Oxygen supply member

Abstract

To provide an oxygen supply member suitable for use in the fabrication of three-dimensional tissue structures.SOLUTION: An oxygen supply member comprises a carrier, modified calcium peroxide, and a hydrogen peroxide remover. The modified calcium peroxide is calcium peroxide with its surface covered by a coating layer. The modified calcium peroxide and the hydrogen peroxide remover are supported on the carrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oxygen supply member. [Background technology]

[0002] A three-dimensional tissue structure has a structure in which cells are arranged three-dimensionally, and is used as a biological tissue model, etc. When producing a three-dimensional tissue structure, a problem may occur in which the supply of oxygen to the inside of the tissue is insufficient, causing cell death due to oxygen deficiency.

[0003] Calcium peroxide (CaO2) generates oxygen by reacting with water. Attempts have been made to utilize this property to solve the above-mentioned problem of oxygen deficiency. For example, Non-Patent Document 1 discloses a carrier produced by mixing polycaprolactone, a biodegradable polymer, and calcium peroxide using fused deposition modeling, and the use of the carrier to culture mouse pancreatic β cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Japan Society of Mechanical Engineers, Proceedings of the 30th Bioengineering Conference, 2017, 2G14 Summary of the Invention [Problem to be solved by the invention]

[0005] When calcium peroxide reacts with water, it releases oxygen in a short time (initial burst release), making it difficult to continuously supply oxygen during cell culture over several days to several weeks. In response to this, the present inventors have discovered that this initial burst release can be suppressed by coating the surface of calcium peroxide with hydroxyapatite.

[0006] In view of the above-mentioned circumstances, the present invention has an object to provide an oxygen supplying member suitable for producing a three-dimensional tissue structure. Another object of the present invention is to provide a method for producing a three-dimensional tissue structure using the oxygen supplying member. [Means for solving the problem]

[0007] The present invention includes, for example, the following inventions. [1] A carrier, a modified calcium peroxide, and a hydrogen peroxide scavenger, The modified calcium peroxide is calcium peroxide whose surface is coated with a coating film, The oxygen supplying member, wherein the modified calcium peroxide and the hydrogen peroxide removing agent are supported on the carrier. [2] The oxygen supplying member according to [1], wherein the coating contains hydroxyapatite. [3] The oxygen supplying member according to [1], wherein the coating contains amorphous calcium carbonate. [4] The oxygen supplying member according to any one of [1] to [3], wherein the carrier is a hydrogel. [5] The oxygen supplying member according to [4], wherein the modified calcium peroxide and the hydrogen peroxide remover are embedded in the hydrogel. [6] The oxygen supplying member according to [4] or [5], wherein the hydrogel contains cross-linked gelatin. [7] The oxygen supplying member according to any one of [1] to [6], wherein the hydrogen peroxide remover contains catalase. [8] The oxygen supplying member according to any one of [1] to [7], which is for cell culture. [9] The oxygen supplying member according to any one of [1] to [8], which is used for producing a three-dimensional tissue structure.

[10] A method for producing a three-dimensional tissue structure, comprising culturing at least one type of cell in a culture medium while the oxygen supplying member according to any one of [1] to [9] is in contact with the culture medium. Effect of the Invention

[0008] According to the present invention, it is possible to provide an oxygen supplying member suitable for producing a three-dimensional tissue structure. According to the present invention, it is also possible to provide a method for producing a three-dimensional tissue structure using the oxygen supplying member. [Brief description of the drawings]

[0009] [Figure 1] 1A and 1B are diagrams showing the results of producing an oxygen supplying member (hydrogel) in Test Example 1-1. Fig. 1(a) is a photograph showing the formation of a hydrogel. Fig. 1(b) is a photograph showing a scanning electron microscope (SEM) image of the hydrogel. [Diagram 2] 2A and 2B are diagrams showing the evaluation results of the oxygen supply member (hydrogel) in Test Example 1-2. FIG. 2(a) is a diagram showing the evaluation results of the hydrogen peroxide concentration. FIG. 2(b) and FIG. 2(d) are diagrams showing the evaluation results of the oxygen release behavior. FIG. 2(c) and FIG. 2(e) are enlarged views of parts of FIG. 2(b) and FIG. 2(d), respectively. [Diagram 3] FIG. 3 is a diagram showing the results of evaluating cell proliferation under hypoxic conditions in Test Example 2-1. FIG. 3(a) is a diagram showing the cell culture state in this Test Example. FIG. 3(b) is a graph showing the results of evaluating the mitochondrial activity of NHDF. FIG. 3(c) is a graph showing the results of evaluating the mitochondrial activity of MSC. FIG. 3(d) is a graph showing the results of evaluating the mitochondrial activity of ADSC. FIG. 3(e) is a graph showing the results of evaluating the mitochondrial activity of HepG2. FIG. 3(f) is a photograph showing the results of photographing cells after cell culture for 7 days. [Figure 4]FIG. 4 is a diagram showing the results of evaluating cell proliferation under hypoxic conditions in Test Example 2-2. FIG. 4(a) is a diagram showing a schematic diagram of cell culture in a Test Example for evaluating the content of modified calcium peroxide. FIG. 4(b) is a graph showing the results of evaluating the mitochondrial activity of NHDF. FIG. 4(c) is a photograph showing the results of photographing cells after 7 days of cell culture. FIG. 4(d) is a diagram showing a schematic diagram of cell culture in a Test Example for evaluating long-term culture. FIG. 4(e) is a graph showing the results of measuring the number of viable cells. FIG. 4(f) is a photograph showing the results of photographing cells after 14 days of culture. [Diagram 5] 5A and 5B are diagrams showing the results of the production and evaluation of a three-dimensional tissue structure in Test Example 3. FIG. 5(a) is a schematic diagram showing the state of cell culture in this Test Example. FIG. 5(b) is a hematoxylin-eosin (HE) stained image of a control (sample not containing hydrogel). FIG. 5(c) is a HE stained image of a sample using a hydrogel containing HAp-CaO2. FIG. 5(d) is a graph showing the measurement results of cell density. FIG. 5(e) is a photograph showing a confocal laser scanning microscope (CSLM) image of a three-dimensional tissue structure stained with DAPI and phalloidin. [Figure 6] 1 is a graph showing the results of measuring the change over time in the dissolved oxygen concentration in various solutions when CaO2 was immersed in the solutions in Test Example 4-1. [Figure 7] FIG. 1 shows scanning electron microscope (SEM) images of modified calcium peroxide (NaH2PO4 0.1 mg / mL) and control calcium peroxide (NaH2PO4 0 mg / mL) in Test Example 4-1, and spectra obtained by X-ray diffraction (XRD). [Figure 8] 1 is a graph showing the results of measuring the change in dissolved oxygen concentration over time in Test Example 4-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0011] [Oxygen supply member] The oxygen supplying member according to the present embodiment includes a carrier, a modified calcium peroxide having a surface coated with a coating film, and a hydrogen peroxide removing agent. In the oxygen supplying member, the modified calcium peroxide and the hydrogen peroxide removing agent are both supported on the carrier.

