Apparatus for manufacturing cell-containing three-dimensional gel molding and method for manufacturing the same

Abstract

To provide an apparatus capable of easily manufacturing even a cell-containing three-dimensional gel having a complicated curved shape.SOLUTION: An apparatus for manufacturing a cell-containing three-dimensional gel has a recessed mold 100 having a recessed surface having a curved shape, a projecting mold 200 which projects so as to face the recessed surface and has a projecting surface having a curved shape, and an alginic acid aqueous solution injection part 300 for injecting an alginic acid aqueous solution containing cells between the recessed surface and the projecting surface. The recessed mold 100 and / or the projecting mold 200 are formed of a hygroscopic material containing a calcium salt aqueous solution. The alginic acid aqueous solution is an aqueous solution of sodium alginate. The hygroscopic material is gelatin. The included cell is a cartilage cell. The cell-containing three-dimensional gel molding is configured to form tissues such as articular, auricular, or tracheal cartilage.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an apparatus and method for producing a cell-containing three-dimensional gel molded object. [Background technology]

[0002] Biomolecular cartilage has a complex shape with curvature. For example, articular cartilage and auricular cartilage have irregular shapes, while tracheal cartilage is cylindrical. The thickness, shape, and size vary from patient to patient. Furthermore, cartilage tissue is primarily composed of extracellular matrix (ECM) and lacks blood vessels, nerves, and lymphatic vessels, making spontaneous healing difficult. Therefore, there is a need for a treatment method for damage and defects of biomolecular cartilage caused by disease or injury. Autologous cultured cartilage transplantation is performed as a treatment for cartilage diseases. In this treatment, chondrocytes collected from the patient are seeded and cultured on a scaffold, and the resulting cultured cartilage is then transplanted into the affected area. Generally, transplantation of cultured cartilage in sheet form is performed. It has been reported that cartilage defects have been treated using chondrocyte cell sheets and synovial cell sheets, and tissue formation has been promoted, resulting in the construction of cartilage tissue by the chondrocyte cell sheets (Non-Patent Documents 1 and 2). Thus, sheet-form cultured cartilage successfully forms tissue after transplantation. However, it is difficult to prepare sheet-form cultured cartilage into a three-dimensional shape of a predetermined size that fits the treatment site before transplantation.

[0003] Therefore, it is hoped that transplanting cultured cartilage of a predetermined size that matches the shape of biological cartilage will promote faster tissue regeneration. Currently, cultured cartilage that mimics the shape of biological cartilage is produced using 3D printing and bioabsorbable materials. 3D printing is a technology that creates biological structures by stacking successive layers and placing cells on a biocompatible scaffold to create a three-dimensional tissue structure (Patent Document 1, Non-Patent Document 3).

[0004] However, discontinuous areas were observed during tissue formation, making it difficult to uniformly create a scaffold of a specified size in a complex shape like an auricle. Furthermore, because 3D printing involves seeding cells onto a prefabricated scaffold, the cells only reach the surface of the scaffold. Furthermore, antibacterial agents may be added to the scaffold, raising concerns about foreign body reactions and inflammation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-182047 [Non-patent literature]

[0006] [Non-Patent Document 1] Masato Sato, Masayuki Yamato, Genya Mitani, Tomonori Takagaki, Kosuke Hamahashi, Yoshihiko Nakamura, Miya Ishihara, Ryo Matoba, Hiroyuki Kobayashi, Teruo Okano, Joji Mochida and Masahiko Watanabe, Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis, npj Regenerative Medicine, Vol.4, No.4 (2019), pp.1-4. [Non-patent document 2] Kosuke Hamahashi, Eriko Toyoda, Miya Ishihara, Genya Mitani, Tomonori Takagaki, Nagatoshi Kaneshiro, Miki Maehara, Takumi Takahashi, Eri Okada, Ayako Watanabe, Yoshihiko Nakamura, Reiko Kato, Ryo Matoba, Takehiko Takagi, Hidenori Akutsu, Akihiro Umezawa, Hiroyuki Kobayashi, Tadashi Akamatsu, Masayuki Yamato, Teruo Okano, Masahiko Watanabe and Masato Sato, Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis, npj Regenerative Medicine, Vol.7, No.71 (2022), pp.1-12. [Non-patent document 3] Nitin Sahai, Manashjit Gogoi, Ravi Prakash Tewari, 3D Printed Chitosan Composite Scaffold for Chondrocyte Differentiation, Curr Med Imaging. 2021;17(7):832-842 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above problems, and aims to provide an apparatus and method that can produce a cell-containing three-dimensional gel even if it has a complex curved shape. [Means for solving the problem]

[0008] The apparatus for manufacturing a cell-containing three-dimensional gel molding according to the present invention comprises: a concave mold having a curved concave surface; a convex mold having a curved convex surface that protrudes opposite the concave surface; and an alginic acid aqueous solution injection unit for injecting a cell-containing alginic acid aqueous solution between the concave and convex surfaces, wherein the concave and / or convex molds are formed from a hygroscopic material containing a calcium salt aqueous solution; the apparatus is characterized in that the apparatus injects the alginic acid aqueous solution between the concave and convex surfaces, brings the concave and convex molds close to each other, and maintains the alginic acid aqueous solution sandwiched between the concave and convex surfaces until gel molding is completed by a chemical gelation reaction between the calcium salt aqueous solution and the alginic acid aqueous solution, thereby manufacturing a cell-containing three-dimensional gel molding having a curved shape that corresponds to the space between the concave and convex surfaces.

