Closed culture vessel, cell culture device, and cell culture method

Abstract

The following configuration is used to provide: a closed cell culture vessel equipped with a mechanism capable of reliably discharging the old culture liquid in a culture vessel; a cell culture apparatus; and a cell culture method.　Provided is a closed culture vessel that has an open culture vessel, a closed vessel for sealing the open culture vessel, a liquid feed port for supplying liquid to at least the open culture vessel, and a liquid discharge port for discharging the liquid in the open culture vessel, each above the closed vessel. The liquid feed port is located above the open surface of the open culture vessel, and the tip of a drainage pipe connected to the liquid discharge port is positioned in the vicinity of the bottom surface of the open culture vessel. Also provided are a cell culture device in which the cell culture vessel can be installed, and a cell culture method.

Description

Closed culture container, cell culture device, cell culture method

[0001] The present invention relates to a closed culture vessel, a cell culture device, and a cell culture method.

[0002] In regenerative medicine, which uses a patient's own cells or cells from other people to treat disease, cells taken from the living body are cultured to increase cell numbers or to create tissues with appropriate morphology, which are then used for therapeutic transplants. The cells and tissues used for these transplants are cultured in clean rooms for cell culture called cell processing centers (CPCs). Because cell culture in the centers is performed manually by technicians, there are challenges, including the significant labor and cost required to prepare a single patient's worth of cells, and the risk of biological contamination due to the manual process.

[0003] To address these challenges, automated cell culture devices have been developed to automate cell culture in closed systems. In these devices, sealed bottles or culture vessels are connected by tubes to create a biologically closed space, which is then placed in a temperature-controlled space suitable for cell culture. The device then performs the necessary medium changes for cell culture, as well as gas exchange and humidification within the culture vessels. This device configuration achieves automation of cell culture and reduces the risk of biological contamination. However, the use of dedicated culture vessels poses the challenge of high manufacturing costs.

[0004] To solve this problem, the invention described in Patent Document 1 discloses a method for inexpensively and easily converting various open culture vessels (which may be commercially available) into closed culture vessels by sealing them in a sealed container and sterilizing them.

[0005] International Publication No. 2015 / 190090

[0006] In the invention described in Patent Document 1, a culture medium inlet for introducing culture medium into the sealed container and a culture medium outlet for discharging culture medium from the sealed container are provided on the side of the sealed container. With this structure, in order to supply culture medium to a culture vessel placed inside the sealed container, the culture medium must be supplied into the sealed container so that its height is equal to or exceeds the height of the culture vessel. Furthermore, when discharging culture medium from the sealed container, overflowing culture medium is discharged through the outlet. In this case, new culture medium supplied through the inlet and old culture medium overflowing from the culture vessel may mix, potentially interfering with cell culture. Furthermore, there is also a concern that culture costs may increase because more medium than the capacity of the culture vessel placed inside is used.

[0007] In view of the above problems, an object of the present invention is to provide a closed system culture vessel, a cell culture device, and a cell culture method that are equipped with a mechanism that can reliably discharge old culture medium from within the culture vessel.

[0008] The present invention, which achieves the above object, is configured as follows: A closed-system culture vessel having, above the sealed container, an open-system culture vessel, an airtight container that airtightly seals the open-system culture vessel, a liquid supply port that supplies at least a liquid to the open-system culture vessel, and a liquid drainage port that drains the liquid in the open-system culture vessel, the liquid supply port being located above the open surface of the open-system culture vessel, and the tip of a drainage pipe connected to the liquid drainage port being positioned near the bottom surface of the open-system culture vessel.

[0009] The cell culture device is also equipped with the above-mentioned closed-system culture vessel, and has a pressure sensor that measures the pressure inside the closed-system culture vessel, and a control unit that controls the amount of air pressure supplied through the air supply port or the amount of gas exhausted from the closed-system culture vessel so that the pressure measured by the pressure sensor becomes a predetermined pressure.

