Cell culture container and cell culture method using same

Abstract

Provided is a cell culture container that ensures a broad cell adhesion surface for adhesion and proliferation of cells inside the container. This cell culture container comprises a bottomed container body 1 that has a polygonal, columnar internal space, a polygonal, columnar first cylindrical body 2 that is provided inside the container body 1 so as to be coaxial with the container body 1, a lid body 4 that is attached to an axial end part of the container body, a cap 5 that is attached to a cylindrical neck part of the lid body 4, and first spacers 21, 22 that are corner parts of an outer circumferential surface of the first cylindrical body 2 at an axial end part of the first cylindrical body 2. The first spacers 21, 22 are fitted into corner parts of an inner circumferential surface of the container body 1.

Description

Cell culture vessel and cell culture method using the same

[0001] The present invention relates to a cell culture vessel.

[0002] The vessels commonly used for cell culture are multi-well plates or T-flasks. Culture vessels based on T-flasks have been developed by various manufacturers and are used for large-scale cell culture.

[0003] US2017 / 0029756 US Patent 5,010,013 US Patent 4,317,886

[0004] However, conventional cell culture vessels are focused on research culture, and among them, roller bottles are the most practical for mass production culture.

[0005] Patent Document 1 proposes an octagonal column-type cell culture vessel in which adherent mammalian cells grow only on the inner surface of the vessel. The inner surface of the vessel functions as a scaffold for cell growth and attachment. The area of ​​the inner surface on which mammalian cells attach increases only when the vessel size is enlarged. Therefore, although mass-production culture is possible by increasing the diameter of the vessel bottle, there is a problem in that the bottle becomes too large.

[0006] In response to these issues, Patent Document 2 proposes increasing the internal surface area by structuring the container in a bellows or accordion shape. However, this method is also insufficient to support large-scale culture and has its own drawbacks. Specifically, while this structure can increase the internal surface area of ​​the container, it does not allow cells to attach efficiently. Cells are suspended in the medium within the container, but they fall due to gravity while the container is rotated for culture. The bellows or accordion structure is a structure in which peaks and valleys are connected to form curved surfaces, and cells fall vertically, which may result in the accumulation of cells at the bottom of the bellows. As a result, cells cannot grow in the accumulated areas. Therefore, even if the internal surface area is increased by structuring the container in a bellows or accordion shape, the effective area for cell attachment is actually limited.

[0007] Furthermore, Patent Document 3 describes an invention relating to a roller bottle having a large internal surface area and multiple inner surfaces. However, because the multiple inner surfaces provided inside the roller bottle are in contact with the bottom of the bottle, it is difficult to collect the cells cultured on the multiple inner wall surfaces of the bottle in one place and extract the cells, resulting in a low cell recovery rate.

[0008] Therefore, an object of the present invention is to provide a cell culture vessel that can ensure a wide cell adhesion surface for cells to attach and grow within the vessel, and that can increase the cell recovery rate.

[0009] The cell culture vessel disclosed in the present application comprises a bottomed vessel body having a polygonal pillar-shaped internal space, a plurality of polygonal pillar-shaped cylinders arranged coaxially with the vessel body inside the vessel body, a lid attached to the axial end of the vessel body, a cap attached to the cylindrical neck portion of the lid, and spacers provided at corners of the outer peripheral surfaces of the plurality of polygonal pillar-shaped cylinders and at the axial end of the plurality of polygonal pillar-shaped cylinders, wherein the spacers are fitted into corners of the inner peripheral surface of the vessel body or corners of the inner peripheral surface of the cylinders, and the bottom of the vessel body is conical or polygonal pyramidal.

[0010] According to the above configuration, by placing multiple polygonal cylindrical bodies inside the vessel body, both the inner and outer surfaces of the cylinders can be immersed in the culture medium, thereby ensuring a wide cell adhesion surface for cells to adhere and grow in the vessel. Furthermore, by providing the spacers at the corners of the outer surface of the cylinders and at the axial end of the cylinders, it is possible to reduce the occurrence of uneven cell adhesion due to liquid flow during rotational culture.

[0011] In the cell culture vessel, the plurality of polygonal cylindrical bodies are a first cylindrical body and a second cylindrical body, the second cylindrical body is provided inside the first cylindrical body and is arranged coaxially with the first cylindrical body, and further includes a first spacer provided at a corner of the outer peripheral surface of the first cylindrical body and at an axial end of the first cylindrical body, and a second spacer provided at a corner of the outer peripheral surface of the second cylindrical body and at an axial end of the second cylindrical body, and the first spacer may be fitted into a corner of the inner peripheral surface of the vessel body, and the second spacer may be fitted into a corner of the inner peripheral surface of the first cylindrical body.

[0012] According to the above configuration, by placing the second cylindrical body inside the first cylindrical body, both the inner and outer surfaces of the second cylindrical body can be immersed in the culture medium, thereby ensuring a larger cell adhesion surface for cells to adhere and grow in the container. Furthermore, by providing the second spacer at a corner of the outer surface of the second cylindrical body and at the axial end of the second cylindrical body, it is possible to reduce the occurrence of uneven cell adhesion due to liquid flow during rotary culture.

[0013] In the cell culture vessel, the spacer may have a V-shaped end that fits into a corner of the inner circumferential surface of the vessel body or a corner of the inner circumferential surface of the cylindrical body.

[0014] According to the above configuration, it is possible to fix the cylindrical body at a fixed distance from the container body in the internal space of the container body.

[0015] In the cell culture vessel, the first spacer may have a V-shaped end that fits into a corner of the inner surface of the vessel body, and the second spacer may have a V-shaped end that fits into a corner of the inner surface of the first cylindrical body.

[0016] According to the above configuration, it is possible to fix the second cylindrical body at a fixed distance from the first cylindrical body in the internal space of the container body.

[0017] In the cell culture vessel, the vessel body, the first cylindrical body, and the second cylindrical body are all hexagonal prism-shaped, the first spacers are provided at three corners of the outer peripheral surface of the first cylindrical body and at equal intervals on the axial end of the first cylindrical body, the second spacers are provided at three corners of the outer peripheral surface of the second cylindrical body and at equal intervals on the axial end of the second cylindrical body, and the second spacers are fitted inside the corners of the outer peripheral surface of the first cylindrical body where the first spacers are not provided, and may be used to detach, separate, and disperse target cells adhering to the inner surface of the vessel body, both the inner and outer surfaces of the first cylindrical body, and both the inner and outer surfaces of the second cylindrical body.

[0018] According to the above configuration, by providing spacers alternately at the corners of the first cylindrical body and the second cylindrical body, it is possible to maintain strength with the minimum number of spacers.

[0019] In the cell culture vessel, the longitudinal length of the first spacer in the first cylindrical body may be smaller than the longitudinal length of the first cylindrical body, and a passage at a fixed interval may be secured between the inner surface of the vessel body and the outer surface of the first cylindrical body for the flow of cell culture medium.

[0020] According to the above configuration, a passage is formed at regular intervals between the inner surface of the container body and the outer surface of the first cylindrical body for the flow of cell culture medium, thereby reducing the obstruction of the flow of culture medium and the uniform flow of cell suspension when culturing cells, and creating a structure that makes it easy for the cell suspension to flow to the bottom when recovering the cultured cells in the cell detachment process.

[0021] In the cell culture vessel, a support that supports the second cylindrical body when the cell culture vessel is placed upright is provided at the axial end of the first cylindrical body on the bottom side of the vessel body, and a groove into which the support fits may be provided at the axial end of the second cylindrical body on the bottom side of the vessel body.

[0022] According to the above configuration, the first cylindrical body and the second cylindrical body can be reliably fixed in the internal space of the container body.

[0023] In the cell culture vessel, at least two linear marks extending in the axial direction of the vessel body may be provided on at least one flat surface of the outer surface of the vessel body.

[0024] According to the above configuration, by physically indicating the imaging start point and the focus adjustment start point with two linear marks, it is possible to perform microscopic imaging of the same area even if the cell culture vessel is moved without having to place the cell culture vessel stationary in the imaging device. As a result, it is possible to accurately determine the state of the cells and to decide whether to continue cell culture.

[0025] In the cell culture vessel, the cap comprises a cap body and a first valve attached to the cap body that can supply and discharge fluid to the internal space of the vessel body, and the first valve may have a first valve body made of an elastic material that has a slit portion that opens when a supply and discharge tool is inserted.