[0009] Furthermore, the method for producing a cell-containing three-dimensional gel molding according to the present invention is characterized by comprising: a preparation step of preparing a molding set consisting of a pair of concave and convex molds, the concave mold having a curved concave surface and a convex mold having a curved convex surface that protrudes opposite the concave surface, the concave mold and / or the convex mold being formed from a hygroscopic material containing an aqueous calcium salt solution; an alginic acid aqueous solution injection step of injecting a cell-containing alginic acid aqueous solution between the concave surface of the concave mold and the convex surface of the convex mold; and a gel molding step of bringing the concave mold and the convex mold close to each other, maintaining the alginic acid aqueous solution sandwiched between the concave and convex surfaces, and completing gel molding by a chemical gelation reaction between the calcium salt aqueous solution and the alginic acid aqueous solution, thereby molding a cell-containing three-dimensional gel molding having a curved shape that corresponds to the space between the concave and convex surfaces. [Effects of the Invention]

[0010] According to the present invention, a manufacturing apparatus and method are provided that can easily mold a cell-containing three-dimensional gel molded body, even one with a complex curved shape, to a predetermined size before transplantation. Furthermore, unlike 3D printing, the manufacturing apparatus and method of the present invention can uniformly position cells not only on the surface but also in the center of the molded body. Furthermore, alginate is a substance that has not been recognized as toxic to living organisms and has high biocompatibility, so there is no risk of substances that cause inflammation or other problems remaining in the transplanted tissue. Furthermore, if necessary, alginate gel can be dissolved with a chelating agent such as EDTA, allowing for the production of scaffold-free tissue. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram illustrating the principle of producing a cell-containing three-dimensional gel molded article. [Figure 2] FIG. 1 is a diagram illustrating an outline of a manufacturing apparatus for a cell-containing three-dimensional gel molding. [Figure 3] FIG. 1 is a diagram illustrating a manufacturing device for a cell-containing three-dimensional gel molding in which a concave mold and a convex mold are placed close to each other. [Figure 4] FIG. 1 is a diagram illustrating a curved cell-containing three-dimensional gel molded body produced by the three-dimensional gel molded body production apparatus. [Figure 5] FIG. 1 is a photograph showing a specific example of a manufacturing apparatus for a cell-containing three-dimensional gel molded article. [Figure 6] FIG. 1 is a photograph of a cross section of a gel formed at an alginate concentration of 1.0 w / v % taken with a digital microscope. [Figure 7] FIG. 1 is a photograph of a cross section of a gel formed at an alginate concentration of 2.0 w / v % taken with a digital microscope. [Figure 8] FIG. 1 shows the relationship between the thickness of an alginate gel formed at an alginate concentration of 1.0 w / v % and the gelation time. [Figure 9] FIG. 1 shows the relationship between the thickness of an alginate gel formed at an alginate concentration of 2.0 w / v % and the gelation time. [Figure 10] FIG. 2 is a diagram illustrating a measurement site. [Figure 11] FIG. 1 is a photograph of a concave mold made with gelatin gel at gelatin concentrations of 8, 12, 16, and 20 w / v%. [Figure 12] FIG. 1 shows the compressive rigidity of gelatin gels at gelatin concentrations of 8, 12, 16, and 20 w / v%. [Figure 13] Figure 1 shows photographs of the cross sections of gels made with gelatin concentrations of 12, 16, and 20 w / v% at different indentation speeds. [Figure 14] This figure shows the distribution of gel thicknesses created at indentation speeds of 1, 3, 5, and 7 mm / s with a gelatin concentration of 12 w / v%. [Figure 15] This figure shows the distribution of gel thicknesses created at indentation speeds of 1, 3, 5, and 7 mm / s with a gelatin concentration of 16 w / v%. [Figure 16] This figure shows the distribution of gel thicknesses created at indentation speeds of 1, 3, 5, and 7 mm / s with a gelatin concentration of 20 w / v%. [Figure 17] FIG. 1 is a photograph of a chondrocyte-mixed alginate gel molded article taken with a phase-contrast microscope. [Figure 18] FIG. 1 is a photograph of a bright-field image of a cross section of a cartilage cell-mixed alginate gel molded body. [Figure 19] FIG. 10 is a diagram showing the thickness of a cartilage cell-mixed alginate gel molded article. [Figure 20] FIG. 1 is a photograph of a fluorescent stained image of a gel stained with Calcein AM and Ethidium homodimer. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings. However, the embodiment is intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiment. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiment are also included in the scope of the present invention.

[0013] As shown in Figure 2, the apparatus for producing a cell-containing three-dimensional gel molded article according to the present invention includes a concave mold 100 having a curved concave surface, a convex mold 200 having a curved convex surface that protrudes opposite the concave surface, and an alginic acid aqueous solution injection unit 300 for injecting a cell-containing alginic acid aqueous solution between the concave and convex surfaces. The concave mold 100 and / or the convex mold 200 are formed from a hygroscopic material containing a calcium salt aqueous solution. That is, the concave mold 100 may be formed from a hygroscopic material containing a calcium salt aqueous solution and the convex mold 200 from a polymeric material such as polycarbonate, or the concave mold 100 may be formed from a polymeric material and the convex mold 200 from a hygroscopic material containing a calcium salt aqueous solution, or the concave mold 100 may be formed from a hygroscopic material containing a calcium salt aqueous solution and the convex mold 200 from a hygroscopic material containing a calcium salt aqueous solution.

