Container and kit for preparing cell cultures in a highly clean space

Abstract

To provide a tool that can simply prepare a cell culture under a clean environment without introducing a large-scale installation for preparing a space of high cleanliness in preparing a cell culture.SOLUTION: Provided is an airtight bag-shaped container having: an internal space having the cleanliness of Class 1 to 9 regulated in ISO14644-1 by an air filter; an operation part which facilitates an operation inside a container from outside the container; and a housing part which houses materials necessary for preparing various kinds of cell culture.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a sealed bag-like container for preparing cell cultures, which contains an internal material used for preparing cell cultures and has a clean interior. [Background technology]

[0002] In recent years, attempts have been made to transplant various cells to repair damaged tissues, etc. For example, attempts have been made to use fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction (Non-Patent Document 1).

[0003] As an example of such an attempt, a sheet-shaped cell culture was developed, in which cells were formed into a sheet. Some of the uses of such cell cultures have reached the stage of clinical application, and they are already being used in regenerative medicine and other medical settings (Non-Patent Document 2).

[0004] The preparation of cell cultures for clinical applications requires work in a highly clean space, such as a sterile area (Non-Patent Document 2). For example, Patent Document 1 discloses a cell culture system that reduces the risk of contamination within a sterile area. Patent Document 2 discloses a cell culture system that reduces the risk of cross-contamination. However, both systems require the introduction of large-scale equipment such as an isolator. Furthermore, the system described in Patent Document 1 poses the risk of passing through a non-sterile area when introducing cell cultures into a centrifuge or incubator. In the system described in Patent Document 2, machines such as a centrifuge are installed in a sterile area, which increases the risk of damage to the equipment during the sterilization procedure. There is also a system described in Patent Document 3 that does not require large-scale equipment, but this system only thaws frozen cells and does not include culturing. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5592080 specification [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-259378 [Patent Document 3] Japanese Patent Application Laid-Open No. 2001-70402 [Non-patent literature]

[0006] [Non-Patent Document 1] Haraguchi et al., Stem Cells Transl Med. 2012 Feb;1(2):136-41 [Non-patent document 2] HeartSheet (registered trademark) package insert (revised February 2019) Summary of the Invention [Problem to be solved by the invention]

[0007] As the clinical application of cell cultures for transplantation advances, there is a growing demand for more widespread treatments using cell cultures. Because such cell cultures for transplantation must be prepared in the hospital where the transplant surgery is performed, handling these cell cultures for clinical applications requires a highly clean space within the hospital for preparing these cell cultures. Preparing such a space requires the introduction of large-scale equipment, which requires significant capital investment. Therefore, the introduction of such equipment and the handling of cell cultures for transplantation have been limited to a select few highly specialized hospitals. The present invention addresses the challenge of minimizing the need for large-scale equipment and enabling cell culture preparation in a simple, clean environment. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems and have found that, instead of using large facilities such as isolators and safety cabinets that have been conventionally used when preparing cell cultures, a space that is kept sufficiently clean for preparing cell cultures can be secured by using a sealed bag-like container whose internal space has been made highly clean by an air filter or the like. By preparing cell cultures inside such containers, it has been found that cell cultures can be prepared in a clean space without the need for large-scale facilities, and this has led to the completion of the present invention.

[0009] That is, the present invention relates to the following. [1] A sealed bag-like container, Air filters to maintain the cleanliness of the internal space within the specified range of Class 1 to 9 specified in ISO14644-1, one or more storage compartments for storing items used in the preparation of cell cultures; an operating unit capable of operating items used in the preparation of the cell culture from the outside; A container comprising: [2] The container described in [1], which has an expandable structure. [3] A container according to [1] or [2], which is made of a material that can be frozen. [4] The container according to any one of [1] to [3], wherein the operating part has a glove-like structure that protrudes into the inside of the container.

[0010] [5] The housing comprises: a cell culture section for accommodating and culturing the seeded cells; a liquid storage section for storing a plurality of liquids; a centrifugation processing section for accommodating the cell suspension to be centrifuged; a cell storage unit for storing cells to be cultured; a waste liquid section for storing waste liquid; The container according to any one of [1] to [4], comprising one or more selected from the group consisting of: [6] The container according to any one of [1] to [5], wherein the storage section has a structure that allows it to be separated from the container. [7] The container according to [5] or [6], wherein the cell culture section has a temperature-responsive cell culture substrate. [8] A kit for preparing a cell culture, comprising the container according to any one of [1] to [7]. [9] The cell culture preparation kit according to [8], further comprising a culture vessel having a temperature-responsive cell culture substrate.

