Bioink, molded body, product, and method for manufacturing a molded body

Abstract

Provided are a bioink that contains collagen fibers having an average fiber length of 0.5-1,000 μm, a molded body molded from the bioink, an article and a method for producing a molded body. This bioink, which comprises collagen fibers formed of collagen and / or a collagen derivative and a solvent, is characterized in that the average fiber length of the collagen fibers is 0.5-1000 μm. This bioink can be discharged from a 3D printer even in the case where the collagen fiber concentration is 5-30 w / w%. A molded body obtained by the bioprinting has excellent resolution, high heat stability and high structural stability.

Description

Technical Field

[0001] The present disclosure relates to a bioink containing collagen fibers with an average fiber length of 0.5 to 1,000 μm, a molded body using the bioink, products such as a cell culture substrate including the molded body, etc., and a method for manufacturing a molded body.

Background Art

[0002] Structures composed of extracellular matrix (ECM) molecules such as collagen are excellent in tissue regenerative ability when filled in damaged parts of a living body and can be suitably used for artificial materials for regenerative medicine, etc. (Patent Document 1). However, shaping is not easy, and Patent Document 1 only discloses a sheet-shaped object obtained by filtering a material and shaping it into a flat shape, and a cubic object obtained by filling a columnar mold with the material and shaping it.

[0003] On the other hand, there is 3D bioprinting as a technique for shaping an arbitrary shape of a living tissue using a bioink containing ECM molecules such as collagen. If the bioink is laminated to shape a tissue or an organ, it can be applied to medical research such as regenerative medicine and replacement of functional organs. Also, a 3D molded body in which cells are deposited using the bioink as a support material can be manufactured. Such a 3D molded body can be used as a scaffold for cells during in vitro or in vivo culture. In addition, an alternative method for evaluating the effects of cosmetics and pharmaceuticals using a 3D molded body can also contribute to reducing the use of experimental animals.

[0004] As a bioink containing collagen, a bioink containing native collagen, which has a static rigidity of about 100 to about 150,000 Pa and 0.001 sec at room temperature -1There is a bioink having a shear modulus of less than about 50 Pa at a higher shear rate (Patent Document 2). The bioink described in Patent Document 2 addresses the conventional problems, namely, that the molded body made of the conventional bioink gels at 37°C to promote cell adhesion, but when the collagen concentration is low, it can only provide minimal structural integrity to the 3D structure, and also lacks the ability to shear-thin and regain rigidity when printed, and the neutralized collagen ink gels in the syringe and clogs the printer. The bioink described in Patent Document 2 can be mixed with cells at neutral pH, can be printed in a cell culture medium, and when printed in a cell culture medium, it has excellent working time and rigidity.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] Collagen exists extracellularly in fibrous form and, in vivo, constitutes various tissues at high concentrations of 25% in skin, 32% in tendon, 16% in cartilage, 23% in bone, and 18% in dentin per wet weight. However, collagen solutions have a high viscosity and it is difficult to dissolve them at the same high concentrations as in vivo. Patent Document 2 states that a collagen concentration of 50 mg / mL may be used, but there are no examples with a collagen concentration of 50 mg / mL. On the other hand, when a 3D molded body made of collagen is used as an organ substitute, it is preferable that the collagen density of the molded body is close to the collagen density of living tissue. Usually, since the collagen density of a 3D molded body depends on the collagen concentration of the bioink used as a raw material, the development of a bioink with a higher collagen concentration is desired.

[0007] On the other hand, since a collagen solution gels at 37°C under neutral salt conditions, when molding with a 3D printer, it is necessary to avoid clogging due to gelation and printing must be performed at a temperature of 1 to 10°C. If the printing operation is carried out at room temperature, the temperature of the bioink will rise over time, resulting in a limited working time. Also, when the collagen concentration is low, it is necessary to eject it into a supporting medium such as a cell culture medium, but since the bioink diffuses into the supporting medium before gelling, the resolution of the resulting 3D molded body may be reduced. Therefore, the development of a bioink that can ensure fluidity when ejecting the bioink from a 3D printer and can perform 3D molding even without a supporting medium such as a culture solution is desired.

[0008] In view of the above situation, an object of the present disclosure is to provide a bioink that can contain collagen fibers at a high concentration.

[0009] Another object of the present disclosure is to provide a molded body molded using the above bioink.

[0010] Another object of the present disclosure is to provide a product such as a cell culture substrate including the above molded body.

[0011] Another object of the present disclosure is to provide a method for manufacturing a molded article using the above bioink.

Means for Solving the Problems

[0012] The present inventors focused on the average fiber length of collagen and found that when using collagen with an average fiber length shorter than about 2,000 μm of conventional collagen fibers, even a bioink with a high collagen concentration can be discharged without clogging the discharge holes of a 3D printer, and that 3D molding is possible by discharging into the atmosphere without using a support medium during bioprinting because of the high collagen concentration, and thus completed the present disclosure.

[0013] That is, the present disclosure provides a bioink for bioprinting, which comprises collagen fibers composed of collagen and / or a collagen derivative and a solvent having a pH of 5.0 to 9.0, wherein the average fiber length of the collagen fibers is 0.5 to 1,000 μm, and the concentration of the collagen fibers in the solvent is 12.5~30 w / w% and the collagen fibers are dispersed in the solvent without being dissolved, and provides a bioink.

[0015] The present disclosure also provides the above bioink, wherein the solvent is one or more selected from the group consisting of pure water, a buffer solution, physiological saline, and a cell culture medium.

[0016] The present disclosure also further provides the above bioink, which contains one or more compounds selected from the group consisting of extracellular matrix molecules, decellularized tissues, growth factors 、 cytokines and a crosslinking agent The present disclosure also further provides the above bioink, which contains cells.

[0017] The present disclosure also provides the above bioink, which is a bioink for a 3D printer.

[0018] The present disclosure also provides the above bioink, which is a bioink for a 3D printer.

[0019] The present disclosure also provides a molded article made of the bioink.

[0020] The present disclosure also provides a crosslinked molded article, which is crosslinked by one or more selected from the group consisting of riboflavin, methacrylated gelatin (GelMA), polyethylene glycol diacrylate (PEGDA), glutaraldehyde, formaldehyde, genipin, ammonium derivatives, photoinitiators, Irgacure (registered trademark), lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and ruthenium, and the molded article is made of the bioink.

[0021] The present disclosure also provides one or more products selected from the group consisting of a cell culture substrate, a substrate for transplantation, a tissue structure, an organ model, and a substrate for regenerative medicine, which contain the molded article or the crosslinked molded article.

[0022] The present disclosure also provides a method for manufacturing a molded article, which is characterized by discharging the bioink from a nozzle with a diameter of 0.2 to 1 mm into the air by pressing.

Advantages of the Invention

[0023] According to the present disclosure, there are provided a bioink having an average fiber length of collagen fibers of 0.5 to 1,000 μm, a molded article made of the bioink, a product containing the molded article, and a method for manufacturing a molded article using the bioink.

