Cell culture method, cell culture vessel, method for manufacturing a cell culture vessel, and cell-containing structure

Abstract

A cell culture method including a step for placing a culture medium and cells in a cell culture container and performing the adherent culture of the cells on a coat layer layered on the surface of the cell culture container, in which the coat layer comprises a layer (i) including a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material and a layer (ii) layered on the layer (i) and containing a cell adhesion factor, the layer (ii) is arranged at a position at which the layer (ii) comes into contact with the cells or alternatively the coat layer includes the layer (i) and the layer (i) is arranged at a position at which the layer (i) comes into contact with the cells, and the culture medium contains a cell adhesion factor.

Description

Technical Field

[0001] The present invention relates to a cell culture method, a container for cell culture, a method for producing a container for cell culture, and a cell-containing structure. The present application claims priority based on Japanese Patent Application No. 2021-125556 filed in Japan on July 30, 2021, and Japanese Patent Application No. 2022-044329 filed in Japan on March 18, 2022, the contents of which are incorporated herein by reference. Background Art

[0002] In recent years, along with expectations for the realization of regenerative medicine for the nervous system, research on nerve regenerative medicine has been actively conducted. For this purpose, it is necessary to evaluate the function of cell aggregates containing nerve cells and investigate pharmacological effects. Among these, functional evaluation of neural networks composed of aggregates of nerve cells is particularly important because it can provide electrophysiological findings.

[0003] Currently, functional evaluation of neural networks is performed not only by detecting changes in Ca concentration and observing synaptic activity in general-purpose planar culture plates, but also by extracellular potential measurement using a planar microelectrode array (Microelectrode Array, MEA). In order to perform functional evaluation of these neural networks, it is necessary to culture nerve cell aggregates for a long period of time. Furthermore, in the case of human iPS cell-derived nerve cells, it is necessary to culture nerve cells at a higher density and for a longer period of time than in the case of nerve cells derived from non-human animals.

[0004] On the other hand, it is known that during long-term culture, nerve cell aggregates are easily detached from the culture surface of the culture container. This is a major factor that causes variations in the results of functional evaluation and pharmacological effects of nerve cell aggregates.

[0005] Generally, in order to suppress the detachment of nerve cell aggregates from the culture surface of a culture container, it is practiced to coat the culture surface of the culture container with a base (polycation material) before culture, and then coat a cell adhesion factor thereon. These procedures are publicly disclosed by companies that commercially supply nerve cells and companies that commercially supply MEA measurement instruments.

[0006] Furthermore, if the effect is insufficient with just the primer and cell adhesion factor coating, methods for pre-treating the culture surface before coating with the primer and cell adhesion factor, such as exposing it to ozone plasma or immersing it in concentrated sterile protein solution, have also been published.

[0007] For example, Non-Patent Document 1 discloses a method in which the culture surface of a cell culture vessel is exposed to ozone plasma for one minute. However, this method requires an expensive ozone plasma device. Furthermore, because the culture surface becomes extremely hydrophilic, the cell suspension spreads throughout when cells are seeded, requiring a large number of cells in the case of MEA, and there is a problem that cells may be seeded onto the reference electrode.

[0008] Non-patent document 2 discloses a method of immersing a culture surface in a concentrated sterile protein solution. However, it only lists serum culture medium, albumin solution, etc., as examples of concentrated sterile protein solutions, and does not specify conditions such as the concentration of the concentrated sterile protein solution, making it unclear.

[0009] Non-patent document 3 discloses a method in which serum culture medium is immersed in the culture surface for 1 to 2 minutes. This method does indeed show an effect in suppressing cell detachment from the culture surface. However, our own verification results showed that there is room for further improvement in order to suppress the detachment of cell aggregates, including nerve cells, and reduce the variability in the results of functional evaluation.

[0010] Furthermore, methods have been published for giving the substrate of cell culture containers properties that suppress cell detachment. For example, Patent Document 1 describes a cell culture container having a laminated membrane in which a collagen layer is provided on a silicone rubber membrane. However, this method cannot be applied to existing cell culture containers that are generally widely available, and it is not practical due to the cost involved. [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention aims to provide a cell culture method, a cell culture container, and a method for manufacturing the same, which can suppress the detachment of cell aggregates from the culture surface and obtain stable functional evaluation results. [Means for solving the problem]

[0012] The cell culture method according to the present invention comprises the steps of placing a culture medium and cells in a cell culture container and adhering the cells on a coating layer laminated on the surface of the cell culture container, wherein the coating layer comprises a layer (i) comprising a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and a layer (ii) containing a cell adhesion factor laminated on layer (i), wherein layer (ii) is positioned in contact with the cells, or the coating layer comprises layer (i), and layer (i) is positioned in contact with the cells, and the culture medium contains a cell adhesion factor.

[0013] The cell culture container according to the present invention has a coating layer laminated on its surface, and the coating layer comprises layer (i) which includes a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and layer (ii) which is laminated on layer (i) and contains a cell adhesion factor, and layer (ii) is positioned to come into contact with cells during cell culture.

[0014] The present invention provides a method for manufacturing a cell culture container with a modified culture surface, comprising the steps of: 1) contacting the surface of the cell culture container with a solution containing gelatin or casein; 2) removing the solution containing gelatin or casein; 3) contacting the surface of the cell culture container with a solution containing a polycationic material; 4) removing the solution containing the polycationic material; 5) contacting the surface of the cell culture container with a solution containing a cell adhesion factor; and 6) removing the solution containing the cell adhesion factor, in this order; or comprising the steps of 3, 4, 1, 2, 5, and 6 in this order.

[0015] The cell-containing structure according to the present invention comprises a cell culture container, cells, and a culture medium, wherein the cell culture container contains the culture medium and the cells, the cell culture container has a coated layer laminated on its surface, the cells are adhered to the coated layer, and the coated layer comprises a layer (i) comprising a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and a layer (ii) laminated on layer (i) containing a cell adhesion factor, wherein layer (ii) is positioned in contact with the cells, or the coated layer comprises layer (i) and is positioned in contact with the cells, and the culture medium contains a cell adhesion factor. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a cell culture method, a cell culture container, and a method for manufacturing the same, which can suppress the detachment of cell aggregates from the culture surface and obtain stable functional evaluation results. [Brief explanation of the drawing]

[0017] [Figure 1] Figures 1(a) to 1(d) are schematic diagrams of a cell culture method according to one embodiment. [Figure 2] Figures 2(a) and 2(b) are schematic cross-sectional views of a cell culture vessel according to one embodiment. [Figure 3] Figures 3(a) to 3(h) are representative images of cell aggregates cultured in the cell culture vessels of Examples 1 and 2 and Comparative Examples 1 and 2. [Figure 4] Figure 4 shows representative images of cell aggregates cultured in cell culture vessels from Example 3 and Comparative Examples 3-4, transported by air, and the number of effective electrodes that detected their action potentials. [Figure 5] Figures 5(a) to 5(c) are graphs showing the variability (CV value) of spontaneous firing number, burst number, and synchronous burst number of cell aggregates cultured in the cell culture vessels of Example 3 and Comparative Examples 3 to 4 and transported by air. [Figure 6]Figures 6(a) to (c) are graphs showing variations (CV values) in the number of spontaneous firings, the number of bursts, and the number of synchronous bursts of cell aggregates cultured in the cell culture containers of Example 3 and Comparative Examples 3 to 5 and allowed to stand overnight at room temperature. [Figure 7] Figures 7(a) to (b) are representative images obtained by capturing cell aggregates cultured in the cell culture containers of Example 4 and Comparative Example 6. [Figure 8] Figures 8(a) to (b) are a mapping image of effective electrodes that detected action potentials of cell aggregates cultured in the cell culture containers of Example 4 and Comparative Example 6, and an image showing the proportion of the number of effective electrodes. [Figure 9] Figure 9 is a representative image obtained by capturing cell aggregates cultured in the cell culture containers of Example 5 and Comparative Example 7. [Figure 10] Figures 10(a) to (d) are representative photographs obtained by capturing cell aggregates cultured in the cell culture containers of Example 6 and Comparative Example 8. [Figure 11] Figure 11 is a representative photograph obtained by capturing cell aggregates cultured in the cell culture containers of Examples 6 to 7 and Comparative Examples 8 to 16. [Figure 12] Figure 12 is a representative photograph obtained by capturing cell aggregates cultured in the cell culture containers of Examples 6 to 7 and Comparative Examples 8 to 13. [Figure 13] Figure 13 is a representative photograph obtained by capturing cell aggregates cultured in the cell culture containers of Example 10 and Comparative Example 21. MODE FOR CARRYING OUT THE INVENTION

[0018] Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate. Note that dimensional ratios in each drawing are exaggerated for explanatory purposes in some parts, and do not necessarily match the actual dimensional ratios.

[0019] [Cell culture method using modified culture surface] A cell culture method in one embodiment includes the steps of placing a culture medium and cells in a cell culture container and adhering the cells to a coating layer laminated on the surface of the cell culture container, wherein the coating layer comprises a layer (i) comprising a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and a layer (ii) laminated on layer (i) containing a cell adhesion factor, wherein layer (ii) is positioned in contact with the cells, or the coating layer comprises layer (i) and layer (i) is positioned in contact with the cells, and the culture medium contains a cell adhesion factor.

[0020] Figures 1(a) to 1(d) are schematic diagrams showing examples of the cell culture method of this embodiment. The cell culture method shown in Figure 1(a) is an example of a method in which a culture medium and cells are placed in a cell culture container and the cells are adherently cultured on a coating layer laminated on the surface of the cell culture container. In this example, a coating layer is laminated on the surface of the cell culture container, and the cells are adherently cultured on the coating layer. On the surface of the substrate 11 of the cell culture container, a layer 12 containing gelatin or casein, a layer 13 containing a polycationic material, and a layer 14 containing a cell adhesion factor are laminated in this order. The cells 15 are adhered to layer 14.

[0021] The cell culture method shown in Figure 1(b) is an example of a method in which a culture medium and cells are placed in a cell culture container, and the cells are cultured on a coated layer laminated on the surface of the cell culture container. In this example, a coated layer is laminated on the surface of the cell culture container, and the cells are cultured on the coated layer. On the surface of the substrate 11 of the cell culture container, a layer 12 containing gelatin or casein and a layer 13 containing a polycationic material are laminated in that order. The cells 15 are adhered to layer 13. The culture medium 16 also contains cell adhesion factors.

[0022] The cell culture method shown in Figure 1(c) is an example of a method in which a culture medium and cells are placed in a cell culture container, and the cells are cultured on a coated layer laminated on the surface of the cell culture container. In this example, a coated layer is laminated on the surface of the cell culture container, and the cells are cultured on the coated layer. On the surface of the substrate 11 of the cell culture container, a layer 13 containing a polycationic material, a layer 12 containing gelatin or casein, and a layer 14 containing a cell adhesion factor are laminated in this order. The cells 15 are adhered to layer 14.

