WO2019021528A1 - Oxygen supply mechanism - Google Patents

Oxygen supply mechanism Download PDF

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Publication number
WO2019021528A1
WO2019021528A1 PCT/JP2018/011305 JP2018011305W WO2019021528A1 WO 2019021528 A1 WO2019021528 A1 WO 2019021528A1 JP 2018011305 W JP2018011305 W JP 2018011305W WO 2019021528 A1 WO2019021528 A1 WO 2019021528A1
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Prior art keywords
oxygen
oxygen supply
mediator
supply mechanism
mediator fluid
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PCT/JP2018/011305
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French (fr)
Japanese (ja)
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優史 丸山
正樹 松森
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株式会社日立製作所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a mechanism that allows oxygen supply to cells.
  • Non-patent Document 1 As a means for supplying oxygen to transplanted cells / tissues, for example, prevascularization (prevascularization) around the transplantation site (Non-patent Document 1), for example, combined use of an oxygen-releasing material such as calcium peroxide (Patent Document 1), Oxygen generation in vivo (Patent Document 2), supply of gaseous oxygen via a gas permeable membrane (Patent Document 3), and the like are known.
  • an oxygen-releasing material such as calcium peroxide
  • Patent Document 2 Oxygen generation in vivo
  • Patent Document 3 gas permeable membrane
  • the present invention has an object to provide a closed system oxygen supply mechanism capable of long-term operation with low power and low contamination risk.
  • the present inventor has taken a closed system that continuously takes in oxygen to cells by taking in oxygen from outside air into the mediator fluid through the oxygen permeable membrane and circulating the oxygenated mediator fluid.
  • the inventors have found that the above problems can be solved by the fluid circulation mechanism of the present invention, and complete the invention.
  • the oxygen supply mechanism comprises an oxygen supply site, a mediator fluid holding vessel, and a flow path connecting the oxygen supply site and the mediator fluid holding vessel, and the oxygen supply site and the mediator fluid holding vessel are oxygen It is characterized by comprising a permeable membrane.
  • the present specification includes the disclosure content of Japanese Patent Application No. 201-147140 based on which the priority of the present application is based.
  • the present invention allows oxygen to be supplied to cells with low power for a long period of time.
  • the risk of contamination is low due to the closed system.
  • it since it has a simple structure, it has high maintainability. Problems, configurations, and effects other than those described above will be apparent from the description of the embodiments below.
  • FIG. 1 is a schematic view of an oxygen supply mechanism having one oxygen supply site, one mediator fluid holding container, and one flow passage as one embodiment of the present invention and its operation pattern.
  • FIG. 1 is a schematic view of an oxygen delivery mechanism comprising one oxygen delivery site, two mediator fluid holding vessels, and two flow paths as one embodiment of the present invention and its operating pattern.
  • FIG. 1 is a schematic view of an oxygen supply mechanism comprising one oxygen supply site, two mediator fluid holding containers, and two flow paths as one embodiment of the present invention and its operation pattern.
  • An oxygen supply mechanism comprising one oxygen supply site, two mediator fluid holding containers, and three flow paths (including a flow path with two check valves) as one embodiment of the present invention and a schematic diagram of its operation pattern FIG.
  • the oxygen supply mechanism provided by the present invention includes one or more oxygen supply sites for supplying oxygen to cells via an oxygen permeable membrane, and one or more mediator fluid holding containers for taking in oxygen from the open air via an oxygen permeable membrane. And one or more flow paths connecting the oxygen supply site and the mediator fluid holding vessel, and can operate in a closed system fluid circulation mechanism.
  • the "closed system” in the present invention refers to a system in which the transfer of substances to the outside and the transfer of substances from the outside are not performed except through the oxygen permeable membrane.
  • the shapes and materials of the oxygen supply site, the mediator fluid holding container, the flow path and the like are the oxygen taken from the outside air through the oxygen permeable membrane in the closed system fluid circulation mechanism.
  • the material of each component is desirably a highly biocompatible material such as PTFE, silicone, polyurethane, PE, PP and the like.
  • the oxygen supply site and the mediator fluid holding vessel comprise an oxygen permeable membrane.
  • the oxygen delivery site and the mediator fluid reservoir may be formed solely of an oxygen permeable membrane.
  • the oxygen permeable membrane part may be one or more places, and the size of the entire oxygen permeable membrane is not limited. Is typically 30% to 99%, preferably 60% to 95%, of the surface area of the entire oxygen delivery site, eg, 20% to 99% of the surface area of the entire mediator fluid holding vessel, eg, for a mediator fluid holding vessel. %, Preferably 40% to 95%.
  • the material of the oxygen permeable membrane is not limited.
  • a material having high oxygen permeability is desirable.
  • Such materials are, for example, polydimethylsiloxane, fluorine-based polymers and the like.
  • the oxygen permeability of the oxygen permeable membrane is more advantageous as the surface area is larger and the thickness is smaller.
  • the thickness of the oxygen permeable membrane is not limited.
  • the thickness of the oxygen permeable membrane is usually about 10 ⁇ m to 1 mm, preferably 20 ⁇ m to 0.2 mm, from the viewpoint of achieving both oxygen permeability and strength reliability.
  • the oxygen permeable membrane can also be used by being bonded to a support material to reinforce the oxygen permeable membrane.
  • the support material include PTFE, PVDF, nylon, polyamide, polyether sulfone, PE, PP, cellulose, cellulose derivatives (cellulose acetate, nitrocellulose and the like), cellulose mixed ester and the like.
  • a porous membrane as the oxygen permeable membrane, the strength can be improved without significantly reducing the oxygen permeability of the oxygen permeable membrane.
  • an oxygen permeable membrane with high strength can also be realized.
  • the mediator fluid transports oxygen taken from the outside air through the oxygen permeable membrane to the oxygen supply site and supplies the cells through the oxygen permeable membrane in the closed fluid circulation mechanism.
  • the substance is not limited as long as it can be used.
  • the mediator fluid may be gaseous or liquid.
  • As a mediator fluid if it is gas, for example, air etc. will be mentioned, if it is liquid, for example, the liquid containing an artificial red blood cell etc. will be mentioned.
  • the mediator fluid is preferably a gas in terms of the oxygen content per volume.
  • one or more oxygen supply sites may be provided.
  • the number of mediator fluid holding containers may be one or more.
  • the flow path connecting the oxygen supply site and the mediator fluid holding vessel and the flow path connecting the mediator fluid holding vessels may be one or two or more.
  • the oxygen supply mechanism provided by the present invention may include a portion that is not directly related to the supply and / or uptake of oxygen, such as a valve, a check valve, a sensor, an adsorbent, and a filter.
  • the mediator fluid can be externally replenished when the amount of mediator fluid in the mediator fluid holding container decreases.
  • the oxygen supply mechanism provided by the present invention includes a check valve in the flow path, control of the flow of the fluid is facilitated, and unnecessary mixing of the low oxygen concentration fluid and the high oxygen concentration fluid is facilitated. Can be prevented.
  • the oxygen supply mechanism provided by the present invention includes a sensor, for example, it is possible to detect a shortage of the oxygen supply amount and perform feedback control to increase the oxygen supply amount.
  • the oxygen supply mechanism provided by the present invention includes a hygroscopic agent as an adsorbent
  • the water vapor mixed in the mediator fluid can be adsorbed to prevent the flow path from being blocked due to condensation of the water vapor.
  • the power and mechanism for sending oxygen from the mediator fluid holding vessel to the oxygen supply site is not limited. It is desirable that the power and mechanism be lightweight, highly reliable, long-term usable, easily waterproof, and low sound from the viewpoint of convenience at the time of use.
  • the oxygen supply mechanism provided by the present invention is used for a human (wearer)
  • a mechanism that changes the movement of the wearer to power may be used as the mechanism, or the wearer or an assistant can manually operate. May be used.
  • the power and mechanism are at least one selected from the group consisting of power, air pressure, manual (including the use of a spring and the like), inertia force accompanying the movement of the wearer, and mechanical action accompanying the movement of the wearer. Is preferred.
  • the above-described power and mechanism can easily operate the oxygen supply mechanism of the present invention.
  • the origin, type, phenotype, presence or absence of genetic modification, passage number, etc. of cells supplying oxygen using the oxygen supply mechanism provided by the present invention are not limited. Furthermore, the nature and form of floating cells, adherent cells, single sheets, sheets and organoids are not limited. In addition, the use of the cells to which oxygen is supplied is not limited.
  • the cells are preferably transplanted cells, cells cultured for transplantation, cells cultured for useful substance production, and cells cultured for research. By using the above-mentioned cells, the effect of the improvement of the cell proliferation and function by the increase of the oxygen supply amount can be particularly remarkably obtained.
  • the use of the oxygen supply mechanism provided by the present invention is not limited, and can be used, for example, for transplantation, cell culture, cell storage, cell transport, microorganism culture, plant cultivation and the like.
