moves as the film boiling bubble 13 shrinks. That is, in the process of the film boiling bubble 13 shrinking, the gas-dissolved liquid 3 at the point where the unfoamed negative pressure region 15 passes through is precipitated one after another, becoming the second UFB 11B.

Figure 8 (c) shows the state just before the film boiling bubbles 13 disappear. Due to the accelerated contraction of the film boiling bubbles 13, the movement speed of the surrounding liquid W also increases, but pressure loss occurs due to the flow path resistance in the chamber 301. As a result, the area occupied by the unfoamed negative pressure region 15 becomes even larger, and a large number of second UFBs 11B are generated.

Figures 9(a) to (c) are diagrams showing how UFB is generated by reheating the liquid W when the film boiling bubble 13 contracts. Figure 9(a) shows the state in which the surface of the heating element 10 is covered with the contracting film boiling bubble 13.

Figure 9(b) shows a state in which the film boiling bubble 13 has contracted and part of the surface of the heating element 10 has come into contact with the liquid W. At this time, there is still enough heat remaining on the surface of the heating element 10 that film boiling will not occur even if the liquid W comes into contact with it. In the figure, the region of the liquid that is heated by contact with the surface of the heating element 10 is shown as the unfoamed reheated region 16. Although film boiling will not occur, the gas-dissolved liquid 3 contained in the unfoamed reheated region 16 will precipitate beyond its thermal solubility limit. In this embodiment, the bubbles generated by reheating the liquid W when the film boiling bubble 13 contracts are referred to as the third UFB 11C.

Figure 9 (c) shows a state in which the film boiling bubble 13 has further contracted. As the film boiling bubble 13 becomes smaller, the area of the heating element 10 in contact with the liquid W becomes larger, so the third UFB 11C is generated until the film boiling bubble 13 disappears.

Figures 10(a) and (b) show how UFB is generated by the impact (a type of cavitation) that occurs when film boiling bubbles 13 generated by film boiling collapse. Figure 10(a) shows the state just before the film boiling bubble 13 disappears. The film boiling bubble 13 contracts rapidly due to the internal negative pressure, and is surrounded by an unfoamed negative pressure region 15.

Figure 10(b) shows the state immediately after the film boiling bubble 13 disappears at point P. When the film boiling bubble 13 disappears, the impact causes an acoustic wave to spread concentrically starting from point P. An acoustic wave is a general term for elastic waves that propagate regardless of whether they are gas, liquid, or solid, and in this embodiment, they are propagated alternately between the dense and dense liquid W, i.e., the high pressure surface 17A and the low pressure surface 17B of the liquid W.

In this case, the gas-dissolved liquid 3 contained in the unfoamed negative pressure region 15 is resonated by the shock waves generated when the film boiling bubbles 13 collapse, and undergoes a phase transition beyond the pressure dissolution limit when the low pressure surface 17B passes. In other words, at the same time as the film boiling bubbles 13 collapse, a large number of bubbles precipitate in the unfoamed negative pressure region 15. In this embodiment, the bubbles generated by the shock waves generated when the film boiling bubbles 13 collapse are referred to as the fourth UFB 11D.

The fourth UFB 11D, which is generated by the shock wave generated when the film boiling bubble 13 collapses, appears suddenly in an extremely narrow thin-film region in an extremely short time (less than 1 μS). Its diameter is much smaller than the first to third UFBs, and its gas-liquid interfacial energy is higher than the first to third UFBs. For this reason, the fourth UFB 11D is thought to have different properties from the first to third UFBs 11A to 11C, and to produce different effects.

In addition, the fourth UFB11D is generated uniformly throughout the concentric spherical region through which the shock wave propagates, and therefore exists uniformly within the chamber 301 from the time it is generated. At the time the fourth UFB11D is generated, a large number of the first to third UFBs are already present, but the presence of these first to third UFBs does not significantly affect the generation of the fourth UFB11D. Furthermore, the generation of the fourth UFB11D does not cause the first to third UFBs to disappear.

As described above, UFB 11 is generated in multiple stages from when film boiling bubbles 13 are generated by heat generation of the heating element 10 until they disappear. In the above example, an example up to when the film boiling bubbles 13 disappear is shown, but this is not the only way to generate UFB. For example, by communicating with the atmosphere before the generated film boiling bubbles 13 disappear, it is possible to generate UFB even if the film boiling bubbles 13 are not consumed.

Next, we will explain the residual characteristics of UFB. The higher the temperature of the liquid, the lower the solubility of the gas components, and the lower the temperature, the higher the solubility of the gas components. In other words, the higher the temperature of the liquid, the more the phase transition of the dissolved gas components is promoted, making it easier for UFB to be generated. The temperature of the liquid and the solubility of gas are inversely proportional, and as the temperature of the liquid rises, gas that exceeds its saturated solubility becomes bubbles and precipitates in the liquid.

For this reason, when the temperature of the liquid rises suddenly from room temperature, the solubility characteristics drop dramatically and UFBs begin to be produced. And the higher the temperature, the lower the thermal solubility characteristics become, resulting in a situation where a lot of UFBs are produced.

Conversely, when the temperature of the liquid drops from room temperature, the solubility of the gas increases, making the generated UFB more likely to liquefy. However, this temperature is well below room temperature. Furthermore, even if the temperature of the liquid drops, once generated UFB has high internal pressure and high gas-liquid interfacial energy, so it is extremely unlikely that high pressure will act on the gas-liquid interface to destroy it. In other words, once generated, UFB will not disappear easily as long as the liquid is stored at room temperature and pressure.

In this embodiment, the first UFB 11A described in Figures 7(a) to (c) and the third UFB 11C described in Figures 9(a) to (c) can be said to be UFBs generated by utilizing the thermal dissolution properties of such gases.

On the other hand, in terms of the relationship between liquid pressure and solubility characteristics, the higher the liquid pressure, the higher the gas solubility characteristics, and the lower the pressure, the lower the solubility characteristics. In other words, the lower the liquid pressure, the more the gas dissolved in the liquid is promoted to undergo a phase transition to gas, making it easier for UFB to be generated. When the liquid pressure is reduced from normal pressure, the solubility characteristics drop sharply and UFB begins to be generated. And the lower the pressure, the lower the pressure solubility characteristics become, resulting in a situation where a lot of UFB is generated.

Conversely, when the pressure of the liquid increases from normal pressure, the solubility properties of the gas increase, making the generated UFB more likely to liquefy. However, this pressure is much higher than atmospheric pressure, and even if the pressure of the liquid increases, the UFB once generated has high internal pressure and high gas-liquid interfacial energy, so it is highly unlikely that high pressure will act to destroy the gas-liquid interface. In other words, once generated, UFB will not easily disappear as long as the liquid is stored at room temperature and pressure.

In this embodiment, the second UFB 11B described in Figures 8(a) to (c) and the fourth UFB 11D described in Figures 10(a) to (c) can be said to be UFBs generated by utilizing the pressure dissolution characteristics of such gases.

