Oxygen supplying agent

Abstract

To provide an oxygen supplying agent capable of efficiently forming oxygen from a peroxide produced by the reaction of water with a metal peroxide.SOLUTION: An oxygen supplying agent comprising: a metal peroxide; and a first anionic coating located on at least a part of the metal peroxide.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to oxygenates. [Background technology]

[0002] Regenerative medicine has been studied for some time, in which compositions are constructed ex vivo and then transplanted into the living body. However, constructing tissues ex vivo is difficult, and constructing thick tissues (e.g., 200 μm or thicker) has been particularly challenging. One of the reasons for this is the insufficient supply of oxygen to the interior of the tissue. For this reason, it is expected that if sufficient oxygen can be supplied to the interior of the tissue, it will be possible to construct three-dimensional tissues thicker than a millimeter.

[0003] In recent years, cell scaffold materials that release oxygen sustainedly have been investigated. For example, materials using hemoproteins (hemoglobin, myoglobin) as oxygen sources (hereinafter sometimes referred to as "oxygen sources") have been investigated (Non-Patent Document 1). However, materials using hemoproteins have a low oxygen storage capacity, and therefore, there is a need to increase the oxygen storage capacity. Furthermore, protein stability may remain an issue.

[0004] Furthermore, materials using calcium peroxide (CaO2) as an oxygen source have been reported (Non-Patent Documents 2 to 4). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] D. Tomioka et al., Chem. Commun. 2021, 57, 5131. [Non-patent document 2] K. Park et al., Biomaterials 2018, 182, 234. [Non-patent document 3] D. Tomioka et al., Chem. Mater. 2023, 35, 5378. [Non-patent document 4] D. Tomioka et al., ACS Omega 2024, 9, 5903. Summary of the Invention [Problem to be solved by the invention]

[0006] Metal peroxides such as CaO2 react with water to produce hydrogen peroxide and oxygen, but it is difficult to control the amount or timing of contact between the metal peroxide and water, and it is also difficult to control the amount of oxygen released. Furthermore, the generation of hydrogen peroxide is undesirable, so it was necessary to consider ways to reduce hydrogen peroxide.

[0007] An object of the present disclosure is to provide an oxygen supplying agent capable of efficiently producing oxygen from peroxides produced by the reaction of water with metal peroxides. [Means for solving the problem]

[0008] The present disclosure provides the following [1] to

[12] . [1] A metal peroxide, a first anionic coating located at least in part on the metal peroxide; and An oxygen supplying agent comprising: [2] The oxygen supply agent according to [1], wherein the particle diameter D50 of the metal peroxide is 1 mm or less. [3] The oxygen supplying agent according to [1] or [2], wherein the metal atom of the metal peroxide is a divalent cation. [4] The oxygen supplying agent according to any one of [1] to [3], wherein the metal peroxide contains at least one of Mg and Ca. [5] The oxygen supplying agent according to any one of [1] to [4], wherein the first anionic coating contains at least one of catalase, peroxidase, and glutathione peroxidase. [6] The oxygen supplying agent according to any one of [1] to [5], further comprising a first cationic coating on at least a portion of the first anionic coating. [7] The oxygen supplying agent according to any one of [1] to [6], wherein the first cationic coating comprises at least one of poly-L-lysine, polyethyleneimine, polyallylamine, and polydiallyldimethylammonium chloride. [8] The oxygen supplying agent according to any one of [1] to [8], further comprising an intermediate film at least partially between the metal peroxide and the first anionic coating. [9] The oxygen supplying agent according to [8], wherein the intermediate film is formed using at least one of a phosphate buffer solution and a carbonate buffer solution.

[10] The oxygen supplying agent according to [8] or [9], wherein the intermediate film contains hydroxyapatite and calcium carbonate.

[11] moreover, an intermediate film at least partially between the metal peroxide and the first anionic coating; a first cationic coating on at least a portion of the first anionic coating; The oxygen supplying agent according to any one of [1] to

[10] .

[12] further comprising a coating layer consisting of a second anionic coating and a second cationic coating; The oxygen supplying agent according to

[11] , wherein at least one coating layer is present on at least a portion of the first cationic coating. [Effects of the Invention]

[0009] According to the present disclosure, it is possible to provide an oxygen supplying agent that can efficiently generate oxygen from peroxides generated by the reaction of water with metal peroxides. [Brief explanation of the drawings]