[0014] When the concave mold 100 or the convex mold 200 is formed from a hygroscopic material containing an aqueous calcium salt solution, it is desirable that it has a predetermined rigidity, which is not particularly limited, but is, for example, 1 to 50 kPa, preferably 2 to 40 kPa, more preferably 3 to 30 kPa, and most preferably 3.2 to 28 kPa.

[0015] In Figure 2, the concave surface of the concave mold 100 and the convex surface of the convex mold 200 are configured to fit together, but this is not limited to such a combination, and it is also possible to combine shapes in which the concave surface of the concave mold 100 and the convex surface of the convex mold 200 do not correspond to each other. The concave mold 100 has a curved concave surface, but the entire concave surface does not have to be curved, and it is also possible for a portion of the concave surface to be flat. Furthermore, the convex mold 200 has a curved convex surface, but the entire convex surface does not have to be curved, and it is also possible for a portion of the convex surface to be flat.

[0016] The aqueous alginic acid solution is not particularly limited and includes, for example, an aqueous solution of at least one alginate selected from the group consisting of sodium alginate, potassium alginate, and ammonium alginate, and is preferably an aqueous solution of sodium alginate.

[0017] The alginic acid concentration of the aqueous alginic acid solution is not particularly limited, and is, for example, 0.1 w / v% to 10 w / v%, preferably 0.5 w / v% to 5 w / v%, more preferably 0.8 w / v% to 3 w / v%, and most preferably 1 w / v% to 2 w / v%.

[0018] The calcium salt aqueous solution is not particularly limited, and is, for example, an aqueous solution containing at least one calcium salt selected from the group consisting of calcium chloride, calcium lactate, calcium gluconate, calcium fumarate, calcium citrate, calcium succinate, calcium acetate, and calcium hydroxide, and is preferably an aqueous solution of calcium chloride.

[0019] The hygroscopic material is not particularly limited and includes, for example, at least one selected from the group consisting of gelatin, agar, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, methyl cellulose, pullulan, carboxymethyl cellulose, dextrin, pectin, sodium polyacrylate, and polyvinylpyrrolidone, and is preferably gelatin.

[0020] When the hygroscopic material is gelatin, the gelatin concentration is not particularly limited as long as the physical strength is maintained, and is, for example, 6 w / v% to 60 w / v%, preferably 8 w / v% to 50 w / v%, more preferably 12 w / v% to 40 w / v%, and most preferably 16 w / v% to 20 w / v% gelatin.

[0021] The cells contained in the three-dimensional gel molding are not particularly limited, and may be any one selected from the group consisting of, for example, chondrocytes, myoblasts, mesenchymal stem cells, cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, epithelial cells, endothelial cells, hepatic cells, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, and skin cells, and are preferably chondrocytes.

[0022] When the cells contained in the three-dimensional gel molding are chondrocytes, the cell-containing three-dimensional gel molding is used to damage any one type of tissue selected from the group consisting of, but not limited to, articular cartilage, auricular cartilage, tracheal cartilage, nasal cartilage, elbow cartilage, knee meniscus, costal cartilage, ankle cartilage, laryngeal cartilage, and vertebral cartilage.

[0023] Next, the manner of use of the device for producing a cell-containing three-dimensional gel molded article of the present invention will be described.

[0024] First, as shown in Fig. 2, a pair of mold sets is prepared (preparation step) consisting of a concave mold 100 having a curved concave surface and a convex mold 200 having a curved convex surface that protrudes opposite the concave surface. For example, the concave mold 100 is formed from a hygroscopic material containing a calcium salt aqueous solution, and the convex mold 200 is formed from polycarbonate.

[0025] Next, an alginic acid aqueous solution containing cells is injected between the concave surface of the concave mold 100 and the convex surface of the convex mold 200 (alginic acid aqueous solution injection step). The method for injecting the alginic acid aqueous solution is not particularly limited, and for example, the alginic acid aqueous solution can be dripped onto the concave surface of the concave mold 100 using a dropper-type dripping device. Alternatively, for example, an injection hole penetrating the convex mold 200 can be provided, and the alginic acid aqueous solution can be injected between the concave surface of the concave mold 100 and the convex surface of the convex mold 200 through the injection hole using an injection syringe device.

[0026] Next, the concave mold 100 and the convex mold 200 are brought close to each other, and the alginic acid aqueous solution is held between the concave and convex surfaces. The gel formation is completed by a chemical gelation reaction between the calcium salt aqueous solution and the alginic acid aqueous solution, thereby forming a cell-containing three-dimensional gel object with a curved shape corresponding to the space between the concave and convex surfaces (gel forming step). The method for bringing the concave mold 100 and the convex mold 200 close to each other is not particularly limited. For example, the concave mold 100 can be fixed, and the convex mold 200 can be pressed into the concave mold 100 at a constant pressing speed. Alternatively, the convex mold 200 can be fixed, and the concave mold 100 can be pressed into the convex mold 200 at a constant pressing speed. The indentation speed is not particularly limited as long as it does not adversely affect the shape of the three-dimensional gel molding and does not excessively reduce the viability of the contained cells, and is, for example, 0.5 to 15 mm / s, preferably 1 to 12 mm / s, more preferably 3 to 10 mm / s, and most preferably 3 to 7 mm / s.

[0027] Also, for example, it is possible to press both the concave mold 100 and the convex mold 200 close to each other at a constant pressing speed. The pressing speed in this case is the relative speed between the concave mold 100 and the convex mold 200.