[10] The cell culture preparation kit according to [8] or [9], further comprising an instrument used for preparing the cell culture.

[0011]

[11] A method for preparing a cell culture, comprising the steps of: (i) seeding the cells to be cultured; (ii) culturing the cells; The method comprising:

[12] A method for preparing a cell culture according to

[11] , comprising the steps of: a) heating the bag-shaped container in a contracted state; b) placing the container in a centrifuge bucket; or c) placing the container in an incubator.

[0012]

[13] A method for preparing a cell culture according to

[11] or

[12] , comprising a step of making the operating section and each storage section usable when the bag-shaped container is in an extended state.

[14] The method according to any one of

[11] to

[13] , further comprising the step of removing the contents of the container without affecting the cleanliness of the interior of the container.

[15] The method according to any one of

[11] to

[14] , wherein the cell culture to be prepared is a cell culture for transplantation.

[16] The method according to

[15] , wherein the cell culture for transplantation is a sheet-shaped cell culture.

[17] A cell culture for transplantation prepared by the method according to any one of

[11] to

[16] . [Effects of the Invention]

[0013] According to the present invention, cell cultures for transplantation can be handled without the need for large-scale equipment, simply by preparing the bag-shaped container according to the present invention and, if necessary, a centrifuge, an incubator, etc. In other words, even if a hospital is not large enough to invest in large-scale equipment, it can handle cell cultures for transplantation as long as it has the space to deploy the container according to the present invention and equipment such as a centrifuge and an incubator.

[0014] Furthermore, when the container according to the present invention is used, the cell culture remains in the container, which is a clean space, from the beginning of preparation until it is removed for use, so there is no opportunity for the cell culture to come into contact with the outside world, and the risk of contamination is reduced. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows a schematic diagram of a bag-like container according to an embodiment of the present invention. [Figure 2] FIG. 2 shows a schematic diagram of the bag-shaped container being deflated and placed in the centrifuge bucket. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. In one aspect, the present invention relates to a bag-like container for preparing a cell culture. In the present invention, a "cell culture" is prepared by cell culture, and the cell culture may be an adherent culture or a suspension culture. Examples of cell cultures include, but are not limited to, sheet-shaped cell cultures, cell cultures for injection, and bulk cell cultures. In one embodiment, the cell culture is used for cell transplantation. Preferably, the cell culture is a sheet-shaped cell culture for transplantation or cultured cartilage (e.g., JACK (registered trademark)). A sheet-shaped cell culture is a sheet-like structure in which cells are linked to one another. The cells may be directly linked to one another via cell adhesion factors or indirectly linked via extracellular matrices. The sheet-shaped cell culture may be a single layer, or may have multiple layers such as two, three, four, or more layers, or may have a three-dimensional structure without a layered structure.

[0017] In the present invention, a "bag-like container" is a container capable of creating a sealed space separating the interior of the container from the outside and large enough to allow cell culture preparation inside. In one embodiment, the container has an expandable structure. Here, an expandable structure includes, but is not limited to, a container with a bellows-like structure that can expand and contract. In one embodiment, the container has a structure that is resistant to holes and tears during use in cell culture preparation in order to maintain internal airtightness. Here, an expandable structure includes, but is not limited to, a container with a double structure. In one embodiment, the container is made of a freezable material. Here, freezable materials include, but are not limited to, polyester, polyethylene, polypropylene, and polyurethane. Preferably, the freezable material is polyester. For example, the freezable container allows frozen cells or the like to be stored in the container in a frozen state.

[0018] Generally, air filters are used to remove dirt and dust from the air to make it clean. Air filters include, but are not limited to, HEPA filters, ULPA filters, gas removal filters, etc., and are used in air purifiers, clean benches, etc. In the present invention, the "air filter" is incorporated into the surface of the container and is the only part connecting the inside of the container to the outside world. When taking in outside air, the air filter removes fine particles from the air and prevents them from being brought into the container. When discharging air from inside the container, the air filter captures substances inside and prevents them from escaping to the outside world. This function allows the air filter to keep the internal space of the container clean. This function also prevents internal factors from contaminating the outside world.

