Biological tissue forming device and biological tissue forming method

Abstract

A biological tissue formation device capable of efficiently performing cell-to-cell interaction and liquid component exchange between cell layers of a formed biological tissue is provided. A biological tissue formation device (1) is provided for forming a biological tissue having multiple cell layers composed of adherent cells (30, 31), comprising: a culture membrane (10) having culture areas for adherent cells (30, 31) on both sides, wherein the cultured adherent cells (30, 31) are placed between the multiple cell layers, and multiple flow paths (110, 130) are divided by the culture membrane (10); the culture membrane (10) is composed of a soluble material (101).

Description

Technical Field

[0001] The present invention relates to a cell culture technology, and in particular to a device for forming biological tissue by culturing cells and a method for forming biological tissue. Background Art

[0002] Conventionally, in pharmaceutical development, if the human pharmacokinetics of a new drug candidate cannot be predicted before preclinical studies, and if toxicity is identified during clinical studies and development is halted, there is a problem that the research and development costs to date have been wasted.

[0003] Specifically, in preclinical studies, cell assays and animal experiments have been conducted. However, cell assays cannot reproduce in vivo blood flow, and the expression of cell-specific functions is insufficient, making it impossible to evaluate pharmacokinetics.

[0004] In addition, in animal experiments, due to species differences, the results of human pharmacokinetics may not be consistent with those of animal pharmacokinetics. Therefore, there is a problem of difficulty in predicting human pharmacokinetics.

[0005] In this context, in recent years, organ chips are expected to eliminate these problems and improve the prediction accuracy of human pharmacokinetics.

[0006] Organ chips are microfluidic devices that reproduce the tissue structure of organs. Lung chips that model the structure of the lung, chips that combine small intestine models and liver models to model the enterohepatic circulation structure, and chips that model the structure of the kidney, such as the glomerulus model and proximal convoluted tubule model, have been proposed.

[0007] Here, in conventional in vitro culture systems using culture dishes, etc., the culture environment is quasi-static, there is no reproduction of blood flow, and the supply of oxygen and nutrients and the removal of waste rely solely on diffusion, making it difficult to conduct experiments that consider cell-specific functions and interactions between organs.

[0008] In contrast, with organ chips, blood flow is reproduced by pumping fluid, and the supply of oxygen and nutrients can be changed by adjusting the flow rate, making it possible to conduct experiments involving cell-specific functions and inter-organ interactions in an appropriate manner.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application No. 2017-504320 Summary of the Invention

[0012] Technical problem to be solved by the invention

[0013] Such organ chips have a flow path divided into two by a membrane. Cells are cultured on both surfaces of the membrane, forming two cell layers sandwiched between them. A semipermeable membrane is used, and liquid components such as nutrients and salts are exchanged between the two cell layers through the pores of the semipermeable membrane.

[0014] However, when the pore size of the semipermeable membrane is too large for cells to pass through, there is a problem of hindering contact between cells between cell layers and reducing the interaction between cell layers. As the cell layer is formed through cell culture, there is a problem that the pores are easily clogged and the exchange efficiency of liquid components is reduced.

[0015] Therefore, the inventors of the present invention conducted intensive research and developed an organ chip (biological tissue formation device) that can efficiently perform intercellular interactions and exchange of liquid components between cell layers of formed biological tissue.

[0016] Specifically, a soluble material is first used as the membrane of the organ chip. After cell layers are formed on both surfaces of the membrane, the membrane is dissolved to prevent intercellular interactions between the two cell layers and a decrease in the efficiency of liquid component exchange.

[0017] However, a membrane composed of a readily soluble material functions as a scaffold for the cells when a cell layer is formed on both surfaces. However, after the readily soluble material is dissolved, the scaffold disappears, and the formed cell layer may be easily damaged.

[0018] Therefore, the inventors of the present invention further configured the membrane to be composed of a readily soluble material and a poorly soluble material. After dissolving the readily soluble material, the poorly soluble material was allowed to function as a support for the cell layer, thereby making it difficult to damage the cell layer even after dissolving the readily soluble material.

[0019] Patent Document 1 discloses a lung chip and states that a biocompatible polymer can be used as the membrane in the lung chip (Claim 53). It also states that a biocompatible polymer is any material that does not significantly degrade when implanted in or near target biological tissue, does not induce a significant immune response or adverse tissue reaction (e.g., toxic reaction or significant pain) over time, or does not induce thrombosis or coagulation when in contact with blood (Paragraph 0055), and that biocompatible and biodegradable materials can be used in the device to facilitate in vivo implantation of the device (Paragraph 0316).

[0020] However, there is no description or suggestion regarding the structure of the dissolvable membrane in this lung chip. Furthermore, there is no disclosure of a structure that prevents damage to the cell layer after the membrane dissolves.

[0021] The present invention is completed in view of the above situation, and its purpose is to provide a biological tissue formation device and a biological tissue formation method that can efficiently carry out intercellular interaction and exchange of liquid components between cell layers of the formed biological tissue, and can make the cell layers difficult to be damaged.

[0022] Technical means to solve problems

[0023] In order to achieve the above-mentioned objectives, the biological tissue formation device of the present invention is a biological tissue formation device for forming biological tissue having multiple cell layers composed of adherent cells, and is constructed to include: a culture membrane having culture areas for the adherent cells on both sides, the adherent cells being cultured and placed between the multiple cell layers, and multiple flow paths divided by the culture membrane; the culture membrane is composed of a soluble material.

[0024] In addition, another embodiment of the biological tissue formation device of the present invention is a biological tissue formation device for forming biological tissue having multiple cell layers composed of adherent cells, and is constructed to include: a culture membrane having culture areas for adherent cells on both sides, the adherent cells being cultured and placed between the multiple cell layers, and multiple flow paths divided by the culture membrane; the culture membrane is composed of a soluble material and a poorly soluble material.