[0028] When the alginic acid aqueous solution is held between the concave and convex surfaces, the distance between the concave and convex surfaces is not particularly limited and is, for example, 0.1 mm to 10 mm, preferably 0.5 mm to 5 mm, more preferably 0.8 mm to 2 mm, and most preferably 1 mm. The distance between the concave and convex surfaces can be constant over the entire surface area, or can be non-uniform.

[0029] The chemical gelation reaction between an aqueous calcium salt solution and an aqueous alginate solution occurs as shown in Figure 1. 2+The gelation reaction is between alginic acid and calcium ions, which are gelation factors. Because the reaction between alginic acid and calcium ions, which are gelation factors, occurs in a short time, it is possible to efficiently produce a cell-containing three-dimensional gel molded article with a curved shape that corresponds to the space between the concave and convex surfaces. The gelation reaction time is not particularly limited, and is, for example, 0.5 to 10 minutes, preferably 2 to 8 minutes, more preferably 4 to 7 minutes, and most preferably 5 to 6 minutes.

[0030] Alginate is water-soluble, dissolves even in cold water, has smooth fluidity, and is viscous. Therefore, when cells are added to an alginate solution, the cells can be uniformly distributed throughout the solution. Therefore, in the present invention, cells can be uniformly distributed throughout the 3D gel molded object. While 3D printing creates a 3D tissue structure by stacking layers, which has the disadvantage of varying cell concentrations in each layer, the present invention has the significant advantage of being able to uniformly distribute cells throughout the 3D gel molded object. [Example]

[0031] (1-1) Manufacturing equipment for cell-containing three-dimensional gel moldings Figure 1 shows a schematic diagram of the fabrication principle of a cell-containing 3D gel molded object. 2+ It reacts with EDTA to form a gel. It also becomes a water-soluble salt and solates, making it possible to decompose and remove alginic acid. Utilizing this phenomenon, the alginic acid solution is gelled by dropping it into a concave mold and supplying calcium chloride (CaCl2) from the mold.

[0032] Figure 2 shows a manufacturing apparatus for cell-containing 3D gel molded articles. The manufacturing apparatus for cell-containing 3D gel molded articles includes a concave mold 100 with a curved concave surface, a convex mold 200 with a curved convex surface that protrudes opposite the concave surface, and an alginic acid aqueous solution injection unit 300 for injecting a cell-containing alginic acid aqueous solution between the concave and convex surfaces. In this example, the concave mold 100 was formed from a hygroscopic material containing a calcium salt aqueous solution. Specifically, to supply CaCl2 from the concave mold 100, the mold material used was a CaCl2-containing gelatin gel prepared by mixing a CaCl2 solution with gelatin powder. That is, the concave mold 100 was a hemispherical convex mold with a radius of 10 mm, and was used to form a concave mold for the CaCl2-containing gelatin gel. In this example, the alginic acid aqueous solution injection unit 300 was a dropper-type dripping device capable of dripping the alginic acid aqueous solution. An alginic acid solution was dripped onto the concave surface of the concave mold 100 using the alginic acid solution injection part 300 .

[0033] An alginic acid aqueous solution is poured between the concave surface of the concave mold 100 and the convex surface of the convex mold 200. Specifically, the alginic acid aqueous solution is sodium alginate. Then, as shown in Figure 3, the concave and convex molds are brought close to each other, and the sodium alginate is held between the concave and convex surfaces until gel formation is completed by a chemical gelation reaction between the calcium chloride aqueous solution and the sodium alginate aqueous solution.

[0034] As a result, as shown in FIG. 4, a cell-containing three-dimensional gel molding 310 is produced that has a curved shape corresponding to the space between the concave surfaces.

[0035] Figure 5 shows a photograph of a specific example of a manufacturing device for cell-containing 3D gel molds. This device uses a compression device (EMX-500N, Imada Co., Ltd.) to compress a convex mold 200 at a constant speed. A polycarbonate convex mold 200 for molding alginate was attached to the compression device's load cell (ZTA-500N, Imada Co., Ltd.) using a joint. In addition, a positioning pin was installed in the concave mold 100 to align the positions of the concave mold 100 and convex mold 200. To prevent damage to the mold, a spring mechanism consisting of a spring attached to a polycarbonate rod was installed between the measurement stand and base of the compression device.

[0036] (1-2) Evaluation of alginate gelation using CaCl2-containing gelatin gel In this example, the manufacturing device for cell-filled 3D gel bodies was designed so that CaCl2 was supplied to the alginate solution only from one side. That is, the alginate solution was dripped onto the concave surface of the mold 100 using the alginate solution injector 300. The thickness of the alginate gel formed by the single-sided CaCl2 supply was observed. Calcium chloride dihydrate (C7902-500G, SIGMA-ALDLICH) was dissolved in distilled water to prepare a 1.2 w / v% CaCl2 solution. Pigskin gelatin powder (Type A, Nitta Gelatin Co., Ltd.) was added to the CaCl2 solution, and the mixture was heated in a hot bath at 120 rpm and 50°C to prepare 8, 12, 16, and 20 w / v% CaCl2 gelatin solutions. Physiological saline (3311401A7010, Otsuka Pharmaceutical Factory, Inc.) was mixed with sodium alginate (I-1G, Kimica Co., Ltd.). The alginate-mixed saline solution was then stirred at 2000 rpm for 30 minutes using a stirrer / deaerator (HM-500, KEYENCE) to prepare 1.0 and 2.0 w / v% alginate solutions. To remove impurities, the alginate was filtered through a 1 μm mesh filter (#508 / 585-1μ, tantore). 2 ml of CaCl2 gelatin solution was poured into a 35 mm dish and allowed to stand at 4°C for 30 minutes. A 3 mm-thick PDMS frame, cut with an 8 mm diameter biopsy trephine, was placed on top of the CaCl2-containing gelatin gel. 200 μl of alginate was poured into the PDMS sheet frame. The alginate gelation time was set to 1, 3, or 5 minutes. After gelation, the prepared alginate gel was washed with distilled water, and the center of the sheet was cut with a scalpel blade to measure its cross section. The cross section of the sheet was photographed using a digital microscope (VHX-5000, KEYENCE). From the image, the thickness of the sheet cross section was measured at two edges and at the center, and the average value obtained was used as the thickness of the alginate gel under each condition.

