Apparatus for forming biological tissue, and method for forming biological tissue.

Abstract

The present invention makes it possible to provide a biological tissue forming device that can efficiently carry out intercellular interaction and liquid component exchanging between cell layers of a formed biological tissue. This biological tissue forming device 1 forms a biological tissue having a plurality of cell layers composed of adherent cells 30, 31, and comprises: a culture membrane 10 comprising culture regions of the adherent cells 30, 31 on both surfaces, and disposed, after the adherent cells 30, 31 have been cultured, between a plurality of cell layers; and a plurality of flow paths 110, 130 divided via the culture membrane 10. The culture membrane 10 is composed of an easily soluble material 101.

Description

Technical Field

[0001] The present invention relates to cell culture technology, and particularly to an apparatus for forming a biological tissue by culturing cells and a method for forming a biological tissue.

Background Art

[0002] Conventionally, in the development of pharmaceuticals and the like, when the pharmacokinetics of a drug candidate substance cannot be predicted up to preclinical trials and its toxicity is revealed in clinical trials, resulting in the suspension of development, the research and development costs up to that point are wasted. That is, in preclinical trials, cell assays, animal experiments, etc. are conducted. However, in cell assays, blood flow in the body cannot be reproduced, the expression of cell-specific functions is insufficient, and there is a problem that pharmacokinetics cannot be evaluated. In addition, in animal experiments, due to species differences, the results of pharmacokinetics do not always match those of humans, so there is a problem that it is difficult to predict human pharmacokinetics.

[0003] In such a situation, in recent years, organ chips have been expected to solve these various problems and improve the prediction accuracy of human pharmacokinetics. An organ chip is a reproduction of the tissue structure of an organ in a microfluidic device. There have already been proposed a lung chip that models the structure of the lung, a combination of a small intestine model and a liver model that models the structure of enterohepatic circulation, and a model of the structure of the kidney such as a glomerulus model and a proximal tubule model.

[0004] [[ID=*]] Here, in an in vitro culture system using a conventional culture dish or the like, the culture environment is quiescent, there is no reproduction of blood flow, and the supply of oxygen and nutrients and the removal of waste products depend only on diffusion. Therefore, it has been difficult to conduct tests considering cell-specific functions and interactions between organs. In contrast, organ chips allow for the reproduction of blood flow through pump delivery, and enable the adjustment of oxygen and nutrient supply based on flow rate. This makes it possible to suitably conduct tests that include specific cell functions and inter-organ interactions. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Special Publication No. 2017-504320 [Overview of the project] [Problems that the invention aims to solve]

[0006] These organ chips are equipped with a channel divided into two sections by a membrane, where cells are cultured on both surfaces of the membrane, forming two cell layers separated by the membrane. A semipermeable membrane is used for this purpose, and exchange of nutrients and other liquid components such as salts occurs between the two cell layers through the pores of the semipermeable membrane. However, the pore size of the semipermeable membrane is too small for cells to pass through, which hinders cell-to-cell contact between cell layers and reduces inter-cell interaction. Additionally, as cell layers are formed through cell culture, the pores easily become blocked, reducing the efficiency of fluid exchange.

[0007] Therefore, the inventors diligently conducted research and developed an organ chip (biological tissue formation device) that can efficiently facilitate intercellular interactions and exchange of fluid components between cell layers in the formed biological tissue. Specifically, by first using an easily soluble material as the membrane for the organ chip, and then forming cell layers on both surfaces of this membrane, and then dissolving the membrane, it became possible to prevent a decrease in the efficiency of intercellular interactions and exchange of liquid components between the two cell layers.

[0008] Incidentally, while membranes made of easily soluble materials function as a scaffold for cells in the formation of cell layers on both surfaces, after the easily soluble material is dissolved, this scaffold disappears, which can sometimes make the formed cell layers more susceptible to damage. Therefore, the inventors have further made it possible to make the membrane composed of an easily soluble material and a poorly soluble material, and to make the poorly soluble material function as a support for the cell layer after the easily soluble material has been dissolved, thereby making the cell layer less prone to damage even after the easily soluble material has been dissolved.

[0009] Herein, Patent Document 1 discloses a lung chip, and it is stated that a biocompatible polymer can be used as the membrane in this lung chip (Claim 53). It is also stated that biocompatibility means that the material does not significantly degrade when implanted or placed adjacent to the target biological tissue, does not induce a significant immune response or harmful tissue reaction (e.g., a toxic reaction or significant pain) over time, or does not induce thrombosis or coagulation when in contact with blood (Paragraph 0055), and that a biocompatible and biodegradable material may be used in the device to facilitate in vivo implantation of the device (Paragraph 0316). However, this lung chip does not describe or suggest any mechanism for dissolving the membrane. Nor does it disclose any mechanism for making the cell layer less susceptible to damage after the membrane has been dissolved.

[0010] The present invention has been made in view of the above circumstances, and aims to provide a biological tissue formation device and a method for forming biological tissue that can efficiently perform intercellular interactions and exchange of liquid components between cell layers of the formed biological tissue, and that can make the cell layers less susceptible to damage. [Means for solving the problem]

[0011] To achieve the above objective, the present invention provides a biological tissue forming apparatus for forming a biological tissue having a plurality of cell layers composed of adherent cells, comprising a culture area for the adherent cells on both sides, a culture membrane placed between the plurality of cell layers after the adherent cells have been cultured, and a plurality of channels separated by the culture membrane, wherein the culture membrane is made of an easily soluble material.