[0023] The cell culture method shown in Figure 1(d) is an example of a method in which a culture medium and cells are placed in a cell culture container, and the cells are cultured on a coated layer laminated on the surface of the cell culture container. In this example, a coated layer is laminated on the surface of the cell culture container, and the cells are cultured on the coated layer. On the surface of the substrate 11 of the cell culture container, a layer 13 containing a polycationic material and a layer 12 containing gelatin or casein are laminated in this order. The cells 15 are adhered to layer 12. The culture medium 16 also contains cell adhesion factors.

[0024] A coating layer is laminated on the surface of the substrate of the cell culture container. The coating layer includes layer (i). Layer (i) includes layer (i-1) containing gelatin or casein, and layer (i-2) containing a polycationic material. These layers are arranged in the order of substrate of the cell culture container, layer (i-1), layer (i-2), or substrate of the cell culture container, layer (i-2), layer (i-1).

[0025] When the layers are arranged in the order of substrate, layer (i-1), and layer (i-2) of the cell culture vessel, it is preferable that the substrate and layer (i-1) are adjacent and that no other material is present between the surface of the substrate and layer (i-1). Furthermore, it is preferable that layer (i-1) and layer (i-2) are adjacent and that no other material is present between layer (i-1) and layer (i-2).

[0026] When the layers are arranged in the order of substrate, layer (i-2), and layer (i-1) of the cell culture vessel, it is preferable that the substrate and layer (i-2) are adjacent and that no other material exists between the surface of the substrate and layer (i-2). Furthermore, it is preferable that layer (i-2) and layer (i-1) are adjacent and that no other material exists between layer (i-2) and layer (i-1).

[0027] The layer (i) is positioned so as to be in contact with the cells, and the culture medium may contain cell adhesion factors. In this case, it is preferable that the layer (i) and the cells are adjacent and that no other material is present between the layer (i) and the cells.

[0028] A layer (ii) containing cell adhesion factors may be further stacked on layer (i). In this case, the arrangement of these layers is in the order of the cell culture container substrate, layer (i), and layer (ii). Preferably, layer (i) and layer (ii) are adjacent to each other, and no other material is present between them. In this case, layer (ii) is positioned so as to be in contact with the cells. Furthermore, preferably, layer (ii) and the cells are adjacent to each other, and no other material is present between layer (ii) and the cells.

[0029] As described later in the examples, the inventors have found that a cell culture method in which a coating layer having the above configuration is laminated on the surface of a cell culture container and cells are cultured on the coating layer can significantly suppress the peeling of cells from the culture surface, even when the cells are prone to peeling.

[0030] Therefore, the cell culture method of this embodiment is particularly suitable for culturing cells that easily detach. Examples of cells that easily detach include cells that detach from the culture surface of a cell culture vessel simply by applying shock or vibration to the vessel. Specific examples of cells that easily detach include cell aggregates containing nerve cells and the human embryonic kidney cell line HEK293. Cells that detach to a similar or greater degree (or less with similar or less severe shock or vibration) are included in the definition of "easily detachable cells" in this disclosure.

[0031] When the detachment of cell aggregates containing nerve cells from the culture surface is suppressed, the action potentials of nerve cells can be stably detected and evaluated using MEA or other methods. Furthermore, stable evaluation results can be obtained through other analytical methods such as detection of changes in Ca concentration, observation of synaptic activity, and immunohistochemistry.

[0032] As described later in the examples, the inventors have demonstrated that the cell culture method of this embodiment has a sufficiently large effect in suppressing cell detachment from the culture surface. Specifically, compared to the cell culture method described in Non-Patent Document 3 above, which uses a cell culture container immersed in serum medium on the culture surface, the inventors have demonstrated that the variability between wells in extracellular potential measurement of MEA is reduced, and stable cell functionality evaluation results can be obtained.

[0033] As described above, the cell culture container used in the cell culture method of this embodiment includes a coating layer, and the coating layer includes a layer (i-1) containing gelatin or casein. Examples of gelatin include bovine bone-derived gelatin, bovine hide-derived gelatin, pigskin-derived gelatin, donkey hide-derived gelatin, chicken skin-derived gelatin, and fish-derived gelatin. Examples of fish-derived gelatin include fish scale-derived gelatin and fish skin-derived gelatin. Among these, fish-derived gelatin is particularly preferred because it has a low gelling temperature and is easy to handle. These may be used individually or in combination of two or more types.

[0034] Examples of casein include bovine casein, goat casein, sheep casein, and human casein. Among these, bovine casein is preferred because it is abundant in milk, inexpensive, and readily available. These can be used individually or in combination of two or more types.

[0035] As described above, the cell culture container used in the cell culture method of this embodiment includes a coating layer, and the coating layer includes a layer (i-2) containing a polycationic material. There are no particular restrictions on the polycationic material, and it can be appropriately selected depending on the purpose, but polyethyleneimine (PEI), polylysine (PLL or PDL), and polyornithine (PLO or PDLO), which have many examples of use in cell culture, are preferred. These may be used individually or in combination of two or more.

[0036] As described above, the cell culture container used in the cell culture method of this embodiment includes a coating layer. The coating layer includes layer (i) and layer (ii) which is laminated on layer (i) and contains cell adhesion factors, or the coating layer includes layer (i) and the culture medium contains cell adhesion factors.

[0037] There are no particular restrictions on the cell adhesion factors, and they can be appropriately selected depending on the purpose, but extracellular matrix is ​​preferred. The extracellular matrix, also called the extracellular matrix, is a substance that serves as a scaffold for cells. Examples of extracellular matrix include laminin, collagen, fibronectin, fibrinogen, Matrigel (registered trademark, Corning), and Geltrex (Thermo Fisher Scientific). These may be used individually or in combination of two or more.

[0038] In particular, when the cells to be cultured are a cell aggregate including nerve cells, laminin and Matrigel® are preferred from the viewpoint of promoting the maturation of nerve cells.

[0039] [Cell culture vessel with modified culture surface] In one embodiment, the cell culture container is a cell culture container with a modified culture surface. The cell culture container of this embodiment has a coating layer laminated on its surface, and the coating layer includes layer (i) which includes a layer containing gelatin or casein (i-1) and a layer containing a polycationic material (i-2), and layer (ii) which is laminated on layer (i) and contains a cell adhesion factor, with layer (ii) positioned to come into contact with cells during cell culture. The culture surface refers to the surface of the cell culture container to which cells adhere during cell culture. When the coating layer is laminated on the surface of the substrate of the cell culture container, the culture surface is the surface of the coating layer.

[0040] Figures 2(a) and 2(b) are schematic cross-sectional views of the cell culture surface portion of a cell culture container, each representing an example of a cell culture container according to this embodiment. In the cell culture container shown in Figure 2(a), a layer 22 containing gelatin or casein, a layer 23 containing a polycationic material, and a layer 24 containing a cell adhesion factor are laminated on the surface of a base material 21 in this order.

[0041] Here, it is preferable that the base material 21 and layer 22 are adjacent, and that no other material exists between the surface of the base material 21 and layer 22. Furthermore, it is preferable that layer 22 and layer 23 are adjacent, and that no other material exists between layer 22 and layer 23. Also, it is preferable that layer 23 and layer 24 are adjacent, and that no other material exists between layer 23 and layer 24.

[0042] The cell culture container shown in Figure 2(b) has a base material 21 on which layers 23 containing a polycationic material, 22 containing gelatin or casein, and 24 containing a cell adhesion factor are stacked in that order on the surface.

[0043] Here, it is preferable that the base material 21 and layer 23 are adjacent, and that no other material exists between the surface of the base material 21 and layer 23. Furthermore, it is preferable that layer 23 and layer 22 are adjacent, and that no other material exists between layer 23 and layer 22. Also, it is preferable that layer 22 and layer 24 are adjacent, and that no other material exists between layer 22 and layer 24.

[0044] As will be described later in the examples, the present inventors have found that when cells are cultured using a cell culture container on which a coating layer having the above configuration is laminated on the surface of the cell culture container, the peeling of cells from the culture surface can be significantly suppressed, even when the cells are prone to peeling.

[0045] Therefore, the cell culture vessel of this embodiment is suitable for culturing easily detachable cells. Examples of easily detachable cells include cells that detach from the culture surface of the cell culture vessel simply by applying shock or vibration to the vessel. Specific examples of easily detachable cells include cell aggregates containing nerve cells and the human embryonic kidney cell line HEK293.

[0046] As described above, suppressing the detachment of cell aggregates containing nerve cells from the culture surface allows for stable detection and evaluation of nerve cell action potentials using MEA, etc. Furthermore, stable evaluation results can be obtained through other analytical methods such as detection of Ca concentration changes, observation of synaptic activity, and immunohistochemistry.

[0047] As described later in the examples, the inventors have demonstrated that the cell culture container of this embodiment has a sufficiently large effect in suppressing cell detachment from the culture surface. Specifically, compared to the cell culture container described in Non-Patent Document 3 above, in which serum medium is immersed in the culture surface, the variability between wells in extracellular potential measurement of MEA is reduced, and stable cell functionality evaluation results can be obtained.

[0048] The cell culture container may be any cell culture container commonly used for cell culture, such as a dish or a well plate. The diameter of the dish, the number of wells in the well plate, etc., can be appropriately selected according to the application. The culture surface refers to the surface of the cell culture container to which cells adhere during cell culture.

[0049] The cell culture vessel of this embodiment may have an electrode array arranged on its culture surface. In other words, the cell culture vessel of this embodiment may be an MEA plate. The number of electrodes in the MEA can be appropriately selected depending on the application.

[0050] Examples of materials for the substrate of cell culture containers include the organic and inorganic materials described below. These may be used individually or in combination of two or more materials.

[0051] There are no particular restrictions on the organic materials used, and they can be appropriately selected according to the purpose. Examples include polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetylcellulose), polyimide (PI), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, polyacrylamide, urethane acrylate, and other acrylic materials; cellulose, polydimethylsiloxane (PDMS), and other silicone materials; polyvinyl alcohol (PVA), alginate metal salts such as calcium alginate, methylcellulose, agarose, and other gel-like materials.

[0052] There are no particular restrictions on the inorganic materials used; they can be appropriately selected according to the purpose, and examples include glass and ceramics.

[0053] As described above, the cell culture container of this embodiment includes a layer containing gelatin or casein. The gelatin is as described above, and is preferably fish-derived gelatin. The casein is also as described above. Furthermore, as described above, the cell culture container of this embodiment includes a layer containing a polycationic material. The polycationic material is as described above, and is preferably polyethyleneimine, polylysine, or polyornithine. Furthermore, as described above, the cell culture container of this embodiment includes a layer containing a cell adhesion molecule. The cell adhesion molecule is as described above, and is preferably laminin or Matrigel®.