Above, the first to fourth UFBs, which are generated by different factors, have been explained individually, but the above-mentioned generation factors occur simultaneously in association with the phenomenon of film boiling. For this reason, at least two or more types of UFBs among the first to fourth UFBs may be generated simultaneously, and these generation factors may cooperate with each other to generate UFBs. However, all generation factors have in common that they are caused by the film boiling phenomenon. Hereinafter, in this specification, such a method of generating UFBs using film boiling caused by sudden heat generation will be referred to as a T-UFB (Thermal-Ultra Fine Bubble) generation method. In addition, UFB generated by the T-UFB generation method will be referred to as T-UFB, and a liquid containing T-UFB generated by the T-UFB generation method will be referred to as a T-UFB-containing liquid.

Most of the bubbles generated by the T-UFB generation method are 1.0 um or less, and millibubbles and microbubbles are difficult to generate. In other words, the T-UFB generation method efficiently generates only UFB. In addition, the T-UFB generated by the T-UFB generation method has a higher gas-liquid interfacial energy than the UFB generated by the conventional method, and does not disappear easily as long as it is stored at room temperature and normal pressure. Furthermore, even if new T-UFB is generated by new film boiling, the previously generated T-UFB does not disappear due to the impact. In other words, it can be said that the number and concentration of T-UFB contained in the T-UFB-containing liquid have hysteresis characteristics with respect to the number of times film boiling occurs in the T-UFB-containing liquid. In other words, the concentration of T-UFB contained in the T-UFB-containing liquid can be adjusted by controlling the number of heating elements arranged in the T-UFB generation unit 300 and the number of times voltage pulses are applied to the heating elements.

Referring again to FIG. 1, once the T-UFB-containing solution W having the desired UFB concentration is produced in the T-UFB production unit 300, the UFB-containing solution W is supplied to the post-treatment unit 400.

Figures 11(a) to (c) are diagrams showing an example of the configuration of the post-treatment unit 400 of this embodiment. The post-treatment unit 400 of this embodiment removes impurities contained in the UFB-containing liquid W in stages, in the order of inorganic ions, organic matter, and insoluble solids.

11A shows a first post-treatment mechanism 410 for removing inorganic ions. The first post-treatment mechanism 410 includes an exchange container 411, a cation exchange resin 412, a liquid introduction passage 413, a water collection pipe 414, and a liquid discharge passage 415. The exchange container 411 contains the cation exchange resin 412. The UFB-containing liquid W generated in the T-UFB generation unit 300 is injected into the exchange container 411 via the liquid introduction passage 413 and absorbed into the cation exchange resin 412, where cations as impurities are removed. Such impurities include metal materials peeled off from the element substrate 12 of the T-UFB generation unit 300, such as SiO ₂ , SiN, SiC, Ta, Al ₂ O ₃ , Ta ₂ O ₅ , and Ir.

The cation exchange resin 412 is a synthetic resin in which functional groups (ion exchange groups) have been introduced into a polymer matrix having a three-dimensional mesh structure, and the synthetic resin has spherical particles of about 0.4 to 0.7 mm. The polymer matrix is generally a copolymer of styrene-divinylbenzene, and the functional groups can be, for example, methacrylic acid and acrylic acid. However, the above material is only an example. As long as the desired inorganic ions can be effectively removed, the above material can be changed in various ways. The UFB-containing liquid W that has been absorbed by the cation exchange resin 412 and from which the inorganic ions have been removed is collected by the water collection tube 414 and sent to the next process via the liquid outlet 415.

11B shows a second post-treatment mechanism 420 for removing organic matter. The second post-treatment mechanism 420 includes a storage container 421, a filter 422, a vacuum pump 423, a valve 424, a liquid inlet passage 425, a liquid outlet passage 426, and an air suction passage 427. The inside of the storage container 421 is divided into two regions, upper and lower, by the filter 422. The liquid inlet passage 425 is connected to the upper region of the two regions, and the air suction passage 427 and the liquid outlet passage 426 are connected to the lower region. When the vacuum pump 423 is driven with the valve 424 closed, the air in the storage container 421 is discharged through the air suction passage 427, the inside of the storage container 421 becomes negative pressure, and the UFB-containing liquid W is introduced from the liquid inlet passage 425. Then, the UFB-containing liquid W in a state in which impurities have been removed by the filter 422 is stored in the storage container 421.

Impurities removed by the filtration filter 422 include organic materials that may be mixed in the tubes and each unit, such as organic compounds containing silicon, siloxane, and epoxy. Filter membranes that can be used for the filtration filter 422 include sub-μm mesh filters that can remove even bacteria, and nm mesh filters that can remove even viruses.

After a certain amount of UFB-containing liquid W is stored in the storage container 421, the vacuum pump 423 is stopped and the valve 424 is opened, and the T-UFB-containing liquid in the storage container 421 is sent to the next process via the liquid outlet path 426. Note that, although vacuum filtration is used here as a method for removing organic impurities, other filtration methods using a filter, such as gravity filtration or pressure filtration, can also be used.

Figure 11 (c) shows a third post-treatment mechanism 430 for removing insoluble solids. The third post-treatment mechanism 430 includes a settling vessel 431, a liquid inlet passage 432, a valve 433, and a liquid outlet passage 434.

First, with the valve 433 closed, a predetermined amount of UFB-containing liquid W is stored in the settling container 431 from the liquid inlet path 432 and left for a while. During this time, solid matter contained in the UFB-containing liquid W settles to the bottom of the settling container 431 due to gravity. Furthermore, among the bubbles contained in the UFB-containing liquid, relatively large bubbles such as microbubbles also rise to the liquid surface due to buoyancy and are removed from the UFB-containing liquid. When the valve 433 is opened after a sufficient amount of time has passed, the UFB-containing liquid W from which solid matter and large bubbles have been removed is sent to the recovery unit 500 via the liquid outlet path 434.

Refer to FIG. 1 again. The T-UFB-containing solution W from which impurities have been removed in the post-treatment unit 400 may be sent directly to the recovery unit 500, or may be returned to the dissolution unit 200. In the latter case, the gas dissolved concentration of the T-UFB-containing solution W, which has decreased due to the generation of T-UFB, can be replenished to a saturated state in the dissolution unit 200. If new T-UFB is then generated in the T-UFB generation unit 300, the UFB content concentration of the T-UFB-containing solution can be further increased based on the above-mentioned characteristics. In other words, the UFB content concentration can be increased by the number of times the solution circulates through the dissolution unit 200, the T-UFB generation unit 300, and the post-treatment unit 400, and after the desired UFB content concentration is obtained, the UFB-containing solution W can be sent to the recovery unit 500.

Here, the effect of returning the generated T-UFB-containing liquid W back to the dissolving unit 200 will be briefly described in accordance with the details of the verification specifically conducted by the present inventors. First, in the T-UFB generation unit 300, 10,000 heating elements 10 were arranged on the element substrate 12. Industrial pure water was used as the liquid W, and was caused to flow at a flow rate of 1.0 liter/hour in the chamber 301 of the T-UFB generation unit 300. In this state, a voltage pulse with a voltage of 24 V and a pulse width of 1.0 μs was applied to each heating element at a drive frequency of 10 kHz .