[0019] In one embodiment, the air filter enables the space within the container to be kept highly clean. Preferably, the air filter provides the space within the container with a cleanliness level of Class 1 to 9 as specified in ISO 14644-1 or JIS B9920, or a cleanliness level of Class 1 to 100,000 as specified in Federal Standard 209E, and more preferably, the space within the container with a cleanliness level of Class 1 to 5 as specified in ISO 14644-1. In one embodiment, the container may be manufactured in a highly clean space, and the air filter may maintain the space within the container at the high level of cleanliness at the time of manufacture. In one embodiment, the air filter may be incorporated into an air purifier or the like to automatically increase the cleanliness of the space within the container. In one embodiment, the air filter is a vent filter. In one embodiment, when the container is extended, air is drawn into the container through an air filter. As the air passes through the air filter, dust, particles, etc. are removed from the air, making it clean. In another embodiment, when the container is deflated, air is discharged from the container through an air filter. As the air passes through the air filter, factors present in the container are removed from the air, preventing them from being carried out to the outside world.

[0020] In the present invention, the "operating part" is a part provided on the surface of the container that allows the inside of the container to be operated from the outside. In one embodiment, the operating part may have a glove-like structure that protrudes into the inside of the container, allowing a hand to be inserted into it to operate parts of the container or tools inside. In one embodiment, the operating part may be contracted together with the container when it is contracted, and may only become usable when the container is expanded and the space inside the container expands enough to allow the operating part to be expanded.

[0021] In the present invention, a "reservoir" refers to a portion provided inside a container for containing items used in the preparation of a cell culture or waste liquid generated during the preparation. Here, the reservoir has a structure that can be opened and closed to prevent items contained therein from leaking into the container during cell culture preparation. Items used in the preparation of a cell culture include, but are not limited to, prepared medium, washing solution, buffer solution, cell suspension, cells to be cultured, and tools used in the preparation of cell culture. Here, known tools used in cell culture can be used for the prepared medium, washing solution, and buffer solution. Furthermore, tools used in the preparation of a cell culture refer to all tools used in the preparation of a cell culture, excluding the reservoir itself, and include, but are not limited to, pipettes, tips, droppers, tweezers, and spatulas. In one embodiment, there may be multiple reservoirs.

[0022] The storage section may include one or more storage sections selected from the group consisting of a storage section functioning as a "cell culture section," a storage section functioning as a "liquid storage section," a storage section functioning as a "centrifugation processing section," a storage section functioning as a "cell storage section," and a storage section functioning as a "waste liquid section." In one embodiment, the storage section may have a structure that allows it to be separated from the container without affecting the cleanliness of the interior of the container. A structure that allows it to be separated from the container refers to a structure in which the space inside the container and the storage section are separated by, for example, heat sealing from the outside of the container, without affecting the space inside the container. In one embodiment, the storage section may contain a portion of the cell suspension and then be separated in order to measure the concentration of the cell suspension outside the container.

[0023] Here, the "cell culture section" refers to a section that accommodates seeded cells and prepares a cell culture. In one embodiment, the cell culture section may be a culture vessel. The culture vessel may be any known vessel used for cell culture, including, but not limited to, a petri dish, a culture flask, or a microplate. In a preferred embodiment, the bottom surface of the cell culture section is coated with a culture substrate. The culture substrate may be any known vessel used for cell culture, including, but not limited to, collagen, fibronectin, laminin, etc. In a more preferred embodiment, the bottom surface of the cell culture section is coated with a temperature-responsive cell culture substrate, such as one commercially available as UpCell (registered trademark) from CellSeed Co., Ltd.

[0024] Here, the "liquid storage section" refers to a storage section that stores various liquids other than cells used in the preparation of a cell culture, and is a section that can store culture media, washing solutions, buffer solutions, etc. In one aspect, the liquid storage section may have an openable and closable structure that can seal the liquid inside to prevent leakage. Such a structure is, for example, a zipper. Here, the "centrifugation processing section" refers to a section having a structure suitable for containing a cell suspension and centrifuging it during the preparation of a cell culture. The structure suitable for centrifugation here refers to, but is not limited to, a structure that is not damaged or deformed by the centrifugal force applied during centrifugation and has a pointed tip that allows the cells and the supernatant to easily separate after centrifugation.

[0025] Here, the "cell storage section" refers to a section for storing cells to be cultured prior to the preparation of a cell culture. In one embodiment, the cell storage section may be a container in which cryovials containing cells are stored. Here, the "waste liquid section" is a section that stores waste liquid generated during the preparation of cell cultures, and is structured so that the stored waste liquid does not leak out of the waste liquid section and does not affect the liquids or cultures stored in other storage sections.