[0010] In addition, the cell culture device is equipped with the above-mentioned closed system culture vessel and is provided with a control unit that controls the amount of air pressure supplied through the air supply port so that it is approximately equal to the amount of gas exhausted from the closed system culture vessel.

[0011] and a drainage port for discharging liquid from the open system culture vessel. The method includes the steps of: placing the open system culture vessel in the sealed container from the open end of the sealed container in a highly sterile area; positioning the liquid supply port above the open system culture vessel and positioning the tip of a drainage tube connected to the drainage port near the bottom of the open system culture vessel; heat-sealing the open end of the sealed container to seal the sealed container; connecting the liquid supply port and the drainage port to specified piping of a cell culture device;

[0012] It is possible to provide a closed system culture vessel, a cell culture device, and a cell culture method that are equipped with a mechanism that can reliably discharge old culture medium from within the culture vessel.

[0013] FIG. 1 is a schematic diagram of a cell culture device in an embodiment of the present invention. FIG. 2 is an overall diagram of a cell culture device in an embodiment of the present invention. FIG. 3 is a flowchart of the overall operation of cell culture in an embodiment of the present invention. FIG. 4 is a schematic diagram of a closed culture vessel in an embodiment of the present invention. FIG. 5 is a cross-sectional view of a closed culture vessel during culture. FIG. 6 is a diagram showing a cell culture process in an embodiment of the present invention. FIG. 7 is a diagram showing a usage form of a clip applicable to a closed culture vessel in an embodiment of the present invention. FIG. 8 is a schematic diagram of a closed culture vessel in an embodiment of the present invention. FIG. 9 is a schematic diagram of a closed culture vessel in an embodiment of the present invention. FIG. 10 is a diagram showing a cell culture procedure in an embodiment of the present invention.

[0014] Hereinafter, examples of the closed culture vessel, cell culture device, and cell culture method of the present invention will be described with reference to the drawings. However, these examples are merely examples for carrying out the present invention and do not limit the technical scope of the present invention. Furthermore, the same reference numerals are used to designate common components in each drawing.

[0015] (1) Basic Configuration of the Cell Culture Device Figure 1 shows the peripheral configuration of a closed-system culture vessel (hereinafter also referred to as "cell culture vessel") of the cell culture device. The internal space of the entire system of the cell culture device is biologically closed.

[0016] (1-1) Cell Culture Vessel The cell culture vessel 1 of this embodiment is an open culture vessel 3 sealed within a closed vessel 2 (details of the cell culture vessel 1 will be described later).

[0017] During cell culture, the cell culture vessel is kept at a CO2 concentration of approximately 5-10% (generally approximately 5%). ２ Since it is necessary to maintain the concentration of 1000 ppm or more, this cell culture device is provided with a liquid / gas supply port 5, a liquid drain port 6, and an exhaust port 12 for exchanging gas and liquid in the gas phase within the cell culture vessel 1. In Fig. 1, the liquid supply port and the gas supply port are shared to simplify the structure of the cell culture vessel 1 (to reduce costs), but they can also be provided as separate ports if necessary.

[0018] (1-2) Air supply section The liquid / air supply pipe 7 is connected to the liquid / air supply port 5, and liquid and gas are supplied to the open culture vessel 3 installed in the cell culture vessel 1 via the liquid / air supply port 5.

[0019] The liquid / air supply pipe 7 is connected to a cylinder 201 filled with the gas to be ventilated. For example, it may be directly connected to the cylinder 201 through a first filter 501, but Figure 1 shows an example in which it is indirectly connected by providing a humidifying bottle 601.

