Cell culture vessel, cell detachment method, and cell production method

Abstract

To provide a cell culture vessel and a cell separation method using the same, capable of facilitating cell separation without making separability and proliferation compatible, that is, without reducing proliferation power of cells.SOLUTION: When an observation image of a cross section perpendicular to the culture surface is obtained, in a state where a length Lcp of a contour of the cell on the culture surface side in a measurement region of the observation image and a length Lf of the measurement region in a direction parallel to the culture surface satisfy Lcp> Lf, generating ultrasonic vibration in a vibration unit disposed to be capable of applying vibration to the culture container; And a separation step of separating the cells from the culture surface by transmitting the ultrasonic vibration to the cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a cell culture vessel, a cell detachment method, and a cell production method. [Background technology]

[0002] In the fields of cell medicine and regenerative medicine, large amounts of cells are required, and there is a particular need for an efficient and stable supply of adhesive cells, which account for the majority of biological tissues. Adherent cells are produced by culturing them in a culture vessel, such as a polystyrene dish, and then detaching and recovering them from the culture vessel. To efficiently and stably supply adherent cells, a method for easily detaching adherent cells is needed. Patent Documents 1 and 2 propose a method of forming a group of protrusions on the culture surface to bring the cells into point contact with the culture surface, thereby reducing the adhesion area and weakening the adhesion strength, thereby making detachment easier. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-168494 [Patent Document 2] Japanese Patent Application Publication No. 2019-37151 Summary of the Invention [Problem to be solved by the invention]

[0004] The inventors have found that weakening the adhesive strength of cells, as in conventional techniques, can reduce the proliferation ability of cells when dealing with adhesive cells that exhibit proliferation ability through strong adhesion, and can make it impossible to obtain a sufficient number of cells. The present disclosure aims to provide a cell culture vessel that achieves both detachment and proliferation, i.e., that facilitates cell detachment without reducing the proliferation ability of cells, and a cell detachment method using the same. [Means for solving the problem]

[0005] In contrast to the prior art method of facilitating detachment by reducing the contact area, the present inventors have discovered a method for detachment when the adhesion area is large. That is, the present inventors have found that applying ultrasonic vibrations when the cell adhesion area per unit area of ​​the culture surface is large makes it possible to achieve both cell detachment and proliferation.

[0006] The present disclosure provides the following cell detachment method. Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell detachment method comprising a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibrations to the cells.

[0007] The present disclosure also provides the following cell detachment method. Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibration to the cells, The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, A cell detachment method characterized in that the culture surface has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

[0008] The present disclosure also provides the following cell production method. a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell production method characterized by transmitting the ultrasonic vibration to the cells, thereby detaching the cells from the culture surface.

[0009] The present disclosure also provides the following cell production method. a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; The ultrasonic vibration is transmitted to the cells, thereby detaching the cells from the culture surface. The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, Furthermore, the cell production method is characterized in that the concave-convex pattern has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

[0010] The present disclosure also provides the following cell culture vessel. A cell culture vessel having a periodic uneven pattern on the culture surface, The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, A cell culture vessel characterized in that the concave-convex pattern has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied. [Effects of the Invention]

[0011] According to the present disclosure, it is possible to provide a cell culture vessel that enables both detachment and proliferation, a cell detachment method that enables detachment while maintaining proliferation, and a method for producing cells that achieves both proliferation and detachment. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows examples of cross-sectional observation images of a concave-convex pattern and cells. [Figure 2A] 1 shows an example of a concave-convex pattern having convex portions. [Figure 2B] 1 shows an example of a concave-convex pattern having recesses. [Figure 3A] An example of a concave-convex pattern is shown below. [Figure 3B] An example in which the concave-convex pattern is formed of cylindrical convex portions will be shown. [Figure 3C] An example in which the concave-convex pattern is formed of cylindrical recesses is shown. [Figure 3D] An example in which the concave-convex pattern is formed of convex portions in the shape of a truncated cone is shown. [Figure 3E] An example in which the concave-convex pattern is formed of truncated cone-shaped recesses is shown. [Figure 4] FIG. 1 is a flow chart of a cell detachment method and a cell production method. [Figure 5] FIG. 2 is a schematic diagram of a mechanism for applying ultrasonic vibrations. [Figure 6A] FIG. 10 is a diagram for explaining evaluation of the adhesion state of cells from a cross-sectional observation image. [Figure 6B] FIG. 10 is a diagram for explaining evaluation of the adhesion state of cells from a cross-sectional observation image. [Figure 7A] FIG. 1 is a schematic diagram of a separation mechanism using a jet. [Figure 7B] FIG. 1 is a schematic diagram of a separation mechanism using a jet. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present inventors believe that the reason why the culture vessel, cell detachment method, and cell production method of the present disclosure are able to achieve both detachment and proliferation is as follows. When cells are cultured in the culture vessel of the present disclosure, as shown in FIG. 1 , the cells 11 penetrate into the gaps 120 of the uneven pattern 100 and adhere thereto, enabling culturing without reducing proliferation. Furthermore, in the present disclosure, the cells 11 are detached by applying ultrasonic vibrations rather than by a detachment method using a flow such as a jet. When ultrasonic vibrations are applied to a culture surface with a large adhesion area of ​​the cells 11 per unit area, the cells are subjected to ultrasonic vibrations from various angles, allowing the energy required for detachment to be efficiently imparted to the adhesion surface. This makes it possible to detach the cells 11 more efficiently than by applying ultrasonic vibrations to a normal culture surface without the uneven pattern 100. Therefore, it is believed that detachability can be improved without reducing the adhesion area of ​​the cells 11, and that it has been possible to achieve both detachability and proliferation of the cells 11, which was previously difficult.

[0014] Hereinafter, embodiments relating to a cell culture vessel, a cell detachment method, and a cell manufacturing method according to first and second embodiments of the present disclosure will be described. The following embodiments do not limit the present disclosure according to the claims.

[0015] First Embodiment As a first embodiment, the present disclosure provides the following culture vessel. A culture vessel having a periodic uneven pattern on the culture surface, The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, A culture vessel characterized in that the concave-convex pattern has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

[0016] (Culture container) The culture vessel in this embodiment is not particularly limited as long as it has a cell-adhesive culture surface, and examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, multi-well plates, multi-plates, Petri dishes, culture bags, bottles, etc. The culture surface refers to the surface that is located inside the culture vessel and to which cells can mainly adhere and grow, among the surfaces that make up the culture vessel.

[0017] The material of the culture vessel in this embodiment may be any material that is chemically stable and capable of culturing the desired cells, and examples thereof include polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(meth)acrylic acid, poly(meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, glass, metal, etc. Among these, polystyrene is preferred.

