JP5319607B2 - Liquid composition containing microbubbles and method for producing composition containing microbubbles - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve a cell manipulation effect by micro- and/or nano-bubbles Mnb. <P>SOLUTION: A "P type" Mnb are a mixture of Mnb containing single and separate component gases of nitrogen N<SB POS="POST">2</SB>, oxygen O<SB POS="POST">2</SB>, carbon dioxide CO<SB POS="POST">2</SB>and the like, each having a 90% or higher purity. A "C type" Mnb contain component-conditioned gas mixture of nitrogen N<SB POS="POST">2</SB>, oxygen O<SB POS="POST">2</SB>, carbon dioxide and the like. The effect of the a cell manipulation is improved with a "P/C mixed type" Mnb being a mixture of the "P type" and the "C type" Mnb. The contained gases are not limited of these, but may be a gas existing in the natural growing environment of a target cell such as Argon Ar, and/or a gas contributing to biological reaction in the target cell such as nitrogen monoxide NO and carbon monoxide CO. <P>COPYRIGHT: (C)2012,JPO&amp;INPIT

Description

  The present invention relates to a liquid composition containing microbubbles called microbubbles, nanobubbles, and micro / nanobubbles, and a method for producing a composition containing microbubbles.

  In particular, the present invention supplements the unclear part of the microbubble technology described in Patent Document 1 and Patent Document 2 described later, and is an improved invention of the microbubble technology in Patent Document 1 and Patent Document 2.

  In the present specification, microbubbles are abbreviated as “Mnb” which abbreviates micro and / or nanobubbles. A bubble of normal size or larger is abbreviated as “Mm”.

  The Mnb and the utilization technique of Mnb are described in Patent Document 1 to Patent Document 4 and are described in the following Reference Documents 01 to 22, and therefore will be omitted.

<References 01 to 22>
01 JP-A-2009-189307 “Methods for sterilization or inactivation of spore bacteria”
02 JP2009-131770 "Method for producing carbon dioxide nanobubble water"
03 JP 2009-131769 “Method for producing nitrogen nanobubble water”
04 JP 2009-084258 A "Drug for treatment or prevention of cancer containing nanobubbles"
05 Japanese Unexamined Patent Application Publication No. 2009-039600 “Ultrafine Bubble Generator”
06 JP 2008-259456 A “Method for Preserving Seafood”
07 JP 2008-237950 A “Method for producing water containing hydroxyl radicals and water containing hydroxyl radicals”
JP 2008-093612 A "Method for producing water containing reactive active species and water containing reactive active species"
09 Japanese Patent Application Laid-Open No. 2008-093611 “Method for producing water containing ultrafine bubbles and water containing ultrafine bubbles”
10 JP 2008-063258 A "Tissue preservation solution"
11 Japanese Patent Application Laid-Open No. 2007-275089 “Long-lasting ozone water, environmental sterilization / deodorizing purification method using long-lasting ozone water”
12 Japanese Unexamined Patent Publication No. 2006-223239 “A method for extracting active ingredients of roasted seafood using oxygen nanobubbles and a processed fish meat material to which active ingredients obtained by the extraction method are added”
13 Japanese Unexamined Patent Application Publication No. 2005-246294 “Oxygen Nano Bubble Water and Method for Producing the Same”
14 Japanese Unexamined Patent Application Publication No. 2005-246293 “Ozone water and method for producing the same”
15 Japanese Patent Laid-Open No. 2005-245817 “Method for producing nanobubbles”
16 Japanese Unexamined Patent Application Publication No. 2005-110552 “Pressurized Multilayer Micro-Ozone Sterilization / Purification / Animal Sterilization System”
No. 17 Reissue 2005/030649 “Crushing of microbubbles”
18 Ichiro Nagao, Ken Muramatsu, Kenji Sato “Histological study on tissue preservation of oxygen nanobubble water (1st report)” The 111th Annual Meeting of the Japanese Anatomy and Academic Meeting 19 2005 New Energy and Industrial Technology Development Organization Commissioned research Research on the industrial use of nanobubble water in the bio field Result report 38-40 “Evaluation research on preservation of living tissue using nanobubble water”
10 Hojo Y, et al. “Anti-inflammability Property of Oxygen Nano-bubbles” Circulation Journal vol. 70, supplement I, p276 (The 70th Annual Meeting of the Japanese Circulation Society)
21 2005 New Energy and Industrial Technology Development Organization Commissioned Research Research Survey on Industrial Use of Nanobubble Water in Biotechnology Results Report 40-45 “Evaluation of Nanobubble Water on Cell Physiology”
22 The Multiphase Flow Lecture Series 35th "The Characteristics of Micro / Nano Bubbles and Their Applications" December 5, 2009 (distributed at Kansai University)

  The microbubble technology of Patent Document 1 and Patent Document 2 is a technique for the purpose of manipulating useful cells or promoting useful cell changes, and provides a large effect that Mnb cannot obtain with conventional Mm on cells. Is used. The microbubble technology of Patent Document 1 and Patent Document 2 will be described below.

FIG. 1 is a schematic diagram illustrating the microbubble technology disclosed in Patent Document 1 and Patent Document 2. That is, FIG. 1 is a schematic diagram showing that the micro / nano bubble Mnb is larger than that of a bubble Mm of a millimeter size or more, and FIG. 1 (a) shows a hydrophilic cell membrane on the Mm surface. It is shown that M m does not have a great influence on the cells.