[0054] [Method for manufacturing cell culture vessels with modified culture surfaces] In one embodiment, the present invention provides a method for manufacturing a cell culture container with a modified culture surface, comprising the steps of: 1) contacting the surface of a cell culture container with a solution containing gelatin or casein; 2) removing the solution containing gelatin or casein; 3) contacting the surface of the cell culture container with a solution containing a polycationic material; 4) removing the solution containing the polycationic material; 5) contacting the surface of the cell culture container with a solution containing a cell adhesion factor; and 6) removing the solution containing the cell adhesion factor, in this order; or comprising the steps of 3, 4, 1, 2, 5, and 6 in this order.

[0055] The cell culture container described above can be manufactured by the manufacturing method of this embodiment. As will be described later in the examples, by culturing a cell aggregate containing nerve cells in a cell culture container manufactured by the manufacturing method of this embodiment, the detachment of cells from the culture surface can be significantly suppressed.

[0056] The cell culture vessels are the same as those described above. The cell culture vessels may be dishes, well plates, or MEA plates with an electrode array placed on the culture surface.

[0057] If the manufacturing method of this embodiment includes the above steps in the order of step 1, step 2, step 3, step 4, step 5, and step 6, it may further include a step of hydrophilizing the surface of the cell culture container before step 1. Alternatively, if the manufacturing method of this embodiment includes the above steps in the order of step 3, step 4, step 1, step 2, step 5, and step 6, it may further include a step of hydrophilizing the surface of the cell culture container before step 3.

[0058] By hydrophilizing the cell culture surface, surface modification with gelatin, casein, or polycationic materials, as described later, becomes easier. Hydrophilizing the cell culture surface may be carried out by methods known in the art, and is not limited thereto, but examples include treating the cell culture surface with ethanol or pure water, or treating the cell culture surface with ozone plasma. Below, the hydrophilizing treatment will be described using the method of treating the cell culture surface with ethanol as an example.

[0059] The step of hydrophilizing the surface of the cell culture container may include a step of bringing a solution containing ethanol into contact with the surface of the cell culture container (a) and a step of removing the solution containing ethanol (b).

[0060] In step (a), the surface of the cell culture vessel is brought into contact with a solution containing ethanol. Specifically, the solution containing ethanol may be added to the cell culture vessel, or the entire cell culture vessel may be immersed in the solution containing ethanol.

[0061] As the solution containing ethanol, an aqueous ethanol solution is preferred. The concentration of ethanol is preferably about 70% by mass. There are no particular restrictions on the aqueous ethanol solution, but in order to minimize the impact on cells, it is preferable to prepare it from a highly purified ethanol solution and ultrapure water.

[0062] The contact time for the ethanol-containing solution is preferably 2 to 10 minutes. Furthermore, the temperature at which the ethanol-containing solution is applied is preferably room temperature, around 18 to 25°C.

[0063] Next, in step (b), the solution containing ethanol is removed. After removing the solution containing ethanol, it is preferable to dry the culture surface. The drying method is not particularly limited, but examples include air drying in a sterile environment.

[0064] Next, in step 1, a solution containing gelatin or casein is brought into contact with the surface of the cell culture container. Here, the surface of the cell culture container refers to the surface that has been modified by contact with the solution containing ethanol. Specifically, the solution containing gelatin or casein may be added to the cell culture container, or the entire cell culture container may be immersed in the solution containing gelatin or casein.

[0065] The gelatin is the same as described above, and fish-derived gelatin is preferred. Examples of gelatin-containing solutions include aqueous gelatin solutions and gelatin buffer solutions. The materials contained in the gelatin-containing solution are not particularly limited as long as they are not toxic to cells, and can be appropriately selected according to the purpose. However, the concentration of gelatin in the solution is preferably 0.05 to 2% by mass, from the viewpoint of being able to uniformly layer it on the surface of the cell culture vessel and manufacturing it inexpensively.

[0066] The same applies to casein as described above. Examples of casesin-containing solutions include aqueous casein solutions and buffer solutions of casein. The materials included in the casein-containing solution are not particularly limited as long as they are not toxic to cells, and can be appropriately selected according to the purpose. However, the concentration of casein in the solution is preferably 0.01 to 2% by mass, from the viewpoint of being able to uniformly layer it on the surface of the cell culture vessel and manufacturing it inexpensively.

[0067] It is preferable that the contact time for the solution containing gelatin or casein is 1 hour or more. Furthermore, it is preferable that the temperature when the solution containing gelatin or casein is contacted be less than 40°C so as not to lose the properties of the gelatin or casein. Specifically, for example, by letting it stand at approximately 37°C for 1 hour, the gelatin or casein can be efficiently layered onto the surface of the cell culture container.

[0068] Next, in step 2, the solution containing gelatin or casein is removed. After removing the solution containing gelatin or casein, the surface of the cell culture vessel may be washed with water or buffer solution. The number of washes may be one to several times. For example, as is common in cell culture, it may be two to four times. Subsequently, it is preferable to dry the surface of the cell culture vessel. The drying method is not particularly limited, but examples include air drying in a sterile environment.

[0069] Next, in step 3, a solution containing the polycation material is brought into contact with the surface of the cell culture container. Specifically, the solution containing the polycation material may be added to the cell culture container, or the entire cell culture container may be immersed in the solution containing the polycation material.

[0070] The polycation material is the same as described above, and is preferably polyethyleneimine, polylysine, or polyornithine. Examples of solutions containing the polycation material include aqueous solutions of the polycation material and buffer solutions of the polycation material. The concentration of the polycation material in the solution is preferably 0.001 to 0.2% by mass, as has been observed in numerous cell culture applications.

[0071] It is preferable that the contact time for the solution containing the polycation material is one hour or more. Furthermore, there are no particular restrictions on the temperature at which the solution containing the polycation material is contacted. Specifically, for example, as has been done in many cell culture applications, the polycation material can be efficiently deposited onto the surface of the cell culture vessel by standing it at approximately 37°C for one hour.

[0072] Next, in step 4, the solution containing the polycationic material is removed. After removing the solution containing the polycationic material, the washing can be performed one to several times. For example, as is common in cell culture, it may be performed two to four times. Subsequently, it is preferable to dry the surface of the cell culture container. The drying method is not particularly limited, but examples include air drying in a sterile environment.

[0073] Next, in step 5, the surface of the cell culture container is brought into contact with a solution containing cell adhesion factors. Here, the surface of the cell culture container refers to the surface that has been modified by carrying out steps 1, 2, 3, and 4 described above. Specifically, the solution containing cell adhesion factors may be added to the surface of the cell culture container, or the entire cell culture container may be immersed in the solution containing cell adhesion factors.

[0074] The cell adhesion factors are the same as those described above, and are preferably extracellular matrix. Examples of solutions containing cell adhesion factors include aqueous solutions of cell adhesion factors and buffer solutions of cell adhesion factors. There are many examples of cell culture in which the concentration of cell adhesion factors in the solution is used, but it is preferably 5-20 μg / mL for laminin and 10-50 μg / mL for Matrigel.

[0075] It is preferable that the solution containing the cell adhesion factors be left in contact for at least one hour. Furthermore, there are no particular restrictions on the temperature at which the solution containing the cell adhesion factors is left in contact. Specifically, for example, as has been done in numerous cell culture applications, leaving the solution at approximately 37°C for one hour allows for efficient deposition of the cell adhesion factors onto the surface of the cell culture vessel.

[0076] Next, in step 6, the solution containing the cell adhesion molecule is removed. After removing the solution containing the cell adhesion molecule, the surface of the cell culture vessel may be washed with water or buffer solution. The washing may be done one to several times. If the cell adhesion molecule is Matrigel, the surface of the cell culture vessel may then be dried. The drying method is not particularly limited, but examples include air drying in a sterile environment.

[0077] In the manufacturing method of this embodiment, steps 1 to 6 described above may be carried out in this order. Alternatively, the above steps may be carried out in the order of step 3, step 4, step 1, step 2, step 5, and step 6. Furthermore, when performing hydrophilization treatment, the above steps may be carried out in the order of step (a), step (b), step 1, step 2, step 3, step 4, step 5, and step 6. Alternatively, the above steps may be carried out in the order of step (a), step (b), step 3, step 4, step 1, step 2, step 5, and step 6. In other words, the order in which the contact and removal of the solution containing gelatin or casein and the contact and removal of the solution containing the polycationic material are carried out may be reversed. As will be described later in the examples, in any case, it is possible to manufacture a cell culture container that can significantly suppress the peeling of cells from the culture surface.

[0078] [Cells and culture media] The cells cultured in the cell culture vessel of this embodiment are not particularly limited as long as they are adherent cells and can be appropriately selected according to the purpose. Examples include endothelial cells such as hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, endothelial cells, and corneal endothelial cells; epidermal cells such as fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, and epidermal keratinocytes; epithelial cells such as tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells; muscle cells such as mammary gland cells, pericytes, smooth muscle cells, and cardiomyocytes; nerve cells such as renal cells, pancreatic islet cells, peripheral nerve cells, and optic nerve cells; chondrocytes; and osteocytes. Among these, nerve cells are preferred as adherent cells.

[0079] The nerve cells may be primary cells directly collected from tissues or organs, or they may be primary cells that have been passaged through several generations. Furthermore, from the perspective of easily obtaining a cell population containing a large number of the desired nerve cells, cells differentiated from stem cells may also be used; in other words, stem cell-derived nerve cells may be used.

[0080] Examples of stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells, mesenchymal stem cells, umbilical cord blood-derived stem cells, and neural stem cells. Examples of induced pluripotent stem cells include nuclear transfer embryonic stem cells (ntES cells) and induced pluripotent stem cells (iPS cells). Examples of mesenchymal stem cells include bone marrow mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells. Among these, iPS cells are preferred as stem cells.

[0081] iPS cells may be derived from healthy individuals or from patients with various neurological diseases. They may also be genetically edited, for example, cells that have been modified to possess genes that cause or are risk factors for various neurological diseases.

[0082] If iPS cells are derived from patients with various neurological disorders, they can be used to construct disease models of those disorders. Neurological disorders, while not limited to those mentioned above, include, for example, neurodegenerative diseases, autism, epilepsy, attention-deficit hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder. Examples of neurodegenerative diseases include, for example, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS).

[0083] The animal species from which nerve cells originate are not particularly limited, and examples include humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, and hamsters. Among these, humans are preferred.

[0084] Furthermore, nerve cells may consist of a single type or a mixture of two or more types. Nerve cells can be broadly classified into peripheral nerves and central nerves. Examples of peripheral nerves include sensory nerves, motor nerves, and autonomic nerves. Examples of central nerves include interneurons and projection neurons. Examples of projection neurons include cortical neurons, hippocampal neurons, and amygdala neurons. Central nerve cells can also be broadly classified into excitatory neurons and inhibitory neurons.

[0085] Examples of neurons in the central nervous system that primarily handle excitatory transmission include glutamatergic neurons and GABAergic (γ-aminobutyric acid) neurons that primarily handle inhibitory transmission. Other types of neurons that release neuromodulatory substances include cholinergic neurons, dopaminergic neurons, noradrenergic neurons, serotonergic neurons, and histaminergic neurons.