[0012] In the oxygen supply member of this embodiment, the modified calcium peroxide and the hydrogen peroxide remover are supported on a carrier and are therefore located in close proximity to each other, so that hydrogen peroxide generated as a by-product in the reaction between calcium peroxide and water can be efficiently removed.

[0013] In this specification, the term "support" refers to a substance capable of supporting the modified calcium peroxide and the hydrogen peroxide removing agent. "Supported on a support" means that the modified calcium peroxide and the hydrogen peroxide removing agent are held on or near the support by adhering or bonding to the support. The "bond" may be any of a covalent bond, an ionic bond, a hydrogen bond, a bond due to an intermolecular force, etc.

[0014] The carrier is preferably biocompatible. "Biocompatible" means that it does not cause excessive inflammation or the like when it comes into contact with living tissue.

[0015] The shape of the carrier is not particularly limited, and may be any shape such as fibrous, flat, ellipsoidal, or spherical.

[0016] The carrier may be a porous body. When the carrier is a porous body, it becomes possible to embed the modified calcium peroxide and the hydrogen peroxide removing agent inside the carrier, and the efficiency of removing hydrogen peroxide becomes higher.

[0017] Specific examples of the carrier include hydrogel, hydrogel fiber, and hydrogel film.

[0018] "Hydrogel" means a three-dimensional network structure formed by crosslinking polymers through hydrogen bonds, ionic bonds, coordinate bonds, covalent bonds, etc., and the three-dimensional network structure contains a liquid such as water. "Hydrogel fiber" means a fiber formed from hydrogel. "Hydrogel film" means a film formed from hydrogel.

[0019] The hydrogel may be a gel composed of biocompatible molecules such as fibrin, collagen, gelatin, hyaluronic acid, alginic acid, pectin, chitosan, etc. The hydrogel may also be a gel composed of crosslinked biocompatible molecules obtained by crosslinking these biocompatible molecules intermolecularly and / or intramolecularly. Examples of the crosslinking method include physical crosslinking by application of heat, ultraviolet light, radiation, etc., and chemical crosslinking by a crosslinking agent or an enzyme reaction (e.g., a reaction by transglutaminase).

[0020] In the oxygen supply member according to the present embodiment, the modified calcium peroxide and the hydrogen peroxide remover may be supported on the surface of the carrier, or may be supported inside the carrier. In the latter case, it is preferable that the carrier is porous, and it is also preferable that the modified calcium peroxide and the hydrogen peroxide remover are embedded in the carrier. Here, "embedded" means that 90% or more or the entirety of the surface of the oxygen supply member is the carrier. This allows hydrogen peroxide to be removed more efficiently while maintaining oxygen supply.

[0021] In this specification, "modified calcium peroxide" means calcium peroxide whose surface is coated with a coating. In the modified calcium peroxide according to this embodiment, the coating acts as a barrier against surrounding water, suppressing the movement of water or blocking the movement of water under specific conditions. As a result, the reaction between calcium peroxide and water gradually occurs to release oxygen, or when the specific conditions are released, the reaction between calcium peroxide and water occurs to release oxygen.

[0022] The shape of the modified calcium peroxide is not particularly limited, but may be particulate from the viewpoint of easy handling. The particle size of the particles may be, for example, 150 mesh or more and 250 mesh or less, or 170 mesh or more and 200 mesh or less.

[0023] The modified calcium peroxide may be calcium peroxide having a part or all of its surface covered with a coating, preferably having 90% or more of its surface covered with a coating, more preferably having 95% or more of its surface covered with a coating, and particularly preferably having the entire surface covered with a coating.

[0024] The thickness of the coating of the modified calcium peroxide is not particularly limited, but may be, for example, 0.1 μm or more and 10 μm or less.

[0025] In one embodiment, the coating may contain hydroxyapatite (Ca5(PO4)3(OH)). In this specification, modified calcium peroxide coating containing hydroxyapatite may be specifically referred to as "HAp-CaO2". By containing hydroxyapatite, the coating becomes porous, and the diffusion and movement of surrounding water is suppressed. Therefore, calcium peroxide and water react gradually, and the initial burst release of oxygen is suppressed, and oxygen is gradually released. At the same time, the initial burst release of hydrogen peroxide is also suppressed, so that hydrogen peroxide can be removed until it reaches a sufficiently low concentration by the hydrogen peroxide remover in the oxygen supply member. The coating according to this embodiment is preferably substantially made of hydroxyapatite, and more preferably made of hydroxyapatite, since the above-mentioned effects are more significantly exhibited.

[0026] The modified calcium peroxide (HAp-CaO2) according to the present embodiment may be obtained by immersing calcium peroxide in an aqueous solution containing phosphate ions. The phosphate ion concentration in the aqueous solution containing phosphate ions is not particularly limited, but may be, for example, 50 mM or more and 1 M or less, or 100 mM or more and 500 mM or less. A specific example of an aqueous solution containing phosphate ions is an aqueous solution prepared by mixing an aqueous solution of sodium dihydrogen phosphate and an aqueous solution of disodium hydrogen phosphate of the same concentration so that the pH becomes 7.0. The temperature condition when immersing calcium peroxide is not particularly limited, but may be, for example, 20°C or more and 50°C or less, preferably 30°C or more and 40°C or less, and more preferably 37°C. The time for immersing calcium peroxide is not particularly limited, but may be, for example, 30 minutes or more, 45 minutes or more, 1 hour or more, or 2 hours or less.

[0027] In another embodiment, the coating may contain amorphous calcium carbonate (amorphous CaCO3). In this specification, modified calcium peroxide containing amorphous calcium carbonate in the coating may be specifically referred to as "ACC-CaO2". By containing amorphous calcium carbonate, the diffusion movement of surrounding water is substantially blocked. In addition, since the solubility of amorphous calcium carbonate increases as the pH decreases, the coating gradually dissolves by lowering the pH (for example, to pH 5 or less), and oxygen is gradually released. Therefore, the oxygen supply member containing modified calcium peroxide (ACC-CaO2) can be used as a pH-responsive oxygen supply member. The coating according to this embodiment is preferably substantially made of amorphous calcium carbonate, and more preferably made of amorphous calcium carbonate, since the above-mentioned effects are more significantly exhibited.

[0028] The modified calcium peroxide (ACC-CaO2) according to the present embodiment may be obtained by immersing calcium peroxide in an aqueous solution containing phosphate ions and carbonate ions. The phosphate ion concentration in the aqueous solution containing phosphate ions and carbonate ions may be, for example, 1 mM or more and 50 mM or less, or 1 mM or more and 10 mM or less. The carbonate ion concentration in the aqueous solution containing phosphate ions and carbonate ions may be, for example, 5 mM or more and 1 M or less, or 10 mM or more and 500 mM or less, or 10 mM or more and 100 mM or less. A specific example of an aqueous solution containing phosphate ions and carbonate ions is an aqueous solution containing sodium dihydrogen phosphate and sodium hydrogen carbonate. The temperature condition when immersing calcium peroxide is not particularly limited, but may be, for example, 20°C or more and 50°C or less, preferably 30°C or more and 40°C or less, and more preferably 37°C. The time for immersing calcium peroxide is not particularly limited, but may be, for example, 30 minutes or more, 45 minutes or more, 1 hour or more, or 2 hours or less.