[0012] Another embodiment of the present invention is a biological tissue forming apparatus for forming a biological tissue having multiple cell layers composed of adherent cells, comprising a culture area for adherent cells on both sides, a culture membrane placed between the multiple cell layers after the adherent cells have been cultured, and multiple channels separated by the culture membrane, wherein the culture membrane is composed of an easily soluble material and a poorly soluble material.

[0013] It is also preferable that the biological tissue formation apparatus of the present invention be configured such that holes penetrating the culture membrane are formed when the easily soluble material in the culture membrane is dissolved. Furthermore, it is also preferable that the biological tissue forming apparatus of the present invention be configured such that the pore size of the porous membrane made of the poorly soluble material is 10 microns or larger in diameter. Furthermore, it is also preferable that the biological tissue formation apparatus of the present invention be configured such that the easily soluble material and the poorly soluble material serve as a scaffold in the culture of the adherent cells.

[0014] Furthermore, it is also preferable that the biological tissue formation apparatus of the present invention be configured such that the poorly soluble material serves as a support for the cell layer after the easily soluble material in the culture membrane has been dissolved. Furthermore, it is also preferable that the bio-tissue forming apparatus of the present invention be configured such that the poorly soluble material consists of polyethylene terephthalate, polylactic acid, or an ultraviolet-curable resin. Furthermore, it is also preferable that the biological tissue formation apparatus of the present invention be configured such that the culture membrane is a porous membrane.

[0015] Furthermore, it is also preferable that the biological tissue formation apparatus of the present invention be configured to include one culture membrane and two channels separated by the culture membrane, thereby forming a biological tissue having two cell layers. Furthermore, it is also preferable that the biological tissue formation apparatus of the present invention be configured such that the two channels are formed by adhering each channel-side surface of two plates equipped with channels to both sides of the culture membrane.

[0016] Furthermore, it is also preferable to configure the biological tissue formation device of the present invention such that cell layers consisting of different types of adherent cells are formed in the two channels. Furthermore, it is also preferable that the bio-tissue forming apparatus of the present invention be configured such that the easily soluble material consists of a water-soluble polymer. Furthermore, it is also preferable that the bio-tissue forming apparatus of the present invention be configured such that the water-soluble polymer is polyvinyl alcohol, alginic acid, or methylcellulose.

[0017] Furthermore, the present invention relates to a method for forming a biological tissue having multiple cell layers made of adherent cells, comprising the steps of supplying adherent cells and culture medium to the two channels of a biological tissue forming apparatus having a culture membrane on both sides having culture regions for adherent cells and being placed between the multiple cell layers after the adherent cells have been cultured, and having two channels separated by the culture membrane, the culture membrane being made of an easily soluble material, culturing the adherent cells in the two channels to form cell layers on both sides of the culture membrane, and dissolving the culture membrane.

[0018] Another form of the method for forming a biological tissue of the present invention is a method for forming a biological tissue having a plurality of cell layers composed of adhesive cells, the method comprising: a culture membrane having culture regions for adhesive cells on both sides, the culture membrane being disposed between the plurality of cell layers after the adhesive cells are cultured; and two flow paths defined by the culture membrane, the method further comprising the steps of: supplying adhesive cells and a culture solution to the two flow paths in a biological tissue forming apparatus including the culture membrane made of an easily soluble material and a hardly soluble material; culturing the adhesive cells in the two flow paths to form cell layers on both sides of the culture membrane; and dissolving the easily soluble material in the culture membrane.

[0019] It is also preferable that, in the method for forming a biological tissue of the present invention, the easily soluble material is alginic acid, and in the step of dissolving, an alginic acid degrading enzyme is supplied to at least one of the two flow paths to dissolve the easily soluble material in the culture membrane. Furthermore, it is also preferable that, in the method for forming a biological tissue of the present invention, the easily soluble material is polyvinyl alcohol, and in the step of dissolving, the biological tissue forming apparatus is heated to dissolve the easily soluble material in the culture membrane. It is also preferable that, in the method for forming a biological tissue of the present invention, the easily soluble material is methyl cellulose, and in the step of dissolving, the biological tissue forming apparatus is cooled to dissolve the easily soluble material in the culture membrane.

Advantages of the Invention

[0020] According to the present invention, it is possible to provide a biological tissue forming apparatus and a method for forming a biological tissue that can efficiently perform cell-cell interactions and exchange of humoral components between cell layers of the formed biological tissue and can make the cell layers difficult to break.

Brief Description of the Drawings

[0021] [Figure 1] It is a schematic diagram showing the constituent members of a biological tissue forming apparatus according to each embodiment of the present invention. [Figure 2]This is a schematic diagram showing the configuration of a biological tissue formation apparatus according to each embodiment of the present invention. [Figure 3] This is a schematic diagram showing cross-section AA of a biological tissue formation apparatus according to each embodiment of the present invention. [Figure 4] This is a schematic diagram showing the cell layer formation process using a biological tissue formation apparatus according to the first embodiment of the present invention. [Figure 5] This is a schematic diagram showing the process of preparing a culture membrane in a biological tissue formation apparatus according to the second embodiment of the present invention. [Figure 6] This is a schematic diagram showing the cell layer formation process using a biological tissue formation apparatus according to the second embodiment of this present invention. [Figure 7] This is a schematic diagram illustrating the cell layer formation process using a conventional biological tissue formation device. [Figure 8] This figure shows the results of an experiment conducted to confirm the dissolution of easily soluble materials used in the biological tissue formation apparatus according to the first embodiment of the present invention. [Modes for carrying out the invention]

[0022] The embodiments of the biological tissue formation apparatus and the biological tissue formation method of the present invention will be described in detail below. However, the present invention is not limited to the specific details of the following embodiments.