When the generated T-UFB-containing liquid W was not returned to the dissolution unit 200 but was recovered by the recovery unit 500, i.e., when the number of circulations was set to one, 3.6 billion UFBs were confirmed per 1.0 mL in the T-UFB-containing liquid W recovered by the recovery unit 500. On the other hand, when the operation of returning the T-UFB-containing liquid W to the dissolution unit 200 was performed 10 times, i.e., when the number of circulations was set to 10, 36 billion UFBs were confirmed per 1.0 mL in the T-UFB-containing liquid W recovered by the recovery unit 500. In other words, it was confirmed that the UFB content concentration increases in proportion to the number of circulations. The number density of UFBs as described above was obtained by counting UFBs 41 with a diameter of less than 1.0 μm contained in a specified volume of UFB-containing liquid W using a measuring device (model number SALD-7500) manufactured by Shimadzu Corporation.

The recovery unit 500 recovers and stores the UFB-containing liquid W sent from the post-treatment unit 400. The T-UFB-containing liquid recovered by the recovery unit 500 becomes a highly pure UFB-containing liquid from which various impurities have been removed.

In the recovery unit 500, several stages of filtering may be performed to classify the UFB-containing liquid W according to the size of the T-UFB. In addition, since the T-UFB-containing liquid W obtained by the T-UFB production method is expected to be at a temperature higher than room temperature, the recovery unit 500 may be provided with a cooling means. Such a cooling means may be provided as part of the post-processing unit 400.

The above is an overview of the UFB generating device 1, but the multiple units shown in the figure can of course be modified, and it is not necessary to prepare all of them. Depending on the type of liquid W and gas G used, and the intended use of the T-UFB-containing liquid to be generated, some of the above-mentioned units may be omitted, or further units may be added in addition to the above-mentioned units.

For example, if the gas contained in the UFB is atmospheric air, the degassing unit 100 and the dissolving unit 200 can be omitted. Conversely, if it is desired to contain multiple types of gas in the UFB, a dissolving unit 200 may be further added.

The functions of several units shown in FIG. 1 can also be integrated into one unit. For example, by arranging a heating element 10 in the dissolving vessel 201 shown in FIG. 3(a) and (b), the dissolving unit 200 and the T-UFB production unit 300 can be integrated. Specifically, an electrode-type T-UFB module is built into the gas dissolving vessel (high-pressure chamber), and multiple heaters arranged in the module are driven to generate film boiling. In this way, it is possible to dissolve gas in one unit and generate T-UFB containing the gas. In this case, by arranging the T-UFB module at the bottom of the gas dissolving vessel, the heat generated by the heater causes Marangori convection, and the liquid in the vessel can be stirred to a certain extent without providing a circulation/stirring means.

In addition, the removal unit for removing impurities as shown in Figures 11(a) to (c) may be provided as part of a pretreatment unit upstream of the T-UFB generation unit 300, or may be provided both upstream and downstream. When the liquid supplied to the UFB generation device is tap water, rainwater, or contaminated water, the liquid may contain organic or inorganic impurities. If liquid W containing such impurities is supplied to the T-UFB generation unit 300, there is a risk that the heating element 10 may be altered or salting out may occur. By providing a mechanism as shown in Figures 11(a) to (c) upstream of the T-UFB generation unit 300, it is possible to remove the above-mentioned impurities in advance and more efficiently generate a UFB-containing liquid with higher purity.

In particular, when the impurity removal unit using ion exchange resin as shown in FIG. 11(a) is provided in the pretreatment unit, the placement of an anion exchange resin contributes to the efficient generation of T-UFB water. This is because it has been confirmed that the ultra-fine bubbles generated by the T-UFB generation unit 300 have a negative charge. Therefore, by removing impurities with the same negative charge in the pretreatment unit, it is possible to generate T-UFB water with high purity. As the anion exchange resin used here, both a strongly basic anion exchange resin with a quaternary ammonium group and a weakly basic anion exchange resin with a primary to tertiary amine group are suitable. Which is optimal depends on the type of liquid used. Usually, when tap water or pure water is used as the liquid, the latter weakly basic anion exchange resin alone can fully fulfill the function.

<<Liquids and gases that can be used for T-UFB-containing liquid>>
Here, the liquid W that can be used to generate the T-UFB-containing liquid will be described. Examples of the liquid W that can be used in this embodiment include pure water, ion-exchanged water, distilled water, physiologically active water, magnetically active water, lotion, tap water, seawater, river water, sewage water, lake water, groundwater, and rainwater. Mixed liquids containing these liquids can also be used. Mixed solvents of water and water-soluble organic solvents can also be used. The water-soluble organic solvent to be used in a mixture with water is not particularly limited, but specific examples include the following. Alkyl alcohols having 1 to 4 carbon atoms, such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, and tert-butyl alcohol. Amides, such as N-methyl-2-pyrrolidone, 2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, N,N-dimethylformamide, and N,N-dimethylacetamide. Ketones or ketoalcohols, such as acetone and diacetone alcohol. Cyclic ethers, such as tetrahydrofuran and dioxane. Ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol. Glycols such as 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol, 1,2-hexanediol, 1,6-hexanediol, 3-methyl-1,5-pentanediol, diethylene glycol, triethylene glycol, and thiodiglycol. Lower alkyl ethers of polyhydric alcohols such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, triethylene glycol monomethyl ether, triethylene glycol monoethyl ether, and triethylene glycol monobutyl ether. Polyalkylene glycols such as polyethylene glycol and polypropylene glycol. Triols such as glycerin, 1,2,6-hexanetriol, and trimethylolpropane. These water-soluble organic solvents may be used alone or in combination of two or more.

Examples of gas components that can be introduced by the dissolution unit 200 include hydrogen, helium, oxygen, nitrogen, methane, fluorine, neon, carbon dioxide, ozone, argon, chlorine, ethane, propane, and air. Mixtures of some of the above may also be used. Furthermore, the dissolution unit 200 does not necessarily have to dissolve substances in a gaseous state, and may melt liquids or solids composed of desired components into liquid W. Dissolution in this case may be natural dissolution, dissolution by application of pressure, or dissolution involving hydration, ionization, or chemical reaction due to ionization.

<<Specific example when ozone gas is used>>
Here, as a specific example, a case where ozone gas is used as the gas component will be described. First, methods for generating ozone gas include a discharge method, an electrolysis method, and an ultraviolet lamp method. Below, these will be described in order.

(1) Electric discharge method There are two types of electric discharge method: silent discharge method and surface discharge method. In the silent discharge method, a high AC voltage is applied while oxygen-containing gas is flowing between a pair of electrodes arranged in the shape of parallel plates or coaxial cylinders. This causes a discharge in the oxygen-containing gas, generating ozone gas. One or both of the surfaces of the pair of electrodes must be covered with a dielectric material such as glass. An electric discharge occurs in the gas (air or oxygen) as the charge on the surface of this dielectric material alternates between positive and negative.

On the other hand, in the surface discharge method, the surface of a flat electrode is covered with a dielectric material such as ceramics, a linear electrode is placed on the surface of the dielectric, and a high AC voltage is applied between the flat electrode and the linear electrode. This causes a discharge on the surface of the dielectric, generating ozone gas.