[0029] The content of the modified calcium peroxide in the oxygen supply member according to the present embodiment can be set as an amount sufficient for oxygen supply depending on the application of the oxygen supply member, the type of modified calcium peroxide, etc. Specifically, for example, based on the capacity of the oxygen supply member, the content may be, for example, 1 mg / mL or more and 50 mg / mL or less, 2.5 mg / mL or more and 25 mg / mL or less, 5 mg / mL or more and 20 mg / mL or less, or 10 mg / mL or more and 15 mg / mL or less.

[0030] The hydrogen peroxide remover can be used without any particular limitation as long as it is a substance that can remove hydrogen peroxide by decomposing hydrogen peroxide, catalyzing the decomposition of hydrogen peroxide, etc. The hydrogen peroxide remover is preferably biocompatible.

[0031] Examples of hydrogen peroxide removers include enzymes such as catalase, peroxidase, and glutathione peroxidase, compounds such as ascorbic acid and its salts, and inorganic compounds such as manganese oxide (MnO2) and iron chloride (FeCl3). Among these, from the viewpoint of excellent biocompatibility, enzymes such as catalase, peroxidase, and glutathione peroxidase, and compounds such as ascorbic acid and its salts are preferred, and from the viewpoint of excellent hydrogen peroxide removal ability, enzymes such as catalase, peroxidase, and glutathione peroxidase are more preferred.

[0032] The content of the hydrogen peroxide remover in the oxygen supply member according to the present embodiment can be set to an amount sufficient for removing hydrogen peroxide depending on the type of hydrogen peroxide remover, the type and content of modified calcium peroxide, etc. Specifically, for example, when the hydrogen peroxide remover is an enzyme, it may be, for example, 50 U / mL or more and 200 U / mL or less, or 80 U / mL or more and 120 U / mL or less, based on the volume of the oxygen supply member. Also, for example, it may be 5 U / mg or more and 100 U / mg or less, or 10 U / mg or more and 40 U / mg or less, based on the weight of the modified calcium peroxide. Note that 1 U is the amount of enzyme required to decompose 1 micromole of hydrogen peroxide per minute under conditions of 25°C and pH 7.0 using 10 mM hydrogen peroxide as a substrate.

[0033] The oxygen supplying member according to the present embodiment may contain other components in addition to the carrier, the modified calcium peroxide, and the hydrogen peroxide remover, such as glucose, fatty acids, and proteins.

[0034] The oxygen supply member according to the present embodiment can be manufactured by mixing a carrier, modified calcium peroxide, and a hydrogen peroxide remover, and other components as necessary. For example, when the carrier is a hydrogel, the oxygen supply member can be manufactured by mixing a hydrogel raw material (e.g., a non-crosslinked biocompatible molecule) and a crosslinker (e.g., an enzyme such as transglutaminase) with the modified calcium peroxide and the hydrogen peroxide remover, and carrying out a crosslinking reaction (e.g., at 37°C for 1 hour). Note that, if necessary, a reaction for forming a bond between the carrier and the modified calcium peroxide and / or the hydrogen peroxide remover may be carried out.

[0035] The oxygen supplying member according to the present embodiment is used so as to come into contact with a culture medium, thereby supplying oxygen into the culture medium. The arrangement of the oxygen supplying member, the culture medium, and the cells is not particularly limited, and for example, the cells and the culture medium may be present outside the oxygen supplying member, the cells and the culture medium may be present inside the oxygen supplying member, the cells and the culture medium may be present both inside and outside the oxygen supplying member, or the culture medium may be present inside the oxygen supplying member and the cells and the culture medium may be present outside the oxygen supplying member.

[0036] The oxygen supplying member according to an embodiment may be an oxygen gradual release member that gradually releases oxygen. The oxygen gradual release member may not release oxygen under a specific condition, and gradually release oxygen when the specific condition is released. The specific condition may be, for example, pH.

[0037] The oxygen supplying member according to this embodiment can be suitably used for cell culture applications, three-dimensional tissue structure manufacturing applications, and the like.

[0038] [Method for producing a three-dimensional structure] The manufacturing method for a three-dimensional tissue structure according to this embodiment includes a step of culturing at least one type of cell in a culture medium while the oxygen supply member according to the present invention is in contact with the culture medium (hereinafter also referred to as the "culture step").

[0039] The three-dimensional tissue structure may be, for example, skeletal muscle tissue, adipose tissue, vascular tissue, nerve tissue, epidermal tissue, epithelial tissue, cardiac muscle tissue, cartilage tissue, etc. The three-dimensional tissue structure may also be a structure (cell population) in which at least one type of cell is arranged three-dimensionally and does not have a specific tissue structure.

[0040] The shape of the three-dimensional tissue structure is not particularly limited, and may be, for example, a sphere, an ellipsoid, a rectangular parallelepiped, a cube, a fiber, or the like.

[0041] The cells are not particularly limited, and may be cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats. The site of origin of the cells is also not particularly limited, and may be somatic cells derived from bones, muscles, internal organs, nerves, brains, bones, skin, blood, etc., or may be germ cells. Furthermore, the cells may be stem cells, or cultured cells such as primary cultured cells, subcultured cells, and cell line cells.

[0042] Specific examples of the cells include skeletal muscle cells, smooth muscle cells (e.g., aortic smooth muscle cells, cardiomyocytes (e.g., human iPS cell-derived cardiomyocytes), adipocytes (e.g., mature adipocytes), vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells), vascular pericytes, lymphatic endothelial cells, nerve cells, dendritic cells, immune cells, fibroblasts, chondrocytes, osteoblasts, epithelial cells (e.g., human gingival epithelial cells), keratinocytes, hepatocytes, pancreatic islet cells, tissue stem cells (e.g., satellite cells, mesenchymal stem cells), astrocytes, colon cancer cells (e.g., human colon cancer cells), and cancer cells such as hepatic cancer cells. One type of cell may be used alone, or multiple types of cells may be used in combination.

[0043] The medium can be appropriately selected depending on the type of cells to be cultured. Examples of the medium include liquid media such as Eagle's MEM medium, Dulbecco's modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RPMI medium, GlutaMax medium, EGM2, etc. The medium may be a medium containing serum or a serum-free medium. The medium may be a mixed medium in which two types of media are mixed.

[0044] The method for contacting the oxygen supplying member with the culture medium is not particularly limited, and for example, a method in which the oxygen supplying member is added to the culture medium directly or together with a support (e.g., a 24-well plate insert), a method in which the culture medium is added to a culture vessel having an oxygen supplying member attached or unattached thereto, etc. can be used.