Brief Description of the Drawings

[0024]

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Mode for Carrying Out the Invention

[0025] A first aspect of the present disclosure is a bioink for bioprinting, which comprises collagen fibers composed of collagen and / or a collagen derivative and a solvent, and is characterized in that the average fiber length of the collagen fibers is 0.5 to 1,000 μm.

[0026] In the present disclosure, bioprinting means a technique for manufacturing a molded body using biomaterials by using the technology of a 3D printer. Also, bioink means a modeling raw material used in bioprinting. Also, a 3D printer means an apparatus for modeling a molded body based on a three-dimensional digital model.

[0027] The bioink in the present disclosure is characterized by containing collagen fibers composed of collagen and / or a collagen derivative as a biomaterial. Collagen is one of the proteins that mainly constitute the dermis, ligaments, tendons, bones, cartilage, and various internal organs of vertebrates, and is also included in crustaceans, mollusks, etc. The collagen used in the present disclosure may be derived from any tissue or organ of any animal. Collagen is classified into type I, type II, etc. in the order of discovery, and any type may be used. Also, there is no limitation on the method for preparing collagen. Although most of the collagen is insolubilized in vivo, solubilized collagen originally contained in the living body may be extracted and used. Also, it may be obtained by solubilizing an insoluble collagen-containing tissue by adding an enzyme such as protease, an acid, an alkali, or a neutral salt and then extracting it. To isolate collagen from a collagen solubilized solution, generally, a salting-out method, an isoelectric point precipitation method, etc. may be used, and any method may be used. On the other hand, when the amino acid residues of the collagen used in the present disclosure are represented by X and Y, it is -(Gly-X-Y) nIt has a triple-helix structure in which three polypeptides having a collagen-like sequence represented by - are helically bound. If it has a triple-helix structure, it may be atelocollagen that does not contain telopeptides at both ends or telocollagen that contains telopeptides. In addition, when solubilized by alkali treatment, for example, asparagine residues or glutamine residues may be changed to aspartic acid residues or glutamic acid residues respectively by deamidation reaction. In addition, the amino acids contained in collagen may be chemically modified. Further, it is not limited to extracts from animal tissues, and may be purified from collagen-high expressing cells using known techniques, or produced as a recombinant protein. For example, it may be synthesized by genetic recombination techniques in CHO cells or tobacco cells.

[0028] In the present disclosure, the "collagen derivative" means a modification of the amino acids constituting the collagen with other functional groups. For example, there are acylated collagen and esterified collagen. For example, there is a collagen derivative obtained by acylating or esterifying collagen isolated from a tissue. On the other hand, it may be collagen in which a functional group is esterified or acylated during salting out or isoelectric precipitation of collagen. For example, when extracting solubilized collagen from a collagen-containing tissue, collagen may be acylated in advance and an acylated collagen may be solubilized for preparation. Similarly, insoluble collagen contained in a collagen-containing tissue may be esterified in advance and an esterified collagen may be solubilized for preparation. The collagen derivative may be one acylated or esterified during the collagen extraction process.

[0029] Examples of acylated collagen include succinylated collagen, phthalylated collagen, maleylated collagen, etc. For example, there are acylated collagens such as succinylated collagen, phthalylated collagen, and maleylated collagen, which are obtained by adjusting the pH of an atelocollagen solution extracted by enzymatic treatment to 9 - 12 and then adding acid anhydrides such as succinic acid, phthalic anhydride, and maleic anhydride. Examples of esterified collagen include those obtained by esterifying solubilized collagen, as well as esterified collagen that is solubilized by an enzymatic reaction or the like after esterifying insoluble collagen. Alcohols used to react with collagen to obtain esterified collagen may include, in addition to primary alcohols, secondary alcohols and tertiary alcohols. Also, they are not limited to monohydric alcohols, and may be dihydric alcohols, trihydric alcohols, or other polyhydric alcohols.

[0030] The collagen fibers composed of collagen and / or collagen derivatives used in the present disclosure are characterized in that their average fiber length is 0.5 to 1,000 μm. Preferably, the average fiber length is 10 to 700 μm, more preferably 100 to 500 μm, and particularly preferably 100 to 250 μm. Fibrous collagen is formed by multiple collagen molecules shifting by 67 nm each and associating in a neutral aqueous solution to form collagen fibrils, and further multiple collagen fibrils associating to form collagen fibers. When salt is added to a collagen solution, the collagen precipitates by salting out. Also, when an acid or an alkali is added to adjust the pH near the isoelectric point of collagen, isoelectric precipitation occurs. When the collagen precipitate thus formed is dissolved in an aqueous solution and brought to 37°C and neutral salt conditions, collagen fibrils and collagen fibers are formed by the associative force of collagen molecules. Usually, the formed collagen fibers have an average fiber length of about 2,000 μm from the balance with their solubility and the like. On the other hand, by reducing the associative force of collagen molecules in the solution or physically cutting the fibers formed by association, collagen fibers with an average fiber length of 0.5 to 1,000 μm can be prepared. In the present disclosure, collagen fibers with the average fiber length thus adjusted can be used. As such a method for adjusting the fiber length of collagen, for example, there is a method for adjusting the average fiber length in accordance with the description of Patent Document 3. For example, a predetermined amount of an alkali-solubilized collagen precipitate obtained from the dermis layer of porcine skin by solubilization, salting out, etc. is dispersed in distilled water, adjusted to pH 4.5, and stirred with a mortar-type grinder such as a mascoloider and the collagen fibers are crushed to obtain an isoelectric precipitate. When the solution is stirred with a mortar-type grinder or the like during the formation of the collagen precipitate and the precipitate is ground and crushed, the association of collagen molecules is inhibited by strong stirring, and the associated collagen fibers are cut by crushing. By adjusting the degree of stirring and crushing, collagen fibers with an average fiber length of 0.5 to 1,000 μm can be produced.

[0031] On the one hand, non-fibrillar collagens such as type IV collagen differ from fibrillar collagen in that they form fine network-like aggregates. However, similar to fibrillar collagen, when salt is added to the solution, the collagen precipitates by salting out, and when an acid or an alkali is added and the pH is adjusted to the isoelectric point range, isoelectric precipitation occurs. When generating a collagen precipitate, collagen fibers with an average fiber length of 0.5 to 1,000 μm can be produced by stirring the solution with a mortar-type grinder such as a mascolloidator and crushing the precipitate by grinding.

[0032] In the present disclosure, the fiber length of collagen shall be measured by the method shown in the examples described later. Specifically, for dried collagen fibers, a scanning electron microscope is used, and when measuring using a liquid in which collagen fibers are dispersed, a phase-contrast microscope is used. Twenty or more collagen fibers are randomly selected and the fiber length is measured, and the value calculated as the average thereof is defined as the average fiber length of the collagen fibers.