  On the other hand, FIG. 1 (b) shows that the micro / nano bubble Mnb is smaller than the scale of the cell C, FIG. 1 (c) is an enlarged schematic diagram of (b), and the substance having Mnb on the cell membrane surface CS. It indicates that there is a case where it influences by binding to or affinity with a channel constituent substance CP (cell membrane protein etc.).

  If the gas forming Mnb elutes and forms a high-concentration gas dissolved state around it, it may permeate the cell membrane by diffusion.

  FIG. 5 and FIG. 6 schematically show examples of permeation through the cell membrane by the diffusion phenomenon caused by gas elution forming Mnb. That is, FIG. 5 is a schematic diagram showing the influence of Mnb on the carbon dioxide (CO2) absorption of the pores.

Here, Mnb is Young-Laplace's formula [P = Pl + 2σ / r (P = gas pressure, Pl =
liquid pressure, σ = surface tension, r = radius of the
bubble)] reduces the diameter and increases the pressure. Therefore, the dissolved amount of the gas forming Mnb locally increases at the gas-liquid interface between Mnb and the liquid, and the gas is eluted into the liquid. Therefore, if there is Mnb containing carbon dioxide in the vicinity of the pores, the amount of carbon dioxide dissolved in the vicinity of the pores becomes extremely high, and the amount of cell membrane permeation through the pores due to diffusion increases, thereby increasing the amount of carbon dioxide absorption.

  FIG. 6 is a schematic diagram showing the influence of Mnb on the oxygen (O2) absorption of cells. Here, Mnb is reduced in diameter by the Young-Laplace equation as in FIG. 5, and the dissolved amount of the inclusion gas locally increases at the gas-liquid interface due to the increase in pressure, and gas is eluted. Therefore, it is considered that when there is Mnb containing oxygen in the vicinity of the cell, the amount of dissolved oxygen becomes extremely high in the vicinity of the cell, the amount of cell membrane permeation due to diffusion increases, and the amount of oxygen absorbed by the cell increases. The “high concentration supersaturation zone” shown in FIG. 5 and FIG. 6 shows a collection of gas molecules that are locally concentrated in the vicinity of the cell and permeate the cell membrane. It is thought that this gas molecule is taken into the cell by diffusion or the like and triggers various actions.

  Patent Document 1 discloses the Mnb manufacturing method shown by the time charts in FIGS. 8 (a), 8 (b), and 8 (c).

  That is, in FIG. 8A, a plurality of gases sequentially selected by the gas selection means are sucked from the gas suction means, and the plurality of gases are made into microbubbles. This is a production method for obtaining a composition containing a plurality of gaseous microbubbles obtained by sequentially and continuously discharging.

  FIG. 8 (b) shows that the cell culture solution is sucked into a plurality of microbubble generators, and a plurality of gases sucked from a plurality of gas suction means are formed as microbubbles and continuously discharged to a plurality of liquid discharge means. Is a method for producing a composition for cell culture containing a plurality of gaseous microbubbles obtained by the operation described above.

  FIG. 8C shows a manufacturing method in which the time sequential gas switching of FIG. 8A is combined with the manufacturing apparatus of FIG. 8B.

  Mnb by the manufacturing method of these FIG. 8 (a) (b) (c) is "P type" Mnb shown to (a) of FIG. P of “P type” means pure gas (pure gas), and is a single gas having a purity of 90% or more. Specifically, nitrogen (N2) gas having a purity of 90% or more, oxygen (O2) gas having a purity of 90% or more, carbon dioxide (CO2) gas having a purity of 90% or more, or the like.

  FIG. 2 (a) is a diagram showing a “P type” Mnb including 100% of each single component gas of nitrogen (N2), oxygen (O2), and carbon dioxide (CO2).

On the other hand, Patent Document 2 discloses the Mnb manufacturing method shown in the time chart schematic diagram of FIG.

  That is, FIG. 8 (d) shows the components of the suction gas upstream of the gas suction means and the means for adjusting and controlling the components, and the means for adjusting and controlling the components of the suction gas and the concentrations thereof. This is a manufacturing method using an apparatus including a gas component for outputting a concentration adjustment control target command and a means for outputting a concentration adjustment control target command.

  The Mnb by the manufacturing method of FIG. 8D is a “C type” Mnb shown in FIG. C of “C type” means conditioned gas (regulated gas), which is a mixture of a plurality of gases (which may or may not be pure gas (pure gas)), and a component ratio of atmospheric components; It may be adjusted close to this component ratio. More specifically, if there is a change of 5% relative to the component ratio of normal atmospheric components, the effect on cells will appear, so it is preferable to mix one or more of each component with a component change of 5% or more. It is. FIG. 16 is a reference diagram showing the ratio of atmospheric components.

  FIG. 2B is a view showing a “C type” Mnb including a gas in which components are adjusted by mixing nitrogen (N 2), oxygen (O 2), and carbon dioxide (CO 2).

  3 and FIG. 4, supplementary explanation will be given for Mnb of “C type” and “P type”. FIG. 3 is a schematic diagram for explaining the “C type” Mnb. In FIG. 3, Gas “CA” and Gas “CB” in the first row have different component ratios of contained gas (second row in FIG. 3) and bubble particle size distribution (third row in FIG. 3). The effect (third line in FIG. 3) differs between Gas “CA” and Gas “CB”. [CA ≠ CB]

  FIG. 4 is a schematic diagram for explaining the “P type” Mnb. The gas “PA” and Gas “PB” in the first row in FIG. 4 have different component ratios of the contained gas (second to fourth rows in FIG. 4) and bubble particle size distributions (third row in FIG. 4). The component ratio of inclusion gas (FIG. 4, second to fourth lines) is as follows: nitrogen (N 2) single component gas amount (FIG. 4 second row), oxygen (O 2) single component gas amount (FIG. 4 third row), If the ratio of the carbon (CO2) single component gas amount (the fourth line in FIG. 4) is adjusted, it can be made substantially equal to Gas “CA” or Gas “CB” in FIG.