[0025] It is also preferable that the biological tissue forming device of the present invention is configured so that pores penetrating the culture membrane are formed by dissolving the easily soluble material in the culture membrane.

[0026] Furthermore, the biological tissue forming device of the present invention is preferably configured such that the pore size of the porous membrane formed of the poorly soluble material is 10 μm or larger.

[0027] Furthermore, it is also preferable that the biological tissue formation device of the present invention is configured such that the easily soluble material and the poorly soluble material are used as a scaffold when culturing the adherent cells.

[0028] Furthermore, the biological tissue forming apparatus of the present invention is preferably configured such that the poorly soluble material is used as a support for the cell layer after the easily soluble material in the culture membrane is dissolved.

[0029] Furthermore, it is also preferable that the biological tissue forming device of the present invention is configured such that the poorly soluble material is composed of any one of polyethylene terephthalate, polylactic acid, and ultraviolet curable resin.

[0030] Furthermore, it is also preferable that the biological tissue forming apparatus of the present invention is configured such that the culture membrane is a porous membrane.

[0031] Furthermore, it is also preferable that the biological tissue forming apparatus of the present invention is configured to include one culture membrane and two flow paths divided by the culture membrane, thereby forming a biological tissue having two cell layers.

[0032] Furthermore, the biological tissue forming apparatus of the present invention is preferably configured such that the two flow paths are formed by adhering the flow path-side surfaces of two plates having the flow paths to the two surfaces of the culture membrane, respectively.

[0033] Furthermore, the biological tissue forming apparatus of the present invention is preferably configured so that cell layers composed of different types of adherent cells are formed in each of the two flow channels.

[0034] Furthermore, it is also preferable that the biological tissue forming device of the present invention is configured such that the easily soluble material is composed of a water-soluble polymer.

[0035] Furthermore, the biological tissue forming device of the present invention is preferably configured such that the water-soluble polymer is polyvinyl alcohol, alginic acid, or methylcellulose.

[0036] In addition, the method for forming a biological tissue of the present invention is a method for forming a biological tissue having multiple cell layers composed of adherent cells, comprising: a step of supplying adherent cells and a culture medium to the two flow paths in a biological tissue forming device, the biological tissue forming device comprising: a culture membrane having culture areas for adherent cells on both sides, the adherent cells being cultured and then placed between the multiple cell layers, two flow paths divided by the culture membrane, the culture membrane being composed of a soluble material; a step of culturing the adherent cells in the two flow paths to form cell layers on both sides of the culture membrane; and a step of dissolving the culture membrane.

[0037] In addition, another embodiment of the method for forming a biological tissue of the present invention is a method for forming a biological tissue having multiple cell layers composed of adherent cells, comprising: a step of supplying adherent cells and a culture medium to the two flow paths in a biological tissue forming device, the biological tissue forming device comprising: a culture membrane having culture areas for adherent cells on both sides, the adherent cells being cultured and then placed between the multiple cell layers, two flow paths divided by the culture membrane, the culture membrane being composed of a soluble material and a poorly soluble material; a step of culturing the adherent cells in the two flow paths to form cell layers on both sides of the culture membrane; and a step of dissolving the soluble material in the culture membrane.

[0038] Furthermore, the biological tissue forming method of the present invention is preferably configured such that the soluble material is alginic acid, and in the dissolution step, an alginate-degrading enzyme is supplied to at least one of the two flow channels to dissolve the soluble material in the culture membrane.

[0039] Furthermore, the biological tissue forming method of the present invention is preferably configured such that the readily soluble material is polyvinyl alcohol, and in the dissolving step, the biological tissue forming apparatus is heated to dissolve the readily soluble material in the culture membrane.

[0040] Furthermore, it is also preferable that the biological tissue forming method of the present invention is configured such that the readily soluble material is methylcellulose, and in the dissolving step, the biological tissue forming apparatus is cooled to dissolve the readily soluble material in the culture membrane.

[0041] Effects of the Invention

[0042] According to the present invention, a biological tissue forming apparatus and a biological tissue forming method can be provided, which can efficiently perform intercellular interaction and exchange of liquid components between cell layers of formed biological tissue and can prevent the cell layers from being damaged. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagrams showing components of a biological tissue forming apparatus according to various embodiments of the present invention.

[0044] Figure 2 Schematic diagrams showing the configuration of biological tissue forming devices according to various embodiments of the present invention.

[0045] Figure 3 Schematic diagram showing an AA cross section of a biological tissue forming device according to each embodiment of the present invention.

[0046] Figure 4 Schematic diagram showing the process of forming a cell layer using the biological tissue formation apparatus according to the first embodiment of the present invention.

[0047] Figure 5 Schematic diagram showing a process for producing a culture membrane in a biological tissue formation apparatus according to a second embodiment of the present invention.

[0048] Figure 6 Schematic diagram showing the process of forming a cell layer using the biological tissue formation apparatus according to the second embodiment of the present embodiment.

[0049] Figure 7 This is a schematic diagram showing the process of forming a cell layer using a conventional biological tissue formation device.

[0050] Figure 8 This is a diagram showing the results of an experiment conducted to confirm the dissolution of the easily soluble material used in the biological tissue forming apparatus according to the first embodiment of the present invention. DETAILED DESCRIPTION

[0051] Hereinafter, embodiments of the biological tissue forming apparatus and biological tissue forming method of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments.

[0052] [First embodiment]

[0053] First, a biological tissue forming apparatus according to a first embodiment of the present invention will be described.

[0054] The biological tissue formation device of this embodiment is a device for forming biological tissue having a plurality of cell layers composed of adherent cells, and can be configured as a so-called organ chip or the like.