[0018] (convex and concave pattern) Specific examples of the concave-convex pattern in this embodiment are shown in Figures 2A and 2B. In each figure, a perspective view of the concave-convex pattern is shown at the top, a top view in the middle, and a cross-sectional view at the bottom. The culture surface 10, indicated by the bold line in Figures 2A and 2B, has a concave-convex pattern 100. The culture surface 10 having a concave-convex pattern 100 refers to the culture surface 10 having a periodic concave-convex pattern (design) with a height difference of at least 10 nm (the height difference HD will be described later). The periodic concave-convex pattern 100 is a pattern in which concaves and convexities are repeated at approximately regular intervals. The terms "convex" and "convex" are relative terms. The terms "convex" and "convex" refer not to relative terms but to a reference plane 105. The convex portions 101 are portions of the culture surface 10 that protrude from the reference plane 105, and the concave portions 102 are portions of the culture surface 10 that are recessed from the reference plane 105. The reference plane 105 is used for convenience and can be determined arbitrarily. Furthermore, the periodic uneven pattern allows cells to grow uniformly and allows for uniform exfoliation by applying ultrasonic vibrations.

[0019] 2A and 2B, 131 indicates the highest surface of the culture surface 10, 132 indicates the lowest surface of the culture surface 10, and 135 indicates the side of the uneven portion (concave portion 102 and / or convex portion 101). In FIG. 2A, the lowest surface 132 is the same as the reference surface 105, and in FIG. 2B, the highest surface 131 is the same as the reference surface 105.

[0020] The concave-convex pattern 100 is formed by periodically arranged convex portions 101 and / or periodically arranged concave portions 102. Fig. 2A shows an example in which the concave-convex pattern 100 is formed by convex portions 101, and Fig. 2B shows an example in which the concave-convex pattern 100 is formed by concave portions 102.

[0021] Gap 120, or simply "recess", refers to a relative depression regardless of reference surface 105. For example, as shown in Fig. 2A, the space sandwiched between multiple protrusions 101 is gap (recess) 120, and as shown in Fig. 2B, recess 102 is also gap 120. The same is true for protrusions; both the portion sandwiched between multiple recesses 102 and protrusion 101 can be said to be protrusions.

[0022] In the example shown in FIG. 2A, the convex portions 101 protrude from the reference surface 105 of the concave-convex pattern 100, and are periodically arranged at a height H and an interval I. 100nm≦I≦2000nm, 20nm≦H≦400nm, and H / I≦1.1 Meet the following.

[0023] In the example shown in FIG. 2B, the recesses 102 are recessed from the reference surface 105 of the recess-protrusion pattern 100, are periodically arranged, have a depth D, and have a hole diameter S. 100nm≦S≦2000nm, 20nm≦D≦400nm, and D / S≦1.1 Meet the following.

[0024] The pitch P is the distance from the center of a depression to the center of an adjacent depression, or from the center of a protrusion to the center of an adjacent protrusion, in a top view of the concave-convex pattern 100.

[0025] It is preferable that the pitch P satisfies 100 nm≦P≦2000 nm. By keeping the pitch P within this range, the surface area of ​​the culture surface 10 is increased, and cells (not shown in FIGS. 2A and 2B) can easily enter the gaps 120, thereby increasing the cell adhesion area per unit area of ​​the culture surface. This allows ultrasonic vibrations to be applied to the cells from all directions during detachment by applying ultrasonic vibrations, improving detachability. Furthermore, during culture, the increased adhesion area makes it difficult for adhesive strength to decrease, resulting in high proliferation. Therefore, it is possible to achieve both detachability and proliferation.

[0026] On the other hand, if the pitch P is less than 100 nm, the openings of the gaps 120 are small, making it difficult for cells to enter the gaps 120, resulting in a small adhesion area and making it difficult to increase proliferation. Also, if the pitch P is greater than 2000 nm, the spacing I between the convex portions 101 or the pore diameter S of the concave portions 102 becomes too wide, causing most of the cells to adhere to one uneven portion, making it difficult to increase the cell adhesion area per unit area of ​​the culture surface, and therefore making it difficult to achieve the release effect that can be obtained by applying ultrasonic vibrations when the cell adhesion area is large.

[0027] Furthermore, the pitch P is preferably within a range that allows cells to easily enter the gaps 120 and is sufficiently small relative to the size of the cells used, and more preferably, the pitch P satisfies 100 nm≦P≦1000 nm.

[0028] The uneven pattern height difference HD refers to the difference in height between the highest surface 131 and the lowest surface 132 of the uneven pattern 100. When the uneven pattern 100 is composed of convex portions 101, the height H of the convex portions 101 can be the uneven pattern height difference HD. When the uneven pattern 100 is composed of concave portions 102, the depth D of the concave portions 102 can be the uneven pattern height difference HD. When both convex portions 101 and concave portions 102 are present, the height H + depth D can be the uneven pattern height difference HD. The uneven pattern height difference HD preferably satisfies 20 nm ≦ HD ≦ 400 nm. When the uneven pattern height difference HD is within this range, the surface area of ​​the culture surface 10 is increased, and cells can easily reach and adhere to the lowest surface 132 of the concave-convex pattern 100, thereby increasing the cell adhesion area per unit area of ​​the culture surface. This allows ultrasonic vibrations to be applied to cells from all directions during detachment by applying ultrasonic vibrations, improving detachability. Furthermore, the adhesive strength is less likely to decrease during culture, resulting in high proliferation. Therefore, both detachability and proliferation can be achieved.

[0029] On the other hand, if the height difference HD of the uneven pattern is less than 20 nm, the surface area of ​​the culture surface 10 will not be large, and the cell adhesion area per unit area of ​​the culture surface will not be large, making it difficult to achieve the release effect that can be achieved by applying ultrasonic vibrations when the cell adhesion area is large.If the height difference HD of the uneven pattern is more than 400 nm, it will be difficult for cells to adhere to the entire structure that makes up the uneven pattern, resulting in a small adhesion area and making it difficult to increase proliferation.

[0030] By setting the height difference HD of the concave-convex pattern to a range of 20 nm to 400 nm, the initial adhesiveness of the recovered cells is likely to be improved. Conventional cell detachment methods use enzymes such as trypsin to detach cells, which tends to reduce membrane proteins and reduce the initial adhesiveness of the recovered cells. In contrast, in the present disclosure, the culture surface 10 having the concave-convex pattern 100 increases the adhesion area per unit area of ​​the cell culture surface, and by applying ultrasonic vibrations to the cells, cells can be easily detached, allowing cells with high membrane protein content to be recovered. Therefore, it is expected that cells detached using the detachment method of the present disclosure have higher initial adhesiveness than cells detached using conventional methods. For example, using cells detached using the detachment method of the present disclosure can improve culture efficiency, making it particularly effective for large-scale culture.

[0031] The height difference HD of the concave-convex pattern is preferably 20% or less of the thickness of the cell at the time of adhesion, and from this point of view, it is preferable to satisfy 50 nm≦HD≦300 nm, and more preferably to satisfy 100 nm≦HD≦300 nm.