[0026] According to the above configuration, the cell culture vessel can inject gas or liquid into the cell culture vessel while preventing contamination by foreign matter (bacteria, etc.), and can also replace the gas inside the cell culture vessel.

[0027] In the cell culture vessel, the cap may further include a second valve attached to the cap body that can supply and discharge fluid to and from the internal space of the vessel body, and the second valve may have a second valve body made of an elastic material that has a slit portion that opens when a supply and discharge tool is inserted.

[0028] According to the above configuration, gas or liquid can be introduced from the outside through one valve, and gas or liquid can be extracted from the cell culture vessel through the other valve.

[0029] The cell culture method disclosed in the present application is a cell culture method using any of the cell culture vessels described above, and includes a culture step of culturing cells in the cell culture vessel by rotating the cell culture vessel in a horizontal position around the axial direction of the cell culture vessel at a predetermined rotation speed; a confirmation step of microscopically inspecting the state of the cells in the cell culture vessel after each predetermined culture period; an exchange step of exchanging the liquid medium and / or gas in the cell culture vessel via a valve attached to the cap after each predetermined culture period; and a recovery step of recovering target cells adhered to the inner surface of the vessel body and both the inner and outer surfaces of the cylindrical body after each predetermined culture period.

[0030] According to the above configuration, cell culture can be continuously performed in a closed system, which enables mass production of useful cells while preventing contamination by foreign substances (bacteria, etc.). Furthermore, regular medium and gas exchange can be performed automatically via valves, which makes cell culture more efficient.

[0031] The cell culture vessel disclosed in the present application comprises a bottomed vessel body having a polygonal internal space, a polygonal cylinder arranged coaxially with the vessel body inside the vessel body, a lid attached to the axial end of the vessel body, a cap attached to the neck portion of the lid, and a spacer provided at a corner of the outer peripheral surface of the polygonal cylinder and at the axial end of the polygonal cylinder, the spacer being fitted into a corner of the inner peripheral surface of the vessel body, and a receiving portion abutting against the spacer being provided at the bottom of the vessel body.

[0032] According to the above configuration, by placing a cylindrical body inside the container body, both the inner and outer surfaces of the cylindrical body can be immersed in the culture medium, thereby ensuring a wide cell adhesion surface for cells to attach and grow within the container. Furthermore, by providing the spacers at the corners of the outer surface of the cylindrical body and at the axial end of the cylindrical body, the occurrence of uneven cell adhesion due to liquid flow during rotary culture can be reduced. Furthermore, by providing a receiving portion at the bottom of the container body that abuts the spacer, the spacer can be prevented from falling toward the bottom of the cylindrical body, and a space for storing cells with a predetermined gap between the bottom of the container body and the spacer can be formed. As a result, cells cultured on both the inner and outer surfaces of the cylindrical body can be easily collected in this space and removed to the outside, thereby increasing the cell recovery rate.

[0033] In the cell culture vessel, a single space may be formed between the bottom of the vessel body and the axial lower end of the polygonal prism-shaped cylindrical body.

[0034] According to the above configuration, cells cultured on the inner surface of the container body and on both the inner and outer surfaces of the cylindrical body can be collected and recovered in the space formed between the bottom of the container body and the lower axial end of the polygonal cylindrical body.

[0035] Furthermore, in the cell culture vessel, a groove into which a part of the spacer fits may be provided on the side of the receiving portion that abuts against the spacer.

[0036] According to the above configuration, since a part of the spacer fits into the groove provided in the receiving part, the positioning of the cylinder inside the container body is facilitated, which improves the efficiency of assembling the cylinder to the container body and also strengthens the fixing force of the cylinder to the container body. As a result, the attachment strength of the cylinder to the container body can be ensured without providing spacers at all corners of the cylinder, thereby increasing the degree of freedom in design.

[0037] In the cell culture vessel, the spacer has an end that is rectangular in top view and fits into a corner of the inner surface of the vessel body, and the corner of the inner surface of the vessel body may have a recessed groove into which the end fits.

[0038] According to the above configuration, the end of the spacer fits into a recessed groove at the corner of the inner surface of the container body, making it easier to position the cylindrical body inside the container body, thereby improving the efficiency of assembling the cylindrical body to the container body.

[0039] According to the present invention, a wide cell adhesion surface for cells to attach and grow in a cell culture vessel can be secured, and the cell recovery rate can be increased.

[0040] FIG. 1 shows a front view of a cell culture vessel according to one embodiment. FIG. 2 shows an exploded perspective view of a cell culture vessel according to one embodiment. FIG. 3 shows a cross-sectional view of the cell culture vessel along line P-P in FIG. 1. FIG. 4 shows a top view of the cell culture vessel according to one embodiment. FIG. 5 shows a cross-sectional view of the cell culture vessel along line Q'-Q' in FIG. 1. FIG. 6 shows a cross-sectional view of the cell culture vessel along line Q-Q in FIG. 1. FIG. 7(a) shows a top view of a first cylindrical body of a cell culture vessel according to one embodiment. FIG. 7(b) shows a bottom view of a first cylindrical body of a cell culture vessel according to one embodiment. FIG. 8(a) shows a top view of a second cylindrical body of a cell culture vessel according to one embodiment. FIG. 8(b) shows a bottom view of a second cylindrical body of a cell culture vessel according to one embodiment. FIG. 9 shows a side view of a cell culture vessel according to one embodiment. FIG. 10 shows a perspective view of a first cylindrical body of a cell culture vessel according to one embodiment. FIG. 11 shows a perspective view of a second cylindrical body of a cell culture vessel according to one embodiment. FIG. 12 shows a cross-sectional view of the cell culture vessel taken along line P'-P' in FIG. 9. FIG. 13 shows a cross-sectional view of the cell culture vessel taken along line Q-Q in FIG. 9. FIG. 14 shows a cross-sectional view of the cell culture vessel taken along line P-P in one embodiment. FIG. 15 shows a cross-sectional view of the cell culture vessel taken along line Q-Q in one embodiment. FIG. 16 shows a cross-sectional view of the cell culture vessel taken along line Q-Q in one embodiment. FIG. 17 shows a cross-sectional view of the cell culture vessel taken along line Q-Q in one embodiment. FIG. 18 shows an example of a cell culture vessel in which two linear marks extending in the axial direction of the vessel body are provided on the plane of the vessel body of the cell culture vessel according to one embodiment. FIG. 19 shows a perspective view of a cap of a cell culture vessel according to one embodiment. FIG. 20 shows a top view of a cap of a cell culture vessel according to one embodiment. FIG. 21 shows a front view of a cap of a cell culture vessel according to one embodiment. Fig. 22 shows an exploded perspective view of a valve attached to a cap of a cell culture vessel according to one embodiment. Fig. 23 shows a cross-sectional perspective view of the internal structure of a valve attached to a cap of a cell culture vessel according to one embodiment. Fig. 24 is a schematic diagram showing a state in which a syringe is connected via a valve attached to a cap of a cell culture vessel according to one embodiment.FIG. 25 is a schematic diagram illustrating a state in which a syringe is connected via a Luer lock needle hub to a valve attached to a cap of a cell culture vessel according to an embodiment. FIG. 26 is a schematic diagram illustrating a state in which a rubber tube is connected via a Luer lock connector to a valve attached to a cap of a cell culture vessel according to an embodiment. FIG. 27 is a front view of a modified cap of a cell culture vessel according to an embodiment. FIG. 28 is a flow chart illustrating steps of a cell culture method and a cell recovery method using a cell culture vessel according to an embodiment. FIG. 29 is an explanatory diagram illustrating a gripping device attached to a cell culture vessel according to an embodiment. FIG. 30 is a flow chart illustrating steps of a cell culture method and a method for producing a supernatant fluid using a cell culture vessel according to an embodiment. FIG. 31 is a cross-sectional view of a cell culture vessel according to a modified (first modified) embodiment. FIG. 32 is a top view of the vessel body of FIG. 31 formed from a transparent material, with a cylindrical body not inserted inside the vessel body. FIG. 33 is a partial cross-sectional view of a cell culture vessel according to a modified (second modified) embodiment. Fig. 34 shows a partial cross-sectional view of the cell culture vessel taken along line R-R in Fig. 33. Fig. 35 shows a partial cross-sectional view of the cell culture vessel of a modified example (third modified example) of one embodiment. Fig. 36 shows a cross-sectional view of the cell culture vessel of the third modified example.

[0041]

[0023] The embodiments will be described in more detail below with reference to the accompanying drawings. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways, all without departing from the spirit and scope of the present disclosure.

