Cell culture substrate, cell culture container, kit, method for detaching adherent cells, method for culturing adherent cells, cell recovery device, and cell culture system

Abstract

Problem: To provide a cell culture substrate, a cell culture container, a kit, a method for detaching adherent cells, a method for culturing adherent cells, a cell recovery device, and a cell culture system, with each of which adherent cells can be detached more easily compared with the cases where trypsin is used and the adherent cells can be cultured more efficiently. Solution: A substrate for coating a cell culture container to be used for culturing adherent cells, the substrate comprising a first scaffold component being one or more proteins and a second scaffold component having a composition different from that of the first scaffold component, the second scaffold component being one or more proteins contained in an extracellular matrix.

Description

Cell culture substrate, cell culture vessel, kit, method for detaching adherent cells, method for culturing adherent cells, cell recovery device, and cell culture system

[0001] The present invention relates to a cell culture substrate, a cell culture vessel, a kit, a method for detaching adherent cells, a method for culturing adherent cells, a cell recovery device, and a cell culture system.

[0002] Conventionally, there has been known a technique for recovering cultured adherent cells by culturing them and then detaching the cells adhered to a cell culture vessel using trypsin. Furthermore, there has also been known a technique for performing a surface treatment, such as plasma treatment, gelatin coating, or collagen coating, on the surface of the cell culture vessel in order to enhance the adhesiveness of the adherent cells to the cell culture vessel (see, for example, Patent Document 1).

[0003] Conventional cell detachment methods for adherent cells involve trypsin treatment to detach adherent cells from cell culture vessels. Trypsin treatment requires steps to remove the liquid medium, wash with a buffer solution, and then remove the buffer solution before adding trypsin to prevent trypsin from reacting with components in the liquid medium. Furthermore, because trypsin also damages the surface of cultured adherent cells, a step of inactivating trypsin by adding liquid medium after a certain period of trypsin reaction is required. Thus, detachment of adherent cells using trypsin is labor-intensive, and a simpler method for detaching adherent cells is desired to reduce manufacturing costs. Furthermore, cell culture vessels that have undergone surface treatments such as plasma treatment, gelatin coating, and collagen coating to enhance the adhesiveness of adherent cells have been provided. However, these surface-treated cell culture vessels are expensive, which also contributes to increased manufacturing costs.

[0004] Therefore, the inventor discovered that by culturing cells using a cell culture substrate containing one or more proteins selected from silk protein, silk protein hydrolysate, and fibroin as active ingredients, and then decomposing the cultured cells with a silk protein-degrading enzyme such as protease 14, the cultured cells can be detached without using trypsin, and filed a patent application (see Patent Document 2).

[0005] Japanese Patent Application Publication No. 5-260950, Patent Application No. 2023-507329

[0006] However, cell culture substrates containing one or more of the above-mentioned silk proteins, silk protein hydrolysates, and fibroins as active ingredients have the problem that they have low adhesiveness to some adherent cells, such as iPS cells and mesenchymal stem cells (hereinafter referred to as MSCs), resulting in reduced culture efficiency of these adherent cells.

[0007] The present invention aims to provide a cell culture substrate, a cell culture vessel, a kit, a method for detaching adherent cells, a method for culturing adherent cells, a cell recovery device, and a cell culture system that enable adherent cells to be detached more easily than when trypsin is used and that enable adherent cells to be cultured more efficiently.

[0008] The present invention provides cell culture substrates as described below in (1) to (3). (1) A cell culture substrate for coating a cell culture vessel for culturing adherent cells, the cell culture substrate comprising a first scaffold component comprising one or more proteins and a second scaffold component having a different composition from the first scaffold component, the second scaffold component being one or more proteins contained in an extracellular matrix. (2) The cell culture substrate described above in (1), the first scaffold component being one or more proteins not contained in an extracellular matrix that function as a scaffold material. (3) The cell culture substrate described above in (2), the first scaffold component containing, as active ingredients, one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, and fibroin, and an adhesion factor for target adherent cells. The present invention also provides cell culture vessels as described below in (4) to (6). (4) A cell culture vessel coated with the cell culture substrate described above in any one of (1) to (3). (5) The cell culture vessel according to (4) above, which may have a first coating layer formed by coating the cell culture vessel with a first cell culture substrate containing the first scaffold substrate component as an active ingredient, and a second coating layer formed by coating the first coating layer with a second cell culture substrate containing the second scaffold substrate component as an active ingredient. (6) The cell culture vessel according to (4) above, which may have a coating layer formed by coating a cell culture substrate comprising a mixture of a first cell culture substrate containing the first scaffold substrate component as an active ingredient and a second cell culture substrate containing the second scaffold substrate component as an active ingredient. The present invention further encompasses a kit for preparing a cell culture substrate as described below in (7). (7) A kit for preparing a cell culture substrate, in which the first scaffold substrate component and the second scaffold substrate component are enclosed or packaged separately. Additionally, the present invention encompasses methods for detaching adherent cells as described below in (8) to (12).(8) A method for detaching adherent cells cultured using the cell culture vessel according to any one of (4) to (6) above, from the cell culture vessel, comprising adding a protease that specifically degrades the first scaffold substrate component rather than the second scaffold substrate component and allowing it to react with the cell culture substrate to detach the adherent cells from the cell culture vessel. (9) The method for detaching adherent cells according to (8) above, which can be configured to add the protease after removing the liquid medium used for culture, without washing the cell culture substrate, to detach the adherent cells from the cell culture vessel. (10) The method for detaching adherent cells according to (8) above, which can be configured to add the protease without removing the liquid medium used for culture, to detach the adherent cells from the cell culture vessel. (11) A method for detaching adherent cells cultured using the cell culture vessel according to any one of (4) to (6) above, from the cell culture vessel, comprising adding a protease that specifically degrades the first scaffold substrate component rather than the second scaffold substrate component and allowing it to react with the cell culture vessel, thereby detaching the adherent cells in a clumped state from the cell culture vessel without pipetting. (12) The method for detaching adherent cells according to any one of (8) to (11) above, wherein the first scaffold substrate component is one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, and fibroin, and the protease is selected from the group consisting of a Streptomyces griseus-derived protease or a mixture thereof, a Bacillus Licheniformis-derived protease or a mixture thereof, α-chymotrypsin, and collagenase IA. The present invention also encompasses a method for culturing adherent cells as set forth in (13) below. (13) A method for culturing adherent cells, comprising a culture step of culturing adherent cells in a cell culture vessel as set forth in any one of (4) to (6) above, and a detachment step of detaching the adherent cells cultured in the culture step by the method for detaching adherent cells as set forth in any one of (8) to (12) above. Furthermore, the present invention also encompasses a cell recovery device as set forth in (14) below.(14) A cell recovery device for recovering adherent cells cultured using the cell culture vessel described in any one of (4) to (6) above, the cell recovery device performing enzyme addition control for adding a protease that specifically degrades the first scaffold base material component rather than the second scaffold base material component to the cell culture vessel in which the adherent cells have been cultured, and cell recovery control for recovering the adherent cells without performing a pipetting operation after the enzyme addition control. Additionally, the present invention also provides a cell culture system described in (15) below. (15) A cell culture system comprising: a culture device for culturing adherent cells using the cell culture vessel described in any one of (4) to (6) above; and the cell recovery device described in (14) above for recovering the adherent cells cultured in the culture device.

[0009] The present invention can also include the following cell detachment methods (16) to (21): (16) A method for detaching adherent cells, which comprises detaching adherent cells cultured in a cell culture vessel coated with a cell culture substrate containing, as an active ingredient, any one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, fibroin, and fibroin hydrolysate, from the cell culture substrate, wherein the detachment agent for the adherent cells is a protease derived from Streptomyces griseus or a mixture thereof, a protease derived from Bacillus Licheniformis or a mixture thereof, α-chymotrypsin, or collagenase IA. (17) A cell detachment method comprising the steps of culturing adherent cells in a cell culture vessel coated with a cell culture substrate containing, as an active ingredient, any one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, fibroin, and fibroin hydrolysate, and adding a Streptomyces griseus-derived protease or a mixture thereof, a Bacillus Licheniformis-derived protease or a mixture thereof, α-chymotrypsin, or collagenase IA to detach the adherent cells from the cell culture substrate. (18) The cell detachment method according to (16) or (17), comprising removing the liquid medium used to culture the adherent cells, and, after removing the liquid medium, adding the detachment agent without washing the cell culture substrate to detach the adherent cells. (19) The cell detachment method according to (16) or (17), comprising adding the detachment agent without removing the liquid medium used to culture the adherent cells to detach the adherent cells. (20) The cell detachment method according to any one of (16) to (19) above, wherein the detached adherent cells are recovered without any treatment to inactivate the added detachment agent.(21) The protease derived from Streptomyces griseus or a mixture thereof is Empynase PD, Pronase, Pronase A, Pronase E, Pronase-p, Actinase E, Protease type XIV, Proteinine, Proteinaza 1, Streptomyces griseus protease, or Streptomyces griseus proteinase, and the Bacillus The cell detachment method according to any one of (16) to (20) above, wherein the Licheniformis-derived protease or a mixture thereof is protease type 8 (protease type XIII). The present invention can also include the following method for culturing adherent cells (22). (22) A method for culturing adherent cells, comprising a culture step of culturing adherent cells in the cell culture vessel, and a detachment step using the cell detachment method for adherent cells according to any one of (16) to (21) above. The present invention can also include the following kit for culturing adherent cells (23). (23) A kit for culturing adherent cells, comprising a cell culture substrate containing, as an active ingredient, any one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, fibroin, and fibroin hydrolysate, or a cell culture vessel coated with the cell culture substrate, and a detachment agent for adherent cells adhered to the cell culture substrate, which is a Streptomyces griseus-derived protease or a mixture thereof, a Bacillus Licheniformis-derived protease or a mixture thereof, α-chymotrypsin, or collagenase IA.

[0010] According to the present invention, the scaffold material contains as an active ingredient one of silk protein, silk protein hydrolysate, and fibroin, which function as a scaffold material.Therefore, by using a silk protein-degrading enzyme, adherent cells can be detached from the culture vessel, and detachment of adherent cells can be easily performed without using trypsin.In addition, by containing adhesion factors of the target adherent cells as a scaffold material, the adhesiveness of adherent cells that have low adhesiveness with the above protein alone can be increased.

[0011] FIG. 1 is a schematic side view showing a double-coated container and a mixed-coated container according to the first embodiment. FIG. 2 is a flowchart illustrating the procedure of a conventional method for detaching adherent cells using trypsin. FIG. 3 is a flowchart illustrating the procedure of a method for detaching adherent cells according to the first embodiment. FIG. 4 is a diagram illustrating the detachment of adherent cells in a conventional cell culture container and the detachment of cells cultured in a cell culture container according to the present embodiment. FIG. 5 is a micrograph of human iPS cells in Example 1. FIG. 6 is a micrograph of human iPS cells in Example 2. FIG. 7 is a micrograph of human iPS cells in Example 3. FIG. 8 is a graph showing the results of counting the number of adherent cells recovered in Example 3. FIG. 9 is a micrograph of MSCs in Example 4. FIG. 10 is a schematic side view showing a cell culture container according to the second embodiment. FIG. 11 is an image of Petri dishes showing the verification results of cell culture in Example 5, where (A) is an image after culturing adherent cells for three days using a Petri dish not coated with a cell culture substrate according to the present embodiment, and (B) is an image after culturing adherent cells for three days using a Petri dish coated with a cell culture substrate according to the present embodiment. FIG. 12 is a graph showing the growth curves of adherent cells cultured by the method according to the present embodiment and the growth curves of adherent cells cultured by a conventional method in Example 6. 1 is a diagram showing a captured image of adherent cells passaged after culture by the method according to the present embodiment and a captured image of adherent cells passaged after culture by a conventional method in Example 7. FIG. 1 is a diagram showing verification results in Example 8. FIG. 1 is a diagram showing verification results in Example 9. FIG. 1 is a diagram showing verification results in Example 10, illustrating the case where insolubilization treatment was performed using methanol. FIG. 1 is a diagram showing verification results in Example 10, illustrating the case where insolubilization treatment was performed using methanol. FIG. 1 is a diagram showing verification results in Example 10, illustrating the case where insolubilization treatment was performed by leaving in a water vapor saturated state. FIG. 1 is a diagram showing verification results in Example 10, illustrating the case where insolubilization treatment was performed by leaving in a water vapor saturated state. FIG. 1 is a diagram showing verification results in Example 11. FIG. 12 is a diagram showing a captured image of adherent cells cultured using a cell culture vessel produced by autoclave sterilizing the cell culture substrate according to this example.12 is a diagram showing an image of an observation of the detached state of adherent cells cultured using a cell culture vessel produced by autoclave sterilization of the cell culture substrate according to this example in Example 12. FIG. 13 is a diagram showing the verification results of Example 13. FIG. 14 is a diagram showing the verification results of Example 14, illustrating the case where the cell culture vessel according to this example was used. FIG. 14 is a diagram showing the verification results of Example 14, illustrating the case where a conventional cell culture vessel was used. FIG. 15 is a diagram for explaining the verification results of cell culture using the cell culture vessel produced in Example 15. FIG. 16 is a diagram for explaining the verification results of cell culture using the cell culture vessel produced in Example 16. FIG. 17 is a diagram for explaining the verification results of cell detachment using the cell culture vessel produced in Example 17. FIG. 18 is a diagram showing the results of detaching adherent cells using multiple types of proteases derived from Streptomyces griseus, as shown in Example 18. FIG. 11 shows the results of detaching adherent cells using a protease derived from Bacillus Licheniformis, as shown in Example 19.