  Similarly, the bubble particle size distribution (third line in FIG. 4) can also be made substantially equal to Gas “CA” Gas “CB” in FIG. But their effects are not equal. (Effect CA ≠ PA, Effect CB ≠ PB)

  In addition, Patent Document 3 shows a description similar to the microbubble technology described in Patent Document 1 and Patent Document 2 described above. That is, in Patent Document 3 [Claim 3], “the hydrogen generation means includes a gas distribution means capable of changing a supply ratio according to the type of gas to be generated. Nanobubble liquid manufacturing apparatus. ”[0010] of Patent Document 3“ (3) The hydrogen generation means includes gas distribution means that can change the supply ratio according to the type of gas to be generated. It is possible to provide the nanobubble liquid manufacturing apparatus according to the above (1) or (2), which is characterized. " [0011] of Patent Document 3 states that "... the gas distribution means may change the ratio of various gases by a combination of variable valves, and adjusts the flow rate of each gas (for example, a mass flow controller). May be used. " Furthermore, there is a description of “16a gas distribution means” in the drawing of Patent Document 3.

  However, the gas distribution, gas ratio change, and the like in Patent Document 3 are unclear with respect to the purpose and effect as compared with those described in Patent Document 1 and Patent Document 2. That is, Patent Document 3 discloses nanobubble water containing hydrogen and the nanobubble liquid production apparatus, but there is no description of gas distribution means other than hydrogen, no description of gas supply means other than hydrogen, and the inclusion of microbubbles. It does not specifically show the adjustment.

  Similarly, Patent Document 4 and Patent Document 5 also show a description similar to the microbubble technology of Patent Document 1 and Patent Document 2, but the manufacturing procedure and the like are different in apparatus components. (Details omitted)

  Furthermore, similarly, Patent Document 6 shows a description similar to the microbubble technology of Patent Document 1 and Patent Document 2. The main point of Patent Document 6 is “a technique for dissolving a large amount of gas in a liquid in a supersaturated state”, but generation of microbubbles (Mnb) due to a plurality of gases generated by supersaturation is shown. (Refer to Patent Document 6 [Claim 2] and [Claim 11]) However, the manufacturing procedure and the like of Patent Document 6 are also different from the microbubble technology of Patent Document 1 and Patent Document 2 in detail. It is. (Details omitted)

  Here, with reference to FIG. 11 to FIG. 15, a technique that should be additionally supplemented by the microbubble technique of Patent Document 1 and Patent Document 2 and a combination technique of Patent Document 7 will be described.

  FIG. 11 is data of an Mnb generator provided by Kyowa Kikai Co., Ltd., and is a schematic diagram showing that the Mnb particle size decreases with time after Mnb generation. The measuring instrument is a Coulter type fine particle measuring instrument manufactured by Beckman Coulter.

As shown in FIG. 5 and FIG. 6, the reduction change in the Mnb particle size shown in FIG. 11 is the Young-Laplace equation [P = Pl + 2σ / r (P = gas pressure, Pl =
liquid pressure, σ = surface tension, r = radius of the
bubble)].

  In cell manipulation, this time change in particle size must be taken into account. In particular, it should be noted that the change in particle size with time varies depending on the type of gas.

  Specifically, since carbon dioxide (CO2) has a higher solubility than oxygen / nitrogen, a large amount is eluted after Mnb generation. Therefore, Mnb containing carbon dioxide (CO2) shrinks relatively early (see FIG. 12).

  FIG. 12A is a schematic diagram showing that carbon dioxide (CO2) elutes more with time and the inclusion amount decreases. FIG.12 (b) is a reference figure which shows the temperature change of the solubility with respect to the water of each gas. It is necessary to devise a technique to collect experimental data in anticipation of the difference in temporal change due to such gas species so that the difference between the experiment and the actual application does not occur.

  Simply, the Mnb supply time from the generation of Mmb in the experiment to the vicinity of the cell may be matched with that in the practical apparatus. Of course, incidental environmental conditions such as liquid temperature must be matched.

  Actually, since it is difficult to match the Mnb supply time from the generation of Mmb in the experiment to the vicinity of the cell and that in the practical apparatus, the change in the particle size distribution time of FIG. 11 is measured for each Mnb generated. In addition, it is preferable to carry out by estimating the Mnb particle size distribution, dissolved amount, gas elution amount, etc. in the vicinity of the cells in the practical apparatus in consideration of the dissolved amount change for each gas type and application temperature in FIG.

  FIG. 13 is a reference diagram and is a schematic diagram of the Mnb generator exemplified in Patent Document 1. FIG. (Modified drawing of Patent Document 1 [FIG. 2]) With the multi-nozzle device configuration as shown in FIG. 13, Mnb including a plurality of different gases shown in FIG. 8 (a) and FIG. 8 (b) is generated simultaneously. Can be made. Here, 10 is a fluid holding container H or a combination of a fluid holding container H and a Taylor reactor TR (described later with reference to FIG. 14). It is a culture vessel (bioreactor).

  FIG. 14 is also a reference diagram and is a schematic diagram of the Mnb generator exemplified in Patent Document 2. FIG. (Correction of Patent Document 2 [FIG. 4]] 17, 18, 18A, 30, and 31 in FIG. 14 are as follows.