[0042] The drawings and description are illustrative and not restrictive. Like reference numerals refer to like elements throughout the specification.

[0043] The size and thickness of each component shown in the drawings are arbitrarily shown for better understanding and ease of explanation. Unless otherwise stated, the present disclosure is not limited thereto. For example, when specific size and positional relationships between elements are described with reference to drawings, the drawings are used to illustrate such size and positional relationships. The thickness of layers, plates, panels, regions, etc. may be exaggerated for clarity unless otherwise stated. For example, when specific thickness relationships between elements are described with reference to drawings, the drawings are used to illustrate such thickness relationships. The thickness of some layers or regions may be exaggerated.

[0044] The singular forms include the plural forms unless the context clearly indicates otherwise.

[0045] In the specification and claims, the term "and / or" is intended for purposes of meaning and interpretation to include any combination of the terms "and" and "or." For example, the phrase "A and / or B" shall be interpreted to mean "A, B, or A and B."

[0046] In the specification and claims, the phrase "at least one" is intended to mean and be interpreted as "at least one selected from a group." For example, the phrase "at least one of A and B" is to be interpreted as meaning "A, B, or A and B."

[0047] Terms such as "first," "second," and the like are used only to describe various components and are not limiting of these components. These terms are used merely to distinguish between different components. For example, a first component may be referred to as a second component, and similarly, a second component may be referred to as a first component, without departing from the scope of the present disclosure.

[0048] When an element, such as a layer, plate, panel, region, or substrate, is described as being "on" another element, it may be directly on the other element, or there may be intervening elements. Conversely, when a first element is described as being "directly on" a second element, there are no intervening elements. Throughout the specification, an element that is "on" an object is understood to be disposed above or below the object, and does not necessarily refer to "on" relative to the opposite direction of gravity.

[0049] For example, the spatially relative terms "below" or "above" can be used to describe the relationship of one element or component to another, as shown in the drawings. Spatially relative terms are intended to encompass other orientations during use or operation in addition to the orientation depicted in the drawings. For example, if a device depicted in the drawings is inverted, a device that was positioned below another device may now be positioned "above" the other device. Thus, the exemplary term "below" can encompass lower and upper positions. Devices may be oriented in other ways, and the spatially relative terms may be interpreted differently depending on the orientation.

[0050] In this specification, when an element (or region, layer, portion, etc.) is described as being "connected" or "coupled" to another element, it may be directly positioned, connected, or coupled, or there may be elements disposed therebetween.

[0051] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Furthermore, terms defined in commonly used dictionaries should be interpreted in a manner consistent with the meaning in the context of the relevant art and / or this specification, and should not be interpreted in an idealized or overly formal sense unless expressly defined as such in this specification. Embodiment

[0052] FIG. 1 shows a front view of a cell culture vessel according to one embodiment. FIG. 2 shows an exploded perspective view of a cell culture vessel according to one embodiment. FIG. 3 shows a cross-sectional view of the cell culture vessel along line P-P in FIG. 1. FIG. 4 shows a top view of the cell culture vessel according to one embodiment. FIG. 5 shows a cross-sectional view of the cell culture vessel along line Q'-Q' in FIG. 1. FIG. 6 shows a cross-sectional view of the cell culture vessel along line Q-Q in FIG. 1. In FIG. 7, FIG. 7(a) shows a top view of a first cylindrical body of a cell culture vessel according to one embodiment. FIG. 7(b) shows a bottom view of the first cylindrical body of a cell culture vessel according to one embodiment. In FIG. 8, FIG. 8(a) shows a top view of a second cylindrical body of a cell culture vessel according to one embodiment. FIG. 8(b) shows a bottom view of the second cylindrical body of a cell culture vessel according to one embodiment.

[0053] 1 to 6, a cell culture vessel according to one embodiment includes a vessel body 1 with a bottom, a first cylindrical body 2, a second cylindrical body 3, a lid body 4, a bottom 11, and a cap 5.

[0054] The container body 1 has a polygonal prism shape with an empty interior space. Therefore, the container body 1 is composed of multiple rectangular flat panels 1A. In this embodiment, the container body 1 is composed of six rectangular flat panels 1A. In this embodiment, the container body 1 is hexagonal prism-shaped, but is not limited thereto. In some embodiments, the container body 1 may have various polygonal prism shapes, such as a pentagonal prism or an octagonal prism. The container body 1 may be made of various materials, such as plastic or glass, and may be transparent, colored, or opaque. These materials may be selected to ensure transparency. However, the container body 1 may also be made of an opaque resin that can block light depending on the culture conditions. Furthermore, a material that selectively absorbs or transmits light in a specific wavelength range may also be used. As described above, the container body 1 may be hexagonal prism-shaped or polygonal prism-shaped, but it is necessary to configure it to have at least one flat panel 1A. This allows the culture state of the cells to be observed by observing the inner surface to which the cells adhere with a microscope R, and determining whether to continue the culture. If the container body 1 is cylindrical, it is difficult to observe the culture state of the cells with the microscope R during the culture process because there is no flat panel 1A.

[0055] Although the present invention is embodied as an embodiment employing a microscope, an inverted microscope is suitable for observing cell culture when the culture state is continued while observing the bottle serving as the vessel body 1 using the microscope R. In a method using the microscope R as an inverted microscope, an eyepiece lens is placed on the front side of the bottle serving as the culture vessel, and an objective lens is placed on the back side of the bottle, and observation is required by sandwiching the object between the two lenses. Therefore, when attempting to automate culture using multiple culture vessels of this embodiment, areas for placing lenses on both the front and back sides of the bottle are required, which poses a problem of increasing the size of the culture device for automated culture using multiple culture vessels.

[0056] Therefore, the present inventors have discovered that using a digital camera such as a CCD camera or CMOS camera, which can magnify and display the captured image itself, is extremely effective in terms of miniaturization when assembling an automatic culture device. This is because, in this method of monitoring the culture state using a digital camera, the light source required for capturing and observing the image can be integrally provided in the same position as the digital camera. As shown in Figures 13 and 14 of Japanese Patent Application Laid-Open No. 2022-23903, an apparatus for monitoring such a culture state using a digital camera comprising a CCD sensor, a CMOS sensor, or a line sensor is configured such that the digital camera includes a semiconductor sensor comprising a CCD sensor, a CMOS sensor, or a line sensor, and is integrally configured with the light source. Therefore, such a digital camera can be provided on either the front or back side of the bottle, thereby enabling miniaturization. Such monitoring devices integrated with a light source are well known, and for example, as such a digital camera, it is preferable to use the Incubation Monitoring System CM manufactured by Evident (formerly Olympus Corporation) or the Cell Recorder manufactured by Cytronix.

[0057] When using such a digital camera to observe the culture state of the multilayer culture vessel of the present invention, the focus position of the digital camera can be appropriately changed to appropriately focus the first cylindrical body 2 and the second cylindrical body 3 on the culture areas on the front and back surfaces of the multiple rectangular flat panels 2A and 3A, thereby enabling the culture state of the first cylindrical body 2 and the second cylindrical body 3 to be observed on the culture areas on the front and back surfaces of the flat panels 2A and 3A.

[0058] Next, we will explain why it is preferable to observe the culture state of each layer individually by appropriately changing the focal position of the digital camera to observe the culture state on the front and back surfaces of flat panel 2A and flat panel 3A of the multi-layered bottle-shaped culture vessel in this embodiment.

[0059] When culturing cells, the first step in culturing cells is seeding, in which the original cells are placed in a culture vessel together with a culture medium.

[0060] Because the culture vessel of the present invention has multiple cylindrical bodies (first cylindrical body 2, second cylindrical body 3), the behavior of the cell suspension of seeded cells differs depending on whether the cell suspension contacts the inner circumferential surface of a cylindrical body with a concave outer shape (first cylindrical body 2, second cylindrical body 3) or the outer circumferential surface of a cylindrical body with a convex outer shape (first cylindrical body 2, second cylindrical body 3). To non-invasively measure the cell count and confluency rate more accurately, it is necessary to observe all cell adhesion surfaces.

[0061] In a multi-layered bottle-shaped culture vessel such as that of the present invention, it is possible to grasp the culture state by more accurately monitoring the cell adhesion surface of each layer, and it is possible to calculate and determine the appropriate timing for detaching and dispersing expanded cells.