[0010] [Figure 1]FIG. 1 is a schematic diagram showing an oxygen supplying agent (LbL-HAp-CaO2) formed by laminating a hydroxyapatite film (HAp), a first anionic coating, a first cationic coating, a second anionic coating, and a second cationic coating in this order on a metal peroxide CaO2 (also referred to as Layer by Layer coating, LbL). [Figure 2] FIG. 2 is a graph showing the results of Example 1, showing the relationship between the total number of layers of catalase and poly-L-lysine and the frequency shift (Hz) and total mass (ng / cm 2 ) at that time. [Figure 3] FIG. 3 is a graph showing the zeta potential of catalase and poly-L-lysine. [Figure 4] FIG. 4 is a graph showing the results of Example 2, in which the horizontal axis of the graph shows time and the vertical axis shows the content of H2O2 over time. [Figure 5] FIG. 5 shows the results of Example 3. The upper row shows the fluorescent signal immediately after the oxygen-supplying agent was formed, and the lower row shows the fluorescent signal of the oxygen-supplying agent after 14 days. [Figure 6] FIG. 6 is a graph showing the results of Example 4 and Comparative Example 1, with the horizontal axis representing time and the vertical axis representing the H2O2 content. [Figure 7A] FIG. 7A is an illustration for explaining the fifth embodiment. [Figure 7B] The left image of FIG. 7B shows a slice image of a three-dimensional tissue without an oxygen-supplying agent, and the right image shows a slice image of a three-dimensional tissue with an oxygen-supplying agent. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments. The drawings include some schematic views and may not reflect actual dimensions or proportions.

[0012] [Oxygen supply agent] The oxygen supplying agent of the present disclosure comprises: A metal peroxide, a first anionic coating located at least in part on the metal peroxide; and It has. By adopting the above-mentioned embodiment, oxygen can be efficiently produced from hydrogen peroxide produced by reacting a metal peroxide with water.

[0013] Preferably, the oxygen supplying agent of the present disclosure further comprises a first cationic coating on at least a portion of the first anionic coating. The above-mentioned embodiment improves the stability of the oxygen supplying agent, and the first cationic coating improves cell adhesion to the oxygen supplying agent.

[0014] Preferably, the oxygen supplying agent of the present disclosure further comprises an intermediate film at least partially between the metal peroxide and the first anionic coating. By adopting the above-mentioned embodiment, the reaction between the metal peroxide and water can be suppressed, the stability of the oxygen supplying agent can be improved, and the sustained oxygen-releasing ability of the oxygen supplying agent can be maintained for a long period of time.

[0015] Preferably, the oxygen supplying agent of the present disclosure further has a coating layer consisting of a second anionic coating and a second cationic coating, and at least one of the coating layers is present on at least a portion of the first cationic coating. That is, the coating layer has an LbL of 2 or more. The second anionic coating and the second cationic coating may be the same as the first anionic coating and the first cationic coating, respectively. The coating layer may be, for example, 1 to 10 layers. The above-described embodiment allows the metal peroxide to react with water to generate hydrogen peroxide, which is then efficiently converted into oxygen in the first and second anionic coatings. Furthermore, the intermediate film can inhibit the reaction between the metal peroxide and water, improving the stability of the oxygen-supplying agent. Furthermore, the second cationic coating, specifically the outermost second cationic coating of the oxygen-supplying agent, improves cell adhesion to the oxygen-supplying agent.

[0016] Preferably, as shown in FIG. 1, the oxygen supplying agent (for example, the oxygen supplying agent LbL-HAp-CaO when CaO is used as the metal peroxide) is a metal peroxide (e.g., CaO2), an intermediate film (e.g., a hydroxyapatite film HAp) located at least partially on the metal peroxide; a first anionic coating located at least partially on the intermediate film; a first cationic coating located at least partially on the first anionic coating; and Furthermore, at least one coating layer consisting of a second anionic coating and a second cationic coating is provided on at least a portion of the first cationic coating. The above-described embodiment allows efficient production of oxygen from hydrogen peroxide produced by the reaction of metal peroxide with water in the first and second anionic coatings. Furthermore, the presence of an intermediate film can suppress the reaction between metal peroxide and water, improving the stability of the oxygen supply agent. In addition, although CaO2 is shown as an example of a metal peroxide in Figure 1, other metal peroxides as shown below may also be used. Also, although a coating layer is shown in Figure 1, the coating layer may not be included, and only a first anionic coating film and a first cationic coating film may be included. In other words, LbL may be 1 only.

[0017] The oxygen supplying agent is preferably biocompatible. "Biocompatible" means that it does not cause excessive inflammation or the like when it comes into contact with living tissue.

[0018] The shape of the oxygen supplying agent is not particularly limited, but examples thereof include fibrous, flat, ellipsoidal, and spherical shapes, and specifically, the shape is ellipsoidal or spherical.

[0019] The particle diameter D50 of the oxygen supplying agent is not particularly limited, but may be 75 μm or more, 10 μm or more, 200 μm or less, or 75 μm or less. In this specification, the particle diameter D50 can be measured using a phase contrast microscope or a scanning electron microscope, specifically, a scanning electron microscope. D50 is the median diameter, and refers to the particle diameter at which 50% of particles have a particle diameter of D50 or less.

[0020] The particle size of the oxygen supplying agent may be, for example, in the range of 10 to 100 μm. The oxygen supplying agent may contain particles with a large particle size. The large particle size may be, for example, 200 μm or less. The particle size may be measured using a phase contrast microscope or a scanning electron microscope, and specifically, may be measured using a scanning electron microscope.