  • the size of the oxygen supply mechanism provided by the present invention is not limited and can be varied depending on the application.
  • the mediator fluid having the oxygen concentration lowered by the oxygen consumption of the cells at the oxygen supply site is blown with the mediator fluid having the oxygen concentration higher than that in the mediator fluid holding container. Is supplied.
  • the oxygen concentration of the mediator fluid in the mediator fluid holding vessel temporarily decreases by mixing with the mediator fluid near the oxygen supply site, but recovers by replenishing oxygen from the outside air through the oxygen permeable membrane.
  • Such oxygen flow is completed in a closed system through the oxygen permeable membrane. That is, the isolated mediator fluid which is not in direct contact with the outside air takes in oxygen from the outside air through the oxygen permeable membrane of the mediator fluid holding container, is carried to the oxygen supplying site, and cells through the oxygen permeable membrane of the oxygen supplying site.
  • the source of oxygen supplied to the cells is not limited, but may be oxygen in the atmosphere, so a mechanism such as a gas cylinder, a gas syringe, or a gas generator may not be used, and an oxygen supply mechanism The whole can be simplified.
  • the oxygen supply mechanism includes one oxygen supply site G having an oxygen permeable membrane B1, one mediator fluid holding vessel H having an oxygen permeable membrane B2, an oxygen supply site G and a mediator fluid holding vessel H. And one channel C to be connected.
  • the high oxygen fluid E (mediator fluid with high oxygen concentration) is supplied to the oxygen supply site G through the channel C.
  • the oxygen supply site G preferably includes an expandable site, such as a stretchable membrane, a folding mechanism, a bellows mechanism, a sliding mechanism, etc., in order to receive the hyperoxic fluid E.
  • the highly oxygenated fluid E supplied to the oxygen supply site G returns to the inside of the mediator fluid holding container H again by releasing the compression of the mediator fluid holding container H.
  • the high oxygen fluid E is mixed with the low oxygen fluid D in the vicinity of the oxygen supply site G (a mediator fluid whose oxygen concentration is lowered by supplying oxygen to the cell A through the oxygen permeable membrane B1).
  • the oxygen concentration is reduced and becomes medium oxygen fluid F (mediator fluid having oxygen concentration between high oxygen fluid and low oxygen fluid).
  • medium oxygen fluid F becomes the high oxygen fluid E again by the oxygen supply from the outside air through the oxygen permeable membrane B 2 in the mediator fluid holding container H. By repeating this, oxygen can be continuously supplied to the cell A.
  • the expansion of the oxygen supplying site G may stir the liquid near the cell A. While this can be a stress to the cell A, there is also a possibility that a high efficiency liquid exchange effect can be obtained by liquid agitation. Under conditions where stress on cell A is sufficiently small, such as when using cell A that is resistant to shear stress or when cell A is in a state in which shear stress is less likely to occur due to the shape of the scaffold (three-dimensional structure)
  • the expansion of the oxygen supply site G may be able to receive only the merit of highly efficient liquid exchange, and is preferable not only from oxygen supply but also from the viewpoint of nutrient supply and metabolite removal.
  • the oxygen supply mechanism includes one oxygen supply site G having an oxygen permeable membrane B1 and two mediator fluid holding vessels having an oxygen permeable membrane B2, ie, a first mediator fluid holding vessel H1 and a second A mediator fluid holding vessel H2 and two flow paths, ie, a first flow path C1 connecting the oxygen supplying site and the first mediator fluid holding vessel H1, and an oxygen supplying site and a second mediator fluid holding vessel H2 And a second channel C2 connecting the two.
  • the high oxygen fluid E moves to the oxygen supply site G through the first flow channel C1, and the oxygen supply site G
  • the low oxygen fluid D mixes with the high oxygen fluid E to become a medium oxygen fluid F, and moves to the second mediator fluid holding container H2 through the second flow passage C2.
  • the second mediator fluid holding vessel H2 preferably includes an expandable portion to receive the high oxygen fluid E.
  • the oxygen supplying site G does not necessarily have to be an expandable site, but can be provided to receive the merits of the liquid exchange as described above.
  • the oxygen supply site G and the second mediator fluid holding vessel H1 is returned to the first mediator fluid holding vessel H1 through the first channel C1.
  • the oxygen concentration of the first mediator fluid holding vessel H1, the oxygen supplying site G and the second mediator fluid holding vessel H2 is relatively averaged, and the mediator fluid becomes the medium oxygen fluid F throughout.
  • oxygen is supplied from the outside air through the oxygen permeable membrane B2, and the medium oxygen fluid F becomes the high oxygen fluid E.
  • the cycle of operation is a repetition of the compression of the first mediator fluid holding container H1 and the compression of the second mediator fluid holding container H2. If it is possible to wait for the oxygen concentration in the mediator fluid holding containers H1 and H2 to become sufficiently high, the high oxygen fluid E is continuously sent to the oxygen supply site G by such a cycle of operation. Can.
  • the oxygen supply mechanism is one oxygen supply site G having an oxygen permeable membrane B1 and two mediator fluid holding vessels having an oxygen permeable membrane B2, ie, a first mediator fluid holding vessel H1 and a second A first channel C1 connecting the mediator fluid holding vessel H2 and three flow paths, ie, the oxygen supplying site G and the first mediator fluid holding vessel H1, an oxygen supplying site G and a second mediator fluid holding vessel A second channel C2 (with a check valve) connecting H2 and a third channel C3 (a check valve connecting the first mediator fluid holding container H1 and the second mediator fluid holding container H2 And B.) can be provided.
  • the medium oxygen fluid F that has entered the second mediator fluid holding container H2 becomes the second flow passage C2 and the third flow passage C3.
  • the non-return valve returns to the first mediator fluid holding container H1 without returning to the oxygen supply site G.
  • the high oxygen fluid E can be continuously sent to the oxygen supply site G by adopting such a cycle of operation.
  • Example 1 In Example 1, an oxygen supply mechanism including one oxygen supply site G, one mediator fluid holding vessel H, and one flow path C connecting them was studied (see FIG. 1). Molding the PDMS film having a thickness of 0.03 mm (oxygen permeable membrane), and the volume change can oxygenation site G in the range of 5 cm 3 ⁇ 10 cm 3 surface area 70cm 2, range in surface area 70cm 2 of 45cm 3 ⁇ 50 cm 3 An oxygen supply mechanism 1 including a volume changeable mediator fluid holding container H and a flow path C was produced.
  • Example 2 In the second embodiment, an oxygen supply comprising two flow paths C1 and C2 connecting one oxygen supply site G, two mediator fluid holding vessels H1 and H2, an oxygen supply site G and a mediator fluid holding vessels H1 and H2
  • the mechanism was considered (see FIGS. 2 and 3). Molding the PDMS film having a thickness of 0.03 mm (oxygen permeable membrane), an oxygen supply portion G of volume 7 cm 3 surface area 70cm 2, volume change possible mediators fluid maintained within the range of 15cm 3 ⁇ 30 cm 3 surface area 70cm 2
  • the oxygen supply mechanism 2 provided with the containers H1 and H2 and the flow paths C1 and C2 was produced.
  • Example 3 In the third embodiment, two flow paths C1 and C2 and two mediator fluids connecting one oxygen supplying site G, two mediator fluid holding vessels H1 and H2, oxygen supplying site G and mediator fluid holding vessels H1 and H2 An oxygen supply mechanism was studied which was provided with a flow path C3 connecting the holding containers H1 and H2 with each other, and one check valve in each of the flow paths C2 and C3 (see FIG. 4).
  • the oxygen supply mechanism 3 including the containers H1 and H2, the flow path C1 and the flow paths C2 and C3 with a check valve was manufactured.
  • Example 4 the oxygen supply rate to deoxygenated water was examined using the oxygen supply mechanisms 1, 2 and 3.
  • the oxygen supply mechanism 1 After the mediator fluid holding vessel H is compressed so that the flow rate of the mediator fluid passing through the flow path C is 6.0 mL / min, the flow rate from the oxygen supply site G to the mediator fluid holding vessel H The cycle of decompression was repeated so that the mediator fluid returned at 30 mL / min.
  • the fluid was circulated by alternately compressing the mediator fluid holding containers H1 and H2 so that the flow rate of the mediator fluid passing through the flow channels C1 and C2 would be 3.0 mL / min.
  • Figure 3 operation method
  • the average oxygen supply rate was 1.9 ⁇ mol / min for the oxygen supply mechanism 1, 2.4 ⁇ mol / min for the oxygen supply mechanism 2, and 2.4 ⁇ mol / min for the oxygen supply mechanism 3. These are, depending on the type of cells, an oxygen supply rate sufficient to maintain cell numbers of up to about 10 9 to 10 10 .