[0037] (2-1) Study on gelatin concentration that can form concave mold 100 made of CaCl2-containing gelatin gel Because it was thought that the gelatin concentration was involved in the formability of the concave mold 100 for CaCl2-containing gelatin gel, concave molds for CaCl2-containing gelatin gel were prepared with gelatin concentrations of 8, 12, 16, and 20 w / v%. The CaCl2 gelatin solution was poured into a mold for preparing CaCl2-containing gelatin gel, similar to that described in (2), through the injection port. The CaCl2 gelatin was allowed to gel by standing at 4°C for 30 minutes, and the convex mold 200 for preparing CaCl2-containing gelatin gel was then removed. The prepared concave molds 100 for CaCl2-containing gelatin gel were observed and their shapes were evaluated.

[0038] Figures 6 and 7 show bright-field images of cross sections of gels formed at alginate concentrations of 1.0 w / v% and 2.0 w / v% taken with a digital microscope. Figures 8 and 9 show the relationship between gelation time and thickness for alginate gels at 1.0 w / v% and 2.0 w / v% alginate concentrations. Regardless of the alginate or gelatin concentration, thickness increased with gelation time. Furthermore, regardless of gelation time, gelatin concentration had little effect on thickness at either alginate concentration. Table 1 shows the thickness of gels formed at a 1.0 w / v% alginate concentration. At a gelation time of 5 minutes, the thickness was approximately 0.9 mm, regardless of gelatin concentration. At a gelation time of 20 w / v% gelatin and a gelation time of 5 minutes, the thickness reached 1.04 mm, the thickest gel formed at a 1.0 w / v% alginate concentration.

[0039] [Table 1]

[0040] Table 2 shows the thickness of gels formed at an alginate concentration of 2.0 w / v%. At a gelation time of 5 minutes, the thickness was approximately 1.2 mm regardless of the gelatin concentration. At a gelatin concentration of 16 w / v% and a gelation time of 5 minutes, the thickness was 1.39 mm, the thickest gel formed at an alginate concentration of 2.0 w / v%. Furthermore, at the same gelatin concentration and gelation time, the thickness of 2.0 w / v% alginate gels was greater than that of 1.0 w / v% alginate gels. At all gelatin concentrations, alginate gels with a thickness of 1 mm or more were obtained at an alginate concentration of 2.0 w / v% and a gelation time of 5 minutes.

[0041] [Table 2]

[0042] In Figures 8 and 9, the thickness of the gel formed was greater when the alginic acid concentration was 2.0 w / v%. When the alginic acid solution was dropped into the CaCl2 solution, Na + and Ca 2+ The exchange of Ca occurs and gelation occurs instantly at the contact point between the alginate solution and the gelatin gel. 2+Gelation progresses as the alginate diffuses into the interior. As gelation progresses, the gel shrinks and dehydrates. It has been reported that the lower the alginate concentration, the higher the shrinkage rate. Therefore, it is thought that the 1.0 w / v% alginate gel had a higher shrinkage rate and smaller thickness than the 2.0 w / v% alginate gel. This suggests that increasing the alginate concentration can produce thicker gels. Furthermore, regardless of alginate concentration, the thickness of the formed gel increased with gelation time. When a core solution containing CaCl2 was dropped into an alginate solution, a gel coating formed around the core liquid, which gradually increased in thickness with gelation time. This suggests that gel thickness can be increased by extending the gelation time. At an alginate concentration of 2.0 w / v% and a gelation time of 5 minutes, the gel thickness exceeded 1 mm at all gelatin concentrations. To produce gels thicker than 1 mm, a gelation time of 5 minutes or longer is required. Furthermore, at the same alginate concentration and gelation time, there was no difference in the thickness of the gel formed regardless of the gelatin concentration, which suggests that the Ca necessary for alginate gelation 2+ It can be said that Ca was supplied sufficiently from the gelatin gel. Gelatin is usually in a state of being flexible in warm water above 40oC, and maintains a random coil structure. When cooled, the gelatin molecules partially create a helical structure, and these parts join together to form a network, forming a gel. The bonds between gelatin molecules change depending on the gelatin concentration, so Ca 2+ However, regardless of the gelatin concentration, the surface of the gelatin gel contains calcium phosphate, which is necessary for the gelation of the alginate solution. 2+ There is sufficient Ca 2+ It was shown that it is possible to produce alginate gels with a thickness of 1 mm or more by supplying

[0043] (2-2) Measurement of stiffness of CaCl2-containing gelatin gel To form a cell-containing 3D gel object, we dropped an alginate solution onto a CaCl2-containing gelatin gel and pressed a polycarbonate convex mold 200 at a constant speed. We considered that the stiffness of the CaCl2-containing gelatin gel and the pressing speed would affect the formability of the alginate gel. Therefore, we measured the compressive stiffness of the CaCl2-containing gelatin gel. The CaCl2-containing gelatin solution was prepared as described in (1-2). The gelatin concentrations were 8, 12, 16, and 20 w / v%. The CaCl2-containing gelatin gel was poured into a 3 mm thick, 7 mm diameter mold and allowed to stand at 4°C for 30 minutes. The CaCl2-containing gelatin gel was then extruded using a 6 mm diameter biopsy trephine. The cylindrical CaCl2-containing gelatin gel was subjected to stiffness testing using a Shimadzu EZ Graph (SHIMADZU) compact benchtop testing machine. The compressive stiffness at 15% strain was calculated from the results of indenting 1 mm at an indentation speed of 1 mm / s.

