Methods for manufacturing foods and cell complexes containing designed three-dimensional structures, apparatus therefor, and 3D foods and 3D cell complex structures manufactured by such methods

Abstract

This invention provides a method for efficiently manufacturing three-dimensional structures using food ink (or cell aggregate ink) containing impurities such as granular or fibrous fragments, and also provides food three-dimensional structures or cell aggregate structures manufactured by these methods. [Solution] Multiple frames prepared for molding can be fixed to each other by inserting fixing rods or the like into common positioning holes provided in the frames. Food ink is dispensed into each filling area within the frames, spread and filled by a spreading means, and the surface is smoothed. Then the upper frames are stacked sequentially. These operations are repeated to form a composite. After the composite is fixed, the frames can be removed to obtain a three-dimensional structure (M). M can be used as is, decorated with a food 3D printer, or heated in a microwave or steamed to make it suitable for eating.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing foods having artificially designed structures and the appliances necessary for their manufacture. It also relates to 3D foods (3D foods) and 3D cell mass structures manufactured thereby.

Background Art

[0002] A food 3D printer is known as a device that applies 3D printer technology of the type that heat-melts a plastic resin and stacks linear inks to stack food materials and manufacture foods with complex shapes and designs. This printer has attracted recent attention as a technology that can create visually unique dishes, promote manufacturing efficiency, and also affect sustainable food supply and nutrition management. The basic process of manufacturing by current layer-type food 3D printers proceeds in the following steps. 1) Design as a digitalized model of the food shape on a computer (implemented using 3D modeling software that handles CAD software or STL files, etc.). 2) As food ink, separately process and prepare food materials into paste, liquid, or powder (currently, chocolate, wheat or rice flour dough, fluidized cheese, fish powder or vegetable powder, pureed fruits, etc. are used as food ink). 3) Set the food ink in the food 3D printer. 4) When the printer starts operating, the continuously discharged food ink is printed according to the designed shape and sequentially stacked from the lower layer to the upper layer to construct a 3D food. 5) The manufactured 3D food may be eaten as it is in some cases, but in other cases, it may be cooked by heating, etc., and further finished and processed to improve the appearance. Food 3D printing technology continues to evolve, and some models are already commercially available, leading to an increase in the provision of highly designed dishes (chocolate, desserts, pasta, etc.) in certain restaurants and cafes. Small-scale food 3D printers are also being used at home for personal use, allowing for the creation of unique foods for home cooking and parties (Non-Patent Documents 1 and 2). Furthermore, by adding vitamins and proteins that are often lacking in the human diet to food ink, it is possible to create foods that promote improved nutritional status and the maintenance of nutritional balance (Patent Document 1), and there is also potential for use in the production of food provided in nursing care and medical settings. Food produced using food 3D printers is also being explored as a potential future space food or as disaster relief food for use in special restricted environments such as during disasters. However, there are drawbacks such as the relative difficulty in mass-producing 3D food, limitations on the properties of usable food inks, and high manufacturing costs, so further development and improvement are ongoing (Patent Document 2). Most of the food 3D printers mentioned above use food ink, which is made by processing food ingredients into a fluid, and encapsulate it in a cylinder. The food ink is then extruded. In most cases, the printer's computer controls the extrusion of thin, thread-like food ink from the end of the cylinder, creating layers of material.

[0003] The 3D cell aggregate complex targeted by this invention is an object being studied in the field of cell printing. Cell printing, also known as bioprinting, is one example of the application of 3D printer technology, and involves creating living tissues and organs by layering living cells, cell aggregates, and biomaterials. This technology can be used in regenerative medicine and is attracting attention as an alternative to organ transplantation. One method of cell or cell aggregate printing involves designing the shape of the target tissue or organ using 3D models created from CAD data, CT scans, or MRI data, or proprietary 3D application software. Then, a bio-ink, which is a mixture of living cells and biomaterials such as hydrogel, is set in a bioprinter, and a 3D composite is formed by layering according to the design conditions to obtain a tissue or organ model. Currently, there have been successful cases with relatively simple tissues such as skin, cartilage, and bone, and practical applications are progressing through preclinical trials and animal experiments. Investigations are also underway for organs with complex vascular networks, such as the liver, heart, and kidneys, and the development of biomaterials for use is continuing. However, a bio-ink with sufficient performance has not yet been completed, and development is still in progress. Furthermore, even in successful cases, the process often remains at the stage where small tissue samples can be obtained, and human organ-sized samples have not yet been produced, highlighting the need for scaling up. Currently, bio-inks are mostly used in animal experiments and preclinical trials (including toxicity assessments), and there are not many cases that have progressed to full-scale human clinical trials. Advances in cell or cell aggregate (bio)printing technology are expected to enable transplant surgery using a patient's own cells to replace tissue in pathological areas, overcoming difficulties such as rejection reactions. Furthermore, the created tissues can be applied to new drug development and toxicity testing, potentially replacing animal testing and reducing the number of animals used in experiments. Furthermore, cell or cell aggregate printing is often on a smaller scale than the objects targeted by food 3D printers, and in this invention as well, it is necessary to adapt the frame and all elements to these scale sizes. Currently, the technology for printing these biological tissues and organs is closely related to ethical issues. In particular, regulations are needed when creating human organs. Furthermore, there are few high-precision bioprinters, and the cost of bio-inks is high, so many challenges still need to be overcome before widespread adoption is possible. Like food 3D printers, bioprinters are still relatively few in number, but some are commercially available. Examples include devices from CELLINK, Organovo, 3D Discovery Evolution, RegenHU, the former Allevi (acquired by 3D Systems), EnvisionTEC, and ROKIT Healthcare. However, the methods of forming structures vary (Non-Patent Literature 3). Many of these devices, like food 3D printers, use a bio-ink extrusion method. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] GB2580194B [Patent Document 2] Japanese Patent Publication No. 2024-45891 [Patent Document 3] Patent No. 7203737 [Non-patent literature]

