Cell-culturing container

Abstract

The present invention addresses the problem of simplifying the structure of a cell-culturing container (1) and suppressing the generation of a dead space. A cell-culturing container (1) is provided with a container body (2) having a bag part (20) and a culture chamber (21) which is partitioned inside the bag part (20) and in which a content liquid (A) containing cells and a culture solution for culturing cells is stored. The bag part (20) has: an expansion / contraction section (200) that has expansion / contraction properties and gas permeability over the entire part; and a port section (201) that is contiguous with the expansion / contraction section (200) and has an opening (201c) that is in communication with the culture chamber (21). The volume of the culture chamber (21) is variable in association with the expansion and contraction of the expansion / contraction section (200).

Description

cell culture container

[0001] The present disclosure relates to a cell culture vessel used for culturing cells in fields such as drug discovery, regenerative medicine, and genetic engineering.

[0002] A culture medium (medium) containing dispersed cells is stored inside a cell culture vessel. The culture medium contains various components necessary for cell growth, proliferation, and differentiation. As cells proliferate, i.e., as the cell density in the culture medium increases, the nutrients supplied to the cells become insufficient. Furthermore, cells interfere with each other, limiting their proliferation. For this reason, cells are transferred (passaged). Specifically, cells are transferred from a cell culture vessel with a given volume to another cell culture vessel with a larger volume. However, the transfer process is complicated. Furthermore, there is a risk of contamination during transfer due to the introduction of bacteria, yeast, viruses, chemicals, etc.

[0003] Therefore, Patent Document 1 discloses a cell culture device in which the volume of the culture section can be changed. The cell culture device described in this document includes a culture vessel, a partition member, and a vessel loading platform. The culture vessel is made of a soft packaging material and is flexible and pliable. The culture vessel is bag-shaped. The culture vessel is placed on the vessel loading platform. A partition member (roller) is linearly pressed against the culture vessel from above. The partition member separates the culture vessel into two chambers (a culture section and an expandable section). The partition member is movable horizontally.

[0004] The culture section stores cells and culture solution. A tube is connected to the culture section. The partition member can be moved horizontally to slide the partition position (boundary line) between the culture section and the expandable section. Specifically, by moving the partition member, the volume of the culture section can be expanded and the volume of the expandable section can be reduced accordingly. This allows the volume of the culture section to be increased without the need for transplantation. In other words, the culture solution can be replenished to the culture section via the tube without the need for transplantation. This prevents contamination from occurring.

[0005] JP 2011-239734 A

[0006] In the case of the cell culture device described in the same document, the volume of the culture section is changed by moving a partition member (i.e., the partition position). Therefore, the partition member is essential. This results in a large number of parts and a complex structure. Furthermore, the volume of the culture section can be expanded by the volume of the expandable section. Therefore, the expandable section is essential. However, before the volume of the culture section is expanded (before cell proliferation), the expandable section becomes dead space. Therefore, the cell culture vessel disclosed herein aims to simplify the structure of the cell culture vessel and suppress the occurrence of dead space.

[0007] (1) In order to solve the above problem, the cell culture container of the present disclosure comprises a container body having a bag portion and a culture chamber partitioned inside the bag portion in which a content liquid containing cells and a culture medium for culturing the cells is stored, and the bag portion has an expansion / contraction portion that is expandable and gas permeable throughout, and a port portion that is connected to the expansion / contraction portion and has an opening that communicates with the culture chamber, and the volume of the culture chamber can be changed as the expansion / contraction portion expands and contracts.

[0008] According to this configuration, the volume of the culture chamber can be changed (expanded or contracted) in accordance with the expansion or contraction (expansion or contraction) of the expansion / contraction portion of the bag portion. This allows the culture scale to be changed without the need for cell transplantation (passage). Alternatively, the frequency of transplantation can be reduced. This reduces the complexity of the transplantation work. Furthermore, the occurrence of contamination due to transplantation can be suppressed.

[0009] Furthermore, with this configuration, the volume of the culture chamber can be changed as the expansion / contraction portion of the bag expands or contracts. Therefore, there is no need to add a separate component (such as the partition member of the cell culture device of Patent Document 1) to change the volume of the culture chamber. This reduces the number of components in the cell culture vessel, thereby simplifying the structure of the cell culture vessel.

[0010] Furthermore, with this configuration, the volume of the culture chamber can be changed by expanding and contracting the expansion / contraction section of the bag section, which makes it possible to suppress the occurrence of dead space regardless of the volume of the culture chamber.

[0011] Furthermore, with this configuration, the expansion / contraction section has expansion / contraction properties (expansion and contraction properties) throughout the entirety, which makes it possible to increase the rate of change in the volume of the culture chamber (the rate of change in volume relative to the initial volume (the volume when the culture chamber is empty)) compared to when the expansion / contraction section has localized expansion / contraction properties.

[0012] Furthermore, with this configuration, the expansion / contraction section is entirely gas permeable. Therefore, gas necessary for cell culture can be supplied from the outside to the inside of the culture chamber via the expansion / contraction section. Also, gas unnecessary for cell culture can be discharged from the inside to the outside of the culture chamber via the expansion / contraction section.

[0013] (2) In the configuration of (1) above, it is preferable that the larger the volume of the culture chamber, the smaller the wall thickness of the expansion / contraction section, and the higher the gas permeability of the expansion / contraction section. This configuration allows the gas permeability of the expansion / contraction section to be adjusted depending on the scale of culture (the degree of cell growth). For example, when the culture scale is small (when the number of cells is small), the gas permeability of the expansion / contraction section can be reduced. Furthermore, when the culture scale is large (when the number of cells is large), the gas permeability of the expansion / contraction section can be increased. In this way, sufficient gas can be supplied to the cells in the culture chamber regardless of the culture scale (regardless of the number of cells). In other words, healthy cell growth can be promoted regardless of the culture scale.

