Tissue manufacturing methods

Abstract

The present invention provides a method for producing tissue that can gel a sol-like substrate (viscous mixed liquid) while maintaining its shape, and that can control the orientation of cells contained in the substrate. [Solution] The tissue manufacturing method comprises: step 1, discharging a viscous mixture containing animal cells, heparin, and collagen in a strip from a discharging device; step 2, incubating the viscous mixture discharged in step 1 with an aqueous solution containing metal ions at a concentration of 1.25 mM or higher; and step 3, after incubation, replacing the aqueous solution with a culture medium and culturing the animal cells. The metal ions are one or more metal ions selected from the group consisting of platinum ions, nickel ions, palladium ions, titanium ions, gold ions, copper ions, and zinc ions. The discharging device is equipped with a discharging port for discharging the viscous mixture, and in step 1, the viscous mixture is discharged from the discharging port while changing the horizontal position of the discharging port in a direction that orients the animal cells.

Description

[Technical Field]

[0001] This invention relates to a method for producing tissue. [Background technology]

[0002] Traditionally, animal tissues, formed by organizing animal cells, have been used in various fields, such as drug assay systems and cultured meat. In forming animal tissues, it is common practice to include an extracellular matrix (ECM) that can transfer from sol to gel in a substrate (a viscous mixture) to ensure cell adhesion to the substrate and maintain cell arrangement. By forming the substrate into a desired shape in the sol state and then gelling it, that shape can be maintained.

[0003] For example, Patent Document 1 discloses a technique in which fibrinogen and thrombin are incorporated into a substrate along with cells, and the substrate is then coated onto a surface. In this technique, the enzymatic activity of thrombin converts fibrinogen into fibrin, causing gelation, which in turn allows the substrate to maintain its shape.

[0004] Furthermore, Patent Document 2 discloses a technique in which collagen is incorporated into a substrate and heated to approximately 37°C to gel the substrate. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Republished Gazette WO2019-088224 [Patent Document 2] Japanese Patent Publication No. 2023-007495 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] When forming tissues, there is a need to control the orientation of the cells contained within the tissue. However, in conventional tissue formation techniques described in Patent Documents 1 and 2, cells tend to orient in random directions, making it difficult to control their orientation to a specific direction.

[0007] The present invention has been made in view of these circumstances, and aims to provide a method for producing tissue that can gel a sol-like substrate (mixture) while maintaining its shape, and that can control the orientation of cells contained in the substrate. [Means for solving the problem]

[0008] To solve the above problems, one aspect of the present invention includes the following aspects. [1] A method for producing tissue, comprising: step 1, discharging a viscous mixture containing animal cells, heparin, and collagen in a strip from a discharging device; step 2, incubating the viscous mixture discharged in step 1 in contact with an aqueous solution containing metal ions at a concentration of 1.25 mmol / L or more; and step 3, after incubation, replacing the aqueous solution with a culture medium and culturing the animal cells, wherein the metal ions are one or more metal ions selected from the group consisting of platinum ions, nickel ions, palladium ions, titanium ions, gold ions, copper ions, and zinc ions; the discharging device is equipped with a discharging port for discharging the viscous mixture; and in step 1, the viscous mixture is discharged from the discharging port while changing the horizontal position of the discharging port in a direction that orients the animal cells.

[0009] [2] The method for producing the structure according to [1], wherein the metal ion is one or more metal ions selected from the group consisting of platinum ions, nickel ions, and gold ions.

[0010] [3] The method for producing the tissue according to [1] or [2], wherein the metal ion is a platinum ion.

[0011] [4] The method for producing tissue according to [3], wherein the concentration of the metal ions in the aqueous solution is 6 mmol / L or more.

[0012] [5] The method for producing the tissue according to [1], wherein the concentration of collagen in the residue obtained by removing the animal cells from the viscous mixture is 5 mg / mL or more.

[0013] [6] The method for producing the tissue according to any one of [1] to [5], comprising a step of centrifuging a liquid mixture of heparin and collagen prior to step 1, removing the supernatant, and mixing the precipitate with the animal cells to obtain the viscous mixture.

[0014] [7] The method for producing the tissue according to any one of [1] to [6], wherein the ratio of the concentration of heparin to the concentration of collagen in the viscous mixture is 3:1 to 1:4.

[0015] [8] The method for producing the tissue according to any one of [1] to [7], wherein the ratio of the concentration of heparin to the concentration of collagen in the mixture is 1:1 to 1:4.

[0016] [9] The number of cells contained in a lump of the viscous mixture discharged in step 1 is 4.0×10 4 or more. The method for producing the tissue according to any one of [1] to [8].

[0017]

[10] The method for producing the tissue according to any one of [1] to [9], wherein the animal cells contain at least one kind of cells selected from myoblasts, muscle cells, and satellite cells.

[0018]

[11] In step 2, incubate for 25 minutes or more. The method for producing the tissue according to any one of [1] to

[10] .

