Cell culture container

Abstract

To provide a cell culture container that can secure a wide cell adhesion surface for cells to attach and grow within the container.SOLUTION: A cell culture container comprises a bottomed container body 1 having a polygonal column-shaped internal space, a polygonal column-shaped first cylindrical body 2 arranged coaxially with the container body 1 inside the container body 1, a lid body 4 attached to the axial end of the container body, a cap 5 attached to the cylindrical neck portion of the lid body 4, and first spacers 21, 22 provided 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 first spacers 21, 22 being fitted into the corners of the inner peripheral surface of the container body 1.SELECTED DRAWING: Figure 2

Description

Technical Field

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

Background Art

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

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, conventional cell culture containers are focused on research culture. Among them, when considering mass production culture, roller bottles are used as the most practical ones.

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

[0006] To address such problems, Patent Document 2 proposes increasing the internal surface area by making the structure inside the container bellows- or accordion-shaped. However, this method is also insufficient to support large-scale culturing and has its own drawbacks. Specifically, while such a structure can increase the inner area inside the container, it is not a structure that allows cells to efficiently adhere. Cells are suspended in the culture medium inside the container, but during rotation culture of the container, the cells fall according to gravity. The bellows- or accordion-shaped structure has ridges and valleys that are continuous while forming curved surfaces, and since the cells fall vertically, there is a risk that the cells will accumulate in the part corresponding to the bottom of the bellows. As a result, cells cannot grow in the part where the cells have accumulated. Therefore, even if the inner area is increased by making the structure inside the container bellows- or accordion-shaped, the effective area where cells can actually adhere is limited.

[0007] Therefore, an object of the present invention is to provide a cell culture container capable of widely securing a cell adhesion surface for cells to adhere and proliferate inside the container.

Means for Solving the Problems

[0008] The cell culture container disclosed in the present application includes a bottomed container body having a polygonal columnar internal space, a polygonal columnar first cylinder 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 cylindrical neck portion of the lid, and 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 the first spacer is fitted into a corner of the inner peripheral surface of the container body.

[0009] According to the above configuration, by installing the first cylinder inside the container body, both the inner and outer surfaces of the first cylinder can be used as the immersion area of the culture medium. Therefore, a wide cell adhesion surface for cells to adhere and grow in the container can be ensured. In addition, since the first spacer is provided at a corner of the outer peripheral surface of the first cylinder and at an axial end of the first cylinder, the occurrence of non-uniform cell adhesion due to the liquid flow during rotary culture can be reduced.

[0010] The cell culture container further includes a polygonal columnar second cylinder disposed coaxially with the first cylinder inside 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. The second spacer may be fitted into a corner of the inner peripheral surface of the first cylinder.

[0011] According to the above configuration, by installing the second cylinder inside the first cylinder, both the inner and outer surfaces of the second cylinder can be used as the immersion area of the culture medium. Therefore, a wider cell adhesion surface for cells to adhere and grow in the container can be ensured. In addition, since the second spacer is provided at a corner of the outer peripheral surface of the second cylinder and at an axial end of the second cylinder, the occurrence of non-uniform cell adhesion due to the liquid flow during rotary culture can be reduced.

[0012] In the cell culture container, an end portion of the first spacer that fits into a corner of the inner peripheral surface of the container body may be V-shaped.

[0013] According to the above configuration, in the internal space of the container body, it is possible to fix the first cylinder at a certain interval from the container body.

[0014] In the second spacer, an end portion that fits into a corner of the inner peripheral surface of the first cylinder may be V-shaped.

[0015] According to the above configuration, in the internal space of the container body, it is possible to fix the second cylinder at a certain interval from the first cylinder.

[0016] In the cell culture container, a support for supporting the second cylinder is provided at an axial end of the first cylinder on the bottom side of the container body, and when the cell culture container is placed vertically, a groove into which the support fits may be provided at an axial end of the second cylinder on the bottom side of the container body.

[0017] According to the above configuration, in the internal space of the container body, the first cylinder and the second cylinder can be reliably fixed.

[0018] In the cell culture container, at least two linear marks extending in the axial direction of the container body may be provided on at least one plane of the outer surface of the container body.

[0019] According to the above configuration, by physically indicating the shooting start point and the focus adjustment start point with two linear marks, even if the cell culture container is moved without stationary it on the imaging device, microscopic imaging of the same area can be performed. As a result, the state of the cells can be accurately determined, and it is possible to judge whether to continue culturing the cells.

[0020] In the cell culture container, 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. The first valve may have a first valve body made of an elastic material and provided with a slit portion that opens when a supply / discharge instrument is inserted.

[0021] According to the above configuration, the cell culture container can inject gas and liquid into the cell culture container while preventing contamination by foreign substances (such as bacteria). In addition, the gas in the cell culture container can be replaced.

[0022] In the cell culture container, the cap further includes 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. The second valve may have a second valve body made of an elastic material and provided with a slit portion that opens when a supply / discharge instrument is inserted.

[0023] According to the above configuration, gas or liquid can be introduced from the outside through one valve, and gas or liquid in the cell culture container can be drawn out through the other valve.

[0024] The cell culture method disclosed in the present application is a cell culture method using any of the above cell culture containers, comprising: a culturing step of culturing cells in the cell culture container by rotating the horizontally placed cell culture container at a predetermined rotational speed around the axial direction of the cell culture container; a confirming step of examining the state of the cells in the cell culture container every time a predetermined culture period elapses; an exchanging step of exchanging the liquid medium and / or gas in the cell culture container via the valve attached to the cap every time a predetermined culture period elapses; and a recovering step of recovering the target cells attached to the inner surface of the container body and both the inner and outer surfaces of the first cylinder after a predetermined culture period has elapsed.