[0012] The present invention relates to a cell culture substrate and cell culture vessel for culturing adherent cells, a kit for producing the cell culture substrate or cell culture vessel, a method for detaching and culturing adherent cells using the cell culture substrate or cell culture vessel, and a cell collection device and cell culture system. Below, the cell culture substrate, cell culture vessel, kit, method for detaching adherent cells, method for culturing adherent cells, cell collection device, and cell culture system according to the present embodiments will be described with reference to the drawings.

[0013] First Embodiment A first embodiment of the present invention will be described. In the first embodiment, adherent cells are cultured using a cell culture vessel coated with a cell culture substrate containing, as active ingredients, a first scaffold substrate component and a second scaffold substrate component different from the first scaffold substrate component. The cultured adherent cells are then detached using a protease that specifically degrades the first scaffold substrate component rather than the second scaffold substrate component. The first scaffold substrate component is one or more proteins that function as a scaffold material and are not contained in the extracellular matrix. In this embodiment, examples of the first scaffold substrate component include one or more proteins such as silk protein, silk protein hydrolysate, and fibroin. The first scaffold substrate component is preferably a protein not contained in the adherent cells. The first scaffold substrate component is not limited to proteins such as silk protein, as long as it is one or more proteins that function as a scaffold material and are not contained in the extracellular matrix. It is also possible to use animal proteins such as egg protein and milk protein, or plant proteins such as soy protein. The second scaffold substrate component is one or more proteins contained in the extracellular matrix (not limited to the extracellular matrix of the adherent cells to be cultured) that function as a scaffold material. In this embodiment, examples include adhesion factors such as laminin, Matrigel, and collagen. By using one or more proteins as the first scaffold substrate component that are not contained in the adherent cells to be cultured and that are not contained in the extracellular matrix, when detaching the adherent cells, a protease that specifically degrades the proteins contained in the first scaffold substrate component rather than the second scaffold substrate component can be used to detach the adherent cells without significantly damaging the adherent cells by acting on the proteins contained in the adherent cells. Furthermore, by using one or more proteins contained in the extracellular matrix of the adherent cells to be cultured as the second scaffold substrate component, even adherent cells that are difficult to adhere to the first scaffold substrate component alone can be attached to the cell culture substrate, enabling efficient cultivation and detachment of adherent cells.The cell culture substrate, cell culture vessel, kit, method for detaching adherent cells, method for culturing adherent cells, cell recovery device, and cell culture system according to the first embodiment will be described in detail below.

[0014] (Cell Culture Substrate) The cell culture substrate of the present invention functions as a scaffold material for enhancing adhesion of adherent cells to cell culture vessels and can be used as a coating agent for coating cell culture vessels. In a first embodiment, the cell culture substrate is characterized by containing, as active ingredients, a first scaffold component that functions as a scaffold material and is one or more proteins not contained in the extracellular matrix, and a second scaffold component that is one or more proteins contained in the extracellular matrix (not limited to the extracellular matrix of the adherent cells to be cultured). In this embodiment, the first scaffold component is described using one or more proteins such as silk protein, silk protein hydrolysate, or fibroin, and the second scaffold component is described using proteins such as laminin, Matrigel, or collagen, but the first and second scaffold components are not limited to these.

[0015] In this embodiment, a substrate containing one or more proteins, either silk protein, silk protein hydrolysate, or fibroin, as an active ingredient will be referred to as a first cell culture substrate, and a substrate containing an adhesion factor of adherent cells, such as laminin, Matrigel, or collagen, as an active ingredient will be referred to as a second cell culture substrate.

[0016] Silk protein is a type of protein fiber secreted by insects such as silkworms or spiders. The form and refining method of silk protein are not particularly limited. Not only can cocoon silk, raw silk, or silk thread obtained from silkworms be used as raw materials, but also, for example, those physically crushed into powder (ground type) or fibers hydrolyzed with acid or alkali (hydrolyzed type) can be used as raw materials. Fibroin, along with sericin, is a protein that constitutes silk protein. Fibroin can be extracted using known methods. For example, since sericin is a water-soluble protein, the fibrous protein fibroin can be extracted by removing sericin, such as by boiling silk protein in water. Commercially available silk powder can also be used as fibroin. The following description will be given using, as an example, a first cell culture substrate containing fibroin as an active ingredient, among silk protein, silk protein hydrolysate, and fibroin.

[0017] A first cell culture substrate containing fibroin as an active ingredient can be prepared, for example, as follows. Specifically, dried domesticated silkworm cocoons are cut into pieces approximately 1 cm square, heated and stirred in a heated sodium carbonate solution for approximately 30 minutes to remove sericin, and the sodium carbonate is removed. The resulting cocoons (fibroin fibers) are then dried to obtain refined cocoons. The resulting refined cocoons (fibroin fibers) are then dissolved to obtain a fibroin solution. The fibroin solution is then placed in a dialysis film and dialyzed against ion-exchanged water or pure water to remove salts and solvents used for dissolution. In this embodiment, the fibroin solution after dialysis is adjusted to a fibroin concentration of 0.1 to 1.0%, thereby obtaining a first cell culture substrate (fibroin aqueous solution) containing fibroin as an active ingredient.

[0018] The first cell culture substrate according to this embodiment is not limited to those containing silk protein, silk protein hydrolysate, or fibroin as an active ingredient, and may also contain, for example, egg protein, milk protein, or vegetable protein such as soy protein as an active ingredient. The first cell culture substrate according to this embodiment may also contain water or a buffer solution in addition to the protein active ingredient. The first cell culture substrate according to this embodiment may also contain a mixture of two or more of silk protein, silk protein hydrolysate, and fibroin.

[0019] Adhesion factors for adherent cells are proteins directly involved in the adhesion between adherent cells, and examples include the extracellular matrix proteins fibronectin, collagen, gelatin, laminin, vitronectin, pronectin, proteoglycans, hyaluronic acid, tenascin, entactin, elastin, and compounds derived therefrom. These proteins are also sometimes referred to as structural proteins, fibrous proteins, glycosaminoglycans, etc. For example, laminin and collagen are sometimes referred to as fibrous proteins. Matrigel (registered trademark), which contains multiple components constituting the extracellular matrix, can also be used as an adhesion factor. The following describes a second cell culture substrate containing laminin, Matrigel, or gelatin as an active ingredient. The second cell culture substrate may be a solution in which an adhesion factor is dissolved in a buffer solution or liquid medium, or a solution in which adhesive cells such as laminin or Matrigel are dissolved in a fibroin aqueous solution prepared as the first cell culture substrate may be mixed with the second cell culture substrate to form a cell culture substrate.

[0020] The first and second cell culture substrates according to the first embodiment are preferably sterilized, for example, by high-pressure steam sterilization using an autoclave, or by sterilization using gamma rays, electron beams, or filters, which are used for conventional cell culture substrates.

[0021] (Cell Culture Vessel) The cell culture vessel according to the first embodiment is a cell culture vessel in which the above-described cell culture substrate is coated on the bottom surface of the cell culture vessel. As described above, in this embodiment, two coating methods, double coating and mixed coating, can be used. Below, cell culture vessels produced by each coating method will be described with reference to Figure 1. Note that Figure 1 is a schematic side view illustrating the cell culture vessel according to the first embodiment.

[0022] (Double-coated container) First, the double-coated container will be described. Fig. 1(A) is a diagram for explaining the double-coated container. As shown in Fig. 1(A), the double-coated container 1 has a coating layer 20 of a second cell culture substrate formed on a coating layer 10 of a first cell culture substrate.

[0023] A double-coated vessel 1 having such a configuration can be fabricated as follows. For example, a liquid first cell culture substrate containing silk protein, silk protein hydrolysate, or fibroin as an active ingredient is first poured or sprayed onto the bottom surface of an untreated cell culture vessel to coat the bottom surface evenly, and then the bottom surface is dried, for example, by placing the vessel in a dryer. To prevent the active ingredient, such as silk protein, silk protein hydrolysate, or fibroin, from dissolving into the liquid medium during cell culture, the active ingredient, such as silk protein, silk protein hydrolysate, or fibroin, may be insolubilized by adding ethanol or methanol to the bottom surface of the dried cell culture vessel and contacting it, or by leaving it in a saturated water vapor state overnight. After the first cell culture substrate has dried and a first cell culture substrate coating layer 10 has been formed, a liquid second cell culture substrate containing an adhesion factor as an active ingredient is injected or sprayed onto the bottom surface of the cell culture vessel on which the first cell culture substrate coating layer 10 has been formed, so as to evenly coat the bottom surface, and the vessel is then allowed to stand for a certain period of time, thereby forming a second cell culture substrate coating layer 20. This allows the production of a double-coated vessel 1 in which a second cell culture substrate coating layer 20 has been formed on top of the first cell culture substrate coating layer 10.

[0024] (Mixed-coated vessel) Fig. 1(B) is a schematic side view illustrating the mixed-coated vessel 2. As shown in Fig. 1(B), in the mixed-coated vessel 2, the bottom surface 4 of the cell culture vessel 3 is coated with a mixed cell culture substrate obtained by mixing the first cell culture substrate and the second cell culture substrate, thereby forming a single coating layer 30.

[0025] The mixed-coated vessel 2 can be prepared as follows. For example, first, an adhesion factor such as laminin, Matrigel, or collagen is dissolved in a liquid first cell culture substrate containing silk protein, silk protein hydrolysate, or fibroin as an active ingredient to prepare a mixed cell culture substrate by mixing the first cell culture substrate and the second cell culture substrate. The prepared mixed cell culture substrate is then poured or sprayed onto the bottom surface 4 of an untreated cell culture vessel 3 to evenly coat the bottom surface, and then allowed to stand for a certain period of time to form a coating film. This allows the preparation of a mixed-coated vessel 2 on which a single coating layer 30 of mixed cell culture substrates containing the first cell culture substrate and the second cell culture substrate is formed.

[0026] In this embodiment, the type of cell culture vessel is not particularly limited, and can be provided as, for example, a petri dish, a plate, a flask, a multi-layer flask, or the like. The material of the cell culture vessel according to this embodiment is also not particularly limited, and for example, a container made of plastic or glass can be used. Furthermore, the container does not require special surface treatment before being coated with the cell culture substrate according to this embodiment, and inexpensive containers with untreated (hydrophobic) surfaces, such as polystyrene petri dishes, can be used. Furthermore, the cell culture vessel according to this embodiment may have not only the bottom surface but also the sides coated with the cell culture substrate according to this embodiment. In addition, since the cell culture vessel according to this embodiment is produced using a liquid cell culture substrate, it can also have a bottom or side with a complex shape.

[0027] (Cultivation and Detachment of Adherent Cells) Next, a method for culturing and detaching adherent cells using the cell culture vessel according to the present embodiment will be described. As with conventional cell culture vessels, the cell culture vessel according to the present embodiment can be used to seed adherent cells, followed by the addition of a culture medium for culturing the adherent cells. In particular, the cell culture vessels according to the present embodiment (double-coated vessels and mixed-coated vessels) are coated with a first cell culture substrate containing silk protein, a silk protein hydrolysate, or fibroin as an active ingredient, and a second cell culture substrate containing an adhesion factor of adherent cells as an active ingredient. These cell culture substrates serve as scaffolds for the adherent cells to the cell culture vessel, allowing them to proliferate. In particular, the use of double-coated vessels tends to result in higher adhesiveness of adherent cells and higher culturing efficiency of adherent cells than the use of mixed-coated vessels.

[0028] Furthermore, in the cell culture vessel according to the present embodiment, unlike conventional cell detachment methods using trypsin, cell detachment is performed using a protease capable of efficiently degrading silk protein, silk protein hydrolysate, or fibroin contained in the first cell culture substrate (hereinafter simply referred to as a silk protein-degrading enzyme). First, the procedure of the conventional cell detachment method using trypsin and the procedure of the cell detachment method according to the present embodiment will be described with reference to Figures 2 and 3. Figure 2 is a flowchart illustrating the procedure of the conventional method of detaching adherent cells using trypsin, and Figure 3 is a flowchart illustrating the procedure of the method of detaching adherent cells according to the present embodiment.