Advantages of the Invention

[0019] According to the present invention, after dispensing a substrate (viscous mixture) containing animal cells while changing the horizontal position of the dispensing port, the substrate is brought into contact with an aqueous solution containing one or more metal ions selected from the group consisting of platinum ions, nickel ions, palladium ions, titanium ions, gold ions, copper ions, and zinc ions, and incubated. This allows the substrate to gel while maintaining its shape, and the orientation of the animal cells contained in the substrate can be controlled in the direction of the change in the position of the dispensing port during dispensing. [Brief explanation of the drawing]

[0020] [Figure 1] This is a schematic side view of a bio-3D printer as an example of an extrusion device. [Figure 2] These are microscopic images showing the state of the mixture before the addition of the aqueous solution containing platinum ions, and the state of the mixture after incubation with the addition of platinum ions and subsequent stimulation. [Figure 3] This is a microscopic image taken four days after initiating the culture of satellite cells by changing the culture medium from an aqueous solution containing Pt2+. [Figure 4] This is a fluorescence microscope image showing α-actinin and nuclei of satellite cells 10 days after initiating culture by changing from an aqueous solution containing Pt2+ to culture medium. [Figure 5] This is a fluorescence microscope image showing α-actinin in satellite cells 10 days after starting culture by changing from an aqueous solution containing Pt2+ to culture medium. [Figure 6] These graphs show the angular distribution of cell fibers, as shown in Figures 4 and 5, with respect to the direction in which the pipette nozzle was moved while dispensing 2 μL of the viscous mixture. [Figure 7] These graphs show the angular distribution of cell fibers with respect to the direction in which the pipette nozzle was moved when 4 μL of a viscous mixture was dispensed. [Figure 8] This graph shows the viscosity of a viscous material at a shear rate of 1 / second, for different concentrations of heparin and collagen. [Figure 9]This graph shows the viscosity as a function of shear rate for different concentrations of heparin and collagen in a viscous material. [Figure 10] This graph shows the viscosity at a shear rate of 1 / second for each volume ratio of heparin and collagen in a viscous material. [Figure 11] This graph shows the viscosity as a function of shear rate for different volume ratios of heparin and collagen in a viscous material. [Modes for carrying out the invention]

[0021] The embodiments of the present invention will be described in detail below, with reference to the drawings as appropriate.

[0022] [Overview of the manufacturing method] A preferred embodiment of the present invention provides a method for producing tissue (animal tissue) comprising: step 1, discharging a viscous mixture containing animal cells, heparin, and collagen in a strip (viewed from above) from a discharging device; step 2, incubating the viscous mixture (viscous mass) discharged in step 1 in contact with an aqueous solution containing metal ions at a concentration of 1.25 mM (mol / L) or higher; and step 3, after incubation, replacing the aqueous solution with a culture medium and culturing the animal cells. The discharging device is equipped with a discharging port for discharging the viscous mixture, and in step 1, the viscous mixture is discharged from the discharging port while changing the horizontal position of the discharging port in a direction that orients the animal cells.

[0023] The use of the viscous mixture (viscous mixture) extruded by the manufacturing method of this embodiment is not particularly limited, but it may be, for example, a bio-ink extruded by a 3D bioprinter.

[0024] The metal ions contained in the aqueous solution that comes into contact with the viscous mixture in step 2 are platinum ions (for example, Pt 2+ Pt 4+ ), nickel ions (for example, Ni 2+ ), palladium ions (e.g., Pd 2+ , Pd 4+ ), titanium ions (for example, Ti+ , Ti 2+ , Ti 3+ , Ti 4+ ), a metal ion selected from the group consisting of a metal ion (e.g., Au + , Au 2+ , Au 3+ , Au 4+ ), a copper ion (e.g., Cu + , Cu 2+ ), and a zinc ion (e.g., Zn 2+ ). Palladium ions and platinum ions may be divalent ions or tetravalent ions.

[0025] The tissue (animal tissue) obtained by the production method of the present embodiment may be, for example, a three-dimensional tissue. In the present specification, the "three-dimensional tissue" means a three-dimensional cell aggregate containing at least one type of animal cell and having a plurality of layers in the vertical direction. The number of cell layers in the three-dimensional tissue may be 2 to about 100 layers, or 10 to about 100 layers.

[0026] When observing the cell layer in a sectional image of a cross-section in the thickness direction of the three-dimensional tissue, when the magnification at which the cell nucleus can be recognized, that is, the magnification at which the entire thickness of the stained section enters the visual field, for example, when observed at a magnification of 100 to 200 times, another layer is defined when the cell nuclei do not overlap in the vertical direction (height direction). At that time, a visual field in a direction (lateral direction, horizontal direction) perpendicular to the vertical direction is ensured to be at least 200 μm or more.

[0027] When constructing a three-dimensional tissue by the production method of the present embodiment, the thickness of the constructed tissue is not particularly limited, but may be, for example, about 5 to about 300, about 400, or about 500 μm, may be 500 to 1000 μm, may be 1 to 50 mm, may be 150 μm or more, may be 200 μm or more, or may be 250 μm or more.

[0028] The tissue constructed by the manufacturing method of this embodiment may include muscle tissue, or it may include other types of tissue other than muscle tissue, such as vascular structures. "Vascular structures" refers to network-like structures such as vascular networks and lymphatic networks in living tissue.

[0029] Other types of tissues besides muscle tissue include, but are not limited to, living tissues such as fat, skin, hair, bone, cartilage, teeth, cornea, blood vessels, lymphatic vessels, heart, liver, pancreas, nerves, and esophagus, as well as solid cancer models (e.g., gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, liver cancer, etc.).