[0044] (2-3) Evaluation of the effect of CaCl2-containing gelatin gel stiffness and molding speed on the thickness of three-dimensional gel bodies Hemispherical 3D gel molds were fabricated using a molding machine. The CaCl2 gelatin solution and alginate solution were prepared using the same method as in (1-2). Experiments were conducted with gelatin concentrations of 12, 16, and 20 w / v% and alginate concentration of 2.0 w / v%. A concave mold for CaCl2-containing gelatin gel was prepared using the method described in (2-1) and placed on the base of the molding machine. A convex mold for alginate molding was placed on a load cell and alginate solution was added dropwise. The convex mold was pressed at indentation speeds of 1, 3, 5, and 7 mm / s until the convex and concave molds came into contact. After leaving the mold stationary for 5 minutes, the convex mold for alginate molding was lifted, and the 3D gel mold was washed with distilled water. The center of the 3D gel mold was cut using a scalpel blade to measure the cross-sectional thickness. The cross-section of the 3D gel mold was photographed using a digital microscope. From the photographed image, the thickness was measured at five points at 0, ±30, and ±60 degrees from the center of the hemisphere in the cross section, as shown in Figure 10.

[0045] Figure 11 shows a concave mold made with CaCl2-containing gelatin gel. At a gelatin concentration of 8 w / v%, the gelatin gel adhered to the convex mold, causing the concave mold to break. At gelatin concentrations of 12, 16, and 20 w / v%, the hemispherical concave molds could be made without breakage.

[0046] In Figure 11, it was difficult to create a concave mold only at a gelatin concentration of 8 w / v%. The conditions for gelatin gelation in the prior art are to adjust a 4.0 w / v% pigskin gelatin solution to 40°C, then rapidly cool it to 5°C, with the cooling period set to 5 minutes. Therefore, in the present invention, the gelatin gel was created by rapidly cooling a 50°C pigskin gelatin solution to 4°C for 30 minutes, which means that sufficient gelation was achieved. It has been shown that the higher the concentration of gelatin gel, the stronger the jelly becomes. Therefore, it is believed that the jelly strength was insufficient at a gelatin concentration of 8 w / v%, causing the breakage.

[0047] The compressive stiffness of CaCl2-containing gelatin gels at 15% strain is shown in Figure 12. The compressive stiffness increased with increasing gelatin concentration, from 1.69 ± 0.22 kPa at a gelatin concentration of 8 w / v%, to 2.32 ± 0.57 kPa at 12 w / v%, to 3.27 ± 0.46 kPa at 16 w / v%, and to 4.75 ± 0.85 kPa at 20 w / v%.

[0048] Figure 13 shows a bright-field image of a cross section of a gel prepared with an alginate concentration of 2.0 w / v%. Figures 14, 15, and 16 show the thickness distribution of gels with gelatin concentrations of 12, 16, and 20 w / v%. Tables 3, 4, and 5 show the thickness of gels with gelatin concentrations of 12, 16, and 20 w / v%. At an indentation speed of 1 mm / s, the maximum thickness of the drop area was 1.56 mm for gelatin concentrations of 12 w / v%, 1.34 mm for gelatin concentrations of 16 w / v%, and 1.07 mm for gelatin concentrations of 20 w / v%. The thickness of the drop area of ​​the gel formed with the lowest gelatin concentration, 12 w / v%, was the largest and most uneven. Furthermore, regardless of the indentation speed, the CV of gel thickness was large at a gelatin concentration of 12 w / v%. At gelatin concentrations of 16 and 20 w / v%, the CV tended to be large at an indentation speed of 1 mm / s. This indicates that a pushing speed of 3 mm / s or more is suitable for forming a uniform gel at a gelatin concentration of 16 w / v% or more.

[0049] [Table 3]

[0050] [Table 4]

[0051] [Table 5]

[0052] In Figures 14 and 15, the thickness of the drop portion of the gel formed at a displacement speed of 1 mm / s was significantly non-uniform at gelatin concentrations of 12 w / v% and 16 w / v%. In Figure 16, the thickness of the drop portion of the gel formed at a gelatin concentration of 20 w / v% and a displacement speed of 1 mm / s was uniform, but the error bars showed large variations compared to the displacement speeds of 3 mm / s, 5 mm / s, and 7 mm / s. This is thought to be due to changes in the viscosity of the alginate solution and the rigidity of the gelled alginate before gel formation. The Na content of alginate + and Ca2+ It has been reported that when the amount of substitution is increased, the viscosity changes irregularly and then increases. In the present invention, since the alginate gelled, it is believed that the viscosity of the alginate solution gradually increased after the solution was dripped. Therefore, under conditions of slow indentation speed, the time until the convex and concave molds were indented was prolonged, which likely increased the viscosity of the alginate and reduced its fluidity, resulting in an increase in the thickness of the gel dripped portion. Furthermore, only the gel formed with a gelatin concentration of 12 w / v% had a large thickness in the dripped portion. It has been reported that the stiffness of alginate gels increases with increasing added salt content. Therefore, it is possible that the alginate gel, whose stiffness increased due to the slow indentation speed, was indented into the gelatin gel concave mold, which had a low stiffness, resulting in deformation. Therefore, if the increase in the pressing pressure and the increase in the stiffness of the gelled portion are higher than that of gelatin gel, there is a risk of uneven thickness. Therefore, in the present invention, it was shown that a concave mold of gelatin gel with a stiffness of 3.27 kPa or more is suitable for molding a curved shape of biological cartilage at a pressing speed of 3 mm / s or more.