[0020] The humidifying bottle 601 is a sealed bottle that holds sterilized pure water or the like. When the humidifying bottle 601 is provided, the liquid / air supply pipe 7 penetrates the lid of the humidifying bottle 601 and opens to the gas phase inside the humidifying bottle 601. The liquid / air supply pipe 7 is provided with a valve (also referred to as a valve) 80, which serves as a control valve for supplying gas inside the humidifying bottle 601 to the gas phase inside the culture vessel. Meanwhile, a gas pipe 301 is provided from a cylinder 201 that stores the gas to be supplied to the humidifying bottle 601, and its opening opens into the pure water inside the humidifying bottle 601. The gas pipe 301 is provided with a gas flow regulator 401 and a first filter 501.

[0021] A flexible resin material is suitable for the tube used for the gas pipe 301, and since gas must pass through the inside, a material with low gas permeability is also preferable. Materials such as ethyl vinyl alcohol (EVOH), polyester, or polyvinyl chloride may be used, but highly elastic rubber may also be used for the part that is closed by the solenoid valve.

[0022] The first filter 501 is provided to make the system a closed system. For example, a filter with a mesh size of 0.22 μm that prevents the entry of viruses and bacteria can be used.

[0023] (1-3) Liquid Supply Section The liquid / gas supply port 5 can supply not only gas but also liquid to the open culture vessel 3. After closing valve 80 and opening valve 230, pump 240 is operated to supply the liquid in the liquid bottle containing the liquid to be supplied to the open culture vessel 3. At this time, atmospheric gas (air) is introduced into the liquid container 260 via filter 220 to prevent negative pressure from forming inside the liquid container.

[0024] It is desirable that the liquid / gas supply port 5 be provided above the opening of the open culture vessel 3 so that the liquid 200 supplied from the liquid / gas supply port 5 falls into the open culture vessel 3 without any loss of liquid. Note that when the air supply port is provided separately from the liquid supply port, it is not necessary to provide the air supply port above the opening of the open culture vessel 3.

[0025] (1-4) Exhaust Unit and Pressure Regulator The exhaust pipe 120 is open to the environment via a second filter 130. A pressure regulator for regulating the pressure inside the cell culture vessel 1 is provided midway through the exhaust pipe 120, maintaining a constant pressure within the closed system, particularly within the cell culture vessel 1. When the pressure regulator includes a pressure reducer, for example, a suction device 140 may be connected, thereby enabling the gas inside the exhaust pipe 120 to be efficiently discharged to the environment. The suction device 140 may have any structure as long as it can suck the gas inside the exhaust pipe 120. For example, it may be a pressure reducer (ejector) having a mechanism for reducing the pressure on the side of the pipe connected in a T-shape relative to the perpendicular flow by the Venturi effect. To adjust the pressure reduction by the pressure reducer, for example, a pressure source 150 connected to the pressure reducer by a pipe may be provided, or a flow rate regulator 160 may be provided in the pipe connecting the pressure source 150 and the suction device 140. In this case, the exhaust pipe 120 can be depressurized by controlling the flow rate regulator 160 to discharge the air pressurized by the pressure source 150. The suction force in the exhaust pipe 120 can be adjusted by using the flow regulator 160 to control the amount of pressurized air discharged. In addition to pressure reducers with this type of structure, peristaltic pumps, diaphragm pumps, syringe pumps, and the like can also be used as suction devices, but suction devices without a physical valve structure inside are preferable. This is because by turning off the suction device 140 when suction is not required, the exhaust pipe will not be clogged even in the event of a malfunction, and there is no risk of an abnormal increase in internal pressure.

[0026] The tubes used for the liquid / gas supply pipe 7 and the exhaust pipe 120 are preferably made of a flexible resin material, similar to the gas pipe 301, and further preferably have low gas permeability, since gas must pass through the inside. Examples of materials that may be used include ethyl vinyl alcohol (EVOH), polyester, and polyvinyl chloride.

[0027] The second filter 130 is provided to make the system a closed system, similar to the first filter 501. For example, a filter with a mesh size of 0.22 μm that prevents the entry of viruses and bacteria can be used.