[0045] The cells may be contained in the medium. For example, if the medium can permeate the inside of the oxygen supplying member, the cells may be outside the oxygen supplying member, or may be inside the oxygen supplying member together with the medium.

[0046] The amount of the oxygen supply member according to the present invention used can be appropriately set depending on the type of cells, the culture period, the shape and thickness of the desired three-dimensional tissue structure, etc. The oxygen supply member according to the present invention can be used in an amount such that the amount of modified calcium peroxide is, for example, 0.01 mg to 10 mg, 0.05 mg to 5 mg, or 0.1 mg to 2 mg per mL of culture medium, although this is not limited thereto.

[0047] The culture step can be carried out in the same manner as in the usual culture method for a three-dimensional tissue structure, except that the culture is carried out in a state in which the oxygen supply member according to the present invention is in contact with the culture medium.

[0048] The cell density in the medium in the culture step can be appropriately determined depending on the shape and thickness of the intended three-dimensional tissue structure, the size of the culture vessel, etc. For example, the cell density in the medium in the culture step can be set to 1 to 10 8 cells / mL,3 ~10 7 It may be in cells / mL.

[0049] The method for culturing the cells can be a suitable culture method depending on the type of cells to be cultured. The culture temperature may be 20° C. to 40° C., or 30° C. to 37° C. The pH of the medium may be 6.0 to 8.0, or 7.2 to 7.4. The culture time may be 1 day to 2 weeks, or 1 week to 2 weeks.

[0050] The cells to be cultured may be dispersed in a hydrogel. Examples of hydrogels include fibrin gel, Matrigel, collagen gel, and gelatin gel. By dispersing the cells to be cultured in a hydrogel, it becomes easier to form a three-dimensional tissue structure with a more desired shape. In addition, when the oxygen supply member according to the present embodiment is in the form of a hydrogel, the cells to be cultured may be dispersed in the hydrogel serving as the oxygen supply member.

[0051] When the cells to be cultured include stem cells, the culture step may include proliferation culture and differentiation induction culture. Differentiation induction culture can be performed by a conventional method, for example, by replacing the culture medium used in proliferation culture with a specific culture medium capable of inducing differentiation, or by adding a differentiation inducer to the culture medium.

[0052] When the oxygen supplying member used in the method for producing a three-dimensional tissue structure according to this embodiment is pH responsive (for example, when the modified calcium peroxide coating contains amorphous calcium carbonate), the culture step may include adjusting the pH of the medium to a pH (for example, pH 5 or less) at which oxygen can be released from the oxygen supplying member. The pH may be adjusted by adding a pH adjuster (for example, hydrochloric acid, sodium hydroxide) or without adding a pH adjuster. In the latter case, the pH can be adjusted by, for example, a change in the pH of the medium due to lactic acid or the like secreted from cells as the cells grow. EXAMPLES

[0053] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0054] The materials used in this example are as follows: Calcium peroxide (CaO2) (particle size 200 mesh (74 μm)), manufactured by Sigma-Aldrich Catalase from bovine liver, Sigma-Aldrich Transglutaminase, manufactured by Ajinomoto Co., Inc. Sodium dihydrogen phosphate, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Disodium hydrogen phosphate 12-water, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Gelatin (077-03155), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Calcium chloride anhydrous, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. ·Tertiary butyl alcohol, manufactured by Tokyo Chemical Industry Co., Ltd. Dulbecco's Modified Eagle Medium (DMEM) (clear; 08489-45), manufactured by Nacalai Tesque DMEM (cell culture; 08458-16), manufactured by Nacalai Tesque Fetal bovine serum (FBS) (10270), Thermo Fisher Scientific Antibiotics (50 U / mL penicillin and 50 μg / mL streptomycin), Thermo Fisher Scientific Normal human dermal fibroblasts (NHDF, CC-2509), manufactured by LONZA Human mesenchymal stem cells (MSC, PT-2501), manufactured by LONZA HepG2 hepatocellular carcinoma cells (human) (HEPG2-500), manufactured by Cellular Engineering Technologies Trypsin derived from porcine pancreas, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Fibronectin human plasma, manufactured by Sigma-Aldrich Phalloidin-iFluor 594 Reagent, Abcam ·Triton-X 100, made by Sigma-Aldrich Bovine serum albumin (BSA, A3294), from Sigma-Aldrich Normal goat serum, Jackson Immuno Research Laboratories Inc. Dulbecco's phosphate buffered saline (PBS), manufactured by Nacalai Tesque Serum albumin, Sigma-Aldrich 10% Formaldehyde Neutral Buffer, Nacalai Tesque 4',6-diamidino-2-phenylindole (DAPI), Thermo Fisher Scientific Sodium bicarbonate, manufactured by Nacalai Tesque Mineral oil High sensitivity (oxygen barrier oil), manufactured by Abcam

[0055] [Test Example 1: Production and Evaluation of Oxygen Supply Member] [Test Example 1-1: Production of oxygen supply member] (Preparation of Reagents) A 100 U / mL transglutaminase (TG) solution and a 10,000 U / mL catalase solution were prepared using DMEM (transparent). A 0.5 M CaCl2 solution was prepared using ultrapure water (MilliQ). A gelatin solution (20% by mass) was prepared by dissolving 2500 mg of gelatin in 10 mL of DMEM (transparent) by heating.

[0056] (Production of modified calcium peroxide) As modified calcium peroxide, calcium peroxide coated with a film containing hydroxyapatite (hereinafter also referred to as "HAp-CaO2") was produced. Specifically, a phosphate buffer solution (PB) was first prepared by mixing an aqueous solution of sodium dihydrogen phosphate and an aqueous solution of disodium hydrogen phosphate of the same concentration to a pH of 7.0. Next, HAp-CaO2 was produced by immersing 3 mg of CaO2 in 3 mL of 100 mM or 500 mM PB at 37°C for 1 hour. The obtained HAp-CaO2 was collected by centrifuging at 4000 rpm for 3 minutes.

[0057] (Manufacturing of oxygen supply components) CaO2 or HAp-CaO2 was dispersed in gelatin solution, and then catalase solution, TG solution, CaCl2 solution and DMEM (transparent) were mixed to a final concentration of 2.5mg / mL or 10mg / mL CaO2, 16% by mass gelatin, 10U / mL TG, 100U / mL catalase and 2.5mM CaCl2, respectively. The HAp-CaO2 concentration was calculated from the CaO2 concentration before modification. A sample without catalase was also prepared in the same manner. 800μL of the prepared mixed solution was added to a sample bottle and incubated at 37℃ for 1 hour to perform a crosslinking reaction of gelatin with TG. The sample bottle was then tilted to confirm the formation of a hydrogel. The results are shown in Figure 1(a).