[0030] As will be described in detail later in the examples, by contacting the aqueous solution containing the above metal ions with a sol-like viscous mixture containing collagen, the viscous mixture can be rapidly and strongly gelled by metal complexation (crosslinking).

[0031] Therefore, when dispensing the viscous mixture containing collagen and cells in a sol-like state, the cell orientation can be highly controlled by dispensing the mixture while moving the nozzle of the dispensing device at least horizontally, and then gelling it immediately afterward. Step 1 will be explained in detail below.

[0032] [Process 1] In step 1, a viscous mixture containing animal cells, heparin, and collagen is dispensed from the nozzle of the dispensing device in any shape. During the dispensing of the viscous mixture, the horizontal position of the nozzle (the position in the X direction, which is any one direction extending horizontally, and / or the position in the Y direction, which is perpendicular to the X direction and extends horizontally) is changed in a direction that orients the cells. The viscous mixture after dispensing may be linear or curved when viewed from above, for example.

[0033] The direction in which the nozzle is moved during dispensing does not necessarily have to be horizontal; it may be moved diagonally upward or downward so as to change its horizontal position as well as its upward or downward position. By changing at least the horizontal position of the nozzle during the dispensing of the viscous mixture, the cells in the viscous mixture can be highly (in other words, to a high degree) oriented in the changed direction.

[0034] For example, if the operator of the dispensing device wants to orient cells in the left-right direction while dispensing a viscous mixture, they can change the position of the dispensing device's nozzle from one side to the other (move the nozzle to the other side) during dispensing, thereby orienting the cells in the dispensed viscous mixture in the left-right direction.

[0035] The discharge rate of the viscous mixture is not particularly limited, but may be, for example, 0.1 mL / second or more, 0.5 mL / second or more, 1 mL / second or more, or 1.5 mL / second or more. The upper limit of the discharge rate of the viscous mixture may be, for example, 5 mL / second, 3 mL / second or 1 mL / second.

[0036] The more cells a single mass of viscous mixture discharged in step 1 (see Figure 1 for a single mass of viscous mixture discharged), the greater the tensile force acting on the cells, making them more likely to orient in one direction. In particular, the number of cells in a mass of discharged viscous mixture is 4.0 × 10⁶. 4 It is preferable that there be one or more.

[0037] Examples of locations where viscous mixtures are discharged include, but are not limited to, substrates, dishes, tubes, flasks, bottles, and plates.

[0038] The dispensing device for dispensing the viscous mixture in step 1 only needs to be able to dispense the viscous mixture, and its type is not particularly limited. For example, it may be a pipette, syringe, dropper, a so-called dispenser-type bio 3D printer, or an automatic dispensing machine. If a pipette is used as the dispensing device, it may be a manual pipette or an electric pipette.

[0039] The diameter of the discharge port is not particularly limited, but it may be, for example, 0.3 to 50 mm, 0.5 to 15 mm, or 1 to 10 mm.

[0040] The width of the discharged viscous mixture (the width in the horizontal direction perpendicular to the direction of movement of the discharge port and horizontal in the horizontal direction, for example, the width in the shorter direction in the case of a viscous mixture that extends in a long straight line) is not particularly limited, but may be, for example, 0.3 to 50 mm, 0.5 to 15 mm, or 1 to 10 mm.

[0041] The following provides a detailed description of an example of extruding a viscous mixture containing animal cells, heparin, and collagen using a bio-3D printer.

[0042] Figure 1 is a schematic side view of a bio-3D printer 1 as an example of an extrusion device. As shown in Figure 1, a bio3D printer 1 as an example of an extrusion device includes a mixture tank 1a for storing a viscous mixture 2 containing animal cells, heparin, and collagen; a discharge pump 1b for discharging the viscous mixture 2 stored in the mixture tank 1a; a discharge unit 1e for discharging the viscous mixture 2 discharged from the mixture tank 1a by the discharge pump 1b; a tube 1c connecting the mixture tank 1a, the discharge pump 1b, and the discharge unit 1e; a control device 1d for controlling the discharge pump 1b; and a motor 1f for moving the discharge unit 1e horizontally.

[0043] Examples of known moving mechanisms for the discharge section 1e, including the motor 1f, include a rack and pinion mechanism. The tube 1c is made of a flexible material.

[0044] The control device 1d is an information processing device composed of a well-known microcomputer including a CPU and its peripheral circuits. The discharge pump 1b (more specifically, the motor of the discharge pump 1b, which is not shown) and motor 1f are connected to the output side of the control device 1d.

[0045] The discharge section 1e is a so-called dispenser nozzle, and is equipped with a discharge port 1e1 at its lower end for discharging a viscous mixed liquid 2, and a solenoid valve 1e2 is located just above the discharge port 1e1.

[0046] When the control device 1d outputs to the discharge pump 1b, solenoid valve 1e2, and motor 1f, the viscous mixed liquid stored in the mixed liquid tank 1a is supplied to the discharge unit 1e through the tube 1c, and at the same time, the solenoid valve 1e2 opens, and the viscous mixed liquid 2 is discharged onto the substrate 3 below through the discharge port 1e1. In addition, the motor 1f drives the discharge unit 1e, and the discharge port 1e1 moves horizontally, for example, with the direction of the discharge port 1e1 relative to the discharge pump 1b being forward, and left and right being left and right in the forward and / or left and right directions.