[0053] (3-1) Chondrocyte isolation method 7.5 g of cartilage tissue was minced and collected from the knee joint of a 6-month-old, 100 kg pig. 10 ml of 0.25% trypsin solution (35554-64, Nacalai Tesque, Inc.) and 10 ml of phosphate-buffered saline (PBS) (166-23555, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the collected cartilage tissue and agitated at 37°C for 30 minutes using a shaker (SN-100SD, Nisshin Rika Co., Ltd.). 45 ml of 0.15% collagenase solution was added to the cartilage tissue and agitated for 12 hours in an incubator (MCO-18AIC, Sanyo Electric Co., Ltd.) at 100% humidity, 37°C, and 5% CO2. A 0.15% collagenase solution was prepared by dissolving COLLAGENASE TYPE 2 (WOR-CLS-2, Funakoshi Co., Ltd.) at a concentration of 0.15 w / v% in Dulbecco's Modified Eagle Medium (DMEM) (08459-35, Nacalai Tesque, Inc.) containing 1% antibiotic (A5955, Sigma-Aldrich). After 10 h of stirring, the ECM degraded by the collagenase solution was removed using a 40 μm cell strainer (REF352340, BD Falcon), and chondrocytes were isolated.

[0054] (3-2) Preparation of chondrocyte-containing 3D gel molded body By mixing chondrocytes with alginate solution, the alginate concentration was 2.0 w / v%, and the cell suspension density was 1.0 × 10 6 A chondrocyte-containing alginate solution was prepared at 1000 cells / ml. A chondrocyte-containing 3D gel molded object was fabricated using the same method as in (2-3). Experiments were performed at a gelatin concentration of 20 w / v% and an indentation speed of 3 mm / s. Images of the center and two edges of the chondrocyte-containing 3D gel molded object were taken using a phase-contrast microscope (IX71, OLYMPUS). The cross section of the chondrocyte-containing 3D gel molded object was then photographed using a digital microscope. The thickness was measured at five points, 0, ±30, and ±60 degrees from the center of the hemisphere of the cross section, from the photographed images, as shown in Figure 10.

[0055] (3-3) Viability assessment of encapsulated chondrocytes To assess cell viability immediately after the formation of cell-loaded 3D gel bodies, cell viability was assessed by fluorescent staining. Because culture medium was still attached, the cell-loaded 3D gel bodies were immersed in 7 ml of PBS and washed twice. The cell-loaded 3D gel bodies were cut in half, and 300 μl of PBS containing 0.1% Calcein AM solution (C1359-100UL, SIGMA-ALDLICH) and 0.3% Ethidium homodimer solution (E1903-.5ML, SIGMA-ALDLICH) was added and allowed to stand for 5 min. After the incubation, the gels were washed twice with 300 μl of PBS. The stained cells were observed using a multiphoton excitation fluorescence microscope (MPM) (TCS SP8 Multi-Photone, Leica) in an observation area measuring 450 × 450 μm in the center of the gel and 1.20 mm deep from the surface. The viability of cells was calculated based on the number of live and dead cells in the three cross sections of the stereoscopic image, and cell activity in the cell-containing three-dimensional gel molding was evaluated.

[0056] Figure 17 shows a cell-filled 3D gel molded article photographed with a phase-contrast microscope. The image shows that the cells are uniformly distributed in two locations, the center and the edge. Figure 18 shows a bright-field image of the cross section of the cell-filled 3D gel molded article. Figure 19 shows the thickness of the cell-filled 3D gel molded article. These results demonstrate that it is possible to mold a uniform gel with a thickness of nearly 1 mm.

[0057] 17 and 18, a gel of uniform thickness could be produced using an alginate solution containing suspended cells, suggesting that the encapsulation of cells has little effect on molding. Furthermore, since uniform cell distribution was possible, it is believed that in gels molded using this method, cells can be uniformly seeded throughout the entire structure without uneven distribution. Therefore, it was suggested that the molding method developed in this invention can reproduce the curved shape of living cartilage and mold a cell-filled 3D gel object with uniformly distributed cells.

[0058] A fluorescent stained image of the gel stained with Calcein AM and Ethidium homodimer is shown in Figure 20. The cell viability was 86.0%.