  That is, 17 is a sensor for measuring 10 liquid turbidity by transmitted light or scattered light measurement method, and 18 is an environmental operation such as 10 temperature, pressure, hydrogen ion concentration, biological / chemical oxygen demand, etc. Means 18A is an environment control means 10 such as 10 temperature, pressure, hydrogen ion concentration, biological / chemical oxygen demand, etc. 30 is a means 18A for adjusting and controlling the environment of the liquid tank; The liquid tank environment adjustment control target command output means 31 for issuing a control target command, each of which is an individual gas corresponding to the individual uptake of each of a plurality of substances required to promote the change of the cell itself with respect to the target cell A database of data obtained by the process of experimentally determining the components and their concentrations in advance, and the ease of uptake of each of the multiple substances required for the cells to promote their changes Environmental data of the liquid tank for advancing a database of data obtained by the step of determining in advance experimentally.

  A bioreactor function such as cell culture and iPS cell conversion operation is realized with the configuration including these 17, 18, 18A, 30, and 31.

  FIG. 15 is also a reference diagram, and is a schematic diagram of a process apparatus in which a CT (Couette-Taylor) reactor (Taylor reactor) TR described in Patent Document 7 and a microbubble generator are combined. When the microbubble technology of Patent Document 1 and Patent Document 2 is implemented, the bioreactor functions such as cell culture and iPS cell conversion operation can be macro-efficiency by combination with Taylor reactor TR.

  Here, “macro” means that the flow environment inside the reactor is made uniform. For example, the time for supplying Mnb to the vicinity of the cells explained in FIG. 11 and FIG. Macroscopically, the conditions in the vicinity of the "microscopic" cell and the conditions in the vicinity of the "microscopic" cell that match the incidental environmental conditions such as the liquid temperature are macroscopically the same at any position inside the reactor. It can be realized. (See Patent Document 7)

The additional supplementary techniques of Patent Document 1 and Patent Document 2 described with reference to FIGS. 11 to 15 and the technique of Patent Document 7 can be implemented in combination in the implementation of the present invention.
Japanese Patent Application No. 2009-234683 “A composition containing microbubbles for promoting cell change, an apparatus for producing the composition containing microbubbles, and a method for promoting cell change using the composition containing microbubbles” Tomotaka Marui Japanese Patent Application No. 2009-282840 “Apparatus for promoting cell change with liquid composition containing microbubbles, Method for promoting cell change with liquid composition containing microbubbles” Tomotaka Marui Patent Publication No. 2008-6397 “Nano Bubble Liquid Manufacturing Apparatus and Nano Bubble Liquid” Opt Creation Japanese Patent Publication No. 2009-195889 “Microbubble generator” Aisin Seiki Co., Ltd. Patent Publication No. 2007-75785 “Water Treatment Method and Water Treatment Apparatus” Sharp Corporation Published Patent Application No. 2009-19589 (WO 2006/12779) “System and Method for Dissolving Gas into Liquid and Supplying Dissolved Gas” University of Arkansas Japanese Patent Application No. 2010-012727 “Process apparatus using a CT (Couette-Taylor) reactor, process apparatus combining the apparatus with a microbubble generator.” Tomotaka Marui

  The present invention proposes an invention in which the microbubble technology described in Patent Document 1 and Patent Document 2 is supplemented, and the microbubble technology in Patent Document 1 and Patent Document 2 is improved.

  The present invention is a “P / C mixed type” Mnb obtained by mixing “P type” and “C type” Mnb shown in Patent Document 1 and Patent Document 2 shown in FIG. The present invention will be described with reference to FIG. 7 (effect), FIG. 9 and FIG. 10 (time chart of the present invention).

  First, FIG. 7 is a summary table of Experiment 1) to Experiment 6) for comparing the effects of “P type” Mnb · “C type” Mnb · “P / C mixed type” Mnb on cells or organisms. In general, it can be seen that the “P / C mixed type” Mnb has a large influence on cells or organisms.

  Experiment 1) <Yeast Growth> in FIG. 7 When yeast (S. cerevisiae) was grown for 48 hours in a medium in which three kinds of Mnb groups having an oxygen component 10% larger than the atmospheric composition were added to the room temperature culture solution It is a comparison of the number of colonies.

  Experiment 2 of FIG. 7) <Escherichia coli growth> When E. coli was grown for 48 hours in a medium in which three kinds of Mnb groups having an oxygen component 10% larger than the atmospheric composition were added to the room temperature culture solution It is a comparison of the number of colonies.

  Experiment 3) in Fig. 7 <Porosity Absorption> Three kinds of Mnb groups having a carbon dioxide component 10% larger than the atmospheric composition were added to room temperature distilled water to form a mist and sprayed to Arabidopsis every 6 hours. It is a comparison of a change in plant weight 30 days after spraying.

  Experiment 4 in Fig. 7) <Methane fermentation> The amount of methane produced when methane bacteria were cultured for 48 hours at room temperature by adding three Mnb groups of zero oxygen, 99% nitrogen and argon, and 1% carbon dioxide to the room temperature culture solution. It is a comparison.

  Experiment 5 in Fig. 7 <Blood flow of human body> Three kinds of Mnb groups having a carbon dioxide component 10% larger than the atmospheric composition are added to 38 ° C. warm bath water, and the upper arm of the human body bathes in the warm bath water for 30 minutes. It is a comparison of blood flow elevation values.

  Experiment 6 in FIG. 7 <Animal cell growth> Standard Chinese hamster (CHO) cells were grown for 48 hours in a medium in which three kinds of Mnb groups having an oxygen component 10% larger than the atmospheric composition were added to the room temperature culture solution. It is a comparison of the number of cells.