[0055] Organ-on-a-chips use microfluidics to recreate the tissue structure of an organ within the device, allowing precise control of the cell culture environment. This has the advantage of enabling cells to express functions close to those found in the body.

[0056] In addition, the biological tissue formation device of this embodiment is characterized by comprising: a culture membrane having culture areas for adherent cells on both sides, cultured adherent cells placed between multiple cell layers, and multiple flow paths divided by the culture membrane; the culture membrane is made of a soluble material.

[0057] For example, Figures 1 to 3 As shown, the biological tissue formation device 1 of this embodiment can be manufactured by placing a culture membrane 10 having a surface for culturing adherent cells between a flow channel plate 11 and a flow channel plate 13, and adhering them together via adhesion layers 12 and 14. It should be noted that the biological tissue formation device 1 can be configured to form three or more cell layers by further including a culture membrane and an adhesion layer.

[0058] The flow path plate 11 is provided with a flow path 110, while the flow path plate 13 is provided with a flow path 130. The flow path-side surfaces of these two plates are adhered to the two surfaces of the culture membrane 10 via adhesive layers, thereby forming two flow paths in the biological tissue formation apparatus 1, which are divided by the culture membrane 10. These flow paths serve as culture chambers.

[0059] Then, cell layers are formed on both sides of the culture membrane 10 using these flow channels, and a biological tissue having two cell layers is formed in the biological tissue forming apparatus 1 .

[0060] The two cell layers may be composed of the same type of adhesion cells or different types of adhesion cells. In particular, it is advantageous if the two cell layers are composed of different types of adhesion cells.

[0061] In the biological tissue formation device 1 of this embodiment, the culture membrane 10 is composed of a readily soluble material 101. The culture membrane 10 can be either porous or non-porous, but preferably has pores that prevent cells from passing through until the readily soluble material 101 dissolves. The pores are preferably 0.4 to 8 microns in diameter, more preferably 3 to 6 microns.

[0062] As the easily soluble material 101 , a water-soluble polymer or the like can be used. For example, polyvinyl alcohol (PVA), alginic acid, methylcellulose, or the like can be used appropriately.

[0063] When alginic acid is used as the easily soluble material 101 , the culture membrane 10 in the biological tissue forming apparatus 1 can be dissolved by supplying an alginate degrading enzyme to the flow channel 110 or the flow channel 130 .

[0064] Furthermore, when polyvinyl alcohol is used as the readily soluble material 101, after the cell layer is formed, the readily soluble material 101 in the culture membrane 10 within the biological tissue formation apparatus 1 can be dissolved by heating the biological tissue formation apparatus 1 to, for example, 37 to 50°C. It should be noted that the dissolution temperature of polyvinyl alcohol is not limited to this range, and it can also be dissolved by heating to 37 to 80°C.

[0065] Furthermore, when methylcellulose is used as the readily soluble material 101, after the cell layer is formed, the readily soluble material 101 in the culture membrane 10 within the biological tissue formation apparatus 1 can be dissolved by cooling the biological tissue formation apparatus 1 to, for example, 5 to 10°C. It should be noted that the dissolution temperature of methylcellulose is not limited to this range, and it can also be dissolved by cooling to 4 to 25°C.

[0066] Here, methylcellulose is a material that gels at high temperatures and liquefies when cooled, and can be dissolved at low temperatures. Furthermore, it is preferable to add additives to adjust the dissolution temperature of methylcellulose. Adding additives to methylcellulose can increase its dissolution temperature. For example, sodium styrene sulfonate (NaSS) can be suitably used as such an additive.

[0067] As described above, according to the biological tissue forming apparatus 1 of this embodiment, by dissolving the culture membrane 10 after the cell layer is formed, it is possible to appropriately perform permeation of liquid components between the plurality of cell layers.

[0068] As cells cultured using the biological tissue formation apparatus 1 of the present embodiment, for example, pluripotent stem cells (iPS cells, etc.) and embryonic stem cells (ES cells) can be used.

[0069] In addition, in the biological tissue formation apparatus 1 of this embodiment, it is preferable to provide a cell scaffold material in order to allow cells to adhere appropriately to the surface of the culture membrane. This also applies to the second embodiment described below.

[0070] Specifically, it is preferred to attach a cell scaffold material to the surface of the culture membrane and then allow the cells to adhere and be cultured.

[0071] As a scaffold material for cells, for example, collagen, matrigel, fibronectin, laminin, or chitosan can be used as appropriate, or two or more of these materials may be used.

[0072] Methods for attaching cell scaffold materials to the surface of the culture membrane include, for example, a method of coating the surface of the culture membrane, a method of mixing a soluble material and a cell scaffold material, a method of chemically combining the cell scaffold material and the soluble material using reagents such as coupling agents, and a method of pre-processing collagen and chitosan into fibrous materials and attaching them to the surface of the culture membrane.

[0073] Furthermore, it is also preferred that the cells be allowed to adhere to a poorly soluble material used in the second embodiment described later, thereby allowing the cells to adhere to the surface of the culture membrane.

[0074] The culture membrane 10 in the biological tissue formation apparatus 1 of this embodiment can be produced by, for example, coating a substrate with a readily soluble material 101 , peeling the obtained culture membrane 10 from the substrate, cutting the resulting culture membrane 10 into a desired shape, and placing it in the biological tissue formation apparatus 1 for use.

[0075] It should be noted that the shape of the culture membrane 10 is of course not limited to Figure 1 The shape shown may be, for example, a shape arranged on the entire lower surface of the flow path plates 11 and 13 .

[0076] Furthermore, it is also preferable to subject the surface of the culture membrane 10 to surface treatment such as corona treatment, excimer treatment, or plasma treatment.