[0023] [First Embodiment] First, a biological tissue formation apparatus according to the first embodiment of the present invention will be described. The biological tissue formation apparatus of this embodiment is a device for forming biological tissue having multiple cell layers composed of adherent cells, and can be configured as a so-called organ chip or the like. Organ chips utilize microfluidic devices to replicate the tissue structure of organs within the device, allowing for precise control of the cell culture environment. This offers the advantage of enabling cells to express cellular functions similar to those found in vivo.

[0024] Furthermore, the biological tissue formation apparatus of this embodiment is characterized by having culture regions for adherent cells on both sides, a culture membrane that is placed between multiple cell layers after the adherent cells have been cultured, and multiple channels separated by the culture membrane, wherein the culture membrane is made of an easily soluble material. For example, as shown in Figures 1-3, the biological tissue formation apparatus 1 of this embodiment can be manufactured by placing a culture membrane 10 having a surface for culturing adherent cells between a channel plate 11 and a channel plate 13, and then bonding and integrating them with adhesive layers 12 and 14. Furthermore, the biological tissue formation apparatus 1 can also be configured to form three or more cell layers by further including a culture membrane and adhesive layers.

[0025] The flow channel plate 11 is provided with a flow channel 110, and the flow channel plate 13 is provided with a flow channel 130. The flow channel sides of these two plates are bonded to both sides of the culture membrane 10 via adhesive layers, thereby forming two separate flow channels in the biological tissue formation apparatus 1, separated by the culture membrane 10. These flow channels are used as culture chambers. Then, cell layers are formed on both sides of the culture membrane 10 through these channels, and a biological tissue having two cell layers is formed in the biological tissue formation device 1.

[0026] These two cell layers may consist of the same type of adherent cells, or they may consist of different types of adherent cells; in particular, it is beneficial for them to consist of different types of adherent cells.

[0027] In the biological tissue formation apparatus 1 of this embodiment, the culture membrane 10 is made of an easily soluble material 101. The culture membrane 10 may be a porous membrane or a non-porous membrane, but it is preferable that it be a porous membrane with a pore size such that cells cannot pass through until the easily soluble material 101 is dissolved. This pore size is preferably 0.4 to 8 microns in diameter, and more preferably 3 to 6 microns.

[0028] As the easily soluble material 101, water-soluble polymers can be used, and for example, polyvinyl alcohol (PVA), alginic acid, methylcellulose, etc., can be suitably used. When alginic acid is used as the easily soluble material 101, the culture membrane 10 in the biological tissue formation apparatus 1 can be dissolved by delivering alginate-degrading enzyme to the flow path 110 or flow path 130.

[0029] Furthermore, when polyvinyl alcohol is used as the easily soluble material 101, the easily 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, 37-50°C after the formation of the cell layer. Note that the dissolution temperature of polyvinyl alcohol is not limited to this range; it can also be dissolved by heating to 37-80°C.

[0030] Furthermore, when methylcellulose is used as the easily soluble material 101, the easily 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 after the formation of the cell layer. Note that the dissolution temperature of methylcellulose is not limited to this range; it can also be dissolved by cooling to 4 to 25°C.

[0031] Here, methylcellulose is a material that gels at high temperatures and liquefies when cooled, and can be dissolved by lowering the temperature. It is also preferable to add an additive to adjust the dissolution temperature of methylcellulose. By adding an additive to methylcellulose, its dissolution temperature can be increased. For example, sodium styrenesulfonate (NaSS) can be suitably used as this additive.

[0032] Thus, according to the biological tissue formation apparatus 1 of this embodiment, by dissolving the culture membrane 10 after the formation of the cell layer, it is possible to suitably allow liquid components to permeate between multiple cell layers.

[0033] Cells cultured using the biological tissue formation apparatus 1 of this embodiment can include, for example, pluripotent stem cells (such as iPS cells) or embryonic stem cells (ES cells). Furthermore, in the biological tissue formation apparatus 1 of this embodiment, it is also preferable to provide a cell scaffold material in order to suitably adhere cells to the surface of the culture membrane. The same applies to the second embodiment described later. Specifically, it is preferable to attach a cell scaffold material to the surface of the culture membrane and then adhere the cells to it before culturing.

[0034] Suitable cell scaffold materials include, for example, collagen, Matrigel, fibronectin, laminin, and chitosan, and two or more of these materials can also be used. Methods for attaching cell scaffolding material to the surface of a culture membrane include, for example, coating the surface of the culture membrane, mixing a readily soluble material with the cell scaffolding material, chemically bonding the cell scaffolding material to the readily soluble material using reagents such as coupling agents, and attaching pre-processed fibers of collagen or chitosan to the surface of the culture membrane. Furthermore, it is also preferable to adhere the cells to the surface of the culture membrane by adhering them to a poorly soluble material used in the second embodiment described later.

[0035] The culture membrane 10 in the biological tissue formation apparatus 1 of this embodiment can be used by, for example, applying an easily soluble material 101 to a substrate, peeling the resulting culture membrane 10 from the substrate and cutting it into a desired shape, and then placing it inside the biological tissue formation apparatus 1. It goes without saying that the shape of the culture membrane 10 is not limited to the shape shown in Figure 1; for example, it can be shaped to cover the entire lower surface of the flow channel plates 11 and 13.