[0025] According to the above configuration, since cell culture can be continuously performed in a closed system, mass culture of useful cells becomes possible while preventing contamination by foreign substances (such as bacteria). Furthermore, since it is also possible to automatically perform regular medium exchange and gas exchange via the valve, cell culture can be made more efficient.

Effects of the Invention

[0026] According to the present invention, a wide cell adhesion surface for cells to adhere and grow in the cell culture container can be ensured.

Brief Description of the Drawings

[0027]

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Embodiments for Carrying Out the Invention

[0028] Hereinafter, embodiments will be described in more detail with reference to the accompanying drawings. As will be understood by those skilled in the art, the described embodiments are subject to various modifications, all of which can be made without departing from the spirit and scope of the present disclosure.

[0029] The drawings and the description are illustrative and not restrictive. Throughout this specification, the same reference numerals indicate the same components.

[0030] The size and thickness of each configuration shown in the drawings are arbitrarily shown for better understanding and easier explanation. Unless otherwise specifically stated, the present disclosure is not limited thereto. For example, when a specific size or positional relationship between elements is described with reference to the drawings, the drawings are used to show such size or positional relationship. The thickness of layers, plates, panels, regions, etc. may be exaggerated for clarity unless otherwise specifically stated. For example, when a specific thickness relationship between elements is described with reference to the drawings, the drawings are used to show such thickness relationship. The thickness of some layers or regions may be exaggerated.

[0031] The singular form includes the plural form unless the context clearly dictates otherwise.

[0032] In the specification and claims, the term "and / or" is intended to include any combination of the terms "and" and "or" for purposes of meaning and interpretation. For example, the expression "A and / or B" is to be interpreted as meaning "A, B, or A and B".

[0033] In the specification and claims, the phrase "at least one" is included for purposes of meaning and interpretation as "at least one selected from the group". For example, the expression "at least one of A and B" is to be interpreted as meaning "A, B, or A and B".

[0034] Terms such as "first" and "second" are used only to describe various components and do not limit these components. These terms are simply used to distinguish different components. For example, the first component may be called the second component, and similarly, the second component may be called the first component, but this does not deviate from the scope of the present disclosure.

[0035] When an element such as a layer, plate, panel, region, or substrate is described as "on" another element, it may be directly on the other element or there may be an element in between. On the other hand, when the first element is described as "directly on" the second element, there is no element in between. Throughout the specification, an element "on" an object is understood to be located above or below the object element and does not necessarily indicate "on" in the opposite direction of gravity.

[0036] For example, the spatially relative terms "lower" or "upper" can be used to describe the relationship between one element or component and another as shown in the drawings. The spatially relative terms are intended to include other directions during use or operation in addition to the directions shown in the drawings. For example, if the device shown in the drawing is inverted, a device that was located below another device may be located "above" the other device. Thus, the exemplary term "lower" may include lower and upper positions. The device may be oriented in other directions, and the spatially relative terms may be interpreted differently depending on the direction.

[0037] In this specification, when an element (or region, layer, part, etc.) is described as "connected" or "coupled" to another element, it may be directly disposed, connected, or coupled, or there may be an element disposed therebetween.

[0038] 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. Further, terms defined in commonly used dictionaries are to be interpreted as having a meaning that coincides with the meaning in the context of the relevant art and / or this specification, and are not to be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Example

[0039] FIG. 1 shows a front view of a cell culture container according to an embodiment. FIG. 2 shows an exploded perspective view of a cell culture container according to an embodiment. FIG. 3 shows a cross-sectional view of the cell culture container along the line P-P of FIG. 1. FIG. 4 shows a top view of a cell culture container according to an embodiment. FIG. 5 shows a cross-sectional view of the cell culture container along the line Q'-Q' of FIG. 1. FIG. 6 shows a cross-sectional view of the cell culture container along the line Q-Q of FIG. 1. In FIG. 7, FIG. 7(a) shows a top view of the first cylinder of the cell culture container according to an embodiment. FIG. 7(b) shows a bottom view of the first cylinder of the cell culture container according to an embodiment. In FIG. 8, FIG. 8(a) shows a top view of the second cylinder of the cell culture container according to an embodiment. FIG. 8(b) shows a bottom view of the second cylinder of the cell culture container according to an embodiment.

[0040] Referring to FIGS. 1 to 6, a cell culture container according to an embodiment includes a bottomed container body 1, a first cylinder 2, a second cylinder 3, a lid 4, a bottom 11, and a cap 5.

[0041] The container body 1 has a polygonal columnar shape with an empty internal space. Therefore, the container body 1 is composed of a plurality of quadrilateral flat panels 1A. In this embodiment, the container body 1 is composed of six quadrilateral flat panels 1A. In this embodiment, the container body 1 is hexagonal columnar, but it is not limited thereto. In some embodiments, the container body 1 may be various polygonal columnar shapes such as pentagonal columnar or octagonal columnar. The container body 1 is made of various materials such as plastic and glass, and may be transparent, colored, or opaque. These materials may be selected to ensure transparency. However, an opaque resin that can block light according to the culture conditions may also be used. Furthermore, a material that selectively absorbs or transmits a specific wavelength range may be used. As described above, the container body 1 may be hexagonal columnar or polygonal columnar, but it is necessary to be configured to have at least one flat panel 1A. Thereby, while observing the internal surface to which the cells adhere with the microscope M, the culture state of the cells can be observed, and it can be determined whether to continue the culture. When the container body 1 is cylindrical, since there is no flat panel 1A, it becomes difficult to observe the culture state of the cells with the microscope M during the culture process.