[0077] By performing such a surface treatment, the hydrophilicity of the surface of the culture membrane 10 can be increased, and the adhesion of the adherent cells to the surface of the culture membrane 10 can be improved.

[0078] As materials for the flow channel plates 11 and 13 in the biological tissue formation apparatus 1 of this embodiment, for example, cycloolefin polymer, polymethyl methacrylate, and polycarbonate can be used. The flow channel plates 11 and 13 can be manufactured by injection molding or the like.

[0079] In addition, as the material of the adhesive layer 12 and the adhesive layer 14 in the biological tissue forming device 1 of this embodiment, for example, an adhesive or the like can be used, and the adhesive layer 12 and the adhesive layer 14 can be formed by, for example, stamping. Figure 1 Made in the shape shown.

[0080] Furthermore, as the material of the culture membrane 10 in the biological tissue formation apparatus 1 of this embodiment, as described above, alginic acid, polyvinyl alcohol, or the like can be used, and the obtained culture membrane 10 can be cut into a desired shape for use.

[0081] Furthermore, by bonding the flow channel plates 11 and 13 and the culture membrane 10 together using the adhesive layers 12 and 14 , the biological tissue forming device 1 of this embodiment can be manufactured.

[0082] The biological tissue forming apparatus 1 of this embodiment may be in a state before a cell layer is formed on the culture membrane 10 , or may be in a state after a cell layer is formed on the culture membrane 10 .

[0083] Furthermore, the biological tissue formation apparatus 1 of this embodiment further includes a device for dissolving the easily soluble material 101 after forming the cell layer on the culture membrane 10 .

[0084] Furthermore, the biological tissue formation device 1 of this embodiment is preferably configured such that the culture membrane 10 contains adherent cells. In this case, the adherent cells may be immobilized on the surface of the culture membrane 10 or contained within the culture membrane 10. When the culture membrane 10 is made of, for example, alginic acid, the adherent cells can survive within the culture membrane 10.

[0085] If the biological tissue forming apparatus 1 of the present embodiment has such a configuration, supply of adherent cells to the flow channel can be omitted in the biological tissue forming method of the present embodiment described later.

[0086] The method for forming biological tissue of this embodiment is a method for forming biological tissue having multiple cell layers composed of adherent cells, and is characterized by comprising: a process of providing adherent cells and culture fluid to two flow paths in a biological tissue forming device, wherein the biological tissue forming device comprises: a culture membrane having culture areas for adherent cells on both sides, wherein the cultured adherent cells are placed between the multiple cell layers, and two flow paths are divided by the culture membrane, wherein the culture membrane is composed of a soluble material; a process of culturing the adherent cells in the two flow paths to form cell layers on both sides of the culture membrane; and a process of dissolving the culture membrane.

[0087] Here, as Figure 7 As shown, a conventional biological tissue formation device 20 generally uses a semi-permeable membrane such as polyester as a culture membrane 210, through which cell interaction and exchange of liquid components are performed between two flow paths.

[0088] That is, in a conventional biological tissue formation device 20, the flow path between the flow path plate 211 and the semipermeable membrane 210 is filled with culture fluid 40, and cells 30 are cultured on one surface of the semipermeable membrane 210, while the flow path between the flow path plate 213 and the semipermeable membrane 210 is filled with culture fluid 41, and cells 31 are cultured on the other surface of the semipermeable membrane 210, thereby forming two cell layers.

[0089] However, the size of the cells cultured in this embodiment is generally 8 to 10 μm. On the other hand, the pore size of the semipermeable membrane 210 in the conventional biological tissue formation device 20 is about 3 μm. Therefore, when using the conventional biological tissue formation device 20, there are problems of hindering the contact between cells between cell layers and reducing the interaction between cell layers. In addition, as the cells proliferate, there is a problem of the pores of the semipermeable membrane 210 being blocked, and the exchange efficiency of the liquid components becoming low.

[0090] In contrast, in the biological tissue forming apparatus 1 of this embodiment, as Figure 4 As shown, the flow path 110 between the flow path plate 11 and the culture membrane 10 is filled with culture fluid 40, and cells 30 are cultured on one surface of the culture membrane 10. At the same time, the flow path 130 between the flow path plate 13 and the culture membrane 10 is filled with culture fluid 41, and cells 31 are cultured on the other surface of the culture membrane 10, thereby forming two cell layers.

[0091] Furthermore, after forming two cell layers, dissolving the soluble material 101 in the culture membrane 10 allows cells to contact each other between the cell layers, eliminating the problem of reduced interaction between the cell layers. Furthermore, liquid components can be exchanged efficiently, eliminating the problem of reduced efficiency in liquid component exchange between the two cell layers.

[0092] In this case, when the easily soluble material 101 is alginic acid, in the step of dissolving the culture membrane 10 , an alginate degrading enzyme may be supplied to at least one of the flow channel 110 and the flow channel 130 to dissolve the culture membrane 10 .

[0093] Furthermore, when the easily soluble material 101 is polyvinyl alcohol, the culture membrane 10 can be dissolved by heating the biological tissue forming apparatus 1 in the step of dissolving the culture membrane 10 .

[0094] The step of dissolving the culture membrane 10 is preferably performed when a cell layer is formed over the entire culture area.

[0095] Furthermore, it is preferable to form cell layers composed of different types of adherent cells in the flow channel 110 and the flow channel 130 .

[0096] If the biological tissue forming method of the present embodiment is such a method, a biological tissue having a cell layer composed of a plurality of different cells can be appropriately formed.

[0097] Here, when culturing adherent cells in the two flow channels of the biological tissue formation apparatus 1 , it is necessary to allow the adherent cells to adhere to both surfaces of the culture membrane 10 .