[0036] Furthermore, it is preferable to perform surface treatments such as corona treatment, excimer treatment, or plasma treatment on the surface of the culture membrane 10. By performing this surface treatment, the hydrophilicity of the surface of the culture membrane 10 can be improved, thereby improving the adhesion of adherent cells to the surface of the culture membrane 10.

[0037] In the biological tissue forming apparatus 1 of this embodiment, the materials for the channel plate 11 and channel plate 13 can be, for example, cycloolefin polymer, polymethyl methacrylate, or polycarbonate, and the channel plate 11 and channel plate 13 can be manufactured by injection molding or the like. Furthermore, the adhesive layers 12 and 14 in the biological tissue forming apparatus 1 of this embodiment can be made of materials such as adhesives, and the adhesive layers 12 and 14 can be manufactured by punching them out into shapes such as those shown in Figure 1.

[0038] Furthermore, as described above, alginic acid, polyvinyl alcohol, and the like can be used as the material for the culture membrane 10 in the biological tissue formation apparatus 1 of this embodiment, and the obtained culture membrane 10 can be cut into a desired shape for use. Then, by bonding the flow channel plates 11 and 13 and the culture membrane 10 together with adhesive layers 12 and 14, the biological tissue formation apparatus 1 of this embodiment can be fabricated.

[0039] The biological tissue formation apparatus 1 of this embodiment may be in a state before a cell layer is formed on the culture membrane 10, or in a state after a cell layer has been formed on the culture membrane 10. Furthermore, the biological tissue formation apparatus 1 of this embodiment also includes a case in which the easily soluble material 101 is dissolved after a cell layer has been formed on the culture membrane 10.

[0040] Furthermore, it is also preferable that the biological tissue formation apparatus 1 of this embodiment be formed in which the culture membrane 10 contains adherent cells. In this case, the adherent cells may be fixed to the surface of the culture membrane 10, or they may be encapsulated within the culture membrane 10. When the culture membrane 10 is made of, for example, alginate, adherent cells can survive within the culture membrane 10. By configuring the biological tissue formation apparatus 1 of this embodiment in this way, the supply of adhesive cells to the channel can be omitted in the biological tissue formation method of this embodiment, which will be described later.

[0041] The method for forming biological tissue according to this embodiment is a method for forming biological tissue having multiple cell layers made of adherent cells, and is characterized by comprising the steps of supplying adherent cells and culture medium to two channels in a biological tissue forming apparatus having a culture membrane on both sides having culture regions for adherent cells and being placed between the multiple cell layers after the adherent cells have been cultured, and two channels separated by the culture membrane, the culture membrane being made of an easily soluble material, culturing adherent cells in the two channels and forming cell layers on both sides of the culture membrane, and dissolving the culture membrane.

[0042] As shown in Figure 7, in conventional biological tissue formation devices 20, a semipermeable membrane such as polyester is generally used as the culture membrane 210, and intercellular interactions and exchange of liquid components occur between the two channels through this membrane. In other words, in the conventional biological tissue formation apparatus 20, culture medium 40 was filled into the channel between the channel plate 211 and the semipermeable membrane 210 to culture cells 30 on one surface of the semipermeable membrane 210, and culture medium 41 was filled into the channel between the channel plate 213 and the semipermeable membrane 210 to culture cells 31 on the other surface of the semipermeable membrane 210, thereby forming two cell layers.

[0043] Incidentally, in this embodiment, the size of the cells cultured is generally 8-10 μm, while 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 such as the obstruction of cell-cell contact between cell layers and a decrease in interaction between cell layers, and the fact that as the cells proliferate, the pores of the semipermeable membrane 210 become blocked, resulting in a decrease in the efficiency of exchange of liquid components.

[0044] In contrast, in the biological tissue formation apparatus 1 of this embodiment, as shown in Figure 4, culture medium 40 is filled into the channel 110 between the channel plate 11 and the culture membrane 10 to culture cells 30 on one surface of the culture membrane 10, and culture medium 41 is filled into the channel 130 between the channel plate 13 and the culture membrane 10 to culture cells 31 on the other surface of the culture membrane 10, thereby forming two cell layers. Furthermore, after the two cell layers are formed, dissolving the easily soluble material 101 in the culture membrane 10 allows cells to come into contact with each other between the cell layers, thus resolving the problem of reduced interaction between cell layers. In addition, the exchange of liquid components can be made highly efficient, resolving the problem of low efficiency in the exchange of liquid components between the two cell layers.

[0045] In this case, if the easily soluble material 101 is alginic acid, the culture membrane 10 can be dissolved by supplying alginate-degrading enzyme to at least one of the channels 110 and 130 during the process of dissolving the culture membrane 10. Furthermore, if the easily soluble material 101 is polyvinyl alcohol, the culture membrane 10 can be dissolved by heating the biological tissue formation apparatus 1 during the process of dissolving the culture membrane 10.

[0046] The step of dissolving the culture membrane 10 is preferably performed when a cell layer has formed throughout the entire culture area. Furthermore, it is preferable to form cell layers consisting of different types of adherent cells in the channel 110 and the channel 130, respectively. By using this method for forming biological tissue according to this embodiment, it is possible to suitably form biological tissue having a cell layer composed of multiple different cells.