[0032] In this embodiment, it is preferable that the ratio H / I of the height H of the convex portions 101 to the interval I, and the ratio D / S of the depth D of the concave portions 102 to the hole diameter S are both 1.1 or less. When a concave-convex pattern is formed by the convex portions, the interval I is the length obtained by subtracting the distance including the convex portions 101 from the pitch P in a top view of the concave-convex pattern. For example, if the convex portions 101 are cylindrical, the interval I is the length obtained by subtracting the diameter of the cylinder from the pitch P. If the convex portions 101 are elliptical cylindrical, the interval I is the length obtained by subtracting the minor axis length of the elliptical cylinder (the length of the shorter of the axes perpendicular to the ellipse). The hole diameter S is the diameter of the concave portions when they are cylindrical. If the concave portions are elliptical cylindrical, the hole diameter S is the minor axis length. If the side surface 135 of the uneven portion is not perpendicular to the culture surface 10 (for example, if the uneven portion is a truncated cone), the pore diameter of the uneven portion is the pore diameter at the point where the depth D of the recess is half the depth or the point where the height H of the projection is half the height. If H / I and D / S are 1.1 or less, cells can easily enter the recess and the adhesion area is large, making it possible to achieve both detachment and proliferation.

[0033] The shape of the concave-convex portions that form the concave-convex pattern 100 is not particularly limited, and the concave-convex pattern 100 can have a pillar structure, a moth-eye structure, or a hole structure, and the concave-convex portions can have a columnar, cone-like, or frustum-like shape.

[0034] 3A, the highest surface 131 of the concave-convex pattern 100 preferably has a flat portion 410. Meanwhile, the highest surface 131 of the concave-convex pattern 100 and the side surface 135 of the concave-convex portion are preferably connected via a curved portion 420. Furthermore, the lowest surface 132 of the concave-convex pattern 100 preferably has a flat portion 410. Meanwhile, the lowest surface 132 of the concave-convex pattern 100 and the side surface 135 of the concave-convex portion are preferably connected via a curved portion 420.

[0035] The uneven pattern 100 has flat portions 410, which allow cells (not shown in FIGS. 3A to 3E) to adhere easily, and the uneven pattern 100 has curved portions 420, which allow cells to easily follow the uneven pattern 100. As a result, the adhesion area of ​​cells increases, making it easier to achieve both detachment and proliferation.

[0036] Furthermore, as shown in Figures 3B to 3E, the concave-convex portions forming the concave-convex pattern 100 are preferably cylindrical or truncated cone shaped. Figure 3B shows an example in which the concave-convex pattern 100 is formed with cylindrical convex portions 101, Figure 3C shows an example in which the concave-convex pattern 100 is formed with cylindrical concave portions 102, Figure 3D shows an example in which the concave-convex pattern 100 is formed with truncated cone shaped convex portions 101, and Figure 3E shows an example in which the concave-convex pattern 100 is formed with truncated cone shaped concave portions 102. When the concave-convex portion side surface 135 is curved or has a curved surface, cells can easily follow the concave-convex pattern 100. As a result, the adhesion area of ​​cells is increased, making it easier to achieve both detachment and proliferation.

[0037] From the viewpoint of strength and transmission of ultrasonic vibrations, the concave-convex pattern 100 is preferably formed integrally with the culture vessel, and from the viewpoint of processing, the culture surface having the concave-convex pattern 100 is preferably made of resin. However, the material is not particularly limited as long as it is a material to which cells can adhere.

[0038] The method for forming the concave-convex pattern on the culture surface is not particularly limited, but from the viewpoint of mass production, shape transfer methods using a mold on which a concave-convex pattern is formed, such as nanoimprinting and injection molding, are preferred. Methods for forming the concave-convex pattern on a mold or culture surface include microfabrication methods such as photolithography, electron beam direct writing, particle beam processing, and scanning probe processing. The culture surface containing the concave-convex pattern may be subjected to a surface treatment suitable for cell culture.

[0039] (cell) The cells used in the present disclosure are not particularly limited as long as they can be cultured in vitro on the culture surface of a culture vessel. For example, various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), human alveolar basal epithelial adenocarcinoma-derived A549 cells, mouse macrophage-like cells (RAW264.7), and human cervical cancer-derived HeLa cells can be used. In addition, for example, epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile cells, and the like can also be used. Examples of cells that can differentiate include skeletal muscle cells, smooth muscle cells, cardiac muscle cells, neuron cells that make up the nervous system, glial cells, and fibroblasts, hepatic parenchymal cells that are involved in the metabolism of the living body, non-parenchymal liver cells, and adipocytes, as well as various stem cells such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, as well as progenitor cells of various tissues, and cells induced to differentiate from these.

[0040] (Cell detachment method and cell culture method) A first embodiment of the present disclosure provides the following cell detachment method. Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibration to the cells, The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, A cell detachment method characterized in that the culture surface has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

[0041] The cell detachment method of this embodiment is shown in Fig. 4 and may include a detachment step S320, a culture step S310 for culturing the cells, and a recovery step S330 for recovering the detached cells. However, the culture step S310 and the recovery step S330 are not essential. A specific example will be described with reference to FIGS. First, in the culture step, cells are cultured in a culture vessel having a culture surface with the concave-convex pattern (step S310).

[0042] Next, the detachment process of applying ultrasonic vibrations is initiated (step S320). For example, as shown in FIG. 5, a mechanism for applying ultrasonic vibrations to a culture vessel can be used to apply ultrasonic vibrations. In FIG. 5, the mechanism for applying ultrasonic vibrations comprises a vibrating body (ring device) 4101, a vibration transmission member 413, and a cover 415. The vibrating body (ring device) 4101 comprises a diaphragm 411 and a piezoelectric body 412. The culture vessel 421 is placed on the vibration transmission member 413. In the example shown in FIG. 5, the culture vessel 421 is covered with a culture vessel top lid 422, and a weight 414 is placed on top of the lid 422. Covering the culture vessel 421 with the culture vessel top lid 422 and further placing the weight 414 in this manner may allow for efficient application of ultrasonic vibrations. Finally, the detached cells are collected (step S330).

[0043] The first embodiment of the present disclosure further provides the following cell production method. a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; The ultrasonic vibration is transmitted to the cells, thereby detaching the cells from the culture surface. The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, Furthermore, the cell production method is characterized in that the concave-convex pattern has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

[0044] The method for producing cells according to this embodiment is also shown in Fig. 4 and includes a culturing step S310 and a detaching step S320. It may further include a recovery step S330 for recovering the detached cells, but the recovery step S330 is not essential. Each step will be described below.

[0045] (Culture process) The culturing step can be carried out by a known cell culturing method, which will be described in detail below.

[0046] (Cell culture method) The cell culture conditions can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to a culture vessel, and 1.0 × 10 1 ~5.0×10 4 cells / cm 2 The cells are seeded in an amount of about 1000 cells per well and cultured in an environment at 37°C and a CO2 concentration of 5%. At this time, it is preferable to culture the cells until they occupy about 70 to 80% of the culture surface, that is, until the cells reach a subconfluent state.

[0047] (Culture medium) The type of culture medium is not particularly limited, and examples thereof include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemSpan H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma-Aldrich), Endothelial Cell Growth Examples of suitable mediums include Medium 2 Kit (Promocell), Mesenchymal Stem Cell Growth Medium 2 (Promocell), MSCGM Bullet Kit (Lonza), mTeSR1 or 2 medium (Stem Cell Technology), Repro FF or Repro FF2 (ReproCell), NutriStem medium (Biological Industries), and MF-Medium mesenchymal stem cell growth medium (Toyobo Co., Ltd.). Among these, it is preferable to use a medium suitable for culturing each cell.