[0098] As a method for this, adherent cells and culture fluid are supplied to two flow channels of the biological tissue formation apparatus 1 , and then culture is performed while the biological tissue formation apparatus 1 is turned upside down, thereby allowing the adherent cells to adhere to both surfaces of the culture membrane 10 .

[0099] In addition, when adherent cells and culture fluid are provided to the two flow paths of the biological tissue forming device 1 without turning the biological tissue forming device 1 upside down, by filling the flow path 130 below the biological tissue forming device 1 with adherent cells, the adherent cells can be attached not only to the upper surface of the culture membrane 10, but also to the lower surface of the culture membrane 10.

[0100] In this case, after the adhered cells are attached to the lower surface of the culture membrane 10 , the cells remaining in the flow channel 130 can be washed with a culture solution or the like, so that the adhered cells on the lower surface of the culture membrane 10 remain in an attached state.

[0101] Although this method wastes cells, it has the advantage of being able to attach adherent cells in a shorter time compared to the former method.

[0102] In the method for forming a biological tissue of the present embodiment, as adherent cells, for example, pluripotent stem cells (iPS cells, etc.) and embryonic stem cells (ES cells) can be appropriately used.

[0103] In addition, the type of biological tissue to be produced is not particularly limited, and various tissues can be used, such as biological tissue in a proximal tubule model comprising tissue composed of renal tubular epithelial cells and tissue composed of vascular endothelial cells, a glomerular model, a small intestine model, a liver model, and a lung model.

[0104] Furthermore, the culture medium used and its flow rate in the flow channel are not particularly limited and can be appropriately set according to the cells and tissues to be cultured.

[0105] As described above, according to the biological tissue formation device and biological tissue formation method of this embodiment, in an organ chip, etc., the culture membrane can be composed of a soluble material. After the cell layers are formed on both sides of the culture membrane, the soluble material is dissolved, so that the cell-to-cell interaction and the exchange of liquid components between the cell layers of the formed biological tissue can be carried out efficiently.

[0106] [Second embodiment]

[0107] Next, a biological tissue forming apparatus according to a second embodiment of the present invention will be described.

[0108] The biological tissue formation device of this embodiment differs from the first embodiment in that the culture membrane is composed of a readily soluble material and a poorly soluble material. The other configurations are the same as those of the first embodiment except for the points described below.

[0109] That is, the biological tissue formation device of this embodiment is characterized by comprising: a culture membrane having culture areas for adherent cells on both sides, wherein the cultured adherent cells are placed between multiple cell layers, and multiple flow paths are divided by the culture membrane; the culture membrane is composed of a soluble material and an insoluble material.

[0110] By Figures 1 to 3 The biological tissue forming device of this embodiment will be described by adding a to the reference numerals of the first embodiment having the same configuration as that of the first embodiment.

[0111] In the biological tissue forming apparatus 1 a of this embodiment, the culture membrane 10 a is composed of a readily soluble material 101 a and a poorly soluble material 102 a .

[0112] After the cell layer is formed, the easily soluble material 101a in the culture membrane 10a is dissolved, leaving only the poorly soluble material 102a in the culture membrane 10a as a support for the cell layer, thereby forming a pore penetrating the culture membrane 10a.

[0113] That is, in the process of culturing adherent cells, the readily soluble material 101a and the poorly soluble material 102a in the culture membrane 10a serve as a scaffold for the adherent cells. Furthermore, after the readily soluble material 101a dissolves, the poorly soluble material 102a serves as a support for the cell layer.

[0114] As the easily soluble material 101 a , a water-soluble polymer or the like can be used. For example, polyvinyl alcohol (PVA), alginic acid, methylcellulose, or the like can be used appropriately.

[0115] When alginic acid is used as the easily soluble material 101 a , the easily soluble material 101 a in the culture membrane 10 a in the biological tissue forming apparatus 1 can be dissolved by supplying an alginate degrading enzyme to the flow channel 110 a or the flow channel 130 a .

[0116] When polyvinyl alcohol is used as the easily soluble material 101a, the easily soluble material 101a in the culture membrane 10a in the biological tissue formation apparatus 1a can be dissolved by heating the biological tissue formation apparatus 1a to, for example, 37 to 50°C after the cell layer is formed.

[0117] Furthermore, when methylcellulose is used as the easily soluble material 101a, the easily soluble material 101a in the culture membrane 10a in the biological tissue formation apparatus 1a can be dissolved by cooling the biological tissue formation apparatus 1a to, for example, 5 to 10°C after the cell layer is formed.

[0118] As the poorly soluble material 102 a , polyethylene terephthalate (PET), polylactic acid (PLA), ultraviolet (UV) curable resin, or the like can be used.

[0119] The pore size of the porous membrane made of a poorly soluble material is preferably 10 micrometers or larger. If the pore size is set to this level, the intercellular interaction and the exchange of liquid components between the two cell layers can be made more efficient.

[0120] As described above, according to the biological tissue formation apparatus 1a of this embodiment, after the cell layer is formed, the soluble material 101a in the culture membrane 10a is dissolved, thereby enabling appropriate cell-to-cell interaction and permeation of liquid components between the plurality of cell layers.

[0121] In addition, the poorly soluble material 102a in the culture membrane 10a can remain between the multiple cell layers. Using the poorly soluble material 102a as a support can make the cell layers less susceptible to damage.

[0122] The culture membrane 10a in the biological tissue formation apparatus 1a of this embodiment can be produced and used, for example, by the following method.

[0123] That is, Figure 5 As shown, a readily soluble material 101a is first applied to a substrate 50. Next, a poorly soluble material 102a is layered, followed by a further application of the readily soluble material 101a. The resulting culture membrane 10a is then peeled from the substrate 50, cut into a desired shape, and placed in the biological tissue formation apparatus 1.