[0005] [Non-Patent Document 1] NAUKI INZYNIERSKIE I TECHNOLOGI—E ENGINEERING SCIENCES AND TECHNOLOGIES 1(38), 2022, Joanna Harasym et al. (2022) [Non-Patent Document 2] Nutrients 2021, 13, 3617, Rodica—Anita Varvara et al. (2021) [Non-Patent Document 3] Nature Biotechnology VOL 32, No.8, 773-785 (2014), Sean V Murphy et al. (2014) [Overview of the project] [Problems that the invention aims to solve]

[0006] When conventional food 3D printers or cell 3D printers (bio 3D printers) are used, the food ink or cell ink (bio ink) is extruded in a thread-like form, which takes a long time when the drawing area is large, and has the problem of slowing down the overall layering speed of the three-dimensional structure (Problem 1). In addition, there is a problem that clogging and breakage are likely to occur during the stage of forming the encapsulated ink into a thread (Problem 2). Food inks need to be uniform; non-uniformity greatly increases the likelihood of clogging and breakage. While increasing the nozzle diameter of the food ink can somewhat prevent clogging in the presence of granular material, generally, the presence of granular material or atypical particles in the ink leads to frequent clogging and breakage, making 3D food production difficult. For example, there are very few examples of layering food inks containing microparticles such as cooked rice grains or caviar (egg). Furthermore, limitations on the types of food ingredients, textures, or fluid solutions containing cells suitable for the ink limit the range of inks that can be used, which is another challenge (Challenge 3). In cell inks containing cell aggregates, the likelihood of clogging increases if the cell aggregates or polymer material aggregates are larger than expected, similar to food inks. In this invention, a cell aggregate refers to a group consisting of two or more cells, preferably a group of 10 or more cells. In many cases, it takes a long time for the ejected ink to solidify or become fixed, and the resulting laminate is unstable and prone to collapse during the process (Problem 4). [Means for solving the problem]

[0007] The inventors of this application, after diligent research to solve the above-mentioned problems, have found that instead of extruding food ink (or cell aggregate ink) in a thread-like manner for molding, by extruding a fixed amount of consolidated ink into a predetermined filling area and spreading it to fill the entire area, it is possible to avoid problems such as ink clogging and breaking, as well as limitations on the ink (difficulty in containing properties, particles, or irregular shapes, etc.) (solving of problems 1 and 2 above). This method allows for a wider range of types and conditions of food ingredients that can be used as ink, and a wider range of conditions for applicable cell aggregate inks (solving of problem 3 above). Furthermore, by using the frame required during molding as is during the overall solidification, it is possible to prevent the collapse of the composite with the frame and stabilize the composite. Although it is necessary to select the frame material, after the solidification of the three-dimensional food composite, it becomes possible to cook the composite by steaming or heating it (solving problem 4). In other words, the present invention is A method for producing a food or cell aggregate complex containing a designed three-dimensional structure, comprising: 1) setting up a frame having a filling space of a specific shape and size at a specific position and a fixing space at a predetermined position; 2) pre-storing a fluid food ink or cell aggregate ink in a container; 3) dispensing the food ink or cell aggregate ink taken from the container into the filling space in the frame; 4) spreading and filling the dispensed food ink or cell aggregate ink in the filling space; 5) setting up a new separate frame on top of the frame filled with food ink or cell aggregate ink; 6) repeating steps 1 to 5 until the target structure is formed; 7) leaving the composite consisting of the frame and the target structure to stand until the target structure solidifies or is fixed; and 8) removing the used frame after the target structure has solidified or is fixed. This concerns... Furthermore, while steps 1) to 8) above can be performed manually, the entire process, including frame setup, dispensing the required food ink (or cell aggregate ink) into the frame, paste application within the frame, stacking upper frames, and frame removal, can be automated using robotic arms, automatic pipetting devices, etc. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a method for manufacturing 3D food (or cell aggregate 3D structures) in a form different from conventional food 3D printers (or bio 3D printers). Furthermore, it is possible to improve clogging and cutting of food ink (or cell aggregate ink) when making 3D food, and to further expand the acceptable range of materials that can be handled as ink. Even if the ink contains granular substances or fine irregularities, it can still be used as ink. In addition, since a three-dimensional composite is formed by stacking frames during the manufacturing process, the frames act as support materials for the composite, improving the physical and morphological stability during and after manufacturing. The lamination method of the present invention can be likened to a plastering method, in contrast to conventional printing methods. Plastering is an operation used in construction sites and artwork production where the target material to be applied is placed within a predetermined frame and then spread to fill the target area to its edges. The main part of the present invention can be called a 3D food plastering method. [Brief explanation of the drawing]