  Here, the reason why the “P / C mixed type” Mnb has a larger influence on cells or organisms than the “P type” Mnb and “C type” Mnb is not clear at this point, and must be studied in the future. .

  The hypothesis is that the gas molecules that cells or organisms act sensitively in the above experiments are oxygen in experiment 1), experiment 2), carbon dioxide in experiment 3), nitrogen and argon in experiment 4), In Experiment 5), carbon dioxide, in Experiment 6), oxygen, etc., these working gases are locally present at high concentrations in the vicinity of the cells, and other gas molecules are dissolved in the normal environment such as the atmosphere. You should be in a state. Therefore, in “P type” Mnb, the concentration of other adverse gas molecules is too high, and thus the effect is suppressed, and in “C type” Mnb, the degree of local high concentration is low. Therefore, the “P / C mixed type” Mnb, which is between the two, has a relatively good effect. (The “high-concentration supersaturation zone” in FIGS. 5 and 6 indicates a set of locally-enriched working gas molecules in the vicinity of the cell)

  Now, the manufacturing method of the “P / C mixed type” Mnb of the present invention will be described with reference to FIGS.

  FIG. 9 is a time chart schematic diagram of the manufacturing method of the “P / C mixed type” Mnb according to the present invention. After the “C type” Mnb is generated, the “P type” Mnb is generated in the same liquid container H. To obtain “P / C mixed type” Mnb.

  In contrast, FIG. 10 shows that “P type” Mnb is generated after “P type” Mnb is generated and mixed to obtain “P / C mixed type” Mnb. In this way, the “P type” / “C type” Mnb mixed type manufacturing method is easy.

That is, the “P / C mixed type” Mnb of the present invention includes a plurality of single-component gas supply means having a purity of approximately 90% or more, a setting means for setting a mixing ratio including zero for each of the plurality of gases, and the set mixing ratio. What is necessary is just to manufacture using the fine bubble generator provided with the main body which produces | generates the gas adjustment means which mixes the said multiple gas, this adjusted gas suction means, a liquid suction means, a liquid discharge means, and a micro gas.

Further, "P / C mixing type" Mnb of the present invention, generally feed means purity of 90% or more of the single component gas supply means, a gas obtained by mixing a plurality of gases (premix control gas), the approximately 90% pure Provided with means for selectively sucking either the above single component gas or a gas (premix adjusting gas) mixed with the plurality of gases, a liquid suction means, a liquid discharge means, and a main body for generating a micro gas What is necessary is just to manufacture using the produced microbubble generator.

  Here, the premix of the “premix adjustment gas” means “Prepared Mix = premixed,” meaning that the premix adjustment gas is a mixture gas whose components have been adjusted in advance or an atmosphere such as the atmosphere. It is a gas that has already been mixed with a specific mixing ratio.

A is "P / C mixing type" Mnb the compositions of the present invention in any way (claim 1 -3), a composition liquid containing microbubbles, the microbubble is at least a particle size The composition contains microbubbles of 30 μm or less, and the gas included in the microbubbles is a mixture of at least the following two types. 1 The gas is a single component gas (with a purity of 90% or more ) . 2 Gas is a mixed gas in which a plurality of gases are mixed.

And the manufacturing method of the composition of the present invention (Claim 4) comprises a plurality of single component gas supply means, a mixing ratio setting means including zero of the plurality of gases, and the plurality of gases at the set mixing ratio. A composition containing micro bubbles using a gas adjusting means for mixing, a suction means for the adjusted gas, a liquid suction means, a liquid discharging means, and a micro bubble generator having a main body for generating micro gas A manufacturing method, wherein the microbubbles include at least microbubbles having a particle size of 30 μm or less, and have at least the following two steps. 1 The setting of the mixing ratio setting means is set to zero except for one of a plurality of gases, and is sucked by the gas suction means, and bubbles including a single component gas are discharged into the liquid sucked by the liquid suction means. Process generated by discharging by means. 2. The setting of the setting means for the mixing ratio is set to a non-zero setting for a plurality of gases, and is sucked by the gas suction means, and bubbles including the mixed gas are discharged into the liquid sucked by the liquid suction means by the liquid discharge means. Process.

Moreover, the manufacturing method of the composition of this invention using the said premix adjustment gas (Claim 5), supply means of single component gas, supply means of the gas (premix adjustment gas) which mixed several gas, this, Contains microbubbles using a means for selectively sucking either a single component gas or a mixed gas , a liquid suction means, a liquid discharge means, and a microbubble generator having a main body that generates microgas The microbubbles are at least two microbubbles having a particle size of 30 μm or less, and have at least the following two steps. 1. A step of selectively sucking the single component gas by the gas suction means and generating bubbles including the single component gas by discharging the liquid suction means into the liquid sucked by the liquid suction means. (2) A step of generating by selectively sucking the mixed gas by the gas sucking means and discharging bubbles including the mixed gas into the liquid sucked by the liquid sucking means by the liquid discharging means.

  Further, the effect of the present invention can be obtained by mixing the liquid of the composition of the present invention with the cell change promoting substances described in [Claim 13] of Patent Document 1 and [0048] to [0050] of Patent Document 1 which is the description thereof. Can be increased.