[0021] (metal peroxide) The shape of the metal peroxide is not particularly limited, but may be, for example, an ellipsoid, a sphere, or a particle. When the metal peroxide has the above-mentioned shape, it is easy to handle.

[0022] The particle diameter D50 of the metal peroxide is preferably 1 mm or less, for example, 75 μm or less. The lower limit of the particle diameter D50 of the metal peroxide is not particularly limited, but is, for example, 5 μm. The particle diameter of the metal peroxide can be measured before the formation of the coating. By adopting the above-mentioned embodiment, it becomes easier to apply the composition to a three-dimensional tissue having a thickness of several hundred micrometers.

[0023] The metal peroxide may be any suitable material as long as it can supply sufficient oxygen depending on the application of the oxygen supply agent, etc. The content of the metal peroxide is not particularly limited, but may be, for example, in the range of 10 to 90% of the particle diameter D50 of the oxygen supply agent, in the range of 20 to 80%, or in the range of 30 to 60%.

[0024] The metal peroxide contains a metal atom, which is preferably a divalent cation, such as at least one of Mg and Ca, for example, Ca.

[0025] The metal peroxide is preferably MgO2, CaO2, for example CaO2.

[0026] (interlayer film) By providing the intermediate film, a porous film is formed on the surface of the metal peroxide, which can suppress the diffusion and movement of surrounding water. Furthermore, the reactivity of the metal peroxide (e.g., CaO2) with water is reduced, preventing the metal peroxide from coming into contact with water. Furthermore, since the intermediate film can suppress the reaction between the metal peroxide and water, a larger amount of metal peroxide can be used as the oxygen supply agent.

[0027] The interlayer is formed on at least a portion of the metal peroxide, and may cover the entire metal peroxide.

[0028] The interlayer film may be a single layer or a laminate of multiple layers.

[0029] The average thickness of the interlayer is, for example, 0.5 to 20 μm, and preferably 1 to 5 μm. The average thickness can be measured by energy dispersive X-ray spectroscopy or a scanning electron microscope, and preferably by a scanning electron microscope.

[0030] The average thickness of the intermediate film is not particularly limited, but may be, for example, in the range of 5 to 90%, 5 to 60%, or 5 to 50% of the particle diameter D50 of the oxygen supplying agent.

[0031] The intermediate film is preferably formed using at least one of a phosphate buffer solution and a carbonate buffer solution, and is formed, for example, from a phosphate buffer solution.

[0032] The buffer solution may be one whose pH is adjusted, for example, to a value between 5 and 10, specifically 7.0.

[0033] By immersing the metal peroxide in the buffer solution, an interlayer film can be formed on the surface of the metal peroxide. The temperature conditions during immersion are not particularly limited, but may be, for example, in the range of 20 to 50°C, specifically in the range of 30 to 40°C, more specifically, 37°C. The immersion time 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.

[0034] In one embodiment, the interlayer film is obtained by impregnating calcium peroxide with an aqueous solution containing phosphate ions. The concentration of phosphate ions in the aqueous solution is not particularly limited, but may be, for example, in the range of 50 mM (i.e., mol / L) to 1 M, or in the range of 100 mM to 500 mM. An example of the aqueous solution is an aqueous solution obtained by mixing an aqueous solution of sodium dihydrogen phosphate and an aqueous solution of disodium hydrogen phosphate at the same concentration so that the pH is 7.0.

[0035] In one embodiment, the interlayer film is obtained by immersing calcium peroxide in an aqueous solution containing phosphate ions and carbonate ions. The concentration of phosphate ions in the aqueous solution is not particularly limited, but may be, for example, in the range of 0 mM to 50 mM, or in the range of 0.5 mM to 10 mM. The concentration of carbonate ions in the aqueous solution is not particularly limited, but may be, for example, in the range of 5 mM to 1 M, or in the range of 10 mM to 500 mM, or in the range of 10 mM to 100 mM. The aqueous solution may be, for example, an aqueous solution containing at least one of sodium dihydrogen phosphate and sodium hydrogen carbonate, and may have a pH between 5 and 10, preferably 7.

[0036] The aqueous solution may contain, in addition to water, other solvents such as ethanol and dimethyl sulfoxide (DMSO), in which case the other solvent may be contained in the water in an amount of 0 to 50% by volume.

[0037] The buffer solution may further contain other ions and / or other compounds. Examples of other ions include calcium ions, sodium ions, carbonate ions, and phosphate ions. Examples of other compounds include buffering agents, polymers, and surfactants.

[0038] The interlayer preferably contains at least one of hydroxyapatite (Ca5(PO4)3(OH), sometimes referred to as "HAp") and calcium carbonate (CaCO3). In one embodiment, the interlayer contains hydroxyapatite. In another embodiment, the interlayer contains calcium carbonate. The calcium carbonate may be in an amorphous state stabilized with phosphoric acid.

[0039] (First anionic coating) The first anionic coating is not particularly limited as long as it can remove hydrogen peroxide, but may contain, for example, at least one enzyme selected from catalase, peroxylase, and glutathione peroxidase, and more preferably at least one of catalase and peroxylase. In one embodiment, the first anionic coating contains catalase. In another embodiment, the first anionic coating contains peroxylase. The first anionic coating may be formed from an anionic polymer.