[0033] Note that the collagen fibers to be used may be commercially available products. For example, commercially available products such as pepsin-solubilized collagen (PSC) powder manufactured by Nippi Co., Ltd. and acid-solubilized collagen (ASC) powder manufactured by Nippi Co., Ltd. may be selected and used as long as the average fiber length of the collagen is 0.5 to 1,000 μm. Specifically, there are powder collagen type I bovine dermis-derived (pepsin solubilized): PSC-1-100-500PW, powder collagen type I bovine dermis-derived (acid extraction) ASC-1-100-500PW, and the like.

[0034] As described above, collagen molecules have a triple helix structure, and collagen molecules having a triple helix structure are called native collagen. Since collagen fibers are composed of native collagen, according to the bioink of the present disclosure, a molded article molded with native collagen can be prepared. Even when the obtained molded article is moistened with a cell culture medium or the like, the collagen fiber structure composed of native collagen can be maintained in the molded body.

[0035] As the solvent constituting the bioink, those that can be uniformly dissolved, dispersed, and kneaded with collagen fibers having an average fiber length of 0.5 to 1,000 μm can be widely used. The solvent preferably has a pH of 5.0 to 9.0. For example, pure water; buffer solutions such as phosphate buffered saline and Tris buffer; physiological saline; cell culture media, etc. can be preferably used.

[0036] The concentration of collagen fibers contained in the bioink is 5 to 30 w / w%. The concentration of collagen fibers can be appropriately selected according to the use of the molded body. To produce a molded body with high resolution, it is preferably 10 to 30 w / w%, more preferably 10 to 25 w / w%. On the other hand, when the resolution of the molded body does not affect its use, a molded body can be produced according to the use within the range of 5 to 30 w / w%.

[0037] In the bioink of the present disclosure, it is not necessary for the collagen fibers to be dissolved in the solvent, and it is sufficient that they are uniformly dispersed in the solvent. If the average fiber length of the collagen fibers is 0.5 to 1,000 μm, they can be uniformly dispersed in the solvent and clogging of the nozzles of the 3D printer can be avoided.

[0038] The bioink of the present disclosure may further contain extracellular matrix molecules such as proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, elastin, fibrillin, glycosaminoglycan, etc.; decellularized tissue; growth factors such as EGF, IGF, TGF, bFGF, NGF, BDNF, VEGF, G-CSF, GM-CSF, PDGF, EPO, TPO, HGF, etc.; and cytokines such as chemokines, interferons, interleukins, lymphokines, etc., as long as the properties of the bioink and the resulting molded body are not impaired. The blending amounts of the extracellular matrix, cytokines, and growth factors are 0.1 ng / mL to 10 mg / mL, preferably 0.1 ng / mL to 1 mg / mL, and more preferably 1 ng / mL to 100 ng / mL in the bioink. The extracellular matrix and growth factors can bring about physiological effects such as specific differentiation and proliferation of the cells derived from the tissue. For example, by adding growth factors, cell differentiation, proliferation, migration, etc. can be regulated. When adding decellularized tissue, it can be added in the range of 0.5 to 2 weight times, more preferably 0.7 to 1.2 weight times, based on the weight of collagen. If the amount of decellularized tissue is within a range less than that of the bioink, the influence on the moldability by collagen or collagen derivatives is small.

[0039] The bioink of the present disclosure may include inorganic salts such as hydroxyapatite and tricalcium phosphate; synthetic polymers such as polyglycolic acid, polylactic acid, poly(lactide-co-glycolide) copolymer, polydioxanone, poly(methyl acrylate), and poly(methyl methacrylate); nanomaterials such as gold nanoparticles, silver nanoparticles, iron oxide nanoparticles, silica nanoparticles, and carbon nanoparticles; various nutritional components; fluorescent labeling compounds such as Fluorescein derivatives, rhodamine derivatives, and Cy dyes; riboflavin, GelMA, PEGDA, glutaraldehyde, formaldehyde, genipin, ammonium derivatives, photoinitiators, Irgacure (registered trademark), lithium phenyl-2,4,6-trimethylbenzoylphosphinate, ruthenium, and other crosslinking agents. Inorganic substances contribute to increasing the rigidity of the bioink. Also, the mixing of metal-based nanoparticles contributes to increasing conductivity. Further, a crosslinked molded article can be prepared by blending a crosslinking agent. When using hydroxyapatite in combination, it is preferably used at a concentration of 0.3 mg / mL or less of the dissolution concentration in water.

[0040] Furthermore, the bioink can also include chemical materials such as nylon and polypropylene, and biological materials such as enzymes, spores, and hyphae. A molded article containing these can be manufactured.

[0041] Furthermore, the bioink can also include various stem cells such as epithelial cells, endothelial cells, fibroblasts, cardiomyocytes, hepatocytes, smooth muscle cells, skeletal muscle cells, satellite cells, Schwann cells, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hepatic stem cells, epithelial stem cells, germ stem cells, and neural stem cells, and pluripotent stem cells such as ES cells and iPS cells. A cell-embedded 3D molded article can be manufactured using such cell-containing bioink.

[0042] There is no limitation on the method for preparing the bioink. For example, when using dried collagen fibers such as collagen powder containing collagen fibers with an average fiber length of 0.5 to 1,000 μm, it can be prepared by uniformly mixing and kneading it with a solvent. The pH of the solvent is preferably 5.0 to 9.0, more preferably 6.0 to 8.0. The temperature during mixing and kneading can be in the range of 4°C to 37°C, preferably at room temperature of 25°C. The kneading time can be in the range of 30 seconds to 10 minutes, preferably 30 seconds to 5 minutes, more preferably 1 minute to 2 minutes. Also, as the hydrated collagen fibers, when extracting collagen fibers with an average fiber length of 0.5 to 1,000 μm from animal tissues, the collagen precipitate obtained as a salting-out or isoelectric point precipitate can be used as the collagen fibers. These collagen precipitates can be concentrated by centrifugation or diluted by adding various solvents such as pure water, etc., to adjust to the desired collagen fiber concentration, and this can be directly used as the bioink.

[0043] Since collagen fibers are highly hydrophilic and have high viscosity, air bubbles may be mixed in when mixing and kneading with the solvent. Similarly, the paste-like collagen precipitate obtained by concentrating the salting-out product or isoelectric point precipitate may also contain air bubbles. Since air bubbles will cause obstacles during the modeling by a 3D printer, it is preferable to degas after mixing and kneading, etc. There is no limitation on the degassing method, for example, there are ultrasonic degassing, vacuum decompression degassing, centrifugal degassing, etc. The degassing time can be appropriately selected according to the equipment and method used, usually 30 seconds to 10 minutes, preferably 30 seconds to 5 minutes, more preferably 1 minute to 2 minutes. In addition, when the bioink is passed through a needle with an inner diameter of 0.3 mm to 1 mm after mixing and kneading and degassing as necessary, the bioink can be made more homogeneous. Also, when the bioink contains other components besides collagen fibers, these components can be mixed with the collagen fibers and kneaded and degassed in the same manner as above. Also, after preparing the bioink with collagen fibers in advance, a solvent containing other components can be mixed with the bioink to prepare the desired bioink.