[0014] (3) In any of the above configurations, it is preferable that the state in which the culture chamber is empty be defined as the initial state, the volume of the culture chamber in the initial state be defined as the initial volume, the state in which an arbitrary amount of the content liquid is stored in the culture chamber be defined as the arbitrary state, and the volume of the culture liquid in the arbitrary state be defined as the arbitrary volume, and that the arbitrary volume be 15.0 times or less of the initial volume.

[0015] This configuration allows the volume of the culture chamber to be changed significantly in response to changes in the culture scale. Furthermore, this configuration prevents excessive expansion of the culture medium volume and suppresses leakage from the culture chamber, compared to when the arbitrary volume exceeds 15.0 times the initial volume.

[0016] (3-1) In any of the above configurations, it is preferable that the internal pressure of the culture chamber be kept below a predetermined pressure regardless of the volume of the liquid content. With this configuration, the internal pressure of the culture chamber can be kept below a predetermined pressure regardless of the volume (amount) of the liquid content. Therefore, stress on the cells can be reduced compared to when the internal pressure increases with an increase in the liquid content.

[0017] (3-2) In the configuration of (3-1) above, it is preferable that the predetermined pressure is 15 kPa. With this configuration, the internal pressure of the culture chamber can be kept below 15 kPa regardless of the volume of the liquid contained therein.

[0018] (4) In any of the configurations (3) to (3-2) above, the arbitrary volume is preferably 5.0 times or less the initial volume. With this configuration, excessive expansion of the volume of the culture medium can be suppressed, and leakage of the culture medium from the culture chamber can be suppressed, compared to when the arbitrary volume is more than 5.0 times the initial volume.

[0019] (5) In any of the above configurations, it is preferable that the state in which the culture chamber is empty be defined as the initial state, the wall thickness of the expansion / contraction section in the initial state be defined as the initial wall thickness, the state in which an arbitrary amount of the content liquid is stored in the culture chamber be defined as the arbitrary state, and the wall thickness of the expansion / contraction section in the arbitrary state be defined as the arbitrary wall thickness, and that the arbitrary wall thickness be 0.5 to 1.0 times the initial wall thickness.

[0020] According to this configuration, the wall thickness of the expansion / contraction section can be significantly changed depending on the change in the culture scale. Furthermore, according to this configuration, the wall thickness can be prevented from becoming excessively small compared to when the arbitrary wall thickness is less than 0.5 times the initial wall thickness. For example, the gas permeability can be prevented from becoming excessively high. Furthermore, according to this configuration, the wall thickness can be prevented from becoming excessively large compared to when the arbitrary wall thickness is more than 1.0 times the initial wall thickness. For example, the gas permeability can be prevented from becoming excessively low.

[0021] (6) In any of the above configurations, the expansion / contraction section is preferably made of silicone rubber. Silicone rubber has high expansion / contraction properties. Therefore, with this configuration, high expansion / contraction properties can be ensured throughout the entire expansion / contraction section. Furthermore, silicone rubber has high gas permeability. Therefore, with this configuration, high gas permeability can be ensured throughout the entire expansion / contraction section.

[0022] Furthermore, silicone rubber has high heat resistance. Therefore, this configuration ensures high heat resistance throughout the entire expansion / contraction section. Therefore, a low-cost sterilization method such as high-pressure steam sterilization (autoclave sterilization) can be used to sterilize the cell culture vessel.

[0023] (6-1) In the configuration of (6) above, it is preferable that the expansion / contraction section be made of PDMS (polydimethylsiloxane). PDMS has high expansion / contraction properties, high gas permeability, and high heat resistance. Therefore, with this configuration, high expansion / contraction properties, high gas permeability, and high heat resistance can be ensured throughout the entire expansion / contraction section.

[0024] (7) In any of the above configurations, it is preferable to further include a flow path member that is detachably arranged in the port portion and has a flow path that connects an external device to the culture chamber, and the flow path member has a tubular member that opens into the interior of the culture chamber and partitions at least a portion of the flow path.

[0025] According to this configuration, the flow path member includes a flow path and a tubular member. The flow path connects the external device to the culture chamber. Therefore, the volume of the culture chamber can be changed depending on the liquid flow direction and amount in the flow path. Furthermore, the tubular member opens into the interior of the culture chamber. That is, the tubular member extends into the interior of the culture chamber. Therefore, the flow path can be reliably reached inside the culture chamber.

[0026] (7-1) In the configuration of (7) above, it is preferable that the flow path member further includes a check valve disposed in the flow path to restrict the flow of liquid from the culture chamber toward the external device. With this configuration, the flow direction of liquid in the flow path can be restricted to only the direction from the external device toward the culture chamber. Therefore, the flow of culture liquid from the culture chamber toward the external device, i.e., backflow, can be suppressed.

[0027] (7-2) In the configuration of (7) or (7-1) above, the port preferably has a cylindrical port body having the opening and a flange protruding radially outward from the opening, and the flow path member has a cap receiver annularly mounted on the port body and a cap attached to the cap receiver to cover the opening, with the flange sandwiched between the cap and the cap receiver. According to this configuration, the flange is sandwiched between the cap and the cap receiver, thereby sealing the opening of the port in a liquid-tight manner. In other words, the liquid-tightness of the culture chamber can be ensured.