[0009] [Figure 1] This is one example diagram of a frame (F1) for manufacturing a three-dimensional structure (M). [Figure 2] This is one example diagram of the frame (F0) that forms the bottom surface during manufacturing of the M model. [Figure 3] The diagram and photograph show the state in which each of the five frames (F1, 2, 3, and 4, which have an ink filling area, and F0, which does not have an ink filling area) is fixed with an aluminum rod. [Figure 4] Examples 4a, 4b, and 4c show frames with a separate area set up within the frame for separation purposes. [Figure 5]This is an example where fixtures are not provided inside the frame, and each frame is surrounded and supported by fixing blocks (an example where each frame is fixed by fixing blocks X, X2, and X3). [Figure 6] It is a diagram showing Examples 6D and 6E in a state where ink is ejected into a frame having a filling area. [Figure 7] It is a diagram showing Examples 7G and 7H of an ink storage container for use in manufacturing M. [Figure 8] It is a diagram showing Examples 8I, 8J, and 8K showing the form of an ink ejection device for manufacturing M. [Figure 9] It is an explanatory diagram of an example of a process in which ink is spread in a filling area inside a frame in the manufacturing process of M. [Figure 10] It is an exterior photograph of the manufactured M as a whole (10 L, 10 M, 10 N, 10O). [Figure 11] It is an overall diagram of the manufacturing process of M. [Figure 12] It is a conceptual diagram of an automatic device for manufacturing M.

Embodiments for Carrying Out the Invention

[0010] In the present invention, the frame used has a thickness of 0.1 mm to 100 mm, and the material is preferably plastic (acrylic, polyethylene, polystyrene, ABS, polyethylene terephthalate, etc.), metal (stainless steel, iron, aluminum, copper, alloy), wood (including plywood), ceramic, or a plate-like combination thereof. Furthermore, the operability is improved by using a frame with a thickness of 0.5 mm to 10 mm. Considering the case of cooking a food containing a three-dimensional structure after molding, it is desirable that these frames have heat resistance, fire resistance, antibacterial properties, etc., but they should be selected according to the cooking method of the target food. Also, the affinity between these frame materials and the hood ink and hood raw materials is important, and it is more preferable to use materials that are easy to mold and separate. Furthermore, since the frame shapes are expected to be extremely diverse and come in multiple types, while mold-based molding methods can be used for frame production, flexible methods such as laser cutting, which can quickly and easily produce the desired frame, should also be considered. These frames must have some kind of fixing marker to ensure positional alignment with the upper frame. A fixing marker is a physical, definite mark that will not disappear during the manufacturing process of the three-dimensional structure. To prevent misalignment of each frame, various shaped holes, such as circles, triangles, and squares, can be provided as spaces. These holes can be circular, triangular, square, or star-shaped, and by inserting rods made of metals such as stainless steel or aluminum, or wood, bamboo, ceramics, etc., the position of each frame or the entire structure can be fixed. Alternatively, instead of using a combination of holes and rods, it is also possible to physically clamp and fix the frames together from the outside according to the fixing markers. If the fixing marker is a hole, the size of the hole is preferably about 0.1 mm to 10 mm in diameter on each side. It is necessary to pre-prepare a food ink filling area of ​​a predetermined shape and volume within the frame. This filling area is filled with food ink (or cell aggregate ink) and becomes one layer constituting the three-dimensional structure. By stacking all the layers of each frame and filling each with ink, the desired three-dimensional structure is formed. Furthermore, in order to prevent misalignment of multiple frames, uneven surfaces may be provided on the upper and lower frame surfaces in addition to the fixing marks mentioned above. The filler area designed for each frame is generally continuous with the filler area of ​​the adjacent frame in the axial direction of the stacking direction, but its size may be larger or smaller than the filler area of ​​the adjacent frame. However, depending on the novelty of the 3D hood's shape design, the filler area may be discontinuous with the filler area of ​​the adjacent frame, and a large step difference may occur. If there are significant differences in the size of the filling areas of adjacent frames, or if there are faults, the process of removing the frames after the 3D composite is completed may become complicated. To address such cases, it is preferable to have separation areas for separating the frames from the composite. These separation areas are for dividing the frame and are essentially cuts within the frame; it is preferable to have two or more of these. These shapes can be set in various ways, such as stepped, wavy, or acute-angled shapes. Furthermore, when manufacturing a composite using a frame and cell aggregate ink, the frame must be adjusted to an appropriate size according to the desired three-dimensional composite of cell aggregates. Furthermore, frames using cell aggregate ink should preferably be cleaned, sterilized, and disinfected, and when using cell aggregate ink, these procedures must be carried out more thoroughly.

[0011] For the food ink (or cell aggregate ink) used in this invention, a syringe capable of extruding ink or a container cup capable of holding the ink in an open-top state can be used as the container for storing the ink. The syringe may be made of metal or plastic, but it must be able to maintain the ink stored inside at an appropriate temperature and keep it in a uniform state. In addition, to ensure smooth ink discharge and prevent clogging, the inside of the syringe must have a smooth surface. Common plastic materials such as polyethylene, polystyrene, ABS resin, and acrylic can be used for the syringe and container components. As for metal materials, stainless steel, aluminum, iron, copper, and ceramics can be used, but materials with durability and rust resistance are desirable.

[0012] Methods for dispensing food ink (or cell aggregate ink) from a container include using a syringe capable of extruding (dispensing) the ink, using a scoop or spoon capable of scraping the ink from the container, or using a manually or automatically controlled pipette capable of aspirating and dispensing the ink. The amount of ink that is aspirated or collected and ejected must be an amount calculated in advance from the volume of the filling area to which the ink will be spread. To streamline subsequent processes, the locations where the ink is ejected are preferably near the center of a predetermined area within the frame, or at multiple locations within that area.