[0040] The first anionic coating is formed on at least a portion of the metal peroxide. The first anionic coating may be formed to cover the entire interlayer. For example, the first anionic coating may cover the entire interlayer or a portion thereof; it may cover the exposed portions of the interlayer and the metal peroxide.

[0041] The first anionic coating may be a single layer or a laminate of multiple layers.

[0042] The average thickness of the first anionic coating is, for example, 1 to 100 nm, and preferably 5 to 20 nm. The average thickness of the first anionic coating can be measured using a quartz crystal microbalance.

[0043] The first anionic coating can be used, for example, as a composition prepared by mixing (e.g., dispersing) a compound for forming the first anionic coating (e.g., catalase, peroxidase, glutathione peroxidase) in a solvent. The compound can be contained in the composition at a concentration of 0.1 to 5 mg / mL, for example, 1 mg / mL. Examples of the solvent include those that do not affect living organisms, and are not particularly limited, but include water. The pH of the composition is, for example, 5 to 10, specifically 7. The composition may contain a buffer and ions in addition to the compound. Examples of ions include phosphate ions, chloride ions, sodium ions, potassium ions, hydrogen ions, carbonate ions, etc. For example, a phosphate buffer can be used as the composition. Specifically, a phosphate buffer of pH 5 to 10 (specifically, pH 7.0) can be used, for example, at 10 mM.

[0044] (First cationic coating) The first cationic coating preferably comprises a cationic polymer, more preferably at least one of poly-L-lysine (PLL), polyethyleneimine, polyallylamine, and polydiallyldimethylammonium chloride, for example, poly-L-lysine. The first cationic coating may include one formed from a cationic polymer.

[0045] The poly-L-lysine may be a polymer of 50 to 2,500 L-lysine molecules. Polyethyleneimine is a compound in which amines are linked by ethylene chains, and may be a polymer of 50 to 500 ethyleneimines. The polyallylamine may be one obtained by polymerizing 100 to 1,500 allylamine units. The polydiallyldimethylammonium chloride may be a polymer of 100 to 5,000 units of diallyldimethylammonium chloride.

[0046] The weight average molecular weight Mw of the first cationic coating may be in the range of 10,000 to 300,000, or in the range of 15,000 to 30,000.

[0047] The first cationic coating may be provided on at least a portion of the first anionic coating. The first cationic coating may be formed to cover the entire first anionic coating. For example, the first cationic coating may cover the entire first anionic coating, and the first anionic coating may cover all or part of the metal peroxide; it may cover the exposed portions of the first anionic coating and the metal peroxide; or it may cover all or part of the first anionic coating, the intermediate layer, and / or the metal peroxide.

[0048] The first cationic coating may be a single layer or a laminate of multiple layers.

[0049] The average thickness of the first cationic coating is, for example, 1 to 100 nm, and preferably 5 to 20 nm.The average thickness of the first cationic coating can be measured by a quartz crystal microbalance.

[0050] The first cationic coating can be used, for example, as a composition prepared by mixing (e.g., dispersing) a compound (e.g., a cationic polymer) for forming the first cationic coating in a solvent. The compound can be contained in the composition at a concentration of 0.1 to 5 mg / mL, for example, 1 mg / mL. Examples of the solvent include those that do not affect living organisms, and include, but are not limited to, water. The pH of the composition is, for example, 5 to 10, specifically 7. The composition may contain a buffer and ions in addition to the compound. Examples of ions include phosphate ions, chloride ions, sodium ions, potassium ions, hydrogen ions, and carbonate ions. For example, a phosphate buffer can be used as the composition. Specifically, a phosphate buffer of pH 5 to 10 (specifically, pH 7.0) can be used, for example, at 10 mM.

[0051] (Second anionic coating) The second anionic coating can be formed on at least a portion of the first cationic coating. The second anionic coating can be formed to cover the entire first cationic coating. For example, the second anionic coating can cover the entire first cationic coating; can cover a portion of the first cationic coating; or can cover the first cationic coating, the first anionic coating, and / or the metal peroxide.

[0052] The second anionic coating may be the same as the first anionic coating. However, the second anionic coating may be different from the first anionic coating. When there are multiple second anionic coatings, they may be different from each other.

[0053] (Second cationic coating) The second cationic coating can be formed on at least a portion of the second anionic coating. The second cationic coating can be formed to cover the entire second anionic coating. For example, the second cationic coating can cover the entire second anionic coating; it can cover a portion of the second anionic coating; or it can cover the second anionic coating, the first cationic coating, the first anionic coating, and / or the metal peroxide.

[0054] The second cationic coating may be the same as the first cationic coating. Alternatively, the second cationic coating may be different from the first cationic coating. When there are multiple second cationic coatings, they may be different from each other.