[0048] (serum) Serum may be added to the above-mentioned medium. Examples of serum include fetal bovine serum (FBS), baby calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum, with FBS being commonly used due to its ease of availability. Alternatively, the medium may be serum-free, not containing raw or unpurified serum, but containing purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0049] (antibiotics) The medium may contain antibiotics, such as penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, and amphotericin B.

[0050] (peeling process) The detachment step includes a vibration application step in which cells are detached by applying ultrasonic vibrations to the cells, as will be described in detail below.

[0051] (Ultrasonic vibration applied) By generating ultrasonic vibrations in a vibration means arranged so as to be able to impart vibrations to the culture vessel, ultrasonic vibrations are imparted to the culture vessel and transmitted to the cells. Ultrasonic vibrations are vibrations in the ultrasonic band, with a frequency of 10 kHz to 1 MHz. The vibration means is not particularly limited as long as it is capable of imparting ultrasonic vibrations to the culture vessel and transmitting the ultrasonic vibrations to the cells. For example, a vibrating body such as an ultrasonic oscillator can be used. Further, specific examples of vibrating bodies include a ring device in which a ring-shaped piezoelectric body is adhered to a glass plate or a metal plate, and a Langevin-type oscillator (Langevin device).

[0052] Ultrasonic vibrations can be applied by directly contacting the vibrator with the outer surface of the culture vessel, or by using a vibration transmission member installed between the vibrator and the outer surface of the culture vessel. This allows the ultrasonic vibrations to be transmitted to the cells to be detached via the culture vessel. Since high detachability is easily achieved by transmitting ultrasonic vibrations to the cells via a culture surface having a concave-convex pattern, it is preferable to apply vibrations from the back side of the culture surface, even among the outer surfaces of the culture vessel. The vibration transmission member may be made of any material that can transmit ultrasonic vibrations to the culture vessel, including liquid materials such as water and glycerin, and solid materials such as rubber and gel.

[0053] Although there are no particular limitations on the maximum amplitude of the vibration surface of ultrasonic vibrations, a value of 10 μm or less is desirable from the viewpoint of maintaining cell viability. This range makes it possible to easily detach cells while suppressing the effects of heat generation from the vibrator. Furthermore, an amplitude of 10 μm or less is preferable because it makes it difficult for cavitation to occur when ultrasonic vibrations are applied, making it difficult for cell viability to decrease.

[0054] The vibration pattern of the ultrasonic vibration can use various vibration modes depending on the vibrator used as the vibration means and the type of ultrasonic device.

[0055] Furthermore, depending on the type of vibrator and the driving method, heat may be generated during driving. This tendency tends to intensify as the vibrator continues to be driven at the resonant frequency, depending on the applied voltage, the frequency band used, and the device environment. To address this, the amount of heat generated can be controlled by repeating driving and pausing at regular intervals (burst driving), or by repeatedly and continuously changing the driving frequency within a certain frequency range (sweep driving), either alone or in combination. Furthermore, to suppress heat generation, a cooling mechanism can be provided around the vibrating means to control the vibrator directly or indirectly via the ambient temperature. Furthermore, the resonant frequency may change due to heat generated by the vibrator during driving. In such cases, known methods, such as resonant frequency tracking control using current detection, can be used.

[0056] The environmental temperature when applying ultrasonic vibrations is not particularly limited and can be set appropriately depending on the cell type used. From the viewpoint of maintaining cell viability, a temperature between 20°C and 40°C is preferable, and in the case of mammalian cells, for example, a temperature between 30.0°C and 37.5°C is particularly preferable. This range is because the environmental temperature is close to the temperature during culture, which reduces the impact of temperature changes on the cells and allows for maintaining a high viability. Furthermore, as with cell culture, the temperature may be set in an environment with a CO2 concentration of 5%. The environmental conditions when applying ultrasonic vibrations can be set appropriately depending on the type and characteristics of the cells and the method of using the cells after detachment.

[0057] In addition to the vibration applying step, the detachment step may include a liquid replacement step of replacing the medium in the culture vessel with a buffer solution or the like immediately before the vibration applying step in order to promote cell detachment. Alternatively, the liquid replacement step may be performed before the detachment step.

[0058] Any buffer solution can be used without limitation as long as it can maintain a neutral pH range. Examples include Tris buffer solutions such as Tris-HCl buffer solution, phosphate buffer solution, HEPES buffer solution, citrate-phosphate buffer solution, glycylglycine-sodium hydroxide buffer solution, Britton-Robinson buffer solution, GTA buffer solution, etc. Among these, phosphate buffer solutions that are close to the in vivo environment are preferred, and phosphate buffered saline (PBS) adjusted to be isotonic with intracellular fluid is more preferably used. When using PBS, Ca 2+ , Mg 2+ It is preferable to use PBS(-) that does not contain divalent cations such as:

[0059] The detachment step may also include a step of applying an external stimulus to the culture surface to generate convection or impact, in order to promote cell detachment. Methods for generating convection include pipetting, or methods using a pump, a stirring blade, or a shaker, and methods for generating impact include tapping on the wall or bottom surface.

[0060] Second Embodiment As a second embodiment of the present disclosure, there is provided the following cell detachment method. Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell detachment method comprising a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibrations to the cells.

[0061] Furthermore, as a second embodiment of the present disclosure, there is provided the following cell production method. a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell production method characterized by transmitting the ultrasonic vibration to the cells, thereby detaching the cells from the culture surface.

[0062] (convex and concave pattern) Regarding the uneven pattern, the explanation in the first embodiment can be referred to. In this embodiment, the pitch P of the uneven portion preferably satisfies 100 nm≦P≦2000 nm. When the pitch P is within this range, the surface area of ​​the culture surface 10 is increased and cells can easily enter the gaps 120, thereby increasing the cell adhesion area per unit area of ​​the culture surface. This allows ultrasonic vibrations to be applied to the cells from all directions during detachment by applying ultrasonic vibrations, improving detachability. Furthermore, during culture, the increased adhesion area makes it difficult for adhesive force to decrease, resulting in high proliferation. Therefore, it is possible to achieve both detachability and proliferation.

[0063] Furthermore, the pitch P is preferably within a range that allows cells to easily enter the gaps 120 and is sufficiently small relative to the size of the cells used, and more preferably, the pitch P satisfies 100 nm≦P≦1000 nm.

[0064] In this embodiment, the pattern height difference HD of the uneven pattern preferably satisfies 20 nm≦HD≦400 nm. When the uneven pattern height difference HD is within this range, the surface area of ​​the culture surface 10 is increased, and cells can easily reach the lowest point of the uneven pattern and adhere thereto, thereby increasing the cell adhesion area per unit area of ​​the culture surface. This allows ultrasonic vibrations to act on the cells from all directions during detachment by applying ultrasonic vibrations, improving detachability. Furthermore, adhesive strength is less likely to decrease during culture, resulting in high proliferation. Therefore, both detachability and proliferation are achieved.

[0065] In this embodiment, the pattern height difference HD more preferably satisfies 20 nm≦HD≦400 nm, and further preferably satisfies 50 nm≦HD≦300 nm.