[0059] As shown in Figure 20, the viability of the cells was 86.0%, suggesting that the molding technique developed in this invention has little effect on cell activity. Increasing the indentation speed increases shear stress, which is thought to affect cell viability, so it is advisable to mold the chondrocyte-mixed gel at an indentation speed of 3 to 7 mm / s and evaluate the viability. [Industrial Applicability]

[0060] It can be used in the field of regenerative medicine. [Explanation of symbols]

[0061] 100: Concave 200: Convex 300: Alginate solution injection part 310: Cell-containing three-dimensional gel molding

Claims

1. a concave mold having a curved concave surface; a convex mold having a curved convex surface that protrudes opposite to the concave surface; an alginic acid aqueous solution injection section for injecting an alginic acid aqueous solution containing cells between the concave surface and the convex surface; the concave mold and / or the convex mold are formed of a hygroscopic material containing an aqueous calcium salt solution; Injecting the alginic acid aqueous solution between the concave surface and the convex surface; the concave mold and the convex mold are brought close to each other, and the alginic acid aqueous solution is held between the concave surface and the convex surface, and this state is maintained until gel formation is completed by a chemical gelation reaction between the calcium salt aqueous solution and the alginic acid aqueous solution; The apparatus for producing a cell-containing three-dimensional gel molded body is characterized by producing a cell-containing three-dimensional gel molded body having a curved shape corresponding to the space between the concave surfaces.

2. The apparatus for manufacturing a cell-containing three-dimensional gel molding according to claim 1, characterized in that the alginic acid aqueous solution contains an aqueous solution of at least one alginate salt selected from the group consisting of sodium alginate, potassium alginate, and ammonium alginate.

3. 2. The apparatus for manufacturing a cell-containing three-dimensional gel molding according to claim 1, wherein the calcium salt aqueous solution is an aqueous solution containing at least one calcium salt selected from the group consisting of calcium chloride, calcium lactate, calcium gluconate, calcium fumarate, calcium citrate, calcium succinate, calcium acetate, and calcium hydroxide.

4. 2. The apparatus for manufacturing a cell-containing three-dimensional gel molding according to claim 1, wherein the hygroscopic material comprises at least one selected from the group consisting of gelatin, agar, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, methyl cellulose, pullulan, carboxymethyl cellulose, dextrin, pectin, sodium polyacrylate, and polyvinylpyrrolidone.

5. 2. The apparatus for producing a cell-containing three-dimensional gel molding according to claim 1, wherein the hygroscopic material is 16 w / v % to 20 w / v % gelatin.

6. 2. The apparatus for manufacturing a cell-containing three-dimensional gel molding according to claim 1, wherein the cells are any one selected from the group consisting of chondrocytes, myoblasts, mesenchymal stem cells, cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, epithelial cells, endothelial cells, hepatic cells, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, and skin cells.

7. 2. The apparatus for manufacturing a cell-containing three-dimensional gel molded body according to claim 1, wherein the cells are chondrocytes, and the cell-containing three-dimensional gel molded body is used to treat damage to any one type of tissue selected from the group consisting of articular cartilage, auricular cartilage, tracheal cartilage, nasal cartilage, elbow cartilage, knee meniscus, costal cartilage, ankle cartilage, laryngeal cartilage, and vertebral cartilage.

8. a preparation step of preparing a forming mold set consisting of a pair of concave and convex molds, the concave mold having a curved concave surface and a convex mold having a curved convex surface that protrudes opposite to the concave surface, the concave mold and / or the convex mold being formed from a hygroscopic material containing a calcium salt aqueous solution; an alginic acid aqueous solution injection step of injecting an alginic acid aqueous solution containing cells between the concave surface of the concave mold and the convex surface of the convex mold; a gel molding step of bringing the concave mold and the convex mold close to each other, maintaining the alginic acid aqueous solution sandwiched between the concave surface and the convex surface, and completing gel molding by a chemical gelation reaction between the calcium salt aqueous solution and the alginic acid aqueous solution, thereby molding a cell-containing three-dimensional gel molded object having a curved shape corresponding to the space between the concave surfaces; A method for producing a cell-containing three-dimensional gel molded article, comprising:

9. 9. The method for producing a cell-containing three-dimensional gel molding according to claim 8, wherein the alginic acid aqueous solution contains an aqueous solution of at least one alginate salt selected from the group consisting of sodium alginate, potassium alginate, and ammonium alginate.

10. 9. The method for producing a cell-containing three-dimensional gel molding according to claim 8, wherein the calcium salt aqueous solution is an aqueous solution containing at least one calcium salt selected from the group consisting of calcium chloride, calcium lactate, calcium gluconate, calcium fumarate, calcium citrate, calcium succinate, calcium acetate, and calcium hydroxide.

11. 9. The method for producing a cell-containing three-dimensional gel molding according to claim 8, wherein the hygroscopic material comprises at least one selected from the group consisting of gelatin, agar, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, methyl cellulose, pullulan, carboxymethyl cellulose, dextrin, pectin, sodium polyacrylate, and polyvinylpyrrolidone.

12. 9. The method for producing a cell-containing three-dimensional gel molded object according to claim 8, wherein the hygroscopic material is 16 w / v % to 20 w / v % gelatin.

13. 9. The method for producing a cell-containing three-dimensional gel molding according to claim 8, wherein the cells are any one selected from the group consisting of chondrocytes, myoblasts, mesenchymal stem cells, cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, epithelial cells, endothelial cells, hepatic cells, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, and skin cells.

14. 9. The method for producing a cell-containing three-dimensional gel molded body according to claim 8, wherein the cells are chondrocytes, and the cell-containing three-dimensional gel molded body is used to treat damage to any one tissue selected from the group consisting of articular cartilage, auricular cartilage, tracheal cartilage, nasal cartilage, elbow cartilage, knee meniscus, costal cartilage, ankle cartilage, laryngeal cartilage, and vertebral cartilage.

Images