[0055] The coating layer consisting of the second anionic coating and the second cationic coating may be present on at least a portion of the first cationic coating, and may include, for example, 1 to 10 layers. That is, the second anionic coating and the second cationic coating may be provided alternately on the first anionic coating and the first cationic coating. By adopting the above-described embodiment, oxygen can be efficiently produced from hydrogen peroxide produced by reacting metal peroxide with water, and the stability of the layer-by-layer nanothin film can be improved, resulting in good stability of the oxygen supply agent.

[0056] The oxygen supplying agent may include 1 to 10 coating layers each consisting of a second anionic coating and a second cationic coating on at least a portion of the first cationic coating, and may further include a second anionic coating. In other words, the outermost layer of the oxygen supplying agent may not have a second cationic coating.

[0057] [Composition] The oxygen supplying agent can be used as a composition dispersed in a solvent or the like.

[0058] The oxygen supplying agent may be contained in the composition at 0.1 to 100 mg / mL, or may be contained at 1 to 10 mg / mL.

[0059] The solvent may include water.

[0060] The composition may further contain a compound that constitutes an intermediate film, a first anionic coating, a first cationic coating, a second anionic coating, and / or a second cationic coating, where the intermediate film, the first anionic coating, the first cationic coating, the second anionic coating, and the second cationic coating are each defined as above.

[0061] The composition may further comprise buffers, ions, proteins, amino acids, glucose, and the like.

[0062] Examples of buffering agents include phosphoric acid.

[0063] (Method of manufacturing oxygen supply agent) First, a metal peroxide is prepared.

[0064] Separately, a buffer solution for forming an interlayer film, such as a phosphate buffer solution, is prepared. A metal oxide is added to the buffer solution at, for example, 0.1 to 10 mg / mL, specifically 1 mg / mL. The mixture is then mixed for 30 minutes or longer, for example, at a temperature in the range of 20 to 50°C, specifically 30 to 40°C, and more specifically 37°C. Mixing may be continued for, for example, 45 minutes or longer, 1 hour or longer, and / or 2 hours or shorter. The metal oxide having the interlayer film is then recovered by centrifugation and dried under reduced pressure. An anionic coating and / or cationic coating may be formed directly without drying under reduced pressure.

[0065] A solution (e.g., phosphate buffer) containing a compound for forming a first anionic coating film is prepared, for example, at a concentration of 0.1 to 10 mg / mL, specifically 1 mg / mL, and a metal peroxide having an intermediate film is dispersed therein at a concentration of, for example, 0.1 to 10 mg / mL, specifically 1 mg / mL. The mixture is mixed at a temperature of, for example, 4 to 40°C, specifically 25°C, for example, for 5 to 30 minutes, specifically 15 minutes. The pH of the solution is, for example, 5 to 10, specifically 7. For example, a 10 mM phosphate buffer solution having a pH of 5 to 10 (specifically, pH 7.0) can be used. The oxygen supply member on which the first anionic coating film is formed is then recovered by centrifugation.

[0066] Furthermore, the oxygen supply agent having the first anionic coating formed thereon is dispersed in a solution of the first cationic coating, for example, a 0.1 to 10 mg / mL solution, specifically 1 mg / mL solution, at a concentration of, for example, 0.1 to 10 mg / mL, specifically 1 mg / mL, and mixed for, for example, 5 to 30 minutes, specifically 15 minutes, at, for example, 4 to 40° C., specifically 25° C. The pH of the above solution is, for example, 5 to 10, specifically 7, and for example, 10 mM phosphate buffer of pH 5 to 10 (specifically pH 7.0) can be used.

[0067] Thereafter, if necessary, a second anionic coating and / or a second cationic coating is formed in the same manner as the first anionic coating and / or the first cationic coating.

[0068] This produces a composition containing an oxygen supplying agent.

[0069] [Application] The oxygen supplying agent can be suitably used for cell culture, production of three-dimensional tissue structures, and the like.

[0070] The method for producing a three-dimensional tissue structure according to this embodiment comprises a step of culturing at least one type of cell using the oxygen supplying agent according to the present invention (hereinafter also referred to as a "culturing step").

[0071] The three-dimensional tissue structure may be, for example, skeletal muscle tissue, adipose tissue, vascular tissue, nervous tissue, epidermal tissue, epithelial tissue, cardiac muscle tissue, cartilage tissue, etc. The three-dimensional tissue structure may also be a structure (cell mass) in which at least one type of cell is arranged three-dimensionally and does not have a specific tissue structure.

[0072] The shape of the three-dimensional tissue structure is not particularly limited, and may be, for example, spherical, ellipsoidal, rectangular, cubic, fibrous, or the like.

[0073] 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 they may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, etc., or germ cells. Furthermore, the cells may be stem cells, or cultured cells such as primary cultured cells, subcultured cells, and cell line cells.

[0074] Specific examples of 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.

[0075] The medium can be selected appropriately depending on the type of cells to be cultured, etc. 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, and EGM2. The medium may be a serum-supplemented medium or a serum-free medium. The medium may also be a mixed medium comprising two types of media.