  • Example 5 In Example 5, the in vitro cell culture was examined using the oxygen supply mechanism 2. HepG2 cells were used as the cells, and the proliferative ability of the cells was evaluated in a state where the oxygen supply mechanism 2 was placed near the cells and operated. As a result, cell death was observed until the cell number 10 7. This is considered to be an effect derived from oxygen supply by the oxygen supply mechanism 2.
  • Comparative Example 1 In Comparative Example 1, in the same manner as Example 5, examination of in vitro cell culture was conducted without oxygen supply by the oxygen supply mechanism 2. As a result, from the cell number of about 10 6 growth arrest of cells considered to be caused by oxygen deficiency was observed.
  • Example 6 In Example 6, the waterproofness (contamination risk of water) of the oxygen supply mechanism 2 was examined. After the oxygen supply mechanism 2 was put in water and a load was applied for 24 hours, water which entered the inside of the oxygen supply mechanism 2 was quantified. As a result, the amount of water entering the inside of the oxygen supply mechanism 2 was below the detection limit.
  • Comparative Example 2 In Comparative Example 2, the waterproofness of the non-closed oxygen supply mechanism was examined. In the same structure as the oxygen supply mechanism 1, the comparative supply mechanism 1 attached with a filter instead of the mediator fluid holding container was prepared, put into water and loaded for 24 hours, and then entered the inside of the comparative supply mechanism 1. Water was quantified. As a result, about 0.2 g of water entered the inside of the comparative supply mechanism 1 with the fluctuation of the internal pressure due to the application of the load.
  • the present invention is not limited to the above-described embodiment, but includes various modifications. For example, with respect to a part of the configuration of the embodiment, it is possible to add, delete, and replace other configurations.
  • A cell (consumption of oxygen), B1 and B2: oxygen permeable membrane, C and C1 to C3: flow path, D: hypoxic fluid, E: hyperoxic fluid, F: medium oxygen fluid, G: oxygen supplying site, H and H1 and H2: mediator fluid holding container

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Abstract

The present invention addresses the problem of providing a closed system oxygen supply mechanism with which long-term operation with low power is possible and contamination risk is low. The present invention relates to an oxygen supply mechanism characterized in: being provided with an oxygen supply region, a mediator fluid-holding container, and a flow channel connecting the oxygen supply region with the mediator fluid-holding container; and the oxygen supply region and mediator fluid-holding container being provided with oxygen-permeable membranes.

Description

酸素供給機構Oxygen supply mechanism
　本発明は、細胞への酸素供給を可能にする機構（デバイス）に関する。 The present invention relates to a mechanism that allows oxygen supply to cells.
　再生医療等における細胞・組織移植では、移植された細胞・組織への酸素供給が不足することによる細胞死が課題となっている。移植細胞・組織への酸素供給手段としては、例えば、移植部位周辺におけるプレ血管新生法（ｐｒｅｖａｓｃｕｌａｒｉｚａｔｉｏｎ）（非特許文献１）、例えば過酸化カルシウム等の酸素徐放材の併用（特許文献１）、ｉｎ　ｖｉｖｏでの酸素発生（特許文献２）、気体透過膜を介した気体状酸素の供給（特許文献３）等が知られている。 In cell and tissue transplantation in regenerative medicine and the like, cell death due to lack of oxygen supply to the transplanted cells and tissue has been a problem. As a means for supplying oxygen to transplanted cells / tissues, for example, prevascularization (prevascularization) around the transplantation site (Non-patent Document 1), for example, combined use of an oxygen-releasing material such as calcium peroxide (Patent Document 1), Oxygen generation in vivo (Patent Document 2), supply of gaseous oxygen via a gas permeable membrane (Patent Document 3), and the like are known.
米国特許出願公開第２０１２／０１１４７２９号明細書US Patent Application Publication No. 2012/0114729 米国特許出願公開第２００３／００８７４２７号明細書US Patent Application Publication No. 2003/0087427 米国特許第８７８４３８９号明細書U.S. Patent No. 8784389
　これらの中でも、気体透過膜を介した気体状酸素の供給は、他手法と比較して簡便である。 Among these, supply of gaseous oxygen via a gas permeable membrane is simple as compared with other methods.
　しかしながら、気体透過膜を介した気体状酸素の供給では、定期的な流体の再充填が必要であり、さらには、外部との接触によるデバイス内部のコンタミネーションリスクもある。 However, the supply of gaseous oxygen through the gas permeable membrane requires periodical fluid refilling, and there is also the risk of contamination inside the device due to contact with the outside.
　上記従来の状況に鑑み、本発明は、低動力で長期稼働が可能であり、コンタミネーションリスクの低い閉鎖系酸素供給機構を提供することを課題とする。 SUMMARY OF THE INVENTION In view of the above-described conventional situation, the present invention has an object to provide a closed system oxygen supply mechanism capable of long-term operation with low power and low contamination risk.
　本発明者は、鋭意研究を重ねた結果、酸素透過膜を介して外気から酸素をメディエータ流体中に取り込み、酸素を取り込んだメディエータ流体を循環させることで細胞に連続して酸素を供給する閉鎖系の流体循環機構によって、上記課題を解決できることを見出し、発明を完成した。 As a result of intensive studies, the present inventor has taken a closed system that continuously takes in oxygen to cells by taking in oxygen from outside air into the mediator fluid through the oxygen permeable membrane and circulating the oxygenated mediator fluid. The inventors have found that the above problems can be solved by the fluid circulation mechanism of the present invention, and complete the invention.
　すなわち、本発明に係る酸素供給機構は、酸素供給部位と、メディエータ流体保持容器と、酸素供給部位とメディエータ流体保持容器とを接続する流路とを備え、酸素供給部位及びメディエータ流体保持容器が酸素透過膜を備えることを特徴とする。 That is, the oxygen supply mechanism according to the present invention comprises an oxygen supply site, a mediator fluid holding vessel, and a flow path connecting the oxygen supply site and the mediator fluid holding vessel, and the oxygen supply site and the mediator fluid holding vessel are oxygen It is characterized by comprising a permeable membrane.
　本明細書は本願の優先権の基礎となる日本国特許出願番号２０１７－１４７１４０号の開示内容を包含する。 The present specification includes the disclosure content of Japanese Patent Application No. 201-147140 based on which the priority of the present application is based.
　本発明により、細胞に対して低動力で長期間にわたって酸素供給が可能になる。また、閉鎖系のためコンタミネーションリスクが低い。さらに、単純な構造であるため、メンテナンス性が高い。上記した以外の課題、構成及び効果は、以下の実施形態の説明により明らかにされる。 The present invention allows oxygen to be supplied to cells with low power for a long period of time. In addition, the risk of contamination is low due to the closed system. Furthermore, since it has a simple structure, it has high maintainability. Problems, configurations, and effects other than those described above will be apparent from the description of the embodiments below.
本発明の一実施形態としての、１つの酸素供給部位と１つのメディエータ流体保持容器と１つの流路とを備える酸素供給機構及びその動作パターンの模式図である。FIG. 1 is a schematic view of an oxygen supply mechanism having one oxygen supply site, one mediator fluid holding container, and one flow passage as one embodiment of the present invention and its operation pattern. 本発明の一実施形態としての、１つの酸素供給部位と２つのメディエータ流体保持容器と２つの流路とを備える酸素供給機構及ぶその動作パターンの模式図である。FIG. 1 is a schematic view of an oxygen delivery mechanism comprising one oxygen delivery site, two mediator fluid holding vessels, and two flow paths as one embodiment of the present invention and its operating pattern. 本発明の一実施形態としての、１つの酸素供給部位と２つのメディエータ流体保持容器と２つの流路とを備える酸素供給機構及びその動作パターンの模式図である。FIG. 1 is a schematic view of an oxygen supply mechanism comprising one oxygen supply site, two mediator fluid holding containers, and two flow paths as one embodiment of the present invention and its operation pattern. 本発明の一実施形態としての、１つの酸素供給部位と２つのメディエータ流体保持容器と３つの流路（２つの逆止弁付き流路を含む）とを備える酸素供給機構及びその動作パターンの模式図である。An oxygen supply mechanism comprising one oxygen supply site, two mediator fluid holding containers, and three flow paths (including a flow path with two check valves) as one embodiment of the present invention and a schematic diagram of its operation pattern FIG.
　以下、図面等を用いて、本発明の実施形態について説明する。以下の説明は本発明の内容の具体例を示すものであり、本発明がこれらの説明に限定されるものではなく、本明細書に開示される技術的思想の範囲内において当業者による様々な変更及び修正が可能である。また、本発明を説明するための全図において、同一の機能を有するものは、同一の符号を付け、その繰り返しの説明は省略する場合がある。 Hereinafter, embodiments of the present invention will be described using the drawings and the like. The following description shows specific examples of the content of the present invention, and the present invention is not limited to these descriptions, and various modifications by those skilled in the art can be made within the scope of the technical idea disclosed herein. Changes and modifications are possible. Moreover, in all the drawings for explaining the present invention, what has the same function may attach the same numerals, and may omit explanation of the repetition.