[0047] As a result, the viscous mixture 2 is ejected in a strip (linear) shape, extending in the direction in which the ejection port 1e1 moves. The above describes in detail one example of ejecting a viscous mixture using a bio 3D printer, but the configuration of the ejection device is not limited to this example.

[0048] On the other hand, the types of animal cells contained in the viscous mixture discharged in step 1 are not particularly limited, but the animal cells may include, for example, at least one type of cell from myoblasts, muscle cells and satellite cells, and may also include one or more cells selected from the group consisting of fibroblasts, osteoblasts, vascular endothelial cells, lymphatic endothelial cells, adipose-derived stem cells, nerve cells, epithelial cells, cardiomyocytes, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells, dendritic cells and immune cells.

[0049] The cells contained in the viscous mixture can be any mammal, and the type of mammal is not particularly limited; for example, it could be a human, or a non-human mammal. Examples of non-human mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, hamsters, hedgehogs, dogs, cats, monkeys, horses, cows, pigs, and sheep.

[0050] Examples of collagen in the viscous mixture include fibrous collagen and / or non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. Examples of non-fibrous collagen include type IV collagen. The collagen component is preferably fibrous collagen, and more preferably type I collagen or type II collagen. Commercially available collagen may be used as the fibrous collagen. A specific example of commercially available collagen is type I collagen derived from pig skin manufactured by Nippon Ham. The collagen component may also be fragmented using a homogenizer or the like.

[0051] The mass ratio of heparin to collagen in the viscous mixture is not particularly limited, but may be, for example, 1:10 to 10:1, 1:8 to 8:1, 1:8 to 3:1, 2:1 to 1:8, 1:5 to 5:1, 1:4 to 4:1, 1:4 to 2:1, 1:1 to 1:4, 3:1 to 1:4, 1:2 to 2:1, 1.5:1 to 1:1.5, 1.2:1 to 1:1.2, 1.1:1 to 1:1.1, or 1:1. The upper and lower limits of each of these ranges can be combined as appropriate. Note that the mass ratio of heparin to collagen in the mixture is the same as the mass ratio of heparin to collagen in the mixture in step 0, which will be explained later.

[0052] The concentration of heparin in the remainder of the viscous mixture after removing the animal cells (for example, the mixture in which only the cells have not yet been added when preparing the viscous mixture) is not particularly limited, but may be, for example, 0.5 mg / mL or more, 1 mg / mL or more, 1.1 mg / mL or more, 1.3 mg / mL or more, 1.5 mg / mL or more, 2.5 mg / mL or more, 3 mg / mL or more, 5 mg / mL or more, or 6 mg / mL or more.

[0053] Furthermore, the concentration of heparin in the remainder after removing the animal cells from the viscous mixture may be 50 mg / mL or less, 30 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, or 8 mg / mL or less. These upper and lower limits for heparin concentration can be combined in any way.

[0054] The concentration of collagen in the remainder of the viscous mixture after removing the animal cells is not particularly limited, but may be, for example, 0.5 mg / mL or more, 1 mg / mL or more, 1.1 mg / mL or more, 1.3 mg / mL or more, 1.5 mg / mL or more, preferably 2.5 mg / mL or more, more preferably 3 mg / mL or more, even more preferably 5 mg / mL or more, and particularly preferably 6 mg / mL or more. As will be described in detail in the examples later, the higher the collagen concentration in the viscous mixture, the higher the viscosity of the viscous mixture, and the easier it is to maintain the shape of the viscous mixture after dispensing.

[0055] Furthermore, the concentration of collagen in the remainder of the viscous mixture after removing the animal cells may be 50 mg / mL or less, 30 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, or 8 mg / mL or less. These upper and lower limits for collagen concentration can be combined in any way.

[0056] (Process 0) In preparing a viscous mixture containing animal cells, heparin, and collagen, one may first centrifuge a liquid mixture containing components other than cells, such as heparin and collagen, discard the supernatant (aqueous fraction), and mix the precipitate with animal cells to obtain the viscous mixture, which is more viscous than the original mixture. In other words, the tissue manufacturing method of this embodiment may include step 0, prior to step 1, in which a liquid mixture of heparin and collagen is subjected to centrifugal separation, and the precipitate obtained by removing the supernatant is mixed with animal cells to obtain the above-mentioned viscous mixture.

[0057] By centrifuging a mixture containing heparin and collagen and reducing the amount of water, the viscosity of the mixture can be increased, thus shortening the time required for gelation in step 2 and increasing the strength of the gel. As a result, it becomes possible to maintain the shape of the viscous mixture immediately after it is dispensed in step 1.

[0058] The conditions for the centrifugation process are not particularly limited, as long as they do not adversely affect the gelation in step 2. For example, a microcentrifugation tube containing the heparin and collagen mixture may be centrifuged at room temperature at 400 × g for 1 minute. Alternatively, the mixture can be processed by removing water through filtration instead of centrifugation.

[0059] The above mixture is preferably a solution in which heparin and collagen are dissolved in water, a buffer solution, or an aqueous solution.

[0060] (Other ingredients) The above mixture may contain other components besides heparin and collagen, to the extent that they do not inhibit cell growth or gelation in step 2. Furthermore, the viscous mixture discharged by the dispensing device may contain other components besides animal cells, heparin, and collagen, to the extent that they do not inhibit cell growth or gelation in step 2.