[0026] In one embodiment, the storage unit may have multiple functions. For example, a storage unit may have the functions of both a centrifugation unit and a cell storage unit. That is, a storage unit storing cells to be cultured may have a structure suitable for centrifugation, and a washing solution may be added thereto to form a cell suspension, which may then be centrifuged. For example, a storage unit storing instruments used in preparing cell cultures may be used as a waste liquid unit after the instruments are removed.

[0027] In one aspect, the present invention relates to a kit for preparing a cell culture, which includes the above-mentioned bag-like container. The kit of the present invention may include, for example, cells for preparing a cell culture, a prepared medium, a washing solution, a buffer solution, a culture vessel, a centrifuge tube, a tube, an instrument used for cell culture, a transport container, an instruction manual, and the like, but is not limited thereto.

[0028] In another aspect, the present invention relates to a method for preparing a cell culture using a sealed, expandable bag-like container in which cleanliness is maintained. Here, "being able to expand and contract" means that the container can be expanded and / or contracted without affecting the internal conditions such as cleanliness. In the present invention, "preparation of cell culture" includes, but is not limited to, the following steps: (i) warming and thawing frozen cells; (ii) transferring the cells to a centrifugable container; (iii) adding a washing solution to the transferred cells; (iv) centrifuging to separate the supernatant from the cells; (v) discarding the supernatant, where steps (iii) to (v) may be repeated multiple times as necessary, and steps (i) to (v) are not necessary if the cells are not frozen; (vi) adding a medium to suspend the cells and seeding them into a container suitable for culture; (vii) incubating the seeded cells; (viii) washing the cell culture completed by the incubation in the above steps with a buffer solution; and (ix) recovering the cell culture.

[0029] In one embodiment, the "preparation of cell culture" may continue from step (vi) after step (i), in which the components of the solution storing the frozen cells are removed by medium exchange.

[0030] In one aspect, "cell culture preparation" may include a step of heating the bag-shaped container in a contracted state, placing the container in a centrifuge bucket, or placing the container in an incubator. In one aspect, "cell culture preparation" may include a step of making the operating unit and each storage unit usable in an extended state of the bag-shaped container. For example, by placing the container in a contracted state, the volume occupied by the container can be reduced, making it possible to store the container in an existing centrifuge or incubator for cell culture preparation. For example, by placing the container in an extended state, the volume inside the container can be increased, providing sufficient working space for cell culture preparation. In one embodiment, "preparing a cell culture" may further include a step of removing the contents of the container without affecting the cleanliness of the container. For example, this step allows the container containing the used liquid and the used waste liquid to be separated from the container. Furthermore, for example, this step allows the cell suspension obtained in steps (iii) to (v) to be removed from the container and the cell concentration to be measured.

[0031] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. 1 is a conceptual diagram of a container according to an embodiment of the present invention. In the drawings in this application, the size and location of each part are appropriately exaggerated for ease of explanation, and the size and location of each part in the drawing do not represent the actual size or location of each part. As shown in FIG. 1, a container 1 according to an embodiment of the present invention includes an air filter 2, an operating unit 3, and a storage unit 4. The container 1 may further include cells 5 to be cultured and tools 6 used to prepare a cell culture. The operating unit 3 is configured so that a user can insert his or her hand into the operating unit 3 to perform operations using each storage unit. The storage unit 4 may optionally be configured to include a cell culture unit 41, a centrifugation unit 42, and a liquid storage unit 43. The storage unit 4 may also optionally be configured to further include a cell storage unit 44.

[0032] Next, an outline of the operations for preparing a cell culture using the vessel 1 will be described. The user heats the container 1 in its shipped state or in its contracted state to thaw the frozen cells in the cell storage unit 44. For example, heating can be performed by immersing the container in a constant temperature water bath set at 37°C. Heating the container while contracting it also reduces the size of the heating equipment and makes it easier for heat to be transferred to the frozen cells. Next, the user extends the container 1, expanding the space within the container and making the operation unit 3 and each storage unit 4 available for use. The user inserts his / her hand into the operation unit 3 and transfers the thawed frozen cells to the centrifugation unit 42. Then, a washing liquid (hereinafter referred to as washing liquid) stored in one of the liquid storage units 43 is added thereto. If necessary, the addition of the washing liquid is carried out in several stages.

[0033] The user contracts the container 1 and places it in the centrifuge bucket of the centrifuge to perform centrifugation. Figure 2 shows a schematic diagram of how the contracted container 1 is placed in the centrifuge. By contracting the container at this time, it can be shrunk to a size that can be stored in an existing centrifuge bucket, making it possible to use an existing centrifuge. After centrifugation, the container 1 is extended, the operation unit 3 and each storage unit 4 are made usable, and the supernatant from the centrifugation unit 42 is discarded into the waste liquid unit 45 via the operation unit 3. Further washing liquid is added, and the above centrifugation operation is repeated. After the final centrifugation operation is completed, the container 1 is extended again, the supernatant is discarded, and then the prepared medium contained in one of the liquid containers 43 is added to the cells to suspend them.