[0047] In this case, when culturing adherent cells in the two channels of the biological tissue formation device 1, it is necessary to attach adherent cells to both sides of the culture membrane 10. The method involves supplying adherent cells and culture medium to two channels in the tissue formation device 1, and then performing culture while inverting the tissue formation device 1 vertically, thereby allowing adherent cells to attach to both sides of the culture membrane 10.

[0048] Furthermore, without inverting the tissue formation device 1, when supplying adherent cells and culture medium to the two channels in the tissue formation device 1, the adherent cells can be filled into the lower channel 130 of the tissue formation device 1, thereby allowing adherent cells to adhere to both the upper and lower surfaces of the culture membrane 10.

[0049] In this case, after the adherent cells are attached to the underside of the culture membrane 10, the cells remaining in the channel 130 can be washed away with culture medium or the like, thereby leaving the adherent cells attached to the underside of the culture membrane 10. Although this method results in some waste of cells, it has the advantage of allowing adhesive cells to attach in a shorter time compared to the former method.

[0050] In the method for forming biological tissue according to this embodiment, pluripotent stem cells (such as iPS cells) and embryonic stem cells (ES cells) can be suitably used as adherent cells. Furthermore, the type of biological tissue to be created is not particularly limited, and various types can be used, such as biological tissue in a proximal tubule model that includes tissue composed of tubular epithelial cells and tissue composed of vascular endothelial cells, as well as biological tissue in glomerular models, small intestine models, liver models, and lung models. Furthermore, there are no particular limitations on the culture medium used or its flow rate in the flow path; these can be set appropriately depending on the cells or tissues being cultured.

[0051] As described above, according to the biological tissue formation apparatus and method of biological tissue formation of this embodiment, the culture membrane in organ chips and the like can be constructed using an easily soluble material. After forming cell layers on both sides of this culture membrane, the easily soluble material is dissolved, making it possible to efficiently perform intercellular interactions and exchange of liquid components between the cell layers of the formed biological tissue.

[0052] [Second Embodiment] Next, a biological tissue formation apparatus according to a second embodiment of the present invention will be described. The biological tissue formation apparatus of this embodiment differs from the first embodiment in that the culture membrane consists of an easily soluble material and a poorly soluble material. Other configurations are the same as those of the first embodiment, except for the points described below.

[0053] In other words, the biological tissue formation apparatus of this embodiment is characterized by having culture regions for adherent cells on both sides, a culture membrane that is placed between multiple cell layers after the adherent cells have been cultured, and multiple channels separated by the culture membrane, wherein the culture membrane is made of an easily soluble material and a poorly soluble material. The biological tissue formation apparatus of this embodiment will be described using the same reference numerals as those used in the first embodiment shown in Figures 1 to 3, with the numeral 'a' added to indicate the same configuration.

[0054] In the biological tissue formation apparatus 1a of this embodiment, the culture membrane 10a consists of an easily soluble material 101a and a poorly soluble material 102a. Then, after the cell layer is formed, the easily soluble material 101a in the culture membrane 10a is dissolved, so that only the poorly soluble material 102a in the culture membrane 10a remains as a support for the cell layer. This creates pores that penetrate the culture membrane 10a. In other words, in the culture process of adherent cells, the easily soluble material 101a and the poorly soluble material 102a in the culture membrane 10a serve as a scaffold for adherent cells. Furthermore, after the easily soluble material 101a is dissolved, the poorly soluble material 102a serves as a support for the cell layer.

[0055] As the easily soluble material 101a, water-soluble polymers can be used, and for example, polyvinyl alcohol (PVA), alginic acid, methylcellulose, etc., can be suitably used. When alginic acid is used as the easily soluble material 101a, the easily soluble material 101a in the culture membrane 10a within the biological tissue formation apparatus 1 can be dissolved by delivering alginate-degrading enzyme to the flow path 110a or flow path 130a.

[0056] Furthermore, when polyvinyl alcohol is used as the easily soluble material 101a, the easily soluble material 101a in the culture membrane 10a within the tissue formation apparatus 1a can be dissolved by heating the tissue formation apparatus 1a to, for example, 37-50°C after the formation of the cell layer.

[0057] Furthermore, when methylcellulose is used as the easily soluble material 101a, the easily soluble material 101a in the culture membrane 10a within the tissue formation apparatus 1a can be dissolved by cooling the tissue formation apparatus 1a to, for example, 5-10°C after the formation of the cell layer.

[0058] As the poorly soluble material 102a, polyethylene terephthalate (PET), polylactic acid (PLA), ultraviolet (UV) curing resins, etc., can be used. The pore size of the porous membrane made of a poorly soluble material is preferably 10 microns or larger in diameter. This pore size allows for highly efficient intercellular interactions and exchange of liquid components between two cell layers.

[0059] Thus, according to the biological tissue formation apparatus 1a of this embodiment, by dissolving the easily soluble material 101a in the culture membrane 10a after the formation of the cell layer, it is possible to suitably facilitate intercellular interactions and the permeation of liquid components between multiple cell layers. Furthermore, the poorly soluble material 102a in the culture membrane 10a can be left between multiple cell layers, and the poorly soluble material 102a can be used as a support to make the cell layers less prone to breakage.

[0060] The culture membrane 10a in the biological tissue formation apparatus 1a of this embodiment can be prepared and used, for example, by the following method. Specifically, as shown in Figure 5, first, an easily soluble material 101a is applied to the substrate 50. Next, a poorly soluble material 102a is layered on top, and then the easily soluble material 101a is applied again. Then, the resulting culture film 10a is peeled off the substrate 50, cut into the desired shape, and placed inside the biological tissue formation apparatus 1.