　本発明で提供する酸素供給機構は、酸素透過膜を介して細胞に酸素を供給する１つ以上の酸素供給部位、酸素透過膜を介して外気から酸素を取り込む１つ以上のメディエータ流体保持容器、及び酸素供給部位とメディエータ流体保持容器とを接続する１つ以上の流路を備え、閉鎖系の流体循環機構の状態で稼働できることを特徴とする。 The oxygen supply mechanism provided by the present invention includes one or more oxygen supply sites for supplying oxygen to cells via an oxygen permeable membrane, and one or more mediator fluid holding containers for taking in oxygen from the open air via an oxygen permeable membrane. And one or more flow paths connecting the oxygen supply site and the mediator fluid holding vessel, and can operate in a closed system fluid circulation mechanism.
　本発明における「閉鎖系」とは、酸素透過膜を介する以外に、外部への物質の移動及び外部からの物質の移動が行われない系を指す。 The "closed system" in the present invention refers to a system in which the transfer of substances to the outside and the transfer of substances from the outside are not performed except through the oxygen permeable membrane.
　本発明で提供する酸素供給機構において、酸素供給部位、メディエータ流体保持容器、流路等の形状及び素材は、閉鎖系の流体循環機構において、酸素透過膜を介して外気から取り込んだ酸素を、酸素供給部位まで運び、酸素透過膜を介して細胞に供給することが可能であれば、限定されない。本発明で提供する酸素供給機構において、各部品の素材としては、生体適合性の高い素材、例えばＰＴＦＥ、シリコーン、ポリウレタン、ＰＥ、ＰＰ等であることが望ましい。 In the oxygen supply mechanism provided by the present invention, the shapes and materials of the oxygen supply site, the mediator fluid holding container, the flow path and the like are the oxygen taken from the outside air through the oxygen permeable membrane in the closed system fluid circulation mechanism. There is no limitation as long as it can be delivered to the delivery site and delivered to the cell via the oxygen permeable membrane. In the oxygen supply mechanism provided by the present invention, the material of each component is desirably a highly biocompatible material such as PTFE, silicone, polyurethane, PE, PP and the like.
　本発明で提供する酸素供給機構において、酸素供給部位及びメディエータ流体保持容器は、酸素透過膜を備える。酸素供給部位及びメディエータ流体保持容器は、酸素透過膜のみから形成されていてもよい。酸素供給部位及びメディエータ流体保持容器の一部が酸素透過膜により形成される場合、酸素透過膜部分は、一箇所以上でよく、酸素透過膜全体の大きさは、限定されないが、例えば酸素供給部位であれば、酸素供給部位全体の表面積の通常３０％～９９％、好ましくは６０％～９５％であり、例えばメディエータ流体保持容器であれば、メディエータ流体保持容器全体の表面積の通常２０％～９９％、好ましくは４０％～９５％である。 In the oxygen supply mechanism provided by the present invention, the oxygen supply site and the mediator fluid holding vessel comprise an oxygen permeable membrane. The oxygen delivery site and the mediator fluid reservoir may be formed solely of an oxygen permeable membrane. When the oxygen supply site and a part of the mediator fluid holding container are formed of an oxygen permeable membrane, the oxygen permeable membrane part may be one or more places, and the size of the entire oxygen permeable membrane is not limited. Is typically 30% to 99%, preferably 60% to 95%, of the surface area of the entire oxygen delivery site, eg, 20% to 99% of the surface area of the entire mediator fluid holding vessel, eg, for a mediator fluid holding vessel. %, Preferably 40% to 95%.
　本発明で提供する酸素供給機構において、酸素透過膜の素材は限定されない。酸素透過膜としては、酸素透過性が高い素材が望ましい。このような素材は、例えばポリジメチルシロキサン、フッ素系ポリマー等である。酸素透過膜の酸素透過性は、表面積が大きく、厚みが小さいほど有利である。 In the oxygen supply mechanism provided by the present invention, the material of the oxygen permeable membrane is not limited. As the oxygen permeable membrane, a material having high oxygen permeability is desirable. Such materials are, for example, polydimethylsiloxane, fluorine-based polymers and the like. The oxygen permeability of the oxygen permeable membrane is more advantageous as the surface area is larger and the thickness is smaller.
　本発明で提供する酸素供給機構において、酸素透過膜の厚みは限定されない。素材としてポリジメチルシロキサンを用いる場合、酸素透過膜の厚みは、酸素透過性と強度信頼性の両立の観点から通常１０μｍ～１ｍｍ程度、好ましくは２０μｍ～０．２ｍｍの範囲が望ましい。 In the oxygen supply mechanism provided by the present invention, the thickness of the oxygen permeable membrane is not limited. When polydimethylsiloxane is used as the material, the thickness of the oxygen permeable membrane is usually about 10 μm to 1 mm, preferably 20 μm to 0.2 mm, from the viewpoint of achieving both oxygen permeability and strength reliability.
　本発明で提供する酸素供給機構において、酸素透過膜は、酸素透過膜の補強のためにサポート材と貼り合わせて使用することもできる。サポート材としては、例えばＰＴＦＥ、ＰＶＤＦ、ナイロン、ポリアミド、ポリエーテルスルホン、ＰＥ、ＰＰ、セルロース、セルロース誘導体（酢酸セルロース、ニトロセルロース等）、セルロース混合エステル等が挙げられる。また、酸素透過膜として多孔質膜を用いることで、酸素透過膜の酸素透過性を大きく下げることなく、強度を向上することができる。さらに、酸素透過膜の構造を、多孔質材の細孔内に酸素透過性の素材を導入した構造にすることで、強度の高い酸素透過膜を実現することもできる。 In the oxygen supply mechanism provided by the present invention, the oxygen permeable membrane can also be used by being bonded to a support material to reinforce the oxygen permeable membrane. Examples of the support material include PTFE, PVDF, nylon, polyamide, polyether sulfone, PE, PP, cellulose, cellulose derivatives (cellulose acetate, nitrocellulose and the like), cellulose mixed ester and the like. Further, by using a porous membrane as the oxygen permeable membrane, the strength can be improved without significantly reducing the oxygen permeability of the oxygen permeable membrane. Furthermore, by making the structure of the oxygen permeable membrane into a structure in which an oxygen permeable material is introduced into the pores of the porous material, an oxygen permeable membrane with high strength can also be realized.
　本発明が提供する酸素供給機構において、メディエータ流体は、閉鎖系の流体循環機構において、酸素透過膜を介して外気から取り込んだ酸素を、酸素供給部位まで運び、酸素透過膜を介して細胞に供給することができる物質であれば、限定されない。メディエータ流体は、気体でも液体でもよい。メディエータ流体としては、気体であれば、例えば空気等、液体であれば、例えば人工赤血球を含む液体等が挙げられる。メディエータ流体は、体積あたりの酸素含有可能量の観点から、気体であることが好ましい。 In the oxygen supply mechanism provided by the present invention, the mediator fluid transports oxygen taken from the outside air through the oxygen permeable membrane to the oxygen supply site and supplies the cells through the oxygen permeable membrane in the closed fluid circulation mechanism. The substance is not limited as long as it can be used. The mediator fluid may be gaseous or liquid. As a mediator fluid, if it is gas, for example, air etc. will be mentioned, if it is liquid, for example, the liquid containing an artificial red blood cell etc. will be mentioned. The mediator fluid is preferably a gas in terms of the oxygen content per volume.
　本発明が提供する酸素供給機構において、酸素供給部位は、１つでも２つ以上でもよい。 In the oxygen supply mechanism provided by the present invention, one or more oxygen supply sites may be provided.
　本発明が提供する酸素供給機構において、メディエータ流体保持容器は、１つでも２つ以上でもよい。 In the oxygen supply mechanism provided by the present invention, the number of mediator fluid holding containers may be one or more.
　本発明が提供する酸素供給機構において、酸素供給部位とメディエータ流体保持容器とを接続する流路及びメディエータ流体保持容器同士を接続する流路は、１つでも２つ以上でもよい。 In the oxygen supply mechanism provided by the present invention, the flow path connecting the oxygen supply site and the mediator fluid holding vessel and the flow path connecting the mediator fluid holding vessels may be one or two or more.
　本発明が提供する酸素供給機構は、バルブ、逆止弁、センサ、吸着材、フィルタ等の酸素の供給及び／又は取り込みに直接関係しない部位を含んでいてもよい。 The oxygen supply mechanism provided by the present invention may include a portion that is not directly related to the supply and / or uptake of oxygen, such as a valve, a check valve, a sensor, an adsorbent, and a filter.