[0034] Before the final centrifugation step, a detachable container 4 can be used to contain the cell suspension without affecting the cleanliness of the container, and the cell suspension can be removed from the container. The cell concentration of the cell suspension can then be measured, allowing the amount of cell suspension to be added to the prepared medium to be calculated. For example, the cell concentration can be measured outside the container using a hemocytometer.

[0035] The user seeds cells suspended in a prepared medium into the cell culture section 41. Then, the container 1 is deflated and placed in an incubator for cultivation in the deflated state. For example, cultivation is carried out for 2 to 26 hours in a CO2 incubator. By shrinking the container, the volume occupied by the container in the incubator can be reduced, allowing multiple containers to be placed in one incubator. After the culture is completed, the culture supernatant is discarded in the waste liquid section 45, and the cells are washed with a buffer solution contained in one of the liquid storage sections 43. Washing is carried out multiple times as necessary. The cell culture is allowed to stand as needed, and then the container 1 is opened in a highly clean environment, such as an operating room, and the cell culture is collected. [Industrial Applicability]

[0036] By using the container of the present invention, it is possible to prepare cell cultures without installing large-scale facilities to prepare a highly clean space. Therefore, the large investment required for installing large-scale facilities is not necessary, and even medium-sized hospitals can handle cell cultures for transplantation, which have a short shelf life, and regenerative medicine using cell cultures for transplantation can become even more widespread. [Explanation of symbols]

[0037] 1 container 2 Air Filter 3 Control section 4. Storage section 41 Cell Culture Department 42 Centrifugation processing section 43 Liquid storage section 44 Cell storage section 45 Waste liquid section 5. Cell culture 6. Equipment used in the preparation of cell cultures

Claims

1. A sealed, expandable bag-like container for preparing cell cultures, comprising: an air filter provided to connect the inside of the container to the outside of the container in order to maintain the cleanliness inside the container within a predetermined range of Class 1 to 9 specified in ISO 14644-1; and one or more containers provided within a container for use in preparing a cell culture; Equipped with At least one of the one or more storage sections has an opening and is connected to an inner surface of the container, and is configured so that the opening faces upward when the container is extended; The container is configured so that a portion of the container including the container portion connected to the inner surface of the container can be separated by heat sealing from the outside of the container without affecting the cleanliness of the other portion of the container. The container.

2. The container of claim 1 , wherein the opening is configured to be openable and closable.

3. 3. The container according to claim 1, further comprising an operating portion protruding into the container, which allows an object used in the preparation of the cell culture to be operated from the outside.

4. The container according to any one of claims 1 to 3, wherein the container is made of a freezable material.

5. The housing comprises: a cell culture section for accommodating and culturing the seeded cells; a liquid reservoir for storing different liquids; a centrifugation processing section for accommodating the cell suspension to be centrifuged; a cell storage unit for storing cells to be cultured; and a waste liquid section for storing waste liquid; The container according to any one of claims 1 to 4, comprising one or more selected from the group consisting of:

6. A container as described in claim 5, wherein the cell storage section contains frozen cells.

7. A container as described in claim 5 or 6, wherein the cell culture section has a temperature-responsive cell culture substrate.

8. A sealed, expandable bag-like container for preparing cell cultures, comprising: an air filter provided to connect the inside of the container to the outside of the container in order to maintain the cleanliness inside the container within a predetermined range of Class 1 to 9 specified in ISO 14644-1; and one or more containers provided within a container for use in preparing a cell culture; Equipped with At least one of the one or more storage sections has an opening, is connected to the inner surface of the container, and is configured so that the opening faces upward when the container is extended. The container.

9. A kit for preparing a cell culture, comprising the container according to any one of claims 1 to 8, and at least one of cells, a prepared medium, a washing solution, a buffer solution, a culture vessel, a centrifuge tube, a tube, an instrument used in preparing the cell culture, a transport container, and an instruction manual.

10. 1. A method for preparing a cell culture, comprising: Providing a container according to any one of claims 1 to 8 or a kit for preparing a cell culture according to claim 9; seeding the cells to be cultured; and Culturing the seeded cells; The method comprising:

11. The method of claim 10, further comprising the step of detaching at least one of the one or more storage sections.

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