[0028] (8) In any of the above configurations, the hardness of the material forming the expansion / contraction portion is preferably 10° or more and 30° or less. Here, "hardness" refers to the hardness measured using a Type A durometer in accordance with JIS K 6253-3.

[0029] This configuration can prevent the expansion / contraction section from becoming excessively flexible, compared to when the hardness is less than 10°. For example, the arbitrary volume of the configuration (3) can be set to 5 times or less the initial volume. Furthermore, this configuration can suppress the internal pressure of the culture chamber when filling the content liquid to 13 kPa or less, compared to when the hardness is more than 30°.

[0030] According to the cell culture vessel of the present disclosure, the structure of the cell culture vessel can be simplified and the occurrence of dead space can be suppressed.

[0031] Fig. 1 is a cross-sectional view in the front-rear direction of a cell culture vessel of a first embodiment. Fig. 2 is an exploded cross-sectional view in the front-rear direction of the same cell culture vessel. Fig. 3 is an enlarged view of the inside of a frame III in Fig. 1. Fig. 4 is a cross-sectional view in the front-rear direction of a cell culture vessel of a second embodiment. Fig. 5 is a graph showing the results of a culture test.

[0032] Hereinafter, an embodiment of the cell culture vessel of the present disclosure will be described.

[0033] First Embodiment Fig. 1 shows a cross-sectional view in the front-rear direction (axial direction) of a cell culture vessel of this embodiment. Fig. 2 shows an exploded cross-sectional view in the front-rear direction of the same cell culture vessel. Fig. 3 shows an enlarged view of the area within box III in Fig. 1. In Fig. 1, the initial state is indicated by a solid line, the first state by a dashed dotted line, and the second state by a dotted line. The initial state refers to a state in which the culture chamber 21 is empty (the state before use of the cell culture vessel 1). The first state and the second state refer to states in which an arbitrary amount of content liquid A is stored in the culture chamber 21. The first state and the second state are included in the concept of "arbitrary state" in the present disclosure. The amount of content liquid A stored in the culture chamber 21 is greater in the second state than in the first state.

[0034] [Configuration of Cell Culture Vessel] First, the configuration of the cell culture vessel of this embodiment will be described. As shown in Figures 1 and 2, the cell culture vessel 1 includes a vessel body 2 and a flow path member 5.

[0035] (Container body 2) The container body 2 is a single piece made of PDMS (polydimethylsiloxane, silicone rubber). The container body 2 includes a bag portion 20 and a culture chamber 21. The bag portion 20 includes an expansion / contraction portion 200 and a port portion 201.

[0036] The bag portion 20 is bag-shaped and gas permeable. The inflation / deflation portion 200 extends in the front-to-rear direction (axial direction) and has a cylindrical shape with a bottom that opens to the rear (one axial side). The inflation / deflation portion 200 includes an end wall portion 200a and a peripheral side wall portion 200b.

[0037] The side peripheral wall portion 200b has a cylindrical shape extending in the front-to-rear direction. The end wall portion 200a seals the opening at the front end (the other axial end) of the side peripheral wall portion 200b. The expansion / contraction portion 200 is elastically deformable (expandable and contractible) throughout its entirety. The larger the volume of the culture chamber 21 (described below), the smaller the wall thickness of the expansion / contraction portion 200. The smaller the wall thickness of the expansion / contraction portion 200, the higher the gas permeability of the expansion / contraction portion 200.

[0038] As shown in FIG. 3 , the port portion 201 includes a port portion main body 201a and a flange portion 201b. The port portion main body 201a is integrally connected to the rear end (one axial end) of the side peripheral wall portion 200b. The port portion main body 201a has a cylindrical shape extending in the front-to-rear direction. An opening 201c is formed at the rear end of the port portion main body 201a. The flange portion 201b is disposed on the periphery of the opening 201c. The flange portion 201b protrudes radially outward from the opening 201c. The flange portion 201b has an annular shape.

[0039] The culture chamber 21 is partitioned inside the bag portion 20. A content liquid A is stored in the culture chamber 21. The content liquid A contains a large number of cells and culture solution. The large number of cells are dispersed and suspended in the culture solution. The volume of the culture chamber 21 can change (expand or contract) as the expansion / contraction portion 200 expands or contracts.

[0040] As shown in FIG. 1 , the initial volume V0 is the volume of the culture chamber 21 in the initial state (shown by the solid line in FIG. 1 ). The first volume V1 is the volume of the culture solution in the first state (shown by the dashed line in FIG. 1 ) (when the culture chamber 21 is filled with content liquid A, this is approximately equal to the volume of the culture chamber 21; the same applies below). The second volume V2 is the volume of the culture solution in the second state (shown by the dotted line in FIG. 1 ). The first volume V1 and the second volume V2 are each within the range of 0.6 to 5.0 times the initial volume V0. The volumes of the culture chamber 21 increase in the order of the initial volume V0, the first volume V1, and the second volume V2.

[0041] Note that when the first volume V1 and the second volume V2 are within a range of less than 1.0 times the initial volume V0 (for example, when the first volume V1 and the second volume V2 are 0.6 times the initial volume V0), the culture chamber 21 is not filled with the content liquid A. In other words, the culture chamber 21 is not full. In this case, the content liquid A occupies a portion of the culture chamber 21, and space is secured in the remaining part of the culture chamber 21.

[0042] In this way, the cell culture vessel 1 can be used not only when the culture chamber 21 is filled with the liquid content A, but also when the culture chamber 21 is filled with the liquid content A with gaps therein.