[0042] In the present invention, an embodiment employing a microscope is used. However, when observing the cells in the culture container using the microscope M and continuing the culture state, the one suitable for observing cell culture is an inverted microscope. In the method using the microscope M as this inverted microscope, it is necessary to arrange the eyepiece lens on the surface side of the culture container and the objective lens on the back side of the culture container, and observe by sandwiching with the two lenses. Therefore, when attempting to automate the culture using a plurality of the culture containers of this embodiment, since areas for arranging the lenses on the surface side and the back side of the culture container are essential, there has been a problem that the culture apparatus for automatically culturing using a plurality of culture containers becomes large-sized.

[0043] Therefore, the present inventors have found that when assembling an automatic culture apparatus using a method that uses a digital camera such as a CCD camera or a CMOS camera and enables the captured image itself to be enlarged and observed, it is very effective in terms of miniaturization. This is because in the method of monitoring the culture state with this digital camera, it is possible to integrally provide the light source necessary for imaging and observation at the same position as the digital camera.

[0044] An apparatus for monitoring such a culture state using a digital camera composed of a CCD sensor, a CMOS sensor, or a line sensor is shown in FIGS. 13 and 14 of JP-A-2022-023903. Since a digital camera including a semiconductor sensor such as a CCD sensor, a CMOS sensor, or a line sensor is integrally configured with the light source 24, it can be provided on either the front surface side or the back surface side of the culture vessel, enabling miniaturization. Such a monitoring apparatus integrated with a light source is known. For example, as such a digital camera, the Incubation Monitoring System CM manufactured by Evident Corporation (formerly Olympus Corporation) and the Cell Recorder manufactured by Cytronics Corporation are suitable.

[0045] Further, when observing the culture state of cells in the multi-layer culture vessel of the present invention using such a digital camera, by appropriately changing the focal position of the digital camera, the culture states of the front and back surfaces of the plurality of rectangular flat panels 2A and flat panels 3A of the first cylinder 2 and the second cylinder 3 can be achieved by appropriately focusing on the culture regions of the front and back surfaces of the flat panels 2A and flat panels 3A.

[0046] Next, the reason why it is preferable to individually observe the culture states of each layer by appropriately changing the focal position of the digital camera for the culture states of the front and back surfaces of the flat panels 2A and flat panels 3A of the multi-layer bottle-shaped culture vessel in the present embodiment will be described below.

[0047] When performing cell culture, an operation called seeding is carried out at the first stage of culturing the cells, and this operation is performed by introducing the original cells into a culture vessel together with a culture medium.

[0048] Since the culture vessel of the present invention has a plurality of inner cylinders (the first cylinder 2 and the second cylinder 3), the behavior of the cell suspension differs depending on whether the seeded cells come into contact with the surface of the inner cylinder (the first cylinder 2 and the second cylinder 3) whose outer shape is concave or with the back surface of the inner cylinder having a convex shape (the first cylinder 2 and the second cylinder 3). In order to non-invasively measure more accurate cell numbers and cell occupation area ratios, it is preferable to observe all cell adhesion surfaces.

[0049] In a multi-layer bottle-type culture vessel such as that of the present invention, in order to accurately monitor the cell culture state, by monitoring the cell adhesion surfaces of each layer, it becomes possible to accurately calculate and determine the timing of detaching and dispersing the expanded cells by grasping a more accurate culture state.

[0050] When adopting the digital camera shown in FIGS. 13 and 14 of Japanese Patent Application Laid-Open No. 2022-023903 described above, in a conventional single-phase octagonal bottle, the distance between the surface opposite to the cell adhesion surface to be observed is too wide, so it is necessary to sufficiently ensure the illuminance of the light source irradiating the cell adhesion surface to be observed. On the other hand, in the case of the culture vessel (multi-layer bottle-type vessel) described in the embodiment of the present invention, the distance to the opposite surface for generating reflected light is sufficiently reduced, and within the range without phototoxicity, it is possible to ensure the illuminance sufficient to facilitate observation without increasing the output of the light source.

[0051] Furthermore, as merits of miniaturizing the culture apparatus as described above, the following points can also be pointed out. That is, when observing culture in a culture vessel, two methods are assumed: observing inside the incubator or taking it out of the incubator for observation and then returning it to the inside of the incubator again. In such a case, by making it a miniaturized automatic culture apparatus, since either method can be easily applied, there is a merit that the degree of freedom in system design when constructing a cell culture system using the automatic culture apparatus is increased.

[0052] For example, as the concept of the cell culture system of the automatic culture device of the present inventor, an arm, a centrifuge, and a photographing device are arranged in a safety cabinet, and for a plurality of culture vessels, the culture process is advanced simultaneously. And with one device, a plurality of culture vessels are processed simultaneously, and at the timing of completion of the culture, they are packed in the dosage form of the final packaging of the automated operation and discharged. The culture process does not proceed while being transferred by a plurality of individual devices, and a configuration is assumed in which it always stays within the device in the same clean space.

[0053] As shown in FIG. 1, the container body 1 is connected to the bottom 11 at the lower end. The bottom 11 may be conical or polygonal pyramidal. The lower side of the container body 1 is closed by the bottom 11. The bottom 11 is made of various materials such as plastic or glass, and may be transparent, colored, or opaque. The bottom 11 may be integrally formed with the container body 1.

[0054] As shown in FIG. 3, in the first cylindrical body 2, first spacers 21 and 22 are provided at the corners of the outer peripheral surface of the first cylindrical body 2 and at the upper and lower axial ends of the first cylindrical body 2. Further, in the second cylindrical body 3, a second spacer 31 is provided at the corner of the outer peripheral surface of the second cylindrical body 3 and at the upper axial end 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 certain interval L for the cells to flow during rotational culture can be secured between the inner surface of the container body 1 and the outer surface of the first cylindrical body 2. Also, a certain interval L can be provided between the inner surface of the container 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 inhibiting the fluidity of the cells during culture.