[0086] The cells cultured in the cell culture vessel of this embodiment may include astrocytes, microglia, etc., along with nerve cells. Depending on the purpose, it is preferable that the nerve cells are mature, and for example, it is preferable that they are positive for expression of one of the following marker genes: Tubulin beta3, MAP2, NeuN, 160kDa Neurofilament, 200kDa Neurofilament, NSE, PSD93, PSD95, etc.

[0087] There are no particular restrictions on the culture medium used in the cell culture container of this embodiment, and it can be appropriately selected according to the purpose. Examples include culture media classified by composition such as natural media, semi-synthetic media, and synthetic media; and culture media classified by form such as semi-solid media, liquid media, and powder media. These may be used individually or in combination of two or more. If the cells are of animal origin, any culture medium used for culturing animal cells can be used.

[0088] Culture media used for nerve cells include media prepared by adding necessary components to a basal culture medium. Examples of basal media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, D-MEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha-Modified Eagle's Minimum Essential Medium (αMEM), MEM medium (Minimum Essential Medium), RPMI1640 (Roswell Park Memorial Institute-1640) medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, M199 medium, High Performance Medium 199, StemPro34 (Thermo Fisher Scientific), and X-VIVO. 10 (Chembrex), X-VIVO 15 (Chembrex), HPGM (Chembrex), StemSpan H3000 (Stem Cell Technologies), StemSpanSFEM (Stem Cell Technologies), StemlineII (Sigma-Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Thermo Fisher Scientific), Essential8® medium (Thermo Fisher Scientific), mTeSR1 or mTeSR2 medium (Stem Cell Technologies), ReproFF or ReproFF2 (ReproCELL), PSGro hESC / iPSC medium (System Biosciences), NutriStem® medium (Biological Industries), CSTI-7 medium (Cell Science Institute), MesenPROExamples include RS medium (Thermo Fisher Scientific), MF-Medium® mesenchymal stem cell proliferation medium (Toyobo Co., Ltd.), Sf-900II (Thermo Fisher Scientific), Opti-Pro (Thermo Fisher Scientific), etc. These may be used individually or in combination of two or more.

[0089] In addition, additives to be added to the basal culture medium include those commonly used for culturing nerve cells, such as Component N (Elixirgen Scientific), Component G2 (Elixirgen Scientific), N2 Supplement (Thermo Fisher Scientific), iCell Neural Supplement B (CDI), and iCell Neuvous System Supplement, B-27 plus (Thermo Fisher Scientific).

[0090] Other ingredients are not particularly limited and can be selected as appropriate depending on the purpose. Examples include culture media, crosslinking agents, pH adjusters, preservatives, and antioxidants.

[0091] There are no particular restrictions on the carbon dioxide concentration in the culture medium, and it can be appropriately selected depending on the purpose, but a concentration of 2% to 5% is preferred, and a concentration of 3% to 4% is more preferred. When the carbon dioxide concentration is 2% to 5%, cells can be cultured suitably. [Examples]

[0092] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0093] [Example 1] (Preparation of cell culture vessels for Example 1) 《Cell culture container》 A 96-well Black / Clear Bottom Plate, TC Surface (model number "165305", Thermo Fisher Scientific) was used.

[0094] 《70% by mass ethanol aqueous solution》 A 70% by mass ethanol solution was prepared using Ethanol (99.5%) (model number "08948-25", Nacalai Tesque) and ultrapure water.

[0095] Aqueous solution containing gelatin Gelatin from cold water fish skin (model number "G7041", Sigma-Aldrich) was dissolved in ultrapure water to prepare an aqueous solution containing gelatin with a fish-derived gelatin concentration of 1% by mass.

[0096] Aqueous solutions containing polycationic materials Poly-L-ornithine solution (PLO, model number "P4957-50ML", Sigma-Aldrich) was diluted with DPBS, no calcium, no magnesium (DPBS, model number "14190250", Thermo Fisher Scientific) to prepare an aqueous solution containing a polycationic material with a polyornithine concentration of 0.002% by mass.

[0097] Aqueous solution containing cell adhesion factors Laminin Mouse Protein, Natural (model number "23017015", Thermo Fisher Scientific) was diluted with DPBS to prepare an aqueous solution containing 10 μg / mL of cell adhesion factor protein.

[0098] Preparation of the cell culture vessel for Example 1 A 70% by mass ethanol aqueous solution was placed in a cell culture container and allowed to stand at room temperature for 5 minutes. Subsequently, the 70% by mass ethanol aqueous solution was removed from the cell culture container and dried in a safety cabinet. After drying, an aqueous solution containing gelatin was placed in the cell culture container and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing gelatin was removed, washed once with ultrapure water, and dried in a safety cabinet. After drying, an aqueous solution containing a polycationic material was placed in the cell culture container and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing the polycationic material was removed, washed twice with DPBS and once with ultrapure water, and dried in a safety cabinet. After drying, an aqueous solution containing a cell adhesion molecule was placed in the cell culture container and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing the cell adhesion molecule was removed, washed once with DPBS, and a cell culture container of Example 1 with a modified culture surface was obtained.

[0099] [Example 2] (Preparation of cell culture vessels in Example 2) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessel, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 1.

[0100] Aqueous solution containing casein Blocker TM Casein in PBS (model number "37582", Thermo Fisher Scientific) was diluted with DPBS to prepare an aqueous solution containing casein with a casein concentration of 0.1% by mass.

[0101] Preparation of the cell culture vessel for Example 2 A cell culture vessel for Example 2, with a modified culture surface, was obtained in the same manner as in Example 1, except that an aqueous solution containing casein was used instead of an aqueous solution containing gelatin.

[0102] [Comparative Example 1] (Preparation of cell culture vessels for Comparative Example 1) Cell culture containers, aqueous solutions containing polycationic materials, aqueous solutions containing cell adhesion factors The cell culture vessel, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 1.

[0103] Preparation of the cell culture vessel for Comparative Example 1 A cell culture container of Comparative Example 1, in which the culture surface was modified, was obtained in the same manner as in Example 1, except that the steps of contacting the culture surface of the cell culture container with a 70% by mass ethanol aqueous solution and contacting it with an aqueous solution containing gelatin were omitted.

[0104] [Comparative Example 2] (Preparation of cell culture vessels for Comparative Example 2) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessel, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 1.

[0105] Serum culture medium A serum culture medium was prepared by mixing 10% by volume of fetal bovine serum and 1% by volume of a 100-fold concentrated antibiotic-antifungal solution (model number 02892-54, Nacalai Tesque) with DMEM, low glucose, GlutaMAX™ Supplement, pyruvate (model number "10567014", Thermo Fisher Scientific).

[0106] Preparation of cell culture vessels for Comparative Example 2 A cell culture container for Comparative Example 2, with a modified culture surface, was obtained in the same manner as in Example 1, except that serum medium was used instead of an aqueous solution containing gelatin, and the conditions for contacting the surface of the cell culture container with serum medium were set to 2 minutes at room temperature.

[0107] [Experimental Example 1] (Verification of cell aggregate detachment in 96-well plates) Neurons were seeded in 24 wells of each cell culture vessel used in Examples 1-2 and Comparative Examples 1-2. The neurons used were a mixture of cells derived from iPS cells differentiated into neurons from a healthy donor (product name "Human iPSC-derived GABAergic Neurons", Elixargen Scientific) and astrocytes (product name "Human Primary Astrocyte", Thermo Fisher Scientific).

[0108] After culturing nerve cells for 5 weeks, each well was observed under a microscope, and the number of wells in which more than half of the culture area had detached was calculated. The results are shown in Table 1 below.

[0109] [Table 1]

[0110] As a result, it was confirmed that the cell culture method using the cell culture containers in Examples 1 and 2 resulted in fewer wells from which cell aggregates detached, compared to the cell culture method using the cell culture containers in Comparative Examples 1 and 2.

[0111] Figures 3(a) to (h) are representative micrographs of cell aggregates containing nerve cells after 5 weeks of culture. Figures 3(a) to (b) are micrographs of cell aggregates cultured in the cell culture container of Example 1, Figures 3(c) to (d) are micrographs of cell aggregates cultured in the cell culture container of Example 2, Figures 3(e) to (f) are micrographs of cell aggregates cultured in the cell culture container of Comparative Example 1, and Figures 3(g) to (h) are micrographs of cell aggregates cultured in the cell culture container of Comparative Example 2.

[0112] As a result, in the cell culture methods using the cell culture containers of Comparative Examples 1 and 2, some wells showed significant detachment of cell aggregates, while in the cell culture methods using the cell culture containers of Examples 1 and 2, it was observed that the cell aggregates did not detach.

[0113] From the results in Table 1 and Figure 3 above, it was confirmed that the cell culture method using a cell culture container, such as in Examples 1 and 2, in which a first layer containing gelatin or casein is laminated on the culture surface, a second layer containing a polycationic material is laminated on the first layer, and a third layer containing a cell adhesion factor is laminated on that layer, can suppress the detachment of cell aggregates from the culture surface compared to the cell culture method using the cell culture containers of Comparative Examples 1 and 2. Furthermore, it was confirmed that the cell culture containers of Examples 1 and 2 can be manufactured by an inexpensive and simple method.

[0114] [Example 3] (Preparation of cell culture vessels in Example 3) 《70% by mass ethanol aqueous solution, aqueous solution containing gelatin》 The 70% by mass ethanol aqueous solution and the gelatin aqueous solution were the same as those used in Example 1.

[0115] 《Cell culture container》 A microelectrode array (MEA) plate (model number "M768-tMEA-48W", Axion Biosystems) was used.

[0116] Aqueous solutions containing polycationic materials A 20-fold dilution of polyethyleneimine solution (PEI, model number "181978", Thermo Fisher Scientific) is used to prepare Pierce TM An aqueous solution containing a polycationic material with a polyethyleneimine concentration of 0.1% by mass was prepared by dilution with 20X Borate Buffer (model number "28341", Thermo Fisher Scientific).

[0117] Preparation of the cell culture vessel for Example 3 The above 70% by mass ethanol aqueous solution was placed in a cell culture container and left to stand at room temperature for 5 minutes. Subsequently, the 70% by mass ethanol aqueous solution was removed from the cell culture container and dried in a safety cabinet. After drying, an aqueous solution containing gelatin was placed in the cell culture container and left to stand at 37°C for 1 hour. After that, the aqueous solution containing gelatin was removed, washed once with ultrapure water, and dried in a safety cabinet. After drying, an aqueous solution containing polycationic material was placed in the cell culture container and left to stand at 37°C for 1 hour. After that, the aqueous solution containing polycationic material was removed, washed four times with ultrapure water, and dried in a safety cabinet to obtain the cell culture container of Example 3 with a modified culture surface.