[0043] As shown in Figure 1, the initial wall thickness T0 is the wall thickness of the expansion / contraction portion 200 in the initial state. The first wall thickness T1 is the wall thickness of the expansion / contraction portion 200 in the first state. The second wall thickness T2 is the wall thickness of the expansion / contraction portion 200 in the second state. The first wall thickness T1 and the second wall thickness T2 are each within a range of 0.5 to 1.0 times the initial wall thickness T0. The wall thickness of the expansion / contraction portion 200 decreases and the gas permeability increases in the order of the initial wall thickness T0, the first wall thickness T1, and the second wall thickness T2.

[0044] (Flow Channel Member 5) As shown in FIGS. 1 to 3 , the flow channel member 5 is detachably disposed in the port portion 201. The flow channel member 5 includes a flow channel B, a cap receiver 50, a tubular member 51, a check valve 52, and a cap 53. The flow channel B communicates between the syringe 90 and the culture chamber 21. The syringe 90 is included in the concept of "external equipment" in the present disclosure. In the flow channel B, the syringe 90 side is referred to as the "upstream side," and the culture chamber 21 side is referred to as the "downstream side." Furthermore, the direction from the syringe 90 toward the culture chamber 21 is referred to as the "forward direction," and the direction from the culture chamber 21 toward the syringe 90 is referred to as the "reverse direction."

[0045] The cap receiver 50 is made of resin (e.g., PP (polypropylene)) and is annularly mounted on the port portion main body 201a. The cap receiver 50 abuts against the flange portion 201b from the front side. The cap receiver 50 has a cylindrical shape that extends in the front-to-rear direction. A cap receiver side thread portion 500 is threaded on the outer peripheral surface of the cap receiver 50.

[0046] The tubular member 51 is made of PDMS and opens into the interior of the culture chamber 21. The tubular member 51 defines a part of the flow path B. The check valve 52 is disposed in the flow path B. The check valve 52 allows the liquid to be sent in the forward direction and prohibits the liquid to be sent in the reverse direction.

[0047] The cap 53 is made of resin (e.g., PP) and includes an end wall portion 530 and a side peripheral wall portion 531. The side peripheral wall portion 531 has a cylindrical shape extending in the front-to-rear direction (axial direction). The side peripheral wall portion 531 is disposed radially outward of the flange portion 201b and the cap receiver 50. A reduced diameter portion 531a that protrudes radially inward is disposed at the rear end of the side peripheral wall portion 531. A cap side thread portion 531b is threadedly provided on the inner circumferential surface of the side peripheral wall portion 531. The cap side thread portion 531b is threadedly engaged with the cap receiver side thread portion 500.

[0048] The end wall portion 530 is disk-shaped. An expanded diameter portion 530a that protrudes radially outward is disposed at the front end of the end wall portion 530. The end wall portion 530 seals the opening at the rear end (one axial end) of the side circumferential wall portion 531. Specifically, the reduced diameter portion 531a of the side circumferential wall portion 531 and the expanded diameter portion 530a of the end wall portion 530 are joined in a state of engagement in the front-to-rear direction. Therefore, the reduced diameter portion 531a (side circumferential wall portion 531) supports the expanded diameter portion 530a (end wall portion 530) from the rear side.

[0049] The end wall portion 530 abuts against the flange portion 201b from the rear side. The end wall portion 530 covers the opening 201c from the rear side. A pipe portion 530b is disposed at the radial center of the end wall portion 530. The pipe portion 530b penetrates the end wall portion 530 in the front-rear direction. The pipe portion 530b defines a part of the flow path B. The pipe member 51 is connected to the downstream end of the pipe portion 530b. The check valve 52 is connected to the upstream end of the pipe portion 530b.

[0050] When the cap 53 is fitted to the cap receiver 50 and the cap-side threaded portion 531b is screwed into the cap receiver-side threaded portion 500, the end wall portion 530 of the cap 53 presses against the flange portion 201b from the rear side. At the same time, the cap receiver 50 presses against the flange portion 201b from the front side. That is, the flange portion 201b is sandwiched between the cap 53 and the cap receiver 50. As a result, the opening 201c is sealed.

[0051] [Movement of Cell Culture Vessel] Next, the movement of the cell culture vessel of this embodiment will be described. First, content liquid A is injected from syringe 90 via flow path B into culture chamber 21 in its initial state. The injection pressure of content liquid A causes bag portion 20 to expand. Culture chamber 21 transitions from the initial state to the first state (shown by the dashed line in FIG. 1 ).

[0052] Next, the syringe 90 is removed from the flow path B, and the cell culture vessel 1 is placed in an incubator under a predetermined environment (a predetermined temperature, a predetermined atmosphere, etc.). The culture solution (medium) in the content liquid A contains various components (carbohydrates, inorganic salts, amino acids, vitamins, etc.) necessary for cell growth, proliferation, and differentiation. The bag portion 20 is gas permeable, and gases (oxygen, etc.) necessary for cell growth are supplied from the outside (inside the incubator) to the culture chamber 21 through the bag portion 20. Unnecessary gases (e.g., excess carbon dioxide mixed in the content liquid A) are also discharged from the culture chamber 21 to the outside through the bag portion 20. Cells grow and proliferate in the culture chamber 21.

[0053] Subsequently, depending on the proliferation of the cells, the culture medium is replenished into the culture chamber 21. Specifically, a syringe 90 is connected to the flow path B, the culture medium is replenished from the syringe 90 into the culture chamber 21 in the first state, and the syringe 90 is removed from the flow path B.

[0054] The supply of culture medium expands the expansion / contraction section 200. The culture chamber 21 transitions from the first state to the second state (shown by the dotted line in FIG. 1).