[0013] As means of spreading food ink (or cell aggregate ink) within the frame, spatulas, scrapers, cards, palette knives, dowels, rollers, brushes, etc., made of plastic or silicone rubber can be used. In the case of rollers and dowels, the material can be selected from plastic, rubber, metal, wood, etc. These means can be miniaturized according to the size of the target three-dimensional structure, and their shape, size, and properties can be appropriately changed. Using these, the work of filling the space of a predetermined area on the frame with food ink (pasting or painting) is carried out, and it is preferable that the surface of the food ink in the area after the work is in a smooth state.

[0014] The bottom frame on which the ink is layered is preferably a platform-like frame that can support and fix the entire set of frames. When fixing this platform frame to the bottom frame with the filling area, the provided markings (holes, etc.) can be used, just as with the other frames. In other words, the platform frame and the bottom frame can be fixed by inserting a round bar or the like into the respective holes (preferably three or more) in the positioning space. The operation of selecting and grasping the platform frame and each frame, moving the frames to the predetermined position, and stacking the frames can be done manually, but it can be made more efficient by using a robotic arm or the like.

[0015] The composite structure, consisting of a constructed three-dimensional structure and multiple frames, allows for frame removal once the three-dimensional structure has solidified. Removal is performed starting from the top frame, and can be done one frame at a time or in batches of two to three frames. In the case of three-dimensional food structures, frame removal can also be performed after the structure has been cooked.

[0016] The series of operations described above can be carried out by computer-controlled automated processes, but they can also be carried out by manual operation. However, in order to increase the speed of composite manufacturing and to prioritize efficiency and labor saving, it is preferable that all processes be automatically controlled by machines. In particular, when creating three-dimensional cell aggregate structures using cell aggregate ink, it is essential that the entire process be controlled under completely sterile conditions.

[0017] The main ingredients of food ink can be those currently used in food 3D printers, such as chocolate, cocoa, cocoa butter, grain (including wheat and rice) powder, corn syrup, plant-derived starch and oil components, egg components, animal meat, meat substitutes, vegetables, fruits, and frozen desserts (ice cream, gelato, etc.). Food ink should preferably have a moderate viscosity and harden quickly after extrusion. For this reason, in addition to the main ingredients, food additives such as excipients and thickeners can be added to facilitate molding and solidification. However, these food additives must be free from food safety issues, and it is preferable to use designated additives, existing additives, natural flavors, and general food additives that have a long history of consumption. In particular, substances used in molding include gelling agents (agar, gelatin, pectin, carrageenan, etc.), thickening polysaccharides (xanthan gum, guar gum, locust bean gum, etc.), binding agents (phosphates, modified starch, etc.), emulsifiers (soy lecithin, monoglycerides, diglycerides, etc.), coagulants (calcium chloride, glucono delta-lactone: GDL, etc.), and foaming agents (baking soda: sodium bicarbonate, ammonium carbonate, etc.). As food additives, preservatives that improve shelf life (such as benzoic acid, sodium benzoate, shirako protein extract (shirako protein, shirako hydrolysate, protamine, nucleoprotein), sorbic acid, potassium sorbate, propionic acid, calcium propionate, sodium propionate, polylysine, glycine, lysozyme, etc.) may be added. Furthermore, in the present invention, the food ink material may include granular particles such as rice grains, sesame seeds, quinoa, and millet, as well as fine fragments (irregularly shaped fine materials) such as chocolate and candy.

[0018] The cell aggregate ink in this invention may include animal or plant cells, microbial cells, or cell aggregates thereof. Furthermore, the retainer that makes the cell aggregate fluid may contain a substance selected from agarose, alginic acid, chitosan, collagen, decellularized extracellular matrix (ECM), fibrin or fibrinogen, gelatin, graphene, hyaluronic acid (HA), hydroxyapatite, polycaprolactone (PCL), polylactic acid (PLA), poly(lactidoco-glycolide) copolymer (PLGA), nonionic polymer polymers such as Pluronic F127 (Pluronic is a registered trademark) or F68, or mixtures thereof, in the ink. Cell aggregate ink can contain culture medium components suitable for the target cells (RPMI-1640 medium, DMEM medium, IMDM medium, EMEM medium, α-MEM medium, etc.), as well as growth factors and cytokines such as L-glutamine, animal serum (fetal bovine serum, etc.), β-mercaptoethanol, interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF), and erythropoietin (EPO), and antibiotics (penicillin, streptomycin, etc.).

[0019] Three-dimensional structures manufactured using food ink can be cooked using methods such as baking, stir-frying, deep-frying, boiling, simmering, steaming, heating, and microwave processing, but in some cases they can be eaten without cooking after manufacturing. Furthermore, the manufactured three-dimensional structures and the cooked products made from them can be processed using conventional food 3D printers during or after the manufacturing process. In other words, patterns, messages, names, illustrations, etc., can be added to the surface of the three-dimensional structure by drawing them with a conventional printer during or after the manufacturing process, thereby creating the final 3D food.

[0020] The aforementioned food inks may contain food additives such as lubricants. Examples of these lubricants include lecithin (soy lecithin), palm oil, canola oil, sunflower oil, vegetable oil mix, olive oil, avocado oil, flaxseed oil, vinegar, and grain vinegar. These substances can mix the materials in food ink, smooth the surface, and improve the flow of the ink even if the particles or irregular substances are viscous. These lubricants and the raw materials for the food inks mentioned above are preferably derived from naturally occurring substances, have a long history of being consumed by humans, and are even more preferably free from pesticides and artificial substances (such as organic chemicals and genetically modified organisms).