[0066] (Process) The cell detachment method and cell production method of this embodiment are shown in Figure 4, and both include a detachment step S320, and may further include a culture step S310 for culturing the cells and a recovery step S330 for recovering the detached cells. However, the culture step S310 and the recovery step S330 are not essential. For details of each step, please refer to the explanation in the first embodiment.

[0067] In the peeling step of this embodiment, when an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image and the length Lf of the measurement region in the direction parallel to the culture surface are expressed as follows: Lcp>Lf When the above condition is satisfied, ultrasonic vibrations are generated in the vibration means, and the ultrasonic vibrations are transmitted to the cells, thereby detaching the cells from the culture surface. And more preferably, in the peeling step, Lcp / Lf≧1.2 The vibration means generates ultrasonic vibrations in a state where the above condition is satisfied. Preferably, in the peeling step, when the length of the contour of the culture surface in the measurement region is Lp, Lcp / Lp≧0.8 The vibration means generates ultrasonic vibrations in a state where the above condition is satisfied.

[0068] 6A and 6B show a schematic diagram of cells cultured in a culture vessel with an uneven surface on the upper side, and an enlarged view of the culture surface on the lower side. As shown in FIGS. 6A and 6B, Lcp, Lf, and Lp are the cell contour line Lcp (shown by a dotted line in the figures), the line Lf parallel to the culture surface (shown by a dashed line in the figures), and the contour Lp of the culture surface (shown by a solid line in the figures). However, Lcp, Lf, and Lp do not need to be measured every time during the detachment process; they can be verified using the Lcp, Lf, and Lp verification method described below when necessary.

[0069] Lcp>Lf (the state in which the outline of the cell on the culture surface side is longer than the length in the direction parallel to the culture surface) preferably means that Lcp is at least 5% longer than Lf, i.e., preferably Lcp>Lf×1.05. An Lcp longer than Lf indicates that the cell adhesion area per unit area of ​​the culture surface is larger than when cultured on a conventional culture surface without a concave-convex pattern. By culturing in this state and detaching by applying ultrasonic vibrations, both cell detachability and proliferation can be achieved. More preferably, Lcp and Lf satisfy Lcp / Lf≧1.2, and even more preferably, Lcp / Lf≧1.7. Furthermore, it is preferable that cells adhere to 30% or more of the height H or depth D of the concave-convex portions that make up the concave-convex pattern, and when the height H or depth D is in the range of 400 nm or more and 1000 nm or less, it is also preferable that the ratio Lcp / Lf > height H [nm] / 1000 + 0.8 or the ratio Lcp / Lf > depth D [nm] / 1000 + 0.8 is satisfied.

[0070] It is preferable that Lcp and Lp satisfy Lcp / Lp≧0.8 or more. A large Lcp / Lp indicates that the cells can follow the uneven pattern 100, and by culturing and detaching using ultrasonic vibration in this state, it is easy to achieve both detachability and proliferation. More preferably, Lcp / Lp≧0.9 or more is satisfied. The more the cells can follow the uneven pattern with no gaps, the more stable the cells will adhere and the easier it will be to maintain proliferation. In Figure 6A, the cells can follow the uneven pattern, but in Figure 6B, they do not follow as well as in Figure 6A.

[0071] (How to verify the relationship between Lcp, Lf, and Lp) The length Lcp of the contour of the cell on the culture surface side in the measurement area of ​​the observation image, the length Lf of the measurement area in the direction parallel to the culture surface, and the length Lp of the contour of the culture surface in the measurement area are determined by acquiring an observation image as described below, and determining Lcp, Lf, and Lp based on the acquired observation image, and using the determined Lcp, Lf, and Lp, verifying whether the respective relationships regarding these are satisfied.

[0072] 1) Observation image acquisition The observed image is a cross-section perpendicular to the culture surface (cross-sectional image). Cross-sectional images are obtained by cryo-SEM observation of a single cell and the cross-section of the culture surface where the cell is attached, and the image used clearly shows the interface between the cell and the culture surface where it is attached. To obtain cross-sectional observation images of the cells and the culture surface, it is necessary to fix the cells. The following is an example of a specific operation method from fixing the cells to obtaining the observation image.

[0073] 1-1) Immobilization After culturing, the medium was removed from the culture vessel to which the cells had adhered, and the cells were washed three times with PBS. 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) was added to fix the cells. In the following examples, cells were fixed by immersing them in 4% paraformaldehyde for 15 minutes, but the optimal fixation time should be selected depending on the cell type. For example, if the cells are disintegrated during image acquisition and a clear image cannot be obtained, this indicates insufficient or excessive fixation, and the immersion time should be adjusted.

[0074] 1-2) Dehydration Dehydrate the cells using a mixture of ethanol and pure water cooled to 4°C. First, remove the 4% paraformaldehyde used for fixation, and wash the cells three times with PBS. Add a 50% aqueous solution of ethanol and let stand for 10 minutes while cooling at 4°C. Remove the 50% aqueous solution of ethanol, then add a 60% aqueous solution of ethanol and let stand for 10 minutes while cooling at 4°C. Repeat the same procedure using 70%, 80%, 90%, and 100% aqueous solutions of ethanol. After removing the 100% ethanol, let stand overnight to dry.

[0075] 1-3) Observation image acquisition Observation images were obtained using the cryo-SEM method using an FEI FIB-SEM (Helios G4UC). The cryo-temperature was set to -140°C, and after cross-section processing using an FIB, the cross-section was observed. The cells to be observed were not those that had just divided or were densely clustered, but rather those that were firmly adhered, with only one cell visible within the field of view, or eight cells with clearly distinguishable boundaries. A cross-section was cut using the FIB so that it passed near the center of the selected cell and near the center of the structure that formed the concave-convex pattern, and a cross-sectional observation image was obtained using an SEM. The observation image was obtained with the cells facing upward relative to the culture surface.

[0076] 2) Determination of Lcp, Lf, and Lp As shown in Figures 6A and 6B, the cell contour line Lcp (shown by the dotted line in the figures), the line Lf parallel to the culture surface (shown by the dashed line in the figures), and the contour Lp of the culture surface (shown by the solid line in the figures) are measured to determine Lcp, Lf, and Lp. Measurement of Lcp, Lf, and Lp is performed by extracting the vicinity of the adhesion interface between the cell 11 and the culture surface 10 from the observation image as a measurement region. The measurement region is defined as the area inside a rectangle formed by line c, which will be described below, in the observation image. In FIG. 6B, an upper side 601, a lower side 602, a left side 603, and a right side 604 are indicated by two-dot chain lines.

[0077] Upper edge 601: a line parallel to the culture surface 10 that passes 300 nm above the highest surface 131 at the adhesion interface between the cell 11 and the culture surface 10 in the observed image Lower edge 602: A line parallel to the culture surface that passes 300 nm below the lowest surface 132 of the concave-convex pattern in the observed image Left side 603: A line perpendicular to the culture surface that passes 600 nm to the right of the leftmost position of the adhesion interface between the cell 11 and the culture surface 10 in the observed image. Right side 604: A line perpendicular to the culture surface that passes 600 nm to the left of the rightmost part of the adhesion interface between the cell 11 and the culture surface 10 in the observed image. Lcp is the length of the line on the culture surface side (lower side) of the contour of the cell 11 in the measurement area image. Lf is the length of the upper or lower side of the measurement area image. Lp is the length of the line on the cell side (upper side) of the contour of the culture surface in the measurement area image. Here, "parallel to the culture surface" refers to "parallel to the culture surface." "Parallel to the culture surface" can be said to be "parallel to the surface when the culture surface including the concave-convex pattern is viewed as a macroscopically smooth surface," or "parallel to the reference plane of the concave-convex pattern." For the acquired observation images, Lcp, Lf, and Lp are measured, and their average values ​​are calculated to be Lcp, Lf, and Lp.