[0118] [Comparative Example 3] (Preparation of cell culture vessels for Comparative Example 3) Cell culture containers, aqueous solutions containing polycationic materials The cell culture vessel and aqueous solution containing the polycationic material were the same as those used in Example 3.

[0119] Preparation of the cell culture vessel for Comparative Example 3 A cell culture container of Comparative Example 3, in which the culture surface was modified, was obtained in the same manner as in Example 3, except that the steps of contacting the culture surface of the cell culture container with a 70% by mass ethanol aqueous solution and contacting it with an aqueous solution containing gelatin were omitted.

[0120] [Comparative Example 4] (Preparation of cell culture vessels for Comparative Example 4) Cell culture container, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material The cell culture vessels, 70% by mass ethanol aqueous solution, and aqueous solution containing polycationic material were the same as those used in Example 3. The serum culture medium was the same as that used in Comparative Example 2.

[0121] Preparation of the cell culture vessel for Comparative Example 4 A cell culture container for Comparative Example 4, with a modified culture surface, was obtained in the same manner as in Example 3, except that serum medium was used instead of an aqueous solution containing gelatin, and the conditions for contacting the surface of the cell culture container with serum medium were set to 2 minutes at room temperature.

[0122] [Experimental Example 2] (Verification of the effect of MEA plate (Axion) transport on cell aggregate detachment and potential detection) Nerve cells were seeded in eight wells of each cell culture vessel used in Example 3 and Comparative Examples 3-4. The same nerve cells as in Experimental Example 1 were used. Laminin was mixed into the cell suspension before seeding in the center of each well.

[0123] After culturing nerve cells for 7 weeks, each cell culture vessel was placed in a container capable of maintaining a suitable temperature and CO2 concentration for cell culture, and transported by air for 16 hours. Subsequently, each well in the cell culture vessel was observed under a microscope, and action potentials were measured. An Axion Meastro microscope was used to measure action potentials. Action potentials were measured for 10 minutes in each well.

[0124] Figure 4 shows representative well images of each cell culture vessel after transport, and the number of effective electrodes in each well after transport. The number of effective electrodes is defined as the number of electrodes in which spontaneous firing of cell aggregates could be detected in each well. The maximum number of effective electrodes is 16.

[0125] Figures 5(a) to (c) are graphs showing the variability (CV value) of spontaneous firing number, burst number, and synchronous burst number in the culture method using each cell culture vessel after transport. The CV value was calculated using the following formula (1). CV value = (Standard deviation of spontaneous firings, bursts, or synchronous bursts) / (Mean value of spontaneous firings, bursts, or synchronous bursts) ... (1)

[0126] Figure 4 shows images of the wells after transport, revealing that in the culture method using the cell culture container of Comparative Example 4, wells with detached cell aggregates were observed. On the other hand, in the culture methods using the cell culture container of Example 3 and Comparative Example 3, no wells with detached cell aggregates were observed.

[0127] From the number of active electrodes in Figure 4, it was found that in the culture method using the cell culture container of Comparative Example 3, there was a well with only one active electrode. It was inferred that minute cell detachment from the culture surface occurred, but was not detectable by microscopic observation. On the other hand, in the culture method using the cell culture container of Example 3 and the cell culture container of Comparative Example 4, the number of active electrodes was the maximum value of 16 in all wells.

[0128] Figure 5 shows that the cell culture method using the cell culture vessel of Example 3 exhibited less variability in spontaneous firing count, burst count, and synchronous burst count compared to the cell culture methods using the cell culture vessels of Comparative Example 3 and Comparative Example 4. The results summarized above are shown in Table 2 below.

[0129] [Table 2]

[0130] From Table 2 above, it was confirmed that even when subjected to external environmental stress such as long-term transport, the culture method in Example 3, in which a first layer containing gelatin is laminated on the culture surface, a second layer containing a polycationic material is laminated on the first layer, and a liquid (culture medium) containing cell adhesion factors is placed on the laminated layers, can suppress the detachment of cell aggregates from the culture surface and obtain stable functional evaluation results compared to the culture methods using cell culture containers in Comparative Examples 3 and 4.

[0131] [Comparative Example 5] (Preparation of cell culture vessels for Comparative Example 5) Cell culture container, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material The cell culture vessel, 70% by mass ethanol aqueous solution, and aqueous solution containing polycationic material were the same as those used in Example 3.

[0132] Aqueous solution containing albumin Blocker TM BSA (10X) in PBS (model number "37525", Thermo Fisher Scientific) was diluted with DPBS to prepare an albumin-containing aqueous solution with an albumin concentration of 3% by mass.

[0133] Preparation of the cell culture vessel for Comparative Example 5 A cell culture container for Comparative Example 5, with a modified culture surface, was obtained in the same manner as in Example 3, except that an aqueous solution containing albumin was used instead of an aqueous solution containing gelatin.

[0134] [Experimental Example 3] (Verification of the effect of leaving MEA plates (Axion) at room temperature on the potential detection of cell aggregates) Nerve cells were seeded in 5 wells each of the cell culture vessels used in Example 3 and Comparative Examples 3-5. The same nerve cells as in Experimental Example 1 were used. Laminin was mixed into the cell suspension before seeding in the center of each well.

[0135] After culturing nerve cells for 5 weeks, each cell culture vessel was left to stand overnight at room temperature, and then action potentials were measured. An Axion Meastro was used to measure action potentials. Action potentials were measured for 10 minutes in each well.

[0136] Table 3 below shows the average number of effective electrodes in each cell culture vessel after being left to stand overnight at room temperature. The maximum number of effective electrodes is 16.

[0137] [Table 3]

[0138] From Table 3 above, it was confirmed that the cell culture method using the cell culture vessel of Example 3 had a higher average number of effective electrodes and the least effect of cell aggregate detachment compared to the cell culture methods using the cell culture vessels of Comparative Examples 3 to 5.

[0139] Figures 6(a) to (c) are graphs showing the variability (CV value) of spontaneous firing number, burst number, and synchronous burst number in each cell culture method using different cell culture vessels after being left to stand overnight at room temperature. The CV value was calculated using the above formula (1).

[0140] Figure 6 shows that the cell culture method using the cell culture vessel of Example 3 showed less variation in the number of synchronous bursts compared to the cell culture methods using the cell culture vessels of Comparative Examples 3 to 5. Furthermore, the variation in the number of spontaneous firings and bursts was comparable to that of the cell culture method using the cell culture vessel of Comparative Example 4, which showed the smallest variation.

[0141] From Table 3 and Figure 6 above, it was confirmed that even when subjected to the external environmental burden of being left undisturbed overnight at room temperature, the culture method as in Example 3, in which a first layer containing gelatin is laminated on the culture surface, a second layer containing a polycationic material is laminated on the first layer, and a liquid (culture medium) containing cell adhesion factors is placed on the laminated layers, can suppress the detachment of cell aggregates from the culture surface and obtain stable functional evaluation results compared to the culture methods using cell culture containers in Comparative Examples 3 to 5.

[0142] [Example 4] (Preparation of cell culture vessels in Example 4) 《70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factor》 The 70% by mass ethanol aqueous solution, the aqueous solution containing gelatin, and the aqueous solution containing a polycationic material were the same as those used in Example 1.

[0143] 《Cell culture container》 A MEA plate (model number "MaxTwo", MaxWell Biosystems) with a CMOS-type planar microelectrode array (MEA) was used.

[0144] Aqueous solution containing cell adhesion factors Laminin was diluted with DPBS to prepare an aqueous solution containing 80 μg / mL of cell adhesion molecule.

[0145] Preparation of the cell culture vessel for Example 4 The above 70% by mass ethanol aqueous solution was placed in a cell culture container and left to stand at room temperature for 30 minutes. Subsequently, the 70% by mass ethanol aqueous solution was removed from the cell culture container and dried in a safety cabinet. After drying, an aqueous solution containing gelatin was placed in the cell culture container and left to stand at 37°C for 1 hour. After that, the aqueous solution containing gelatin was removed, washed once with ultrapure water, and dried in a safety cabinet. After drying, an aqueous solution containing a polycationic material was placed in the cell culture container and left to stand at 37°C for 2 hours. After that, the aqueous solution containing the polycationic material was removed, washed twice with DPBS and once with ultrapure water, and dried in a safety cabinet. After drying, an aqueous solution containing a cell adhesion molecule was placed in the cell culture container and left to stand at 37°C for 30 minutes to obtain the cell culture container of Example 4 with a modified culture surface.

[0146] [Comparative Example 6] (Preparation of cell culture vessels for Comparative Example 6) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessel, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 4.

[0147] Preparation of the cell culture vessel for Comparative Example 6 A cell culture container for Comparative Example 6, with a modified culture surface, was obtained in the same manner as in Example 4, except that the step of contacting the surface of the cell culture container with an aqueous solution containing gelatin was omitted.

[0148] [Experimental Example 4] (Verification of cell aggregate detachment using MEA plates (Maxwell)) Nerve cells were seeded in the wells of the cell culture vessels used in Example 4 and Comparative Example 6. The same nerve cells as in Experimental Example 1 were used. Laminin was mixed into the cell suspension before seeding in the center of the wells.

[0149] After culturing nerve cells for 33 days, each well was observed under a microscope and action potentials were measured. The MaxTwo system from MaxWell Biosystems was used to measure action potentials.

[0150] Figures 7(a) and 7(b) show images of the wells in each cell culture vessel. Figures 8(a) and 8(b) show mapping images of the active electrodes in the wells of each cell culture vessel and the percentage of active electrodes.

[0151] Figure 7 shows that after 33 days of culture, the cell aggregates in the culture method using the cell culture container of Comparative Example 6 were almost completely detached and lost, while in the culture method using the cell culture container of Example 4, the cell aggregates remained without detaching. Furthermore, Figure 8 shows that the ratio of effective electrodes in the culture method using the cell culture container of Example 4 was greater than the ratio of effective electrodes in the culture method using the cell culture container of Comparative Example 6, indicating that more action potentials could be detected.

[0152] From Figures 7 and 8, it was confirmed that even in a CMOS-type MEA plate, the culture method described in Example 4, in which a first layer containing gelatin is laminated on the culture surface, a second layer containing a polycationic material is laminated on the first layer, and a liquid (culture medium) containing cell adhesion factors is placed on the laminated layers, can suppress the detachment of cell aggregates from the culture surface and obtain stable functional evaluation results compared to the culture method using the cell culture container of Comparative Example 6.

[0153] [Example 5] (Preparation of cell culture vessels in Example 5) 《70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material》 The 70% by mass ethanol aqueous solution, the aqueous solution containing gelatin, and the aqueous solution containing a polycationic material were the same as those used in Example 3.

[0154] 《Cell culture container》 A dish-shaped planar microelectrode array (MEA) MEA dish (model number "MED-R5155", AlphaMed Scientific) was used.