[0062] When using a digital camera such as that shown in Figures 13 and 14 of the above-mentioned Patent Publication No. 2022-023903, the distance between the cell adhesion surface to be observed and the opposing surface is too great in a conventional single-phase octagonal bottle, so it is necessary to ensure sufficient illuminance from the light source irradiating the cell adhesion surface to be observed.However, with the culture vessel (multilayer bottle-shaped vessel) described in the embodiment of the present invention, the distance from the opposing surface to generate reflected light is sufficiently reduced, and sufficient illuminance can be ensured to facilitate observation within a range that does not cause phototoxicity, without increasing the output of the light source.

[0063] Furthermore, the following advantages can be pointed out from the miniaturization of the aforementioned culture device. Specifically, when observing cultures in culture vessels, two methods are conceivable: observing them inside the incubator, or observing them outside the incubator and then returning them to the incubator after observation. In such cases, a compact automated culture device can be easily applied to either method, thereby offering the advantage of increased system design flexibility when constructing a cell culture system using the automated culture device. For example, the inventor's cell culture system concept for an automated culture device is one in which an arm, centrifuge, and imaging device are placed inside a safety cabinet, and multiple culture vessels are cultured simultaneously. Multiple culture vessels are processed simultaneously in a single device, and upon completion of the culture, they are packaged and discharged in the final automated packaging. Rather than proceeding through multiple separate devices with transfers, the culture process is envisioned to remain within the same clean space throughout the entire process.

[0064] As shown in FIG. 1 , the container body 1 is connected to a bottom 11 at its lower end. The conical bottom 11 may be conical or pyramidal, and functions as a space M in which cultured cells cultured on both the inner and outer surfaces of the first cylindrical body 2 and the second cylindrical body 3 are collected and stored. The lower side of the container body 1 is closed by the bottom 11. The bottom 11 may be made of various materials such as plastic or glass, and may be transparent, colored, or opaque. The bottom 11 may be formed integrally with the container body 1.

[0065] As shown in FIG. 3 , first spacers 21 and 22 are provided at the corners (2B and 2C) of the outer peripheral surface of the first cylindrical body 2, at the upper axial end (corner 2B side) and lower axial end (corner 2C side) of the first cylindrical body 2. Furthermore, second spacers 31 are provided at the corners (3B) of the outer peripheral surface of the second cylindrical body 3, at the upper axial end (corner 3B side) of the second cylindrical body 3. When the longitudinal lengths of the first spacers 21 and 22 in the first cylindrical body 2 are h and h', and the longitudinal length of the first cylindrical body 2 is H, the first spacers 21 and 22 are provided so that h and h' are sufficiently smaller than H. Therefore, a passage S with a constant gap L can be secured between the inner surface of the vessel body 1 and the outer surface of the first cylindrical body 2 for cell flow during rotational culture. Furthermore, a constant gap L can be secured between the inner surface of the vessel body 1 and the outer surface of the first cylindrical body 2. In this way, by providing the first spacers 21 and 22, it is possible to avoid impeding the fluidity of the cells during culture.

[0066] Furthermore, the diameter of the container body 1 is narrowed to form a conical portion, and a space M for storing cells is formed with a predetermined gap between the bottom 11 of the container body 1 and the first spacer 22. As a result, cells cultured on both the inside and outside of the cylindrical body can be easily collected in the space and removed to the outside, thereby increasing the cell recovery rate.

[0067] 5 and 6, the first cylindrical body 2 and the second cylindrical body 3 are polygonal pillars with empty internal spaces, and each has a plurality of rectangular flat panels 2A and 3A. In this embodiment, the first cylindrical body 2 and the second cylindrical body 3 each have six rectangular flat panels. The container body 1, the first cylindrical body 2, and the second cylindrical body 3 are fixed at a fixed interval.

[0068] The first cylindrical body 2 and the second cylindrical body 3 share the same rotation axis X as the container body 1. In other words, the first cylindrical body 2 and the second cylindrical body 3 and the container body 1 are coaxial cylinders. Here, "coaxial" means "approximately coaxial," but also includes, for example, a state in which one axis and the other axis are misaligned by several millimeters. In this embodiment, the first cylindrical body 2 and the second cylindrical body 3 are hexagonal prisms, but are not limited thereto. In other embodiments, the first cylindrical body 2 and the second cylindrical body 3 may have various polygonal prism shapes, such as pentagonal prisms or octagonal prisms. It is preferable, but not limited thereto, that the first cylindrical body 2 and the second cylindrical body 3 have the same polygonal prism shape as the container body 1. By placing the first cylindrical body 2 and the second cylindrical body 3 inside the container body 1, both the inner and outer surfaces of the first cylindrical body 2 and the second cylindrical body 3 can be immersed in the culture medium, thereby ensuring a wide cell adhesion surface for cells to attach and grow within the container.

[0069] As shown in Figures 2 and 5, in this embodiment, the first spacers 21 are provided at all corners of the outer peripheral surface of the first cylindrical body 2 and at the upper axial end of the first cylindrical body 2. As shown in Figures 2 and 6, the first spacers 22 are provided at all corners of the outer peripheral surface of the first cylindrical body 2 and at the lower axial end of the first cylindrical body 2. The first spacers 21 and 22 may be provided at some corners of the outer peripheral surface of the first cylindrical body 2 rather than at all corners. The first spacers 21 and 22 may be provided at the center of the first cylindrical body 2 rather than at the axial end. By providing the first spacers 21 and 22 at the corners of the outer peripheral surface of the first cylindrical body 2 and at the axial end of the first cylindrical body 2, the occurrence of uneven cell adhesion due to liquid flow during rotary culture can be reduced. Furthermore, by providing the first spacer 22 at the lower axial end of the first cylindrical body 2, the first cylindrical body 2 can be prevented from falling to the bottom 11 of the vessel body 1.

[0070] 7(a) and 7(b), the first spacers 21, 22 preferably have V-shaped ends 21a, 22a so as to fit into the corners of the inner circumferential surface of the container body 1. By making the ends 21a, 22a of the first spacers 21, 22 V-shaped, it becomes possible to fix the first cylindrical body 2 in the internal space of the container body 1 at a certain distance L from the container body 1.

[0071] 2 and 6 , in this embodiment, the second spacers 31 are provided at all corners of the outer peripheral surface of the second cylindrical body 3 and at the upper axial end of the second cylindrical body 3. The second spacers 31 may be provided at some of the corners rather than at all corners of the outer peripheral surface of the second cylindrical body 3. The second spacers 31 may be provided at the lower axial end or the center axial portion of the second cylindrical body 3 rather than at the upper axial end. By providing the second spacers 31 at the corners of the outer peripheral surface of the second cylindrical body 3 and at the axial end of the second cylindrical body 3, it is possible to reduce the occurrence of uneven cell adhesion due to liquid flow during rotary culture.

[0072] As shown in Fig. 8(a), it is preferable that the second spacer 31 has a V-shaped end 31a so as to fit into a corner of the inner circumferential surface of the first cylindrical body 2. By forming the V-shaped end 31a of the second spacer 31, it becomes possible to fix the second cylindrical body 3 at a fixed distance from the first cylindrical body 2 in the internal space of the container body 1. As shown in Fig. 8(b), in this embodiment, a second spacer is not provided at the lower axial end of the second cylindrical body 3, but a second spacer may be provided at the lower axial end of the second cylindrical body 3.

[0073] In this embodiment, the cell culture vessel is provided with two cylinders, namely, the first cylinder 2 and the second cylinder 3, but is not limited to this. In other embodiments, the cell culture vessel may be provided with one cylinder or three or more cylinders.

[0074] The first spacers 21, 22 may be provided at both axial ends of the first cylindrical body 2, or at the upper or lower end. When the first cylindrical body 2 is molded from resin by die molding, providing the first spacer 21 or the first spacer 22 at the upper or lower end of the first cylindrical body 2 simplifies the structure of the first cylindrical body 2 as much as possible, making it easier to remove from the mold. On the other hand, if the first spacers 21 and the first spacers 22 are provided at the upper and lower ends of the first cylindrical body 2, there is a risk that the first spacers 21 or the first spacers 22 provided at both ends of the first cylindrical body 2 may become caught in the mold when removing the first cylindrical body 2 from the mold, making removal difficult. However, if molding is performed using a 3D printer, this demolding problem is eliminated, so the first spacers 21 and the first spacers 22 may be provided at both axial ends of the first cylindrical body 2. Similarly, the second spacers 31 and 32 may be provided at both axial ends of the second cylindrical body 3, or at the upper or lower end.