[0061] Furthermore, as the easily soluble material 101a in the culture membrane 10a of the biological tissue formation apparatus 1a of this embodiment, alginic acid, polyvinyl alcohol, etc., can be used. Also, as the poorly soluble material 102a in the culture membrane 10a, polyethylene terephthalate (PET), polylactic acid (PLA), ultraviolet (UV) curing resin, etc., can be used. Furthermore, the obtained culture membrane 10a can be cut into the desired shape as described above and used. Then, by bonding the flow channel plates 11a and 13a and the culture membrane 10a together with adhesive layers 12a and 14a, the biological tissue formation apparatus 1a of this embodiment can be fabricated.

[0062] The biological tissue formation apparatus 1a of this embodiment may be in a state before a cell layer is formed on the culture membrane 10a, or it may be in a state after a cell layer has been formed on the culture membrane 10a. Furthermore, the biological tissue formation apparatus 1a of this embodiment also includes a configuration in which, after a cell layer is formed on the culture membrane 10a, the easily soluble material 101a is dissolved, leaving the poorly soluble material 102a, and pores penetrating the culture membrane 10a are formed.

[0063] Furthermore, it is also preferable that the biological tissue forming apparatus 1a of this embodiment be formed in which the culture membrane 10a contains adherent cells. In this case, the adherent cells may be fixed to the surface of the culture membrane 10a, or the adherent cells may be encapsulated in the easily soluble material 101a of the culture membrane 10a.

[0064] The method for forming biological tissue according to this embodiment is a method for forming biological tissue having multiple cell layers made of adherent cells, and is characterized by comprising the steps of supplying adherent cells and culture medium to two channels in a biological tissue forming apparatus having a culture membrane on both sides having a culture region for adherent cells and being placed between the multiple cell layers after the adherent cells have been cultured, and two channels separated by the culture membrane, the culture membrane being made of an easily soluble material and a poorly soluble material, culturing adherent cells in the two channels and forming cell layers on both sides of the culture membrane, and dissolving the culture membrane.

[0065] In the biological tissue formation apparatus 1a of this embodiment, as shown in Figure 6, the culture medium 40 is filled into the channel 110a between the channel plate 11a and the culture membrane 10a to culture cells 30 on one surface of the culture membrane 10a, and the culture medium 41 is filled into the channel 130a between the channel plate 13a and the culture membrane 10a to culture cells 31 on the other surface of the culture membrane 10a, thereby forming two cell layers.

[0066] Furthermore, after the two cell layers are formed, dissolving the easily soluble material 101a in the culture membrane 10a allows cells to come into contact with each other between the cell layers, thus resolving the problem of reduced interaction between cell layers. In addition, highly efficient exchange of liquid components can be achieved, resolving the problem of low efficiency in the exchange of liquid components between the two cell layers. Furthermore, since the poorly soluble material 102a in the culture membrane 10a does not dissolve, it can remain between the two cell layers, making it possible to use the poorly soluble material 102a as a support to make the cell layers less prone to breakage.

[0067] In this case, if the easily soluble material 101a is alginic acid, in the step of dissolving the easily soluble material in the culture membrane 10a, alginate-degrading enzyme can be supplied to at least one of the channels 110a and 130a to dissolve the culture membrane 10a. Furthermore, if the easily soluble material 101a is polyvinyl alcohol, the culture membrane 10a can be dissolved by heating the biological tissue formation apparatus 1a during the process of dissolving the easily soluble material in the culture membrane 10a.

[0068] As described above, according to the biological tissue formation apparatus and biological tissue formation method of this embodiment, in organ chips and the like, the culture membrane can be constructed using an easily soluble material and a poorly soluble material. After forming cell layers on both sides of this culture membrane, the easily soluble material is dissolved, which allows for highly efficient intercellular interactions and fluid component exchange between the cell layers of the formed biological tissue, and also makes the cell layers less prone to damage. [Examples]

[0069] The following describes experiments conducted to confirm the dissolution of the easily soluble material used in the biological tissue formation apparatus according to an embodiment of the present invention. The easily soluble material is used as a culture membrane placed between multiple cell layers after adherent cells have been cultured in the biological tissue formation apparatus, and is dissolved after the cell layers have been formed. However, in each embodiment, the formation of cell layers was omitted, and only the solubility of the easily soluble material was evaluated. Furthermore, while a culture medium is used when forming a cell layer using a biological tissue formation device, in each example and reference example, phosphate buffer was used instead of the culture medium to evaluate its solubility. The results are shown in Figure 8.

[0070] [Example 1] In this example, alginic acid was used as an easily soluble material in a biological tissue formation device, and an experiment was conducted to dissolve it with sodium citrate. Specifically, sodium alginate (Fujifilm Wako Pure Chemical Industries, Ltd., 194-13321) was dissolved in pure water to prepare a 1% sodium alginate aqueous solution. This sodium alginate aqueous solution was cast onto a PET film and dried overnight at room temperature (25°C).

[0071] Next, the alginate was immersed in a 0.5M calcium chloride aqueous solution for 10 minutes to crosslink, and then rinsed with pure water. After drying overnight at room temperature (25°C), a calcium alginate thin film (thickness approximately 10 μm) was prepared as an easily soluble material. The easily soluble material, cut into 10 mm squares, was immersed in 20 mL of phosphate buffer (Thermo Fisher Scientific Co., Ltd., 10010023), stored at 37°C for one week, and then the state of the easily soluble material and its transparency were checked.