[0055] Thereafter, the above-described replenishment operation is repeated as necessary. That is, the culture medium is replenished appropriately to the culture chamber 21 in any state. As the replenishment proceeds, the volumes of the culture chamber 21 and the content liquid A expand. Furthermore, as the replenishment proceeds, the wall thickness of the expansion / contraction section 200 decreases.

[0056] [Effects] Next, the effects of the cell culture vessel of this embodiment will be described. As shown in FIG. 1 , according to the cell culture vessel 1 of this embodiment, the volume of the culture chamber 21 can be changed (expanded or contracted) as the expansion / contraction section 200 expands or contracts. This allows expansion culture of cells to be performed without the need to transfer (passage) cells to a culture vessel with a larger volume during expansion culture. Alternatively, the frequency of transfer can be reduced. This reduces the complexity of the transfer work. Furthermore, the occurrence of contamination due to transfer can be suppressed.

[0057] According to the cell culture vessel 1 of this embodiment, there is no need to add a separate member (such as the partition member of the cell culture device of Patent Document 1) to change the volume of the culture chamber 21. This reduces the number of parts in the cell culture vessel 1, thereby simplifying the structure of the cell culture vessel 1.

[0058] According to the cell culture vessel 1 of this embodiment, the volume of the culture chamber 21 can be adjusted to the minimum volume required for each phase of cell growth, depending on the phase of cell growth. Therefore, regardless of the volume of the culture chamber 21, the occurrence of dead space can be suppressed.

[0059] According to the cell culture vessel 1 of this embodiment, the expansion / contraction section 200 has expansion / contraction properties throughout the entire section, which allows the rate of change in the volume of the culture chamber 21 (the rate of change in volume relative to the initial volume V0) to be larger than when the expansion / contraction section 200 has localized expansion / contraction properties.

[0060] According to the cell culture vessel 1 of this embodiment, the bag portion 20 (expansion / contraction portion 200, port portion 201) is gas permeable. Therefore, gases necessary for cell culture (e.g., oxygen, etc.) can be supplied from the outside to the inside of the culture chamber 21 via the bag portion 20. In addition, gases unnecessary for cell culture (e.g., excess carbon dioxide mixed in the content liquid A, etc.) can be discharged from the inside of the culture chamber 21 to the outside via the bag portion 20.

[0061] According to the cell culture vessel 1 of this embodiment, the larger the volume of the culture chamber 21, the smaller the wall thickness of the expansion / contraction section 200, and the higher the gas permeability of the expansion / contraction section 200. Therefore, the gas permeability of the expansion / contraction section 200 can be adjusted depending on the culture scale (the degree of cell proliferation). For example, when the culture scale is small (when the number of cells is small), the gas permeability of the expansion / contraction section 200 can be reduced. On the other hand, when the culture scale is large (when the number of cells is large), the gas permeability of the expansion / contraction section 200 can be increased. In this way, a sufficient amount of gas can be supplied to the cells in the culture chamber 21 regardless of the culture scale (regardless of the number of cells). In other words, healthy cell growth can be promoted regardless of the culture scale.

[0062] In the cell culture vessel 1 of this embodiment, the first volume V1 and the second volume V2 are set to be 0.6 times or more and 5.0 times or less the initial volume V0. This allows the volume of the culture chamber 21 to be changed significantly in response to changes in the culture scale. Furthermore, compared to when the first volume V1 and the second volume V2 are less than 0.6 times the initial volume V0, excessive reduction in the volume (liquid amount) of the culture medium can be suppressed. This prevents the generation of dead space in the culture chamber 21 and prevents the culture medium from volatilizing. Furthermore, compared to when the first volume V1 and the second volume V2 are more than 5.0 times the initial volume V0, excessive expansion in the volume of the culture medium can be suppressed, thereby preventing liquid leakage from the culture chamber 21.

[0063] According to the cell culture vessel 1 of this embodiment, the internal pressure of the culture chamber 21 in the initial state, the first state, and the second state is set to 15 kPa (predetermined pressure) or less, regardless of the volume (amount) of the content liquid A. Therefore, compared to a case where the internal pressure increases with an increase in the content liquid A, stress on the cells can be reduced.

[0064] In the cell culture vessel 1 of this embodiment, the first wall thickness T1 and the second wall thickness T2 are set to be 0.5 times or more and 1.0 times or less the initial wall thickness T0. Therefore, the wall thickness of the expansion / contraction section 200 can be changed significantly in response to changes in the culture scale. Furthermore, compared to when the first wall thickness T1 and the second wall thickness T2 are less than 0.5 times the initial wall thickness T0, the wall thickness can be prevented from becoming excessively small. For example, the gas permeability can be prevented from becoming excessively high. Furthermore, compared to when the first wall thickness T1 and the second wall thickness T2 are more than 1.0 times the initial wall thickness T0, the wall thickness can be prevented from becoming excessively large. For example, the gas permeability can be prevented from becoming excessively low.

[0065] In the cell culture vessel 1 of this embodiment, the bag portion 20 is made of PDMS (silicone rubber). PDMS has high expansion and contraction properties. Therefore, high expansion and contraction properties can be ensured throughout the entire expansion and contraction portion 200. PDMS also has high gas permeability. Therefore, high gas permeability can be ensured throughout the entire bag portion 20.

[0066] Furthermore, PDMS has high heat resistance. Therefore, high heat resistance can be ensured throughout the entire bag portion 20. Therefore, as a sterilization method for the cell culture vessel 1, a low-cost heating method (high-pressure steam sterilization (autoclave sterilization), dry heat sterilization, moist heat sterilization, etc.) can be adopted compared to irradiation methods (radiation sterilization, electron beam sterilization, etc.) and gas methods (ethylene oxide gas sterilization, etc.) (of course, these sterilization methods can also be adopted). In this way, the cell culture vessel 1 of this embodiment provides greater freedom in selecting a sterilization method.