That is , a liquid composition containing microbubbles, wherein the liquid is a cell culture solution, a substance present in the natural growth environment of the target cell in the cell culture solution, and / or a biological reaction in the target cell A composition in which at least one of the following substances 1 to 4 which are substances contributing to the above and / or biological substances is mixed is preferable. 1 gene, 2 enzyme / coenzyme / coenzyme (“·” indicates “or”) 3 recombinant protein and amino acid / peptide / glycan biosynthesized by gene (“•” is “or” 4) Substances that induce hormones, pheromones, and plant allelopathies. ("・" Indicates "or")

  A production method to which a step of mixing a cell change promoting substance is added (Claim 6) is a method for producing the composition of any one of Claims 4 and 5, wherein the liquid is a cell culture solution, and the cell culture At least one of the following substances 1 to 4 (a substance present in the natural growth environment of the target cell and / or a substance contributing to a biological reaction in the target cell and / or a biological substance) is mixed with the liquid What is necessary is just to further include a process. 1 gene, 2 enzyme / coenzyme / coenzyme (“·” indicates “or”) 3 recombinant protein and amino acid / peptide / glycan biosynthesized by gene (“•” is “or” 4) Substances that induce hormones, pheromones, and plant allelopathies. ("・" Indicates "or")

  Here, “Allelopathy” is a general term for the effect of a plant releasing substances that suppress the growth of other plants (allelochemical), or preventing or attracting animals and microorganisms. Then, it is “other feeling effect”.

  The gas of the present invention is more specifically described in [Claim 14] of Patent Document 1 and [0051] [0052] of Patent Document 1 which is an explanation thereof. It is preferable that the gas species exist in the environment and / or the gas species contribute to the biological reaction in the target cell. Of course, since the gas present in the natural growth environment is the atmosphere, argon (Ar) is also included. In addition, gas species that contribute to biological reactions in cells include nitric oxide (NO), which is notable for various cell effects, and carbon monoxide (CO), which is a harmful gas.

That is, the compositions, a composition liquid containing microbubbles, the gas species in which said gas is present in the natural habitat of the target cells, and / or, less contributes to the biological response of a subject in a cell Any one of 1 to 7 gases is preferred. 1 Nitrogen (N2), 2 Oxygen (O2), 3 Carbon dioxide (CO2), 4 Argon (Ar), 5 Hydrogen (H2), 6 Nitric oxide (NO), 7 Carbon monoxide (CO).

Similarly, the method for producing such a composition is (Claim 7), a method for producing the composition of any one of Claims 4 and 5, wherein the gas is any one of the following gases 1 to 7 or a mixture thereof: It is preferable that 1 Nitrogen (N2), 2 Oxygen (O2), 3 Carbon dioxide (CO2), 4 Argon (Ar), 5 Hydrogen (H2), 6 Nitric oxide (NO), 7 Carbon monoxide (CO).

  In carrying out the present invention described above, the additional supplementary technology of Patent Documents 1 and 2 described with reference to FIGS. 11 to 14 and the utilization technology of Taylor reactor according to Patent Document 7 of FIG. 15 are implemented in combination. be able to. That is, experimentally grasping the time variation of the particle size distribution of Mnb as shown in FIGS. 11 and 12 and the elution data for each gas molecular species associated therewith, for example, for a gas that is easily eluted, such as carbon dioxide Performing an operation to increase the amount or delaying the gas injection, using the configuration of a plurality of discharge means of FIG. 13, using the configuration of the control system having the sensor and database of FIG. 14, For example, a macro-homogenization effect such as cell manipulation can be obtained by combining with a CT (Couette-Taylor) reactor (Taylor reactor).

  The present invention relates to “P type” Mnb and nitrogen (N2) mixed with Mnb containing a single component gas having a purity of 90% or more such as nitrogen (N2), oxygen (O2), carbon dioxide (CO2) and the like. The effect of cell manipulation was improved with a “P / C mixed type” Mnb mixed with a “C type” Mnb containing a gas prepared by mixing components such as oxygen (O 2) and carbon dioxide (CO 2). .

“P-type” Mnb is described in Patent Document 1 and “C-type” Mnb is described in Patent Document 2, but the present specification clearly shows a combination of these. Of course, the inclusion gas is not limited to nitrogen (N 2), oxygen (O 2), carbon dioxide (CO 2), but includes gaseous species existing in the natural growth environment of the target cell, such as argon (Ar), and / or nitric oxide (NO). ), Carbon monoxide (CO), and other gaseous species that contribute to (influence) the biological reaction in the target cell. (This is also described in Patent Document 1 [Claim 14].)