[0078] 3) Verification Based on the determined Lcp, Lf, and Lp, the relationship between Lcp and Lf, or Lcp / Lp, is determined. [Example]

[0079] The present disclosure will be described in more detail below with reference to examples and comparative examples, but these are not intended to limit the present disclosure in any way. The cells used in the examples of the present disclosure were obtained according to the culture conditions shown below.

[0080] (CHO cell culture) CHO (Chinese Hamster Ovary) cells were cultured in a Φ35 polystyrene dish (Corning) at 10,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium was Ham's F12 (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 80%.

[0081] (MDCK cell culture) MDCK cells (canine renal tubular epithelial cells) were cultured in a Φ35 polystyrene dish (Corning) at 10,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium used was Eagle's MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 80%.

[0082] (A549 cell culture) A549 cells (human alveolar basal epithelial adenocarcinoma cells) were plated on a Φ35 polystyrene dish (Corning) at 10,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium was DMEM (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 80%.

[0083] (MSC cell culture) MSC cells (mesenchymal stem cells) were cultured in a Φ35 polystyrene dish (Corning) at 4000 cells / cm. 2The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium used was Mesenchymal Stem Cell Growth Medium 2 (Promocell) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 168 hours, with medium changes every 72 hours. Cell adhesion and proliferation were confirmed by observing the cells under a phase-contrast microscope. The cell coverage of the dish was approximately 80%.

[0084] (Culture of mouse macrophage-like cells) Mouse macrophage-like cells (RAW264.7) were cultured in a Φ35 polystyrene dish (Corning) at 20,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium was DMEM (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 80%.

[0085] (Removal method by applying ultrasonic vibration) The culture vessel was set on the ultrasonic device using the vibration imparting mechanism shown in Fig. 5 described above. Then, at an ambient temperature of 37°C, the ultrasonic device was driven under the vibration imparting conditions shown in Table 1 to impart ultrasonic vibrations.

[0086] When a ring device is used as an ultrasonic device in a standing wave mode, bending vibrations can be applied in a vibration mode in which the maximum amplitude acts on the center of the culture surface of the culture vessel in the frequency band used.

[0087] When a Langevin device is used as the ultrasonic device, vibrations having a nearly flat vibration distribution with the maximum amplitude at the center can be applied to the entire culture surface of the culture vessel.

[0088] [Table 1]

[0089] (Jet separation means) Using the jet exfoliation device shown in Figures 7A and 7B, exfoliation was performed using a jet without using ultrasonic vibration. As shown in Figure 7A, the jet detachment device was fabricated by combining a silicon tube 701, a tube pump 702, a reservoir tank 703, a needle 704, a three-way valve 705, and a jet mechanism 730 including a nozzle 706. Reference numeral 710 denotes a culture vessel, 720 denotes a holding mechanism for holding the culture vessel 710, 740 denotes a support, and 750 denotes a base. Then, as shown in Figure 7B, the culture vessel 710 in which cells (not shown in Figures 7A and 7B) were cultured was placed so that the distance between the tip of the nozzle 706 (inner diameter 1.04 mm) and the culture surface 10 was 17 mm. PBS was sprayed from the nozzle 706 perpendicularly to the culture surface at 0.65 mL / s for 3 seconds over the entire culture surface, thereby performing jet detachment.

[0090] (Creating culture vessel 1) A Φ35 polystyrene dish (manufactured by Corning) was used as the base of the culture vessel. The culture vessel was softened by heating to 95°C, and then a mold (nickel) corresponding to the concave-convex pattern 1 was pressed against it to transfer the concave-convex pattern to the culture surface. The pressing was performed under the conditions of increasing the pressure to 40 MPa at 10 MPa / min, and then maintaining the pressure at 40 MPa for 3 minutes. After pressing, the dish was immediately cooled and released from the mold to obtain culture vessel 1. The obtained culture vessel 1 was washed and sterilized with ethanol before being used for culture.

[0091] (Creating culture vessels 2 to 30) Culture vessels 2 to 30 were produced in the same manner as culture vessel 1, except that the concave-convex pattern was changed to one corresponding to Table 2. A list of the culture vessels is shown in Table 2.

[0092] (Culture container 31) A Φ35 polystyrene dish (manufactured by Corning), a conventional culture vessel without a concave-convex pattern, was used.

[0093] [Table 2]

[0094] (Evaluation criteria for peelability) Cells were cultured under the same culture conditions in a conventional culture vessel (culture vessel 31) without a concave-convex pattern and in a culture vessel of the present disclosure with a concave-convex pattern, and the cells cultured under the same detachment conditions were then detached. The proportion of detached cells was taken as the detachment rate, and the detachment rate for each vessel was compared to evaluate detachment properties. The evaluation criteria were as follows: A: More than 180% of the detachment rate when using conventional culture vessels B: 150% to 180% of the detachment rate when using conventional culture vessels C: 120% to 150% of the detachment rate when using conventional culture vessels D: Less than 120% of the detachment rate when using conventional culture vessels

[0095] (Evaluation criteria for proliferation) Cells were cultured under the same culture conditions in a conventional culture vessel (culture vessel 31) without a concave-convex pattern and in a culture vessel of the present disclosure with a concave-convex pattern. The proliferation rate was defined as the ratio of the number of cells seeded to the number of cells after culture, and the proliferation rate in each culture vessel was compared to evaluate the proliferation potential. The evaluation criteria were as follows:

[0096] A: 90% or more of the proliferation rate when using conventional culture vessels B: 80% to less than 90% of the proliferation rate when using conventional culture vessels C: 70% to less than 80% of the proliferation rate when using conventional culture vessels D: Less than 70% of the proliferation rate when using conventional culture vessels

[0097] (Evaluation criteria for initial adhesion) A conventional culture vessel (culture vessel 31) without a concave-convex pattern and a culture vessel of the present disclosure with a concave-convex pattern were used, and cells were cultured under the same culture conditions and then detached under the same detachment conditions. The cells recovered by detachment were reseeded into a conventional culture vessel without a concave-convex pattern and left to stand for 1 hour. The percentage of cells that adhered after standing was taken as the initial adhesion rate, and the initial adhesion rates in each vessel were compared to evaluate the initial adhesion. The evaluation criteria were as follows:

[0098] A: The initial adhesion rate is 200% or more compared to the initial adhesion rate when using conventional culture vessels. B: Initial adhesion rate is 150% or more but less than 200% of the initial adhesion rate when using conventional culture vessels C: Initial adhesion rate is 95% or more but less than 150% of the initial adhesion rate when using conventional culture vessels D: Initial adhesion rate is less than 95% of the initial adhesion rate when using conventional culture vessels.