[0029] In conventional methods for detaching adherent cells using trypsin, if trypsin is added without first removing the liquid medium used during cell culture, the trypsin reacts with components of the liquid medium, resulting in insufficient detachment of the adherent cells. Therefore, as shown in Figure 2, after culturing adherent cells in a cell culture vessel (step S101), the liquid medium is removed before adding trypsin (step S102). Furthermore, the dish is washed with phosphate buffer (step S103), and the washed phosphate buffer is removed (step S104). Trypsin is then added (step S105), and the trypsin is allowed to react at a constant temperature for a certain period of time (step S106). Furthermore, after the trypsin treatment, liquid medium is added again (step S107) to prevent damage to the adherent cells caused by trypsin, and the trypsin is inactivated. The adherent cells are then precipitated by centrifugation (step S108), the supernatant liquid medium is removed (step S109), and the precipitated adherent cells can be recovered by adding liquid medium again for storage (step S110) (step S111).

[0030] In contrast, in the cell culture vessels (double-coated vessels and mixed-coated vessels) according to the present embodiment, the silk protein, silk protein hydrolysate, or fibroin contained in the first cell culture substrate can be decomposed by reacting with a silk protein-degrading enzyme that efficiently decomposes these proteins, thereby separating and detaching the adherent cells from the cell culture vessel. Furthermore, because the silk protein-degrading enzyme has the property of being less reactive with the protein components contained in the liquid culture medium for the adherent cells, the silk protein-degrading enzyme can be used without removing the liquid culture medium.

[0031] Therefore, in the adherent cell detachment process according to this embodiment, after culturing the adherent cells (step S201), the silk protein-degrading enzyme is added directly (step S202) as shown in Fig. 3, and the enzyme is allowed to react at a constant temperature for a certain period of time (step S203), without removing the liquid medium, washing with phosphate buffer, and then removing the washed phosphate buffer, as in the case of using trypsin as shown in Fig. 2. In particular, when a mixed-coated vessel is used, the adherent cells tend to be more easily detached than when a double-coated vessel is used, and it becomes possible to detach the grown adherent cells in clumps without pipetting. Furthermore, since silk protein-degrading enzymes react less readily with the proteins of the adherent cells themselves than trypsin, in this embodiment, after adding and reacting the silk protein-degrading enzyme in step S202, there is no need to add liquid culture medium to inhibit the activity of the silk protein-degrading enzyme, as in step S107 of Figure 2. Instead, as shown in Figure 3, centrifugation is performed as is (step S204), the supernatant of the liquid culture medium is removed (step S205), and then the liquid culture medium is added again for storage (step S206), and the settled adherent cells can be recovered (step S207).

[0032] FIG. 4 illustrates the detachment of adherent cells in a conventional cell culture vessel and in a cell culture vessel according to the present embodiment. FIG. 4(A) shows adherent cells cultured in a conventional cell culture vessel (e.g., a vacuum plasma-treated petri dish) detached with trypsin, and FIG. 4(B) shows adherent cells cultured in a cell culture vessel according to the present embodiment detached with a silk protein-degrading enzyme. As shown in FIG. 4(A), conventional cell culture vessels are surface-treated with plasma, gelatin, collagen, or other methods to enhance the adhesiveness of adherent cells, and the addition of trypsin results in the detachment of adherent cells. However, trypsin not only degrades the bonds between adherent cells themselves, but also the bonds between adherent cells and the extracellular matrix, and between the extracellular matrix itself, resulting in the problem of the adherent cells breaking apart, as shown in FIG. 4(A). Another problem is that trypsin acts on the adherent cells, damaging them.

[0033] In contrast, the cell culture vessel according to this embodiment is coated with a first cell culture substrate containing silk protein, a silk protein hydrolysate, or fibroin as an active ingredient, as shown in Figure 4(B), and therefore, by using a silk protein-degrading enzyme to decompose the silk protein, silk protein hydrolysate, or fibroin, adherent cells can be detached from the cell culture vessel. Furthermore, by using the silk protein-degrading enzyme, as shown in Figure 4(B), the adherent cells do not break up, but can be detached in a sheet or clump, and damage to the adherent cells can be suppressed.

[0034] The silk protein decomposing enzyme is an enzyme that decomposes silk protein, a silk protein hydrolysate, or fibroin, and examples thereof include protease 14 derived from Streptomyces griseus or a mixture thereof. Further, examples of silk protein-degrading enzymes include proteases derived from Streptomyces griseus and mixtures thereof such as Empynase PD, Pronase, Pronase A, Pronase E, Pronase-p, Actinase E, Protease type XIV, Proteinine, Proteinaza 1, Streptomyces griseus protease, and Streptomyces griseus proteinase. Specific examples (product examples) of these proteases derived from Streptomyces griseus or mixtures thereof include Protease P8811 (manufactured by Sigma-Aldrich), Protease (manufactured by Wako Pure Chemical Industries, Ltd.), Actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.), Protease P5174 type 14 (manufactured by Sigma-Aldrich), Protease E (manufactured by Sigma-Aldrich), and Pronase (manufactured by Roche). Furthermore, proteases derived from Bacillus Licheniformis or mixtures thereof, α-chymotrypsin, or collagenase IA can also be used as silk protein-degrading enzymes. An example of a protease derived from Bacillus Licheniformis or a mixture thereof is protease type 8 (Protease type XIII), and a specific example (product example) of this is Protease P5380 type 8 (manufactured by Sigma-Aldrich).

[0035] In addition, the silk protein degrading enzyme is preferably an enzyme other than trypsin that does not degrade (or has extremely low degrading ability) the extracellular matrix derived from adherent cells. Furthermore, the silk protein degrading enzyme is preferably an enzyme that has higher reactivity (degrading ability) with silk protein, silk protein hydrolysate, or fibroin than trypsin, and lower reactivity (degrading ability) with the extracellular matrix derived from adherent cells.

[0036] (Cell Culture Substrate Preparation Kit) Furthermore, this embodiment can also provide a cell culture substrate kit for producing a cell culture substrate according to this embodiment. Specifically, the kit according to this embodiment is configured by enclosing or separately packaging a first scaffold substrate component, which functions as a scaffold material and is one or more proteins not contained in the extracellular matrix, and a second scaffold substrate component, which is one or more proteins contained in the extracellular matrix. As an example of a kit according to this embodiment, a first cell culture substrate containing one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, and fibroin as an active ingredient, and a second cell culture substrate containing an adhesion factor of target adherent cells as an active ingredient, mixed and packaged together, or the first cell culture substrate and the second cell culture substrate, individually packaged, can be provided as a kit. This allows those who wish to prepare a cell culture vessel according to this embodiment (double-coated vessel and / or mixed-coated vessel) to prepare the cell culture vessel according to this embodiment using the first cell culture substrate and second cell culture substrate included in the kit.

[0037] (Cell Collection Device) Additionally, this embodiment can provide a cell collection device that automatically collects adherent cells using the adherent cell detachment method according to this embodiment. For example, such a cell collection device is preferably configured to perform enzyme addition control, which adds a silk protein-degrading enzyme to cells cultured in a cell culture vessel according to this embodiment, and cell recovery control, which recovers the detached adherent cells. In particular, by using a mixed-coating container according to this embodiment, the cell collection device can detach and recover adherent cells in clumps without performing operations that are difficult to control mechanically, such as pipetting. Furthermore, by adding a silk protein-degrading enzyme that does not interact easily with liquid culture medium or adherent cells, the cell collection device can detach adherent cells without performing the steps of removing the liquid culture medium, washing the Petri dish with phosphate buffer, and removing the washed phosphate buffer before adding the silk protein-degrading enzyme (steps S102 to S104 in FIG. 2), or the step of adding a liquid culture medium to inhibit the activity of the silk protein-degrading enzyme (step S107 in FIG. 2).

[0038] Next, an example of culturing and detaching adherent cells using the cell culture vessel according to this embodiment will be described.

[0039] Example 1 In Example 1, human iPS cells were used as adherent cells, and whether the human iPS cells adhere to the cell culture substrate when cultured using the double-coated container according to the present embodiment was examined. In Example 1, a relatively large number of human iPS cells were seeded compared to Example 3 described below, and observations were made from the perspective of whether the seeded human iPS cells adhere to the cell culture substrate according to the present embodiment.

[0040] First, we will explain the method for producing the double-coated container used in Example 1. In Example 1, a double-coated container was produced by coating the wells of a well plate with a first cell culture substrate containing fibroin as an active ingredient, and then coating the wells with a second cell culture substrate containing various types of laminin or Matrigel as an active ingredient as an adhesion factor.

[0041] Specifically, a 0.1% fibroin aqueous solution was first prepared as the first cell culture substrate. More specifically, dried domesticated silkworm cocoons were cut into quarters, and 1 L of 0.02 M (mol / L) sodium carbonate solution was added to 10 g of the cut domesticated silkworm cocoons. The mixture was stirred at 95-98°C for approximately 30 minutes to remove sericin from the domesticated silkworm cocoons, yielding fibroin fibers. The resulting fibroin fibers were then washed with ion-exchanged water or ultrapure water, and the sodium carbonate was removed and dried. 50 mL of a solution consisting of calcium chloride, ethanol, and water in a ratio of 1:2:8 M (mol / L) was added to 3 g of dried, refined cocoons (fibroin fibers) to dissolve the refined cocoons. The mixture was then heated to approximately 70°C, stirred for approximately 1 hour, and then cooled to room temperature. Approximately 50 ml of the dissolved fibroin solution was then placed in a dialysis film (approximately 28 mm in diameter, molecular weight cutoff 12,000-14,000) and dialyzed against 8 L of ion-exchanged water or ultrapure water for approximately half a day. This process was repeated six times. This removed calcium and ethanol from the solution. Furthermore, the concentration of the fibroin solution after dialysis was measured, and ion-exchanged water or ultrapure water was added to adjust the fibroin concentration to 0.1%. The concentration of the fibroin solution after dialysis was measured by the direct gravimetric method (a method for measuring the concentration of the fibroin solution by determining the weight of the residue (fibroin) after total evaporation of the fibroin solution after dialysis). In this way, a 0.1% fibroin solution was prepared as a first cell culture substrate containing fibroin as an active ingredient.

[0042] Next, laminin solutions and Matrigel solutions were prepared as second cell culture substrates. In Example 1, laminin solutions containing laminin 111, laminin 121, laminin 211, laminin 221, laminin 332, laminin 411, laminin 511, and laminin 521 were prepared. Specifically, when using various laminins as adhesion factors, 2 μg of each laminin was dissolved in 300 μL of 0.5% BSA / PBS solution to prepare various laminin solutions as second cell culture substrates containing various laminins as the active ingredient. When using Matrigel as the adhesion factor, Matrigel was diluted 60-fold with RPMI medium to prepare Matrigel solutions as second cell culture substrates containing Matrigel as the active ingredient. The various laminins used were proprietary preparations, and commercially available Matrigel was used.

[0043] A first cell culture substrate containing fibroin as an active ingredient was coated as the first layer, and then a second cell culture substrate containing various laminin or Matrigel adhesion factors as an active ingredient was coated as the second layer to prepare a double-coated container. Specifically, 300 μL of the first cell culture substrate was poured into the well of a well plate, left to stand at room temperature for 1 hour, the remaining liquid was removed by suction, and the well was left to dry at room temperature for another 45 minutes in a clean bench, thereby coating the first cell culture substrate as the first layer. Next, 300 μL of the prepared laminin or Matrigel solution was poured onto the well coated with the first cell culture substrate, and left to stand at 37°C for at least 1 hour to prepare a double-coated container.

[0044] In Example 1, as comparative examples, a single-coated container coated only with a first cell culture substrate containing fibroin as an active ingredient and a single-coated container coated only with a second cell culture substrate containing Matrigel as an active ingredient were also prepared. Specifically, 300 μL of fibroin aqueous solution was poured into the well, and the well was left to stand at room temperature for 1 hour. The excess fibroin aqueous solution was then removed by suction and dried in a clean bench for 45 minutes to prepare a single-coated container coated only with the first cell culture substrate containing fibroin as an active ingredient, and 300 μL of Matrigel solution was poured into the well and left to stand at 37° C. for 3 hours to prepare a single-coated container coated only with the second cell culture substrate containing Matrigel as an active ingredient.