[0061] Other components that may be contained in the above mixture and the above-mentioned viscous mixture that is discharged include, but are not limited to, cationic substances, polymer electrolytes other than heparin, extracellular matrix components other than collagen, fibrinogen and thrombin, nutrients for cells to be cultured, pH adjusters, solute components of buffers, solute components of aqueous solutions, etc.

[0062] In this embodiment, any positively charged substance can be used as the cationic substance, as long as it does not adversely affect cell growth and gelation in step 2. Examples of cationic substances include, but are not limited to, cationic buffers such as Tris-HCl buffer, Tris-maleate buffer, bis-Tris-Buffer, and HEPES, as well as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, and polyarginine. The cationic substance is preferably a cationic buffer. The cationic substance is more preferably a Tris-HCl buffer.

[0063] The concentration of the cationic substance in the remainder of the viscous mixture after removing the animal cells is not particularly limited, as long as it does not adversely affect cell growth and gelation in step 2, but for example the concentration may be 1 to 100 mM. For example the concentration of the cationic substance in the mixture may be 1 to 10 mM, 20 to 90 mM, 30 to 80 mM, 40 to 70 mM, or 45 to 60 mM.

[0064] When a cationic buffer is used as the cationic substance, the pH of the cationic buffer is not particularly limited, as long as it does not adversely affect cell growth and gelation in step 2. The pH of the cationic buffer is preferably 6.0 to 8.0, and may be 7.2 to 7.6.

[0065] The polymer electrolyte used in this embodiment is a polymer having dissociable functional groups in its polymer chain. Any polymer electrolyte can be used as long as it does not adversely affect cell growth and gelation in step 2. Examples of polymer electrolytes include, but are not limited to, heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, glycosaminoglycans such as hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, and polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, etc. These polymer electrolytes may be used individually or in combination. The polymer electrolyte may be heparin, dextran sulfate, chondroitin sulfate, or dermatan sulfate. In this embodiment, the polymer electrolyte may be dissolved in a suitable solvent. Examples of solvents include, but are not limited to, water and buffer solutions.

[0066] In this embodiment, any component constituting the extracellular matrix (ECM) can be used as the extracellular matrix component, as long as it does not adversely affect cell growth and gelation in step 2. Examples of extracellular matrix components include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, enteractin, fibrillin, and proteoglycans. These extracellular matrix components may be used individually or in combination. Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans. Modified and variant forms of the above-mentioned extracellular matrix components can also be used, as long as they do not adversely affect cell growth and gelation in step 2.

[0067] The amount of extracellular matrix components in the viscous mixture is not particularly limited, but based on the total amount of the remainder after removing the animal cells from the viscous mixture, it may be, for example, 0.001% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.15% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 10% by mass or more, 15% by mass or more, or 20% by mass or more.

[0068] The amount of extracellular matrix components in the remainder after removing the animal cells from the viscous mixture may be, for example, 50% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, or 0.5% by mass or less, based on the total amount of the remainder after removing the animal cells from the viscous mixture. These lower and upper limits for the amount of extracellular matrix components can be combined arbitrarily.

[0069] The amount of thrombin used may be 3.0 U / mL or less, for example, based on the total volume of the remainder after removing the animal cells from the viscous mixture.

[0070] The fibrinogel content may be, for example, 0.001% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.15% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 2.0% by mass or more, or 3.0% by mass or more, based on the total amount of the remainder after removing the animal cells from the viscous mixture. The upper limit of the amount of precursor used is not particularly limited as long as it can be prepared, but may be, for example, 10.0% by mass or less, 5.0% by mass or less, 3.0% by mass or less, or 1.0% by mass or less, based on the total amount of the gel composition.

[0071] The amount of the other components in the remainder after removing the animal cells from the viscous mixture may be, for example, 50% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, 0.5% by mass or less, or 0.1% by mass or less, based on the total amount of the remainder after removing the animal cells from the viscous mixture.

[0072] Furthermore, the amounts of the other components mentioned above may be 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, or 10% by mass or more, based on the total amount of the remainder after removing the animal cells from the viscous mixture. These lower and upper limits for the amounts of the other components can be combined in any way.

[0073] [Process 2] Aqueous solutions containing metal ions can be obtained by dissolving a metal-containing compound in water or an aqueous solution. In the case of platinum ions, for example, an aqueous solution containing platinum ions can be obtained by dissolving K2PtCl4 (potassium tetrachloroplatinate), Pt(NH3)4Cl2, or K2PtCl6. In the case of nickel ions, for example, an aqueous solution containing nickel ions can be obtained by dissolving NiCl2. In the case of palladium ions, for example, an aqueous solution containing palladium ions can be obtained by dissolving PdCl2. In the case of gold ions, for example, an aqueous solution containing gold ions can be obtained by dissolving HAuCl4. In the case of copper ions, for example, an aqueous solution containing copper ions can be obtained by dissolving CuCl2. In the case of zinc ions, for example, an aqueous solution containing zinc ions can be obtained by dissolving ZnCl2.

[0074] The metal ions contained in the aqueous solution are preferably one or more metal ions selected from the group consisting of platinum ions, nickel ions, and gold ions, and most preferably platinum ions.