[0076] The method for contacting the oxygen supply agent with the culture medium is not particularly limited, and for example, a method in which the oxygen supply agent is added to the culture medium directly or together with a support (e.g., a hydrogel), or a method in which the culture medium is added to a culture vessel having an oxygen supply agent attached or unattached thereto, can be used.

[0077] The cells may be contained in the medium, and for example, the cells may be outside the oxygen supplying agent or may be directly attached to the oxygen supplying agent.

[0078] The amount of oxygen supplying agent used according to the present invention can be appropriately determined depending on the type of cells, the culture period, the shape and thickness of the desired three-dimensional tissue structure, etc. The oxygen supplying member according to the present invention can be used in an amount such that the amount of oxygen supplying agent (e.g., LbL-HAp-CaO2) per mL of culture medium is, for example, 0.0.1 mg to 10 mg, or 0.05 mg to 5 mg, although this is not limited thereto.

[0079] The culturing step can be carried out in the same manner as in the usual culturing method for a three-dimensional tissue structure.

[0080] The cell density in the medium in the culture step can be appropriately determined depending on the shape and thickness of the desired 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, and 3 ~10 7 It may be cells / mL.

[0081] The method for culturing cells can be an appropriate 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.

[0082] The cells to be cultured may be dispersed in a hydrogel together with an oxygen supplying agent. Examples of hydrogels include fibrin gel, Matrigel, collagen gel, and gelatin gel. Dispersing the cells to be cultured in a hydrogel makes it easier to form a three-dimensional tissue structure with a more desired shape.

[0083] When the cells to be cultured include stem cells, the culture step may include proliferation culture and differentiation-inducing culture. Differentiation-inducing 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.

[0084] Although the embodiments of the present disclosure have been described above, it will be understood that various changes in form and details can be made without departing from the spirit and scope of the claims. [Example]

[0085] The present disclosure will be explained in more detail below through examples, but is not limited to these examples.

[0086] [Materials used] Calcium peroxide (CaO2): particle size 200 mesh, 74 μm, manufactured by Sigma-Aldrich First anionic coating, second anionic coating: Catalase from bovine liver, Sigma-Aldrich First cationic coating, second cationic coating: Poly-L-lysine hydrobromide (PLL) (Mw=15,000-30,000), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Sodium dihydrogen phosphate: Fujifilm Wako Pure Chemical Industries, Ltd. Disodium hydrogen phosphate 12-water: Fujifilm Wako Pure Chemical Industries, Ltd. Sulfuric acid: Fujifilm Wako Pure Chemical Industries, Ltd. Hydrogen peroxide: Kishida Chemical Phosphate buffered saline (PBS): Nacalai Tesque Fluorescein Isothiocyanate (FITC), manufactured by Nacalai Tesque Dimethyl sulfoxide (DMSO): Fujifilm Wako Pure Chemical Industries, Ltd. Fibronectin: Sigma-Aldrich Dulbecco's Modified Eagle Medium (DMEM): Nacalai Tesque Fetal bovine serum (FBS): Thermo Fisher Scientific 10% neutral formalin: manufactured by Nacalai Tesque

[0087] [Experimental Example 1: Examination of laminated films] Example 1 In Example 1, an LbL nano-thin film was prepared on a quartz crystal microbalance (QCM) chip, and the formation of the nano-thin film was evaluated based on the change in vibration frequency.

[0088] Phosphate buffer was prepared by mixing equal concentrations of sodium dihydrogen phosphate and disodium hydrogen phosphate solutions to a pH of 7.0. Catalase and PLL were dissolved at 1 mg / mL in 10 mM phosphate buffer (pH 7.0). A QCM chip (QCM01S, ULVAC) washed with piranha solution was placed in a QCM measurement device (affinixQ8, ULVAC). After washing the chip with 10 mM phosphate buffer, catalase solution was added and the frequency change was measured after 15 minutes. The chip was then washed with 10 mM phosphate buffer, PLL solution was added, and the frequency change was measured after 15 minutes. Five similar cycles were performed.

[0089] In Figure 2, the horizontal axis shows the total number of layers of catalase and poly-L-lysine, and the vertical axis shows the frequency shift (Hz) and the total mass (ng / cm) calculated from the frequency shift. 2 ) The first layer on the horizontal axis is the first anionic coating, the second layer is the first cationic coating, the third, fifth, seventh, and ninth layers are the second anionic coating, and the fourth, sixth, eighth, and tenth layers are the second cationic coating. Figure 2 was created based on values ​​measured with n=3.

[0090] As shown in Figure 2, catalase and poly-L-lysine were layered in order. Catalase was 8,797±1,165 ng / cm 2 , and poly-L-lysine was 2,386±788.7ng / cm 2 Stacked.

[0091] As shown in Figure 3, catalase is an anionic protein that exhibits a negative zeta potential, while poly-L-lysine is a cationic polymer that exhibits a positive zeta potential.

[0092] [Method for measuring zeta potential] Catalase was dissolved at 1 mg / mL in 10 mM phosphate buffer (pH 7.0). Separately, PLL was dissolved at 1 mg / mL in 10 mM phosphate buffer (pH 7.0). The zeta potentials of catalase and PLL were measured using a Zetasizer (Malvern).