The schematic diagram which shows that the influence which acts on a cell is larger than that of bubble Mm whose Mnb is millimeter size or more. (A) shows that it is attracting cells with a hydrophilic membrane on Mm surface M m is not in a significant impact on the cell. On the other hand, (b) shows that Mnb is smaller than the scale of cell C, (c) is an enlarged schematic diagram of (b), and the substance channel constituent CP (cell membrane of the cell membrane) where Mnb is on the cell membrane surface CS. This indicates that it may affect the protein by binding it with proximity or affinity. The schematic diagram which shows the difference in the inclusion gas of Mnb. (A) is a figure which shows "P type" Mnb including the single-component single-component gas of each purity 90% or more of nitrogen (N2), oxygen (O2), and carbon dioxide (CO2). (B) is a figure which shows "C type" Mnb including the gas which mixed nitrogen (N2), oxygen (O2), and the carbon dioxide (CO2), and adjusted the component. (C) is a figure which shows "P / C mixed type" Mnb which mixed "P type" "C type" Mnb. The schematic diagram explaining "C type" Mnb. Since Gas “CA” and Gas “CB” have different component ratios of the contained gas (second line in FIG. 3) and bubble particle size distribution (third line in FIG. 3), the effect on the cells (third line in FIG. 3). ) Is different between Gas “CA” and Gas “CB”. [CA ≠ CB] The schematic diagram explaining "P type" Mnb. Gas “PA” and Gas “PB” are different in the contained gas component ratio (second to fourth lines in FIG. 4) and bubble particle size distribution (third line in FIG. 4). 4 (second to fourth lines) are nitrogen (N2) single component gas amount (second row in FIG. 4), oxygen (O2) single component gas amount (third row in FIG. 4), carbon dioxide (CO2) single component gas By adjusting the ratio of the quantity (the fourth line in FIG. 4), it can be made substantially equal to Gas “CA” · Gas “CB”. Similarly, the bubble particle size distribution (the third line in FIG. 4) can be substantially equal to Gas “CA” Gas “CB”. But their effects are not equal. (Effect CA ≠ PA, Effect CB ≠ PB) The schematic diagram which shows the influence of Mnb which gives to the carbon dioxide (CO2) absorption of a pore. Mnb is reduced by the Young-Laplace equation [P = Pl + 2σ / r (P = gas pressure, Pl = liquid pressure, σ = surface tension, r = radius of the bubble)]. Therefore, the dissolved gas content increases locally at the gas-liquid interface and gas is eluted. Therefore, if there is Mnb containing carbon dioxide in the vicinity of the pores, the amount of carbon dioxide dissolved in the vicinity of the pores becomes extremely high, and the amount of cell membrane permeation through the pores due to diffusion increases, thereby increasing the amount of carbon dioxide absorption. The schematic diagram which shows the influence of Mnb which gives to oxygen (O2) absorption of a cell. Mnb is reduced by the Young-Laplace equation [P = Pl + 2σ / r (P = gas pressure, Pl = liquid pressure, σ = surface tension, r = radius of the bubble)]. Therefore, the dissolved gas content increases locally at the gas-liquid interface and gas is eluted. Therefore, if there is Mnb containing oxygen in the vicinity of the cell, it is considered that the amount of dissolved oxygen is extremely high in the vicinity of the cell, the amount of cell membrane permeation due to diffusion increases, and the amount of oxygen absorbed by the cell increases. Summary table of experiments 1) to 6) for comparing the effects of “P type” Mnb, “C type” Mnb, and “P / C mixed type” Mnb on cells (organisms). In general, the influence of “P / C mixed type” Mnb on cells (organisms) is large. The time chart schematic diagram of the manufacturing method of the Mnb containing liquid (liquid composition containing a microbubble) described in patent document 1 and patent document 2. FIG. (A) sucks a plurality of gases sequentially selected by the gas selection means from the gas suction means, forms the plurality of gases into microbubbles, and sequentially and sequentially to the liquid discharge means. A production method for obtaining a composition containing a plurality of gaseous microbubbles obtained by a discharging operation will be described. In (b), the cell culture solution is sucked into a plurality of microbubble generators, and a plurality of gases sucked from a plurality of gas suction means are formed as microbubbles and continuously discharged to a plurality of liquid discharge means. The manufacturing method of the composition for cell culture containing several gas microbubbles obtained by operation is shown. (C) shows the manufacturing method which combined the time sequential gas switching of (a) with the (b) manufacturing apparatus. (D) is a means for adjusting and controlling the component of the suction gas and its concentration upstream of the gas suction means, and a means for adjusting and controlling the component of the suction gas and its concentration. The manufacturing method by the apparatus provided with the output means of the gas component and concentration adjustment control target command which outputs a target command is shown. The types of Mnb by these production methods are shown in the figure. The time chart schematic diagram of the manufacturing method of the Mnb containing liquid (liquid composition containing a microbubble) of this invention. After generating “C type” Mnb, “P type” Mnb is generated and mixed to obtain “P / C mixed type” Mnb. The time chart schematic diagram of the manufacturing method of the Mnb containing liquid (liquid composition containing a microbubble) of this invention. After generating “P type” Mnb, “C type” Mnb is generated and mixed to obtain “P / C mixed type” Mnb. It is the data of the Mnb generator of Kyowa Kikai Co., Ltd., The schematic diagram which shows that a Mnb particle size shrinks with progress of time after Mnb generation | occurrence | production. The measuring instrument is Beckman Coulter's Coulter fine particle measuring instrument. Since carbon dioxide (CO2) has a higher solubility than oxygen and nitrogen, a large amount of carbon dioxide (CO2) is eluted after Mnb generation. (A) is a schematic diagram showing that carbon dioxide (CO2) elutes more with time and the inclusion amount decreases. (B) is a reference figure which shows the temperature change of the solubility with respect to the water of each gas. The schematic diagram of the Mnb generator of patent document 1. FIG. It is a correction figure of patent document 1 [FIG. 2]. The schematic diagram of the Mnb generator of patent document 2. FIG. It is a correction figure of patent document 2 [FIG. 4]. The schematic diagram of the process apparatus which combined CT (Couette-Taylor) reactor and the microbubble generator of patent document 7. FIG. Reference diagram Main components of dry air