[0155] Preparation of the cell culture vessel for Example 5 Except for the difference in the cell culture container, the cell culture container of Example 5, in which the culture surface was modified, was obtained in the same manner as in Example 3.

[0156] [Comparative Example 7] (Preparation of cell culture vessel for Comparative Example 7) Cell culture container, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material The cell culture vessel, 70% by mass ethanol aqueous solution, and aqueous solution containing polycationic material were the same as those used in Example 5.

[0157] Preparation of the cell culture vessel for Comparative Example 7 A cell culture container for Comparative Example 7, in which the culture surface was modified, was obtained in the same manner as in Example 5, except that the step of contacting the surface of the cell culture container with an aqueous solution containing gelatin was not performed.

[0158] [Experimental Example 5] (Verification of cell aggregate detachment using an MEA dish (AlphaMed Scientific)) Nerve cells were seeded in the cell culture vessels of Example 5 and Comparative Example 7. The same nerve cells as in Experimental Example 1 were used. Laminin was mixed into the cell suspension before seeding in the center of the wells.

[0159] After culturing nerve cells for 34 days, each dish was observed under a microscope. Images of each dish are shown in Figures 9(a) and 9(b).

[0160] Figure 9 shows that after 34 days of culture, the cell aggregates were almost completely detached and lost in the culture method using the cell culture vessel of Comparative Example 7, while in the culture method using the cell culture vessel of Example 5, the cell aggregates remained without detaching.

[0161] As shown in Figure 9, it was confirmed that, even in an MEA dish, a culture method in which a first layer containing gelatin is laminated on the culture surface, a second layer containing a polycationic material is laminated on the first layer, and a liquid (culture medium) containing cell adhesion factors is placed on the laminated layers, can suppress the detachment of cell aggregates from the culture surface compared to the culture method using a cell culture container in Comparative Example 7.

[0162] [Example 6] (Preparation of cell culture vessels in Example 6) 《Cell culture container》 A 384-well Black Plate with an Optically Clear Polymer Bottom (model number "142761", Thermo Fisher Scientific) was used.

[0163] 《70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factor》 The 70% by mass ethanol aqueous solution, the aqueous solution containing gelatin, the aqueous solution containing a polycationic material, and the aqueous solution containing a cell adhesion factor were the same as those used in Example 1.

[0164] Preparation of the cell culture vessel for Example 6 Except for the difference in the cell culture container, the cell culture container of Example 6, in which the culture surface was modified, was obtained in the same manner as in Example 1.

[0165] [Comparative Example 8] (Preparation of cell culture vessels for Comparative Example 8) Cell culture containers, aqueous solutions containing polycationic materials, aqueous solutions containing cell adhesion factors The cell culture vessel, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 6.

[0166] Preparation of the cell culture vessel for Comparative Example 8 A cell culture container for Comparative Example 8, in which the culture surface was modified, was obtained in the same manner as in Example 6, except that the step of contacting the culture surface of the cell culture container with an aqueous solution containing gelatin was not performed.

[0167] [Experimental Example 6] (Verification of inhibition of cell aggregate aggregation in 384-well plates) Neurons were seeded in 24 wells of each cell culture vessel used in Example 6 and Comparative Example 8. The neurons used were a mixture of cells derived from iPS cells differentiated into neurons from a healthy donor's cell line (product name "Human iPSC-derived Excitatory Neurons", Elixargen Scientific) and astrocytes (product name "Human Primary Astrocyte", Thermo Fisher Scientific).

[0168] After culturing nerve cells for 32 days, each well was observed under a microscope. Figures 10(a) and (c) show images of each well observed with a 4x objective lens, and (b) and (d) show images of each well observed with a 10x objective lens.

[0169] Figure 10 shows that after 32 days of culture, the culture method using the cell culture vessel of Comparative Example 8 showed numerous areas of localized cell aggregation, as indicated by the arrows in Figure 10(d). In contrast, the culture method using the cell culture vessel of Example 6 did not show any areas of localized cell aggregation, confirming that the cells were cultured uniformly within the wells.

[0170] As shown in Figure 10, even in a 384-well plate, the cell culture method using a cell culture container as in Example 6, in which a first layer containing gelatin is laminated on the culture surface, a second layer containing a polycationic material is laminated on the first layer, and a third layer containing a cell adhesion factor is laminated on that layer, was confirmed to suppress local cell aggregation and enable uniform culture within the wells compared to the cell culture container used in Comparative Example 7. Furthermore, it was confirmed that the cell culture container of Example 6 can be manufactured using an inexpensive and simple method.

[0171] [Example 7] (Preparation of cell culture vessels in Example 7) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessels, 70% by mass ethanol aqueous solution, gelatin aqueous solution, polycationic material aqueous solution, and cell adhesion factor aqueous solution were the same as those used in Example 6.

[0172] Preparation of the cell culture vessel for Example 7 A cell culture container for Example 7, with a modified culture surface, was obtained in the same manner as in Example 1, except that the order of the steps was reversed: 1) Adding an aqueous solution containing gelatin to a cell culture container, letting it stand at 37°C for 1 hour, removing the aqueous solution containing gelatin, washing once with ultrapure water, and drying in a safety cabinet; and 2) Adding an aqueous solution containing a polycationic material to a cell culture container, letting it stand at 37°C for 1 hour, removing the aqueous solution containing the polycationic material, washing twice with DPBS and once with ultrapure water, and drying in a safety cabinet.

[0173] [Comparative Example 9] (Preparation of cell culture vessels for Comparative Example 9) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessels, 70% by mass ethanol aqueous solution, gelatin aqueous solution, polycationic material aqueous solution, and cell adhesion factor aqueous solution were the same as those used in Example 6.

[0174] Preparation of the cell culture vessel for Comparative Example 9 A 70% by mass ethanol aqueous solution was placed in a cell culture vessel and allowed to stand at room temperature for 5 minutes. Subsequently, the 70% by mass ethanol aqueous solution was removed from the cell culture vessel and dried in a safety cabinet. After drying, an aqueous solution containing gelatin was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing gelatin was removed, washed once with ultrapure water, and dried in a safety cabinet. After drying, an aqueous solution containing cell adhesion factors was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing cell adhesion factors was removed and washed once with DPBS. An aqueous solution containing a polycationic material was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing the polycationic material was removed, washed twice with DPBS and once with ultrapure water to obtain a cell culture vessel of Comparative Example 9 with a modified culture surface.

[0175] [Comparative Example 10] (Preparation of cell culture vessels for Comparative Example 10) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessels, 70% by mass ethanol aqueous solution, gelatin aqueous solution, polycationic material aqueous solution, and cell adhesion factor aqueous solution were the same as those used in Example 6.

[0176] Preparation of cell culture vessel for Comparative Example 10 A 70% by mass ethanol aqueous solution was placed in a cell culture vessel and allowed to stand at room temperature for 5 minutes. Subsequently, the 70% by mass ethanol aqueous solution was removed from the cell culture vessel and dried in a safety cabinet. After drying, an aqueous solution containing a polycationic material was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing the polycationic material was removed, washed twice with DPBS and once with ultrapure water, and dried in a safety cabinet. After drying, an aqueous solution containing a cell adhesion factor was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing the cell adhesion factor was removed and washed once with DPBS. An aqueous solution containing gelatin was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. Then, the aqueous solution containing gelatin was removed and washed once with ultrapure water to obtain a cell culture vessel of Comparative Example 10 with a modified culture surface.

[0177] [Comparative Example 11] (Preparation of cell culture vessels in Example 11) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessels, 70% by mass ethanol aqueous solution, gelatin aqueous solution, polycationic material aqueous solution, and cell adhesion factor aqueous solution were the same as those used in Example 6.

[0178] Preparation of cell culture vessels for Comparative Example 11 A 70% by mass ethanol aqueous solution was placed in a cell culture vessel and allowed to stand at room temperature for 5 minutes. Subsequently, the 70% by mass ethanol aqueous solution was removed from the cell culture vessel and dried in a safety cabinet. After drying, an aqueous solution containing cell adhesion factors was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. After that, the aqueous solution containing cell adhesion factors was removed and washed once with DPBS. An aqueous solution containing a polycationic material was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. After that, the aqueous solution containing the polycationic material was removed and washed twice with DPBS and once with ultrapure water. An aqueous solution containing gelatin was placed in the cell culture vessel and allowed to stand at 37°C for 1 hour. After that, the aqueous solution containing gelatin was removed and washed once with ultrapure water to obtain a cell culture vessel of Comparative Example 11 with a modified culture surface.

[0179] [Comparative Example 12] (Preparation of cell culture vessels for Comparative Example 12) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessels, 70% by mass ethanol aqueous solution, gelatin aqueous solution, polycationic material aqueous solution, and cell adhesion factor aqueous solution were the same as those used in Example 6.

[0180] Preparation of cell culture vessels for Comparative Example 12 A cell culture container for Comparative Example 12 with a modified culture surface was obtained in the same manner as in Comparative Example 11, except that the order of the steps was reversed: one step involved placing an aqueous solution containing gelatin into a cell culture container, letting it stand at 37°C for 1 hour, removing the aqueous solution containing gelatin, and washing it once with ultrapure water; the other step involved placing an aqueous solution containing a polycationic material into a cell culture container, letting it stand at 37°C for 1 hour, removing the aqueous solution containing the polycationic material, and washing it twice with DPBS and once with ultrapure water.

[0181] [Comparative Example 13] (Preparation of cell culture vessels for Comparative Example 13) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessel, 70% by mass ethanol aqueous solution, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 6.

[0182] Preparation of the cell culture vessel for Comparative Example 13 A cell culture vessel for Comparative Example 13, with a modified culture surface, was obtained in the same manner as for Comparative Example 11, except for the step of placing an aqueous solution containing gelatin into a cell culture vessel, letting it stand at 37°C for 1 hour, removing the aqueous solution containing gelatin, and washing it once with ultrapure water.

[0183] [Comparative Example 14] (Preparation of cell culture vessels for Comparative Example 14) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing cell adhesion factors The cell culture vessel, 70% by mass ethanol aqueous solution, gelatin aqueous solution, and cell adhesion factor aqueous solution were the same as those used in Example 6.

[0184] Preparation of cell culture vessels for Comparative Example 14 A cell culture vessel for Comparative Example 14, with a modified culture surface, was obtained in the same manner as for Comparative Example 9, except that the following steps were taken: an aqueous solution containing a polycationic material was placed in a cell culture vessel, it was left to stand at 37°C for 1 hour, the aqueous solution containing the polycationic material was removed, and the vessel was washed twice with DPBS and once with ultrapure water.

[0185] [Comparative Example 15] (Preparation of cell culture vessels for Comparative Example 15) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing cell adhesion factors The cell culture vessel, 70% by mass ethanol aqueous solution, gelatin aqueous solution, and cell adhesion factor aqueous solution were the same as those used in Example 6.