[0028] (1-5) Drainage Port The drainage port 6 is a port for discharging waste liquid in the open culture vessel 3 to the outside of the cell culture vessel 1. To improve the efficiency of medium exchange in the open culture vessel 3, it is desirable that the tip of the drainage pipe connected to the drainage port is positioned near the bottom of the culture dish. When draining liquid, the valve 610 is opened and the pump 620 is operated to collect the drainage liquid in the drainage bag 630.

[0029] (1-6) Gas Exchange Method Using the above-described gas exchange device, the inside of the cell culture vessel 1 can be ventilated as follows.

[0030] First, a predetermined amount of liquid medium containing cells is placed in the open culture vessel 3. The cylinder 201 is opened and the air supply valve 80 is opened. The gas flow rate is adjusted using the flow rate regulator 401 so that a predetermined amount of gas from the cylinder 201 enters the pure water in the humidifying bottle 601 through the gas pipe 301. The gas, humidified by passing through the pure water, enters the gas phase inside the cell culture vessel 1 through the liquid / air supply pipe 7. Initially, the gas phase inside the cell culture vessel 1 is filled with air, but this is replaced with humidified gas over time.

[0031] The gas pushed out from the cell culture vessel 1 reaches the suction device 140 through the exhaust pipe 120. When there is no control by the flow rate regulator 160, the gas passes through the inside of the suction device 140 by the pushing force and is exhausted into the environment, but when the flow rate regulator 160 is operated, the gas is efficiently exhausted by the suction force.

[0032] Since the cell culture vessel 1 has a structure in which an open culture vessel 3 is sealed by a sealed vessel 2, when air is supplied to the cell culture vessel 1, the sealed vessel 2 expands. However, by adjusting the pressure of the entire system using the suction device 140, the internal pressure of the sealed vessel 2 can be maintained at normal pressure, and the shape of the sealed vessel 2 and the volume of the gas phase can be maintained constant.

[0033] (2) Overall Configuration of Cell Culture Device Fig. 2 shows an example of the overall configuration of the cell culture device 100. The cell culture device 100 equipped with a liquid transfer control means for supplying or discharging a liquid culture medium will be described below.

[0034] (2-1) Description of Device Configuration The cell culture device 100 is provided with a thermostatic bath 63, and is controlled by a controller (also referred to as a "controller") 65 to maintain the cell culture vessel 1 at an optimum culture temperature for cell culture. A refrigerator 64 is provided to maintain a low temperature for liquid culture medium replacement. The closed cell culture system installed in the thermostatic bath 63 and refrigerator 64 can be replaced as an integrated flow path, and the entire flow path can be removed for each culture operation, and a new flow path can be installed and used.

[0035] The configuration for delivering liquid or gas to the cell culture vessel 1 is described below. First, a sealed cell bottle 20 for holding a cell suspension is provided, and a supply pipe 23 connected to the cell bottle 20 is provided for supplying the cell suspension to the cell culture vessel 1. One end of the supply pipe 23 has an opening inside the cell bottle 20, which contacts the held cell suspension and serves as the opening for delivering the cell suspension. Therefore, the opening is preferably located near the bottom of the cell bottle 20. The supply pipe 23 is connected to a branch point 25 via an on-off valve 24 that controls the supply pipe 23. This branch point 25 is a junction of multiple branches, and is connected to a gas inlet pipe 28 having a gas inlet valve 27 and a supply pipe 34 having a supply valve 35. A ventilation pipe 51 leading to a ventilation branch point 26 is also connected to the branch point 25. The branch point 25 is located above the liquid level held in the cell bottle 20 and above the liquid level held in the culture medium bottle 33.

[0036] The supply pipe 34 is connected to a culture medium bottle 33. The culture medium bottle 33 is a liquid bottle that holds a liquid culture medium for culture medium replacement, and is kept in a refrigerator 64. The lid of the cell bottle 20 is provided with an air pressure adjustment pipe 21 for adjusting the air pressure, and the air pressure adjustment pipe 21 is provided with a filter 22 that controls the opening and closing of the air pressure adjustment pipe 21.