[0055] As shown in FIGS. 5 and 6, the first cylinder 2 and the second cylinder 3 are polygonal columns having an empty internal space, and the first cylinder 2 and the second cylinder 3 have a plurality of square flat panels 2A and flat panels 3A. In this embodiment, the first cylinder 2 and the second cylinder 3 each have six square flat panels. The container body 1, the first cylinder 2, and the second cylinder 3 are fixed at regular intervals.

[0056] The first cylinder 2 and the second cylinder 3 have the same rotation axis X as the container body 1. That is, the first cylinder 2 and the second cylinder 3 and the container body 1 are coaxial cylinders. Here, coaxial means substantially coaxial, but includes, for example, a state where one axis is displaced from the other axis by about several millimeters. In this embodiment, the first cylinder 2 and the second cylinder 3 are hexagonal columns, but are not limited thereto. In other embodiments, the first cylinder 2 and the second cylinder 3 may be various polygonal columns such as pentagonal columns or octagonal columns. The first cylinder 2 and the second cylinder 3 preferably have the same polygonal shape as the container body 1, but are not limited thereto. By installing the first cylinder 2 and the second cylinder 3 inside the container body 1, both the inner and outer surfaces of the first cylinder 2 and the second cylinder 3 can be used as the immersion area of the culture medium, so that a wide cell adhesion surface for cells to adhere and grow in the container can be ensured.

[0057] As shown in FIGS. 2 and 5, in this embodiment, the first spacer 21 is provided at all the corner portions of the outer peripheral surface of the first cylindrical body 2 and at the upper end portion in the axial direction of the first cylindrical body 2. As shown in FIGS. 2 and 6, the first spacer 22 is provided at all the corner portions of the outer peripheral surface of the first cylindrical body 2 and at the lower end portion in the axial direction of the first cylindrical body 2. The first spacers 21 and 22 may be provided at some of the corner portions instead of all the corner portions of the outer peripheral surface of the first cylindrical body 2. The first spacers 21 and 22 may be provided at the central portion in the axial direction instead of the axial end portions of the first cylindrical body 2. By providing the first spacers 21 and 22 at the corner portions of the outer peripheral surface of the first cylindrical body 2 and at the axial end portions of the first cylindrical body 2, the occurrence of non-uniform cell adhesion due to the liquid flow during rotational culture can be reduced. Further, by providing the first spacer 22 at the lower end portion in the axial direction of the first cylindrical body 2, the first cylindrical body 2 can be prevented from falling onto the bottom portion 11 of the container body 1.

[0058] As shown in FIGS. 7(a) and 7(b), it is preferable that the end portions 21a and 22a of the first spacers 21 and 22 are V-shaped so as to fit into the corner portions of the inner peripheral surface of the container body 1. By forming the end portions 21a and 22a of the first spacers 21 and 22 into a V shape, it becomes possible to fix the first cylindrical body 2 at a certain interval L from the container body 1 in the internal space of the container body 1.

[0059] As shown in FIGS. 2 and 5, in this embodiment, the second spacer 31 is provided at all the corner portions of the outer peripheral surface of the second cylindrical body 3 and at the upper end portion in the axial direction of the second cylindrical body 3. The second spacer 31 may be provided at some of the corner portions instead of all the corner portions of the outer peripheral surface of the second cylindrical body 3. The second spacer 31 may be provided at the lower end portion or the central portion in the axial direction instead of the upper end portion in the axial direction of the second cylindrical body 3. By providing the second spacer 31 at the corner portion of the outer peripheral surface of the second cylindrical body 3 and at the axial end portion of the second cylindrical body 3, the occurrence of non-uniform cell adhesion due to the liquid flow during rotational culture can be reduced.

[0060] As shown in Fig. 8(a), it is preferable that the end portion 31a of the second spacer 31 is V-shaped so as to fit into the corner of the inner peripheral surface of the first cylindrical body 2. By making the end portion 31a of the second spacer 31 V-shaped, it becomes possible to fix the second cylindrical body 3 at a certain interval from the first cylindrical body 2 in the internal space of the container body 1. As shown in Fig. 8(b), in this embodiment, although no second spacer is provided at the lower end portion in the axial direction of the second cylindrical body 3, a second spacer may be provided at the lower end portion in the axial direction of the second cylindrical body 3.

[0061] In this embodiment, the cell culture container is provided with the first cylindrical body 2 and the second cylindrical body 3, and two cylindrical bodies are provided, but it is not limited thereto. In other embodiments, the cell culture container may be provided with one cylindrical body or three or more cylindrical bodies.

[0062] 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, it is also possible to use an opaque resin that can block light according to the culture conditions. Also, a material that selectively absorbs or transmits a specific wavelength range may be used.

[0063] The lid body 4 is connected to the upper end of the container body 1. The lid body 4 may be connected to the upper end of the container body 1 by an adhesive, by welding, or by mechanical coupling.

[0064] The lid body 4 includes a neck portion 41 having an opening and an external thread for connection to the cap 5, and a shoulder portion that covers the upper side of the container body 1. The lid body 4 can prevent the first cylindrical body 2 and the second cylindrical body 3 from coming off the container body 1 and fix the first cylindrical body 2 and the second cylindrical body 3 to the container body 1.

[0065] The cap 5 is provided with an internal thread for coupling with the external thread of the neck portion 41 of the lid body 4. The cap 5 is screwed onto the neck portion 41 of the lid body 4, closes the opening of the lid body 4, and is used to seal the cell culture vessel. The cap 5 can be unscrewed from the neck portion 41 of the lid body 4 to open the cell culture vessel.