[0044] The bioink prepared in this way is in the form of a paste in which collagen fibers and a solvent are uniformly kneaded. As shown in the examples described below, the average fiber length of the collagen fibers contained is stably maintained at 0.5 to 1,000 μm.

[0045] On the other hand, the collagen fibers in the bioink are native collagen. Native collagen may be decomposed by heating, pH fluctuations, or the action of enzymes, resulting in the disappearance of the triple helix structure. Therefore, it is preferable to store the bioink of the present disclosure at a pH of 5.0 to 9.0 and a temperature of 4°C. By storing at a low temperature, it is possible to suppress the alteration of the properties due to the fiber formation of collagen and prevent denaturation.

[0046] This bioink can be loaded into a 3D printer and used for manufacturing a molded body during bioprinting. As the 3D printer, either an inkjet method in which the bioink is atomized and ejected for lamination and modeling or a dispensing method in which the bioink is extruded for modeling may be used. Since the inkjet method atomizes and ejects the bioink, it is preferable to use a bioink with low viscosity that instantly solidifies after landing on the modeling surface. On the other hand, the dispensing method can use a bioink with a higher viscosity than the inkjet method. The bioink of the present disclosure has a high concentration of collagen fibers and can be suitably used in a 3D printer of the dispensing method.

[0047] It should be noted that bioprinting can also be performed by increasing the nozzle diameter of the 3D printer, but the resolution will decrease. According to the present disclosure, by specifying the average fiber length and the collagen fiber concentration within the above ranges, a molded body with high speed and excellent resolution can be manufactured. In the present disclosure, the resolution is, for example, obtained by preparing a lattice sheet as a molded body, photographing it from directly above, arbitrarily selecting the lines constituting the lattice sheet, measuring the line widths at five or more locations, and taking the average value. The resolution can be measured in the same way using a lattice cube instead of the lattice sheet.

[0048] The second aspect of the present disclosure is a molded article made of the bioink. The molded article may be a sheet-like article, a cubic article, or other articles with irregular shapes formed by discharging one or more layers of the bioink from a nozzle onto a plane. This molded article may be an undried molded article discharged from a 3D printer, or may be a dried product dehydrated thereafter. The collagen density of the dried molded article varies depending on the collagen fiber concentration of the bioink used and the shape of the molded article, and is usually 0.1 to 0.7 g / cm 2 3. Note that the inside of the dried product obtained by removing moisture from the molded article by freeze-drying or otherwise becomes sponge-like. Although there is no limitation on the drying method, if it is freeze-drying, a molded article in which the collagen fibers are maintained without denaturation is obtained. The molded article of the present disclosure is superior in thermal stability to the collagen solution, as shown in the examples described later. In particular, the dried molded article is also excellent in storage stability. Also, the resolution of the dried molded article depends on the nozzle diameter used in the 3D printer, but is 200 to 1,200 μm.

[0049] Further, when the bioink contains crosslinking agents such as riboflavin, GelMA, PEGDA, glutaraldehyde, formaldehyde, genipin, ammonium derivatives, photoinitiators, Irgacure (registered trademark), lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and ruthenium in advance, a crosslinked structure can be formed according to the crosslinking agent during or after molding by bioprinting. Therefore, the molded article of the present disclosure may be such a crosslinked molded article. For example, light is irradiated during bioprinting to form a crosslinked structure, or after the molded article is bioprinted, the undried molded article is irradiated with light to form a crosslinked structure. Light such as ultraviolet light, visible light, and infrared light is irradiated according to the crosslinking agent. Note that the collagen fibers may be crosslinked by heat, dehydration, or the like in addition to light according to the crosslinking agent.

[0050] The third aspect of the present disclosure is one or more products selected from the group consisting of a cell culture substrate, a transplantation substrate, a tissue structure, an organ model, and a regenerative medicine substrate, including the molded article and the crosslinked molded article. Since the molded body of the present disclosure is composed of collagen fibers, for example, when the molded body is a dried product, it is impregnated with a solution having a pH of 5.0 to 9.0, heated to a temperature of 37° C. to be moistened, and then cultured with cells in a cell culture medium, the cells grow on the molded body using the collagen fibers as a scaffold. Therefore, it can be suitably used as a cell culture substrate. Further, by including cells or other components in the molded body, it can be used as a substitute for tissues such as a transplantation substrate or a regenerative medicine substrate. In addition, by creating a tissue structure similar to that of a living body tissue, it can be used as an in vitro evaluation system such as drug screening. The bioink of the present disclosure is composed of undenatured collagen fibers similar to those in vivo and has a high collagen fiber concentration. Therefore, it is also suitable as a substitute for a part of the missing tissue. In particular, as shown in the examples described later, when cell culture is performed using the molded body, the cells grow gently and have excellent tissue compatibility. Further, when the molded body is immersed in a cell culture medium, a part of the molded body is dissolved over time, but it has been found that collagen fine fibers are generated. When collagen refiberization occurs in the cell culture medium, the formed collagen fibers become long fibers having a fiber length of 2,000 μm or more, have excellent biocompatibility, and the structure is stabilized.

[0051] The molded body can be suitably used as a cell culture substrate even when it is not dried. For example, an undried molded body is immersed in a medium solution for the purpose of use, and the solvent contained in the molded body is replaced with the medium solution. When cells are seeded thereon, the molded body can function as a cell scaffold material. Since the step of freeze-drying is unnecessary, it is advantageous that it can be applied to cell culture immediately after molding.

[0052] Examples of the cells that can be cultured with the molded body of the present disclosure include cells derived from mammals such as humans, mice, rats, cows, and pigs. For example, there are epithelial cells, endothelial cells, fibroblasts, cardiomyocytes, hepatocytes, smooth muscle cells, skeletal muscle cells, muscle satellite cells, Schwann cells of nerves, and adipocytes. In addition, it is also suitable for culturing various stem cells such as mesenchymal stem cells, hematopoietic stem cells, hepatic stem cells, epithelial stem cells, germ stem cells, and neural stem cells, and pluripotent stem cells such as ES cells and iPS cells.

[0053] The molded body of the present disclosure can be used as a tissue substitute in bone transplantation and other applications. In this case, the undried or dried molded body can be directly filled into the defect site and used as a substitute, or the molded body after pre-culturing cells can also be used as a substitute. Since the molded body of the present disclosure functions as a scaffold for cell culture when implanted in a living body, it can also be used as a tissue regeneration induction device.

[0054] In addition, by mixing cells in advance in the bioink and manufacturing the molded body with a 3D printer, a cell structure mimicking the tissue structure of a living body can be prepared. For example, by making the 3D printer multi-nozzle and efficiently arranging various cells at desired sites of the molded body, a tissue structure composed of multiple cells can be formed. Such a tissue structure can be used, for example, in in vitro metabolism tests for a liver tissue structure, and in skin irritation tests and eye irritation tests for cosmetics, etc. for skin and corneal tissue structures. Also, by producing tissue structures mimicking various organs, it can be applied, for example, to drug screening in the field of drug discovery. These are attracting attention as a method to replace animal experiments.