[0075] The first cylindrical body 2 and the second cylindrical body 3 may be made of various materials, such as plastic or glass, and may be transparent, colored, or opaque. These materials may be selected to ensure transparency. However, opaque resins that can block light depending on the culture conditions may also be used. The first cylindrical body 2 and the second cylindrical body 3 may also be made of materials that selectively absorb or transmit light in a specific wavelength range.

[0076] The lid 4 is connected to the top end of the container body 1. The lid 4 may be connected to the top end of the container body 1 by adhesive, welding, or mechanical bonding.

[0077] The lid 4 includes a neck portion 41 with an opening and external threads for connection to the cap 5, and a shoulder portion that covers the upper side of the container body 1. The lid 4 prevents the first cylindrical body 2 and the second cylindrical body 3 from coming off the container body 1 and can fix the first cylindrical body 2 and the second cylindrical body 3 to the container body 1.

[0078] The cap 5 has internal threads for mating with the external threads of the neck 41 of the lid 4. The cap 5 is threaded onto the neck 41 of the lid 4 to close the opening of the lid 4 and seal the cell culture vessel. The cap 5 can be unscrewed from the neck 41 of the lid 4 to open the cell culture vessel.

[0079] The cell culture vessel according to this embodiment includes at least one cylindrical body, and therefore can provide an internal surface area sufficient for large-scale cell culture.

[0080] Figure 9 shows a side view of a cell culture vessel according to one embodiment. Figure 10 shows a perspective view of a first cylindrical body of a cell culture vessel according to one embodiment. Figure 11 shows a perspective view of a second cylindrical body of a cell culture vessel according to one embodiment. Figure 12 shows a cross-sectional view of the cell culture vessel along line P'-P' in Figure 10. Figure 13 shows a cross-sectional view of the cell culture vessel along line Q-Q in Figure 10.

[0081] 9, 10, 12, and 13, the cell culture vessel may be provided with a support 23 at the axial end of the first cylindrical body 2 on the bottom side of the vessel body 1, which supports the second cylindrical body 3 when the cell culture vessel is placed upright. Also, as shown in Fig. 11, a groove 33 into which the support 23 fits may be provided at the axial end of the second cylindrical body 3 on the bottom side of the vessel body 1. By fitting the support 23 of the first cylindrical body 2 into the groove 33 of the second cylindrical body 3, the first cylindrical body 2 and the second cylindrical body 3 can be securely fixed in the internal space of the vessel body 1.

[0082] Figure 14 shows a cross-sectional view of a cell culture vessel along line PP according to one embodiment.Figures 15-17 show cross-sectional views of a cell culture vessel along line QQ according to one embodiment.

[0083] The cell culture vessels shown in FIGS. 14 and 15 to 17 are further provided with a second spacer 32 .

[0084] 15 , the first spacers 22 are provided at all corners of the outer peripheral surface of the first cylindrical body 2 and at the axial lower end of the first cylindrical body 2, but this is not limited to this. As shown in FIGS. 16 and 17 , the first spacers 22 may be provided at some corners of the outer peripheral surface of the first cylindrical body 2, rather than at all corners. Like the first spacer 21, the first spacer 22 may have a V-shaped end so as to fit into the corners of the inner peripheral surface of the container body 1. The first spacer 22, together with the first spacer 21, fixes the first cylindrical body 2 to the container body 1 with a certain distance L therebetween.

[0085] 15 , the second spacers 32 are provided at all corners of the outer circumferential surface of the second cylindrical body 3 and at the axial lower end of the second cylindrical body 3, but this is not limited thereto. As shown in FIGS. 16 and 17 , the second spacers 32 may be provided at some corners of the outer circumferential surface of the second cylindrical body 3, rather than at all corners. Like the second spacer 31, the second spacers 32 may have V-shaped ends so that they fit into the corners of the inner circumferential surface of the first cylindrical body 2. The second spacers 32, together with the second spacer 31, fix the second cylindrical body 3 to the first cylindrical body 2 at a fixed interval.

[0086] Fig. 15 shows an example of a cell culture vessel in which first spacers 22 are provided at all corners of the outer peripheral surface of the first cylindrical body 2 and at the axial lower end of the first cylindrical body 2, and second spacers 32 are provided at all corners of the outer peripheral surface of the second cylindrical body 3 and at the axial lower end of the second cylindrical body 3. Fig. 16 shows an example of a cell culture vessel in which first spacers 22 and second spacers 32 are provided at two opposing corners. Fig. 17 shows an example of a cell culture vessel in which first spacers 22 and second spacers 32 are provided at three corners. In Fig. 17, the second spacers 32 are configured to fit into three corners of the inner peripheral surface of the first cylindrical body 2 where no first spacers 22 are arranged.

[0087] The first and second spacers 22, 32, 21, 31 allow the first and second cylindrical bodies 2, 3 to be securely attached to the container body 1.

[0088] FIG. 18 shows an example of a cell culture vessel according to one embodiment, in which at least two linear marks extending in the axial direction of the vessel body are provided on the outer surface of the flat panel of the vessel body. As shown in FIG. 18, at least two linear marks 13 must be provided on one surface of the flat panel 1A of the vessel body 1, but additional linear marks 13 may be provided. The linear marks 13 may also be dashed lines. The linear marks 13 may be printed or linear protrusions. Providing multiple cut ends inside the linear marks 13 facilitates highly accurate alignment of the field of view each time when automating microscopic imaging using functions such as autofocus. In other words, by physically indicating the imaging start point and focus adjustment start point using the linear marks 13, microscopic imaging of the same area can be performed even if the cell culture vessel is moved without having to place the cell culture vessel stationary in the imaging device. As a result, the state of the cells can be accurately determined, and it is possible to determine whether to continue cell culture.

[0089] The cell culture vessel cap 5 can be equipped with various types of valves. FIG. 19 shows a perspective view of a cell culture vessel cap according to this example. FIG. 20 shows a top view of a cell culture vessel cap according to this example. FIG. 21 shows a front view of a cell culture vessel cap according to this example. FIG. 22 shows an exploded perspective view of a valve attached to a cell culture vessel cap according to one embodiment. FIG. 23 is a cross-sectional perspective view showing the internal structure of a valve attached to a cell culture vessel cap according to one embodiment. FIG. 24 shows a schematic diagram of connecting a syringe via a valve attached to a cell culture vessel cap according to this example. FIG. 25 shows a schematic diagram of connecting a syringe with a Luer lock structure to a valve attached to a cell culture vessel cap according to this example. FIG. 26 shows a schematic diagram of connecting a rubber tube via a Luer lock connector to a valve attached to a cell culture vessel cap according to this example. FIG. 27 shows a front view of a modified cell culture vessel cap according to one embodiment.

[0090] 19 to 21 , the cap 5 includes a top plate 55, a peripheral panel 56 connected to the periphery of the top plate 55 along its periphery, and a first valve 52 and a second valve 52 attached to the top plate 55. Each valve 52 protrudes above the top plate 55 of the cap 5 and has an insertion structure (first valve body, second valve body) 521 into which a supply / discharge tool 6 such as a syringe needle or needle hub is inserted. Each valve 52 may employ a split septum system or may be a mechanical valve. Each valve 52 may be made of isoprene, silicone, a combination thereof, or a similar material.

[0091] 22 to 24, the cap 5 includes a cap body and a valve 52 attached to the cap body that can supply and discharge a fluid to and from the internal space of the container body 1. The valve 52 may have a first valve body 521 and a second valve body 521 made of an elastic material and provided with a slit portion 522 that opens when the supply and discharge tool 6 is inserted. ２ Although open-system culture is performed in an incubator, providing a valve 52 on the cap 5 enables closed-system culture. Replacing the gas phase (gas) within the cell culture vessel is important. Using a supply / discharge device 6, such as a Luer-lock syringe, can release the internal pressure within the cell culture vessel while preventing contamination by foreign substances (e.g., bacteria). Gases and liquids can also be easily injected into the cell culture vessel. This allows for easy replacement of the internal atmosphere of the cell culture vessel. The valve 52 may be connected to the cap 5 by various methods, such as insert molding, Luer-lock, or Luer slip. The valve 52 may be made of isoprene, silicone, a combination thereof, or similar materials.