[0072] The reason for storing the easily soluble material for a week and checking its condition before dissolving it is that a culture period of about one week is generally required for cell layer formation, and it is desirable that it not dissolve during that time. The reason for checking the transparency of the easily soluble material is that since the cell layer is formed on the surface of the easily soluble material, it is desirable for the easily soluble material to be transparent for easier observation of the cell layer. The above findings revealed that the sodium chloride contained in the phosphate buffer slightly dissolved the surface of the easily soluble material, reducing its transparency.

[0073] Furthermore, to dissolve the easily soluble material, sodium citrate was added to phosphate buffer to a concentration of 1%, and the mixture was immersed in the phosphate buffer at 37°C for 6 hours. As a result, the easily soluble material was completely dissolved in the phosphate buffer.

[0074] [Example 2] In this example, alginate was used as an easily soluble material in a biological tissue formation device, and an experiment was conducted to dissolve it using an alginate-degrading enzyme. Specifically, a calcium alginate thin film was prepared in the same manner as in Example 1, and this was used as an easily soluble material. After immersion in phosphate buffer and storage, the state and transparency of the easily soluble material were confirmed. As a result, the state and transparency of the easily soluble material were comparable to those before immersion in phosphate buffer.

[0075] Furthermore, to dissolve this easily soluble material, 100 μg of alginate-degrading enzyme (Nippon Gene Co., Ltd., 319-08261) was added to phosphate buffer, and the mixture was immersed in the phosphate buffer at 37°C for 6 hours. As a result, the easily soluble material was completely dissolved in the phosphate buffer.

[0076] [Example 3] In this example, polyvinyl alcohol was used as an easily soluble material in a biological tissue formation apparatus, and an experiment was conducted to dissolve it by heating. 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 produce a polyvinyl alcohol thin film (film thickness approximately 7 μm), which was then used as an easily soluble material.

[0077] Next, the easily soluble material, cut into 10mm squares, was immersed in 20mL of phosphate buffer (Thermo Fisher Scientific Co., Ltd., 10010023) and stored at 37°C for one week. After storage, the state and transparency of the easily soluble material were checked. As a result, the state and transparency of the easily soluble material were comparable to those before immersion in the phosphate buffer. Furthermore, to dissolve this easily soluble material, it was immersed in a phosphate buffer at 50°C for 3 hours. As a result, the easily soluble material dissolved completely in the phosphate buffer.

[0078] [Example 4] In this example, methylcellulose was used as an easily soluble material in a biological tissue formation apparatus, and an experiment was conducted to dissolve it by cooling. Specifically, methylcellulose (Shin-Etsu Chemical Co., Ltd., MCE-4000) 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 produce a methylcellulose thin film (thickness approximately 20 μm), which was then used as an easily soluble material.

[0079] Next, the easily soluble material, cut into 10mm squares, was immersed in 20mL of phosphate buffer (Thermo Fisher Scientific Co., Ltd., 10010023) and stored at 37°C for one week. After storage, the state and transparency of the easily soluble material were checked. As a result, the state and transparency of the easily soluble material were comparable to those before immersion in the phosphate buffer. Furthermore, to dissolve this easily soluble material, it was immersed in a phosphate buffer at 10°C for 10 minutes. As a result, a portion of the easily soluble material dissolved in the phosphate buffer.

[0080] Furthermore, when this easily soluble material was stored at 5°C to dissolve, it dissolved more readily in the phosphate buffer. Therefore, in order to make the easily soluble material dissolve better in the phosphate buffer at 10°C, the following experiment was conducted to adjust the dissolution temperature of the methylcellulose aqueous solution.

[0081] [Example 5] In this embodiment, methylcellulose was used as the easily soluble material in the biological tissue formation apparatus, and sodium styrene sulfonate was added as an additive. Experiments were conducted to dissolve these materials by cooling them. Specifically, to the methylcellulose aqueous solution prepared in the same manner as in Example 4, sodium styrene sulfonate (Fujifilm Wako Pure Chemical Industries, Ltd., 192-03292) was added to a concentration of 0.1 M to adjust the dissolution temperature. This methylcellulose aqueous solution was cast onto a PET film and dried overnight at room temperature (25°C) to produce a methylcellulose thin film (film thickness approximately 20 μm), which was then used as an easily soluble material.

[0082] Next, the easily soluble material, cut into 10mm squares, was immersed in 20mL of phosphate buffer (Thermo Fisher Scientific Co., Ltd., 10010023) and stored at 37°C for one week. After storage, the state and transparency of the easily soluble material were checked. As a result, the state and transparency of the easily soluble material were comparable to those before immersion in the phosphate buffer. Furthermore, to dissolve this easily soluble material, it was immersed in a phosphate buffer at 10°C for 10 minutes. As a result, the easily soluble material dissolved completely in the phosphate buffer.

[0083] [Reference example 1] In this reference example, instead of the easily soluble material used in the bio-tissue formation apparatus, a commercially available semipermeable membrane, a PET membrane (Corning, 353091), was used, and an experiment was conducted to dissolve it. Specifically, PET membranes cut into 10mm squares were immersed in 20mL of phosphate buffer (Thermo Fisher Scientific Co., Ltd., 10010023), stored at 37°C for one week, and then the state of easily soluble material and transparency were checked. As a result, the state of easily soluble material and transparency were comparable to those before immersion in phosphate buffer.