[0055] When the molded body of the present disclosure is molded into the shape of a predetermined organ, the obtained molded body can also be used as an organ model. Such an organ model can be used, for example, as an optimal training tool for improving the skills of surgical procedures using an endoscope. By using an organ model specialized for the texture of each organ, it becomes possible to conduct training emphasizing touch. In particular, it can be applied to training using a thoracic and abdominal simulator, organ models for surgical training: gastrectomy D2 lymph node dissection model, gastric reconstruction model, partial nephrectomy model, inguinal hernia surgery model (TAPP), mitral valve surgery model, cholecystectomy model, rectal cancer nerve-preserving dissection model, lobectomy model, dissection sheet, blood vessels for microsurgery training, etc. In order to ensure the rigidity of the molded body used as an organ model, the obtained molded body can be crosslinked and adjusted to the desired rigidity.

[0056] A fourth aspect of the present disclosure is a method for manufacturing a molded body, which is characterized by discharging the bioink from a nozzle having a diameter of 0.2 to 1 mm into the atmosphere by pressing. The bioink of the present disclosure is used for bioprinting and is used to manufacture a molded article having a predetermined shape using a 3D printer. Therefore, originally, there is no limitation on the nozzle diameter. However, since the average fiber length of the collagen used in the bioink of the present disclosure is as short as 0.5 to 1,000 μm, even when the collagen fiber concentration is 5 to 30 w / w%, a 3D printer can be used to discharge the bioink from a nozzle having a diameter of 0.2 to 1 mm, more preferably 0.3 to 0.8 mm, for bioprinting. As the collagen fiber concentration increases, the viscosity of the bioink increases, so it is not easy to discharge a 5 to 30 w / w% bioink from a nozzle having a diameter of 0.2 to 1 mm. However, since the average fiber length is as short as 0.5 to 1,000 μm, the bioink can be discharged from the nozzle having the above diameter to manufacture a molded body with high resolution. In addition, since the collagen fiber concentration is as high as 5 to 30 w / w%, the bioink can be discharged from the nozzle into the atmosphere by pressing without discharging the bioink into the support medium, and the molded body can be laminated and formed. When the collagen concentration is low, even when the bioink is discharged into the support medium, molding cannot be performed unless the nozzle diameter is 0.1 to 0.3 mm. According to the present disclosure, a molded body can be manufactured by discharging the bioink into the atmosphere with a nozzle diameter exceeding 0.2 mm. For this reason, a large-sized molded article can be efficiently manufactured in a short time without using a support medium.

[0057] When manufacturing a molded body using the bioink of the present disclosure, the bioink may be discharged from a nozzle with a diameter exceeding 1 mm for molding. Although the resolution decreases, it is excellent in that a large-sized molded body can be formed in a short time. Further, the bioink is not limited to the atmosphere and may be discharged and molded in a support medium. Since the collagen fiber concentration of the bioink of the present disclosure is 5 to 30 w / w%, even when discharged into the support medium, the dispersion of the bioink is small, and a molded body with excellent resolution can be manufactured. Generally, when a collagen solution with a low concentration is used as the bioink, after molding the molded body, it may be heated to 37°C to gelate and increase the rigidity. Similarly, the bioink of the present disclosure may be applied to neutral salt conditions after molding and heated at 37°C for fiber formation treatment. However, since the collagen fiber concentration contained in the bioink is high, it is excellent in that the rigidity of the molded body can be maintained without performing the fiber formation treatment and the decrease in resolution can be suppressed.

[0058] After molding with a 3D printer, the molded body may be used as it is or dehydrated to obtain a dried product. The dehydration method can be appropriately selected according to the shape of the molded body, and examples include freeze-drying, heat-drying, vacuum-drying, infrared-drying, and air-drying. Freeze-drying is suitable for maintaining the collagen fibers in an undenatured state.

[0059] When the bioink contains a crosslinking agent, a crosslinking step may be further included when molding with a 3D printer. Further, a crosslinking step may be performed after molding with a 3D printer. For example, since genipin, a natural crosslinking agent, is known to have low toxicity to cells, it can be mixed with the bioink and used directly for cell culture without washing or removing operations.

Examples

[0060] Next, the present disclosure will be specifically described with reference to examples, but these examples do not limit the present disclosure in any way.

[0061] (Example 1) 2 g of collagen powder (PSC powder, manufactured by Nippi Inc.) with an average fiber length of 142 μm fractionated with a 100 - 250 μm sieve and 8 g of pure water cooled to 4°C were kneaded at 25°C for 1 minute using a planetary mixer (Thinky Corporation: Awatori Rentaro), and then defoamed for 1 minute using the same apparatus to prepare a bioink with a collagen fiber concentration of 20 w / w%. When the average fiber length of the collagen fibers contained in the prepared bioink was measured, it was 150 μm. The average fiber length of the collagen fibers constituting the collagen powder was calculated as the average of measurements of more than 20 randomly selected collagen fibers using a scanning electron microscope. On the other hand, the average fiber length after bioink preparation was calculated as the average of measurements of more than 20 randomly selected collagen fibers. A portion of the bioink was taken, dispersed in 50 mM Tris - HCl buffer (pH 8.0), and the fiber lengths were measured using a phase - contrast microscope.

[0062] (Example 2) The bioink prepared in Example 1 (at a temperature of 25°C) was extruded into the atmosphere from a nozzle with a diameter of 0.4 mm of a dispensing-type 3D printer (Musashi Engineering, SHOT mini, model M22-123) under an air pressure of 50 to 300 kPa to fabricate a lattice sheet of 3 cm × 3 cm × 0.1 cm and an oriented sheet of 3 cm × 3 cm × 0.1 cm. Also, except that an alkali-solubilized collagen powder derived from porcine dermis with a collagen fiber length of about 158 μm was used instead of the collagen powder (PSC powder manufactured by Nippi Co., Ltd.) used in Example 1, the same operations as in Example 1 were performed to prepare a bioink. Using this bioink, the same operations as above were performed to fabricate a lattice cube of 1.7 cm × 1.7 cm × 1 cm and a nose-shaped cube of 2.5 cm × 3 cm × 2.5 cm. The molding time of the lattice sheet was 2 minutes, the molding time of the oriented sheet was 5 minutes, the molding time of the lattice cube was 10 minutes, and the molding time of the nose-shaped cube was 30 minutes. After molding, freeze-drying was performed to obtain a molded body. The oriented sheet is formed by extruding the bioink parallel to a certain direction to form a plane, and then extruding and laminating the bioink parallel to a direction intersecting the said direction on this plane. The molded body after drying is shown in Fig. 1. A is the lattice sheet, B is the oriented sheet, C is the cube (lattice), and D is the nose-shaped molded body. Also, Fig. 2 shows the results of imaging the surface and cross-section of the oriented sheet B with a scanning electron microscope. A are enlarged views of the surface of the oriented sheet at 30 times, 50 times, and 100 times, and B are enlarged views of the longitudinal cross-section of the oriented sheet at 50 times, 100 times, and 200 times. As shown in Fig. 2, orientation in the uniform discharge direction was observed on the surface of the oriented sheet. Also, the longitudinal cross-sectional structure of the oriented sheet was sponge-like, forming uniform cavities. Also, Fig. 3 shows enlarged images of the surface and longitudinal cross-section of the oriented sheet. As shown in Fig. 3, fibrous structures derived from collagen were observed in both the surface and the longitudinal cross-section. The fibrous structures are indicated by arrows.