[0124] As described above, the readily soluble material 101a in the culture membrane 10a of the biological tissue formation device 1a of this embodiment can be made of materials such as alginic acid and polyvinyl alcohol. Furthermore, as the poorly soluble material 102a in the culture membrane 10a, materials such as polyethylene terephthalate (PET), polylactic acid (PLA), and ultraviolet (UV) curable resin can be used. Furthermore, as described above, the resulting culture membrane 10a can be cut into a desired shape for use.

[0125] Furthermore, by bonding the flow channel plates 11a and 13a and the culture membrane 10a together using the adhesive layers 12a and 14a, the biological tissue forming device 1a of this embodiment can be manufactured.

[0126] The biological tissue forming apparatus 1 a of this embodiment may be in a state before or after a cell layer is formed on the culture membrane 10 a .

[0127] Furthermore, the biological tissue forming apparatus 1a of this embodiment further includes an apparatus in which, after forming a cell layer on the culture membrane 10a, the easily soluble material 101a is dissolved, leaving the poorly soluble material 102a, thereby forming pores penetrating the culture membrane 10a.

[0128] Furthermore, the biological tissue formation device 1a of this embodiment is preferably configured such that the culture membrane 10a contains adherent cells. In this case, the adherent cells may be immobilized on the surface of the culture membrane 10a, or the adherent cells may be contained in the soluble material 101a within the culture membrane 10a.

[0129] The method for forming biological tissue of the present embodiment is a method for forming biological tissue having multiple cell layers composed of adherent cells, and is characterized by comprising: a process of providing adherent cells and culture fluid to two flow paths in a biological tissue forming device, wherein the biological tissue forming device comprises: a culture membrane having culture areas for adherent cells on both sides, wherein the cultured adherent cells are placed between the multiple cell layers, and two flow paths are divided by the culture membrane, wherein the culture membrane is composed of a soluble material and an insoluble material; a process of culturing the adherent cells in the two flow paths to form cell layers on both sides of the culture membrane; and a process of dissolving the culture membrane.

[0130] In the biological tissue forming apparatus 1a of this embodiment, as Figure 6 As shown, the flow path 110a between the flow path plate 11a and the culture membrane 10a is filled with culture fluid 40, and cells 30 are cultured on one surface of the culture membrane 10a. At the same time, the flow path 130a between the flow path plate 13a and the culture membrane 10a is filled with culture fluid 41, and cells 31 are cultured on the other surface of the culture membrane 10a, thereby forming two cell layers.

[0131] Furthermore, after forming two cell layers, dissolving the soluble material 101a in the culture membrane 10a allows cells to contact each other between the cell layers, eliminating the problem of reduced interaction between the cell layers. Furthermore, liquid components can be exchanged efficiently, eliminating the problem of reduced efficiency in liquid component exchange between the two cell layers.

[0132] In addition, since the poorly soluble material 102a in the culture membrane 10a is insoluble, it can remain between the two cell layers. Using the poorly soluble material 102a as a support can make the cell layers less susceptible to damage.

[0133] In this case, when the soluble material 101a is alginic acid, in the step of dissolving the soluble material in the culture membrane 10a, an alginate degrading enzyme may be supplied to at least one of the flow channel 110a and the flow channel 130a to dissolve the culture membrane 10a.

[0134] Furthermore, when the easily soluble material 101 a is polyvinyl alcohol, the culture membrane 10 a can be dissolved by heating the biological tissue forming apparatus 1 a in the step of dissolving the easily soluble material in the culture membrane 10 a .

[0135] As described above, according to the biological tissue formation device and biological tissue formation method of this embodiment, in an organ chip, etc., the culture membrane can be composed of soluble materials and insoluble materials. After the cell layers are formed on both sides of the culture membrane, the intercellular interaction and exchange of liquid components between the cell layers of the formed biological tissue can be carried out efficiently by dissolving the soluble materials, and the cell layers can be made less susceptible to damage.

[0136] Example

[0137] The following describes experiments conducted to verify the dissolution of a readily soluble material used in a biological tissue formation device according to an embodiment of the present invention. The readily soluble material is used as a culture membrane placed between multiple cell layers after culturing adherent cells in the biological tissue formation device. It dissolves after the cell layers are formed. However, in each example, the formation of the cell layers was omitted, and only the solubility of the readily soluble material was evaluated.

[0138] In addition, when forming a cell layer using a biological tissue formation device, a culture medium was used. In each example and reference example, phosphate buffer was used instead of the culture medium to evaluate the solubility. Figure 8 shown.

[0139] [Example 1]

[0140] In this example, an experiment was conducted in which alginic acid was used as a readily soluble material in a biological tissue forming device and dissolved with sodium citrate.

[0141] Specifically, sodium alginate (Fujifilm Wako Pure Chemical Industries, Ltd., 194-13321) was dissolved in pure water to prepare a 1% sodium alginate aqueous solution, which was cast onto a PET film and dried overnight at room temperature (25°C).

[0142] Then, soak in 0.5M calcium chloride aqueous solution for 10 minutes to crosslink the alginate and then rinse with pure water. Dry overnight at room temperature (25°C) to prepare a calcium alginate film (film thickness of about 10 μm) as a soluble material. The soluble material cut into 10 mm squares was immersed in 20 mL of phosphate buffer (Thermo Fisher Scientific, Inc., 10010023), stored at a temperature of 37°C for 1 week, and the state and transparency of the soluble material were confirmed.

[0143] The reason for storing the soluble material for one week and confirming its state before dissolution is that the formation of a cell layer generally requires a culture period of about one week, and it is best not to dissolve during this period. Furthermore, the transparency of the soluble material is confirmed because a cell layer forms on the surface of the soluble material, and therefore, it is best if the soluble material is transparent to facilitate identification when observing the cell layer.