(2) Electrolysis method: A pair of electrodes sandwiching an electrolyte membrane is placed in water, and a DC voltage is applied between the two electrodes. This causes electrolysis of water, which generates ozone gas along with oxygen on the oxygen generating side. Ozone generators in practical use include those that use porous titanium with a platinum catalyst layer on the cathode, porous titanium with a lead dioxide catalyst layer on the anode, and those that use a perfluorosulfonic acid cation exchange membrane as the electrolyte membrane. This device can generate high-concentration ozone of 20% by weight or more.

(3) Ultraviolet Lamp Method Using the same principle as the creation of the Earth's ozone layer, ozone gas is generated by exposing the air to ultraviolet light. Mercury lamps are usually used as ultraviolet lamps.

When ozone gas is used as the gas component, an ozone gas generation unit employing the methods (1) to (3) described above may be further added to the UFB generation device 1 in FIG. 1.

Next, a method for dissolving the generated ozone gas will be described. In addition to the pressurized dissolution method shown in Figures 3(a) and (b), other methods suitable for dissolving ozone gas in liquid W include the "bubble dissolution method," the "diaphragm dissolution method," and the "packed bed dissolution method." Below, these three methods will be described in order while comparing them.

(i) Bubble dissolution method This is a method in which ozone gas is mixed as bubbles in the liquid W and dissolved while flowing together with the liquid W. For example, there is a bubbling method in which ozone gas is blown in from the bottom of a container in which the liquid W is stored, an ejector method in which a narrow section is provided in part of the piping through which the liquid W flows and ozone gas is blown into the narrow section, and a method in which the liquid W and ozone gas are stirred with a pump. This is a relatively compact dissolution method and is also useful in water purification plants, etc.

(ii) Diaphragm Dissolution Method In this method, liquid W is made to flow through a porous Teflon membrane, and ozone gas is made to flow on the outside of the membrane, so that the ozone gas is absorbed and dissolved in the liquid W.

(iii) Packed Bed Dissolution Method In this method, liquid W is made to flow from the top of a packed bed and ozone gas is made to flow from the bottom, thereby causing the ozone gas and the liquid to flow in countercurrents, and the ozone gas is dissolved in the liquid W within the packed bed.

When adopting any of the methods (i) to (iii) described above, the dissolving unit 200 of the UFB generator 1 can be changed from the configuration shown in Figures 3(a) and (b) to one that adopts any of the methods (i) to (iii).

In particular, because of its high toxicity, highly pure ozone gas is restricted from being purchased or used in gas cylinders unless a special environment is prepared. For this reason, it is difficult to generate ozone micro- and ultra-fine bubbles using conventional methods for generating micro- and ultra-fine bubbles by introducing gas (for example, the Venturi method, the swirling flow method, the pressurized dissolution method, etc.).

On the other hand, as a method for producing ozone-dissolved water, a method in which ozone is generated from oxygen supplied by the aforementioned discharge method, electrolysis method, or ultraviolet lamp method, and simultaneously dissolved in water, etc., is useful from the standpoint of safety and ease.

However, while it is possible to generate ozone ultra-fine bubbles using water containing dissolved ozone using methods such as cavitation, there are still issues with this method, such as the large size of the equipment and the inability to increase the concentration of ozone ultra-fine bubbles.

In contrast, the T-UFB generation method is superior to other methods in that it can generate high-concentration ozone ultrafine bubbles from ozone-dissolved water using a relatively small device.

<<Effects of the T-UFB generation method>>
Next, the features and effects of the T-UFB generation method described above will be explained in comparison with conventional UFB generation methods. For example, in conventional bubble generation devices, such as the Venturi system, a mechanical pressure reduction structure such as a pressure reduction nozzle is provided in a part of the flow path, and liquid is made to flow through this pressure reduction structure at a predetermined pressure to generate bubbles of various sizes in the area downstream of the pressure reduction structure.

In this case, buoyancy acts on relatively large bubbles such as millibubbles and microbubbles among the bubbles generated, and they eventually rise to the liquid surface and disappear. Furthermore, UFBs that are not buoyant do not have a large gas-liquid interfacial energy and therefore disappear along with millibubbles and microbubbles. In addition, even if the above-mentioned pressure reduction structures are arranged in series and the same liquid is repeatedly passed through the pressure reduction structures, it is not possible to store a number of UFBs corresponding to the number of repetitions for a long period of time. In other words, it was difficult to maintain the UFB concentration at a specified value for a long period of time in UFB-containing liquid generated by conventional UFB generation methods.

In contrast, in the T-UFB generation method of the present embodiment that utilizes film boiling, a sudden temperature change from room temperature to about 300°C and a sudden pressure change from normal pressure to about several megapascals are locally generated in the immediate vicinity of the heating element. The heating element is a quadrilateral with a side of about tens to hundreds of μm. It is about 1/10 to 1/1000 of the size of a conventional UFB generator. Furthermore, the gas-dissolved liquid present in the extremely thin film region on the surface of the film boiling bubble instantaneously (in an extremely short time of less than microseconds) exceeds the thermal solubility limit or pressure solubility limit, causing a phase transition and precipitating as UFB. In this case, bubbles of relatively large size such as millibubbles and microbubbles are hardly generated, and the liquid contains UFB with a diameter of about 100 nm at an extremely high purity. Furthermore, the T-UFB generated in this way has a sufficiently high gas-liquid interface energy, so it is not easily destroyed under normal environments and can be stored for a long period of time.

In particular, this embodiment uses the film boiling phenomenon, which can form a gas interface locally in the liquid, and can form an interface in a portion of the liquid without affecting the entire liquid region, making the area that is thermally and pressure-sensitive to the interface extremely localized. As a result, the desired UFB can be generated stably. In addition, by circulating the liquid and applying further UFB generation conditions to the generated liquid, new UFB can be generated with minimal impact on existing UFB. As a result, UFB liquid of the desired size and concentration can be produced relatively easily.

Furthermore, because the T-UFB production method has the above-mentioned hysteresis characteristics, it is possible to increase the content concentration to the desired concentration while maintaining a high purity. In other words, the T-UFB production method makes it possible to efficiently produce a UFB-containing liquid that is highly pure, highly concentrated, and can be stored for a long period of time.

In this embodiment, the T-UFB-containing solution prepared by the above method is used as a cell culture solution. Below, we will specifically explain cell culture using the T-UFB-containing solution by giving several examples.

Example 1
12(a) to (c) are diagrams showing the state of culturing spheroids using the T-UFB-containing solution of this embodiment, together with a comparative example. All of Fig. 12(a) to (c) show the state in which a culture solution 602 is stored in a culture vessel 601 such as a petri dish, a single human cell 603 is immersed in the culture solution 602, and the culture vessel is left to stand for a certain period of time under a predetermined temperature and humidity.