[0063] (Example 3) The oriented sheet prepared in Example 2 was punched out with a dermal punch having a diameter of 6 mm to produce a disk. This disk was moistened with 2 mL of DMEM medium and incubated at 37°C for 7 days. After 1 day, 3 days, and 7 days of incubation, it was collected, fixed with PBS containing 2.5% glutaraldehyde, washed with distilled water, and then freeze-dried. The scanning electron micrograph of the disk surface is shown in Fig. 4. It was found that the fibrous structure indicated by the arrow was maintained even after incubation for 7 days of moistening. Also, a tendency for an increase in collagen fine fibers was observed as the incubation progressed. It was presumed that refibrillation occurred after a part of the collagen fibers of the disk was dissolved. Here, PBS is phosphate buffered saline.

[0064] (Example 4) 20 mg of the oriented sheet prepared in Example 2 was dissolved in 20 mL of 5 mM acetic acid to obtain a collagen acetic acid solution. To this collagen acetic acid solution at 1 mg / mL, the same amount of 2×PBS (PBS at twice the concentration) as this collagen acetic acid solution was added, adjusted to a collagen fiber concentration of 0.5 mg / mL and pH 7.4, and then incubated at 37°C, and the turbidity (OD520) from the start of incubation to 360 minutes was measured. The results are shown in Fig. 5. It was found that the collagen solution obtained by redissolving the oriented sheet rapidly increased in turbidity at 100 minutes of incubation and had the ability of refibrillation.

[0065] The CD value of the collagen acetic acid solution derived from the oriented sheet prepared above was measured at a temperature of 20 °C using a circular dichroism spectrometer (JASCO Corporation: J-805). As a control, a collagen powder-derived acetic acid solution (collagen fiber concentration 0.5 mg / mL) obtained by dissolving the collagen powder (PSC powder, manufactured by Nippi Co., Ltd.) used in Example 1 in 20 mL of 5 mM acetic acid was used. Further, the collagen acetic acid solution derived from the oriented sheet prepared above was heat-treated at 50 °C for 5 minutes, and the CD value was measured at a temperature of 20 °C using a circular dichroism spectrometer (JASCO Corporation: J-805) in the same manner as above. The results are shown in Fig. 6. A shows the CD value of the collagen acetic acid solution derived from the oriented sheet, B shows the acetic acid solution of the collagen powder, and C shows the CD value of the collagen acetic acid solution derived from the oriented sheet heat-treated at 50 °C. In the collagen acetic acid solution (A) derived from the oriented sheet and the collagen acetic acid solution (B) derived from the collagen powder, a peak at 221 nm indicating the unchanged state was observed. On the other hand, the peak at 221 nm indicating the unchanged state disappeared in the collagen acetic acid solution (C) derived from the oriented sheet after heat treatment, indicating that heat denaturation of collagen occurred by heating at 50 °C.

[0066] (Example 5) After wetting the oriented sheet prepared in Example 2 with PBS, the denaturation temperature was measured using a differential scanning calorimeter (SII Corporation: DSC6100). The results are shown in A of Fig. 7. Further, as a control, the denaturation temperature of a collagen powder-derived acetic acid solution (collagen fiber concentration 0.5 mg / mL) obtained by dissolving the collagen powder (PSC powder, manufactured by Nippi Co., Ltd.) used in Example 1 in 20 mL of 5 mM acetic acid was also measured. The results are shown in B of Fig. 7. A large peak of heat quantity change was confirmed at 52.6 °C for the oriented sheet wetted with PBS, but the denaturation temperature of the collagen acetic acid solution was 42.8 °C. Since the peak of heat quantity change was oriented sheet > collagen acetic acid solution, it was found that the oriented sheet wetted with PBS has higher thermal stability than the collagen acetic acid solution.

[0067] (Example 6) After wetting the lattice sheet prepared in Example 2 with DMEM medium containing 10% FBS, fibroblasts (Human Embryonic Lung-derived Fibroblast) were seeded on its surface at a density of 1×10 4 cells / cm 2 and statically cultured at 37°C. The dish surface was pre-coated with a synthetic phospholipid (Lipidure, NOF Corporation) so that the cells would not adhere to the dish. Phase-contrast microscope images (bright field), fluorescence microscope images after calcein staining, and merged images on the 3rd and 6th days of culture are shown in Fig. 8. On the 3rd day of culture, the cells had already adhered and spread along the lattice. The same result was obtained on the 6th day of culture, and no collapse of the lattice sheet was detected.

[0068] (Example 7) In the same manner as in Example 6, the oriented sheet prepared in Example 2 was wetted with DMEM containing 10% FBS to prepare a DMEM-wetted oriented sheet. This was placed statically in a dish, and fibroblasts were seeded on its surface at a density of 1×10 4 cells / cm 2 and statically cultured at 37°C for 7 days. The dish surface was pre-coated with a synthetic phospholipid (Lipidure, NOF Corporation) so that the cells would not adhere to the dish. As a control, acid-soluble collagen (manufactured by Nippi, Inc., acetic acid solution, concentration 50 μg / mL) was coated on the dish at 16.7 μg / cm 2 , and a collagen gel with a concentration of 1 mg / mL was used. Cells were seeded on their surfaces at a density of 1×10 4 cells / cm 2 respectively. The cell growth rate was evaluated by measuring the change in OD450 of the culture supernatant using Cell Counting Kit-8 (manufactured by Dojindo Laboratories). The results are shown in Fig. 9. In Fig. 9, A is the acid-soluble collagen-coated sample at 16.7 μg / cm 2A shows 1 mg / mL collagen gel, and C shows a DMEM wet oriented sheet. In the culture with the DMEM wet oriented sheet (C), compared with the culture on the acid-solubilized collagen coat (A), the turbidity of the solution during the culture process was lower, indicating cell growth similar to that in the collagen gel culture. Since the collagen gel has a collagen fiber structure similar to that of the living body, it was considered that the DMEM wet oriented sheet also has an environment similar to that of the living body.

[0069] In addition, calcein staining was performed after 1, 3, and 7 days of culture, and the fluorescence microscope images were taken respectively. The results are shown in Fig. 10. In the acid-solubilized collagen coat (A), it was observed that the cells had significantly increased. On the other hand, the DMEM wet oriented sheet (C) was similar to the collagen gel (B), and gentle cell growth was confirmed. This gentle cell growth was considered to reflect the closeness to the living body environment.