[0021] Similar to the food inks and cell aggregate inks described above, the manufacturing process of the present invention can also be carried out using plastics, waxes, or other materials as the fluid capable of forming three-dimensional structures. Preferably, these materials have a relatively low melting point (around 60°C to 100°C) and can solidify at or near room temperature.

[0022] The following describes, with reference to the drawings, three-dimensional structures (M) and 3D hood / 3D cell aggregate complexes containing them according to each embodiment of the present invention. Each embodiment is illustrative of the present invention and is not limited to any particular embodiment.

[0023] Figure 1 shows an overview of the frame (F1) in the present invention as an example. This frame has two circular holes (1A, 1B) with a diameter of 2 mm and one square hole (1C) with sides of 10 mm for positioning, and these are located in the exact same positions on other frames. The thickness of the frame can be arbitrarily selected between 0.1 mm and 100 mm, more preferably between 0.5 mm and 10 mm, but the frame shown in Figure 1 is made of white acrylic and has a thickness of 2 mm per sheet. The frame has a region (filling region: P) that can be filled with food ink or cell mass ink (I), and the volume and shape of this region are predetermined. These thicknesses can be arbitrarily changed, and the stacking order can also be arbitrarily changed. Figure 2 shows a frame (F0) without the above-mentioned P. It is made of black acrylic and is 2 millimeters thick. This will serve as the base supporting the structure M. F0 has circular and square holes of the same size and position as F1. The difference in color between F0 and F1 is not significant and is intended to make them easy to distinguish. Therefore, any color is acceptable, or they could all be the same color or transparent (clear). While frames can be fabricated by various means, this time we used a laser cutter capable of emitting a CO2 laser (TROTEC SPEEDY 300) and cut acrylic sheets into the required shape via dedicated software.

[0024] Figure 3 shows a composite structure of frame, aluminum round bar, and aluminum square bar, combining frame F1 from Figure 1, three frames of the same shape and thickness (F2, F3, F4), and frame F0 from Figure 2 without the filling area P, using two aluminum round bars (3D, 3E) with a diameter of 5 mm and a length of 10 cm, and one aluminum square bar (3F) with sides of 10 mm and a length of 10 cm. As shown in Figure 3, each frame is fixed relative to the others. Frame F0, which does not have P, becomes the part that supports the bottom. In this case, frames F1, F2, F3, and F4 in Figure 3 have exactly the same shape, but by smoothly changing the area of ​​P in these four frames, it is possible to construct different shapes with three-dimensional variations. When using cell aggregate ink, scaffolding materials such as collagen, vitronectin, and laminin can be pre-constructed on the surface of the F0 substrate, which serves as the base. When stacking frames, the stacking thickness can be flexibly changed not only one frame at a time, but also in increments of two or three frames, depending on the desired design of the 3D structure.

[0025] Figure 4 shows a frame equipped with separation areas. Examples include 4a (with a stepped separation area), 4b (with a wavy separation area), and 4c (with three or more separation areas).

[0026] Figure 5 shows an example of a method for securing the entire frame when there are no holes for fasteners (round bars / square bars) within the frame. It illustrates a method of using multiple blocks placed around the frame to hold the entire frame in place and prevent each frame from shifting. To clarify the orientation of the frame, it is also possible to shave off one corner of the frame and prepare a fixing block to match that shape.

[0027] Figure 6 shows the state after ink has been ejected (or scooped up and left to stand) in the space of the filling area of ​​frame 1. Multiple ejection points may be present. Below frame 1, frame 0, which does not have a filling area P, is first attached in close contact via the aforementioned aluminum round bars (3D, 3E) and aluminum square bars (3F). This attachment process involves sliding frame 1 downwards along the fixing aluminum bars and placing it on top of frame 0. It is preferable to calculate the required amount of I so that there is little excess after filling, but if there is some overflow from P, the excess can be removed with a spatula or scraper, or pressure can be applied to the filling area with a round bar or roller to fill it. After this, the required number of new frames are sequentially slid on top from above and attached in close contact with the lower frame, and ink is ejected into P in the next frame and smoothed. This series of operations can be repeated for all frames until the desired three-dimensional composite is formed.

[0028] Figure 7 illustrates an example of a container for storing food ink used in the manufacturing of three-dimensional composites (M). 7G is a syringe-type container, which generally has a larger dispensing capacity than conventionally known food 3D printer syringes. 7H is an example of a container for storing food ink (a triple-compartment container for three types of ink). Food ink can be collected from this container using a pipette and tip or a scoop / spoon and dispensed over the target area P. Food ink can be dispensed from the container by extrusion (dispensing) from a syringe, by scoop / spoon from the container, or by a pipette, and these can be performed manually or automatically controlled by a robotic arm. The amount of food ink to be dispensed or collected must be the amount calculated from the volume of the area to be spread, or slightly more. The dispensed food ink can be dispensed at one location in the center of the filling area P within the frame, or at multiple locations distributed within P. The purpose of these methods is to fill the food ink more efficiently, quickly, and to create a uniform surface. Furthermore, when storing cell aggregate ink in container 7H, it is preferable to have an auxiliary mechanism for continuous, slow stirring in order to maintain the uniformity of the cell aggregate ink.

[0029] Figure 8 shows examples of ink dispensing devices for manufacturing three-dimensional structures (M), namely 8I, 8J, and 8K. 8I is an example of a syringe-type dispensing device, where the ink stored inside is pushed from the top, dispensing the required amount of ink to the target filling area P. 8J is a dispensing device that uses a pipette to collect and dispense ink from an ink container, drawing ink from the storage container 5H via a fitted tip and dispensing a predetermined amount at P. 8K shows an example of a scoop-type device that extracts ink from a container 5H that stores ink inside.