[0144] The above confirmation results showed that sodium chloride contained in the phosphate buffer slightly dissolved the surface of the easily soluble material, thereby reducing transparency.

[0145] In order to dissolve the easily soluble material, sodium citrate was added to the phosphate buffer solution to a concentration of 1%, and the mixture was immersed in the phosphate buffer solution for 6 hours at a temperature of 37° C. As a result, the easily soluble material was completely dissolved in the phosphate buffer solution.

[0146] [Example 2]

[0147] In this example, an experiment was conducted in which alginic acid was used as a readily soluble material in a biological tissue forming device and dissolved with an alginate-degrading enzyme.

[0148] Specifically, as in Example 1, a calcium alginate film was prepared and stored as a readily soluble material in a phosphate buffer solution. The state and transparency of the readily soluble material were then confirmed. The results showed that the state and transparency of the readily soluble material were comparable to those before immersion in the phosphate buffer solution.

[0149] In order to dissolve the easily soluble material, 100 μg of alginate degrading enzyme (Nihon Gene Co., Ltd., 319-08261) was added to the phosphate buffer and the mixture was immersed in the phosphate buffer for 6 hours at 37° C. As a result, the easily soluble material was completely dissolved in the phosphate buffer.

[0150] [Example 3]

[0151] In this example, an experiment was conducted in which polyvinyl alcohol was used as a readily soluble material in a biological tissue forming device and dissolved by heating.

[0152] Specifically, polyvinyl alcohol (Fujifilm Wako Pure Chemical Industries, Ltd., 160-11485) was dissolved in pure water to prepare a 5% polyvinyl alcohol aqueous solution. This polyvinyl alcohol aqueous solution was cast onto a PET film and dried overnight at room temperature (25°C) to prepare a polyvinyl alcohol film (thickness approximately 7 μm) as the readily soluble material.

[0153] Next, the readily soluble material, cut into 10 mm squares, was immersed in 20 mL of phosphate buffer (Thermo Fisher Scientific, Inc., 10010023) and stored at 37°C for one week. The state and transparency of the readily soluble material were then checked. The results showed that the state and transparency of the readily soluble material were comparable to those before immersion in the phosphate buffer.

[0154] In order to dissolve the easily soluble material, the sample was immersed in a phosphate buffer solution at a temperature of 50° C. for 3 hours. As a result, the easily soluble material was completely dissolved in the phosphate buffer solution.

[0155] [Example 4]

[0156] In this example, an experiment was conducted in which methylcellulose was used as a readily soluble material in a biological tissue forming device and dissolved by cooling.

[0157] Specifically, methylcellulose (MCE-4000, manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in pure water to prepare a 1% methylcellulose aqueous solution. This methylcellulose aqueous solution was cast onto a PET film and dried overnight at room temperature (25°C) to prepare a methylcellulose film (thickness approximately 20 μm) as the readily soluble material.

[0158] Next, the readily soluble material, cut into 10 mm squares, was immersed in 20 mL of phosphate buffer (Thermo Fisher Scientific, Inc., 10010023) and stored at 37°C for one week. The state and transparency of the readily soluble material were then checked. The results showed that the state and transparency of the readily soluble material were comparable to those before immersion in the phosphate buffer.

[0159] In order to dissolve the easily soluble material, the sample was immersed in a phosphate buffer solution at a temperature of 10° C. for 10 minutes. As a result, the easily soluble material was partially dissolved in the phosphate buffer solution.

[0160] It should be noted that in order to dissolve the highly soluble material, the highly soluble material dissolved more in the phosphate buffer when stored at 5°C. Therefore, in order to better dissolve the highly soluble material in the phosphate buffer at 10°C, an experiment was conducted to adjust the dissolution temperature of the methylcellulose aqueous solution.

[0161] [Example 5]

[0162] In this example, an experiment was conducted in which methylcellulose was used as a readily soluble material in a biological tissue forming device, sodium styrene sulfonate was added as an additive, and the solution was dissolved by cooling.

[0163] Specifically, to adjust the dissolution temperature, sodium styrene sulfonate (Fujifilm Wako Pure Chemical Industries, Ltd., 192-03292) was added to a methylcellulose aqueous solution prepared in the same manner as in Example 4 to a concentration of 0.1 M. This methylcellulose aqueous solution was cast onto a PET film and dried overnight at room temperature (25°C) to prepare a methylcellulose film (thickness approximately 20 μm) as a readily soluble material.

[0164] Next, the readily soluble material, cut into 10 mm squares, was immersed in 20 mL of phosphate buffer (Thermo Fisher Scientific, Inc., 10010023) and stored at 37°C for one week. The state and transparency of the readily soluble material were then checked. The results showed that the state and transparency of the readily soluble material were comparable to those before immersion in the phosphate buffer.

[0165] In order to dissolve the easily soluble material, the sample was immersed in a phosphate buffer solution at a temperature of 10° C. for 10 minutes. As a result, the easily soluble material was completely dissolved in the phosphate buffer solution.

[0166] [Reference Example 1]

[0167] In this reference example, a commercially available semipermeable PET membrane (Corning, 353091) was used instead of the easily soluble material in the biological tissue formation device, and an experiment on its dissolution was conducted.

[0168] Specifically, PET films cut into 10 mm squares were immersed in 20 mL of phosphate buffer (Thermo Fisher Scientific, Inc., 10010023) and stored at 37°C for one week. The state and transparency of the readily soluble material were then checked. The results showed that the state and transparency of the readily soluble material were comparable to those before immersion in the phosphate buffer.

[0169] Furthermore, the PET film was immersed in a phosphate buffer solution at a temperature of 90° C. for 24 hours. As a result, the PET film did not dissolve and no change was observed.