[0058] As shown in Figure 1(a), the formation of hydrogels was confirmed in all samples using HAp-CaO2 (prepared by immersion in 100 mM PB). On the other hand, when CaO2 was used, no hydrogels were formed in the samples without catalase. It has been reported that TG is inactivated by hydrogen peroxide due to conformational changes in the peptide backbone associated with disulfide bond formation (J.Biol.Chem., 2018, 293, pp.2640-2649). It is considered that TG was inactivated due to the initial burst release of hydrogen peroxide from CaO2, and cross-linking of gelatin did not occur. In the samples using CaO2 and catalase, the formation of hydrogels was confirmed only at low concentrations (2.5 mg / mL CaO2). In the samples with high concentrations (10 mg / mL CaO2), it is considered that TG was inactivated before the decomposition of hydrogen peroxide by catalase.

[0059] [Test Example 1-2: Evaluation of oxygen supplying member] (Confirmation of hydrogel physical properties) The porous structure of the hydrogels was observed by scanning electron microscopy (SEM). 200 μL of catalase-containing hydrogels containing 2.5 mg / mL of CaO2 or HAp-CaO2 (prepared by immersion in 100 mM PB or 500 mM PB) were prepared in a 24-well plate insert (3470, Corning). As a control, hydrogels without CaO2 were similarly prepared. The prepared hydrogels were immersed in ethanol for 1 h, and then in t-butyl alcohol for 1 h three times. After freeze-drying and sectioning the samples, the surface morphology of each sample sputter-coated with osmium was observed by SEM.

[0060] The results of SEM observation are shown in Figure 1(b). The areas indicated by the arrows in Figure 1(b) are the pores used to estimate the pore size. A porous structure with a pore size of approximately 5 to 10 μm was identified in all hydrogels, indicating that there was no significant difference in the hydrogel structures.

[0061] (Evaluation of oxygen supply components 1: Evaluation of hydrogen peroxide concentration) When the hydrogel was placed in DMEM (transparent), the hydrogen peroxide concentration in the DMEM was evaluated using the Oxyselect Hydrogen Peroxide / Peroxidase Assay Kit.

[0062] 200 μL of hydrogels (with or without catalase) containing different concentrations of HAp-CaO2 (prepared by immersion in 100 mM PB) (0 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL) were placed in wells of a 24-well plate (3820-024, IWAKI) with 2 mL DMEM (transparent) and incubated at 37°C in a 5% CO2 incubator. After 3 days of incubation, 100 μL of supernatant was collected from each sample and the hydrogen peroxide concentration was measured using the kit mentioned above. The results are shown in Figure 2(a).

[0063] As shown in FIG. 2(a), hydrogen peroxide was not detected in the hydrogel containing catalase (all less than 1.5 μM). On the other hand, in the hydrogel not containing catalase, the hydrogen peroxide concentration increased with an increase in the HAp-CaO2 concentration (47 μM at 5 mg / mL HAp-CaO2 and 139 μM at 10 mg / mL HAp-CaO2). Hydrogen peroxide is generated by the reaction of CaO2 with water, and is a compound that exhibits cytotoxicity. In the oxygen supply member according to the present invention, the modified calcium peroxide (HAp-CaO2) and the hydrogen peroxide remover (catalase) are supported on the carrier (hydrogel) and are located close to each other, and further, the modified calcium peroxide (HAp-CaO2) and the hydrogen peroxide remover (catalase) are embedded in the carrier (hydrogel), so that it has been revealed that the generated hydrogen peroxide can be removed very effectively. This characteristic is extremely advantageous when used for cell culture applications or three-dimensional tissue structure manufacturing applications.

[0064] (Oxygen supply component evaluation 2: pH evaluation) The change in pH of DMEM (transparent) was evaluated when the hydrogel was placed in DMEM.

[0065] 200 μL of catalase-containing hydrogels containing different concentrations of HAp-CaO2 (prepared by immersion in 100 mM PB) (0 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL) were placed in wells of a 24-well plate together with 2 mL DMEM (transparent) and incubated at 37°C in a 5% CO2 incubator. After incubation for 1 hour, 1 day, 3 days, and 5 days, 100 μL of supernatant was collected from each sample and the pH was measured using a pH meter (LAQUAtwin-pH-22B, HORIBA). After the pH measurement, 100 μL of DMEM (transparent) was added to each sample.

[0066] As a result, no significant change in pH was observed in any of the samples (not shown). The reaction of CaO2 with water also produces the basic substance Ca(OH)2. This result indicates that the effect of Ca(OH)2 can be ignored.

[0067] (Evaluation of oxygen supply components 3: Evaluation of oxygen release characteristics) To evaluate the oxygen release characteristics of the hydrogel, the hydrogel was placed in DMEM (transparent) and the amount of dissolved oxygen in the DMEM was measured under low oxygen conditions using a dissolved oxygen measuring device (SDR Sensor Dish (registered trademark) Reader, manufactured by PreSens).

[0068] 200 μL of catalase-containing hydrogels containing CaO2 (2.5 mg / mL), HAp-CaO2 (prepared by immersion in 100 mM PB) (2.5 mg / mL), or HAp-CaO2 (prepared by immersion in 500 mM PB) (0 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL) were prepared in 24-well plate inserts. Hydrogels without CaO2 were prepared similarly as controls.

[0069] The 24-well plate inserts containing the hydrogel were placed in a 24-well sensor dish (OxoDish® OD24, manufactured by PreSens), and then 2 mL of DMEM (clear) was added to each sample. As an additional control, samples without hydrogel were also prepared. The dissolved oxygen measuring device and the 24-well sensor dish were placed in a hypoxic cell culture airtight bag (6-8669-03, manufactured by AS ONE). To create a hypoxic state, the O2 absorbent Anaeropack® (manufactured by Mitsubishi Gas Chemical) was added to the bag, which was then sealed with parafilm. The samples were then incubated at 37°C, and the amount of dissolved oxygen in the DMEM was monitored after 6 hours, 1 day, and every other day thereafter until 14 days later. The results are shown in Figures 2(b) to 2(e).

[0070] Figure 2(b) is a graph showing the amount of dissolved oxygen in the samples containing a hydrogel-free sample (w / o gel), a hydrogel containing no CaO2, a hydrogel containing CaO2 (2.5 mg / mL), a hydrogel containing HAp-CaO2 (prepared by immersion in 100 mM PB) (2.5 mg / mL), and a hydrogel containing HAp-CaO2 (prepared by immersion in 500 mM PB) (2.5 mg / mL), as a relative value with the oxygen concentration of the solution in equilibrium with atmospheric oxygen taken as 100%. Figure 2(c) is an enlarged view of the graph in Figure 2(b).

[0071] As shown in Fig. 2(b) and Fig. 2(c), the hydrogel containing CaO2 (2.5 mg / mL) reacted quickly with water, causing an initial burst release of oxygen, and the period during which oxygen was released was 3 days. On the other hand, the initial burst release of oxygen was suppressed compared to CaO2 for the hydrogel containing HAp-CaO2 produced by immersion in 100 mM PB, and the period during which oxygen was released increased to 4 days. Furthermore, the initial burst release of oxygen was further suppressed for the hydrogel containing HAp-CaO2 produced by immersion in 500 mM PB, and the period during which oxygen was released increased to about 10 days. From these results, it was confirmed that sustained oxygen release from the oxygen supplying material (hydrogel) is possible by using CaO2 (modified calcium peroxide) whose surface is coated with a coating containing hydroxyapatite.