[0186] Preparation of cell culture vessels for Comparative Example 15 A cell culture vessel for Comparative Example 15 with a modified culture surface was obtained in the same manner as for Comparative Example 12, except that the following steps were taken: an aqueous solution containing a polycationic material was placed in a cell culture vessel, it was left to stand at 37°C for 1 hour, the aqueous solution containing the polycationic material was removed, and the vessel was washed twice with DPBS and once with ultrapure water.

[0187] [Comparative Example 16] (Preparation of cell culture vessels for Comparative Example 16) Cell culture container, 70% by mass ethanol aqueous solution, aqueous solution containing cell adhesion factors The cell culture vessel, 70% by mass ethanol aqueous solution, and aqueous solution containing cell adhesion factors were the same as those used in Example 6.

[0188] Preparation of cell culture vessel for Comparative Example 16 A cell culture vessel for Comparative Example 16 with a modified culture surface was obtained in the same manner as for Comparative Example 15, except that the step of placing an aqueous solution containing gelatin into a cell culture vessel, letting it stand at 37°C for 1 hour, removing the aqueous solution containing gelatin, and washing it once with ultrapure water was omitted. [Experimental Example 7] (Verification of inhibition of cell aggregate aggregation and detachment in 384-well plates) Neurons were seeded in 6 wells of each cell culture vessel used in Examples 6-7 and Comparative Examples 8-16. As for the neurons, iPS cells differentiated from healthy individuals (product name "Human iPSC-derived Excitatory Neurons", Elixargen Scientific) were used alone.

[0189] After culturing nerve cells for 12 days, each well was observed under a microscope. Figure 11 shows representative images of the wells in each cell culture vessel.

[0190] Figure 11 shows that after 12 days of culture, significant cell aggregation was observed in the cell culture methods using the cell culture vessels of Comparative Examples 14-16. On the other hand, no significant cell aggregation was observed in the cell culture methods using the cell culture vessels of Examples 6-7 and Comparative Examples 8-13. Figure 11 confirms that significant cell aggregation occurs when using cell culture vessels that are not modified with a layer containing polycationic material.

[0191] Furthermore, in Examples 6-7 and Comparative Examples 8-13, the culture was continued for 19 days, after which each well was observed under a microscope. Figure 12 shows representative images of the wells in each cell culture vessel.

[0192] Figure 12 shows that after 19 days of culture, cell aggregation in the center of the wells was observed in the cell culture containers used in Comparative Examples 10-13, as indicated by the arrows in Figure 12. On the other hand, cell aggregation in the center of the wells was not observed in the cell culture containers used in Examples 6-7 and Comparative Examples 8-9. Figure 12 confirms that cell culture containers in which the outermost layer is not modified with a cell adhesion factor result in a higher rate of cell aggregation in the center of the wells.

[0193] Furthermore, in Examples 6-7 and Comparative Examples 8-9, the culture was continued for 25 days, and after a complete change of the culture medium, each well was observed under a microscope, and the number of wells in which more than one-quarter of the culture area had detached was calculated. The results are shown in the table below.

[0194] [Table 4]

[0195] Table 4 shows that, compared to the cell culture methods using the cell culture vessels of Comparative Examples 8-9, the cell culture methods using the cell culture vessels of Examples 6-7 resulted in fewer wells from which cell aggregates detached.

[0196] From the results in Figures 11-12 and Table 4 above, it was confirmed that the cell culture method using a cell culture container, such as in Examples 6-7, in which a layer containing gelatin or casein and a layer containing a polycationic material are laminated on the culture surface of the cell culture container, and a layer containing a cell adhesion factor is laminated on top of these laminated layers, can suppress the aggregation and detachment of cell aggregates from the culture surface compared to the cell culture method using the cell culture containers of Comparative Examples 8-16. Furthermore, it was confirmed that the cell culture containers of Examples 6-7 can be manufactured by an inexpensive and simple method.

[0197] [Example 8] (Preparation of cell culture vessels in Example 8) 《Cell culture container》 A Costar(R) 24-well transparent cell culture-treated multiwell plate (24-well plate, model number "3526", Corning) was used.

[0198] 《70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factor》 The 70% by mass ethanol aqueous solution, the aqueous solution containing gelatin, the aqueous solution containing a polycationic material, and the aqueous solution containing a cell adhesion factor were the same as those used in Example 1.

[0199] Preparation of the cell culture vessel for Example 8 Except for the difference in the cell culture container, the cell culture container of Example 8, in which the culture surface was modified, was obtained in the same manner as in Example 1.

[0200] [Comparative Example 17] (Preparation of cell culture vessels for Comparative Example 17) Cell culture containers, aqueous solutions containing polycationic materials, aqueous solutions containing cell adhesion factors The cell culture vessel, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 8.

[0201] Preparation of cell culture vessels for Comparative Example 17 A cell culture container for Comparative Example 17, in which the culture surface was modified, was obtained in the same manner as in Example 8, except that the step of contacting the culture surface of the cell culture container with an aqueous solution containing gelatin was not performed.

[0202] [Comparative Example 18] 《Cell culture container》 The same cell culture vessels as in Example 8 were used. No modifications were made to the culture surface.

[0203] [Experimental Example 8] (Verification of cell aggregate detachment in cells other than nerve cells) HEK293 cells were seeded in 8 wells each of the cell culture vessels used in Example 8 and Comparative Examples 16-17.

[0204] After culturing HEK293 cells for 4 days, the entire volume of culture medium was replaced, followed by three pipetting steps. Each well was then examined under a microscope to evaluate the detachment state. The results are shown in Table 5 below.

[0205] Furthermore, the entire multi-well plate was vibrated at 2.89 G for 8 minutes. A Vortex-Genie2 vortex mixer (Kenis Corporation) was used for the vibration. Afterwards, the detachment state was evaluated by observing each well under a microscope. The results are shown in Table 5 below.

[0206] [Table 5]

[0207] Table 5 shows that, even for easily detachable cells other than nerve cells, the cell culture method using a cell culture container, such as in Example 8, in which a first layer containing gelatin is laminated on the culture surface, a second layer containing a polycationic material is laminated on the first layer, and a third layer containing a cell adhesion factor is laminated on that layer, suppresses the detachment of cell aggregates compared to the cell culture containers used in Comparative Examples 17-18. Furthermore, it was confirmed that the cell culture container of Example 8 can be manufactured using an inexpensive and simple method.

[0208] [Example 9] (Preparation of cell culture vessels in Example 9) Cell culture containers, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, aqueous solution containing cell adhesion factors The cell culture vessels, 70% by mass ethanol aqueous solution, aqueous solution containing gelatin, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 8.

[0209] Preparation of the cell culture vessel for Example 9 A cell culture container for Example 9 with a modified culture surface was obtained in the same manner as in Example 8, except that the order of the steps was reversed: placing an aqueous solution containing gelatin in a cell culture container, letting it stand at 37°C for 1 hour, removing the aqueous solution containing gelatin, washing it once with ultrapure water, and drying it in a safety cabinet; and placing an aqueous solution containing a polycationic material in a cell culture container, letting it stand at 37°C for 1 hour, removing the aqueous solution containing the polycationic material, washing it twice with DPBS and once with ultrapure water, and drying it in a safety cabinet.

[0210] [Comparative Example 19] (Preparation of cell culture vessels for Comparative Example 19) Cell culture containers, aqueous solutions containing polycationic materials, aqueous solutions containing cell adhesion factors The cell culture vessel, aqueous solution containing polycationic material, and aqueous solution containing cell adhesion factors were the same as those used in Example 9.

[0211] Preparation of cell culture vessels for Comparative Example 19 A cell culture container for Comparative Example 19, in which the culture surface was modified, was obtained in the same manner as in Example 9, except that the step of contacting the culture surface of the cell culture container with an aqueous solution containing gelatin was not performed.

[0212] [Comparative Example 20] (Preparation of cell culture vessel for Comparative Example 20) Cell culture containers, aqueous solutions containing gelatin, aqueous solutions containing cell adhesion factors The cell culture vessel, the aqueous solution containing gelatin, and the aqueous solution containing cell adhesion factors were the same as those used in Example 9.

[0213] Preparation of cell culture vessels for Comparative Example 20 A cell culture container for Comparative Example 20, in which the culture surface was modified, was obtained in the same manner as in Example 9, except that the step of contacting the culture surface of the cell culture container with an aqueous solution containing a polycation material was not performed.

[0214] [Experimental Example 9] (Verification of cell aggregate detachment in cells other than nerve cells) HEK293 cells were seeded in three wells of each cell culture vessel used in Example 9 and Comparative Examples 19-20.

[0215] After culturing HEK293 cells for 3 days, the culture medium was changed to one containing Hoechst stain (Hoechst 33342, Thermo Fisher Scientific) after four pipetting operations, and each well was observed under a microscope. Furthermore, the average cell coverage area percentage of the three wells was calculated using imageJ from the images of all wells. The results are shown in Table 6 below.

[0216] [Table 6]

[0217] Table 6 shows that, even for easily detachable cells other than nerve cells, the cell culture method using a cell culture container, such as in Example 9, in which a first layer containing a polycation material is laminated on the culture surface, a second layer containing gelatin is laminated on the first layer, and a third layer containing cell adhesion factors is laminated on that layer, suppresses cell aggregate detachment compared to the cell culture methods using the cell culture containers of Comparative Examples 19-20. Furthermore, it was confirmed that the cell culture container of Example 9 can be manufactured using an inexpensive and simple method.

[0218] [Example 10] (Preparation of cell culture vessels in Example 10) Cell culture containers, aqueous solutions containing gelatin, aqueous solutions containing polycationic materials Cell culture vessels, an aqueous solution containing gelatin, and an aqueous solution containing a polycationic material were used, as in Example 6.

[0219] Preparation of the cell culture vessel for Example 10 A cell culture container of Example 10 with a modified culture surface was obtained in the same manner as in Example 6, except that the step of contacting the culture surface of the cell culture container with an aqueous solution containing cell adhesion factors was not performed.

[0220] [Comparative Example 21] (Preparation of cell culture vessels for Comparative Example 21) A cell culture vessel for Comparative Example 21, with a modified culture surface, was obtained using the exact same method as in Example 10.

[0221] [Experimental Example 10] (Verification of inhibition of cell aggregate aggregation in 384-well plates) Nerve cells were seeded in four wells of each cell culture vessel in Example 10 and Comparative Example 21. The same nerve cells as in Experimental Example 7 were used. Laminin was mixed into the cell suspension in Example 10 before seeding, while laminin was not mixed into the cell suspension in Comparative Example 21 before seeding.

[0222] After culturing nerve cells for 3 days, each well was observed under a microscope. FIG. 13 shows representative images of wells in each cell culture vessel.