[0045] In Example 1, undifferentiated human iPS cells were seeded into each of the prepared double-coated vessels and subcultured. Specifically, human iPS cell line 253G1 (Passage 54), which had been cultured in a conventional manner, was washed once with PBS and incubated with 0.5 mM EDTA at 37°C for 2 minutes to detach the cells, and then collected while maintaining the colony state. The collected human iPS cells were then suspended in an equal volume of undifferentiated cell medium (equivalent to Thermo's E8 Flex medium), and 5 μM Y27632 was added. 500 μL of this cell suspension was seeded per well. Furthermore, on days 2 and 3 of subculture, the medium was replaced with 500 μL of undifferentiated cell medium, and microscopic observation was performed after the third day.

[0046] Figure 5 shows micrographs of human iPS cells observed in Example 1. In Figure 5, Example 1A is a test example using laminin 111 as the adhesion factor; similarly, Example 1B is a test example using laminin 121, Example 1C is a test example using laminin 211, Example 1D is a test example using laminin 221, Example 1E is a test example using laminin 332, Example 1F is a test example using laminin 411, Example 1G is a test example using laminin 511, and Example 1H is a test example using laminin 521. Furthermore, Example 1I is a test example using Matrigel as the adhesion factor. Furthermore, Comparative Example 1J is a test example using a single-coated container coated only with a first cell culture substrate containing fibroin as an active ingredient, and Comparative Example 1K is a test example using a single-coated container coated only with a second cell culture substrate containing Matrigel as an active ingredient.

[0047] Note that the black clumps in Figure 5 (e.g., the black clumps imaged in Example 1D) are shadows resulting from the floating state of the human iPS cells. As shown in Figure 5, there were some areas where the human iPS cells were floating. However, visual observation confirmed that the human iPS cells adhered to the cell culture substrate in the double-coated containers of Examples 1A-1B and 1D-1I, as shown in Table 1 below. Table 1 shows the results of visually evaluating the adhesion area of ​​the adherent cells to the wells on a 6-point scale, with an adhesion area of ​​0% being evaluated as "0," greater than 0% but not greater than 20% being evaluated as "1," greater than 20% but less than 90% being evaluated as "2," greater than 90% but less than 95% being evaluated as "3," greater than 95% but less than 98% being evaluated as "4," and greater than 98% being evaluated as "5." As shown in Table 1 above, in Examples 1B, 1E, 1I, and 1K, which used laminin 121, 332, and MG as adhesion factors, adhesion of adherent cells was confirmed over almost the entire surface. Furthermore, in Examples 1A, 1G, and 1H, which used laminin 111, 511, and 521, adhesion of adherent cells was confirmed over more than 90% of the culture surface. Thus, when the adherent cells are human iPS cells, it was found that using laminins with α-chains of α1, α3, and α5 increases adhesiveness. On the other hand, depending on the type of adherent cells, laminins with α-chains of α2 and α4 are also expected to be effective.

[0048] On the other hand, in Comparative Example 1J, human iPS cells did not adhere to the cell culture substrate, and proliferation of human iPS cells was not confirmed. Comparative Example 1J is a container coated solely with a first cell culture substrate containing fibroin as an active ingredient, and is not coated with a second cell culture substrate containing the adhesion factor laminin or Matrigel as an active ingredient. On the other hand, Comparative Example 1K is a conventional container coated solely with Matrigel, and human iPS cells were observed to adhere to the cell culture substrate and proliferate.

[0049] From this, it was found that it is difficult to attach and proliferate human iPS cells in a single-coated container coated only with the first cell culture substrate containing fibroin as an active ingredient, but by using a double-coated container in which the first layer is coated with the first cell culture substrate containing fibroin as an active ingredient, and the second layer is coated with the second cell culture substrate containing the adhesion factor laminin or Matrigel as an active ingredient, it is possible for human iPS cells to adhere to the cell culture substrate and proliferate.

[0050] Example 2 In Example 2, similar to Example 1, human iPS cells were used as adherent cells, and whether the human iPS cells adhere to a cell culture substrate when cultured using a mixed-coated container according to the present embodiment was examined. Note that, similar to Example 1, in Example 2, a relatively large number of human iPS cells were seeded compared to Example 3 described below, and observations were made from the perspective of whether the seeded human iPS cells adhere to the cell culture substrate according to the present embodiment.

[0051] In Example 2, a 0.1% fibroin aqueous solution was prepared as a first cell culture substrate, similar to Example 1. Meanwhile, in Example 2, a mixed-coated container was prepared as follows: That is, 2 μg of each type of laminin was dissolved in 300 μL of a 0.1% fibroin aqueous solution to prepare a mixed cell culture substrate containing a first cell culture substrate containing fibroin as an active ingredient and a second cell culture substrate containing each type of laminin as an active ingredient, and 300 μL of the prepared mixed cell culture substrate was poured into each well and allowed to stand at 37° C. for 3 hours to produce a mixed-coated container. In Example 2, similarly to Example 1, Matrigel was diluted 3-fold with RPMI medium, and the diluted Matrigel was added to a 0.1% aqueous fibroin solution so that the final Matrigel concentration was 60-fold, thereby preparing a mixed cell culture substrate containing a first cell culture substrate containing fibroin as an active ingredient and a second cell culture substrate containing Matrigel as an active ingredient. 300 μL of the prepared mixed cell culture substrate was poured into a well and allowed to stand at 37° C. for 3 hours, thereby producing a mixed coated container.

[0052] Figure 6 shows micrographs of human iPS cells observed in Example 2. In Figure 6, Example 2A is a test example using laminin 111 as the adhesion factor; similarly, Example 2B is a test example using laminin 121, Example 2C is a test example using laminin 211, Example 2D is a test example using laminin 221, Example 2E is a test example using laminin 332, Example 2F is a test example using laminin 411, Example 2G is a test example using laminin 511, and Example 2H is a test example using laminin 521. Furthermore, Example 2I is a test example using Matrigel as the adhesion factor. Note that, in Figure 6, the areas where the human iPS cells are floating appear as black clumps.

[0053] As shown in Figure 6, when the mixed-coated containers of Examples 2A to 2I were used, similar to Examples 1A to 1I, some floating human iPS cells were observed, but the human iPS cells were visually observed adhering to the cell culture substrate. Table 2 shows the results of visually evaluating the adhesion area of ​​the adherent cells to the wells on a 6-point scale. As with Table 1 of Example 1, an adhesion area of ​​0% was evaluated as "0," a range of 0% to 20% was evaluated as "1," a range of 20% to less than 90% was evaluated as "2," a range of 90% to less than 95% was evaluated as "3," a range of 95% to less than 98% was evaluated as "4," and a range of 98% or greater was evaluated as "5." As shown in Table 2 above, in Examples 2A, 2B, 2E, and 2G, which used laminin 111, 121, 332, and 511 as adhesion factors, adhesion of adherent cells was confirmed on more than 90% of the culture surface, and in Examples 2H and 2I, which used laminin 521 and Matrigel, adhesion of adherent cells was confirmed.

[0054] This indicates that human iPS cells can adhere to the cell culture substrate and proliferate even when a second cell culture substrate containing the adhesion factor laminin or Matrigel as an active ingredient is mixed with a first cell culture substrate containing fibroin as an active ingredient to form a mixed coating container. However, in Example 2, the adhesiveness of the adherent cells tended to be lower overall compared to Example 1.

[0055] Example 3 In Example 3, it was verified whether human iPS cells could be proliferated and whether the proliferated human iPS cells could be detached when they were cultured using a double-coated container and a mixed-coated container. That is, in Examples 1 and 2, a relatively large number of human iPS cells were seeded and whether the human iPS cells adhered to the cell culture substrate according to the present embodiment was verified. In Example 3, however, human iPS cells cultured in the usual manner were grown at a density of 2 x 10^3 cells per well (1 x 10^3 cells / cm). ２ ) to observe whether the human iPS cells proliferate to the extent that they form colonies, and to verify whether the proliferated human iPS cells can be detached by the detachment method according to this embodiment.

[0056] Specifically, in Example 3, human iPS cells seeded at the above concentration were passaged using undifferentiated cell medium, as in Examples 1 and 2. Note that on days 2 and 3 of passage, the medium was replaced with 300 μL of undifferentiated cell medium. After the third day, the cultured human iPS cells were observed under a microscope. Figure 7 shows a micrograph of the human iPS cells observed in Example 3.

[0057] Specifically, Examples 3-1A to 3-1E are test examples in which human iPS cells were cultured and detached using a double-coated container, and in each case, a first cell culture substrate containing fibroin as an active ingredient was used as the first coating layer, and a second cell culture substrate containing a different adhesion factor as an active ingredient was used as the second coating layer. More specifically, Example 3-1A used laminin 121 as the adhesion factor, while Example 3-1B used laminin 332, Example 3-1C used laminin 511, Example 3-1D used laminin 521, and Example 3-1E used Matrigel.

[0058] Examples 3-2A to 3-2E are test examples in which human iPS cells were cultured and detached using a mixed-coated container, in which a mixed cell culture substrate was coated with a first cell culture substrate containing fibroin as an active ingredient and a second cell culture substrate containing the adhesion factor laminin or Matrigel as an active ingredient. Human iPS cells were cultured and detached using a mixed-coated container coated with a mixed cell culture substrate. In Example 3-2A, laminin 121 was used as the adhesion factor; in Example 3-2B, laminin 332 was used; in Example 3-2C, laminin 511 was used; in Example 3-2D, laminin 521 was used; and in Example 3-2E, Matrigel was used.

[0059] Furthermore, in Comparative Examples 3-3A to 3-3E, human iPS cells were cultured and detached using a single-coated container coated with only a second cell culture substrate containing the adhesion factor laminin or Matrigel as an active ingredient. Specifically, human iPS cells were cultured and detached using a single-coated container using a second cell culture substrate containing laminin 121 as an active ingredient in Example 3-3A, laminin 332 as an active ingredient in Example 3-3B, laminin 511 as an active ingredient in Example 3-3C, laminin 521 as an active ingredient in Example 3-3D, and Matrigel as an active ingredient in Comparative Example 3-3E.

[0060] Figure 7 shows micrographs of human iPS cells observed after three days. As shown in Figure 7, in Examples 3-1A to 3-1D, which used double-coated containers, colonies or clumps of human iPS cells were observed, confirming the proliferation of human iPS cells. On the other hand, in Examples 3-2A to 3-2D, which used mixed-coated containers, colonies or clumps of human iPS cells were observed, confirming the proliferation of human iPS cells, but in Example 3-2A, which used laminin 121, no proliferation of human iPS cells was observed. Furthermore, in Comparative Examples 3-3A to 3-3E, which used conventional single-coated containers, colonies or clumps of human iPS cells were observed, confirming the proliferation of human iPS cells. From this, it was found that in a double-coated container or a mixed-coated container made of a first cell culture substrate containing fibroin as an active ingredient and a second cell culture substrate containing laminin or Matrigel as an active ingredient, human iPS cells, which are adhesive cells, generally adhere to the cell culture substrate and can be proliferated.

[0061] In Example 3, on day 4 of subculture, the cultured human iPS cells were detached using protease 14. Specifically, after aspirating the liquid medium from each well, 300 μL of undifferentiated cell medium warmed to 37°C and 30 μL of a protease solution of protease 14 (2.9 mg / mL) were added per well, and the cells were allowed to stand at 37°C for 10 minutes.

[0062] As a result, in Examples 3-2A to 3-2E, which used a mixed-coating container containing a first cell culture substrate containing fibroin as an active ingredient, human iPS cell colonies were detached within 10 minutes simply by adding protease 14 and allowing it to stand (without pipetting), and human iPS cells were recovered in clump form. Furthermore, in Examples 3-1A to 3-1D, which used a double-coating container in which the first cell culture substrate was coated as the first layer, detachment was not achieved simply by adding protease 14 and allowing it to stand, but by pipetting several times with weaker intensity (enough to thoroughly coat the protease 14), human iPS cells were detached in clump form. In contrast, in Comparative Examples 3-3A to 3-3D, which did not contain the first cell culture substrate, human iPS cells could not be detached simply by adding protease 14, but by pipetting several times with stronger intensity. However, in Comparative Examples 3-3A to 3-3D, the human iPS cells were detached by performing strong pipetting multiple times and applying physical shock, and therefore it was not possible to recover the human iPS cells in the form of clumps.

[0063] In Example 3, to count the number of detached human iPS cells, 2 mL of 0.05% trypsin-EDTA was added to each well after cell detachment and shaken at 37°C for 5 minutes to break up the adherent cells into single cells, and the number of cells was then counted. Figure 8 is a graph showing the counting results. Although this varied depending on the type of laminin, as shown in Figure 8, there was a tendency for the number of human iPS cells to be higher and for the culture efficiency to be higher when cultured using a double-coated vessel than when cultured using a mixed-coated vessel. In particular, when using a double-coated vessel, laminin 121 and laminin 332 were used, human iPS cell proliferation was found to be greater than when using a conventional single-coated vessel (comparison example).