[0099] (Evaluation criteria for differentiation potential) Using a conventional culture vessel (culture vessel 31) without a concave-convex pattern and a culture vessel of the present disclosure with a concave-convex pattern, mesenchymal stem cells were cultured under the same culture conditions and then detached under the same detachment conditions. The cells recovered by detachment were differentiated into cartilage. The cartilage differentiation potential was evaluated by comparing the diameter of each cell mass after differentiation. The evaluation criteria were as follows:

[0100] A: The diameter of the obtained cell clusters is 130% or more of the diameter of the cell clusters obtained using conventional culture vessels. B: The diameter of the obtained cell clusters is 120% or more but less than 130% of the diameter of the cell clusters obtained using a conventional culture vessel. C: The diameter of the obtained cell clusters is 105% or more but less than 120% of the diameter of the cell clusters obtained using a conventional culture vessel. D: The diameter of the obtained cell clusters is less than 105% of the diameter of the cell clusters obtained using a conventional culture vessel.

[0101] (Example 1 and Comparative Example 1) CHO cells were cultured in culture vessel 1 in Example 1, and in culture vessel 31 in Comparative Example 1. Culturing was carried out under similar conditions using four culture vessels 1 in Example 1 and three culture vessels 31 in Comparative Example 1, and one of the culture vessels 1 was used for cross-sectional observation, and the cells in the remaining vessels were used to evaluate detachment, proliferation, and initial adhesion.

[0102] (Cross-section observation) In Example 1, the observed images of the cells and the uneven pattern were obtained by the above-mentioned "method for verifying the relationship between Lcp, Lf, and Lp," and Lcp, Lf, and Lcp / Lf and Lcp / Lp were calculated. Lcp / Lf was 1.8, and Lcp / Lp was 1.0.

[0103] (Evaluation of peeling and proliferation) After cell culture, the culture medium was removed from the culture vessel, washed with PBS(-), and then immersed in PBS(-) for 3 minutes. An ultrasonic transducer was then attached to the culture vessel, and ultrasonic vibrations were applied at an ambient temperature of 37°C. Ultrasonic vibrations were applied using a ring device as the transducer, with standing wave sweep vibrations (frequency 29-35 kHz, sweep period 1 second, voltage 30 V) for 5 minutes. After collection, the detached cells were counted using a hemocytometer, and the viability was calculated using a trypan blue staining method to distinguish viability. In addition, all cells that were not detached by ultrasonic vibration were detached from the culture vessel using a cell scraper, and the cell count was measured using a hemocytometer. The total number of cells detached by ultrasonic vibration and the number of cells detached by the cell scraper were combined to calculate the detachment rate using Equation 1, and the proliferation rate was calculated using Equation 2. The detachment rate and proliferation rate were calculated using three culture vessels 1 and three culture vessels 31, and the average values ​​for each vessel type were used.

[0104] Formula 1: Detachment rate (%) = Number of cells detached by ultrasonic vibration / Total number of cells x 100 Formula 2: Proliferation rate (%) = total number of cells / number of seeded cells x 100

[0105] The average detachment rate of cells detached from culture vessel 1 by ultrasonic vibration was 80.0%, which was 212.8% compared to when detached from culture vessel 31. The average proliferation rate of cells cultured in culture vessel 1 was 663%, which was 107.5% compared to when cultured in culture vessel 31. Furthermore, the survival rate of cells detached by ultrasonic vibration was 99% or higher in all cases.

[0106] (Evaluation of initial adhesion) The cells detached from the culture vessel 1 and the culture vessel 31 by ultrasonic vibration were 35,000 cells / cm 2The cells were seeded into a new culture vessel 31 so that the density was 100%. The cells were homogenized by gently shaking and left to stand for 1 hour in a CO2 incubator at 37°C. After standing, the medium was slowly sucked out of the culture vessel, and unadhered cells were collected and counted using a hemocytometer. The cells remaining in the culture vessel were then collected with a cell scraper and counted using a hemocytometer. The initial adhesion rate was calculated using the following formula 3. The initial adhesion rate was calculated using cells collected from three culture vessels each of culture vessel 1 and culture vessel 31, and the average value for each vessel type was used.

[0107] Equation 3: Initial adhesion rate (%) = number of cells attached in 1 hour / (number of unattached cells + number of cells attached in 1 hour) The average initial adhesion rate of the cells recovered from culture vessel 1 was 65%, which was 244% compared to the rate when recovered from culture vessel 31.

[0108] Examples 2 to 7 The detachment, proliferation, and initial adhesion were evaluated in the same manner as in Example 1, except that the culture vessel 1 was changed to one corresponding to Table 3. The results are shown in Table 3.

[0109] (Examples 8 to 33) The detachment and proliferation properties were evaluated in the same manner as in Example 1, except that the cell type, culture vessel, and conditions for applying ultrasonic vibration were changed to those shown in Table 3. The results are shown in Table 3.

[0110] (Comparative Example 2) The detachment and proliferation properties were evaluated in the same manner as in Example 1, except that the cell type and culture vessel were changed to those corresponding to Table 3, and the jet detachment device shown in Figures 7A and 7B was used instead of applying ultrasonic vibration. The results are shown in Table 3.

[0111] (Comparative Examples 3 to 7) The detachment and proliferation properties were evaluated in the same manner as in Example 1, except that the cell type, culture vessel, and conditions for applying ultrasonic vibration were changed to those shown in Table 3. The results are shown in Table 3.

[0112] [Table 3]

[0113] (Evaluation of cartilage differentiation potential in Example 15) In Example 15, the differentiation potential into cartilage was evaluated using cells detached by ultrasonic vibration. Differentiation into cartilage was performed by preparing pellets of 200,000 cells of each type recovered from each culture vessel and culturing them in a 15 mL polypropylene tube for 504 hours. The culture medium used was a chondrogenesis medium (Promocell) supplemented with 1% TGF-β3, and the medium was changed once every three days. The differentiated cell clusters were removed, and images were taken using a phase-contrast microscope. The diameter of the cell clusters was measured from the obtained images, and the differentiation potential was evaluated according to the above-mentioned evaluation criteria. Cell clusters were measured using cells recovered from three culture vessels each, culture vessel 1 and culture vessel 31, and the average value for each vessel type was used.

[0114] When the cells were recovered from culture vessel 1 and subjected to chondrocyte differentiation, the diameter of the cell clusters was 1300 μm, which was 140% of the diameter of the cell clusters recovered from culture vessel 31.

[0115] (Evaluation of cartilage differentiation potential in Examples 16 to 18) In Examples 16 to 18, the evaluation of chondrocyte differentiation potential was carried out in the same manner as in Example 15, except that the culture vessels were changed to those shown in Table 4. The results are shown in Table 4.

[0116] [Table 4]

[0117] Thus, among the culture vessels disclosed herein, cells cultured in a culture vessel having a curved concave-convex pattern and then detached by applying ultrasonic vibrations were shown to have a higher ability to differentiate into cartilage than cells detached from a culture vessel without a curved concave-convex pattern. The inventors believe that the reason for this is that the curved concave-convex pattern creates a scaffold environment similar to that in vivo, possibly increasing the production of extracellular matrix. Using cells recovered according to the present disclosure not only makes differentiation more efficient, but also has the potential to efficiently supply extracellular matrix, which is valuable as a research subject.