[0075] The aqueous solution may be, for example, a buffer solution or physiological saline, but is preferably PBS (phosphate-buffered saline). That is, the aqueous solution may be PBS containing one or more of the above-mentioned metal ions.

[0076] The concentration of the metal ions in the aqueous solution is preferably 3 mM or higher, more preferably 5 mM or higher, even more preferably 6 mM or higher, and particularly preferably 12 mM or higher.

[0077] In step 2, the method of bringing the aqueous solution into contact with the viscous mixture is not particularly limited, but examples include directly adding the aqueous solution to the viscous mixture or supplying the aqueous solution around the viscous mixture and immersing the viscous mixture in the aqueous solution.

[0078] The amount of aqueous solution added to the viscous mixture discharged in step 1 is not particularly limited, but it is preferable that the amount is such that the viscous mixture is completely immersed in the aqueous solution.

[0079] In step 2, the incubation time while the aqueous solution is in contact with the viscous mixture is not particularly limited, but is preferably 10 minutes or more, more preferably 15 minutes or more, and may be 20 minutes or more, or 30 minutes or more. The incubation temperature is preferably 30°C to 39°C, more preferably 35°C to 38°C, and particularly preferably 37°C.

[0080] [Process 3] (Culture solution) The type of culture medium to be replaced from the aqueous solution is not particularly limited, and a person skilled in the art can select an appropriate type of culture medium depending on the type of cells being cultured.

[0081] The culture period is not particularly limited; for example, it may be 1 day to 1 month, 3 to 20 days, or 5 to 12 days.

[0082] The present invention is not limited to the embodiments described above, and it goes without saying that various modifications are possible within the scope of the invention as described in the claims, and these modifications are also included within the scope of the present invention. [Examples]

[0083] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0084] [Experimental Example 1] In this experiment, a sol-like viscous mixture containing heparin, collagen, and myoblasts was dispensed, and an aqueous solution containing platinum ions was added to confirm whether or not the viscous mixture gelled.

[0085] In detail, first, 4 mL of a mixture of 3 mg / mL heparin and 3 mg / mL collagen in a 1:1 volume ratio was centrifuged at 10,000 rpm for 1 minute, the supernatant was removed, and 1 mL of the remaining viscous liquid was used to add 1.0 × 10⁶ C2C12 cells (myoblasts). 7 The ingredients were added to prepare a sol-like viscous mixture. Specifically, the heparin mentioned above is a solution of heparan sulfate added to PBS at a concentration of 0.3% by mass. The collagen mentioned above is a solution of type I collagen added to acetic acid at a concentration of 0.3% by mass.

[0086] Next, 10 μL of viscous mixture was manually pipetted into each well of a 24-well plate in a straight line when viewed from above (Step 1). Subsequently, Pt of various concentrations was added. 2+ (Step 2) A PBS (aqueous solution) containing platinum ions was added and incubated at 37°C for 30 minutes.

[0087] Note: Pt 2+The aqueous solution containing the substance was prepared by dissolving K2PtCl4 (Potassium tetrachloro-platinate (II) Sigma-aldrich / 206075-1G) powder in PBS. During incubation, the final concentrations of heparin and collagen in the viscous mixture (i.e., the viscous liquid described above), excluding the cells, were 6 mg / mL, respectively.

[0088] After incubation, the aqueous solution was replaced with culture medium (the aqueous solution was discarded), and the mixture was cultured for 7 days (Step 3). After culturing, the viscous mixture was stimulated by lightly tapping the 24-well plate 10 times, and the mixture was imaged under a microscope.

[0089] Figure 2 shows microscopic images of the viscous mixture before the addition of the platinum ion-containing aqueous solution and the viscous mixture after incubation with the addition of platinum ions and subsequent stimulation. Figure 2 shows microscopic images of the aqueous solution at different concentrations of platinum ions added in step 2. The 0 mM "Just after Pt" represents a comparative example where an aqueous solution without platinum ions (PBS) was added. In the 0.1 mM system, an aqueous solution with a platinum ion concentration of 0.125 mM was added.

[0090] As shown in Figure 2, in the systems with platinum ion concentrations of 0 mM and 0.1 mM in the aqueous solution added in step 2, the sol-like viscous mixture did not gel sufficiently, and scattering due to tapping was observed.

[0091] In contrast, in systems where the concentration of platinum ions in the aqueous solution added in step 2 was 1.25 mM or higher, no scattering of the viscous mixture due to tapping was observed, indicating that the viscous mixture had sufficiently gelled.

[0092] These results suggest that adding an aqueous solution containing platinum ions at a concentration of 1.25 mM or higher to a viscous mixture containing collagen rapidly and sufficiently promotes gelation.

[0093] [Experimental Example 2] In this experiment, a viscous sol-like mixture containing heparin, collagen, and myoblasts was dispensed, and an aqueous solution containing platinum ions was added to induce gelation. The orientation of the cells during this gelation process was then observed.

[0094] In detail, first, 1 mL of a mixture of 3 mg / mL heparin and 3 mg / mL collagen in a 1:1 volume ratio was centrifuged at 10,000 rpm for 1 minute, the supernatant was removed, and 250 μL of the remaining viscous liquid was mixed with 1.0 × 10⁶ Wagyu-derived satellite cells (bSC(58)). 7 The ingredients were added to prepare a sol-like viscous mixture. Specifically, the heparin was a solution of heparan sulfate added to PBS at a concentration of 0.3% by mass, and the collagen was a solution of type II collagen added to acetic acid at a concentration of 0.3% by mass.