[0066] The cell culture vessel according to this embodiment includes at least one cylindrical body. Therefore, the cell culture vessel can provide an internal surface area sufficient for large-scale culture.

[0067] FIG. 9 shows a side view of a cell culture vessel according to an embodiment. FIG. 10 shows a perspective view of the first cylindrical body of the cell culture vessel according to an embodiment. FIG. 11 shows a perspective view of the second cylindrical body of the cell culture vessel according to an embodiment. FIG. 12 shows a cross-sectional view of the cell culture vessel along the line P'-P' of FIG. 9. FIG. 13 shows a cross-sectional view of the cell culture vessel along the line Q-Q of FIG. 9.

[0068] As shown in FIGS. 9, 10, 12, and 13, the cell culture vessel may be provided with a support 23 for supporting the second cylindrical body 3 when the cell culture vessel is placed vertically at the axial end of the first cylindrical body 2 on the bottom side of the container body 1. 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 container body 1. By fitting the groove 33 of the second cylindrical body 3 into the support 23 of the first cylindrical body 2, the first cylindrical body 2 and the second cylindrical body 3 can be securely fixed in the internal space of the container body 1.

[0069] FIG. 14 shows a cross-sectional view of the cell culture vessel along the line P-P according to an embodiment. FIGS. 15 to 17 show cross-sectional views of the cell culture vessel along the line Q-Q according to an embodiment.

[0070] The cell culture vessel shown in FIGS. 14 and 15 to 17 is further provided with a second spacer 32.

[0071] In FIG. 15, the first spacer 22 is provided at all the corner portions of the outer peripheral surface of the first cylindrical body 2 and at the lower end portion in the axial direction of the first cylindrical body 2, but is not limited thereto. As shown in FIGS. 16 and 17, the first spacer 22 may be provided at some of the corner portions, rather than at all the corner portions of the outer peripheral surface of the first cylindrical body 2. Similar to the first spacer 21, the end portion of the first spacer 22 may be V-shaped so as to fit into the corner portion 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 at a certain interval L with respect to the container body 1.

[0072] In FIG. 15, the second spacer 32 is provided at all the corner portions of the outer peripheral surface of the second cylindrical body 3 and at the lower end portion in the axial direction of the second cylindrical body 3, but is not limited thereto. As shown in FIGS. 16 and 17, the second spacer 32 may be provided at some of the corner portions, rather than at all the corner portions of the outer peripheral surface of the second cylindrical body 3. Similar to the second spacer 31, the end portions of the second spacer 32 may be V-shaped so as to fit into the corner portion of the inner peripheral surface of the first cylindrical body 2. The second spacer 32, together with the second spacer 31, fixes the second cylindrical body 3 at a certain interval with respect to the first cylindrical body 2.

[0073] FIG. 15 shows an embodiment of a cell culture container in which the first spacer 22 is provided at all the corner portions of the outer peripheral surface of the first cylindrical body 2 and at the lower end portion in the axial direction of the first cylindrical body 2, and the second spacer 32 is provided at all the corner portions of the outer peripheral surface of the second cylindrical body 3 and at the lower end portion in the axial direction of the second cylindrical body 3. FIG. 16 shows an embodiment of a cell culture container in which the first spacer 22 and the second spacer 32 are provided at two opposing corner portions. FIG. 17 shows an embodiment of a cell culture container in which the first spacer 22 and the second spacer 32 are provided at three corner portions. In FIG. 17, the second spacer 32 is configured to fit into three corner portions of the inner peripheral surface of the first cylindrical body 2 where the first spacer 22 is not disposed.

[0074] The first cylindrical body 2 and the second cylindrical body 3 are securely attached to the container body 1 by the first spacer 22 and the second spacer 32, and the first spacer 21 and the second spacer 31.

[0075] FIG. 18 shows an example of a cell culture vessel in which at least two linear marks extending in the axial direction of the container body are provided on the outer surface of the flat panel of the container body according to an embodiment. As shown in FIG. 18, it is necessary to provide at least two linear marks 13 on one surface of the flat panel 1A of the container body 1, but additional linear marks 13 may be provided. Further, the linear marks 13 may be in a dashed line form. The linear marks 13 may be printed or may be linear protrusions. By providing a plurality of broken ends inside the linear marks 13, it becomes easy to accurately match the imaging field of view for each imaging when automating microscopy imaging using functions such as autofocus. That is, by physically indicating the imaging start point and the focus adjustment start point by the linear marks 13, even if the cell culture vessel is moved without stationary the cell culture vessel on the imaging device, microscopy imaging in the same region can be performed. As a result, the state of the cells can be accurately determined and it can be determined whether to continue culturing the cells.

[0076] The cap 5 of the cell culture vessel can be provided with various types of valves. FIG. 19 shows a perspective view of the cap of the cell culture vessel according to this embodiment. FIG. 20 shows a top view of the cap of the cell culture vessel according to this embodiment. FIG. 21 shows a front view of the cap of the cell culture vessel according to this embodiment. FIG. 22 shows an exploded perspective view of the valve attached to the cap of the cell culture vessel according to an embodiment. FIG. 23 is a cross-sectional perspective view showing the internal structure of the valve attached to the cap of the cell culture vessel according to an embodiment. FIG. 24 shows a schematic diagram of connecting a syringe through the valve attached to the cap of the cell culture vessel according to this embodiment. FIG. 25 shows a schematic diagram of connecting a syringe with a luer lock structure to the valve attached to the cap of the cell culture vessel according to this embodiment. FIG. 26 shows a schematic diagram of connecting a rubber tube to the valve attached to the cap of the cell culture vessel according to this embodiment via a luer lock connector. FIG. 27 shows a front view of a modified example of the cap of the cell culture vessel according to an embodiment.