　例えば本発明が提供する酸素供給機構がバルブを備える場合、メディエータ流体保持容器内のメディエータ流体の量が低下した場合に、メディエータ流体を外部から補充することができる。また、例えば本発明が提供する酸素供給機構が流路内に逆止弁を備える場合、流体の流れの制御が容易となり、低酸素濃度の流体と高酸素濃度の流体との不要な混合を容易に防ぐことができる。さらに、例えば本発明が提供する酸素供給機構がセンサを備える場合、例えば酸素供給量の不足を検知し、酸素供給量を上昇させるようなフィードバック制御を行うことができる。また、例えば本発明が提供する酸素供給機構が吸着材として吸湿剤を備える場合、メディエータ流体中に混入した水蒸気を吸着し、水蒸気の結露による流路の閉塞を予防することができる。 For example, when the oxygen supply mechanism provided by the present invention includes a valve, the mediator fluid can be externally replenished when the amount of mediator fluid in the mediator fluid holding container decreases. Also, for example, when the oxygen supply mechanism provided by the present invention includes a check valve in the flow path, control of the flow of the fluid is facilitated, and unnecessary mixing of the low oxygen concentration fluid and the high oxygen concentration fluid is facilitated. Can be prevented. Furthermore, for example, when the oxygen supply mechanism provided by the present invention includes a sensor, for example, it is possible to detect a shortage of the oxygen supply amount and perform feedback control to increase the oxygen supply amount. Further, for example, when the oxygen supply mechanism provided by the present invention includes a hygroscopic agent as an adsorbent, the water vapor mixed in the mediator fluid can be adsorbed to prevent the flow path from being blocked due to condensation of the water vapor.
　本発明が提供する酸素供給機構において、メディエータ流体保持容器から酸素供給部位に酸素を送るための動力・機構は限定されない。動力・機構は、使用時の利便性の観点から、軽量・高信頼性・長期使用可能・防水容易・小音であるものが望ましい。例えば、本発明で提供する酸素供給機構をヒト（装着者）に使用する場合、機構として、装着者の動きを動力に変える機構を用いてもよいし、装着者や介助者が手動で動作可能である機構を用いてもよい。動力・機構は、電力、空気圧、手動（ゼンマイバネ等の使用も含む）、装着者の動きに伴う慣性力、及び装着者の動きに伴う力学的な作用からなる群から選択される少なくとも１つ以上であることが好ましい。上記動力・機構により、本発明の酸素供給機構を容易に稼働させることができる。 In the oxygen supply mechanism provided by the present invention, the power and mechanism for sending oxygen from the mediator fluid holding vessel to the oxygen supply site is not limited. It is desirable that the power and mechanism be lightweight, highly reliable, long-term usable, easily waterproof, and low sound from the viewpoint of convenience at the time of use. For example, when the oxygen supply mechanism provided by the present invention is used for a human (wearer), a mechanism that changes the movement of the wearer to power may be used as the mechanism, or the wearer or an assistant can manually operate. May be used. The power and mechanism are at least one selected from the group consisting of power, air pressure, manual (including the use of a spring and the like), inertia force accompanying the movement of the wearer, and mechanical action accompanying the movement of the wearer. Is preferred. The above-described power and mechanism can easily operate the oxygen supply mechanism of the present invention.
　本発明で提供する酸素供給機構を用いて酸素を供給する細胞の由来、種類、フェノタイプ、遺伝子改変の有無、継代数等は限定されない。さらに、浮遊細胞、接着細胞、シングル、シート、オルガノイドの性質・形態も限定されない。また、酸素供給先の細胞の用途も限定されない。細胞は、移植細胞、移植向けに培養される細胞、有用物質産生向けに培養される細胞、及び研究向けに培養される細胞が好ましい。上記細胞を使用することにより、酸素供給量の増大による細胞の増殖性や機能の向上の効果が特に顕著に得られる。 The origin, type, phenotype, presence or absence of genetic modification, passage number, etc. of cells supplying oxygen using the oxygen supply mechanism provided by the present invention are not limited. Furthermore, the nature and form of floating cells, adherent cells, single sheets, sheets and organoids are not limited. In addition, the use of the cells to which oxygen is supplied is not limited. The cells are preferably transplanted cells, cells cultured for transplantation, cells cultured for useful substance production, and cells cultured for research. By using the above-mentioned cells, the effect of the improvement of the cell proliferation and function by the increase of the oxygen supply amount can be particularly remarkably obtained.
　本発明が提供する酸素供給機構の用途は限定されず、例えば移植、細胞培養、細胞保存、細胞輸送、微生物培養、植物育成等に使用することができる。本発明が提供する酸素供給機構の大きさは限定されず、用途に応じて変更することができる。 The use of the oxygen supply mechanism provided by the present invention is not limited, and can be used, for example, for transplantation, cell culture, cell storage, cell transport, microorganism culture, plant cultivation and the like. The size of the oxygen supply mechanism provided by the present invention is not limited and can be varied depending on the application.
　本発明が提供する酸素供給機構では、酸素供給部位における細胞の酸素消費により低下した酸素濃度を有するメディエータ流体に、メディエータ流体保持容器におけるそれより高い酸素濃度を有するメディエータ流体が吹き込まれることで、酸素が供給される。メディエータ流体保持容器中のメディエータ流体の酸素濃度は、酸素供給部位近傍のメディエータ流体と混合されることで一時的に低下するが、酸素透過膜を介して外気から酸素を補充することにより回復する。このような酸素の流れが、酸素透過膜を介することで閉鎖系の中で完結する。すなわち、外気と直接接触しない隔離されたメディエータ流体が、メディエータ流体保持容器の酸素透過膜を介して外気から酸素を取り込み、酸素供給部位まで運ばれて、酸素供給部位の酸素透過膜を介して細胞に酸素を供給する。このことにより、コンタミネーションリスクとなる外気を細胞の近傍まで導入しなくても細胞に酸素を供給することができる。さらに、細胞に供給する酸素の供給源は、限定されないが、大気中の酸素にすることもできるため、ガスボンベやガスシリンジ、ガス発生装置のような機構を使用しなくてもよく、酸素供給機構全体を簡便にすることができる。これらの特徴により、本発明は、移植のような安全性と簡便性が重視される用途において特に効果的である。 In the oxygen supply mechanism provided by the present invention, the mediator fluid having the oxygen concentration lowered by the oxygen consumption of the cells at the oxygen supply site is blown with the mediator fluid having the oxygen concentration higher than that in the mediator fluid holding container. Is supplied. The oxygen concentration of the mediator fluid in the mediator fluid holding vessel temporarily decreases by mixing with the mediator fluid near the oxygen supply site, but recovers by replenishing oxygen from the outside air through the oxygen permeable membrane. Such oxygen flow is completed in a closed system through the oxygen permeable membrane. That is, the isolated mediator fluid which is not in direct contact with the outside air takes in oxygen from the outside air through the oxygen permeable membrane of the mediator fluid holding container, is carried to the oxygen supplying site, and cells through the oxygen permeable membrane of the oxygen supplying site. Supply oxygen to As a result, it is possible to supply oxygen to the cells without introducing the outside air, which is a contamination risk, to the vicinity of the cells. Furthermore, the source of oxygen supplied to the cells is not limited, but may be oxygen in the atmosphere, so a mechanism such as a gas cylinder, a gas syringe, or a gas generator may not be used, and an oxygen supply mechanism The whole can be simplified. These features make the invention particularly effective in applications where safety and simplicity are important, such as transplantation.
　本発明が提供する酸素供給機構の一例を、図１を使用して説明する。
　図１では、酸素供給機構は、酸素透過膜Ｂ１を有する１つの酸素供給部位Ｇと、酸素透過膜Ｂ２を有する１つのメディエータ流体保持容器Ｈと、酸素供給部位Ｇとメディエータ流体保持容器Ｈとを接続する１つの流路Ｃとを備える。 An example of the oxygen supply mechanism provided by the present invention will be described using FIG.
In FIG. 1, the oxygen supply mechanism includes one oxygen supply site G having an oxygen permeable membrane B1, one mediator fluid holding vessel H having an oxygen permeable membrane B2, an oxygen supply site G and a mediator fluid holding vessel H. And one channel C to be connected.