[0092] In this embodiment, the cap 5 includes two valves (first valve and second valve) 52, but is not limited thereto. In another embodiment, the cap 5 may include one valve or three or more valves. When the cap 5 has two or more valves 52, gas or liquid may be introduced from the outside through one valve (first valve or second valve) 52, and gas or liquid may be withdrawn from the cell culture vessel through the other valve (second valve or first valve) 52. To exchange the atmosphere inside the cell culture vessel, gas may be continuously injected into the cell culture vessel through a membrane filter, and simultaneously gas may be periodically exhausted from the inside of the cell culture vessel. Furthermore, when adding supplements or cell suspensions to the medium during culture, using the valves 52 without opening the cap 5 allows for cleaner operation while reducing the risk of contamination.

[0093] 19 to 24 show an example in which the valve 52 uses a split septum system. The valve 52 has a split septum as an insertion structure (first valve body, second valve body) 521. The split septum has a normally closed slit portion 522 that opens when the supply / discharge tool 6 is pushed in. This system allows easy access to the interior of the cell culture vessel via the valve 52 without unscrewing the cap 5. Such split septum and mechanical valves function as check valves, which have the effect of determining the direction of in- and out-flow fluid flow, and can further enhance sealing by being constantly closed. Other known split septum and mechanical valves can also be used for the valve 52.

[0094] 25, each valve 52 attached to the cap 5 of the cell culture vessel according to this embodiment may be connected to a syringe 110 equipped with a Luer Lock needle hub. By making such a connection, it is possible to add a small amount of a substance or sample a small amount of cultured cells without opening the cap 5.

[0095] As shown in FIG. 26 , each valve 52 attached to the cap 5 of the cell culture vessel according to this embodiment may be connected to a rubber tube 120. Such a connection allows for the exchange of gas or liquid without opening the cap 5. Furthermore, by sealing and gas-phase substitution using the valve 52, it is possible to set the atmosphere inside the vessel to a special hypoxic or hyperoxic environment. Because this embodiment uses a septum system, a chemical injection port into the rubber tube 120 ensures airtightness and cleanliness. Changing the gas phase environment inside the cell culture vessel is also an important condition for culturing cells and can significantly change the behavior of the cells.

[0096] The internal thread of the cap 5 can be applied to vessels other than those described in this embodiment. The cap 5 equipped with the valve 52 can be universally used with cell culture vessels by changing the thread pitch and size of the internal thread. In other words, the cap 5 equipped with the valve 52 can be used not only for rotary culture but also for static culture using a T-flask. The cap 5 equipped with the valve 52 can introduce gas of any phase and partial pressure through the valve 52. This allows for easy replacement of the internal atmosphere of the cell culture vessel, creating a culture environment such as hypoxic culture. Specifically, the cap 5 according to this embodiment can be applied to various types of cell culture vessels, such as the T25, T75, T175, and T225.

[0097] Figure 27 shows a cap 50 with two opening sizes. T-flasks, which are widely used as cell culture vessels, come in sizes T175 and T225. For example, by making cap 50a compatible with the T175 size and cap 50b compatible with the T225 size, it is possible to accommodate the two commonly used sizes of T-flasks. This improves user convenience.

[0098] (Cell Culture Method) A cell culture method using a cell culture vessel having the above-described valve 52 on the cap 5 will be described below.

[0099] The cell culture method using the above-mentioned cell culture vessel may include a culture step of culturing cells in the cell culture vessel by rotating the cell culture vessel in a horizontal position around the axial direction of the cell culture vessel at a predetermined rotation speed; a confirmation step of microscopically inspecting the state of the cells in the cell culture vessel after each predetermined culture period; an exchange step of exchanging the liquid medium and / or gas in the cell culture vessel via valve 52 after each predetermined culture period; and a recovery step of recovering the target cells adhered to the inner surface of vessel body 1 and both the inner and outer surfaces of first cylindrical body 2 after each predetermined culture period.

[0100] According to the above configuration, cell culture can be continuously performed in a closed system, preventing contamination by foreign substances (bacteria, etc.), enabling mass production and culture of useful cells. Furthermore, periodic medium and gas exchange can be performed automatically via a valve, making cell culture more efficient. The target cells recovered by this cell culture method are adherent cells or adherent cells, including iPS cells and mesenchymal stem cells, such as adipose tissue-derived stem cells, amniotic tissue-derived stem cells, stem cells derived from human umbilical Wharton's jelly, and chorionic tissue-derived stem cells.

[0101] The above cells can differentiate into tissues such as muscle, bone, nerve, and fat, and are therefore expected to contribute to regenerative medicine.

[0102] The cell culture method using the above-mentioned cell culture vessel may include an exchange step of exchanging the liquid medium and / or gas in the cell culture vessel via valve 52 every predetermined period of time, a useful substance production step of causing the cells in the cell culture vessel to produce a useful substance, and a recovery step of recovering the supernatant liquid in the cell culture vessel via valve 52.

[0103] According to the above configuration, regular medium and gas exchange optimizes the cell condition, allowing the cells to effectively produce useful substances. Furthermore, regular medium and gas exchange can be performed automatically via a valve, making supernatant production more efficient. Cells that produce useful substances using this supernatant production method include adherent cells and cells, including iPS cells and mesenchymal stem cells, such as adipose tissue-derived stem cells, amniotic tissue-derived stem cells, stem cells derived from human umbilical cord Wharton's jelly, and chorionic tissue-derived stem cells. The supernatant contains growth factors and proteins produced by these cells. Specifically, these include cytokines, exosomes, EGF (epidermal growth factor), KGF (keratinocyte growth factor), IGF (insulin-like growth factor), and the like. The resulting supernatant is used for the treatment of rheumatism, skin diseases, anti-inflammatory effects, and the prevention of arteriosclerosis. The resulting supernatant is also used as an ingredient in treatments in the fields of anti-aging medicine and preventive medicine, and as an ingredient in cosmetics and beauty serums.

[0104] Fig. 28 is a flow diagram showing steps of a cell culture method using a cell culture vessel and a cell recovery method according to one embodiment. Fig. 29 is an explanatory diagram showing a cell culture vessel equipped with a gripping device according to one embodiment.

[0105] (Cell recovery method) 1. Primary culture step When culturing adipose tissue-derived stem cells, fat collected from the patient's abdomen or other area is used as the specimen. When culturing amniotic tissue-derived stem cells, the amniotic membrane is used as the specimen. When culturing human umbilical cord Wharton's jelly-derived stem cells, the human umbilical cord is used as the specimen. When culturing chorionic tissue-derived stem cells, the chorion is used as the specimen.

[0106] After transporting and receiving the specimen (S1), pretreatment is performed (S2). In pretreatment, cells are detached using a detachment enzyme (S3), then extracted and recovered (S4). The recovered cells are seeded into a cell culture vessel containing a liquid medium (S5), and static culture is initiated (S6). A commercially available medium, such as DMEM (Dulbecco's Modified Eagle's Medium), may be used as the liquid medium, or a unique liquid medium may be prepared and used. During static culture, periodic microscopic examination is performed every predetermined culture period to confirm the state of the cells in the cell culture vessel (S7). Periodic medium and gas exchange are also performed via valve 52 every predetermined culture period (S8). Periodic microscopic examination is also performed every predetermined culture period (S9). After sufficient cell growth is confirmed, primary culture is completed (S10).

[0107] 2. Expansion Culture Process: Cell counting is performed on the cells obtained from the primary culture (S11). Cell counting is preferably performed non-invasively, with the number of cells measured by microscopic observation. Non-invasive cell counting does not damage the cells and can be performed automatically, making cell culture more efficient. After measuring the cell number, a detachment enzyme is added via valve 52 to detach cells that have grown on the inner surface of the cell culture vessel (S12). Next, the required amount of liquid medium is added (S13), and after suspension, rotary culture is initiated (S14). The liquid medium may be the same as the liquid medium used in the primary culture, or it may be used as a basal medium with additional ingredients added, or a different liquid medium may be used, or a unique liquid medium may be created and used. At this time, if necessary, quality testing of the cell culture medium and cells may be performed (S19). Quality testing is performed using quantitative PCR to check for bacterial infection or other infections.