The diameter of the single cell 603 is about 1 to 100 μm. The culture solution 602 preferably contains a liquid medium. The liquid medium preferably contains amino acids, inorganic salts, and vitamins. As the liquid medium, amino acid-based "StemFit (registered trademark)" AK03 medium (manufactured by Ajinomoto Co., Ltd.) and DMEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) can be used. The culture solution 602 preferably contains animal serum. Furthermore, the culture solution 602 preferably contains an antibiotic. As the animal serum and antibiotic, known substances can be used. When the culture solution contains oxygen UFB, it is preferable that the dissolved oxygen in the culture solution is in a supersaturated state. It is also preferable that the dissolved oxygen concentration in the culture solution is 8.0 ppm or more. The dissolved oxygen in the culture solution can be measured by a dissolved oxygen concentration meter (optical dissolved oxygen concentration meter, FDO Multi3510 type (WTW (W.T.W. Co., Ltd.))). Typically, cell culture is performed at a temperature similar to human body temperature (approximately 37°C). To measure the dissolved oxygen concentration in a culture incubator, we selected a portable optical type that requires only a small amount of solution. Dissolved oxygen concentration meters (DO meters) can also be of the galvanic type (diaphragm electrode type) or other types.

Figure 12(a) shows the case where a general culture vessel 601 and culture solution 602 are used. A single cell 603 grows while absorbing nutrients and oxygen from the culture solution 602, and forms a spheroid 604, which is a three-dimensional cell. However, when the spheroid 604 reaches a certain size, the inner cells, which are less likely to come into contact with the culture solution 602, tend to suffer from necrosis because they are not supplied with enough oxygen necessary for growth. Although such necrosis can be suppressed to some extent by replenishing and replacing the culture solution 602 during culture, according to the inventor's observations, necrotic cells 605 occur after a certain period of time, even if the culture solution 602 is replaced at least every 12 hours.

Figure 12 (b) shows an example in which the bottom of the culture vessel 601 is made of a material with high oxygen permeability. Examples of such materials include gas-liquid separation membranes such as a silicon hollow fiber separation membrane (Nagasep: manufactured by Nagayanagi Kogyo Co., Ltd.) and polydimethylsiloxane (PDMS).

In this case, compared to FIG. 12(a), the area of the culture solution 602 exposed to the atmosphere is increased, making it easier for oxygen to be supplied to each cell. However, it is still difficult for sufficient oxygen to be supplied to cells located near the center of the spheroid 604, resulting in the formation of necrotic cells 605.

Figure 12 (c) shows the culture method of this embodiment. In this embodiment, a mixture of ultra-fine bubble-containing liquid generated by the UFB generator 1 and the above-mentioned commercially available culture liquid is used as the culture liquid 602. Specifically, in the UFB generator 1 described in Figure 1, oxygen-containing gas is dissolved in the dissolution unit 200, and T-UFB is generated in the T-UFB generation unit 300. The T-UFB-containing liquid thus created is then mixed with the above-mentioned commercially available culture liquid, and the resulting mixture is used as the culture liquid 602 of this embodiment.

The size of the UFB11 generated by the T-UFB generation method is 100 nm or less, which is sufficiently smaller than the single cell 603 of about 1 to 100 μm. Therefore, the oxygen-containing UFB11 disperses uniformly while undergoing Brownian motion, and even if the spheroid 604 becomes a relatively large mass, it can pass between the individual cells and approach the cells located near the center. As a result, oxygen is stably supplied to almost all the cells that form the spheroid 604, and the spheroid 604 can grow without generating necrotic cells 605. That is, according to this embodiment, by using a liquid obtained by mixing a commercially available culture medium with a T-UFB-containing liquid generated by the T-UFB generation method as the culture medium, it is possible to stably produce spheroids of the desired size with high reproducibility.

In addition, according to this embodiment, which makes it easier to supply oxygen to individual cells, the frequency of changing the culture medium can be reduced compared to the comparative example shown in Figures 12(a) and (b). This reduces manpower and reduces stress on cells caused by pipetting, etc., which also promotes efficient generation of three-dimensional cells.

A specific method for producing a T-UFB-containing liquid that can be used in this embodiment will now be described with reference to FIG. 1 again. First, in this embodiment, sterilized purified water (ultrapure water) is used as liquid W. This is because tap water or general pure water may be insufficiently sterilized due to contamination, and there is a risk that bacteria such as E. coli that damage cells may be mixed in and inhibit cell culture. Specifically, ultrapure water purified by an ultrapure water device (including UV sterilization and filter filtration) manufactured by Melco can be suitably used.

The gas G to be dissolved is preferably a medical gas. Specifically, medical gas manufactured by Iwatani Corporation that contains at least oxygen and carbon dioxide is useful. In addition to oxygen and carbon dioxide, the gas G may also contain nitrogen, hydrogen, neon, ozone, argon, perfluorocarbon gases, etc.

In the T-UFB generation unit 300, it is preferable that the entire unit is housed in a draft chamber in a sterile environment. Specifically, a Panasonic biohazard cabinet class II (MHE-181AB3) can be suitably used.

As described above, if the T-UFB-containing liquid is generated in the T-UFB generation unit 300 housed in a draft chamber using already-produced ultrapure water and medical gas, the entire device can be made relatively compact. Then, by mixing the generated T-UFB-containing liquid with the commercially available culture liquid, a contaminant-free culture liquid 602 that can be used in this embodiment can be generated.

However, according to the research of the present inventors, it was confirmed that T-UFB produced by the T-UFB production method tends to form aggregates after production. It was also confirmed that such aggregates can be dispersed by applying ultrasonic waves.

Figure 13 is a schematic diagram for explaining the aggregation and dispersion of T-UFB. The individual UFBs 11 generated by the T-UFB generation method are fine bubbles with a diameter of 100 nm or less, with a zeta potential of ±0 to -30 mV, and tend to aggregate with each other. For this reason, in the UFB-containing liquid generated by the T-UFB generation method, the formation of aggregates 11G begins at a relatively early stage after UFB generation.

The more UFB11 contained in the aggregate 11G, the greater the negative charge that the aggregate 11G has. When the diameter of the aggregate 11G is about 100 to 200 nm, the zeta potential of the aggregate also becomes about -60 to -100 mV, and the aggregates 11G begin to repel each other due to electrostatic forces. In other words, after UFB is generated by the T-UFB generation method and left for a while, the T-UFB-containing liquid becomes a state in which multiple aggregates 11G of a certain size are dispersed and suspended in the liquid while undergoing Brownian motion.

However, in the state where the UFB11 floats in units of aggregates 11G, the number of UFBs is suppressed compared to the actual number of UFB11, and the effect of supplying oxygen to cells near the center of the spheroid 604 is reduced. Therefore, in this embodiment, as a post-treatment after the T-UFB generation by the T-UFB generation unit 300, a process of immersing an ultrasonic horn in the T-UFB-containing solution and emitting 150 kHz vibration at 80 W for 30 seconds was added. This allowed the aggregates 11G to be dispersed, and the number of UFB11 to be apparently increased. Then, the UFB-containing solution after such post-treatment was mixed with the above-mentioned commercially available culture solution, and used as the culture solution 602 in this embodiment. Note that the frequency of the vibration can of course be changed depending on the state. According to the study by the present inventors, it was confirmed that the aggregates 11G can be dispersed into individual UFB11 if the ultrasonic waves are in the kHz to MHz band.

In this example, the presence or absence of necrotic cells in the cultured spheroids and the stability of the entire spheroid were confirmed using a three-dimensional imaging device such as Olympus FV3000. In addition, a method was adopted in which the three-dimensional cell spheroids were sliced, the cut surfaces were stained, and the activation of the cell tissue was evaluated.