[0072] Figure 2(d) is a graph showing the amount of dissolved oxygen in samples containing hydrogels containing HAp-CaO2 (prepared by immersion in 500 mM PB) (0 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL) as a relative value, with the oxygen concentration of the solution in equilibrium with atmospheric oxygen taken as 100%. Figure 2(e) is an enlarged view of the graph in Figure 2(d).

[0073] As shown in Figures 2(d) and 2(e), the dissolved oxygen concentration increased with increasing HAp-CaO2 concentration in the hydrogel. For example, the dissolved oxygen concentrations after 5 days were 6.0% (2.5 mg / mL), 8.8% (5 mg / mL), and 15.2% (10 mg / mL). These values ​​are similar to the dissolved oxygen concentrations in heart (9-18%), skeletal muscle (9-18%), brain (16%), and liver (19-28%) tissues, so the oxygen release properties of these hydrogels are extremely advantageous for use in the manufacture of three-dimensional tissue structures.

[0074] [Test Example 2: Cell culture test] [Test Example 2-1: Evaluation of cell proliferation under hypoxic conditions 1] Cell proliferation under hypoxic conditions using an oxygen supplying material (hydrogel) was evaluated by measuring mitochondrial activity using a WST-8 kit (CCK-8, Nacalai Tesque).

[0075] Human adipose tissue-derived hepatocytes (ADSCs) were prepared according to the method described in non-patent literature (Bioact. Mater., 2022, Vol. 7, pp. 227-241).

[0076] 200 μL of catalase-containing hydrogels containing different concentrations of HAp-CaO2 (prepared by immersion in 500 mM PB) (0 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL) were prepared in 24-well plate inserts and washed three times with 2 mL DMEM (cell culture) containing 10% FBS and 1% antibiotics.

[0077] 2×10 4 NHDF, MSC, ADSC and HepG2 cells were seeded on a 24-well plate with 1mL of DMEM (cell culture) containing 10% FBS and 1% antibiotics. After 3 hours of incubation at 37°C in a 5% CO2 incubator, the 24-well plate insert containing the hydrogel was placed into the 24-well plate, and then 1mL of DMEM (cell culture) containing 10% FBS and 1% antibiotics was added. As a control, a cell culture without hydrogel was also prepared. The 24-well plate was incubated at 37°C with Anaeropack (registered trademark) (Mitsubishi Gas Chemical) in a gas barrier box (GB-3.0L, AS ONE) to create a hypoxic condition (O2<0.1%).

[0078] DMEM (transparent) and cell count measurement reagent SF were mixed at a ratio of 9:1 to prepare the WST measurement reagent. After incubation for 7 days under hypoxic conditions, the cells were washed with 1 mL of PBS, and then 200 μL of WST assay reagent was added. The cells were incubated at 37 °C in a 5% CO2 incubator for 1 hour for NHDF and 45 minutes for MSC, ADSC and HepG2. Next, 100 μL of WST solution from each well was collected in a 96-well plate transwell (3860-096, IWAKI), and the absorbance at 450 nm was measured using a microplate reader. The mitochondrial activity of each sample was calculated as the relative value of the absorbance at 450 nm, with the value of the sample without hydrogel set to 100%. In addition, the cells were photographed using an EVOS (registered trademark) XL Core Imaging System (Thermo Fisher Scientific) after 7 days of cell culture. The results are shown in Figure 3 (a) to (f).

[0079] FIG. 3(a) is a schematic diagram showing the state of cell culture in this test example. FIG. 3(b) is a graph showing the results of evaluating the mitochondrial activity of NHDF. FIG. 3(c) is a graph showing the results of evaluating the mitochondrial activity of MSC. FIG. 3(d) is a graph showing the results of evaluating the mitochondrial activity of ADSC. FIG. 3(e) is a graph showing the results of evaluating the mitochondrial activity of HepG2. FIG. 3(f) is a photograph showing the results of photographing cells after 7 days of cell culture.

[0080] Figure 3(b) shows the results of evaluation in normal human dermal fibroblasts (NHDF). The sample using the hydrogel containing 2.5 mg / mL of HAp-CaO2 showed a dramatic increase in mitochondrial activity (518%). In addition, when the HAp-CaO2 concentration in the hydrogel increased from 2.5 mg / mL to 5 mg / mL and 10 mg / mL, the mitochondrial activity also increased from 518% to 567% and 657%. This result is consistent with the tendency that the amount of oxygen released increases with the HAp-CaO2 concentration in the hydrogel (Figures 2(d) and (e)). In addition, the sample using the hydrogel without HAp-CaO2 showed a slight increase in mitochondrial activity compared to the sample without hydrogel. This is thought to be due to the oxygen originally dissolved in the hydrogel being supplied to the cells at the early stage of incubation.

[0081] As shown in Figure 3(c), (d) and (e), in human mesenchymal stem cells (MSCs), adipose tissue-derived stem cells (ADSCs) and HepG2 hepatocellular carcinoma cells, the hydrogel containing HAp-CaO2 increased mitochondrial activity in MSCs by 563%, ADSCs by 164% and HepG2 by 920%, as in the case of NHDF. The difference in mitochondrial activity due to the cell type is thought to be due to the difference in oxygen utilization of each cell.

[0082] Figure 3(f) is a photograph (phase contrast image) of cells after 7 days of cell culture. For all cells, the hydrogel containing HAp-CaO2 promoted cell proliferation and increased the cell adhesion area (cells spread). On the other hand, for all cells, the cells did not spread well in samples that did not contain hydrogel and in hydrogels that did not contain HAp-CaO2. This is thought to be because the lack of oxygen affected cell function.

[0083] [Test Example 2-2: Evaluation of cell proliferation under hypoxic conditions 2] (Evaluation of modified calcium peroxide content) 200 μL of catalase-containing hydrogels containing CaO2 (2.5 mg / mL) or HAp-CaO2 (prepared by immersion in 500 mM PB) (0 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL) were prepared in 24-well plate inserts and then pre-incubated in 2 mL of DMEM (cell culture) containing 10% FBS and 1% antibiotics for 3 days in a 5% CO2 incubator.

[0084] Next, 2 × 10 4 NHDF cells were seeded onto a 24-well plate with 1 mL of DMEM (cell culture) containing 10% FBS and 1% antibiotics. After 3 hours of incubation at 37°C in a 5% CO2 incubator, the 24-well plate insert containing the pre-incubated hydrogel was placed into the 24-well plate, and then 1 mL of DMEM (cell culture) containing 10% FBS and 1% antibiotics was added. As a control, a cell culture without hydrogel was also prepared. The 24-well plate was incubated at 37°C with Anaeropack (registered trademark) (Mitsubishi Gas Chemical) in a gas barrier box (GB-3.0L, AS ONE) to create a hypoxic condition (O2<0.1%).