　図１の場合では、例えば、メディエータ流体保持容器Ｈを圧縮することで高酸素流体Ｅ（酸素濃度が高いメディエータ流体）が流路Ｃを通って、酸素供給部位Ｇに供給される。酸素供給部位Ｇは、高酸素流体Ｅを受け取るために、膨張可能な部位、例えば伸縮可能な膜、折りたたみ機構、蛇腹状機構、スライド式機構等を含むことが好ましい。酸素供給部位Ｇに供給された高酸素流体Ｅは、メディエータ流体保持容器Ｈの圧縮を解除することで再度メディエータ流体保持容器Ｈの内部に戻る。このとき、高酸素流体Ｅは、酸素供給部位Ｇ近傍の低酸素流体Ｄ（酸素透過膜Ｂ１を介して細胞Ａに酸素を供給したことにより酸素濃度が低くなったメディエータ流体）と混合することで酸素濃度が低下し、中酸素流体Ｆ（高酸素流体と低酸素流体の間の酸素濃度を有するメディエータ流体）になっている。この中酸素流体Ｆは、メディエータ流体保持容器Ｈ内において酸素透過膜Ｂ２を介した外気からの酸素供給によって再度高酸素流体Ｅになる。これを繰り返すことで、酸素を細胞Ａに供給し続けることができる。 In the case of FIG. 1, for example, by compressing the mediator fluid holding container H, the high oxygen fluid E (mediator fluid with high oxygen concentration) is supplied to the oxygen supply site G through the channel C. The oxygen supply site G preferably includes an expandable site, such as a stretchable membrane, a folding mechanism, a bellows mechanism, a sliding mechanism, etc., in order to receive the hyperoxic fluid E. The highly oxygenated fluid E supplied to the oxygen supply site G returns to the inside of the mediator fluid holding container H again by releasing the compression of the mediator fluid holding container H. At this time, the high oxygen fluid E is mixed with the low oxygen fluid D in the vicinity of the oxygen supply site G (a mediator fluid whose oxygen concentration is lowered by supplying oxygen to the cell A through the oxygen permeable membrane B1). The oxygen concentration is reduced and becomes medium oxygen fluid F (mediator fluid having oxygen concentration between high oxygen fluid and low oxygen fluid). The medium oxygen fluid F becomes the high oxygen fluid E again by the oxygen supply from the outside air through the oxygen permeable membrane B 2 in the mediator fluid holding container H. By repeating this, oxygen can be continuously supplied to the cell A.
　酸素供給部位Ｇは細胞Ａの近傍に位置するため、酸素供給部位Ｇの膨張は細胞Ａ近傍の液体を撹拌し得る。これは、細胞Ａに対してストレスになり得る一方で、液体の撹拌による高効率な液交換という効果が得られる可能性もある。せん断応力に強い細胞Ａを用いている場合や、足場材（三次元構造体）の形状等によって細胞Ａがせん断応力を受けにくい状態にある場合等の、細胞Ａに対するストレスが十分に小さい条件では、酸素供給部位Ｇの膨張は、高効率な液交換によるメリットのみを受けられる可能性があり、酸素供給だけでなく栄養供給や代謝物除去の観点からも好ましい。 Since the oxygen supplying site G is located in the vicinity of the cell A, the expansion of the oxygen supplying site G may stir the liquid near the cell A. While this can be a stress to the cell A, there is also a possibility that a high efficiency liquid exchange effect can be obtained by liquid agitation. Under conditions where stress on cell A is sufficiently small, such as when using cell A that is resistant to shear stress or when cell A is in a state in which shear stress is less likely to occur due to the shape of the scaffold (three-dimensional structure) The expansion of the oxygen supply site G may be able to receive only the merit of highly efficient liquid exchange, and is preferable not only from oxygen supply but also from the viewpoint of nutrient supply and metabolite removal.
　次に、本発明が提供する酸素供給機構の別の一例を、図２を使用して説明する。
　図２では、酸素供給機構は、酸素透過膜Ｂ１を有する１つの酸素供給部位Ｇと、酸素透過膜Ｂ２を有する２つのメディエータ流体保持容器、すなわち、第１のメディエータ流体保持容器Ｈ１及び第２のメディエータ流体保持容器Ｈ２と、２つの流路、すなわち、酸素供給部位と第１のメディエータ流体保持容器Ｈ１とを接続する第１の流路Ｃ１及び酸素供給部位と第２のメディエータ流体保持容器Ｈ２とを接続する第２の流路Ｃ２とを備える。 Next, another example of the oxygen supply mechanism provided by the present invention will be described using FIG.
In FIG. 2, the oxygen supply mechanism includes one oxygen supply site G having an oxygen permeable membrane B1 and two mediator fluid holding vessels having an oxygen permeable membrane B2, ie, a first mediator fluid holding vessel H1 and a second A mediator fluid holding vessel H2 and two flow paths, ie, a first flow path C1 connecting the oxygen supplying site and the first mediator fluid holding vessel H1, and an oxygen supplying site and a second mediator fluid holding vessel H2 And a second channel C2 connecting the two.
　図２の場合、例えば、第１のメディエータ流体保持容器Ｈ１を圧縮することにより、高酸素流体Ｅは、第１の流路Ｃ１を通って、酸素供給部位Ｇに移動し、酸素供給部位Ｇの低酸素流体Ｄは、高酸素流体Ｅと混合して中酸素流体Ｆになり、第２の流路Ｃ２を通って、第２のメディエータ流体保持容器Ｈ２に移動する。第２のメディエータ流体保持容器Ｈ２は、高酸素流体Ｅを受け取るために、膨張可能な部位を含むことが好ましい。酸素供給部位Ｇには膨張可能な部位が必ずしも必要ではないが、上記のような液交換によるメリットを受けるために備えさせることもできる。次いで、第１のメディエータ流体保持容器Ｈ１の圧縮を解除することで、第２のメディエータ流体保持容器Ｈ２中の中酸素流体Ｆの一部は、第２の流路Ｃ２、酸素供給部位Ｇ及び第１の流路Ｃ１を通って、第１のメディエータ流体保持容器Ｈ１に戻る。このとき、第１のメディエータ流体保持容器Ｈ１、酸素供給部位Ｇ及び第２のメディエータ流体保持容器Ｈ２の酸素濃度は比較的平均化され、メディエータ流体は全体を通して中酸素流体Ｆになる。その後、２つのメディエータ流体保持容器Ｈ１及びＨ２では、酸素透過膜Ｂ２を介して外気から酸素が供給され、中酸素流体Ｆは高酸素流体Ｅになる。 In the case of FIG. 2, for example, by compressing the first mediator fluid holding container H1, the high oxygen fluid E moves to the oxygen supply site G through the first flow channel C1, and the oxygen supply site G The low oxygen fluid D mixes with the high oxygen fluid E to become a medium oxygen fluid F, and moves to the second mediator fluid holding container H2 through the second flow passage C2. The second mediator fluid holding vessel H2 preferably includes an expandable portion to receive the high oxygen fluid E. The oxygen supplying site G does not necessarily have to be an expandable site, but can be provided to receive the merits of the liquid exchange as described above. Then, by releasing the compression of the first mediator fluid holding container H1, a part of the oxygen fluid F in the second mediator fluid holding container H2 is removed from the second flow passage C2, the oxygen supply site G and the second The first mediator fluid holding vessel H1 is returned to the first mediator fluid holding vessel H1 through the first channel C1. At this time, the oxygen concentration of the first mediator fluid holding vessel H1, the oxygen supplying site G and the second mediator fluid holding vessel H2 is relatively averaged, and the mediator fluid becomes the medium oxygen fluid F throughout. Thereafter, in the two mediator fluid holding containers H1 and H2, oxygen is supplied from the outside air through the oxygen permeable membrane B2, and the medium oxygen fluid F becomes the high oxygen fluid E.
　本発明が提供する酸素供給機構のさらに別の一例を、図３を使用して説明する。
　図３では、動作のサイクルを、第１のメディエータ流体保持容器Ｈ１の圧縮と第２のメディエータ流体保持容器Ｈ２の圧縮の繰り返しとしている。メディエータ流体保持容器Ｈ１及びＨ２内の酸素濃度が十分に高くなるのを待つことが出来る場合は、このような動作のサイクルとすることで酸素供給部位Ｇに対して高酸素流体Ｅを送り続けることができる。 Yet another example of the oxygen supply mechanism provided by the present invention will be described using FIG.
In FIG. 3, the cycle of operation is a repetition of the compression of the first mediator fluid holding container H1 and the compression of the second mediator fluid holding container H2. If it is possible to wait for the oxygen concentration in the mediator fluid holding containers H1 and H2 to become sufficiently high, the high oxygen fluid E is continuously sent to the oxygen supply site G by such a cycle of operation. Can.
　本発明が提供する酸素供給機構のさらにまた別の一例を、図４を使用して説明する。
　図４では、酸素供給機構は、酸素透過膜Ｂ１を有する１つの酸素供給部位Ｇと、酸素透過膜Ｂ２を有する２つのメディエータ流体保持容器、すなわち、第１のメディエータ流体保持容器Ｈ１及び第２のメディエータ流体保持容器Ｈ２と、３つの流路、すなわち、酸素供給部位Ｇと第１のメディエータ流体保持容器Ｈ１とを接続する第１の流路Ｃ１、酸素供給部位Ｇと第２のメディエータ流体保持容器Ｈ２とを接続する第２の流路Ｃ２（逆止弁を備える）及び第１のメディエータ流体保持容器Ｈ１と第２のメディエータ流体保持容器Ｈ２とを接続する第３の流路Ｃ３（逆止弁を備える）とを備えることもできる。 Yet another example of the oxygen supply mechanism provided by the present invention will be described using FIG.