[0064] Example 4 In Example 4, MSCs were used as adherent cells, and the ability to proliferate and detach MSCs was examined when they were cultured using a double-coated container according to the present embodiment. Specifically, in Examples 4A and 4B, a double-coated container was fabricated in which a first cell culture substrate containing fibroin as an active ingredient was coated as a first layer, followed by a second cell culture substrate containing gelatin as an active ingredient. Note that in Examples 4A and 4B, a 0.1% aqueous fibroin solution was used as the first cell culture substrate containing fibroin as an active ingredient, as in Examples 1 to 3. Furthermore, a 0.1% aqueous gelatin solution was used as the second cell culture substrate containing gelatin as an active ingredient, prepared by dissolving 0.5 g of gelatin (Sigma-Aldrich G1890) in 500 mL of water, sterilizing the solution with high-pressure steam in an autoclave, and storing it in a refrigerator at 4°C. In Examples 4A and 4B, a 0.1% fibroin aqueous solution, which is a first cell culture substrate, was spread over the entire bottom surface of the wells of a 6-well plate, allowed to stand at room temperature for 15 minutes, after which excess fibroin aqueous solution was aspirated and dried overnight at 50°C to form a first cell culture substrate coating layer 10. A 0.1% gelatin solution was spread over the entire well on the first cell culture substrate coating layer 10, allowed to stand at room temperature for at least one hour, after which excess gelatin solution was aspirated and removed, and a second cell culture substrate coating layer 20 was formed, thereby producing a double-coated container. In Example 4A, when forming the first cell culture substrate coating layer 10, after drying the 0.1% fibroin aqueous solution, an 80 wt% ethanol solution was spread over the entire surface of the coating layer 10, allowed to stand at room temperature for 30 minutes, after which the ethanol solution was aspirated and dried overnight at 50°C, thereby performing an insolubilization treatment. In contrast, in Example 4B, the insolubilization treatment was not performed.

[0065] Additionally, as comparative examples, the single-coated containers of Comparative Examples 4C to 4F were also examined. Specifically, in Comparative Examples 4C and 4D, single-coated containers were prepared by coating only a first cell culture substrate containing fibroin as an active ingredient. In Comparative Example 4C, an insolubilization treatment was performed similarly to Example 4A, while in Comparative Example 4D, no insolubilization treatment was performed. In Comparative Example 4E, a single-coated container was prepared by coating only a second cell culture substrate containing gelatin as an active ingredient. Furthermore, in Comparative Example 4F, a plasma-treated hydrophilic well was used as is without coating the cell culture substrate.

[0066] Then, 1.5 ml of MSC medium was injected into the wells of Examples 4A, 4B, and Comparative Examples 4C to 4E, and subculture was performed. The MSC medium was prepared as follows: Platelet lysate (UltraGRO-PURE GI (AventaCell)) was removed from a freezer kept at -30°C and thawed by warming in a 37°C incubator until completely thawed. The thawed platelet lysate was added to Dulbecco's modified Eagle's medium (4.5 g / L glucose, L-glutamine, pyruvate-free, liquid medium, Nacalai Tesque) to a concentration of 5%. Furthermore, Glutamax supplement (Thermo Fisher Scientific) was added to a concentration of 1% to prepare the MSC medium. Then, MSCs were seeded into each well containing the MSC medium at 1.3 × 10^5 cells / well, and the wells were shaken to ensure uniform dispersion of the MSCs. After that, the wells were incubated at 37°C in 5% CO ２ The cells were left to stand in an incubator, and the MSCs were observed under a microscope on days 1, 3, and 5 of passage without changing the medium.

[0067] Figure 9 shows a micrograph of MSCs in Example 4. As shown in Figure 9, MSC adhesion and proliferation were confirmed in Examples 4A and 4B, which used a double-coated container coated with a first cell culture substrate containing fibroin as an active ingredient and a second cell culture substrate containing gelatin as an active ingredient. No significant difference in the degree of proliferation was observed between Examples 4A and 4B, indicating that MSC adhesion and proliferation could be confirmed regardless of the presence or absence of insolubilization treatment. In contrast, MSC proliferation was not confirmed in Comparative Examples 4C and 4D, which used a single-coated container coated only with the first cell culture substrate containing fibroin as an active ingredient. On the other hand, MSC adhesion and proliferation were also confirmed in Comparative Example 4E, which used a single-coated container coated only with the second cell culture substrate containing gelatin as an active ingredient, and Comparative Example 4F, which used a hydrophilic container without coating. This indicates that, even for MSCs, fibroin alone is not sufficient for effective adhesion and proliferation, but that MSCs can be efficiently adhered and proliferated by using a double-coated container coated with a first cell culture substrate containing fibroin as an active ingredient and a second cell culture substrate containing gelatin as an active ingredient.

[0068] Furthermore, in Example 4, cell detachment of the proliferated MSCs (Examples 4A and 4B and Comparative Examples 4E and 4F) was performed on day 5 of subculture. Specifically, in Examples 4A and 4B, protease 14 was dissolved in PBS to adjust the concentration to 10 U / ml, and 150 μl of 10 U / ml protease 14 was added to the liquid medium without removing the liquid medium (i.e., the protease 14 was added so that the concentration was 1 U / ml), and the cells were incubated at 37° C. in 5% CO ２The reaction was allowed to proceed in an incubator for approximately 10 minutes. As a result, it was confirmed that in Examples 4A and 4B, MSCs could be recovered in the form of clumps. Furthermore, in Comparative Examples 4E and 4F, since a cell culture substrate containing fibroin as an active ingredient was not used, cell detachment was carried out using trypsin-EDTA (trypsin 0.25%, containing phenol red, Thermo Fisher Scientific). Specifically, the MSC medium was removed by suction, and the adherent cells were washed with PBS. Next, trypsin-EDTA was spread throughout the culture, and the cells were incubated at 37°C in 5% CO ２ The mixture was allowed to react in an incubator for approximately 10 minutes. After that, an equal volume of MSC medium as the trypsin-EDTA solution was added, and the MSCs were detached by pipetting. In Comparative Examples 4E and 4F, because trypsin was used to detach the cells, it was not possible to recover the MSCs in clumps.

[0069] As described above, the cell culture substrate of this embodiment has a cell culture vessel and a first cell culture substrate containing one or more proteins selected from silk protein, silk protein hydrolysate, and fibroin as active ingredients, so that cell detachment can be performed using a silk protein-degrading enzyme without using trypsin, and also has a second cell culture substrate containing an adhesion factor of the target adherent cells as an active ingredient, so that the adhesiveness of the adherent cells can be further enhanced.

[0070] 1 , the cell culture vessel according to this embodiment is configured as a double-coated vessel 1 in which a second coating layer 20 formed by coating a second cell culture substrate is formed on a first coating layer 10 formed by coating a first cell culture substrate, thereby enabling higher adhesion of adherent cells such as human iPS cells and MSCs compared to a mixed-coated vessel 2. Furthermore, by configuring the mixed-coated vessel 2 to have a coating layer 30 formed by coating a mixed cell culture substrate in which the first and second cell culture substrates are mixed, adherent cells can be detached in clumps simply by reacting with a silk protein-degrading enzyme without pipetting.

[0071] Second Embodiment A second embodiment of the present invention will be described. The second embodiment is characterized in that adherent cells are cultured using a cell culture substrate containing one or more proteins selected from silk protein, silk protein hydrolysate, and fibroin as active ingredients, and the cultured adherent cells are detached using a protease (details of which will be described later) that degrades one or more proteins selected from silk protein, silk protein hydrolysate, and fibroin. The cell culture substrate, cell culture vessel, cell culture substrate production kit, adherent cell detachment method, and cell recovery device according to the second embodiment will be described below.

[0072] (Cell Culture Substrate) The cell culture substrate according to the second embodiment also functions as a scaffold material for enhancing adhesion of adherent cells to a culture vessel, and can be used as a coating agent for coating a cell culture vessel. Specifically, in the second embodiment, the cell culture substrate is characterized by containing silk protein, a silk protein hydrolysate, fibroin, or a fibroin hydrolysate as an active ingredient.

[0073] Silk proteins such as fibroin can be obtained (extracted, purified, etc.) using methods similar to those used in the first embodiment. Furthermore, the cell culture substrate according to the second embodiment is not limited to those containing silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate as an active ingredient; for example, it can also be configured to contain a vegetable protein such as egg protein, milk protein, or soy protein as an active ingredient. Furthermore, the cell culture substrate according to this embodiment can be configured to contain water or a buffer solution in addition to the protein active ingredient. Furthermore, the cell culture substrate according to this embodiment can also be configured to contain a mixture of two or more of silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate.

[0074] The cell culture substrate according to the second embodiment is preferably one that has been sterilized. Examples of sterilization include high-pressure steam sterilization using an autoclave, and sterilization using gamma rays, electron beams, or filters, which are used for conventional cell culture substrates. Furthermore, sterilization can be omitted by using autoclave-sterilized silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate together with an existing sterilized cell culture substrate. It has also been confirmed that cell culture substrates using autoclave-sterilized silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate are also capable of favorable growth and detachment of adherent cells.

[0075] (Cell Culture Vessel) As shown in FIG. 10 , the cell culture vessel 5 according to the second embodiment is obtained by applying the cell culture substrate according to the second embodiment described above to the bottom surface 4 of an untreated cell culture vessel 3 to form a coating. Specifically, a liquid cell culture substrate containing silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate as an active ingredient is evenly applied to the bottom surface of an untreated cell culture vessel by dripping or spraying it onto the bottom surface, and then the bottom surface is dried, for example, by placing it in a dryer. Alternatively, to prevent the active ingredient, such as silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate, from dissolving into the liquid medium, the active ingredient, such as silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate, is insolubilized by adding ethanol or methanol to the bottom surface of the dried cell culture vessel and contacting it, or by leaving it in a saturated water vapor state overnight. After these treatments, the cell culture vessel of the second embodiment may be dried. This allows the production of a coated vessel 5 having a coating layer (similar to the first coating layer 10 of the first embodiment) formed on the cell culture substrate containing silk protein, a silk protein hydrolysate, fibroin, or a fibroin hydrolysate as an active ingredient. Fig. 10 is a schematic side view showing the cell culture vessel (coated vessel) according to the second embodiment.

[0076] In the second embodiment, the type of cell culture vessel is not particularly limited, and can be provided in the form of, for example, a petri dish, a plate, a flask, or a multi-layer flask. The material of the cell culture vessel according to this embodiment is also not particularly limited, and for example, a container made of plastic or glass can be used. Furthermore, the container does not require special surface treatment before being coated with the cell culture substrate according to this embodiment, and an inexpensive container with an untreated (hydrophobic) surface, such as a polystyrene petri dish, can be used. Furthermore, the cell culture vessel according to this embodiment may have not only the bottom surface but also the sides coated with the cell culture substrate according to this embodiment. In addition, since the cell culture vessel according to this embodiment is produced using a liquid cell culture substrate, it can also have a bottom or side with a complex shape.

[0077] (Cell Detachment Method) Next, a method for detaching adherent cells cultured using a cell culture vessel according to the second embodiment will be described. The cell culture vessel according to the second embodiment is surface-treated by coating with silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate. Adherent cells adhere to the cell culture vessel using the silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate as a scaffold. Because silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate reacts with silk protein-degrading enzymes and decomposes, adding silk protein-degrading enzymes to the liquid culture medium enables adherent cells to be detached. Furthermore, by using silk protein-degrading enzymes that degrade silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate, which do not readily react with the protein components contained in the liquid culture medium for adherent cells, there is no need to remove the liquid culture medium.

[0078] Therefore, in the adhesive cell detachment process according to the second embodiment, as in the first embodiment, after culturing the adhesive cells (step S201), the liquid medium is not removed, the cells are washed with phosphate buffer, and the washed phosphate buffer is not removed, as in the case of using trypsin as shown in FIG. 2. Instead, as shown in FIG. 3, an enzyme that decomposes silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate is added directly (step S202), and the enzyme is allowed to react at a constant temperature for a certain period of time (step S203), thereby enabling the adhesive cells to be detached from the cell culture vessel. Furthermore, since silk protein, silk protein hydrolysate, fibroin, or an enzyme that decomposes fibroin hydrolysate has a lesser effect on the adherent cells themselves than trypsin, in this embodiment, after adding the enzyme and allowing it to react, there is no need to add liquid culture medium to inhibit the action of the enzyme, as in step S107 of Figure 2.As shown in Figure 3, centrifugation is performed as is (step S204), the supernatant of the liquid culture medium is removed (step S205), and then liquid culture medium is added again for storage (step S206), and the settled adherent cells can be recovered (step S207).