[0108] Rotary culture is preferably performed by attaching the cell culture vessel to a gripping device 7 shown in FIG. 29 . The gripping device 7 is composed of a gripping portion 7a that grips the cell culture vessel and a motor connector 7b that connects to a motor (not shown). The gripping portion 7a grips only a portion of the vessel body 1, preventing damage to the area photographed by the microscope R. Furthermore, observation can be performed on the side without the gripping portion 7a. If necessary, all sides can be easily and thoroughly observed by shifting the insertion position of the gripping portion 7a by one side. For example, before and after medium or gas exchange, all sides can be thoroughly observed by shifting the insertion position of the gripping portion 7a by one side. Rotary culture is performed by rotating the cell culture vessel in a horizontal position around the axial direction (circumferential direction) of the cell culture vessel at a predetermined rotational speed. It is preferable to rotate the cell culture vessel at a predetermined rotation speed so that the cells attached to the inner surface of the vessel body 1 and both the inner and outer surfaces of the first cylindrical body 2 are constantly covered with the liquid medium due to surface tension. While Figure 29 shows the cell culture vessel rotating clockwise (as viewed from the top of the cell culture vessel), it may also rotate counterclockwise (as viewed from the left). During the rotary culture, periodic microscopic examination is performed every predetermined culture period to confirm the state of the cells in the cell culture vessel (S15). Furthermore, periodic medium and gas exchange are performed via the valve 52 of the cap 5 every predetermined culture period (S16). Furthermore, periodic microscopic examination is performed every predetermined culture period (S17), and after cell growth is confirmed, the expansion culture is completed (S18).

[0109] 3. Cell Storage Process The number of cells obtained by expansion culture is determined by cell counting (S20). Cell counting is preferably performed non-invasively, as described above. After cell counting, a detachment enzyme is added via valve 52 on cap 5 to detach cells that have adhered to and proliferated inside the cell culture vessel (S21). If necessary, a portion of the cell culture medium may be sampled and counted to adjust the cell count in the liquid. Cell counting is performed using a standard method, such as a hemocytometer or an automated cell counter using 0.3-0.5% trypan blue staining solution. The cell culture medium in the cell culture vessel is dispensed into cryopreservation tubes (S22) and stored in a deep freezer, liquid nitrogen, or liquid nitrogen vapor (S23). The cryopreserved cells are used to produce a supernatant after the dormancy and expansion culture process described below.

[0110] 4. Cell Recovery Process When recovering cells for transplantation, cells that have adhered to and proliferated inside the cell culture vessel are detached (S21), and the required amount of cells is collected (S24). The cells are washed with PBS or the like (S25) and packaged in a predetermined dosage form. When collecting the required amount of cells (S24), the cells are sampled for quality inspection (S27), and quality inspection of predetermined items is performed (S28). After the inspection results are determined (S29), the dosage form of cells that has been confirmed to be safe is delivered to a medical institution, research facility, etc.

[0111] 30 is a flow diagram showing steps of a cell culture method using a cell culture vessel and a method for producing a supernatant fluid according to one embodiment. Steps S1 to S23 in FIG. 30 are the same as steps S1 to S23 in FIG. 28, and therefore will not be described.

[0112] 5. Initiation and Expansion Culture Process: The frozen and stored cell culture solution (S23) is frozen and thawed (S30). If necessary, a cell count is performed to confirm the number of cells in the solution (S31). At this time, the cell count is performed using a conventional method. Next, cells are seeded into a cell culture vessel containing a liquid medium (S32), and rotary culture is initiated (S33). During rotary culture, periodic microscopic examination is performed every predetermined culture period to confirm the state of the cells in the cell culture vessel (S34). Furthermore, periodic medium and gas exchange are performed via valve 52 every predetermined culture period (S35). Furthermore, periodic microscopic examination is performed every predetermined culture period (S36). After sufficient cell proliferation is confirmed, expansion culture is completed (S37). After this, the cell culture conditions may be switched to supernatant liquid production conditions (S38), and upon completion of expansion culture, the cells may be recovered as cells for regenerative medicine treatment involving cell transplantation (S24).

[0113] 6. Supernatant Liquid Production Process When the cell culture conditions are switched to the supernatant liquid production conditions (S38), the supernatant liquid production process is initiated (S39). During the supernatant liquid production process, periodic medium and gas exchange are performed via valve 52 every predetermined period (S40). When useful substances are produced from the cells, supernatant liquid containing the useful substances is collected (S41). The supernatant liquid production process may be performed with the cell culture vessel stationary; however, from the viewpoint of creating an environment more similar to that in a living body with bodily fluid circulation, it is preferable to perform the supernatant liquid production process by rotating the cell culture vessel axially at a predetermined rotation speed.

[0114] 7. Quality inspection process During the quality inspection, the supernatant is checked for foreign matter or infection.

[0115] An endotoxin test is performed (S42). A common method for detecting endotoxin is the gelation method. The gelation method detects endotoxin based on the coagulation reaction of a lysate reagent caused by the presence of endotoxin. A test tube containing the gelation reagent is incubated at 37°C for 60 minutes to determine gelation. If the gel does not collapse, it is considered positive, and if a gel is not formed, it is considered negative.

[0116] A sterility test is carried out (S43). The sterility test is carried out by treating the supernatant in a specified manner, culturing it, and visually checking for the presence or absence of microorganisms growing in the culture medium.

[0117] A mycoplasma test is performed (S44). The mycoplasma test may be performed by any one of the mycoplasma culture method, the DNA staining method, and the PCR (polymerase chain reaction) method, or a combination of these methods. The mycoplasma culture method is a test method in which a test sample is inoculated into a medium optimized for mycoplasma. The DNA staining method is a test method in which mycoplasma nuclei are counterstained with Hoechst stain or DAPI and then imaged using a fluorescent microscope for confirmation. The PCR (polymerase chain reaction) method is a test method based on the amplification of DNA when mycoplasma is present in a sample.

[0118] The supernatant liquid whose safety has been confirmed through the above three quality inspections (S45) is delivered to medical institutions, research facilities, etc.

[0119] Modified examples of the above-mentioned cell culture vessel will be described below with reference to FIGS.

[0120] (First Modification) FIGS. 31 and 32 are diagrams illustrating a cell culture vessel according to a first modification. Components designated by the same reference numerals are similar to those in the previously described embodiment, and their description will be omitted. As shown in FIG. 31 , a receiving portion 60 that abuts against the first spacer 22 is provided on the bottom 11 of the vessel body 1. The receiving portion 60 is provided on the inner circumferential surface of the bottom 11. The receiving portion 60 abuts against the first spacer 22 provided at the axial lower end of the first cylindrical body 2, preventing the first cylindrical body 2 and the second cylindrical body 3 engaged with the first cylindrical body 2 from falling into the bottom 11. This also forms a space M between the bottom 11 of the vessel body 1 and the first spacer 22, with a predetermined gap therebetween, for storing cells. As a result, cells cultured on the inner surface of the vessel body 1 and on both the inner and outer surfaces of the multiple cylindrical bodies (the first cylindrical body 2 and the second cylindrical body 3) can be easily collected in the space M and removed to the outside, thereby increasing the cell recovery rate.

[0121] The receiving portion 60 has a triangular plate shape, one side 61 of which extends toward the bottom 11 along the inclined surface of the conical shape where the diameter narrows, and the other side 62 of which is parallel to the interface between the vessel body 1 and the bottom 11. The side 62 engages with the first spacer 22, preventing the first cylindrical body 2 and the second cylindrical body 3 from sinking toward the bottom 11. Because the receiving portion 60 is not a cell adhesion surface, it is configured to have no physical effect on cell adhesion. However, when the cell culture vessel is rotated around the axis X for cell culture, the receiving portion 60 can function like a fin to promote convection of the culture medium. Furthermore, providing such a receiving portion 60 strengthens the sinking of the first cylindrical body 2 toward the bottom 11, which has the advantage of eliminating the need to provide first spacers 22 at all corners 2C of the first cylindrical body 2 on the bottom 11 side. This can contribute to saving molding materials and improving yield when forming by metal molding.

[0122] Fig. 32 shows a top view of the container body made of a transparent material in Fig. 31 with no cylindrical body inserted therein. As shown in Fig. 32, the receiving portions 60 may be provided on the bottom 11 connected to all corners of the inner circumferential surface of the container body 1, but they do not have to be provided on the bottom 11 connected to all corners. For example, when using the hexagonal prism-shaped container body 1 shown in Fig. 32, the receiving portions 60 may be provided on the bottom 11 connected to six corners, on the bottom 11 connected to two opposing corners, on the bottom 11 connected to three equally spaced corners, or on the bottom 11 connected to four corners other than the two opposing corners.