DESCRIPTION OF SYMBOLS 1 Fixed hollow cylindrical body, or the inner wall of the rotational rotation body 41 of the inner rotation cylindrical body 32 including the inner cylindrical body which rotates coaxially, maintaining the space | interval 4 and the inner wall of the hollow rotational symmetry body 2 including the same And a gap 10 between the side outer walls of the fluid 2 and the fluid holding container H, or the fluid holding container H and the Taylor reactor TR. When cell culture is performed in the container H, H is a cell culture container (bioreactor).
11 Multiple gas supply means (one of them)
12 A means for adjusting and controlling the component of the suction gas and its concentration 12A A gas component / concentration adjustment control target command for issuing an adjustment control target command for the gas component and its concentration to the means 12 for adjusting and controlling the component of the suction gas and its concentration Output means 13 Aspirated gas supply means 17 whose components and concentrations are adjusted and controlled 17 Temperature, pressure, hydrogen ion concentration, biological / chemistry of sensor 18 10 for measuring the liquid turbidity of 10 by transmitted light or scattered light measurement method Environmental control means 18A for environmental oxygen demand, etc. 18A 10 for temperature control, pressure, hydrogen ion concentration, environmental control means for biological / chemical oxygen demand, etc. Liquid tank environment adjustment control target command output means 31 for issuing an environmental adjustment control target command For a target cell, each of a plurality of substances required to promote the change of the cell itself is taken in individually A database of data obtained by the process of experimentally determining each individual gas component and its concentration according to the cheapness, and the individual uptake of each of the substances required for the cell to promote its own change Database C1 of data obtained by a step of experimentally determining the environmental data of the liquid tank that promotes the cheapness C2 Means for supplying the material to be processed to a part of the gap 4 Material to be processed from the other part of the gap 4 Extraction means C Cell CA Effect of C-type bubble-containing gas CA CB Effect of C-type bubble-containing gas CB CS Cell membrane CT Substance that constitutes a substance channel with proteins of the cell membrane D1 Gas-liquid mixed phase containing microbubbles Means for discharging and supplying fluid D2 Means for sucking and discharging fluid H Fluid holding container. When cell culture is performed in the container H, H is a cell culture container (bioreactor).
M Microbubble generation means M4 Gas suction means M10 Eddy current pump M11 Eddy current pump built-in impeller Mnb Micro / Nano bubble Mm Effect of bubble inclusion gas PA of P-type bubble size PB Effect of bubble inclusion gas PB of P-type TR Taylor reactor

Claims (7)

A liquid composition containing microbubbles by a bubble group in which the total amount of the included gas is adjusted so that the oxygen component is 10% larger than the atmospheric composition ,
The microbubbles include at least microbubbles having a particle size of 30 μm or less, and
A composition in which the inclusion gas of the microbubbles is a mixture of at least the following two kinds.
1 Gas is a single component gas .
2 Gas is a mixed gas in which a plurality of gases are mixed. A liquid composition containing microbubbles by a bubble group in which the total amount of contained gas is adjusted so that the component of carbon dioxide is 10% larger than the atmospheric composition ,
The microbubbles include at least microbubbles having a particle size of 30 μm or less, and
A composition in which the inclusion gas of the microbubbles is a mixture of at least the following two kinds.
1 Gas is a single component gas .
2 Gas is a mixed gas in which a plurality of gases are mixed. A liquid composition containing microbubbles by a bubble group in which the total amount of inclusion gas is adjusted to 99% with nitrogen and argon and 1% carbon dioxide ,
A composition in which the inclusion gas of the microbubbles is a mixture of at least the following two kinds.
1 Gas is a single component gas .
2 Gas is a mixed gas in which a plurality of gases are mixed. Supply means of a plurality of single-component gas, setting means of the mixing ratio, including zero single component gas of the plurality of gas adjusting means for mixing the plurality gas at the set mixing ratio, suction of the adjusted gas A method for producing a composition containing microbubbles according to any one of claims 1 to 3 , using a means, a liquid suction means, a liquid discharge means, and a microbubble generator comprising a main body that generates microgas. Because
The microbubble contains at least a microbubble having a particle size of 30 μm or less and has at least the following two steps.
1 The setting of the mixing ratio setting means is set to zero except for one of a plurality of gases, and is sucked by the gas suction means, and bubbles including a single component gas are discharged into the liquid sucked by the liquid suction means. Process generated by discharging by means.
2. The setting of the setting means for the mixing ratio is set to a non-zero setting for a plurality of gases, and is sucked by the gas suction means, and bubbles including the mixed gas are discharged into the liquid sucked by the liquid suction means by the liquid discharge means. Process. Supply means of a single component gas, the supply means of the mixed gas, the single component gas or means for selectively withdrawing one of the mixed gas, a liquid suction means, a liquid discharge means, and a body to produce a small gas A method for producing a composition containing the microbubbles according to any one of claims 1 to 3 , using a microbubble generator comprising:
The microbubble contains at least a microbubble having a particle size of 30 μm or less and has at least the following two steps.
1. A step of selectively sucking the single component gas by the gas suction means and generating bubbles including the single component gas by discharging the liquid suction means into the liquid sucked by the liquid suction means.
(2) A step of generating by selectively sucking the mixed gas by the gas sucking means and discharging bubbles including the mixed gas into the liquid sucked by the liquid sucking means by the liquid discharging means. 6. The method of claim 4 or claim 5 , wherein
A method wherein the liquid is a cell culture medium and further comprises a step of mixing at least one of the following substances into the cell culture liquid.
1 gene,
2 Enzymes / Coenzymes / Coenzymes (“・” indicates “or”)
3 Recombinant proteins, amino acids, peptides, sugar chains, biosynthesized by genes (“•” indicates “or”)
4. Substances that induce allelopathies of hormones, pheromones, and plants. ("・" Indicates "or") 6. The method of claim 4 or claim 5 , wherein
The method in which the single component gas is any of the following and the mixed gas is a mixture of two or more of the following .
1 Nitrogen (N2),
2 Oxygen (O2),
3 Carbon dioxide (CO2)
4 Argon (Ar),
5 Hydrogen (H2),
6 Nitric oxide (NO),
7 Carbon monoxide (CO).

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