[0079] Furthermore, the cell culture vessel according to the second embodiment is coated with silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate, and adherent cells adhere to the silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate as a scaffold. Therefore, by using an enzyme that degrades silk protein, silk protein hydrolysate, fibroin, a mixture of fibroin and sericin, or fibroin hydrolysate, the silk protein, silk protein hydrolysate, fibroin, a mixture of fibroin and sericin, or fibroin hydrolysate, the adherent cells can be detached from the cell culture vessel, as shown in Figure 3(B). Furthermore, by using a silk protein-degrading enzyme (e.g., protease XIV) that efficiently acts on silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate, the adherent cells can be detached in a sheet form without breaking apart, as shown in Figure 3(B), while also minimizing damage to the adherent cells themselves.

[0080] Next, an example of cell detachment using the cell culture vessel according to the second embodiment will be described. First, a method for producing the cell culture substrate and the cell culture vessel used in the second embodiment will be described.

[0081] (Preparation of Cell Culture Substrates) In Examples 5 to 14, 18, and 19 described below, unless otherwise specified, cell culture substrates containing fibroin as an active ingredient were prepared as described below. Specifically, dried domesticated silkworm cocoons were first cut into approximately 1 cm squares. Next, 1 L of 0.02 M (mol / L) sodium carbonate solution was added to 10 g of dried domesticated silkworm cocoons, and the mixture was stirred at 95 to 98°C for approximately 30 minutes to remove sericin from the domesticated silkworm cocoons, yielding fibroin fibers. The resulting fibroin fibers were then washed with ion-exchanged water or ultrapure water, the sodium carbonate was removed, and the fibers were dried. 50 ml of 9 M (mol / L) lithium bromide solution was added to 3 g of dried, refined cocoons (fibroin fibers) to dissolve the refined cocoons. After adding the lithium bromide solution, the mixture was manually stirred for a certain period of time at room temperature of approximately 25°C. After confirming that the refined cocoons had dissolved, the mixture was stirred overnight with a stirrer. Approximately 50 ml of the dissolved fibroin solution was then placed in a dialysis film (approximately 28 mm in diameter, molecular weight cutoff of 12,000-14,000) and dialyzed against 8 L of ion-exchanged water or ultrapure water for approximately half a day. This process was repeated six times. This removed the lithium bromide from the solution. Furthermore, the concentration of the fibroin solution after dialysis was measured and adjusted to 0.1-1.0% by adding ion-exchanged water or ultrapure water. The concentration of the fibroin solution after dialysis was measured by the direct gravimetric method (a method for measuring the concentration of the fibroin solution by determining the weight of the residue (fibroin) after total evaporation of the fibroin solution after dialysis). This resulted in the cell culture substrates used in Examples 5-14 and 18-19 described below (hereinafter also referred to as the cell culture substrates according to this example). Furthermore, in Examples 15 to 17 described below, cell culture substrates containing fibroin and sericin as active ingredients, or cell culture substrates containing a fibroin hydrolysate as an active ingredient, were prepared. The methods for preparing the cell culture substrates in Examples 15 to 17 will be described in Examples 15 to 17, respectively.

[0082] (Fabrication of Cell Culture Vessel) In this example, unless otherwise specified, a cell culture vessel coated with a cell culture substrate was fabricated as follows. Specifically, the cell culture substrate according to this example was applied to the inner bottom of a Petri dish. The cell culture substrate was applied in an amount sufficient to completely cover the bottom surface of the vessel. If a larger amount was applied, the excess was removed using a dropper or similar device. The Petri dish was then placed in a dryer to evaporate the moisture from the cell culture substrate. After the surface of the Petri dish was completely dried, 80% methanol was applied to the bottom surface of the Petri dish until it completely covered the bottom surface. The methanol was then evaporated at room temperature or in a dryer, thereby performing an insolubilization treatment. Alternatively, instead of insolubilization with 80% methanol, the insolubilization treatment was also performed by leaving the container in a saturated state of water vapor overnight. In this manner, the cell culture vessel used in this example (hereinafter also referred to as the cell culture vessel according to this example) was fabricated.

[0083] In the following Examples 5 to 17 and 19, NIH3T3 cells were used as the adherent cells. In the following Example 18, MDCK cells were used as the adherent cells. Furthermore, in the following Examples 5 to 19, unless otherwise specified, adherent cells were cultured using the cell culture vessel according to the present invention, fabricated as described above. Furthermore, in the following Examples 5 to 19, unless otherwise specified, culture and detachment were performed under the same conditions according to the method shown in FIG. 3 above. In the following Examples 5 to 17, unless otherwise specified, Protease XIV (manufactured by SIGMA) was used as an enzyme that degrades silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate to detach adherent cells cultured in the cell culture vessel according to the present invention. Furthermore, Examples 18 and 19 are examples in which it was verified whether adherent cells could be detached using enzymes other than Protease XIV (manufactured by SIGMA). As enzymes for degrading silk protein, silk protein hydrolysates, fibroin, or fibroin hydrolysates, proteases derived from Streptomyces griseus or mixtures thereof, and proteases derived from Bacillus Licheniformis or mixtures thereof, which will be described later, were used.

[0084] Example 5 In Example 5, whether adherent cells can be cultured using the cell culture vessel according to this Example was verified. Specifically, as an Example, adherent cells were cultured using the cell culture vessel according to this Example. As a Comparative Example, adherent cells were cultured using a surface-untreated (hydrophobic) polystyrene Petri dish that was not coated with the cell culture substrate according to this Example. FIG. 11 shows the results of the cell culture verification in Example 5. (A) is an image of a surface-untreated Petri dish that was not coated with the cell culture substrate according to this Example after culturing adherent cells for three days. (B) is an image of a Petri dish coated with the cell culture substrate according to this Example (the cell culture vessel according to this Example) after culturing adherent cells for three days. Note that in (B), the cell culture vessel was fabricated using a cell culture substrate containing 1% fibroin.

[0085] In an example in which adherent cells were cultured using a cell culture vessel coated with the cell culture substrate according to this example, proliferation of adherent cells was confirmed, as shown in Figure 11(B). In contrast, in a comparative example in which adherent cells were cultured using a surface-untreated (hydrophobic) polystyrene dish that was not coated with the cell culture substrate according to this example, proliferation of adherent cells was not confirmed, as shown in Figure 11(A). This demonstrates that adherent cells can be proliferated by using the cell culture vessel according to this embodiment.

[0086] Example 6 In Example 6, the culture of adherent cells according to this Example was compared with conventional culture of adherent cells to verify any differences. Specifically, as an example, adherent cells were cultured for different culture times using the cell culture vessel according to this Example, and then the cultured adherent cells were detached and recovered using an enzyme. The lactate dehydrogenase activity in the recovered adherent cell population was measured, and the number of adherent cells was calculated to create a growth curve for the method according to this Example. Additionally, as a comparative example, adherent cells were cultured for different culture times using a conventional cell culture vessel (a cell culture vessel subjected to vacuum plasma treatment), and then the cultured adherent cells were detached and recovered using trypsin to create a growth curve for the conventional method. Figure 12 shows the growth curves for an example in which adherent cells were cultured using the method according to this Example and a comparative example in which adherent cells were cultured using the conventional method. As shown in Figure 12, when adherent cells were cultured using the cell culture vessel according to this Example, similar (even greater) growth of adherent cells was observed, demonstrating that similar (even greater) growth of adherent cells was obtained compared to conventional methods.

[0087] Example 7 In Example 7, we verified whether adherent cells cultured using the cell culture vessel of this Example could be cultured after passage in the same manner as before passage. Specifically, as an example, adherent cells were cultured using the cell culture vessel of this Example, and then passaged in a new cell culture vessel of this Example. The state of the adherent cells was observed under a microscope on days 1 to 6. As a comparative example, adherent cells were cultured using a conventional cell culture vessel (a cell culture vessel that had been subjected to vacuum plasma treatment), and then passaged in a new conventional cell culture vessel. The state of the adherent cells was observed under a microscope on days 1 to 6. Figure 13 shows the results of the verification of Example 7, showing images of adherent cells cultured using the method of this Example after passage (Example) and images of adherent cells cultured using a conventional method after passage (Comparative Example). As shown in Figure 13, the growth morphology after passage of the adherent cells cultured using the cell culture vessel of this example was similar to that of the adherent cells cultured using a conventional cell culture vessel, even six days after passage. This indicates that the adherent cells cultured using the cell culture vessel of this example can also be cultured appropriately even after passage.

[0088] Example 8 In Example 8, it was verified whether adherent cells cultured using the cell culture vessel according to this example could be detached using an enzyme. Fig. 14(A) is an image (the same image as Fig. 11(B)) of a Petri dish coated with the cell culture substrate according to this example (the cell culture vessel according to this example) in Example 5 after culturing adherent cells for three days, and Fig. 14(B) is an image of the Petri dish after the adherent cells in (A) were detached using 0.1 U / ml of Protease XIV. As shown in Fig. 14(B), it was found that adherent cells cultured using the cell culture vessel according to this example coated with the cell culture substrate according to this example could be detached using an enzyme capable of degrading fibroin (Protease XIV).

[0089] Example 9 In Example 9, it was verified whether adherent cells cultured in a conventional cell culture vessel (a cell culture vessel that had been subjected to vacuum plasma treatment) could be detached using the enzyme according to this example. Figure 15 shows the verification results of Example 9, in which (A) shows a photographic image of a petri dish in which adherent cells cultured in the conventional cell culture vessel were detached using trypsin, and (B) to (F) are photographic images of the petri dishes in which detachment of adherent cells cultured in the conventional cell culture vessel was attempted using an enzyme (Protease XIV). (B) used 0.1 U / ml of Protease XIV, (C) used 0.5 U / ml of Protease XIV, (D) used 1.0 U / ml of Protease XIV, (E) used 2.0 U / ml of Protease XIV, and (F) used 5.0 U / ml of Protease XIV. Each enzyme was incubated at 37°C for 10 minutes in each of (A) to (F). As shown in Figure 15(A), adherent cells cultured in a conventional cell culture vessel could be detached with trypsin, but as shown in Figures 15(B) to (F), they could not be detached with Protease XIV.

[0090] Example 10: In Example 10, we investigated whether the ease of proliferation and detachment of adherent cells changed when the fibroin concentration in the cell culture substrate of this Example was changed and when the insolubilization method used in fabricating the cell culture vessel was changed. Figures 16 to 19 show the results of the investigation in Example 10, with Figures 16 and 17 showing the results when insolubilization was performed using 80% methanol, and Figures 18 and 19 showing the results when insolubilization was performed using water vapor. In Example 10, adherent cells were cultured for 3 days, and then the Petri dish was washed with phosphate buffer. Then, 0.5 ml of 1 U / ml Protease XIV was added, and the mixture was incubated at 37°C for 5 minutes, after which it was washed again with phosphate buffer. 16 to 19, (1) shows the case where the fibroin concentration in the cell culture substrate was 1%, (2) shows the case where the fibroin concentration in the cell culture substrate was 0.5%, (3) shows the case where the fibroin concentration in the cell culture substrate was 0.1%, (4) shows the case where the fibroin concentration in the cell culture substrate was 0.05%, (5) shows the case where the fibroin concentration in the cell culture substrate was 0.01%, and (6) shows the case where the fibroin concentration in the cell culture substrate was 0.005%. In (1) to (6) of Figures 16 to 19, the top images show the state before detachment of adherent cells with Protease XIV, the middle images show the state immediately after detachment of adherent cells with Protease XIV, and the bottom images show the state after detachment of adherent cells with Protease XIV and subsequent washing with phosphate buffer.

[0091] As shown in Figures 16 to 19, it was found that by increasing the fibroin concentration in the cell culture substrate to 0.005% or higher, adherent cells could be adequately grown and detached, and by increasing the fibroin concentration in the cell culture substrate to 0.1% or higher, adherent cells could be more effectively detached. It was also found that adherent cells could be detached using an enzyme (Protease XIV) regardless of the insolubilization treatment method. In Figures 16 to 19, the white spots seen in the images taken after detachment and after washing represent adherent cells that have been completely or partially released from the cell culture vessel, indicating that detachment of the adherent cells has progressed compared to before detachment.