[0123] (Second Modification) Next, a further modification of the first modification will be described. FIG. 33 is a diagram illustrating a cell culture vessel according to the second modification. FIG. 34 is a partial cross-sectional view of the cell culture vessel taken along line R-R in FIG. 33, viewed from the Y direction. The difference from the first modification is that a groove (recess 62A) into which the first spacer 22 fits is provided on an edge 62 of the receiving portion 60, which is parallel to the interface between the vessel body 1 and the bottom 11. A portion of the first spacer 22 engages with this recess 62A, enabling reliable positioning of the first spacer 22. This reduces assembly defects when assembling multiple cylindrical bodies (first cylindrical body 2, second cylindrical body 3) into the vessel body 1. This improves the efficiency of assembling the cylindrical bodies (first cylindrical body 2, second cylindrical body 3) to the vessel body 1, and also strengthens the fixing force of the cylindrical bodies (first cylindrical body 2, second cylindrical body 3) to the vessel body 1. As a result, the strength of the attachment of the first cylindrical body 2 to the container body 1 can be ensured without providing spacers at all corners 2C of the cylindrical body 2, thereby increasing the degree of freedom in design.

[0124] (Third Modification) Next, a further modification of the second modification will be described. Figures 35 and 36 are diagrams illustrating a cell culture vessel in the third modification. The third modification differs from the second modification in that the first spacer 22 has an end 22D that is rectangular in top view and fits into a corner of the inner circumferential surface of the vessel body 1, and a recessed groove (recess 1D) into which the end 22D fits is provided at the corner 1C of the inner circumferential surface of the vessel body 1. In the third modification, when the first cylindrical body 2 is assembled to the vessel body 1, a groove-like recess 1D into which the first spacer 22 can fit is provided at a position facing the corner 1C of the inner circumferential surface of the hexagonal column-shaped vessel body 1, and the end 22D of the first spacer 22 fits into this recess 1D, thereby positioning and fixing the first cylindrical body 2 to the vessel body 1. This reduces assembly defects when assembling multiple cylindrical bodies (first cylindrical body 2, second cylindrical body 3) into the container body 1, as in the second modified example. Therefore, the efficiency of the assembly work of the cylindrical bodies (first cylindrical body 2, second cylindrical body 3) into the container body 1 is improved. With this configuration, it is not necessary to provide the receiving portion 60 as in the second modified example. As described above, in the second and third modified examples, in order to position and fix the first spacer 22 to the container body 1, a groove (recess 1D) into which a portion of the first spacer 22 fits is provided in the receiving portion 60 or the corner 1C of the inner circumferential surface of the container body 1, and the first spacer 22 fits into this groove.

[0125] The present embodiment can be implemented in various different forms. It will be understood by those skilled in the art to which the present disclosure is directed that the present disclosure may be implemented in other specific forms without changing the spirit or essential characteristics thereof. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

[0126] DESCRIPTION OF SYMBOLS 1 Container body 1A Flat panel 1C Corner 1D Recess 11 Bottom 13 Mark 2 First cylindrical body 2A Flat panel 2B, 2C Corner 21, 22 First spacer 21a, 22a End 22D End 23 Support 3 Second cylindrical body 3A Flat panel 3B Corner 31, 32 Second spacer 31a End 33 Groove 4 Lid 41 Neck 5, 50 Cap 52 First valve, second valve 521 First valve body, second valve body 522 Slit 6 Supply / discharge tool 60 Receptacle 62A Recess M Space R Microscope

Claims

1. A cell culture container comprising: a bottomed container body having a polygonal columnar internal space; a plurality of polygonal columnar cylinders arranged coaxially with the container body inside the container body; a lid attached to an axial end of the container body; a cap attached to a cylindrical neck portion of the lid; and a spacer provided at a corner of the outer peripheral surface of the plurality of polygonal columnar cylinders and at an axial end of the plurality of polygonal columnar cylinders, wherein the spacer is fitted into a corner of the inner peripheral surface of the container body or a corner of the inner peripheral surface of the cylinder, and the bottom of the container body is conical or polygonal pyramidal.

2. The cell culture container according to claim 1, wherein the plurality of polygonal columnar cylinders are a first cylinder and a second cylinder, the second cylinder is provided inside the first cylinder coaxially with the first cylinder, a first spacer provided at a corner of the outer peripheral surface of the first cylinder and at an axial end of the first cylinder, and a second spacer provided at a corner of the outer peripheral surface of the second cylinder and at an axial end of the second cylinder are further provided, the first spacer is fitted into a corner of the inner peripheral surface of the container body, and the second spacer is fitted into a corner of the inner peripheral surface of the first cylinder.

3. The cell culture container according to claim 1, wherein an end portion of the spacer that fits into a corner of the inner peripheral surface of the container body or a corner of the inner peripheral surface of the cylinder is V-shaped.

4. The cell culture container according to claim 2, wherein an end portion of the first spacer that fits into a corner of the inner peripheral surface of the container body is V-shaped, and an end portion of the second spacer that fits into a corner of the inner peripheral surface of the first cylinder is V-shaped.

5. The cell culture container according to claim 2 or 4, wherein the container body, the first cylinder, and the second cylinder are all hexagonal prisms, the first spacer is provided at three corner portions of the outer peripheral surface of the first cylinder and at equal intervals at the axial end portions of the first cylinder, the second spacer is provided at three corner portions of the outer peripheral surface of the second cylinder and at equal intervals at the axial end portions of the second cylinder, the second spacer is fitted inside the corner portion of the outer peripheral surface of the first cylinder where the first spacer is not provided, and is used for peeling and separating and dispersing the target cells attached to the inner surface of the container body, both the inner and outer surfaces of the first cylinder, and both the inner and outer surfaces of the second cylinder. Cell culture container.

6. The cell culture container according to claim 2 or 4, wherein the longitudinal length of the first spacer in the first cylinder is smaller than the longitudinal length of the first cylinder, and a passage with a constant interval for the cell culture medium to flow is secured between the inner surface of the container body and the outer surface of the first cylinder. Cell culture container.

7. The cell culture container according to claim 2 or 4, wherein a support for supporting the second cylinder when the cell culture container is placed vertically is provided at the axial end portion of the first cylinder on the bottom side of the container body, and a groove into which the support is fitted is provided at the axial end portion of the second cylinder on the bottom side of the container body. Cell culture container.

8. The cell culture container according to claim 1 or 2, wherein at least two linear marks extending in the axial direction of the container body are provided on at least one plane of the outer surface of the container body. Cell culture container.

9. The cell culture container according to claim 1 or 2, wherein the cap includes a cap body and a first valve attached to the cap body and capable of supplying and discharging fluid to and from the internal space of the container body, and the first valve has a first valve body made of an elastic material provided with a slit portion that opens when a supply and discharge instrument is inserted. Cell culture container.

10. The cell culture container according to claim 9, wherein the cap further comprises a second valve attached to the cap body and capable of supplying and discharging fluid to and from the internal space of the container body, and the second valve has a second valve body made of an elastic material and provided with a slit portion that opens when a supply / discharge instrument is inserted, the cell culture container.

11. A cell culture method using the cell culture container according to claim 1 or 2, comprising a culturing step of rotating the horizontally placed cell culture container at a predetermined rotational speed around the axial direction of the cell culture container to culture the cells in the cell culture container, a confirmation step of examining the state of the cells in the cell culture container every time a predetermined culture period elapses, an exchange step of exchanging the liquid medium and / or gas in the cell culture container via a valve attached to the cap every time a predetermined culture period elapses, and a recovery step of recovering the target cells attached to the inner surface of the container body and both the inner and outer surfaces of the cylindrical body after a predetermined culture period has elapsed, the cell culture method using the cell culture container.

12. A cell culture container comprising a bottomed container body having a polygonal columnar internal space, a polygonal columnar cylindrical body disposed coaxially with the container body inside the container body, a lid attached to an axial end of the container body, a cap attached to a neck portion of the lid, and a spacer provided at a corner of the outer peripheral surface of the polygonal columnar cylindrical body and at an axial end of the polygonal columnar cylindrical body, wherein the spacer is fitted into a corner of the inner peripheral surface of the container body, and a receiving portion that abuts against the spacer is provided at the bottom of the container body.

13. The cell culture container according to claim 12, wherein a single space is formed between the bottom of the container body and the lower axial end of the polygonal columnar cylindrical body.

14. The cell culture container according to claim 12 or 13, wherein a groove into which a part of the spacer fits is provided on the side of the receiving portion that abuts against the spacer.

15. The cell culture container according to claim 12 or 13, wherein the end portion of the spacer that fits into a corner of the inner peripheral surface of the container body is rectangular in top view, and a groove having a concave shape into which the end portion fits is provided at a corner of the inner peripheral surface of the container body.

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