[0085] The WST measurement reagent was prepared by mixing DMEM (transparent) and cell count measurement reagent SF at a ratio of 9:1. After incubation for 7 days under hypoxic conditions, the cells were washed with 1 mL of PBS, and then 200 μL of WST assay reagent was added. The cells were incubated at 37 °C for 45 minutes in a 5% CO2 incubator. Next, 100 μL of WST solution from each well was collected in a 96-well plate transwell (3860-096, IWAKI), and the absorbance at 450 nm was measured using a microplate reader. The mitochondrial activity of each sample was calculated as the relative value of the absorbance at 450 nm, with the value of the sample without hydrogel set to 100%. In addition, the cells were photographed using an EVOS (registered trademark) XL Core Imaging System (Thermo Fisher Scientific) after 7 days of cell culture. The results are shown in Figure 4 (a) to (c).

[0086] FIG. 4(a) is a diagram showing the cell culture in this test example. In cell culture using a hydrogel containing CaO2, oxygen release ends during the 3-day preincubation, and oxygen is not released from the hydrogel during the subsequent cell culture period. On the other hand, in cell culture using a hydrogel containing HAp-CaO2 (manufactured by immersion in 500 mM PB), oxygen is released for about 10 days, so oxygen is released even during the cell culture period after the 3-day preincubation. FIG. 4(b) is a graph showing the results of evaluating the mitochondrial activity of NHDF. FIG. 4(c) is a photograph showing the results of photographing cells after 7 days of cell culture.

[0087] As shown in Figure 4(b), the sample using the hydrogel containing 2.5 mg / mL of HAp-CaO2 showed a dramatic increase in mitochondrial activity to 369%. On the other hand, the sample using the hydrogel containing 2.5 mg / mL of CaO2 showed only 180%. In addition, when the HAp-CaO2 concentration in the hydrogel was increased from 2.5 mg / mL to 5 mg / mL and 10 mg / mL, the mitochondrial activity also increased from 369% to 416% and 543%. Furthermore, as shown in Figure 4(c), significant cell proliferation was confirmed only in the sample using the hydrogel containing HAp-CaO2.

[0088] (Evaluation during long-term culture) As in the above (Evaluation of cell proliferation under hypoxic conditions 1), NHDFs were cultured under hypoxic conditions for 7 days. After 7 days of culture, the medium was replaced with a new one, the hydrogel was removed or retained, and the cells were cultured under hypoxic conditions for another 7 days (total 14 days). After 7 days of culture and after 14 days of culture, the number of live cells was measured by trypan blue staining. After 14 days of culture, the cells were photographed using an EVOS (registered trademark) XL Core Imaging System (manufactured by Thermo Fisher Scientific). The results are shown in Figure 4 (d) to (f).

[0089] Figure 4(d) is a schematic diagram showing the state of cell culture in this test example. After 7 days of culture, the sample with the hydrogel retained continues to receive oxygen, promoting cell proliferation. On the other hand, the sample from which the hydrogel was removed no longer receives oxygen, so cell proliferation is not promoted. Figure 4(e) is a graph showing the results of measuring the number of live cells. Figure 4(f) is a photograph showing the results of photographing the cells after 14 days of culture.

[0090] As shown in Figure 4(e), the number of viable cells after 7 days of culture was 2.0 × 10 4 6.2×10 4 Next, on the 7th day, the medium was replaced and the hydrogel was removed from the sample, and the viable cell count on the 7th day was 6.2 × 10 4 The number of live cells on day 14 is 10.0 x 10 4 This is thought to be due to the supply of oxygen from the fresh medium. On the other hand, the sample in which the medium was replaced on the 7th day and the hydrogel was retained had a viable cell count of 6.2 × 10 4 The number of live cells on day 14 was 12.9 x 10 4 The number of cases had increased by 10.

[0091] As shown in Figure 4(f), cell proliferation was clearly promoted in the sample in which the medium was replaced and the hydrogel was retained (Retain) on the 7th day compared to the sample in which the hydrogel was removed (Remove).

[0092] [Test Example 3: Production and evaluation of three-dimensional tissue structure] 1 mL of 16% by mass gelatin solution (DMEM) containing 10 U / mL TG, 100 U / mL catalase, 2.5 mM CaCl2, and 2.5 mg / mL HAp-CaO2 (prepared by immersion in 500 mM PB) was added to a 24-well plate. Before gelation, a 24-well insert containing 100 μL of 0.2 mg / mL fibronectin solution (50 mM Tris buffer, pH 7.4) was placed on the gelatin solution. Then, after incubation at 37 °C for 1 h to allow gelation, the fibronectin solution was removed and the resulting hydrogel was washed with medium (DMEM containing 10% FBS and 1% antibiotics). 5 × 10 6 Mouse fibroblast cells (L929 cells) were suspended in 200 μL of medium and added to the 24-well insert. Then, 400 μL of medium was added to the 24-well plate. After centrifuging the 24-well plate at 1100 rpm for 15 minutes, 1 mL of medium was added. As a control sample, a sample was prepared using the same procedure except that no hydrogel was prepared.

[0093] The 24-well plate was cultured in a 5% CO2 incubator at 37°C, and the medium was changed every day. After 3 days of culture, the three-dimensional tissue structure was fixed in 10% formaldehyde neutral buffer and embedded in paraffin.

[0094] The sectioned three-dimensional tissue structure was stained with hematoxylin-eosin (HE) and the HE-stained image was captured using an FL EVOS automated microscope (Thermo Fisher Scientific). The number of cells in five areas (200 μm × 200 μm) near the center of the HE-stained image was counted using ImageJ, and the cell density was calculated.

[0095] Sectioned 3D tissue constructs were permeabilized with 0.3% Triton X-100 for 5 min and blocked with 1% bovine serum albumin and 10% normal goat serum in PBS. They were then incubated with staining solution (Phalloidin-iFluor 594, DAPI, diluted 1:500 in PBS). After washing with PBS, they were observed using a confocal laser scanning microscope (CSLM). The results are shown in Figure 5(a)-(e).

[0096] Figure 5(a) is a schematic diagram of the cell culture in this test example. Figure 5(b) is an HE stained image of the control (sample not containing hydrogel). Figure 5(c) is an HE stained image of the sample using hydrogel containing HAp-CaO2. Figure 5(d) is a graph showing the measurement results of cell density. Figure 5(e) is a photograph showing a confocal laser scanning microscope (CSLM) image of the three-dimensional tissue construct stained with DAPI and phalloidin.

[0097] As shown in Figures 5(b) and 5(c), a thick three-dimensional tissue structure with high cell density was obtained in the sample containing hydrogel (Figure 5(c)). On the other hand, in the sample not containing hydrogel (control), there were many gaps (unstained areas) in the HE stained image, which is thought to be due to cell death caused by lack of oxygen. As shown in Figure 5(d), the cell density was significantly higher in the sample containing hydrogel. As shown in Figure 5(e), the phalloidin stained image (fluorescence image) showed that the actin-derived fluorescence was stronger in the sample using hydrogel.