[0030] Figure 9 is a schematic diagram illustrating an example of a process in the manufacturing of a three-dimensional structure (M) in which food ink (I) is spread within a filling area P on frame 1 on frame 0. The I, which is extruded onto P set within the frame, is spread within P using a silicone rubber spatula (R) or the like that can be moved on the frame, and can fill the entire area. In Figure 7, → → <c>Move the spatula from side to side (or repeat this process) in that order to smooth out I.

[0031] Figure 10 shows photographs of examples of fabricated three-dimensional structures (M) (overall appearance) (rice ball: 10L, Japanese confectionery: 10M, dim sum: 10N, pyramid-shaped rice ball: 10O). It is clear that layers have been formed in each of the foods in the photographs. While it is conceivable to construct a multi-layered food product without utilizing the frame group in this invention, one could also consider using a integrally molded container, placing food material to form the first layer, pressing it down, smoothing the surface, and then filling it with food material to form the second and third layers. However, with these methods, it is difficult to precisely control the thickness of each layer to create a final 3D food product. Assuming the size of a typical rice ball, only about two to three layers of the same size and shape can be formed, and it is extremely difficult to change the shape and size of these layers to create variations. Furthermore, extreme compression alters the texture.

[0032] Figure 11 is an overall flowchart summarizing the processes for manufacturing a three-dimensional structure (M) (frame installation process, food ink storage process, I sampling (suction) and discharge process, I filling space deployment process, new frame installation process, circulation process, and frame-I composite solidification process).

[0033] Figure 12 is a conceptual diagram of an automated system intended for the manufacture of three-dimensional structures (M). The automated system consists, for example, of a set area (1) for multiple types of frames, a set area (2) for multiple types of food ink storage containers, a construction area (3) for a three-dimensional structure (M) that can fix the entire frame, a device (4) for adjusting the temperature, humidity, etc. of the construction area (3), and robot arms 1 (ARM1) and 2 (ARM2). ARM1 moves between area (1) and area (3), and ARM2 moves between area (2) and area (3). In area (3), the arms are used to stack the frames and construct M, and after construction, ARM1 sequentially removes each frame. It is also preferable to have a device (4) that can adjust the temperature, humidity, etc. suitable for the composite structure. This example does not specifically limit the present invention, and various other versions can be constructed. [Examples]

[0034] The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0035] [Example 1] 《Introduction of a multi-layered structure to rice balls: Using low-amylose rice (Milky Queen: MQ) and medium-high amylose rice (Akita Komachi: AK) as food inks: Multi-layered rice balls》 150g each of MQ and AK were weighed, washed, and then cooked in a rice cooker with the amount of water used for cooking 1 cup of rice. After cooking, a small amount of olive oil was added to improve the slipperiness between the rice grains. Furthermore, appropriate amounts of various commercially available furikake (such as those made by Mishima Foods) were added to 30g of each to allow for color identification. Food ink consisting of two types of rice grains, colored and with added vinegar, was laminated using 12 frames (each 2 mm thick) of the present invention. A frame without a food ink filling area was used for the bottom surface. All frames were connected with 2 mm diameter aluminum rods and 10 mm square rods, and set so that each frame was in close contact during the manufacturing process. The surface of the filling area was smoothed evenly using a silicone spatula, and the next upper layer frame was set. Frames F0 to F12 were used (two frames per layer), and after filling the food ink up to the top layer, the frames were removed to obtain the desired multi-layered rice ball (6-layer structure). A photograph of this multi-layered rice ball is shown in Figure 10, labeled 10L. The layer configuration was set to alternate between MQ and AK layers. This example is merely one illustration, and by changing the thickness of the frame and the substances added to the rice grains, a variety of structures can be created. For example, by adding vitamins and various protein powders that are often lacking in cooked white rice, nutritional supplementation and adjustment become possible. As a comparative example, we can cite the example of Rem3dy Health, which manufactures gummies with various nutritional supplements added using conventional food 3D printer equipment (see Patent Document 1 mentioned above). The present invention makes manufacturing even simpler, more efficient, and less expensive. The present invention is particularly effective for manufacturing three-dimensional composites with relatively simple structures (such as geometric structures).

[0036] [Example 2] 《Introduction of multi-layered structures to Japanese confectionery: Using a mixture of low-amylose rice flour (Milky Queen: hereinafter abbreviated as MQ) and various commercially available bean pastes as food ink: Multi-layered Japanese confectionery》 120g of commercially available low-amylose rice flour was mixed with 140g of water and thoroughly combined. A portion of this mixture was then combined with the same weight of commercially available bean paste (smooth bean paste, chestnut bean paste, pumpkin bean paste, purple sweet potato bean paste, white bean paste, chunky bean paste, and edamame bean paste from Tomizawa Shoten Co., Ltd.) and cream cheese (Kraft Philadelphia cream cheese from Morinaga Milk Industry Co., Ltd.), and each was used as a food ink material to create a composite. The layers were arranged in the following order: smooth bean paste, chestnut bean paste, pumpkin bean paste, purple sweet potato bean paste, white bean paste, chunky bean paste, and edamame bean paste), a layer of rice flour only without bean paste, and a layer containing cream cheese (9 layers in total). Frames F0 to F18 were used (two 2mm thick frames were used per layer). Similar to Example 1, after layering food ink up to the top layer, the composite structure, integrated with the frame, was transferred to a steamer and cooked by steaming at around 100°C for 30 minutes or more. After cooking, the aluminum rod and frame were removed from the composite structure to obtain the multi-layered Japanese confectionery (9 layers) according to the present invention. A photograph of this is shown at 10M in Figure 10.