[0067] According to the cell culture vessel 1 of this embodiment, the flow path member 5 includes a flow path B and a tubular member 51. The flow path B communicates between the syringe 90 and the culture chamber 21. Therefore, the volume of the culture chamber 21 can be changed depending on the liquid supply direction and amount in the flow path B. The tubular member 51 opens into the interior of the culture chamber 21. That is, the tubular member 51 extends into the interior of the culture chamber 21. Therefore, the flow path B can be reliably reached into the interior of the culture chamber 21.

[0068] According to the cell culture vessel 1 of this embodiment, the flow path member 5 is provided with a check valve 52. The check valve 52 is disposed in the flow path B. This makes it possible to prevent the content liquid A from flowing in the reverse direction (from the culture chamber 21 toward the syringe 90), i.e., to prevent the content liquid A from flowing back. Therefore, when the syringe 90 is attached to or detached from the tube portion 530b, it is possible to prevent the content liquid A from leaking from the culture chamber 21 to the outside via the tube portion 530b.

[0069] According to the cell culture vessel 1 of this embodiment, by encircling the cap 53 on the cap receiver 50 and threading the cap-side threaded portion 531b onto the cap receiver-side threaded portion 500, the end wall portion 530 of the cap 53 is pressed against the flange portion 201b from the rear side. At the same time, the cap receiver 50 is pressed against the flange portion 201b from the front side. That is, the flange portion 201b is sandwiched between the cap 53 and the cap receiver 50. This allows the opening 201c to be liquid-tightly sealed. Thus, according to the cell culture vessel 1 of this embodiment, the opening 201c can be liquid-tightly sealed simply by tightening the cap 53.

[0070] In the cell culture vessel 1 of this embodiment, the port portion 201 is pressed from the radially outer side by the cap receiver 50 while the flange portion 201b is clamped. This makes it possible to suppress expansion of the port portion 201. In other words, the expansion / contraction portion 200 can be expanded preferentially relative to the port portion 201.

[0071] According to the cell culture vessel 1 of this embodiment, the bag portion 20 is an integrated part. That is, the expansion / contraction portion 200 and the port portion 201 are integrally connected. Therefore, compared to when the expansion / contraction portion 200 and the port portion 201 are separate members, the configuration of the bag portion 20 can be simplified. Furthermore, the liquid-tightness of the culture chamber 21 can be improved.

[0072] Second Embodiment The cell culture vessel of this embodiment differs from the cell culture vessel of the first embodiment in that the cell culture vessel includes a case. Here, only the difference will be described. Fig. 4 shows a cross-sectional view in the front-rear direction (axial direction) of the cell culture vessel of this embodiment. Note that parts corresponding to those in Fig. 1 are designated by the same reference numerals. Note that Fig. 1 is a cross-sectional view (horizontal cross-sectional view) as seen from above, whereas Fig. 4 is a cross-sectional view (vertical cross-sectional view) as seen from the right side.

[0073] 4, the cell culture vessel 1 includes a case 6. The case 6 is made of resin (e.g., PP) and has a shallow box (tray) shape that is open at the top. The vessel body 2 and the flow path member 5 are housed in the case 6.

[0074] The cell culture vessel of this embodiment and the cell culture vessel of the first embodiment have similar functions and effects with respect to common configurations. According to the cell culture vessel 1 of this embodiment, when the culture chamber 21 is expanded, the expansion / contraction portion 200 can be expanded along the shape of the bottom surface 60 of the case 6 by utilizing the weight of the container body 2 (including the content liquid A). That is, compared to when the culture chamber 21 expands freely (when the culture chamber 21 expands without any external restriction), the expansion / contraction portion 200 can be expanded into a flat bag shape. That is, in the expansion / contraction portion 200, the bottom wall 200d (the portion of the expansion / contraction portion 200 that abuts the bottom surface 60) can be expanded preferentially relative to the standing wall 200c (the portion of the expansion / contraction portion 200 that rises from the bottom surface 60). In other words, the bottom area (surface area of ​​the culture surface) of the culture chamber 21 can be expanded preferentially. This can improve gas exchange (supply and discharge) with respect to the cells in the culture chamber 21. Therefore, the efficiency of cell culture can be increased.

[0075] <Others> The embodiments of the cell culture vessel of the present disclosure have been described above. However, the embodiments are not particularly limited to the above-described embodiments. Various modifications and improvements that can be made by those skilled in the art are also possible.

[0076] The material of the bag portion 20 of the container body 2 is not particularly limited. The bag portion 20 may be made of a thermosetting elastomer material such as silicone rubber, natural rubber, isoprene rubber, butadiene rubber, styrene-butadiene rubber, butyl rubber, nitrile rubber, ethylene-propylene rubber, chloroprene rubber, acrylic rubber, urethane rubber, or fluororubber. It may also be made of an elastomer, such as a thermoplastic elastomer material, such as polyamide-based thermoplastic elastomer (TPA), polyester-based thermoplastic elastomer (TPC), olefin-based thermoplastic elastomer (TPO), styrene-based thermoplastic elastomer (TPS), urethane-based thermoplastic elastomer (TPU), or dynamically crosslinked thermoplastic elastomer (TPV). Examples of silicone rubber include liquid silicone rubber and millable silicone rubber. Examples of the polymer component of silicone rubber include PDMS. At least the expansion / contraction portion 200 of the bag portion 20 may have expansion / contraction properties (e.g., rubber elasticity). Furthermore, it is sufficient that at least the expansion / contraction section 200 of the bag section 20 has gas permeability (gas permeability to a degree necessary for cell culture). The expansion / contraction section 200 and the port section 201 may be integral or separate. That is, the bag section 20 may be an integral body or an integrated body made up of multiple members.