[0098] These results show that the use of the oxygen supplying member (hydrogel) according to the present invention alleviates oxygen deficiency within the three-dimensional tissue structure, and significantly improves cell viability in the three-dimensional tissue structure.

[0099] [Test Example 4: Production and Evaluation of Modified Calcium Peroxide] [Test Example 4-1: Production and Evaluation of Modified Calcium Peroxide] The oxygen release behavior of the modified calcium peroxide was investigated by measuring the dissolved oxygen concentration using a multi-parameter DO meter (HANNA).

[0100] Various aqueous solutions containing sodium bicarbonate (44 mM, 10 mM, or 1 mM) and sodium dihydrogen phosphate (1 mg / mL, 0.1 mg / mL, 0.01 mg / mL, or 0 mg / mL) were prepared. As controls, HEPES buffer (pH 7.4) containing sodium dihydrogen phosphate (0.1 mg / mL or 0 mg / mL) and ultrapure water (MilliQ) containing sodium dihydrogen phosphate (0.1 mg / mL or 0 mg / mL) were prepared.

[0101] 10 mg of CaO2 was immersed in 10 mL of each of the above solutions, and the DO meter was immersed in the sample solution. The dissolved oxygen concentration was measured at room temperature every 2 minutes while stirring the sample solution. The results are shown in Figure 6.

[0102] Figure 6 is a graph showing the results of measuring the change in dissolved oxygen concentration over time in various solutions when CaO2 was immersed in them. As shown in Figure 6, when CaO2 was immersed in an aqueous solution containing 10 mM or more of sodium bicarbonate and 0.1 mg / mL or more of sodium dihydrogen phosphate, the release of oxygen from CaO2 was suppressed.

[0103] (Analysis of modified calcium peroxide) The surface structures of CaO2 immersed in an aqueous solution containing 44 mM sodium bicarbonate and 0.1 mg / mL sodium dihydrogen phosphate (modified calcium peroxide) and CaO2 immersed in an aqueous solution containing only 44 mM sodium bicarbonate (control calcium peroxide) were analyzed by SEM and X-ray diffraction (XRD).

[0104] The modified calcium peroxide and the control calcium peroxide were collected by centrifugation at 4000 rpm for 3 min, washed three times with ethanol, and then the samples were dried under vacuum at room temperature. The dried modified calcium peroxide and the control calcium peroxide were sputter-coated with osmium using an HPC-30 plasma coater, and the surface morphology was observed with a JSM-6701F device (JEOL Ltd.). The crystal structure of each sample was also evaluated by XRD using an X-ray diffractometer (AERIS, Malvern). The results are shown in Figure 7.

[0105] Figure 7 shows SEM images of modified calcium peroxide (NaH2PO4 0.1 mg / mL) and control calcium peroxide (NaH2PO40 mg / mL) and spectra obtained by XRD. As shown in Figure 7, peaks derived from calcium carbonate crystals (calcite and vaterite) were observed in the XRD spectrum of the control calcium peroxide, whereas these peaks were not observed in the modified calcium peroxide. In addition, calcium carbonate crystals were observed on the CaO2 surface in the SEM image of the control calcium peroxide, whereas they were not observed in the modified calcium peroxide. These results suggest that in the modified calcium peroxide, the surface of CaO2 is covered with amorphous calcium carbonate.

[0106] Calcium carbonate crystals are formed in a supersaturated solution of Ca 2+ and CO3 2- It has been reported that CaO2 is formed from amorphous CaCO3 via a metastable amorphous state (Science, 2015, Vol. 349, aaa6760). In addition, a process called dispersion-re-precipitation has been reported in which amorphous CaCO3 dissolves in the presence of crystal nuclei and crystals grow (Science, 2014, Vol. 345, 1158). Furthermore, it has been reported that phosphoric acid inhibits this process (Cryst. Growth Des., 2021, Vol. 21, 414). Based on these findings, it can be seen that CaO2 (modified calcium peroxide) whose surface is coated with amorphous CaCO3 can be obtained by immersing CaO2 in an aqueous solution containing sodium bicarbonate and sodium dihydrogen phosphate at a specific concentration or higher. Hereinafter, this modified calcium peroxide will also be referred to as ACC-CaO2.

[0107] [Test Example 4-2: Evaluation of modified calcium peroxide (ACC-CaO2)] 10 mg of CaO2 was immersed in 10 mL of DMEM (pH 7.0-8.0, containing 44 mM NaHCO3 and 0.11 mg / mL NaH2PO4), and the DO meter was immersed in the sample solution. The dissolved oxygen concentration was measured at room temperature every 2 minutes while stirring the sample solution. In addition, 30 minutes after the start of the measurement, the modified calcium peroxide was collected by centrifugation at 4000 rpm for 3 minutes, and redispersed in DMEM whose pH had been adjusted to 5.0 with hydrochloric acid, and the dissolved oxygen concentration was measured again. The results are shown in Figure 8.

[0108] FIG. 8 is a graph showing the results of measuring the change in dissolved oxygen concentration over time. As in Test Example 4-1, the surface of CaO2 was covered with amorphous CaCO3, suppressing oxygen release until 30 minutes after the start of the experiment. On the other hand, an increase in the dissolved oxygen concentration was confirmed 30 minutes after the pH was adjusted to 5.0. It is known that the lower the pH, the higher the solubility of amorphous CaCO3. This result indicates that the reaction between CaO2 and water occurred as amorphous CaCO3 dissolved in a weakly acidic environment. This result also indicates that the oxygen supplying material containing modified calcium peroxide (ACC-CaO2) can be used as a pH-responsive oxygen supplying material.

Claims

1. A carrier, a modified calcium peroxide, and a hydrogen peroxide scavenger, The modified calcium peroxide is calcium peroxide whose surface is coated with a coating film, The oxygen supplying member, wherein the modified calcium peroxide and the hydrogen peroxide removing agent are supported on the carrier.

2. 2. The oxygen supply member according to claim 1, wherein the coating contains hydroxyapatite.

3. 2. The oxygen supply member according to claim 1, wherein the coating contains amorphous calcium carbonate.

4. The oxygen supply member according to claim 1 , wherein the carrier is a hydrogel.

5. The oxygen supply member according to claim 4 , wherein the modified calcium peroxide and the hydrogen peroxide scavenger are embedded in the hydrogel.

6. The oxygen delivery member according to claim 4 , wherein the hydrogel comprises cross-linked gelatin.

7. The oxygen supply member according to claim 1 , wherein the hydrogen peroxide remover comprises catalase.

8. The oxygen supply member according to claim 1 , which is for cell culture.

9. The oxygen supplying member according to claim 1 , which is used for producing a three-dimensional tissue structure.

10. A method for producing a three-dimensional tissue structure, comprising culturing at least one type of cell in a culture medium while the oxygen supplying member according to any one of claims 1 to 9 is in contact with the culture medium.

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