[0037] [Example 3] 《Introduction of multi-layered structures to dim sum: Using MQ (Mixed Quotient) and minced beef, minced chicken, minced pork, miso, and dumpling wrappers as food ingredients: multi-layered dim sum》 50g each of commercially available ground beef, chicken, and pork were mixed with 1g of soy sauce, 1g of salt, and 1g of MQ (agar powder), and each was thoroughly mixed to create a food ink (ground beef food ink: O, ground chicken food ink: P, ground pork food ink: Q, dumpling wrapper: R, miso: S). Layering was carried out in the same manner as in Example 1, with the layers in the order of Q→R→P→R→O→R→S→R→Q→R→P→R→O→R from the bottom up. A photograph of the layered product is shown in Figure 10, 10N.

[0038] [Example 4] (Making pyramid-shaped rice balls: Two types of food inks were used: one containing red pigment in MQ, and the other containing green pigment.) 100g of MQ was mixed thoroughly with 8g of furikake containing a red component (Mishima Foods Co., Ltd. "Akari") and 8g of furikake containing a green component (Mishima Foods Co., Ltd. "Hiroshi"). A frame with a square space in the center was prepared for lamination. The largest square was made with sides of 60 mm, and nine different plastic frames (3 mm thick) were also made, each decreasing by 6 mm from 60 mm (the smallest square was 12 mm on each side). Two frames with the largest square spaces were placed on a frame without a square space, their position was fixed, and green MQ was packed in to adjust the shape. Then, two more frames with squares reduced by 6 mm on each side were placed on top, and red MQ was packed in to smooth the surface. Layers of mixed green and red MQ were stacked alternately, and a frame with the smallest square (12 mm on each side) was placed on top, and the final mixed MQ was packed in. After filling with MQ, the frames were removed sequentially from the top layer to form a pyramid-shaped rice ball. A photograph of the stacked structure is shown at 10O in Figure 10. Although this example uses MQ, various other materials such as chocolate, bean paste, and ice cream can be used in the same way.

[0039] [Example 5] Cell culture, cell aggregate ink preparation, and three-dimensional cell aggregate structure preparation were carried out based on the following steps. • Cell collection and culture Knee joint cartilage was harvested from 8-week-old Balb / c mice (body weight 23g) under sterile conditions. After cutting the cartilage tissue into small pieces (approximately 1 mm in diameter or less) using sterile dissection instruments, the following enzymatic treatments were performed. First, the fragmented tissue was immersed in a 2 mg / mL collagenase D (Roche Diagnostics) solution and shaken at 37°C for 3 hours (120 rpm). Next, it was immersed in a 0.25% trypsin-EDTA solution and treated at 37°C for 15 minutes. After treatment, the cell suspension was centrifuged at 1500 rpm for 5 minutes, and the pellet was washed twice with phosphate-buffered saline (PBS). The isolated cells were seeded in a 6 cm culture dish and cultured under the following conditions. The culture was performed at 37°C, 5% CO2, and 95% humidity. The culture medium used was DMEM (Dulbecco's Modified Eagle Medium, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin mixture. After the cells reached confluence, they were harvested using 0.25% trypsin / EDTA and the cell count was measured. Next, 1 × 10⁻⁶ cells were collected. 5 The cells were adjusted to a concentration of cells / mL and reseeded, and cell proliferation was continued. This process was repeated at least twice, resulting in a final cell count of approximately 5 × 10⁶. 7 Individual cells were collected. • Preparation of cell aggregate ink The cultured cells described above were harvested, and the cell density was 2 × 10⁶. 7 The sample was adjusted to a concentration of cells / mL. A 0.5% alginic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the hydrogel base and mixed with the cell suspension in a 1:1 volume ratio. To prevent cell damage, mixing was performed at a low speed of 10 rpm for 30 minutes at 4°C while cooling. The following components were added to the resulting cell aggregate ink: glutamine solution (final concentration 2 mM), growth factor 10 ng / mL TGF-β1 (PeproTech), supplemental nutrients 10 mM HEPES buffer, and 2% B27 supplement (Gibco). The ink was stored at 4°C until immediately before use to maintain sterility. 3) Manufacturing of three-dimensional structures A cylindrical shape with dimensions of 3 mm in length, 3 mm in width, and 5 mm in height was designed as a three-dimensional structure. The filling area was divided into five layers, with each layer having a thickness of 0.2 mm. The frame was fabricated as follows: A frame shaped to correspond to the filling area was cut from a 0.2 mm thick acrylic sheet using a laser cutter (Speedy 300, Trotec). After processing, the frame was immersed in 70% ethanol for 30 minutes, and then sterilized by ultraviolet irradiation (UV-C, 254 nm) for 30 minutes. The cell aggregate ink was filled as follows: Collagen I (concentration 100 μg / mL) was applied to the bottom layer of the frame to form a substrate for cell colony attachment. Then, acrylic frames were stacked sequentially from the first to the fifth layer, and cell aggregate ink was pipettered into each layer. After filling each layer with cell aggregate ink, it was left to stand at 37°C for 30 minutes to confirm solidification. The constructed three-dimensional structures were transferred to a 6-well plate and cultured in 2 mL of culture medium. The culture medium was changed every 48 hours to ensure that the entire structure was fully submerged. The culture period was ultimately 7 days to check cell viability and cartilage matrix formation. [Explanation of Symbols]