Example 2
In Example 2, the case of culturing organoids using the T-UFB-containing solution of this embodiment is described.

Figure 14 is a schematic diagram to explain the difference between spheroids and organoids. While spheroids are three-dimensional cells formed by three-dimensionally culturing a single cell (one type of single cell), organoids are three-dimensional cells formed by three-dimensionally co-culturing two or more types of single cells. A spheroid is a cell mass in which multiple cells of one type are connected to each other by blood vessels, while an organoid is a cell mass in which multiple cells of multiple types are connected to each other by blood vessels.

As with spheroids, the challenge of culturing organoids is the supply of oxygen to the cells inside the cell mass. This challenge becomes even more important with organoids, which require 3D culture until they approach the size of the desired organ, that is, until they grow to a relatively large size.

FIG. 15 is a diagram showing the state in which the UFB-containing solution of this embodiment is used as the culture solution to culture organoids. The culture solution 602 is the same as that in Example 1. That is, in the UFB generating device 1 shown in FIG. 1, ultrapure water is used as the liquid W, medical gas is dissolved as gas G, and T-UFB is generated by the T-UFB generating unit 300 stored in the draft chamber. Then, an ultrasonic horn is inserted into the obtained UFB-containing solution, and after emitting 100 kHz vibration at 80 W for 30 seconds, the mixture is mixed with "StemFit" AK03 medium (manufactured by Ajinomoto Co., Inc.) to form the culture solution 602 used in this embodiment. Then, the culture solution 602 is stored in the culture vessel 601, and multiple types of human cells 606 and 607 are immersed in the culture solution, and the culture solution is left to stand for a certain period of time under an environment in which a predetermined temperature and humidity are maintained.

In this embodiment, UFB11 also passes between the individual cells 606, 607 and can approach the cells located near the center of the organoid 608. As a result, oxygen is stably supplied to almost all the cells that make up the organoid 608, and the organoid 608 can grow without generating necrotic cells. According to the observations of the inventors, no necrotic cells were observed even when the organoid was grown to a size of 200 μm to 1.0 mm.

In other words, according to this embodiment, by using a liquid obtained by mixing a commercially available culture medium with a UFB-containing liquid produced by the T-UFB production method as the culture medium, it is possible to stably produce organoids of the desired size in a highly reproducible state.

Example 3
In Example 3, a case where a plurality of spheroids are cultured at once using the T-UFB-containing solution of this embodiment will be described.

Figure 16 is a diagram showing the culture method of this embodiment. The difference from Figure 12 (c) described in Example 1 is that a microplate 610 with multiple recesses is placed on the bottom of a culture vessel 601. The culture medium 602 used is the same as in Examples 1 and 2. By placing one single cell in each of the recesses, spheroids can be formed in each of the recesses.

In this embodiment, the oxygen-containing UFB11 is uniformly dispersed and suspended in the culture vessel 601. Therefore, even if the spheroid 604 becomes a relatively large mass, the UFB11 can pass between the individual cells and approach the cells near the center. As a result, oxygen is stably supplied to almost all the cells that form each spheroid 609, and each spheroid 609 can grow normally and uniformly without containing necrotic cells 605. In other words, according to this embodiment, by using a liquid obtained by mixing a commercially available culture medium with a T-UFB-containing liquid produced by the T-UFB production method as the culture medium, it has become possible to efficiently produce multiple spheroids of the desired size in large quantities.

In the first to third embodiments described above, a commercially available culture solution was mixed with a T-UFB-containing liquid produced in a T-UFB production unit to produce a culture solution used for culturing spheroids or organoids, but the method of producing the culture solution 602 is not limited to this. In the UFB production device 1 described in FIG. 1, the liquid W supplied to the pretreatment unit 100 or dissolution unit 200 may be a commercially available culture solution. In this case, the T-UFB production unit 300 generates T-UFB by causing film boiling in the commercially available culture solution. In any case, any liquid that contains nutrients necessary for cell culture and T-UFB produced by the T-UFB production method can be considered as the culture solution of this embodiment.

In addition, during the culture process, it is not necessary to always use the culture medium of this embodiment containing T-UFB until the single cells grow into three-dimensional cells of the desired size. The culture medium of this embodiment may be used only during the period of a given culture process when oxygen is particularly required, and then switched to a conventional culture medium once the three-dimensional cells have grown to a certain extent.

Second Embodiment
FIG. 17 is a schematic diagram of the culture device 2000 used in this embodiment. The culture device 2000 of this embodiment mainly includes a liquid supply unit 700, a gas dissolving unit 800, a storage chamber 900, an ultra-fine bubble generating unit (UFB generating unit) 1000, and a culture unit 600. The liquid supply unit 700, the gas dissolving unit 800, and the UFB generating unit 1000 correspond to the pre-treatment unit 100, the dissolving unit 200, and the T-UFB generating unit 300 in FIG. 1, respectively, described in the first embodiment. Each unit is connected to each other by a pipe 1006, and the liquid W is circulated by a pump 1002 arranged in the middle of the pipe 1006. In FIG. 17, solid arrows indicate the flow of the liquid, and dashed arrows indicate the flow of the gas.

The liquid supply unit 700 includes a liquid storage unit 701, pumps 702 and 703, and a degassing unit 704. A commercially available culture solution is stored in the liquid storage unit 701. For example, "StemFit" AK03 medium (manufactured by Ajinomoto Co., Inc.) can be used as the culture solution. The liquid W stored in the liquid storage unit 701 is sent to the storage chamber 900 by the pumps 702 and 703 via the degassing unit 704. Inside the degassing unit 704, a membrane is provided that allows gas to pass but not liquid to pass through. Only the gas passes through the membrane due to the pressure of the pumps 702 and 703, separating the gas and liquid, and the liquid W is directed to the storage chamber 900, while the gas is discharged to the outside.

The gas dissolving section 800 includes a gas supply section 804, a pre-treatment section 801, a junction section 802, and a gas-liquid separation chamber 803. The gas supply section 804 may be a medical gas cylinder as used in the first embodiment, but may also be a device capable of continuously generating gas G containing oxygen. For example, it may be a device that takes in air, removes nitrogen, and continuously pumps in gas with an increased oxygen concentration.

The gas G supplied from the gas supply unit 804 is subjected to discharge and other processes by the pretreatment unit 801, and then merges with the liquid W flowing out of the storage chamber 900 at the merging unit 802. At this time, part of the gas G dissolves in the liquid W. The merged gas G and liquid W are separated again by the gas-liquid separation chamber 803, and only the gas G that has not dissolved in the liquid W is discharged to the outside. The liquid W with the dissolved gas G is then sent to the UFB generation unit 1000 by the pump 1002. A solubility sensor 805 is provided downstream of the gas-liquid separation chamber 803 to detect the solubility of the gas G in the liquid W.

The UFB generating unit 1000 generates UFB in the flowing liquid W. The T-UFB generating method described with reference to Figures 4 to 10 is used as the UFB generating method. A filter 1001 is disposed upstream of the UFB generating unit 1000 to prevent impurities, debris, etc. from flowing into the UFB generating unit 1000. By removing impurities, debris, etc., the efficiency of UFB generation in the UFB generating unit 1000 can be improved. The UFB-containing liquid W generated in the UFB generating unit 1000 is stored in the storage chamber 900 through piping 1006.