[0092] Example 11: The effect of enzyme concentration on detachment of adherent cells was examined in Example 11. Specifically, adherent cells were cultured for 3 days using a cell culture substrate containing 0.1% fibroin and a cell culture vessel insolubilized with methanol, the liquid medium was removed, and the cells were washed with phosphate buffer, after which various concentrations of Protease XIV were added and allowed to react at 37°C for 5 minutes. 20 shows the results of verification of Example 11, where (A) shows the state before detachment with Protease XIV, (B) shows the state after detachment with 0.01 U / ml Protease XIV, (C) shows the state after detachment with 0.05 U / ml Protease XIV, (D) shows the state after detachment with 0.1 U / ml Protease XIV, (E) shows the state after detachment with 0.5 U / ml Protease XIV, and (F) shows the state after detachment with 1.0 U / ml Protease XIV. As shown in Figures 20(B) to (F), it was found that adherent cells could be sufficiently detached by using Protease XIV at a concentration of 0.01 U / ml or more. Furthermore, it was found that adherent cells could be more effectively detached by increasing the concentration of Protease XIV to 0.1 U / ml or more in (D) to (F). Although not shown, results similar to those obtained when using a cell culture substrate containing 0.1% fibroin were obtained when using a cell culture substrate containing 1% fibroin. Furthermore, results similar to those obtained when using methanol for insolubilization were obtained when using water vapor for insolubilization.

[0093] Example 12 In Example 12, the cell culture substrate according to this example was sterilized in an autoclave, and the culturing and detachment of adherent cells was verified using a cell culture vessel coated with the autoclave-sterilized cell culture substrate. Specifically, the cell culture substrate according to this example was autoclaved at 120°C for 20 minutes, and then the sterilized cell culture substrate was applied to a sterilized, surface-untreated (hydrophobic) polystyrene plate, dried, and then insolubilized to produce a cell culture vessel. Adherent cells were then cultured for 4 days using the produced cell culture vessel, and then detached by reacting with 0.1 U / ml of Protease XIV for 5 minutes. 21 and 22 are figures showing the verification results in Example 12. FIG. 21 shows an image obtained by observing adherent cells cultured in a cell culture vessel fabricated using the sterilized cell culture substrate according to this Example after sterilizing the cell culture substrate according to this Example in an autoclave, and FIG. 22 shows an image obtained by observing the detached state of adherent cells cultured in a cell culture vessel fabricated using the sterilized cell culture substrate according to this Example after sterilizing the cell culture substrate according to this Example in an autoclave.

[0094] In Figures 21 and 22, (A) shows the case where a 0.1% fibroin solution was applied and insolubilized with methanol, (B) shows the case where a 1% fibroin solution was applied and insolubilized with methanol, (C) shows the case where a 0.1% fibroin solution was applied and insolubilized with water vapor, and (D) shows the case where a 1% fibroin solution was applied and insolubilized by leaving it in a saturated water vapor state overnight. Figure 21(E) shows the case where adherent cells were cultured on a conventional polystyrene plate for tissue culture. As shown in Figure 21, adherent cells could be cultured on a cell culture vessel in which the cell culture substrate (fibroin solution) according to the present embodiment, which was sterilized in an autoclave as a cell culture vessel, was applied to a sterilized vessel without surface treatment, similar to the case of a conventional polyethylene plate for cell culture. It was also found that detachment tended to be easier when a fibroin solution of 1% or more was used than when a 0.1% fibroin solution was used. Furthermore, as shown in Figure 22, it was found that adherent cells cultured in a cell culture vessel in which the cell culture substrate (fibroin solution) according to this embodiment, which had been sterilized in an autoclave as a cell culture vessel, was applied to a sterilized vessel without surface treatment, could be detached using Protease XIV. Although not shown, adherent cells could also be detached when 0.01 U / ml of Protease XIV was allowed to react for 5 minutes. However, it was observed that adherent cells tended to be more easily detached when Protease XIV was used at a concentration of 0.1 U / ml or higher.

[0095] Example 13 In Example 13, adherent cells were cultured using a cell culture vessel according to this example, and after removing the liquid medium, an enzyme was added to verify whether the adherent cells could be detached without washing with phosphate buffer. Specifically, the cell culture substrate (fibroin solution) according to this example was sterilized by autoclaving at 120°C for 20 minutes, and then the fibroin solution was applied to an untreated (hydrophobic) polystyrene plate for insolubilization, thereby producing a cell culture vessel according to this example. Adherent cells were cultured using this cell culture vessel for four days. After that, the liquid medium was removed from the cell culture vessel, and without washing with phosphate buffer, 0.1 U / ml of protease XIV was added to the dish and allowed to react for 5 minutes to detach the cultured adherent cells. Figure 23 shows the results of Example 13. (A) and (D) show the results of applying a 0.1% fibroin solution and leaving it overnight in a saturated water vapor environment to insolubilize the cells. (B) and (E) show the results of applying a 1% fibroin solution and leaving it overnight in a saturated water vapor environment to insolubilize the cells. Figures 23(C) and (F) show comparative examples in which adherent cells cultured on conventional tissue culture polystyrene plates were detached using trypsin without washing with phosphate buffer after removing the culture medium. As shown in Figures 23(C) and (F), adherent cells cultured on tissue culture polystyrene plates were insufficiently detached using trypsin when not washed with phosphate buffer after removing the liquid culture, and washing with phosphate buffer was therefore necessary. In contrast, as shown in Figures 23(A), (B), (D), and (E), it was found that adherent cells cultured in the cell culture vessel according to this embodiment can be sufficiently detached by Protease XIV after removing the liquid medium, even without washing with phosphate buffer.

[0096] Example 14 In Example 14, we investigated whether adherent cells could be cultured using the cell culture vessel of this example and then detached by adding an enzyme without removing the liquid medium. Specifically, the cell culture substrate (fibroin solution) of this example was sterilized by autoclaving at 120°C for 20 minutes. Then, 1 ml of the fibroin solution was added to a surface-untreated (hydrophobic) polystyrene dish (60 mm diameter). After 30 minutes at room temperature, the fibroin solution was removed and the dish was left overnight in an incubator for insolubilization. Adherent cells (40,000 cells / dish) were then seeded into the resulting cell culture vessel, and the adherent cells were cultured for 3 days. Subsequently, without removing the liquid medium from the cell culture vessel, protease XIV was added to the cell culture vessel and allowed to react for 5 to 30 minutes to detach the cultured adherent cells. Figures 24 and 25 show the results of the verification of Example 14. In Figure 24, adherent cells were cultured using the cell culture vessel of this Example, and then Protease XIV was added to the liquid medium at concentrations of 0.1 U / ml, 0.5 U / ml, or 1.0 U / ml. The cells were then observed 5, 15, and 30 minutes after addition. As shown in Figure 24, when Protease XIV was added at a concentration of 0.1 U / ml or higher, some adherent cells detached approximately 5 minutes after addition. However, when Protease XIV was added at a concentration of 0.1 U / ml, some adherent cells remained adhered even 30 minutes after addition. On the other hand, when Protease XIV was added at a concentration of 0.5 U / ml, many cells detached 15 minutes after addition, and most of the cells detached 30 minutes after addition. When the concentration of Protease XIV was 1.0 U / ml, most of the cells were detached 15 minutes after addition.

[0097] 25 shows that adherent cells were cultured in a conventional cell culture vessel (a cell culture vessel that had been subjected to vacuum plasma treatment), and then Protease XIV was added to the culture medium without removing the liquid medium so that the concentration of Protease XIV in the culture medium was 1.0 U / ml. The state of the cells was observed 5, 15, and 30 minutes after the addition. As shown in Fig. 25, when adherent cells were cultured in a conventional cell culture vessel and reacted with Protease XIV without removing the liquid medium, the adherent cells could not be detached.

[0098] Example 15 In Example 15, cell culture vessels were fabricated using a cell culture substrate containing a mixture of sericin and fibroin. Specifically, fibroin refined from cocoons was dissolved in a solution containing calcium chloride, ethanol, and pure water in an 8:2:1 ratio to prepare a fibroin solution containing 1 wt% fibroin. Approximately 20 ml of pure water was added to 0.25 g of cocoons, and the mixture was extracted by heating at 120°C for 20 minutes to prepare a sericin aqueous solution containing 1 wt% sericin. The fibroin solution and the sericin aqueous solution were then mixed in weight ratios of 7:3, 5:5, 3:7, and 0:10 to prepare mixed solutions. The prepared mixed solutions were autoclave sterilized at 120°C for 20 minutes, and each mixed solution was applied to a Petri dish and then dried overnight at 50°C to prepare the cell culture vessels according to Example 15. Note that no insolubilization treatment was performed in Example 15. Then, adherent cells (HIH3T3 cells) were cultured for 4 days using the completed cell culture vessel.

[0099] Figure 26 shows images of petri dishes after culturing adherent cells using the cell culture vessel of Example 15. As shown in Figure 26, adherent cells were cultured in all cell culture vessels using cell culture substrates containing a fibroin solution and a sericin aqueous solution mixed at weight ratios of 7:3, 5:5, 3:7, and 0:10, respectively. Next, after removing the medium from these four cell culture vessels, 1 U / ml of protease XIV was added, and the state of adherent cell detachment was observed after 5 to 7 minutes. As a result, as shown in Figure 27, it was confirmed that cells could be detached into a sheet-like form using cell culture substrates containing a fibroin solution and a sericin aqueous solution mixed at ratios of 7:3, 5:5, and 3:7, respectively. On the other hand, cell detachment was not observed in cell culture vessels using a cell culture substrate containing only sericin without a fibroin solution (a cell culture substrate containing a fibroin solution and a sericin aqueous solution mixed at a ratio of 0:10). Thus, it was found that cell detachment using protease was possible even on a cell culture substrate in which an aqueous sericin solution and a fibroin solution were mixed. Fig. 27 is a diagram for explaining the results of the cell detachment verification in Example 15, showing a photograph of a Petri dish after detachment of adherent cells cultured in the cell culture vessel according to Example 15. In Fig. 27, only a high-magnification photographic image is shown for the cell culture substrate in which the fibroin solution and the aqueous sericin solution were mixed at a ratio of 0:10.

[0100] Example 16 In Example 15, adherent cells could be cultured in cell culture vessels using cell culture substrates containing only sericin but no fibroin, but protease detachment was not possible. Therefore, in Example 16, the amount of fibroin required (the required fibroin ratio) in a cell culture substrate containing a mixture of sericin and fibroin was examined. Specifically, in Example 16, cell culture substrates were prepared by mixing a fibroin solution with an aqueous sericin solution at weight ratios of 1:9, 0.5:9.5, and 0.1:9.9, respectively. The fibroin solution and the aqueous sericin solution were prepared using the same method as in Example 15. The mixed solution was then autoclaved at 120°C for 20 minutes and applied to a Petri dish to produce the cell culture vessel of Example 16. After application of the cell culture substrate to the cell culture vessel, the cell culture substrate was dried overnight at 50°C without insolubilization treatment. Adherent cells (HIH3T3 cells) were then cultured for three days using the prepared cell culture vessels. Figure 28 shows images of the petri dishes after culturing adherent cells using the cell culture vessel of Example 16. As shown in Figure 28, adherent cells could be cultured in all of the cell culture vessels using cell culture substrates containing mixed solutions in which the fibroin solution and the sericin aqueous solution were mixed at ratios of 1:9, 0.5:9.5, and 0.1:9.9, respectively. However, adhesion of the adherent cells to the cell culture substrate tended to be lower than in Example 15.

[0101] Next, after removing the medium from these cell culture vessels, 1 U / ml of Protease XIV was added, and after 5 minutes, cell detachment was observed. As a result, as shown in Figure 29, in cell culture vessels using cell culture substrates in which the fibroin solution and the sericin aqueous solution were mixed at a ratio of 1:9 and 0.5:9.5, respectively, the detached adherent cells appeared floating and round, confirming that the adherent cells could be detached into a sheet-like form even with the naked eye. On the other hand, as shown in Figure 29, in cell culture vessels using cell culture substrates in which the fibroin solution and the sericin aqueous solution were mixed at a ratio of 0.1:9.9, the adherent cells appeared elongated due to adhesion, and detachment of the adherent cells could not be confirmed. Note that Figure 29 is a diagram illustrating the results of cell detachment verification in Example 16, and is a photograph of a Petri dish after detachment treatment of adherent cells cultured in the cell culture vessel of Example 16.