[0077] Regarding the expansion / contraction property of the expansion / contraction portion 200, it is preferable that the hardness of the material forming the expansion / contraction portion 200 is 10° or more and 30° or less, as measured by a Type A durometer in accordance with JIS K 6253-3. In this way, the internal pressure of the culture chamber 21 when filled with the content liquid A can be kept to 13 kPa or less. In addition, the arbitrary volume can be set to 5 times or less the initial volume.

[0078] Regarding the expansion / contraction property of the expansion / contraction portion 200, it is preferable that the breaking elongation of the material forming the expansion / contraction portion 200 is 700% or more and 900% or less when measured using a measurement method conforming to JIS K 6251 (test piece: dumbbell-shaped No. 3 test piece; test speed: 500±50 mm / min). This allows the internal pressure of the culture chamber 21 when filled with content liquid A to be kept below 13 kPa. Furthermore, the arbitrary volume can be set to 5 times the initial volume or less.

[0079] The material of each component constituting the flow path member 5 (cap receiver 50, pipe member 51, check valve 52, cap 53) is not particularly limited. Each component may be made of resin, elastomer, metal, or the like. The method of attaching the cap 53 to the cap receiver 50 is not particularly limited. It may be by screwing, engaging, or bonding. The cap 53 may also be attached to the cap receiver 50 using a separate component (bolt, clamp, or the like). A seal member (such as a seal ring) may also be interposed between the cap receiver 50 and the cap 53. The cap receiver 50 does not have to be provided. The cap 53 may also be attached to the port portion 201.

[0080] The type of culture medium is not particularly limited. The culture medium may contain inorganic salts, carbohydrates, amino acids, vitamins, proteins, fatty acids, serum, etc. as appropriate. The culture form is not particularly limited. It may be a suspension culture system (a system in which a single cell or a cell mass consisting of a small number of cells is suspended in the culture medium) or an adherent culture system (a system in which a large number of cells adhere to the inner surface of the bag portion 20 in a single layer). The type of cell is not particularly limited. For example, cell lines (passaged cell lines) include adherent cell lines such as MRC-5, HeLa, Vero, NIH3T3, L929, BHK-21, HEK293, HepG2, BAE-1, and SH-SY5Y. Also included are suspension cell lines such as CHO, NS0, U937, Namalwa, HL60, WEHI231, YAC1, and U266B1. The cell culture method is not particularly limited. Static culture (two-dimensional culture, monolayer culture), agitation culture (stirrer agitation, rotation agitation, gyration agitation, shaking agitation), suspension culture, three-dimensional culture using a cell adhesion scaffold, etc. may be used.

[0081] Next, a culture test performed using the cell culture vessel of the present disclosure will be described with reference to FIG.

[0082] <Specifications of Test Vessels> First, the test vessels (cell culture vessels) used in the culture test will be described. A total of 11 test vessels were used, including Examples 1 to 10 and Comparative Example 1. Table 1 shows the specifications and culture results of each test vessel.

[0083] Examples 1 to 10 use the cell culture vessel 1 shown in FIG. 1 . Comparative Example 1 uses a commercially available cell culture vessel made of PE (polyethylene). The bag portion 20 of Comparative Example 1 has a bag shape similar to the bag portion 20 of FIG. 1 . However, the bag portion 20 of Comparative Example 1 does not have expansion / contraction properties. That is, the bag portion 20 of Comparative Example 1 does not have an expansion / contraction portion 200. Therefore, in the case of Comparative Example 1, the "oxygen permeability of the expansion / contraction portion 200" in Table 1 refers to the "oxygen permeability of the bag portion 20." Regarding the "elongation rate" under "material of the bag portion 20" in Table 1, a tensile test was conducted in accordance with JIS K6251:2010 to measure the elongation at break (Eb), which was used as the elongation rate. For the measurement, a dumbbell-shaped No. 3 test piece was used, and the tensile speed was 100 mm / min.

[0084] As shown in Table 1, all test containers (Examples 1 to 10, Comparative Example 1) were identical in the cells to be cultured (CHO-S (Chinese hamster ovary (suspension))). Furthermore, all test containers were identical in the culture medium (a mixture of basal medium D-MEM (containing high glucose, L-glutamine, phenol red, and sodium pyruvate, Fujifilm Wako Pure Chemical Corporation) + 10% FBS (fetal bovine serum, Cosmo Bio Co., Ltd.) + 1% PSA (penicillin-streptomycin-amphotericin B suspension, Fujifilm Wako Pure Chemical Corporation)). Examples 1 to 10 were identical in the material (type) of the bag portion 20, the sterilization method (AC sterilization (autoclave sterilization)), and the initial volume V0 of the culture chamber 21.

[0085] Focusing on Examples 1 to 7, in which the material (type, hardness, and elongation rate) of the bag portion 20 is the same, the amount of culture solution (volume), expansion rate (= amount of culture solution / initial volume V0), and oxygen permeability of the expansion / contraction portion 200 increase from Example 1 to Example 7. The wall thickness of the expansion / contraction portion 200 decreases from Example 1 to Example 7. In other words, as the volume of the culture chamber 21 increases, the wall thickness of the expansion / contraction portion 200 decreases, and the oxygen permeability (gas permeability) of the expansion / contraction portion 200 increases.