The storage chamber 900 contains a mixture of the liquid W supplied from the liquid supply unit 700, the liquid W in which the desired gas G has been dissolved in the gas dissolution unit 800, and the UFB-containing liquid from which T-UFB has been generated in the UFB generation unit 1000.

The temperature sensor 905 detects the temperature of the liquid contained in the storage chamber 900. The liquid level sensor 902 is disposed at a predetermined height in the storage chamber 900 and detects the liquid level of the liquid W in the storage chamber 900. The UFB concentration sensor 906 detects the UFB concentration of the liquid W contained in the storage chamber 900. The valve 904 is opened when the liquid W contained in the storage chamber 900 is supplied to the culture vessel 601.

The temperature adjustment unit 903 manages the temperature of the liquid W contained in the storage chamber 900. When dissolving the desired gas G in the gas dissolving unit 800, it is more efficient for the temperature of the liquid W supplied to the gas dissolving unit 800 to be as low as possible. On the other hand, when supplying the liquid W stored in the storage chamber 900 to the culture vessel 601, it is preferable that the liquid W be adjusted to a temperature suitable for culture. In this embodiment, the temperature of the liquid is detected by the temperature sensor 905, and the temperature of the liquid W when supplied to the gas dissolving unit 800 and the temperature of the liquid when supplied to the culture vessel 601 are appropriately adjusted according to the situation using the temperature adjustment unit 903.

Although not shown in FIG. 17, an ultrasonic horn as described in the first embodiment may be provided inside the storage chamber 900 to impart vibrations to the liquid before it is sent from the storage chamber 900 to the culture vessel 601. Furthermore, the storage chamber 900 may be provided with a stirring means for making the temperature of the liquid W and the distribution of the UFB uniform.

The culture section 600 mainly includes a culture vessel 601, an inflow pipe 1004, and an outflow pipe 1005. The microplate 610 described in Example 3 of the first embodiment is set in the culture vessel 601, and spheroids or organoids are cultured in each recess. Although three recesses are shown in the figure, many more recesses are formed in the microplate 610, so that many spheroids can be cultured simultaneously. The UBB-containing solution contained in the storage chamber 900 is supplied to the culture vessel 601 continuously or as needed via the inflow pipe 1004, and is discharged from the culture vessel 601 continuously or as needed via the outflow pipe 1003.

The UBB-containing liquid (culture solution) contained in the culture vessel 601 consumes oxygen as cells respire and grow, but the culture device of this embodiment can continuously supply UFB-containing liquid with an appropriate UFB concentration to the culture vessel 601. In other words, there is no need to manually replace the culture solution, and the spheroids can be left in a specified environment until they grow to the desired size.

In this embodiment, the method of inflow and outflow of the culture medium 602 into and from the culture vessel 601 is not particularly limited. A pump may be used, or a head difference may be used. In any case, it is preferable to adjust the flow rate to a gentle rate that does not damage the cells placed in the culture vessel 601.

It is preferable that components that come into contact with the liquid W that will become the culture solution, such as the piping 1006, pump 1002, filter 1001, storage chamber 900, and liquid-contacting parts of the UFB generation unit 1000, are made of a material that is highly resistant to corrosion. For example, fluororesins such as polytetrafluoroethylene (PTFE) and perfluoroalkoxyalkane (PFA), metals such as SUS316L, and other inorganic materials can be suitably used.

In the circulation path of this embodiment shown in FIG. 17, the process of dissolving the desired gas G and the process of generating UFB may be performed simultaneously or separately. When performed simultaneously, the liquid is circulated while both the gas dissolving unit 800 and the UFB generating unit 1000 are operating. In this case, the valve 904 may be open during circulation, but it may also be opened after multiple circulations when the UFB concentration sensor detects an appropriate UFB concentration, and an appropriate amount of UFB-containing liquid is sent to the culture unit 600.

On the other hand, if the process of dissolving gas G and the process of generating UFB are performed separately, a circulation in which only the gas dissolving unit 800 is operated can be performed first, followed by a circulation in which only the UFB generating unit 1000 is operated. In this case, in the circulation in which only the gas dissolving unit 800 is operated, when the solubility sensor 805 detects an appropriate solubility, the gas dissolving unit 800 can be stopped and the operation of the UFB generating unit 1000 can be started. Then, when the UFB concentration sensor 906 detects an appropriate UFB concentration, the valve 904 can be opened to supply liquid to the culture unit 600.

In addition, in FIG. 17, a commercially available culture solution is stored in the liquid storage section 701 and T-UFB is generated in this culture solution, but the culture solution supplied to the culture vessel 601 may be prepared by the method described in the first embodiment. Specifically, ultrapure water may be stored in the liquid storage section 701, and a mixing section for mixing the commercially available culture solution with the UFB-containing solution W may be provided between the storage chamber 900 and the culture section 600. Such a mixing section may also be provided between the gas dissolving section 800 and the UFB generating section 1000, or between the UFB generating section 1000 and the storage chamber 900.

Figure 18 is a diagram showing a modified example of the culture device. The difference from the culture device 2000 shown in Figure 17 is that the culture section 600 is placed midway along the circulation path, specifically between the UFB generation section 1000 and the storage chamber 900. In this modified example, the liquid W stored in the storage chamber 900 passes through the gas dissolution section 800, the UFB generation section 1000, and the culture section 600, and then returns to the storage chamber 900.

In the culture unit 600, the spheroids being cultured consume oxygen, so it is assumed that the UFB concentration of the liquid W discharged from the culture unit 600 is lower than the UFB concentration of the liquid W supplied to the culture unit 600. By circulating such liquid W again through a path including the gas dissolution unit 800 and the UFB generation unit 1000, the oxygen UFB concentration in the liquid W can be increased again. As a result, it is possible to always supply culture liquid with an appropriate UFB concentration to the culture unit 600.

As described above, the culture device of this embodiment, which transfers liquid between the gas dissolution unit 800, the UFB generation unit 1000, and the culture unit 600, makes it possible to efficiently produce large quantities of three-dimensional cells of the desired size.

[Third embodiment]
In addition to the three-dimensional cell culture shown in the first and second embodiments, the two-dimensional cell culture can be efficiently produced in large quantities by using the T-UFB-containing solution. The UFB generating device and the T-UFB generating method can be the same as those in the first and second embodiments.

Below, we will provide a detailed explanation of two-dimensional cell culture using T-UFB-containing solution with some examples.

Example 4
The two-dimensional culture of fibroblasts contained in the dermis of the skin is described below. First, animal serum (FBS: manufactured by BioWest) is added to DMEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) which is a liquid medium containing phenol red (PR) at 10% by mass based on the content of DMEM, and penicillin-streptomycin solution (antibiotics: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is added at 1% by mass based on the content of DMEM to prepare a culture solution (medium). This culture solution is placed in a general-purpose T-type flask, and then 1,000 fibroblasts are added, and the number of live cells is increased in a carbon dioxide incubator (passage process). The temperature inside the carbon dioxide incubator is set to 37°C, which is suitable for cell culture, and the humidity is set to 90 to 100% to prevent evaporation of the medium, and the atmosphere is set to 5% carbon dioxide for pH adjustment. By carrying out such cell culture, the number of cells increases to about 10,000 in about one week.