[0170] Furthermore, to decompose the PET film enzymatically, Savinase (Novozymes, 16L) was added to a concentration of 0.5%, and the mixture was stored at 37° C. for 24 hours. As a result, the PET film did not dissolve and no changes were observed.

[0171] The results of Reference Example 1 show that PET film cannot be used to replace the easily soluble material in the biological tissue formation device. On the other hand, PET film can be suitably used as the poorly soluble material in the biological tissue formation device.

[0172] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the present invention.

[0173] For example, the shape of the flow channel and the shape of the culture membrane in the biological tissue production device are not limited to Figure 1 The shapes shown in the figures can also be appropriately changed into various other shapes.

[0174] Industrial Applicability

[0175] The present invention can be suitably applied to the case of forming biological tissue using an organ chip or the like.

[0176] The contents of the documents described in this specification and the Japanese application specification on which the Paris priority claim of this application is based are incorporated herein by reference.

[0177] Description of Reference Numerals

[0178] 1.1a Biological tissue formation device

[0179] 10, 10a culture membrane

[0180] 101, 101a soluble materials

[0181] 102a Insoluble materials

[0182] 11, 11a, 13, 13a flow board

[0183] 110, 110a, 130, 130a flow path

[0184] 12, 12a, 14, 14a Adhesion layer

[0185] 20 Biological tissue formation device

[0186] 210 semipermeable membrane

[0187] 211, 213 flow board

[0188] 30, 31 cells

[0189] 40, 41 culture medium

[0190] 50 base materials

Claims

1. A biological tissue forming device for forming biological tissue having a plurality of cell layers composed of adherent cells, characterized in that: have: A culture membrane having culture areas for the adherent cells on both sides, and placed between the multiple cell layers formed by culturing the adherent cells. a plurality of flow paths divided by the culture membrane; The culture membrane is composed of a readily soluble material and a hardly soluble material. The poorly soluble material is used as a support for the cell layer after the easily soluble material in the culture membrane is dissolved.

2. The biological tissue formation device according to claim 1, characterized in that By dissolving the easily soluble material in the culture membrane, pores penetrating the culture membrane are formed.

3. The biological tissue formation device according to claim 1 or 2, characterized in that: The pore size of the porous membrane composed of the poorly soluble material is 10 micrometers or more.

4. The biological tissue formation device according to claim 1 or 2, characterized in that: The easily soluble material and the poorly soluble material are used as scaffolds when culturing the adherent cells.

5. The biological tissue formation device according to claim 1 or 2, characterized in that: The poorly soluble material is made of any one of polyethylene terephthalate, polylactic acid or ultraviolet curable resin.

6. The biological tissue formation device according to claim 1 or 2, characterized in that: The culture membrane is a porous membrane.

7. The biological tissue formation device according to claim 1 or 2, characterized in that: The biological tissue having two cell layers is formed by providing one culture membrane and two flow paths divided by the culture membrane.

8. The biological tissue formation device according to claim 7, characterized in that: The two flow paths are formed by adhering the flow path-side surfaces of two plates having the flow paths to the two surfaces of the culture membrane, respectively.

9. The biological tissue formation device according to claim 7, characterized in that: Cell layers composed of different types of adherent cells are formed in the two flow channels, respectively.

10. The biological tissue formation device according to claim 1 or 2, characterized in that: The easily soluble material is composed of a water-soluble polymer.

11. The biological tissue forming device according to claim 10, characterized in that: The water-soluble polymer is polyvinyl alcohol, alginic acid or methyl cellulose.

12. The biological tissue formation device according to claim 1 or 2, characterized in that: Cell layers composed of adherent cells are formed on both sides of the culture membrane.

13. The biological tissue forming device according to claim 12, characterized in that: The cell layer is formed, and the readily soluble material is dissolved.

14. The biological tissue formation device according to claim 1 or 2, characterized in that: The culture membrane is formed by containing adherent cells.

15. A method for forming a biological tissue having a plurality of cell layers composed of adherent cells, characterized in that: have: The process of supplying adherent cells and culture medium to two flow channels in the biological tissue formation device, The biological tissue formation device comprises: a culture membrane having culture areas for adherent cells on both sides, placed between the multiple cell layers formed by culturing the adherent cells, and two flow paths divided by the culture membrane, wherein the culture membrane is composed of a soluble material and a poorly soluble material; a cell layer forming step of culturing the adherent cells in the two flow channels to form cell layers on both sides of the culture membrane; and a dissolving step of dissolving the soluble material in the culture membrane, The poorly soluble material is used as a support for the cell layer after the easily soluble material in the culture membrane is dissolved.

16. The method for forming biological tissue according to claim 15, wherein: By dissolving the easily soluble material in the culture membrane, pores penetrating the culture membrane are formed.

17. The method for forming biological tissue according to claim 15 or 16, characterized in that: The soluble material is alginic acid. In the dissolution step, an alginate-degrading enzyme is supplied to at least one of the two flow channels to dissolve the soluble material in the culture membrane.

18. The method for forming biological tissue according to claim 15 or 16, characterized in that: The soluble material is polyvinyl alcohol. In the dissolving step, the biological tissue forming device is heated to dissolve the soluble material in the culture membrane.

19. The method for forming biological tissue according to claim 15 or 16, characterized in that: The easily soluble material is methyl cellulose. In the dissolving step, the biological tissue forming device is cooled to dissolve the easily soluble material in the culture membrane.

20. The method for forming biological tissue according to claim 15 or 16, characterized in that: The lysis step is performed when a cell layer is formed in the entire culture area.

21. The method for forming biological tissue according to claim 15 or 16, wherein: Cell layers composed of different types of adherent cells are formed in the two flow channels, respectively.

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