[0077] Referring to FIGS. 19 to 21, the cap 5 includes a top plate 55, a peripheral panel 56 connected along the periphery of the top plate 55 around it, 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 device 6 such as a syringe needle or a needle hub is inserted. Each valve 52 may adopt a split septum method or may be a mechanical valve. Each valve 52 may be made of isoprene, silicon, a combination thereof, or a similar material.

[0078] As shown in FIGS. 22 to 24, the cap 5 includes a cap body and a valve 52 attached to the cap body and capable of supplying and discharging 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 / discharge device 6 is inserted. Usually, cell culture is carried out in an open system in a CO2 incubator, but by providing the valve 52 on the cap 5, a closed system culture can be performed. Here, replacement of the gas phase (gas) in the cell culture container is important. By using a supply / discharge device 6 such as a luer lock syringe, the internal pressure of the cell culture container can be released while preventing contamination by foreign substances (such as bacteria). In addition, it becomes possible to easily inject gas or liquid into the cell culture container. Thereby, easy replacement of the internal atmosphere of the cell culture container is realized. 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, silicon, a combination thereof, or a similar material.

[0079] In this embodiment, the cap 5 includes two valves (first valve, second valve) 52, but is not limited thereto. In another embodiment, the cap 5 may include one valve or three or more valves. When there are two or more valves 52 in the cap 5, gas or liquid may be introduced from the outside through one valve (first valve or second valve) 52, and the gas or liquid inside the cell culture vessel may be drawn out through the other valve (second valve or first valve) 52. In order to exchange the internal atmosphere of the cell culture vessel, gas may be continuously injected into the cell culture vessel through a membrane filter, and at the same time, gas may be periodically discharged from the inside of the cell culture vessel. Further, when adding supplements or cell suspensions to the medium during culturing, using the valve 52 without opening the cap 5 enables a cleaner operation while reducing the risk of contamination.

[0080] Figures 19 to 24 show that the valve 52 uses an example of the split septum method. The valve 52 has a split septum as an insertion structure (first valve body, second valve body) 521. The split septum has a slit portion 522 that is normally closed and is configured to open when the supply / discharge device 6 is pushed in. With this system, it is possible to easily access the inside of the cell culture vessel via the valve 52 without unscrewing the cap 5. Such a split septum method or mechanical valve serves as a check valve and has the effect of determining the direction of the in-out fluid flow, and since it is always closed, the sealing degree can be further increased. The valve 52 can employ other known split septum methods or mechanical valves.

[0081] As shown in Figure 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 substance or sample a small amount of cultured cells without opening the cap 5.

[0082] 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. By making such a connection, gas and liquid exchange becomes possible without opening the cap 5. Note that by using the valve 52 to seal and perform gas phase replacement, it is possible to set the atmosphere inside the container to a special environment such as a low oxygen state or a high oxygen state. Since this embodiment is a septum system, the chemical solution injection port into the rubber tube 120 can ensure airtightness and cleanliness. Changing the gas phase environment inside the cell culture vessel is also an important condition for culturing cells and can greatly change the behavior of the cells.

[0083] The internal thread of the cap 5 can also be applied to containers other than this embodiment. The cap 5 equipped with the valve 52 can be universally used for 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 gases with arbitrary phases and partial pressures through the valve 52. Therefore, the internal atmosphere of the cell culture vessel can be easily replaced to create a culture environment such as low oxygen culture. Specifically, the cap 5 according to this embodiment can be applied to various types of cell culture vessels, for example, T25, T75, T175, and T225.

[0084] FIG. 27 shows a cap 50 having two diameters. T-flasks widely used as cell culture vessels are of sizes T175 and T225. For example, by corresponding the cap 50a to the size of T175 and the cap 50b to the size of T225, it is possible to correspond to two commonly used sizes of T-flasks. As a result, the convenience for the user can be improved.

[0085] (Cell culture method) A cell culture method using a cell culture vessel equipped with the above valve 52 on the cap 5 will be described below.

[0086] The cell culture method using the above cell culture vessel includes a culturing step of culturing the cells in the cell culture vessel by rotating the horizontally placed cell culture vessel at a predetermined rotational speed around the axial direction of the cell culture vessel, a confirmation step of examining the state of the cells in the cell culture vessel every time a predetermined culture period elapses, an exchange step of exchanging the liquid medium and / or gas in the cell culture vessel via the valve 52 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 1 and both the inner and outer surfaces of the first cylinder 2 after a predetermined culture period has elapsed.

[0087] According to the above configuration, since cell culture can be continuously performed in a closed system, mass culture of useful cells becomes possible while preventing contamination by foreign substances (such as bacteria). Furthermore, since it is possible to automatically perform regular medium exchange and gas exchange via the valve, it becomes possible to make cell culture more efficient. The target cells recovered by this cell culture method are adherent cells and attached cells including iPS cells and mesenchymal stem cells, and examples include stem cells such as adipose tissue-derived stem cells, amniotic tissue-derived stem cells, human umbilical cord Wharton's jelly-derived stem cells, and chorionic tissue-derived stem cells.

[0088] Since the above cells can differentiate into tissues such as muscle, bone, nerve, and fat, they are expected to contribute to regenerative medicine.

[0089] The cell culture method using the above cell culture vessel may include an exchange step of exchanging the liquid medium and / or gas in the cell culture vessel via the valve 52 every time a predetermined period elapses, 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 in the cell culture vessel via the valve 52.