Next, the cells were collected from the T-flasks, 1,000 cells per well were placed in a multi-well plate, and the plates were placed in the carbon dioxide incubator. The cells were then cultured in two dimensions for approximately one week under the same conditions as above.

When two-dimensional cells are grown as described above, a sheet of cells is formed. The number of cells was approximately 10,000.

Next, a culture medium containing the oxygen UFB of the present invention was separately prepared by the following method.

The culture medium (DMEM, 10% FBS by mass, 1% penicillin-streptomycin solution by mass) was supplemented with oxygen UFB (UFB concentration: 1 billion cells/ml). Then, as described above, the fibroblasts were multiplied in a carbon dioxide incubator. As a result, the number of cells reached 10,000 in about two days. The dissolved oxygen concentration in the culture medium was maintained in a supersaturated state (10 ppm) for two days, and this is presumably the result of a constant supply of oxygen to the cells in the carbon dioxide incubator.

(Example 5)
As described above, the cells in the T-flask were scraped together, 1,000 fibroblasts were placed in each multi-well plate, and 2D cell culture was performed in the carbon dioxide incubator. At that time, oxygen UFB (UFB concentration: 1 billion cells/ml) was added to the culture solution. As a result, 10,000 sheet-like cells were formed in two days. As described above, the dissolved oxygen concentration in the culture solution was maintained in a supersaturated state (10 ppm) for two days, and it is presumed that this is the result of constantly supplying oxygen to the cells in the carbon dioxide incubator.

10 Heating element 11 Ultra fine bubble (UFB)
602 Culture medium 603 Cells (single cells)

Claims (34)

A method for culturing cells using a culture solution containing ultra-fine bubbles generated by generating film boiling at an interface between a liquid and the heating element by heating a heating element, comprising:
A culture method characterized in that the liquid is sterilized purified water . The culture method according to claim 1, characterized in that the ultra-fine bubbles contain oxygen. The culture method according to claim 2, characterized in that the ultra-fine bubbles further contain carbon dioxide. The culture method according to any one of claims 1 to 3, characterized in that the cells are three-dimensional cells of at least one of spheroids and organoids. The culture method according to any one of claims 1 to 4, characterized in that the culture solution is subjected to ultrasound in the kHz to MHz range before being used for culturing the cells. 6. The culture method according to claim 1, wherein the culture medium contains a predetermined nutrient. A method for producing a culture medium used for culturing cells, comprising the steps of:
an ultra-fine bubble generating step of generating ultra-fine bubbles in the liquid by generating film boiling at the interface between the liquid and the heating element by heating the heating element;
a mixing step of mixing an ultra-fine bubble-containing liquid containing the ultra-fine bubbles generated in the ultra-fine bubble generating step with a liquid containing predetermined nutrients to generate the culture solution ,
A method for producing a culture solution , wherein the liquid used in the ultra-fine bubble generating step is sterilized purified water . A method for producing a culture medium used for culturing cells, comprising the steps of:
an ultra-fine bubble generating step of generating ultra-fine bubbles in a liquid containing predetermined nutrients by generating film boiling at an interface between the liquid and the heating element through heating of the heating element,
A method for producing a culture medium , wherein the liquid is purified water that has been sterilized . 9. The method for producing a culture solution according to claim 7 , further comprising a gas dissolving step of dissolving a predetermined gas in the liquid prior to the ultra-fine bubble generating step. 10. The method for producing a culture medium according to claim 9 , wherein the predetermined gas is a medical gas. 11. The method for producing a culture medium according to claim 9 or 10, wherein the predetermined gas contains oxygen. The method for producing a culture medium according to any one of claims 7 to 11, characterized in that the ultra-fine bubbles contain oxygen. 13. The method for producing a culture solution according to claim 7 , further comprising a vibration step of imparting vibration to the ultra-fine bubble-containing liquid after the ultra-fine bubble generating step. The method for producing a culture medium according to claim 13 , wherein the vibration is ultrasonic waves in the kHz to MHz band. The method for producing a culture medium according to any one of claims 7 to 14, characterized in that the cells are three-dimensional cells of at least one of spheroids and organoids. A culture medium for culturing cells, comprising:
The present invention includes ultra-fine bubbles generated by generating film boiling at the interface between a liquid and the heating element by heating the heating element, and predetermined nutrients,
A culture medium characterized in that the liquid is sterilized purified water . The culture medium according to claim 16 , characterized in that the ultra-fine bubbles have a zeta potential of ±0 to -30 mV. 18. The culture medium according to claim 16 , wherein the ultra-fine bubbles are suspended in units of aggregates each composed of a plurality of the ultra-fine bubbles. The culture medium according to claim 18 , characterized in that the aggregates have a zeta potential of -60 to -100 mV. The culture medium according to any one of claims 16 to 19 , characterized in that the culture medium contains a liquid medium as a nutrient. The culture medium according to claim 20 , wherein the liquid medium contains amino acids, inorganic salts, and vitamins. The culture medium according to any one of claims 16 to 21 , characterized in that the ultra-fine bubbles contain oxygen. 23. The culture medium according to claim 22 , characterized in that the dissolved oxygen in the culture medium is in a supersaturated state. The culture medium according to claim 23 , characterized in that the dissolved oxygen concentration in the culture medium is 8.0 ppm or more. The culture medium according to any one of claims 16 to 24, characterized in that the cells are three-dimensional cells, such as at least one of spheroids and organoids. A gas dissolving means for dissolving a predetermined gas in a liquid;
an ultra-fine bubble generating means for generating ultra-fine bubbles in the liquid by generating film boiling at the interface between the liquid and the heating element by heating a heating element;
A culture means for culturing cells using a liquid containing the ultra-fine bubbles;
Equipped with
A culture apparatus characterized in that the liquid is sterilized purified water . The culture apparatus according to claim 26, further comprising a liquid transport means for transporting liquid between the gas dissolving means, the ultra-fine bubble generating means, and the culture means so as to supply the liquid in which the predetermined gas has been dissolved by the gas dissolving means and the ultra-fine bubbles have been generated by the ultra-fine bubble generating means to the culture means . 28. The culture apparatus according to claim 27 , wherein the liquid delivery means discharges the liquid to the outside after it has been used in the culture means. The culture apparatus according to claim 27, characterized in that the liquid supplying means returns the liquid after use in the culture means to the gas dissolving means, and circulates the liquid among the gas dissolving means, the ultra-fine bubble generating means, and the culture means. 30. The culture apparatus according to claim 26, wherein the liquid contains predetermined nutrients necessary for culture. 31. The culture apparatus according to any one of claims 26 to 30 , further comprising a mixing means for mixing the liquid with a liquid containing predetermined nutrients necessary for culture. 32. The culture apparatus according to claim 26 or 31, characterized in that the predetermined gas contains oxygen. The culture apparatus according to any one of claims 26 to 32, characterized in that the ultra-fine bubbles contain oxygen. The culture device according to any one of claims 26 to 33, characterized in that the cells are three-dimensional cells, such as at least one of spheroids and organoids.

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