[0095] Next, 2 μL or 4 μL of the viscous mixture was manually pipetted into each well of a 24-well plate in a straight line, viewed from above (Step 1). The dispensing speed was approximately 10 mm / second. Subsequently, Pt was added at a concentration of 6.25 mM. 2+ Adding PBS (aqueous solution) containing [the substance], the mixture was incubated at 37°C for 30 minutes (Step 2).

[0096] Note: Pt 2+ The aqueous solution containing the substance was prepared by dissolving K2PtCl4 (Potassium tetrachloro-platinate (II) Sigma-Aldrich / 206075-1G) powder in PBS. During incubation, the final concentrations of heparin and collagen in the viscous mixture (i.e., the viscous liquid described above), excluding the cells, were 6 mg / mL, respectively.

[0097] Subsequently, the aqueous solution was replaced with DMEM medium containing 20% ​​serum and growth factors, and cultured for 2 days to promote growth. After that, it was replaced again with DMEM medium containing 2% serum and cultured for 8 days until day 10 to induce muscle differentiation and produce three-dimensional muscle fibers (Step 3).

[0098] Next, the obtained muscle fibers were stained with α-actin and DAPI to confirm the orientation of each cell in the tissue. For α-actinin staining, 2 mL of Sigma-Aldrich antibody (model number: A7811) was used as the primary antibody, and Thermo Fisher Scientific's Antibody, Alexa Fluor® Plus 647 (model number: A32728) was used as the secondary antibody. α-actinin is a protein that binds to actin, and staining α-actin allows us to determine the direction in which the cytoskeleton extends, i.e., the orientation of the cells.

[0099] Figure 3 shows Pt 2+ Figure 4 shows a microscopic image taken four days after starting the culture of satellite cells by changing the culture medium from an aqueous solution containing Pt. 2+ Figure 5 shows a fluorescence microscope image of satellite cells 10 days after starting culture by changing the culture medium from an aqueous solution containing Pt. 2+ This is a fluorescence microscope image showing α-actinin in satellite cells 10 days after starting culture by changing the culture medium from an aqueous solution containing [the substance] to a culture medium.

[0100] Figures 3 to 5 show microscopic images of systems in which 2 μL and 4 μL of the viscous mixture were dispensed. Figure 5 shows a microscopic image of the tissue in the same area and angle as in Figure 4.

[0101] As shown in Figure 5, it can be seen that in many cells, the cytoskeleton containing α-actinin (shown in light color) extends in a direction approximately parallel to the direction in which the viscous mixture was spread during dispensing (in other words, the direction in which the pipette nozzle was moved during dispensing). Furthermore, cells from the system that was dispensed in 4 μL volume showed better differentiation.

[0102] Figure 6 is a graph showing the angular distribution of cell fibers shown in Figures 4 and 5 with respect to the direction in which the pipette nozzle was moved while dispensing 2 μL of the viscous mixture. Figure 7 is a graph showing the angular distribution of cell fibers shown in Figures 4 and 5 with respect to the direction in which the pipette nozzle was moved when dispensing 4 μL of the viscous mixture. In Figures 6 and 7, in a top view, the angle at which α-actin extends, as shown in Figures 4 and 5, is shown as the angle of the cell fiber, with the direction in which the viscous mixture was extended during dispensing (in other words, the direction in which the pipette nozzle was moved during dispensing) being defined as 0 degrees.

[0103] As shown in Figures 6 and 7, in both the system where 2 μL of the viscous mixture was dispensed and the system where 4 μL of the viscous mixture was dispensed, a particularly large proportion of cells were found to be extending in the direction in which the viscous mixture was spread during dispensing, i.e., approximately parallel to the direction in which the pipette was moved during dispensing.

[0104] These results suggest that when dispensing a viscous mixture containing heparin, collagen, and cells, the cell orientation can be highly controlled and maintained by moving the nozzle in the direction in which the cells are to be oriented, and then complexing (crosslinking) the mixture with metal ions to form a gel.

[0105] Furthermore, it can be seen that the system with 4 μL of viscous mixture dispensed, as shown in Figure 7, has a higher proportion of cells that extend around 0 degrees, i.e., in a direction approximately parallel to the direction in which the viscous mixture was extended during dispensing, compared to the system with 2 μL of viscous mixture dispensed, as shown in Figure 6.

[0106] These results suggest that a larger absolute number of cells in the syrup of the extruded viscous mixture allows for a higher degree of orientation of the cells in a direction approximately parallel to the direction in which the viscous mixture was spread during extrusion.

[0107] [Experimental Example 3] In this experiment, viscous solutions with final concentrations of heparin and collagen of 0.1, 0.5, 1, 1.1, 1.3, or 1.5 mg / mL were prepared by mixing heparin and collagen in a 1:1 volume ratio. The heparin was prepared by dissolving and diluting it with PBS, and the collagen was prepared by dissolving and diluting it with 5 mM acetic acid. Three hours after the preparation of the viscous material, the viscosity of 100 μL of the material was measured. The measurement conditions were as follows: the shear viscosity was plotted when the shear rate d(gamma) / dt = 1 to 60 1 / s increased linearly, and for comparison, the value at 1 / sec was plotted as a bar graph.