[0084] Furthermore, the PET membrane was immersed in phosphate buffer at 90°C for 24 hours. As a result, the PET membrane did not dissolve, and no changes were observed. Furthermore, to enzymatically decompose the PET membrane, 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 membrane did not dissolve, and no changes were observed.

[0085] The results from Reference Example 1 revealed that PET membranes cannot be used as a substitute for easily soluble materials in a bio-tissue formation apparatus. On the other hand, it was found that PET membranes can be suitably used as poorly soluble materials in a bio-tissue formation apparatus.

[0086] The present invention is not limited to the embodiments and examples described above, and it goes without saying that various modifications can be made within the scope of the present invention. For example, the shape of the flow channels and culture membranes in a biological tissue manufacturing apparatus are not limited to those shown in Figure 1, etc., and can be changed to various other shapes as appropriate. [Industrial applicability]

[0087] The present invention can be suitably used in cases such as when forming biological tissue using organ chips or the like.

[0088] All documents cited in this specification and the contents of the Japanese application specification on which this application has priority in Paris Convention are incorporated herein by reference. [Explanation of Symbols]

[0089] 1,1a Biological tissue formation apparatus 10,10a culture membrane 101,101a Easily soluble materials 102a Hardly soluble materials 11,11a,13,13a Flow channel plate 110, 110a, 130, 130a flow path 12,12a,14,14a Adhesive layer 20. Biological tissue formation device 210 Semi-permeable membrane 211,213 Flow channel plate 30,31 cells 40,41 Culture solution 50 Base material

Claims

1. A biological tissue forming device that forms a biological tissue having multiple cell layers composed of adherent cells, A culture membrane having culture regions for the adherent cells on both sides, which is placed between the plurality of cell layers after the adherent cells have been cultured, The system comprises a plurality of channels separated by the culture membrane, The culture membrane consists of an easily soluble material and a poorly soluble material. The culture membrane contains the poorly soluble material such that after the easily soluble material in the culture membrane is dissolved, pores penetrating the culture membrane are formed. The readily soluble material is polyvinyl alcohol, alginic acid, or methylcellulose. The aforementioned poorly soluble material is polyethylene terephthalate, polylactic acid, or UV-curing resin. The poorly soluble material is used as a support for the cell layer after the easily soluble material in the culture membrane has been dissolved. A biological tissue formation apparatus characterized by the following features.

2. The biological tissue forming apparatus according to claim 1, characterized in that the readily soluble material and the poorly soluble material are used as a scaffold in the culture of the adherent cells.

3. A biological tissue forming apparatus according to claim 1 or 2, characterized in that it comprises one culture membrane and two channels separated by the culture membrane, and a biological tissue having two cell layers is formed.

4. The biological tissue formation apparatus according to claim 3, characterized in that the two channels are formed by the channel-side surfaces of two plates equipped with channels adhering to both surfaces of the culture membrane, respectively.

5. The biological tissue forming apparatus according to claim 3 or 4, characterized in that a cell layer consisting of different types of adherent cells is formed in each of the two channels.

6. The biological tissue forming apparatus according to any one of claims 1 to 5, characterized in that a cell layer consisting of adherent cells is formed on both sides of the culture membrane.

7. The biological tissue forming apparatus according to claim 6, characterized in that the cell layer is formed and the easily soluble material is dissolved.

8. The biological tissue forming apparatus according to claim 1, characterized in that the culture membrane is formed containing adherent cells.

9. A method for forming a biological tissue having multiple cell layers composed of adherent cells, A biological tissue formation apparatus comprising: a culture membrane having culture regions for adherent cells on both sides and being placed between the plurality of cell layers after the adherent cells have been cultured; and two channels separated by the culture membrane, the culture membrane being made of an easily soluble material and a poorly soluble material, wherein adherent cells and culture medium are supplied to the two channels. The steps include culturing the adherent cells in the two channels and forming cell layers on both sides of the culture membrane, The process includes a step of dissolving the easily soluble material in the culture membrane, The culture membrane contains the poorly soluble material such that after the easily soluble material in the culture membrane is dissolved, pores penetrating the culture membrane are formed. The readily soluble material is polyvinyl alcohol, alginic acid, or methylcellulose. The aforementioned poorly soluble material is polyethylene terephthalate, polylactic acid, or UV-curing resin. The poorly soluble material is used as a support for the cell layer after the easily soluble material in the culture membrane has been dissolved. A method for forming biological tissue characterized by the following features.

10. The method for forming biological tissue according to claim 9, characterized in that, when the readily soluble material is alginic acid, in the dissolution step, an alginate-degrading enzyme is supplied to at least one of the two channels to dissolve the readily soluble material in the culture membrane.

11. The method for forming a biological tissue according to claim 9, characterized in that, when the readily soluble material is polyvinyl alcohol, the biological tissue forming apparatus is heated in the dissolution step to dissolve the readily soluble material in the culture membrane.

12. The method for forming a biological tissue according to claim 9, characterized in that, when the readily soluble material is methylcellulose, the biological tissue forming apparatus is cooled in the dissolution step to dissolve the readily soluble material in the culture membrane.

13. A method for forming a biological tissue according to any one of 9 to 12, characterized in that the step of dissolving the culture membrane is performed at the timing when a cell layer has been formed throughout the culture region.

14. A method for forming biological tissue according to any one of 9 to 13, characterized in that a cell layer consisting of different types of adherent cells is formed in each of the two channels.

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