[0070] (Example 8) Similar to Example 1, a collagen powder (PSC powder manufactured by Nippi Co., Ltd.) with an average fiber length of 142 μm and pure water at a temperature of 4 °C were kneaded to prepare bioinks with collagen fiber concentrations of 10, 20, and 30 w / w%. This bioink was incubated at three temperature conditions of 4 °C, 25 °C, and 37 °C for 24 hours, and the change in the collagen fiber length was measured. The measurement method was to take a part of the bioink, disperse it in 50 mM Tris-HCl buffer (pH 8.0), randomly select 20 or more collagen fibers by a phase contrast microscope, measure the fiber length, and calculate the average. The results are shown in Table 1 below. It was confirmed that there was no significant change in the average fiber length of collagen in any of the bioinks within 24 hours, and they were stable. As a control, the raw material PSC powder was dispersed in 50 mM Tris-HCl buffer (pH 8.0), and the average fiber length was measured in the same manner as above, and it was 151 μm.

[0071]

Table 1

[0072] (Example 9) Similar to Example 1, collagen powder with an average fiber length of 142 μm (PSC powder, manufactured by Nippi Co., Ltd.) and pure water at a temperature of 4°C were kneaded to prepare bioinks with collagen fiber concentrations of 5, 10, 15, 20, 25, and 30 w / w%. Using this bioink, at a temperature of 25°C, a 3D printer with the same dispensing method as in Example 2 (Musashi Engineering, SHOT mini, model M22-123) was used to apply pressure using a nozzle with an inner diameter of 0.4 mm or 0.8 mm and extrude it into the atmosphere to form an oriented sheet with a length of 1 cm × a width of 1 cm and a lattice-shaped cube with a length of 1 cm × a width of 1 cm × a height of 1 cm, followed by freeze-drying. Note that for the ones formed using a nozzle with an inner diameter of 0.8 mm, only the shaped objects with a concentration of 30 w / w% were made, and for bioinks with other concentrations, all used a nozzle with an inner diameter of 0.4 mm. Among them, the 3D molded objects with 10 w / w% to 30 w / w% are shown in Figure 11. The bioinks with 5 w / w% and 10 w / w% slightly collapsed during the lamination process, but the bioink could be smoothly discharged from the nozzle into the atmosphere. On the other hand, at collagen fiber concentrations of 15 to 30 w / w%, the bioink could be smoothly discharged from the nozzle into the atmosphere, and a molded object without collapse could be manufactured even during the lamination process. Arbitrarily select 5 or more lines constituting the lattice-shaped cube obtained with bioinks of various collagen concentrations and measure the line width, and use the average line width as the resolution. Since the molded objects with 5 w / w% and 10 w / w% had collapses, the resolution was not measured. On the other hand, the resolutions of the bioinks with 15 w / w%, 20 w / w%, 25 w / w%, and 30 w / w% were 478.0 μm, 471.2 μm, 488.1 μm, and 813.6 μm, respectively. The resolution of the molded object formed with a 0.4 mm nozzle could ensure a high resolution of approximately 480 μm when the collagen fiber concentration was 15 to 25%. Also, the resolution of the molded object formed with a 0.8 mm nozzle was about 810 μm at a collagen concentration of 30 w / w%, and a high resolution corresponding to the nozzle diameter could be ensured.

[0073] (Comparative Example 1) The porcine dermis-derived collagen solution solubilized with pepsin was adjusted to pH 8.0 with a phosphate buffer, and isoelectric precipitation by standing was performed to obtain isoelectric precipitation collagen with an average collagen fiber length of about 1,260 μm. This precipitated collagen was dispersed in pure water to prepare bioinks with collagen fiber concentrations of 5, 10, 15, and 20 w / w%. In the same manner as in Example 8, pressure was applied and extruded using a 0.4 mm diameter nozzle of a dispensing type 3D printer (Musashi Engineering, SHOT mini, model M22-123) to attempt to form a lattice-shaped cube. However, in the range of collagen fiber concentrations of 5 to 20 w / w%, the bioink clogged the nozzle in all cases, and stable extrusion molding could not be achieved, and a molded body could not be manufactured. It was considered that nozzle clogging occurred due to the long collagen fiber length.

[0074] (Comparative Example 2) When the average fiber length of the collagen fibers of Lifeink (registered trademark) 200 (Neutralized Type I Collagen Bioink, 35 mg / ml, Catalog #5278) of Advanced Biomatrix was measured using a phase contrast microscope as described in Example 1, it was 1,933 μm. Using this as a material, in the same manner as in Example 2, pressure was applied and extruded into the atmosphere using a 0.4 mm diameter nozzle of a dispensing type 3D printer (Musashi Engineering, SHOT mini, model M22-123), and it was molded into a lattice-shaped cube with a length of 1 cm × width of 1 cm × height of 0.6 cm at a temperature of 25°C. The viscosity of the ink was very loose, and it was discharged with a line width of 1 mm or more even when the line width was set to 0.5 mm, and it was difficult to perform molding with a 3D printer. Also, the ink was simply laminated, and a lattice shape could not be formed. This molded object is shown in Fig. 12. A is a plan view, and B is an imaging view of the side. The resolution could not be measured.

[0075] (Comparative Example 3) Using the Advance Biomatrix's Lifeink (registered trademark) 200 (Neutralized Type I Collagen Bioink, 35 mg / ml, Catalog #5278) used in Comparative Example 2 as the material, a 3D printer (Musashi Engineering, SHOT mini) was used, and it was ejected into the atmosphere from a 30G needle (Nipro Flowmax 30G×1 / 2) at an ejection air pressure of 353 kPa to form a lattice cube with a length of 1 cm, a width of 1 cm, and a height of 0.6 cm. Although the 30G needle has an inner diameter as small as 0.12 mm, it was difficult to stack, and a lattice structure could not be fabricated. This shaped object is shown in Figure 13.

[0076] (Example 10) The isoelectric precipitation product of chicken-derived type II collagen extracted by enzymatic treatment with protease instead of pepsin was dried using a vibration dryer VU-45 manufactured by Chuo Kako Co., Ltd. at a vacuum degree of 40 Torr and a drying temperature of 40 °C for 4 hours to obtain collagen powder. The average fiber length was 133 μm. Using this collagen powder, a bioink with a collagen fiber concentration of 20 w / w% was prepared in the same manner as in Example 1. Using this bioink, it was ejected into the atmosphere at an ejection air pressure of 59 kPa in the same manner as in Example 2 to produce a lattice sheet with dimensions of 1 cm×1 cm×0.1 cm. The obtained shaped body is shown in Figure 14. The resolution of this lattice sheet was 466 μm. Even for chicken-derived type II collagen, a lattice sheet with excellent resolution could be manufactured.