[0108] Figure 8 is a graph showing the viscosity at a shear rate of 1 / second for each concentration of heparin and collagen in the viscous material. Figure 9 is a graph showing the viscosity as a function of shear rate for each concentration of heparin and collagen in the viscous material. In Figures 8 and 9, "Collagen" is a comparative example in which only collagen is used without mixing with heparin, and "Heparin" is a comparative example in which only heparin is used without mixing with collagen.

[0109] As shown in Figures 8 and 9, the viscosity of the viscous substance containing heparin and collagen depended on the respective concentrations of heparin and collagen (especially the collagen concentration), with the viscosity being highest at the maximum concentration of 1.5 mg / mL. These results suggest that the concentrations of heparin and collagen directly affect the viscosity of viscous materials.

[0110] [Experimental Example 4] In this experiment, viscous materials were prepared by mixing 3 mg / mL heparin and 3 mg / mL collagen in volume ratios of 4:1, 2:1, 1:1, 1:2, or 1:4. The mixed heparin was dissolved and diluted with PBS, and the mixed collagen was dissolved and diluted with 5 mM acetic acid. Table 1 below shows the amount of heparin and collagen (in μL) in the viscous materials of each system.

[0111] [Table 1]

[0112] Three hours after the preparation of the viscous substance, the viscosity of 100 μL of the substance was measured.

[0113] Figure 10 is a graph showing the viscosity at a shear rate of 1 / second for each volume ratio of heparin and collagen in a viscous material. Figure 11 is a graph showing the viscosity as a function of shear rate for each volume ratio of heparin and collagen in a viscous material.

[0114] As shown in Figures 10 and 11, the shear viscosity of the viscous material in systems with a high proportion of heparin, specifically in systems with a heparin:collagen ratio of 2:1 to 4:1, was lower than that of the system with a heparin:collagen ratio of 1:1. This result suggests that heparin has a low contribution to viscous material formation.

[0115] In contrast, in systems with a high proportion of collagen, specifically those with a heparin:collagen ratio of 1:2 to 1:4, the viscosity of the viscous substance increased with a higher proportion of collagen. This result suggests that the amount of collagen significantly contributes to the formation of the viscous substance containing heparin and collagen. [Industrial applicability]

[0116] According to the present invention, when dispensing a viscous mixture containing heparin, collagen, and animal cells, the dispensing nozzle is moved in the direction in which the cells are to be oriented, and then the viscous mixture is gelled by complexation (crosslinking) through the addition of metal ions, thereby enabling highly controlled cell orientation, and thus making it suitable for industrial use. [Explanation of Symbols]

[0117] 1…Bio 3D printer, 1a…Mixing liquid tank, 1b…Discharge pump, 1c…Tubing, 1d…Control device, 1e…Discharge unit, 1e1…Discharge port, 1e2…Solenoid valve, 1f…Motor, 2…Viscous mixed liquid, 3…Substrate

Claims

1. Step 1 involves dispensing a viscous mixture containing animal cells, heparin, and collagen in a strip-like manner from a dispensing device. Step 2 involves incubating the viscous mixture discharged in step 1 with an aqueous solution containing metal ions at a concentration of 1.25 mmol / L or more. The process includes step 3, in which, after incubation, the aqueous solution is replaced with a culture medium and the animal cells are cultured. The aforementioned metal ion is one or more metal ions selected from the group consisting of platinum ions, nickel ions, palladium ions, titanium ions, gold ions, copper ions, and zinc ions. The discharge device is equipped with a discharge port for discharging the viscous mixed liquid, A method for producing tissue, wherein in step 1, the position of the discharge port in the horizontal direction is changed in a direction that orients the animal cells, and the viscous mixture is discharged from the discharge port.

2. The method for producing the tissue according to claim 1, wherein the metal ion is one or more metal ions selected from the group consisting of platinum ions, nickel ions, and gold ions.

3. The method for producing the tissue according to claim 1, wherein the metal ion is a platinum ion.

4. The method for producing tissue according to claim 3, wherein the concentration of the metal ions in the aqueous solution is 6 mmol / L or more.

5. The method for producing tissue according to claim 1, wherein the concentration of collagen in the remainder after removing the animal cells from the viscous mixture is 5 mg / mL or more.

6. The method for producing tissue according to claim 1, further comprising the step of, prior to step 1, centrifuging a liquid mixture of heparin and collagen, removing the supernatant, and mixing the precipitate with the animal cells to obtain the viscous mixture.

7. The method for producing tissue according to claim 1, wherein the ratio of the concentration of heparin to the concentration of collagen in the viscous mixture is 3:1 to 1:

4.

8. The method for producing tissue according to claim 6, wherein the ratio of the concentration of heparin to the concentration of collagen in the viscous mixture is 1:1 to 1:

4.

9. The number of cells contained in one mass of the viscous mixture discharged in step 1 is 4.0 × 10 ４ A method for producing the tissue according to claim 1, wherein there are one or more of these tissues.

10. The method for producing tissue according to claim 1, wherein the animal cells include at least one type of cell selected from myoblasts, muscle cells, and satellite cells.

11. The method for producing tissue according to claim 1, wherein in step 2, incubation is performed for 25 minutes or more.

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