[0223] From FIG. 13, after 3 days of culture, marked cell aggregation was observed in the culture method of Comparative Example 21. On the other hand, no marked cell aggregation was observed in the culture method of Example 10. From FIG. 13, it was confirmed that, as in Example 10, the culture method in which a first layer containing gelatin is laminated on a culture surface, a second layer containing a polycationic material is laminated on the first layer, and a liquid (medium) containing a cell adhesion factor is provided on the layer can suppress aggregation of cell aggregates, as compared with a culture method as in Comparative Example 21 in which no liquid (medium) containing a cell adhesion factor is provided on the layer. It was also confirmed that the cell culture vessel of Example 10 can be produced by an inexpensive and simple method.

[0224] From the above results, it was confirmed that according to the present invention, there can be provided a cell culture method, a cell culture vessel, and a method for producing the same, which can suppress detachment of cell aggregates from a culture surface and obtain stable functional evaluation results.

[0225] The present invention includes the following aspects. [1] A cell culture method comprising the step of housing a medium and cells in a cell culture vessel, and adhesively culturing the cells on a coat layer laminated on a surface of the cell culture vessel, wherein the coat layer comprises a layer (i) including a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and a layer (ii) containing a cell adhesion factor laminated on the layer (i), and the layer (ii) is arranged at a position in contact with the cells; or the coat layer comprises the layer (i), the layer (i) is arranged at a position in contact with the cells, and the medium contains a cell adhesion factor. [2] The cell culture method according to [1], wherein the cell adhesion factor is an extracellular matrix. [3] The cell culture method according to [1] or [2], wherein the gelatin is fish-derived gelatin. [4] The cell culture method according to any one of [1] to [3], wherein the polycation material is polyethyleneimine, polylysine, or polyornithine. [5] The cell culture method according to any one of [1] to [4], wherein the cells are cells that are easily detached. [6] A cell culture container having a coating layer laminated on its surface, wherein the coating layer comprises layer (i) comprising a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and layer (ii) laminated on layer (i) containing a cell adhesion factor, and layer (ii) is positioned to come into contact with cells during cell culture. [7] The cell culture vessel according to [6], wherein the cell adhesion factor is the extracellular matrix. [8] The cell culture container according to [6] or [7], wherein the gelatin is fish-derived gelatin. [9] A cell culture vessel according to any one of [6] to [8], wherein the polycation material is polyethyleneimine, polylysine, or polyornithine.

[10] A cell culture container according to any of [6] to [9], which is for culturing cells that are easily detached.

[11] A method for manufacturing a cell culture container with a modified culture surface, comprising the steps of: 1) contacting the surface of the cell culture container with a solution containing gelatin or casein; 2) removing the solution containing gelatin or casein; 3) contacting the surface of the cell culture container with a solution containing a polycationic material; 4) removing the solution containing the polycationic material; 5) contacting the surface of the cell culture container with a solution containing a cell adhesion factor; and 6) removing the solution containing the cell adhesion factor, in this order; or comprising the steps of 3, 4, 1, 2, 5, and 6 in this order.

[12] The manufacturing method according to

[11] , wherein the steps include, in the order of step 1, step 2, step 3, step 4, step 5, and step 6, a step of hydrophilizing the surface of the cell culture container before step 1, and wherein the steps include, in the order of step 3, step 4, step 1, step 2, step 5, and step 6, a step of hydrophilizing the surface of the cell culture container before step 3.

[13] The manufacturing method according to claim 12, wherein the step of hydrophilizing the cell culture vessel comprises (a) a step of bringing a solution containing ethanol into contact with the surface of the cell culture vessel and (b) a step of removing the solution containing ethanol.

[14] The method for producing a cell adhesion molecule according to any one of

[11] to

[13] , wherein the cell adhesion molecule is an extracellular matrix.

[15] The manufacturing method according to any one of

[11] to

[14] , wherein the gelatin is fish-derived gelatin.

[16] The manufacturing method according to any one of

[11] to

[15] , wherein the polycation material is polyethyleneimine, polylysine, or polyornithine.

[17] A cell-containing structure comprising a cell culture container, cells and a culture medium, wherein the cell culture container contains the culture medium and the cells, the cell culture container has a laminated coating layer on its surface, the cells are adhered to the coating layer, and the coating layer comprises a layer (i) comprising a layer (i-1) comprising gelatin or casein and a layer (i-2) comprising a polycation material, and a layer (ii) laminated on layer (i) comprising a cell adhesion factor, wherein layer (ii) is positioned in contact with the cells, or the coating layer comprises layer (i) and is positioned in contact with the cells, and the culture medium comprises a cell adhesion factor.

[0226] The cell culture method described in any of [1] to [5] above, the cell culture container with a modified culture surface described in any of [6] to

[10] above, the method for manufacturing the cell culture container described in any of

[11] to

[16] above, and the cell-containing structure described in

[17] above can solve the conventional problems and achieve the objectives of the present invention.

[0227] The present invention can also be said to include the following embodiments. [P1] A cell culture vessel with a modified culture surface, comprising a first layer containing gelatin or casein laminated on the culture surface of the cell culture vessel, and a second layer containing a polycationic material laminated on the first layer. [P2] The cell culture vessel according to [P1], further comprising a third layer containing cell adhesion factors, which is laminated on the second layer. [P3] The cell culture vessel according to [P2], wherein the cell adhesion factor is the extracellular matrix. [P4] A cell culture container according to any one of [P1] to [P3], wherein the gelatin is fish-derived gelatin. [P5] A cell culture vessel according to any one of [P1] to [P4], wherein the polycation material is polyethyleneimine, polylysine, or polyornithine. [P6] A cell culture container described in any of [P1] to [P5], for culturing easily detachable cells. [P7] A method for producing a cell culture container with a modified culture surface, comprising the steps of: contacting the culture surface of the cell culture container with a solution containing ethanol; removing the solution containing ethanol; contacting the surface of the cell culture container with a solution containing gelatin or casein; removing the solution containing gelatin or casein; contacting the surface of the cell culture container with a solution containing a polycationic material; and removing the solution containing the polycationic material, in this order. [P8] The manufacturing method according to [P7], further comprising, in this order, the step of removing the solution containing the polycation material, the step of bringing the solution containing the cell adhesion factor into contact with the surface of the cell culture container, and the step of removing the solution containing the cell adhesion factor. [P9] The method for producing the cell adhesion factor according to [P8], wherein the cell adhesion factor is the extracellular matrix. [P10] The method for producing the cell adhesion factor according to any one of [P7] to [P9], wherein the gelatin is fish-derived gelatin. [P11] The manufacturing method according to any one of [P7] to [P10], wherein the polycation material is polyethyleneimine, polylysine, or polyornithine. [Explanation of Symbols]

[0228] 11,21...Substrate, 12,22...Layer containing gelatin or casein, 13,23...Layer containing polycationic material, 14,24...Layer containing cell adhesion factors, 15...Easily detachable cells, 16...Liquid containing easily detachable cells and cell adhesion factors. [Prior art documents] [Patent Documents]

[0229] [Patent Document 1] Japanese Patent Publication No. 2018-166477 [Non-patent literature]

[0230] [Non-Patent Document 1] "Singlewell-M64-GLx_datasheet", Internet<URL:http: / / www.wpi-europe.com / downloads / content / Singlewell-M64-GLx_datasheet.pdf> [Searched on 2022 / 01 / 14] [Non-Patent Document 2] "Microelectrode Array (MEA) Manual", Internet <URL:https: / / www.multichannelsystems.com / sites / multichannelsystems.com / files / documents / manuals / MCS_MEA_Manual.pdf> [Searched on 2022 / 01 / 14] [Non-Patent Document 3] "Primary culture of hippocampal-derived nerve cells", Internet<URL:https: / / alphamedsci.com / common / pdf / dissociated1_190621%20(J).pdf> [Searched on 2021 / 01 / 14]

Claims

1. A cell culture method, The process includes the steps of placing a culture medium and cells in a cell culture container and adhering the cells to a coated layer laminated on the surface of the cell culture container, The coating layer comprises a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and includes a layer (i) arranged in the order of the substrate of the cell culture container, the layer (i-1), and the layer (i-2), and a layer (ii) containing a cell adhesion factor laminated on the layer (i), wherein the layer (ii) is positioned in contact with the cells. Cell culture method.

2. The cell culture method according to claim 1, wherein the cell adhesion factor is an extracellular matrix.

3. The cell culture method according to claim 1 or 2, wherein the gelatin is fish-derived gelatin.

4. The cell culture method according to claim 1 or 2, wherein the polycation material is polyethyleneimine, polylysine, or polyornithine.

5. The cell culture method according to claim 1 or 2, wherein the cells are cells that are easily detached.

6. A container for cell culture, A coating layer is laminated on the surface. A cell culture container comprising a coating layer comprising a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, a substrate of the cell culture container, a layer (i-1) and a layer (i-2) arranged in that order, and a layer (ii) containing a cell adhesion factor laminated on layer (i), wherein layer (ii) is positioned to come into contact with cells during cell culture.

7. The cell culture container according to claim 6, wherein the cell adhesion factor is an extracellular matrix.

8. The cell culture container according to claim 6 or 7, wherein the gelatin is fish-derived gelatin.

9. The cell culture container according to claim 6 or 7, wherein the polycation material is polyethyleneimine, polylysine, or polyornithine.

10. A cell culture container according to claim 6 or 7, which is for culturing cells that are easily detached.

11. Step 1 involves contacting the surface of a cell culture vessel with a solution containing gelatin or casein, Step 2 involves removing the solution containing the gelatin or the casein, Step 3 involves bringing a solution containing a polycation material into contact with the surface of the cell culture container, Step 4 to remove the solution containing the polycation material, Step 5 involves bringing a solution containing cell adhesion factors into contact with the surface of the cell culture container, A method for producing a cell culture vessel with a modified culture surface, comprising the steps of: 1) removing the solution containing the cell adhesion factor; 2) in this order; and 3)

12. The manufacturing method according to claim 11, further comprising the step of hydrophilizing the surface of the cell culture container before step 1.

13. The manufacturing method according to claim 12, wherein the hydrophilization step includes a step of bringing a solution containing ethanol into contact with the surface of the cell culture container (a) and a step of removing the solution containing ethanol (b).

14. The manufacturing method according to any one of claims 11 to 13, wherein the cell adhesion factor is an extracellular matrix.

15. The manufacturing method according to any one of claims 11 to 13, wherein the gelatin is fish-derived gelatin.

16. The manufacturing method according to any one of claims 11 to 13, wherein the polycation material is polyethyleneimine, polylysine, or polyornithine.

17. A cell-containing structure, Includes cell culture vessels, cells, and culture medium, The cell culture container contains the culture medium and the cells. The cell culture container has a coated layer laminated on its surface, and the cells are adhered to the coated layer. The coating layer comprises a layer (i-1) containing gelatin or casein and a layer (i-2) containing a polycationic material, and includes a layer (i) arranged in the order of the substrate of the cell culture container, the layer (i-1), and the layer (i-2), and a layer (ii) containing a cell adhesion factor laminated on the layer (i), wherein the layer (ii) is positioned in contact with the cells. Cell-containing structures.

Images