[0086] The amount of culture solution increases from Example 1 to Example 7. That is, the relationship in magnitude of the culture solution amount is "Example 1 < Example 2 ... < Example 6 < Example 7." In contrast, the internal pressure of the culture chamber 21 is not proportional to the increase in the amount of culture solution, and saturates at a predetermined pressure (12.7 kPa). That is, the relationship in magnitude of the internal pressure is "Example 1 < Example 2 ... Example 6 = Example 7." In this way, even if the amount of culture solution in the culture chamber 21 increases, the increase in the internal pressure of the culture chamber 21 is suppressed.

[0087] <Test Method> Next, the test method will be described. The test was performed by placing each test container in an incubator and culturing the cells in suspension. The environment inside the incubator was set to a temperature of 37°C, humidity of 95%, and carbon dioxide concentration of 5%. The test was left standing for 7 days.

[0088] <Test Results> Next, the test results will be described. Figure 5 shows the results of the culture test in a graph. Comparing the cell density immediately after the start of the test with the cell density after 7 days, it can be seen that cells proliferated over time in each test container (Examples 1 to 10, Comparative Example 1). Furthermore, Figure 5 and Table 1 (see "Day 7 cell density / seeded cell density" in the bottom column) show that Examples 1 to 10 had a higher cell proliferation rate than Comparative Example 1. Comparing Examples 1 to 10, it can be seen that the cell proliferation rates were similar.

[0089] From this, when actually culturing cells, if the culture scale is increased in the order of Example 1 → Example 2 → Example 3 → Example 4 in the same cell culture vessel 1 (by adding culture medium), healthy cell growth can be promoted regardless of the culture scale.

[0090] In Example 10, the culture medium was actually replenished during the test (on the third day). Here, Example 10 is identical to Example 7 (in which the culture medium was not replenished during the test) in terms of the material (type, hardness, elongation rate) of the bag portion 20 and the amount of culture medium (total). Comparing Example 10 with Example 7, even when the initial amount of culture medium is reduced (half the amount of Example 7) and the culture medium is replenished during the test, as in Example 10, a higher cell proliferation rate than Comparative Example 1 can be ensured, as in Example 7.

[0091] The hardness of the bag portion 20 (container body 2) in Examples 1 to 7 and 10 is 30°, the hardness in Example 8 is 10°, and the hardness in Example 9 is 20°. In this way, if the hardness is 10° or more and 30° or less, the internal pressure of the culture chamber 21 can be suppressed to 13 kPa or less (12.7 kPa or less).

[0092] 1: cell culture vessel, 2: vessel body, 20: bag portion, 200: expansion / contraction portion, 200a: end wall portion, 200b: side peripheral wall portion, 200c: standing wall, 200d: bottom wall, 201: port portion, 201a: port portion body, 201b: flange portion, 201c: opening, 21: culture chamber, 5: flow path member, 50: cap receiver, 500: cap receiver side thread portion, 51: tubular member, 52: check valve, 53: cap, 530: end wall portion, 530a: enlarged diameter portion, 530b: tubular portion, 531: side peripheral wall portion, 531a: reduced diameter portion, 531b: cap side thread portion, 6: case, 60: bottom surface, 90: syringe (external device) A: Content liquid, B: Channel, T0: Initial wall thickness, T1: First wall thickness, T2: Second wall thickness, V0: Initial volume, V1: First volume, V2: Second volume

Claims

1. A cell culture container comprising a container body having a bag portion and a culture chamber partitioned inside the bag portion for storing a liquid content containing cells and a culture medium for culturing the cells, wherein the bag portion has an expansion / contraction portion that is expandable and gas permeable throughout, and a port portion that is connected to the expansion / contraction portion and has an opening that communicates with the culture chamber, and wherein the volume of the culture chamber can be changed as the expansion / contraction portion expands and contracts.

2. A cell culture vessel according to claim 1, wherein the larger the volume of the culture chamber, the smaller the wall thickness of the expansion / contraction section, and the higher the gas permeability of the expansion / contraction section.

3. The cell culture vessel according to claim 1, wherein the state in which the culture chamber is empty is defined as an initial state, the volume of the culture chamber in the initial state is defined as an initial volume, the state in which an arbitrary amount of the content liquid is stored in the culture chamber is defined as an arbitrary state, and the volume of the culture liquid in the arbitrary state is defined as an arbitrary volume, and the arbitrary volume is 15.0 times or less of the initial volume.

4. The cell culture vessel according to claim 3, wherein the arbitrary volume is 5.0 times or less the initial volume.

5. A cell culture vessel as described in claim 1, wherein the state in which the culture chamber is empty is defined as an initial state, the wall thickness of the expansion / contraction section in the initial state is defined as an initial wall thickness, the state in which an arbitrary amount of the content liquid is stored in the culture chamber is defined as an arbitrary state, and the wall thickness of the expansion / contraction section in the arbitrary state is defined as an arbitrary wall thickness, and the arbitrary wall thickness is between 0.5 and 1.0 times the initial wall thickness.

6. The cell culture vessel according to claim 1, wherein the expansion / contraction portion is made of silicone rubber.

7. A cell culture vessel as described in claim 1, further comprising a flow path member that is detachably arranged on the port portion and has a flow path that connects an external device to the culture chamber, the flow path member having a tubular member that opens into the interior of the culture chamber and partitions at least a portion of the flow path.

8. The cell culture vessel according to claim 1, wherein the hardness of the material forming the expansion / contraction portion is 10° or more and 30° or less.

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