In FIG. 4, the oxygen supply mechanism is one oxygen supply site G having an oxygen permeable membrane B1 and two mediator fluid holding vessels having an oxygen permeable membrane B2, ie, a first mediator fluid holding vessel H1 and a second A first channel C1 connecting the mediator fluid holding vessel H2 and three flow paths, ie, the oxygen supplying site G and the first mediator fluid holding vessel H1, an oxygen supplying site G and a second mediator fluid holding vessel A second channel C2 (with a check valve) connecting H2 and a third channel C3 (a check valve connecting the first mediator fluid holding container H1 and the second mediator fluid holding container H2 And B.) can be provided.
　図４の場合、第１のメディエータ流体保持容器Ｈ１を圧縮することで、第２のメディエータ流体保持容器Ｈ２に入った中酸素流体Ｆが、第２の流路Ｃ２及び第３の流路Ｃ３の逆止弁により、酸素供給部位Ｇに戻ることなく第１のメディエータ流体保持容器Ｈ１に戻る。このような動作のサイクルとすることで酸素供給部位Ｇに対して高酸素流体Ｅを送り続けることができる。 In the case of FIG. 4, by compressing the first mediator fluid holding container H1, the medium oxygen fluid F that has entered the second mediator fluid holding container H2 becomes the second flow passage C2 and the third flow passage C3. The non-return valve returns to the first mediator fluid holding container H1 without returning to the oxygen supply site G. The high oxygen fluid E can be continuously sent to the oxygen supply site G by adopting such a cycle of operation.
＜実施例１＞
　実施例１では、１つの酸素供給部位Ｇと１つのメディエータ流体保持容器Ｈとそれらを接続する１つの流路Ｃとを備える酸素供給機構を検討した（図１参照）。
　厚み０．０３ｍｍのＰＤＭＳフィルム（酸素透過膜）を成形し、表面積７０ｃｍ２で５ｃｍ３～１０ｃｍ３の範囲内で体積変化可能な酸素供給部位Ｇと、表面積７０ｃｍ２で４５ｃｍ３～５０ｃｍ３の範囲内で体積変化可能なメディエータ流体保持容器Ｈと、流路Ｃとを備える酸素供給機構１を作製した。 Example 1
In Example 1, an oxygen supply mechanism including one oxygen supply site G, one mediator fluid holding vessel H, and one flow path C connecting them was studied (see FIG. 1).
Molding the PDMS film having a thickness of 0.03 mm (oxygen permeable membrane), and the volume change can oxygenation site G in the range of 5 cm 3 ~ 10 cm 3 surface area 70cm 2, range in surface area 70cm 2 of 45cm 3 ~ 50 cm 3 An oxygen supply mechanism 1 including a volume changeable mediator fluid holding container H and a flow path C was produced.
＜実施例２＞
　実施例２では、１つの酸素供給部位Ｇと２つのメディエータ流体保持容器Ｈ１及びＨ２と酸素供給部位Ｇとメディエータ流体保持容器Ｈ１及びＨ２とを接続する２つの流路Ｃ１及びＣ２とを備える酸素供給機構を検討した（図２及び３参照）。
　厚み０．０３ｍｍのＰＤＭＳフィルム（酸素透過膜）を成形し、表面積７０ｃｍ２で体積７ｃｍ３の酸素供給部位Ｇと、表面積７０ｃｍ２で１５ｃｍ３～３０ｃｍ３の範囲内で体積変化可能なメディエータ流体保持容器Ｈ１及びＨ２と、流路Ｃ１及びＣ２とを備える酸素供給機構２を作製した。 Example 2
In the second embodiment, an oxygen supply comprising two flow paths C1 and C2 connecting one oxygen supply site G, two mediator fluid holding vessels H1 and H2, an oxygen supply site G and a mediator fluid holding vessels H1 and H2 The mechanism was considered (see FIGS. 2 and 3).
Molding the PDMS film having a thickness of 0.03 mm (oxygen permeable membrane), an oxygen supply portion G of volume 7 cm 3 surface area 70cm 2, volume change possible mediators fluid maintained within the range of 15cm 3 ~ 30 cm 3 surface area 70cm 2 The oxygen supply mechanism 2 provided with the containers H1 and H2 and the flow paths C1 and C2 was produced.
＜実施例３＞
　実施例３では、１つの酸素供給部位Ｇと２つのメディエータ流体保持容器Ｈ１及びＨ２と酸素供給部位Ｇとメディエータ流体保持容器Ｈ１及びＨ２とを接続する２つの流路Ｃ１及びＣ２と２つのメディエータ流体保持容器Ｈ１とＨ２同士を接続する流路Ｃ３とを備え、流路Ｃ２及びＣ３内にそれぞれ１つずつの逆止弁を備える酸素供給機構を検討した（図４参照）。
　厚み０．０３ｍｍのＰＤＭＳフィルム（酸素透過膜）を成形し、表面積７０ｃｍ２で体積７ｃｍ３の酸素供給部位Ｇと、表面積７０ｃｍ２で１５ｃｍ３～３０ｃｍ３の範囲内で体積変化可能なメディエータ流体保持容器Ｈ１及びＨ２と、流路Ｃ１及び逆止弁付き流路Ｃ２及びＣ３とを備える酸素供給機構３を作製した。 Example 3
In the third embodiment, two flow paths C1 and C2 and two mediator fluids connecting one oxygen supplying site G, two mediator fluid holding vessels H1 and H2, oxygen supplying site G and mediator fluid holding vessels H1 and H2 An oxygen supply mechanism was studied which was provided with a flow path C3 connecting the holding containers H1 and H2 with each other, and one check valve in each of the flow paths C2 and C3 (see FIG. 4).
Molding the PDMS film having a thickness of 0.03 mm (oxygen permeable membrane), an oxygen supply portion G of volume 7 cm 3 surface area 70cm 2, volume change possible mediators fluid maintained within the range of 15cm 3 ~ 30 cm 3 surface area 70cm 2 The oxygen supply mechanism 3 including the containers H1 and H2, the flow path C1 and the flow paths C2 and C3 with a check valve was manufactured.
＜実施例４＞
　実施例４では、酸素供給機構１、２及び３を用いて、脱酸素水への酸素供給速度を検討した。
　酸素供給機構１に関しては、メディエータ流体保持容器Ｈを、流路Ｃ内を通るメディエータ流体の流速が６．０ｍＬ／ｍｉｎとなるように圧縮した後に、酸素供給部位Ｇからメディエータ流体保持容器Ｈに流速３０ｍＬ／ｍｉｎでメディエータ流体が戻るように圧縮を解除するサイクルを繰り返した。 Example 4
In Example 4, the oxygen supply rate to deoxygenated water was examined using the oxygen supply mechanisms 1, 2 and 3.
With regard to the oxygen supply mechanism 1, after the mediator fluid holding vessel H is compressed so that the flow rate of the mediator fluid passing through the flow path C is 6.0 mL / min, the flow rate from the oxygen supply site G to the mediator fluid holding vessel H The cycle of decompression was repeated so that the mediator fluid returned at 30 mL / min.
　酸素供給機構２に関しては、メディエータ流体保持容器Ｈ１及びＨ２を、流路Ｃ１及びＣ２内を通るメディエータ流体の流速が３．０ｍＬ／ｍｉｎとなるように交互に圧縮することで流体を循環させた（図３の動作方法）。 With regard to the oxygen supply mechanism 2, the fluid was circulated by alternately compressing the mediator fluid holding containers H1 and H2 so that the flow rate of the mediator fluid passing through the flow channels C1 and C2 would be 3.0 mL / min. Figure 3 operation method).
　酸素供給機構３に関しては、メディエータ流体保持容器Ｈ１を、流路Ｃ１及びＣ２内を通るメディエータ流体の流速が６．０ｍＬ／ｍｉｎとなるように圧縮した後に、流路Ｃ３内を通るメディエータ流体の流速が３０ｍＬ／ｍｉｎとなるように、メディエータ流体保持容器Ｈ２からメディエータ流体保持容器Ｈ１にメディエータ流体が戻るように圧縮を解除するサイクルを繰り返した。 Regarding the oxygen supply mechanism 3, after compressing the mediator fluid holding container H1 so that the flow rate of the mediator fluid passing through the flow passages C1 and C2 becomes 6.0 mL / min, the flow rate of the mediator fluid passing through the flow passage C3 The cycle of releasing compression was repeated so that the mediator fluid returned from the mediator fluid holding container H2 to the mediator fluid holding container H1 such that the pH of the solution became 30 mL / min.