[0077] (Example 11) Instead of using the bioink containing the collagen powder with an average fiber length of 142 μm (PSC powder, manufactured by Nippi, Inc.) used in Example 1, pure water was added to the isoelectric precipitate of pepsin-solubilized collagen derived from porcine dermis with a collagen fiber length of approximately 131 μm obtained by multiple centrifugal concentration, and the collagen fiber concentration was adjusted to 12.5 w / w% to prepare bioink (A). Bioink (B) containing 20 w / w% of an alkali-solubilized collagen powder derived from porcine dermis with a collagen fiber length of approximately 158 μm in pure water, pure water was added to the isoelectric precipitate of alkali-solubilized collagen derived from porcine dermis with a collagen fiber length of approximately 141 μm obtained by multiple centrifugal concentration, and the collagen fiber concentration was adjusted to 20 w / w% to prepare bioink (C), and bioink (D) with a collagen fiber concentration of 20 w / w% prepared by mixing the PSC powder used in Example 1 and liver decellularized tissue powder fractionated with a 100 - 250 μm sieve in equal weight (1:1). Using these bioinks, in the same manner as in Example 2, pressure was applied using a nozzle with a diameter of 0.4 mm of a dispensing-type 3D printer and extruded into the air to form a sheet-like shape, and the obtained shaped product was freeze-dried. The freeze-dried shaped product is shown in Fig. 15. As shown in Fig. 15, regardless of the solubilization method of the collagen constituting the collagen fibers, the method of preparing the bioink, or even when other formulations were added to the collagen fibers, a 3D molded body could be manufactured using a 3D printer.

[0078] (Example 12) To 0.2 g of the collagen powder with an average fiber length of 142 μm (PSC powder, manufactured by Nippi, Inc.) used in Example 1, 0.75×10 61.8 mL of DMEM medium containing 10% FBS with fibroblasts (Human Embryonic Lung-derived Fibroblast) at a density of

[0079] (Example 13) 0.3 g of the collagen powder (PSC powder, manufactured by Nippi Co., Ltd.) used in Example 12 was taken, and 1 × 10 61.7 mL of DMEM medium containing 10% FBS and fibroblasts (Human Embryonic Lung-derived Fibroblast) at a density of cells / mL was added, and gently mixed with a medicine spoon to prepare a cell-containing bioink with a collagen fiber concentration of 15 w / w%, which is higher than that in Example 12. This cell-containing bioink was filled into a 10 mL syringe and discharged from an 18G (gauge) injection needle into a container to uniformly mix the collagen powder in the cell-containing bioink. Then, the discharged cell-containing bioink was kneaded at 25 °C and 1,000 rpm for 1 minute using a rotation-revolution mixer (manufactured by THINKY Corporation: Awatori Rentaro). This kneaded cell-containing bioink was filled into a 10 mL syringe and discharged onto a 10 cm diameter lipidure-treated dish from an 18G (gauge) injection needle, and 10 mL of DMEM medium containing 10% FBS without fibroblasts was added. Then, it was incubated at 37 °C. Also, on the 4th day from the start of static culture, calcein staining (calcein AM, 1 μg / mL) and propidium iodide staining (PI, 1 μg / mL) were performed, and fluorescence microscope images of each were taken. The fluorescence microscope images after calcein staining and propidium iodide staining on the 4th day of culture, and the merged images with bright field images are shown in Fig. 18. It was confirmed that there were more live cells and fewer dead cells compared to the 4th day of static culture in Example 12.

[0080] (Example 14) To 0.2 g of the collagen powder (PSC powder, manufactured by Nippi Co., Ltd.) used in Example 12, 1.4×10 different from the fibroblasts used in Example 12 61.8 mL of DMEM medium containing 20% FBS with myoblasts (mouse myoblasts, C2C12) at cells / mL was added, gently mixed with a medicine spoon, and a cell-containing bioink with a collagen fiber concentration of 10 w / w% was prepared. This cell-containing bioink was filled into a 10 mL syringe and discharged from an 18G (gauge) injection needle into a container to uniformly mix the collagen powder in the cell-containing bioink. Then, the discharged cell-containing bioink was kneaded at 25 °C and 1,000 rpm for 1 minute using a rotation-revolution mixer (manufactured by THINKY Corporation: Awatori Rentaro). This kneaded cell-containing bioink was filled into a 10 mL syringe and discharged onto a 10 cm diameter lipid-treated dish from an 18G (gauge) injection needle, and 10 mL of DMEM medium containing 20% FBS without myoblasts was added. Then, it was incubated at 37 °C. On the 4th day from the start of static culture, calcein staining (calcein AM, 1 μg / mL) and propidium iodide staining (PI, 1 μg / mL) were performed, and fluorescence microscope images of each were taken. Fluorescence microscope images after calcein staining and propidium iodide staining on the 4th day of culture, as well as merged images with bright-field images, are shown in Fig. 19. In the case of the cell-containing bioink containing myoblasts, it was also confirmed that living cells were present. Thus, it was verified that a cell-embedded type 3D molded body can be produced using the cell-containing bioink containing myoblasts.

[0081] The present invention can be implemented in various embodiments and modifications without departing from the broad spirit and scope of the present invention. Also, the above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. And various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0082] This application is based on Japanese Patent Application No. 2021-022602, filed on February 16, 2021. The entire specification, claims, and drawings of Japanese Patent Application No. 2021-022602 are incorporated herein by reference.

Claims

1. A bioink for bioprinting, comprising collagen fibers composed of collagen and / or a collagen derivative and a solvent having a pH of 5.0 to 9.0, wherein the average fiber length of the collagen fibers is 0.5 to 1,000 μm, and the concentration of the collagen fibers in the solvent is 12.5 to 30 w / w%, and the collagen fibers are dispersed in the solvent without being dissolved, the bioink.

2. The bioink according to claim 1, wherein the solvent is one or more selected from the group consisting of pure water, a buffer solution, physiological saline, and a cell culture medium.

3. Further, the bioink according to claim 1 or 2, containing one or more compounds selected from the group consisting of an extracellular matrix molecule, a decellularized tissue, a growth factor, a cytokine, and a crosslinking agent.

4. Further, the bioink according to any one of claims 1 to 3, containing cells.

5. The bioink according to any one of claims 1 to 4, which is a bioink for a 3D printer.

6. A molded article comprising the bioink according to any one of claims 1 to 5.

7. The molded article according to claim 6, which is a crosslinked molded article crosslinked by one or more selected from the group consisting of riboflavin, methacrylated gelatin (GelMA), polyethylene glycol diacrylate (PEGDA), glutaraldehyde, formaldehyde, genipin, an ammonium derivative, a photoinitiator, Irgacure (registered trademark), lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and ruthenium.

8. One or more products selected from the group consisting of a cell culture substrate, a substrate for transplantation, a tissue structure, an organ model, and a substrate for regenerative medicine, comprising the molded article according to claim 6 or the crosslinked molded article according to claim 7.

9. A method for manufacturing a molded body, characterized in that the bioink according to any one of claims 1 to 5 is discharged into the atmosphere by pressing from a nozzle having a diameter of 0.2 to 1 mm.

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