[0090] According to the above configuration, by optimizing the cell state through regular medium replacement and gas exchange, useful substances can be effectively produced by the cells. In addition, since regular medium replacement and gas exchange can be automatically performed via a valve, the production of the supernatant can be made more efficient. The cells that produce useful substances by this supernatant production method are adherent cells and attached cells including iPS cells and mesenchymal stem cells, and examples thereof include stem cells such as adipose tissue-derived stem cells, amniotic tissue-derived stem cells, human umbilical cord Wharton's jelly-derived stem cells, and chorionic tissue-derived stem cells. The supernatant contains growth factors and proteins produced from these cells. Specifically, it includes cytokines, exosomes, EGF (epidermal growth factor), KGF (keratinocyte growth factor), IGF (insulin-like growth factor), etc. The obtained supernatant is used for improving rheumatism, improving skin diseases, anti-inflammatory effects, preventing arteriosclerosis, etc. It is also used as a component for use in treatments in the fields of anti-aging medicine and preventive medicine, and is also used as a component of cosmetics and beauty liquids.

[0091] FIG. 28 is a flowchart showing the steps of a cell culture method and a cell recovery method using a cell culture container according to an embodiment. FIG. 29 is an explanatory view of a gripping device attached to a cell culture container according to an embodiment.

[0092] (Cell Recovery Method) 1. Primary Culture Step When culturing adipose tissue-derived stem cells, the adipose tissue collected from the patient's abdomen or the like is used as a specimen. When culturing amniotic tissue-derived stem cells, the amnion is used as a specimen. When culturing human umbilical cord Wharton's jelly-derived stem cells, the human umbilical cord is used as a specimen. When culturing chorionic tissue-derived stem cells, the chorion is used as a specimen.

[0093] After the specimen is transported and received (S1), pretreatment is performed (S2). In the pretreatment, after the cells are detached by the detachment enzyme (S3), the cells are extracted and recovered (S4). The recovered cells are seeded in a cell culture container into which a liquid medium has been introduced (S5), and first, static culture is started (S6). As the liquid medium, a commercially available medium, for example, DMEM (Dulbecco's Modified Eagle Medium) may be used, other components may be added using this as a basal medium, or an original liquid medium may be prepared and used. In the static culture, regular microscopy is performed every time a predetermined culture period elapses, and the state of the cells in the cell culture container is confirmed (S7). Also, regular medium exchange and gas exchange are performed via the valve 52 every time a predetermined culture period elapses (S8). Further, regular microscopy is performed every time a predetermined culture period elapses (S9), and after sufficient cell growth is confirmed, the primary culture is completed (S10).

[0094] 2. Expansion culture process For the cells obtained in the primary culture, cell counting is performed (S11). The cell counting is preferably performed by a non-invasive method, and the number of cells is measured by microscopic observation. Non-invasive measurement of cultured cells can improve the efficiency of cell culture because it does not damage the cells and can be performed automatically. After measuring the number of cells, a detachment enzyme is added via the valve 52 to detach the cells attached and grown on the inner surface of the cell culture container (S12). Next, a required amount of liquid medium is added (S13), and after suspension, rotary culture is started (S14). As the liquid medium, the same liquid medium as that used in the primary culture may be used, other components may be added using this as a basal medium, another liquid medium may be used, or an original liquid medium may be prepared and used. Also, at this time, if necessary, quality inspection of the cell culture solution and the cells may be performed (S19). The quality inspection is performed by quantitative PCR to confirm the absence of infection by bacteria or the like.

[0095] Rotary culture is preferably performed by attaching a cell culture vessel to the 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 connection portion 7b that is connected to a motor (not shown). Since the gripping portion 7a grips only a part of the container body 1, it is possible to prevent damage to the area photographed by the microscope M. In addition, observation can be performed by examining the surface without the gripping portion 7a. If necessary, by shifting the insertion of the gripping portion 7a by one surface, all surfaces can be easily and thoroughly observed. For example, before and after medium replacement or gas exchange, all surfaces can be thoroughly observed by shifting the insertion position of the gripping portion 7a by one surface. Rotary culture is performed by rotating the horizontally placed cell culture vessel at a predetermined rotation speed around the axial direction (circumferential direction) of the cell culture vessel. 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 container body 1 and both the inner and outer surfaces of the first cylinder 2 are always covered with the liquid medium by surface tension. Fig. 29 shows the cell culture vessel rotating in the right direction (clockwise) as viewed from the top of the cell culture vessel, but it may rotate in the left direction (counterclockwise). In rotary culture, regular microscopic examination is performed every time a predetermined culture period elapses, and the state of the cells in the cell culture vessel is confirmed (S15). In addition, regular medium replacement and gas exchange are performed via the valve 52 of the cap 5 every time a predetermined culture period elapses (S16). Furthermore, regular microscopic examination is performed every time a predetermined culture period elapses (S17), and after the growth of the cells is confirmed, the scale-up culture is completed (S18).

[0096] 3. Cell storage process Measure the cell count of the cells obtained by expansion culture by cell counting (S20). The cell count is preferably performed by a non-invasive method as described above. After measuring the cell count, a detachment enzyme is added via the valve 52 of the cap 5 to detach the cells attached and grown inside the cell culture vessel (S21). At this time, if necessary, a part of the cell culture solution may be sampled for cell counting to adjust the cell count in the solution. At this time, the cell count is performed by a general method and measured using a hemocytometer or an automatic cell counter using a 0.3 to 0.5% trypan blue staining solution. The cell culture solution in the cell culture vessel is dispensed into a tube for cryopreservation (S22) and stored in a deep freezer, liquid nitrogen, or a liquid nitrogen vapor phase (S23). The cryopreserved cells are used for the production of the supernatant after the revival and expansion culture process described later.