[0040] M: Three-dimensional complex F0: Frame (bottom section) F1~F18: Frame P: Food ink filling area within the frame I: Food Inc. 1A and 1B: Holes (round) inside the frame 1C: Hole (corner) within the frame 3D, 3E: Aluminum round bar (5 mm diameter) 3F: Aluminum square bar (10 mm per side) 4a: Frame with stepped division area 4b: Frame with a wavy division area 4c: Frames with 3 or more divided areas X, X2, X3: Blocks for securing the frame. 7G: Syringe-type food ink container 7H: Container-type food ink storage container 8I: Syringe-type food ink dispensing device 8J: Pipette-type food ink dispensing device 8K: Scoop-type food ink dispenser S: Ink smoothing means (spatula) 10L: Example of a multi-layered rice ball (rice ball with multiple layers) 10M: Examples of multi-layered Japanese confectionery (Japanese sweets with multiple layers) 10N: Examples of multi-layered point centers (point centers with multiple layers) 10O: An example of a pyramid-shaped rice ball (a rice ball with multiple layers in a pyramid shape). ARM: Robotic arm< / c>

Claims

1. A method for producing a food or cell aggregate complex containing a designed three-dimensional structure, comprising the steps of: 1) installing a frame having a filling space of a specific shape and size at a specific position and a fixing space at a predetermined position; 2) pre-storing a fluid food ink or cell aggregate ink in a container; 3) dispensing the food ink or cell aggregate ink taken from the container into the filling space in the frame; 4) spreading and filling the dispensed food ink or cell aggregate ink in the filling space; 5) installing a new separate frame on top of the frame filled with food ink or cell aggregate ink; 6) repeating steps 1 to 5 until the target structure is formed; 7) leaving the composite consisting of the frame and the target structure to stand until the target structure solidifies or is fixed; and 8) removing the used frame after the target structure has solidified or been fixed.

2. A frame according to claim 1, wherein the thickness is 0.1 to 100 millimeters and the filling space has a pre-designed shape and volume, and a method for using the frame.

3. A frame according to claim 1 to 2, wherein the material is plastic, metal (stainless steel, iron, aluminum, copper, alloy), wood, bamboo, or ceramics, and a method of using the frame.

4. A frame according to claim 1, characterized in that a fixing portion is provided on a part of the frame, and a method of using the frame.

5. The frame according to claim 1, and a method of using such frames, characterized in that the fixing portion of the frame is a space or hole through which a specific jig can pass, or a groove provided between the frames.

6. A frame according to claim 1, having a separation area within the frame so that the frame can be separated, and a method of using such frames.

7. A group of frames according to claim 1, characterized in that each frame has a different size and shape, and a method for using such a group of frames.

8. The method according to claim 1, wherein the container for storing food ink or cell aggregate ink is a syringe capable of extruding food ink components, or a container cup with an open top, and the means for dispensing the food ink or cell aggregate ink from the container is extrusion with a syringe, scraping from the container with a spoon or scoop, or aspiration and dispensing with a manually or automatically controlled pipette.

9. The method according to claim 1, wherein the means for spreading food ink or cell mass ink in the filling space within the frame is a spatula, scraper, card, palette knife, round bar, roller, brush, or paintbrush made of plastic or silicone rubber.

10. The method according to claim 1, wherein when stacking another frame on top of a frame, a predetermined position is secured according to marks (signs or holes) that have been pre-installed in the frame.

11. The method according to claim 1, wherein rods and jigs made of metal, plastic, wood, bamboo, pottery, or ceramics, corresponding to marks in the frame, are used to determine and fix the position of the stacked frames.

12. A method for carrying out each step of claim 1 by manual, automatic, or computer control.

13. The method according to claim 1, wherein the main ingredients of the food ink are chocolate, cocoa, cocoa butter, grain (including wheat and rice) powder, starch syrup, plant-derived starch and oil components, gelatin, agar, agarose, egg components, animal meat, meat substitute, vegetables, fruits, and frozen desserts.

14. The method of claim 13, characterized in that the food ink material contains one or more of the following: fine granular material, fine fibrous material, and irregularly shaped fine material.

15. The method of claims 13 and 14, characterized in that the food ink contains an edible lubricant.

16. A food product comprising a three-dimensional structure manufactured by the method of claim 1.

17. Food prepared by one or more methods selected from baking, stir-frying, deep-frying, boiling, simmering, cooking, steaming, heating, and microwave processing, according to claim 16.

18. A method for producing food containing a three-dimensional structure, and a food product completed by combining the method of claim 1 and a method using a 3D food printer that extrudes food ink in a thread-like manner and forms it, either during or after the construction of the three-dimensional structure.

19. The method of claim 1, wherein the cell aggregate ink, consisting of plant and animal cells and microorganisms, comprises a substance selected from agarose, alginic acid, chitosan, collagen, decellularized extracellular matrix (ECM), fibrin or fibrinogen, gelatin, graphene, hyaluronic acid (HA), hydroxyapatite, polycaprolactone (PCL), polylactic acid (PLA), poly(lactidoco-glycolide) copolymer (PLGA), and nonionic polymer polymer (Pronic F127 or F68), or a mixture thereof.

20. A cell aggregate complex comprising a three-dimensional structure of cell aggregates produced by the method of claims 1, 19.

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