[0037] The gas inlet pipe 28 is connected to a gas bag 29 via a filter 30. The gas bag 29 holds an optimum concentration of gas for controlling the culture environment, such as the pH value of the cell suspension or liquid culture medium. A check valve 32 is provided in the gas bag 29 via a pressure adjustment pipe 31, and the open end of the check valve 32 opens into the interior of the thermostatic chamber 63. The check valve 32, also known as a check valve, restricts the flow of fluid to one direction, and in this embodiment, directs the flow of gas from the gas bag 29 to the space inside the thermostatic chamber 63.

[0038] A liquid feed pump 36 is connected to the ventilation branch point 26 via a liquid feed pipe 38. A third on-off valve 37 is provided and connected to the liquid feed pipe 38 so as to bypass the liquid feed pump 36.

[0039] In this embodiment, the cell culture vessel 1 is provided with a liquid / gas supply port 5, a liquid drain port 6, and an exhaust port 12, as described above. A liquid supply pipe 38 is connected to the liquid / gas supply port 5. The exhaust port 12 is connected to an exhaust pipe 43. The exhaust pipe 43 is controlled by an air pressure adjustment valve 44, and is further connected to a suction device 140 via a filter 45 and opens into the interior of the thermostatic bath 63. The functions of the pressure source 15 and the flow rate regulator 16 for the suction device 140 are as described above. The exhaust pipe 43 is also connected to a pressure sensor 69 via a second filter 68. The pressure sensor 69 allows the pressure inside the cell culture vessel 1 to be constantly measured. The liquid drain port 6 is connected to a discharge pipe 46.

[0040] (2-2) Method for Feeding Cell Suspension and Liquid Culture Medium A method for feeding cell suspension and liquid culture medium to cell culture vessel 1 using this cell culture device will be described. First, air pressure adjustment valve 44 is opened and liquid feed pump 36 is operated. When feeding cell suspension from cell bottle 20 to cell culture vessel 1, on-off valve 24 is opened and other valves 27, 35, 37, 52, and 53 are closed. When feeding liquid culture medium from medium bottle 33 to cell culture vessel 1, only supply valve 35 is opened and other valves 24, 27, 52, and 53 are closed. When feeding gas from gas bag 29 to cell culture vessel 1, only gas introduction valve 27 is opened and other valves 24, 35, 37, 52, and 53 are closed. In either case, the original gas in cell culture vessel 1 is released into thermostatic bath 63 through filter 45 by the pressure of liquid feed pump 36.

[0041] (2-3) Method of Supplying a Predetermined Gas to the Gas Phase of the Culture Vessel A method of ventilating the gas phase of the cell culture vessel 1 with a predetermined gas will be described. A cylinder containing the predetermined gas is connected to the gas mixer 59. As an example, the gas mixer 59 is configured to supply 100% CO ２ It is assumed that a cylinder 60, a nitrogen cylinder 61, and a filter 62 are connected. Clean air can be supplied to the gas mixer 59 from the filter 62, which is open to the atmosphere, through the filter 62. For example, 5% CO ２ When air containing 100% CO is required, the air supplied from the filter 62 is ２ CO supplied from the cylinder 60 ２ By diluting the gas, a gas with the desired concentration can be generated. ２ , 1% O ２ When gas of 100% CO is required, nitrogen cylinder 61 is used. ２ CO supplied from the cylinder 60 ２ can be diluted to produce a gas having the desired concentration composition.

[0042] The flow rate regulator 58 is connected to the gas mixer 59 via a filter 54, and can control the flow rate of the gas prepared to the desired concentration by the gas mixer 59 to any amount from 0. The pipe from the flow rate regulator 58 branches and is connected to a first on-off valve 52 that controls the ventilation pipe 51 and a humidification pipe 55. The humidification pipe 55 is connected to a humidification bottle 56 that holds sterilized water therein, and to a second on-off valve 53 that controls a humidification pipe 57 provided in the humidification bottle 56. The pipe having the first on-off valve 52 and the pipe having the second on-off valve 53 join together and are connected to the ventilation branch point 26 described above via the ventilation pipe 51.