[0118] The disclosure of this embodiment includes the following configurations and methods. (Method 1) Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell detachment method comprising a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibrations to the cells. (Method 2) The pitch P of the concave-convex pattern is 100nm≦P≦2000nm The cell detachment method according to Method 1, characterized in that the above-mentioned condition is satisfied. (Method 3) The uneven pattern height difference HD of the uneven pattern is 20nm≦HD≦400nm The cell detachment method according to Method 1 or 2, characterized in that the above condition is satisfied. (Method 4) 4. The cell detachment method according to any one of Methods 1 to 3, wherein the concave-convex pattern has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied. (Method 5) In the peeling step, Lcp / Lf≧1.2 The cell detachment method according to any one of Methods 1 to 4, wherein ultrasonic vibration is generated by the vibration means in a state where the above condition is satisfied. (Method 6) In the peeling step, when the length of the contour of the culture surface in the measurement region is Lp, Lcp / Lp≧0.8 The cell detachment method according to any one of Methods 1 to 5, wherein ultrasonic vibration is generated by the vibration means in a state where the above condition is satisfied. (Method 7) 7. The cell detachment method according to any one of Methods 1 to 6, wherein in the detachment step, the vibration means applies vibration from the back side of the culture surface. (Method 8) Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibration to the cells, The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, A cell detachment method characterized in that the culture surface has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied. (Method 9) The uneven pattern height difference HD of the uneven pattern is 20nm≦HD≦400nm The cell detachment method described in Method 8, characterized in that the above-mentioned condition is satisfied. (Method 10) 10. The cell detachment method according to any one of Methods 8 and 9, wherein in the detachment step, the vibration means applies vibration from the back side of the culture surface. (Method 11) a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell production method characterized by transmitting the ultrasonic vibration to the cells, thereby detaching the cells from the culture surface. (Method 12) a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; The ultrasonic vibration is transmitted to the cells, thereby detaching the cells from the culture surface. The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, Furthermore, the cell production method is characterized in that the concave-convex pattern has at least one of a) and b). A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied. (Configuration 1) A cell culture vessel having a periodic uneven pattern on the culture surface, The pitch P of the uneven pattern on the culture surface is 100 nm ≦ P ≦ 2000 nm is satisfied, A cell characterized in that the concave-convex pattern has at least one of a) and b). Culture container. A) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied. (Configuration 2) The uneven pattern height difference HD of the uneven pattern is 20nm≦HD≦400nm The cell culture vessel according to configuration 1, characterized in that the above conditions are satisfied. (Configuration 3) The culture surface is The top surface and the side surface are connected via a curved surface portion, and The bottom surface and the side surface are connected via a curved section. 3. The cell culture vessel according to claim 1 or 2, wherein at least one of the above is satisfied. (Configuration 4) 4. The cell culture vessel according to any one of configurations 1 to 3, wherein the convex or concave portion has a cylindrical or truncated conical shape. [Explanation of symbols]

[0119] 10 Culture surface 11 cells 100 uneven patterns 101 Convex part 102 recess 421 Culture vessel

Claims

1. Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell detachment method comprising a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibrations to the cells.

2. The pitch P of the concave-convex pattern is 100 nm≦P≦2000 nm The cell detachment method according to claim 1, wherein the following is satisfied:

3. The uneven pattern height difference HD of the uneven pattern is 20 nm≦HD≦400 nm The cell detachment method according to claim 1, wherein the following is satisfied:

4. 4. The cell detachment method according to claim 1, wherein the concave-convex pattern has at least one of a) and b). a) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

5. In the peeling step, Lcp / Lf≧1.2 4. The cell detachment method according to claim 1, wherein the ultrasonic vibration is generated by the vibration means in a state where the above condition is satisfied.

6. In the peeling step, when the length of the contour of the culture surface in the measurement region is Lp, Lcp / Lp≧0.8 4. The cell detachment method according to claim 1, wherein the ultrasonic vibration is generated by the vibration means in a state where the above condition is satisfied.

7. 4. The cell detachment method according to claim 1, wherein in the detachment step, the vibration means applies vibration from the back side of the culture surface.

8. Using a culture vessel having a periodic uneven pattern on the culture surface, A cell detachment method for detaching cells adhered to the culture surface from the culture surface, comprising: generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; a detachment step of detaching the cells from the culture surface by transmitting the ultrasonic vibration to the cells, The pitch P of the uneven pattern on the culture surface is 100 nm≦P≦2000 nm is satisfied, A cell detachment method characterized in that the culture surface has at least one of a) and b). a) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

9. The uneven pattern height difference HD of the uneven pattern is 20 nm≦HD≦400 nm The cell detachment method according to claim 8, wherein the following is satisfied:

10. 10. The cell detachment method according to claim 8, wherein in the detachment step, the vibration means applies vibration to the back side of the culture surface.

11. a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, When an observation image of a cross section perpendicular to the culture surface is obtained, the length Lcp of the contour of the cell on the culture surface side in the measurement region of the observation image; The length Lf of the measurement region in a direction parallel to the culture surface is Lcp>Lf In a state where generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; A cell production method characterized by transmitting the ultrasonic vibration to the cells, thereby detaching the cells from the culture surface.

12. a culturing step of culturing cells in a culture vessel having a periodic uneven pattern on the culture surface; A cell production method comprising a detachment step of detaching the cells adhered to the culture surface from the culture surface, In the peeling step, generating ultrasonic vibrations in a vibration means that is arranged so as to be able to impart vibrations to the culture vessel; The ultrasonic vibration is transmitted to the cells, thereby detaching the cells from the culture surface. The pitch P of the uneven pattern on the culture surface is 100 nm≦P≦2000 nm is satisfied, Furthermore, the cell production method is characterized in that the concave-convex pattern has at least one of a) and b). a) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

13. A cell culture vessel having a periodic uneven pattern on the culture surface, The pitch P of the uneven pattern on the culture surface is 100 nm≦P≦2000 nm is satisfied, A cell culture vessel characterized in that the concave-convex pattern has at least one of a) and b). a) a plurality of periodically arranged convex portions protruding from a reference surface of the concave-convex pattern, the plurality of convex portions having a height H and being arranged at intervals I; The relationship 20 nm≦H≦400 nm and H / I≦1.1 are satisfied. a) a plurality of recesses recessed from a reference surface of the concave-convex pattern and periodically arranged, each having a depth D and a hole diameter S; The relationship 20 nm≦D≦400 nm and D / S≦1.1 are satisfied.

14. The uneven pattern height difference HD of the uneven pattern is 20 nm≦HD≦400 nm 14. The cell culture vessel according to claim 13, wherein the above condition is satisfied.

15. The culture surface is The top surface and the side surface are connected via a curved surface portion, and The bottom surface and the side surface are connected via a curved section.

14. The cell culture vessel according to claim 13, wherein at least one of the following conditions is satisfied:

16. 14. The cell culture vessel according to claim 13, wherein the convex or concave portion has a cylindrical or truncated conical shape.

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