[0097] 4. Cell recovery step When recovering cells for transplantation, after detaching the cells attached and grown inside the cell culture vessel (S21), the required amount of cells is aliquoted (S24). After washing the cells with PBS or the like (S25), they are packaged into a predetermined dosage form. Also, when aliquoting the required amount of cells (S24), cells are sampled for quality inspection (S27), quality inspection of predetermined items is performed (S28), and after determination of the inspection results (S29), the dosage form of the cells with confirmed safety is delivered to medical institutions, research facilities, etc.

[0098] Figure 30 is a flowchart showing the steps of a cell culture method and a supernatant production method using a cell culture vessel according to an embodiment. Since S1 to S23 in Figure 30 are the same as S1 to S23 in Figure 28, the description thereof is omitted.

[0099] 5. Revival and expansion culture step The cryopreserved cell culture solution (S23) is thawed (S30). Cell counting is performed as necessary to confirm the number of cells in the solution (S31). At this time, cell counting is performed by a general method. Next, after seeding the cells in a cell culture container into which a liquid medium has been introduced (S32), rotational culture is started (S33). In the rotational culture, regular microscopy is performed every time a predetermined culture period has elapsed to confirm the state of the cells in the cell culture container (S34). Also, regular medium replacement and gas exchange are performed via valve 52 every time a predetermined culture period has elapsed (S35). Further, regular microscopy is performed every time a predetermined culture period has elapsed (S36). After sufficient cell growth is confirmed, the scale-up culture is completed (S37). Thereafter, it may be switched from the cell culture conditions to the supernatant production conditions (S38), and the cells may be recovered as cells for treatment in regenerative medicine involving cell transplantation upon completion of the scale-up culture (S24).

[0100] 6. Supernatant production step When switched from the cell culture conditions to the supernatant production conditions (S38), the supernatant production step is started (S39). In the supernatant production step, regular medium replacement and gas exchange are performed via valve 52 every time a predetermined period has elapsed (S40). When a useful substance is produced from the cells, the supernatant containing the useful substance is recovered (S41). The supernatant production step may be performed with the cell culture container stationary, but from the viewpoint of creating an environment closer to that in a living body with body fluid circulation, it is preferably performed by rotating the cell culture container at a predetermined rotational speed around its axial direction.

[0101] 7. Quality inspection step In the quality inspection, it is confirmed whether there is any contamination or infection of foreign matter in the supernatant.

[0102] An endotoxin test is performed (S42). As a general method for detecting endotoxin, the gelation method can be mentioned. The gelation method detects endotoxin based on the coagulation reaction of the lysate reagent due to 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 judged positive, and if the gel is not formed, it is judged negative.

[0103] A sterility test is performed (S43). The sterility test is performed by treating the supernatant by a prescribed method and culturing it, and visually confirming the presence or absence of microorganisms that grow in the medium.

[0104] A mycoplasma test is performed (S44). For the mycoplasma test, any one of the mycoplasma culture method, DNA staining method, and PCR (polymerase chain reaction) method may be performed, or these may be used in combination. The mycoplasma culture method is a test method in which a medium optimized for mycoplasma is used and a test sample is inoculated. The DNA staining method is a test method in which the nucleus of mycoplasma is counterstained with Hoechst or DAPI and confirmed by imaging using a fluorescence microscope. The PCR (polymerase chain reaction) method is a test method based on the amplification of the DNA of mycoplasma when it is present in the sample.

[0105] The supernatant whose safety has been confirmed by the above three quality inspections (S45) is delivered to medical institutions, research facilities, etc.

[0106] This embodiment can be implemented in various different forms. It is understood by those skilled in the art to which the present disclosure pertains that it may be implemented in other specific forms without changing its spirit or essential characteristics. Therefore, it should be understood that the foregoing embodiments are illustrative in all aspects and not restrictive.

Description of Reference Numerals

[0107] 1 Container body 2 First cylinder 21, 22 First spacer 21a end 23 support 3 second cylinder 31, 32 second spacer 33 groove 4 lid 41 neck portion 5, 50 cap 52 first valve, second valve 521 first valve body, second valve body 522 slit portion 6 supply and drainage appliance

Claims

1. A bottomed container body having a polygonal columnar internal space, A polygonal columnar first cylinder disposed coaxially with the container body inside the container body, A lid attached to the axial end of the container body, A cap attached to the cylindrical neck portion of the lid, A first spacer provided at a corner of the outer peripheral surface of the first cylinder and at the axial end of the first cylinder, Comprising, The first spacer is fitted into a corner of the inner peripheral surface of the container body, Cell culture container.

2. The cell culture container according to claim 1, A polygonal columnar second cylinder disposed coaxially with the first cylinder inside the first cylinder, A second spacer provided at a corner of the outer peripheral surface of the second cylinder and at the axial end of the second cylinder, Further comprising, The second spacer is fitted into a corner of the inner peripheral surface of the first cylinder, Cell culture container.

3. The cell culture container according to claim 1 or 2, The first spacer has an end portion that fits into a corner of the inner peripheral surface of the container body and is V-shaped, Cell culture container.

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

5. The cell culture container according to claim 2, On the axial end of the first cylinder on the bottom side of the container body, a support for supporting the second cylinder when the cell culture container is placed vertically is provided, On the axial end of the second cylinder on the bottom side of the container body, a groove into which the support fits is provided, Cell culture container.

6. The cell culture container according to claim 1 or 2, 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.

7. The cell culture container according to claim 1 or 2, The cap is, A cap body, 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 comprising, 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.

8. The cell culture container according to claim 7, The cap is, The cell culture container further includes 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. 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. Cell culture container. **Claim 9** 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 observing the state of the cells in the cell culture container under a microscope 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 target cells attached to the inner surface of the container body and both the inner and outer surfaces of the first cylindrical body after a predetermined culture period has elapsed.

Images