[0093] [Experimental Example 2: Decomposition behavior of H2O2] Example 2 400 μL of 1 mg / mL catalase (10 mM phosphate buffer, pH 7.0) was added to a 24-well plate and incubated at room temperature for 15 minutes. The plate was then washed three times with 10 mM phosphate buffer, pH 7.0. 400 μL of 1 mg / mL poly-L-lysine (10 mM phosphate buffer, pH 7.0) was added and incubated at room temperature for 15 minutes. The plate was then washed three times with 10 mM phosphate buffer, pH 7.0. The plate was then treated with 1 mg / mL catalase, washed, and then treated with 1 mg / mL poly-L-lysine, and washed, as described above. Then, PBS containing 1 mM H2O2 was added to the 24-well plate, and the H2O2 concentration (mM) was measured over time (minutes).

[0094] [Hydrogen peroxide (H2O2) concentration measurement] The hydrogen peroxide concentration was evaluated using an Oxyselect Hydrogen Peroxide / Peroxidase Assay Kit. 400 μL of catalase solution was added to a 24-well plate (IWAKI) and allowed to stand at room temperature for 15 minutes. After washing three times with 10 mM phosphate buffer (pH 7.0), 400 μL of PLL solution was added and allowed to stand at room temperature for 15 minutes. This procedure was repeated five times, adding catalase solution and PLL solution, to create a nanofilm on the 24-well plate. PBS containing 1 mM hydrogen peroxide was added to the 24-well plate, and the hydrogen peroxide concentration of the 100-fold diluted solution was measured every 20 minutes using the above kit.

[0095] The results are shown in Figure 4. It was confirmed that by layering catalase and poly-L-lysine, H2O2 was decomposed over time, resulting in the formation of O2.

[0096] [Experimental Example 3] Example 3 · HAp-CaO 2 Layer-by-layer nanofilm formation on A 1 mg / mL catalase solution was prepared using 50 mM phosphate buffer (pH 8.0), and a 1 mg / mL FITC solution was prepared using DMSO as a solvent. 1.5 mL of FITC solution was added to 20 mL of catalase solution and allowed to stand at room temperature for 45 minutes. The solution was dialyzed for 48 hours in 50 mM phosphate buffer (pH 7.0) using a dialysis membrane with a cutoff of 3500 mm, and then lyophilized to obtain FITC-modified catalase.

[0097] 1 mg / mL of CaO2 was added to 500 mM phosphate buffer (pH 7.0) and incubated at 37°C for 1 hour. The particles were collected by centrifugation and washed three times with ethanol. After washing, the particles were dried under reduced pressure at 80°C to obtain CaO2 surface-modified with hydroxyapatite (HAp) (HAp-CaO2).

[0098] 1 mg / mL HAp-CaO2 was immersed in a 1 mg / mL catalase solution containing 20% ​​FITC-modified catalase for 15 minutes at room temperature, and the HAp-CaO2 was recovered by centrifugation. After washing the HAp-CaO2 with 10 mM phosphate buffer (pH 7.0), it was immersed in 1 mg / mL PLL for 15 minutes at room temperature. Five cycles of the same procedure were performed to obtain HAp-CaO2 coated with a layer-by-layer (LbL) nanofilm of catalase and PLL (LbL-HAp-CaO2, oxygen supply agent).

[0099] · Observation of the effect of resting time Fluorescence and phase-contrast images of the prepared oxygen-supplying agent were taken. After that, the oxygen-supplying agent was left to stand in PBS at 37°C for 14 days, and then fluorescence and phase-contrast images were taken in the same manner.

[0100] [Measurement of phase contrast and fluorescence images] A confocal laser scanning microscope (FV3000, manufactured by Olympus) was used.

[0101] The results are shown in Figure 5. The top row shows the results of measuring the oxygen supply agent immediately after LbL formation. From left to right, the images are FITC-Catalase, Ph, and Merge, which show, respectively, a fluorescent image derived from catalase modified with FITC (fluorescein isothiocyanate), a phase-contrast image of LbL-HAp-CaO2, and a merged image of the fluorescent and phase-contrast images. The bottom row shows the results of measuring the oxygen supply agent after 14 days, in the same manner as above. As shown in Figure 5, it was confirmed that the LbL nanofilm remained on the oxygen supplying agent even after 14 days.

[0102] [Experimental Example 4: Effect of time on H2O2 decomposition] Example 4 1 mg / mL HAp-CaO2 was immersed in 1 mg / mL catalase solution at room temperature for 15 minutes, and then centrifuged to recover the HAp-CaO2. After washing the HAp-CaO2 with 10 mM phosphate buffer (pH 7.0), it was immersed in 1 mg / mL PLL at room temperature for 15 minutes. Five cycles of the same procedure were performed to obtain HAp-CaO2 coated with a layer-by-layer (LbL) nanofilm of catalase and PLL (LbL-HAp-CaO2). The obtained oxygen supply agent was dispersed in PBS at 1 mg / mL and allowed to stand at 37° C. The H2O2 concentration was measured every time (days). The measurement was performed in triplicate.