　その結果、平均酸素供給速度は、酸素供給機構１に関して１．９μｍｏｌ／ｍｉｎ、酸素供給機構２に関して２．４μｍｏｌ／ｍｉｎ、酸素供給機構３に関して２．４μｍｏｌ／ｍｉｎとなった。これらは、細胞の種類にもよるが、最大で１０９～１０１０程度の細胞数を生存させ続けるのに十分な酸素供給速度である。 As a result, the average oxygen supply rate was 1.9 μmol / min for the oxygen supply mechanism 1, 2.4 μmol / min for the oxygen supply mechanism 2, and 2.4 μmol / min for the oxygen supply mechanism 3. These are, depending on the type of cells, an oxygen supply rate sufficient to maintain cell numbers of up to about 10 9 to 10 10 .
＜実施例５＞
　実施例５では、酸素供給機構２を用いてｉｎ　ｖｉｔｒｏでの細胞培養の検討を行った。細胞としてはＨｅｐＧ２細胞を用い、細胞の近傍に酸素供給機構２を設置し動作させた状態で細胞の増殖性を評価した。その結果、細胞数１０７まで細胞死が見られなかった。これは、酸素供給機構２による酸素供給に由来する効果と考えられる。 Example 5
In Example 5, the in vitro cell culture was examined using the oxygen supply mechanism 2. HepG2 cells were used as the cells, and the proliferative ability of the cells was evaluated in a state where the oxygen supply mechanism 2 was placed near the cells and operated. As a result, cell death was observed until the cell number 10 7. This is considered to be an effect derived from oxygen supply by the oxygen supply mechanism 2.
＜比較例１＞
　比較例１では、実施例５と同様にした状態で、酸素供給機構２による酸素供給を行わずにｉｎ　ｖｉｔｒｏでの細胞培養の検討を行った。その結果、細胞数１０６程度から酸素不足に起因すると考えられる細胞の増殖停止が見られた。 Comparative Example 1
In Comparative Example 1, in the same manner as Example 5, examination of in vitro cell culture was conducted without oxygen supply by the oxygen supply mechanism 2. As a result, from the cell number of about 10 6 growth arrest of cells considered to be caused by oxygen deficiency was observed.
＜実施例６＞
　実施例６では、酸素供給機構２の防水性（水のコンタミネーションリスク）について検討した。
　酸素供給機構２を水中に入れて２４時間負荷を加えた後に、酸素供給機構２の内部に浸入した水を定量した。
　その結果、酸素供給機構２の内部に浸入した水の量は、検出限界以下であった。 Example 6
In Example 6, the waterproofness (contamination risk of water) of the oxygen supply mechanism 2 was examined.
After the oxygen supply mechanism 2 was put in water and a load was applied for 24 hours, water which entered the inside of the oxygen supply mechanism 2 was quantified.
As a result, the amount of water entering the inside of the oxygen supply mechanism 2 was below the detection limit.
＜比較例２＞
　比較例２では、閉鎖系でない酸素供給機構の防水性について検討した。
　酸素供給機構１と同様の構成で、メディエータ流体保持容器の代わりにフィルタを取り付けた比較供給機構１を作成し、水中に入れて２４時間負荷を加えた後に、比較供給機構１の内部に浸入した水を定量した。
　その結果、負荷の印加による内圧の変動に伴って、約０．２ｇの水が比較供給機構１の内部に入り込んでいた。 Comparative Example 2
In Comparative Example 2, the waterproofness of the non-closed oxygen supply mechanism was examined.
In the same structure as the oxygen supply mechanism 1, the comparative supply mechanism 1 attached with a filter instead of the mediator fluid holding container was prepared, put into water and loaded for 24 hours, and then entered the inside of the comparative supply mechanism 1. Water was quantified.
As a result, about 0.2 g of water entered the inside of the comparative supply mechanism 1 with the fluctuation of the internal pressure due to the application of the load.
　なお、本発明は上記した実施形態に限定されるものではなく、様々な変形例が含まれる。例えば、実施形態の構成の一部について、他の構成の追加・削除・置換をすることが可能である。 The present invention is not limited to the above-described embodiment, but includes various modifications. For example, with respect to a part of the configuration of the embodiment, it is possible to add, delete, and replace other configurations.
Ａ：細胞（酸素を消費）、Ｂ１及びＢ２：酸素透過膜、Ｃ及びＣ１～Ｃ３：流路、Ｄ：低酸素流体、Ｅ：高酸素流体、Ｆ：中酸素流体、Ｇ：酸素供給部位、Ｈ並びにＨ１及びＨ２：メディエータ流体保持容器 A: cell (consumption of oxygen), B1 and B2: oxygen permeable membrane, C and C1 to C3: flow path, D: hypoxic fluid, E: hyperoxic fluid, F: medium oxygen fluid, G: oxygen supplying site, H and H1 and H2: mediator fluid holding container
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (9)

  1. 　細胞に酸素を供給するための酸素供給機構であって、
    　酸素供給機構が、外気から隔離されたメディエータ流体を含む閉鎖系の流体循環機構であり、
    　酸素供給機構が、１つ以上の酸素供給部位と、１つ以上のメディエータ流体保持容器と、酸素供給部位とメディエータ流体保持容器とを接続する１つ以上の流路とを備え、
    　酸素供給部位及びメディエータ流体保持容器が、酸素透過膜を備えることを特徴とする酸素供給機構。     An oxygen supply mechanism for supplying oxygen to cells;
    The oxygen supply mechanism is a closed system fluid circulation mechanism including a mediator fluid isolated from the ambient air,
    The oxygen delivery mechanism comprises one or more oxygen delivery sites, one or more mediator fluid reservoirs, and one or more flow paths connecting the oxygen delivery sites to the mediator fluid reservoirs,
    An oxygen supply mechanism characterized in that the oxygen supply site and the mediator fluid holding vessel comprise an oxygen permeable membrane.
  2. 　メディエータ流体保持容器が、１つであり、酸素供給部位の体積が、可変であることを特徴とする、請求項１に記載の酸素供給機構。 The oxygenation mechanism according to claim 1, characterized in that the mediator fluid holding vessel is one and the volume of the oxygenation site is variable.
  3. 　１つの酸素供給部位と、第１のメディエータ流体保持容器及び第２のメディエータ流体保持容器と、第１のメディエータ流体保持容器と酸素供給部位とを接続する第１の流路及び第２のメディエータ流体保持容器と酸素供給部位とを接続する第２の流路とを備えることを特徴とする、請求項１に記載の酸素供給機構。 First flow path and second mediator fluid connecting one oxygen supply site, first mediator fluid holding container and second mediator fluid holding container, and first mediator fluid holding container and oxygen supply site The oxygen supply mechanism according to claim 1, further comprising a second flow path connecting the holding container and the oxygen supply site.
  4. 　第１のメディエータ流体保持容器と第２のメディエータ流体保持容器とを接続する第３の流路をさらに備え、
    　第１の流路、第２の流路、及び第３の流路のうち少なくとも１つ以上が、逆止弁を備えることを特徴とする、請求項３に記載の酸素供給機構。     And a third flow path connecting the first mediator fluid holding vessel and the second mediator fluid holding vessel,
    The oxygen supply mechanism according to claim 3, wherein at least one or more of the first flow path, the second flow path, and the third flow path includes a check valve.
  5. 　メディエータ流体保持容器を圧縮する機構を備える、請求項１～４のいずれか一項に記載の酸素供給機構。 The oxygen supply mechanism according to any one of claims 1 to 4, comprising a mechanism for compressing the mediator fluid holding container.
  6. 　メディエータ流体保持容器を圧縮する機構の動力が、電力、空気圧、手動、装着者の動きに伴う慣性力、及び装着者の動きに伴う力学的な作用からなる群から選択される少なくとも１つ以上であることを特徴とする、請求項５に記載の酸素供給機構。 The power of the mechanism for compressing the mediator fluid holding container is at least one selected from the group consisting of electric power, air pressure, manual operation, inertial force accompanying movement of the wearer, and mechanical action accompanying movement of the wearer. The oxygen supply mechanism according to claim 5, characterized in that
  7. 　酸素透過膜が、ポリジメチルシロキサン又はフッ素系ポリマーを含み、
    　酸素透過膜の膜厚が、１０μｍ～１ｍｍであることを特徴とする、請求項１～４のいずれか一項に記載の酸素供給機構。     The oxygen permeable membrane comprises polydimethylsiloxane or a fluorine-based polymer,
    The oxygen supply mechanism according to any one of claims 1 to 4, wherein the film thickness of the oxygen permeable membrane is 10 μm to 1 mm.
  8. 　請求項１～４のいずれか一項に記載の酸素供給機構を用いて細胞に酸素を供給する方法であって、ヒトの治療方法を含まない方法。 A method of supplying oxygen to cells using the oxygen supply mechanism according to any one of claims 1 to 4, which does not include a method of treating human.
  9. 　細胞が、移植細胞、移植向けに培養される細胞、有用物質産生向けに培養される細胞又は研究向けに培養される細胞であることを特徴とする、請求項８に記載の方法。 The method according to claim 8, wherein the cell is a transplanted cell, a cell cultured for transplantation, a cell cultured for production of a useful substance, or a cell cultured for research.
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