[0102] Example 17 In Example 17, adherent cells were cultured and detached using a cell culture vessel containing a cell culture substrate containing hydrolyzed fibroin as an active ingredient. Specifically, sodium hydroxide was added to a fibroin aqueous solution containing 8 wt% fibroin to a concentration of 2N, and hydrolysis was carried out at 60°C for 1 hour. The resulting hydrolyzate was then neutralized with hydrochloric acid and dialyzed to produce a hydrolyzed fibroin aqueous solution. The hydrolyzed fibroin aqueous solution was then autoclaved at 120°C for 20 minutes to produce cell culture substrates with concentrations of 1 wt% and 0.1 wt% per fibroin. The cell culture substrates were then applied to cell culture vessels and dried overnight at 50°C. Note that the cell culture vessels using a cell culture substrate with a 0.1 wt% per fibroin concentration were insolubilized with 80% methanol, while the cell culture vessels using a cell culture substrate with a 1 wt% per fibroin concentration were not insolubilized. Adherent cells (HIH3T3 cells) were then cultured for three days using the prepared cell culture vessels. Figure 30 shows a photograph of a petri dish after culturing adherent cells using the cell culture vessel prepared in Example 17. As shown in Figure 30, adherent cells were successfully cultured in all cell culture vessels using a cell culture substrate containing a hydrolyzed fibroin aqueous solution, but the proliferation of adherent cells varied compared to other examples. Furthermore, after removing the medium, 1 U / ml of protease XIV was added, and after 5 minutes, the cell detachment was observed. As a result, as shown in Figure 31, even in the cell culture vessel using a cell culture substrate containing a fibroin hydrolyzate as an active ingredient, the detached adherent cells appeared floating and round, confirming that the adherent cells could be detached. Note that Figure 31 is a diagram illustrating cell detachment using the cell culture vessel prepared in Example 17, and shows a photograph of a petri dish after detaching adherent cells cultured in the cell culture vessel of Example 17. As described above, the cell culture substrate containing fibroin hydrolysate can be used to culture adherent cells and detach cells, but the other examples provided more favorable results for culturing adherent cells.

[0103] Example 18 In Example 18, adherent cells were cultured and detached using a Streptomyces griseus-derived protease other than Protease XIV or a mixture thereof. Specifically, a 0.1% fibroin aqueous solution was dropped into each well of a well plate, and the plate was left to stand at room temperature for 15 minutes. The 0.1% fibroin aqueous solution was then removed from the well using a dropper or the like, and the plate was then placed in a dryer at 50°C overnight to completely dry the bottom of the well, thereby producing a cell culture vessel according to Example 18. In Example 18, MDCK cells were seeded into each well of the well plate, and a liquid culture medium was added to the well, followed by culturing for 3 days.

[0104] In Example 18, cultured MDCK cells were detached using seven types of proteases derived from Streptomyces griseus or a mixture thereof, as follows: (1) Protease P8811 (Sigma-Aldrich), (2) Protease (Wako Pure Chemical Industries, Ltd.), (3) Actinase E (Kaken Pharmaceutical Co., Ltd.), (4) Protease P5174 type 14 (Sigma-Aldrich), (5) Protease E (Sigma-Aldrich), and (6) Pronase (Roche). Note that the proteases (1) to (6) or their mixtures were adjusted to 10 U / mL with physiological saline, filter-sterilized, and then added to the liquid medium without removing it. In this case, the final activity was 0.9 U / mL. ２ After reacting for 10 minutes in an incubator, the state of detachment of the MDCK cells was observed.

[0105] 32 shows the results of detaching adherent cells using multiple proteases derived from Streptomyces griseus other than Protease XIV in Example 18. As shown in Figure 32, MDCK cells were successfully detached in Examples using (1) Protease P8811 (Sigma-Aldrich), (2) Protease (Wako Pure Chemical Industries, Ltd.), (3) Actinase E (Kaken Pharmaceutical Co., Ltd.), (4) Protease P5174 type 14 (Sigma-Aldrich), (5) Protease E (Sigma-Aldrich), and (6) Pronase (Roche).

[0106] Example 19 In Example 19, adherent cells were detached using a protease derived from Bacillus licheniformis or a mixture thereof. Specifically, in Example 19, as in Example 18, a 0.1% fibroin aqueous solution was dropped into each well of a well plate, and the plate was left to stand at room temperature for 15 minutes. The 0.1% fibroin aqueous solution was then removed from the well using a dropper or the like, and the plate was placed in a dryer at 50°C overnight to completely dry the bottom of the well, thereby producing a cell culture vessel according to Example 19. In Example 19, NIH3T3 cells were seeded into each well of the well plate, and a liquid culture medium was added to the well, followed by culturing for 3 days.

[0107] In Example 19, cultured NIH3T3 cells were detached using Protease P5380 type 8 (Sigma-Aldrich), a protease derived from Bacillus licheniformis. Specifically, Protease P5380 type 8 was adjusted to 10 U / mL with HEPES (a physiological buffer), filter-sterilized, and then added to the liquid medium without removing it. In this case, the final activity was 0.9 U / mL. Then, the cells were incubated at 37°C under CO ２ After reacting for 5 minutes in an incubator, the state of detachment of the NIH3T3 cells was observed.

[0108] Figure 33 shows the results of detaching cultured NIH3T3 cells using Protease P5380 type 8 (Sigma-Aldrich), a protease derived from Bacillus licheniformis, in Example 19. As shown in Figure 33, it was found that detachment of NIH3T3 cells was successful even when using a protease derived from Bacillus licheniformis.

[0109] As described above, the second embodiment is characterized in that adherent cells cultured in a cell culture vessel coated with a cell culture substrate containing, as an active ingredient, any one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, fibroin, and fibroin hydrolysate are detached using a Streptomyces griseus-derived protease or a mixture thereof, a Bacillus Licheniformis-derived protease or a mixture thereof, α-chymotrypsin, or collagenase IA as an agent for detaching adherent cells. In this way, by using a protease derived from Streptomyces griseus or a mixture thereof, a protease derived from Bacillus Licheniformis or a mixture thereof, α-chymotrypsin, or collagenase IA as a detachment agent for adherent cells, the silk protein-degrading enzyme can react with silk protein, silk protein hydrolysate, fibroin, or fibroin hydrolysate without being inactivated by protein components in the liquid medium, and, compared to trypsin, can also be inhibited from reacting with the extracellular matrix or the adherent cells themselves. Therefore, the following steps, which are necessary for the detachment of adherent cells using trypsin, are no longer necessary: ​​removing the liquid medium (step S102 in FIG. 2 ), washing with phosphate buffer (step S103 in FIG. 2 ), removing the washed phosphate buffer (step S104 in FIG. 2 ), and adding back the liquid medium to prevent damage to the adherent cells by trypsin (step S107 in FIG. 2 ), thereby simplifying the process, speeding it up, and reducing costs. Furthermore, the cell culture substrate according to this embodiment uses an enzyme that is less likely to react with the extracellular matrix or the adherent cells themselves, so the adherent cells do not break apart after detachment, and it is possible to obtain the adherent cells in a sheet form.

[0110] While the preferred embodiments of the present invention have been described above, the technical scope of the present invention is not limited to the above-described embodiments. Various modifications and improvements can be made to the above-described embodiments, and such modifications and improvements are also included in the technical scope of the present invention.

[0111] For example, in the first embodiment described above, the first cell culture substrate and the second cell culture substrate are not limited to being liquid or semi-liquid, and may be provided in the form of, for example, a sheet or film. In this case, the cell culture vessel can be produced by attaching the sheet or film cell culture substrate to the surface of the cell culture vessel.

[0112] Furthermore, in the first embodiment described above, laminin, Matrigel, and collagen were given as examples of adhesion factors for adherent cells, but the adhesion factors are not limited to these, and other types of adhesion factors may also be used, such as fibronectin, vitronectin, fibrinogen, polylysine, cadherin, hyaluronic acid, gelatin, proteoglycan, etc. Furthermore, the adhesion factor is not limited to a configuration in which only one of the above types is added, and two or more types may be used in combination.

[0113] Furthermore, in the first embodiment described above, the process of detaching adherent cells is exemplified as a process of removing the liquid culture medium, washing with phosphate buffer, and not removing the washed phosphate buffer. However, this configuration is not limited to this, and for example, the process of detaching adherent cells may be performed by removing the liquid culture medium from adherent cells cultured using the cell culture vessel of this embodiment, washing with phosphate buffer, and removing the washed phosphate buffer.

[0114] In the second embodiment described above, the cell culture substrate according to the present invention was described as a liquid or semi-liquid substrate containing silk protein, a silk protein hydrolysate, fibroin, or a fibroin hydrolysate as an active ingredient, but is not limited thereto and can also be provided as, for example, a sheet- or film-like substrate. In this case, the cell culture vessel according to the present invention can be produced by attaching the sheet- or film-like cell culture substrate to the surface of a Petri dish.

[0115] Furthermore, in the second embodiment described above, the process of detaching adherent cells is exemplified as a process of removing the liquid culture medium, washing with phosphate buffer, and not removing the washed phosphate buffer. However, this configuration is not limited to this, and for example, the process of detaching adherent cells may be performed by removing the liquid culture medium from adherent cells cultured using the cell culture vessel of this embodiment, washing with phosphate buffer, and removing the washed phosphate buffer.

[0116] Furthermore, in Example 15 of the second embodiment described above, a fibroin and sericin mixture was prepared by mixing a fibroin solution using fibroin refined from cocoons with a sericin aqueous solution containing sericin extracted from cocoons. However, the present invention is not limited to this configuration. A fibroin and sericin mixture can also be prepared by mixing a fibroin solution using commercially available fibroin with a sericin aqueous solution extracted from cocoons. Alternatively, a fibroin and sericin mixture can be prepared by boiling silk protein directly in water. In this case, the fibroin and sericin will be contained in a weight ratio of 7:3 to 8:2.

[0117] 1, 2, 5... Coated vessel 3... Cell culture vessel 4... Bottom surface 10... First coating layer 20... Second coating layer 30... Single coating layer

Claims

1. A cell culture substrate which is a substrate for coating a cell culture vessel for culturing adherent cells, comprising a first scaffold substrate component which is one or more proteins, and a second scaffold substrate component which has a composition different from that of the first scaffold substrate component, and wherein the second scaffold substrate component is one or more proteins contained in the extracellular matrix.

2. The cell culture substrate of claim 1, wherein the first scaffold substrate component is one or more proteins not contained in the extracellular matrix that function as a scaffold material.

3. The cell culture substrate according to claim 2, wherein the first scaffold substrate component is one or more proteins selected from the group consisting of silk protein, silk protein hydrolysate, and fibroin.

4. A cell culture vessel coated with the cell culture substrate according to claim 1.

5. A cell culture vessel as described in claim 4, having a first coating layer formed by coating a first cell culture substrate containing the first scaffold substrate component as an active ingredient onto the cell culture vessel, and a second coating layer formed by coating a second cell culture substrate containing the second scaffold substrate component as an active ingredient onto the first coating layer.

6. A cell culture vessel as described in claim 4, having a coating layer formed by coating a mixed cell culture substrate obtained by mixing a first cell culture substrate containing the first scaffold substrate component as an active ingredient and a second cell culture substrate containing the second scaffold substrate component as an active ingredient.

7. A kit for producing a cell culture substrate for producing the cell culture substrate described in claim 1, the kit for producing a cell culture substrate including the first scaffold substrate component and the second scaffold substrate component enclosed or packaged separately.

8. A method for detaching adherent cells cultured using the cell culture vessel described in claim 4 from the cell culture vessel, comprising adding a protease that specifically degrades the first scaffold substrate component rather than the second scaffold substrate component, and reacting the protease with the cell culture substrate to detach the adherent cells from the cell culture vessel.

9. A method for detaching adherent cells as described in claim 8, wherein after removing the liquid medium used for culture, the protease is added without washing the cell culture substrate, and the adherent cells are detached from the cell culture vessel.

10. The method for detaching adherent cells according to claim 8, wherein the protease is added without removing the liquid medium used for culture, and the adherent cells are detached from the cell culture vessel.

11. A method for detaching adherent cells cultured using the cell culture vessel described in claim 6 from the cell culture vessel, comprising adding a protease that specifically degrades the first scaffold substrate component rather than the second scaffold substrate component and reacting it with the cell culture substrate, thereby detaching the adherent cells in a clump state from the cell culture vessel without performing a pipetting operation.

12. The method for detaching adherent cells described in claim 8, wherein the first scaffold substrate component is one or more of silk protein, silk protein hydrolysate and fibroin, and the protease is a protease derived from Streptomyces griseus or a mixture thereof, α-chymotrypsin, or collagenase IA.

13. A method for culturing adherent cells, comprising: a culture step of culturing adherent cells in the cell culture vessel described in claim 4; and a detachment step of detaching the adherent cells cultured in the culture step by the method for detaching adherent cells described in claim 8.

14. A cell recovery device for recovering adherent cells cultured using the cell culture vessel described in claim 4, which performs enzyme addition control for adding a protease that specifically degrades the first scaffold substrate component more than the second scaffold substrate component to the cell culture vessel in which the adherent cells have been cultured, and cell recovery control for recovering the adherent cells.

15. A cell culture system comprising: a culture device for culturing adherent cells using the cell culture vessel according to claim 4; and a cell recovery device according to claim 14 for recovering the adherent cells cultured in the culture device.

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