[0103] (Comparative Example 1) CaO2 was added to 500 mM phosphate buffer (pH 7.0) to prepare a solution in which CaO2 was dispersed at 1 mg / mL, and this solution was incubated at 37°C for 1 hour. The resulting particles were dried under reduced pressure to obtain HAp-CaO2. The composition was dispersed in PBS at 1 mg / mL and allowed to stand at 37° C. The H2O2 concentration was measured every time (days). The measurements were performed in triplicate.

[0104] [Hydrogen peroxide (H2O2) concentration measurement] The hydrogen peroxide concentration was evaluated using the Oxyselect Hydrogen Peroxide / Peroxidase Assay Kit in the same manner as in Example 2. The PBS containing the LbL-HAp-CaO2 dispersion obtained in Example 4 was diluted 10-fold, and the PBS containing the HAp-CaO2 dispersion obtained in Comparative Example 1 was diluted 100-fold, and then the hydrogen peroxide concentration in the PBS was measured.

[0105] The results are shown in Figure 6. ● indicates the results of Example 4, and ◯ indicates the results of Comparative Example 1. In Comparative Example 1 (without LbL), the H2O2 concentration maintained a high value, but in Example 4 (with LbL), the H2O2 concentration became close to 0 immediately after measurement. This confirmed that the H2O2 concentration could be reduced in Example 4.

[0106] [Experimental Example 5: Application to cells] Example 5 As shown in Figure 7A, a 24-well plate insert (24-well insert) was immersed in a 0.02 wt% fibronectin solution to coat the insert with fibronectin. The insert was placed in a 24-well plate, and 800 μL of DMEM (10% FBS) was added to the outside of the insert. 5 × 10 6 200 μL of DMEM (10% FBS) containing 5 mg of LbL-HAp-CaO2 was added to the 24-well plate. After centrifugation at 1100 g for 15 minutes, 1 mL of DMEM (10% FBS) was added. Normal oxygen pressure P O2 The cells were cultured at 37°C for 3 days under a 5% CO₂ atmosphere with the medium changed daily. Then, the cells were fixed in 10% neutral formalin. The fixed tissues were sent to Applied Medical Research, Inc., where they were embedded in paraffin and then stained with hematoxylin and eosin.

[0107] (Comparative Example 2) As a control, tissue structures consisting of cells alone were prepared in the same manner. After culturing for 3 days with daily changes of medium, the tissues were fixed in 10% neutral formalin. The fixed tissues were sent to Applied Medical Research, Inc., where they were embedded in paraffin and then stained with hematoxylin and eosin.

[0108] The results are shown in Figure 7B. In Comparative Example 2 (left panel), rounded cells were scattered, whereas in Example 5 (right panel), dense tissue images in which cells were connected to each other were obtained. This suggests that the oxygen supply from the oxygen supply member inhibited necrosis of cells inside the three-dimensional tissue. [Industrial Applicability]

[0109] The present invention can be applied to cell scaffold materials that can efficiently and sustainably release oxygen, and is also expected to be applied to the construction of thick three-dimensional composites.

Claims

1. A metal peroxide, a first anionic coating located at least in part on the metal peroxide; and An oxygen supplying agent comprising:

2. 2. The oxygen supply agent according to claim 1, wherein the particle diameter D50 of the metal peroxide is 1 mm or less.

3. 3. The oxygen supplying agent according to claim 1, wherein the metal atom of the metal peroxide is a divalent cation.

4. 3. The oxygen supplying agent according to claim 1, wherein the metal peroxide contains at least one of Mg and Ca.

5. 3. The oxygen supplying agent of claim 1, wherein the first anionic coating comprises at least one of catalase, peroxidase, and glutathione peroxidase.

6. 3. The oxygen supplying agent according to claim 1, further comprising a first cationic coating on at least a portion of the first anionic coating.

7. 3. The oxygenator of claim 1 or 2, wherein the first cationic coating comprises at least one of poly-L-lysine, polyethyleneimine, polyallylamine, and polydiallyldimethylammonium chloride.

8. 3. The oxygen supplying agent according to claim 1, further comprising an intermediate film at least partially between the metal peroxide and the first anionic coating.

9. 9. The oxygen supplying agent according to claim 8, wherein the intermediate film is formed using at least one of a phosphate buffer solution and a carbonate buffer solution.

10. 9. The oxygen supplying agent according to claim 8, wherein the intermediate film comprises hydroxyapatite and calcium carbonate.

11. moreover, an intermediate film at least partially between the metal peroxide and the first anionic coating; a first cationic coating on at least a portion of the first anionic coating; The oxygen supplying agent according to claim 1 or 2.

12. further comprising a coating layer consisting of a second anionic coating and a second cationic coating; 12. The oxygen supplying agent according to claim 11, wherein at least one coating layer is provided on at least a portion of the first cationic coating.

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