[0043] As mentioned above, in order to prevent the pH value of the liquid medium during cell culture from changing, for example, if a bicarbonate buffer system is used in the medium, CO is periodically or constantly added. ２ It is necessary to supply air with a high concentration. Furthermore, it is necessary to prevent the concentration of liquid culture medium components due to evaporation of water from the liquid culture medium. When gas exchange and humidification are performed for the gas phase of the cell culture vessel 1, the first on-off valve 52 is closed, and the second on-off valve 53, the third on-off valve 37, and the air pressure adjustment valve 44 are opened, and then the gas mixer 59 is operated. The concentration-adjusted gas is adjusted to a predetermined flow rate by the flow regulator 58, and is humidified by passing through the humidifying pipe 55 and sterilized water in the humidifying bottle 56.

[0044] The CO 2 that has become foamy in the humidifying bottle 56 ２ The gas remains in the gas phase in the humidifying bottle 56 and travels from the humidifying pipe 57 through the ventilation pipe 51 and the third on-off valve 37 to reach the cell culture vessel 1 .

[0045] At this time, pressure sensor 69 measures the internal pressure of cell culture vessel 1, and if the internal pressure is higher than the desired pressure, flow regulator 16 is activated to reduce the internal pressure of cell culture vessel 1 via exhaust pipe 43 connected to suction device 140 and maintain the pressure at a predetermined level. Instead of managing the pressure inside cell culture vessel 1 with pressure sensor 69, the amount of gas supplied into cell culture vessel 1 may be measured by flow regulator 40, and the amount of gas discharged from cell culture vessel 1 may be adjusted by flow regulator 160.

[0046] Gas exchange and humidification of the gas phase of the culture vessel may be performed continuously, or the supply of gas may be stopped periodically or when it is no longer necessary, such as when the gas reaches a predetermined concentration. In this example, the case of one culture vessel is used as an example, but if there are multiple culture vessels, gas may be supplied to them simultaneously, or gas may be supplied intermittently to each culture vessel for a certain period of time, thereby supplying gas to multiple culture vessels in a circular manner.

[0047] (2-4) Method for Discharging Liquid from Culture Vessel A method for discharging liquid held in the cell culture vessel 1 will be described. A discharge pipe 46 connected to the cell culture vessel 1 via a discharge port 6 is connected to a discharge pump 48 via a discharge control valve 47, which is connected to a discharge pipe 49 and then to a drainage bag 50. When the second on-off valve 53, the third on-off valve 37, and the discharge control valve 47 are opened, the other valves 24, 27, 35, 44, and 52 are closed, and the discharge pump 48 and the flow rate regulator 58 are simultaneously operated, the liquid held at the bottom of the open culture vessel 3 in the cell culture vessel 1 is sent to the drainage bag 50. At this time, the amount of liquid and gas in the cell culture vessel 1 is reduced by an amount equivalent to the discharge rate of the discharge pump 48, and the mixed gas flows into the cell culture vessel 1 due to the pressure of the flow rate regulator 58, so that the inside of the cell culture vessel 1 is maintained at normal pressure.

[0048] (3) Cell Culture Operation Figure 3 shows a flowchart of the overall cell culture operation in the cell culture device 100 shown in Figure 2, controlled by the controller 65 (controller). First, a flow path is installed in the thermostatic bath 63 (S01), and then a cell bottle 20 holding a separately prepared cell suspension and a medium bottle 33 holding a liquid medium are connected to the flow path (S02). Next, the gas bag 29 is automatically filled with gas (S03). Gas is supplied to the cell culture vessel 1, followed by the cell suspension (S04). Humidified gas is immediately supplied to the cell culture vessel 1 to maintain a constant temperature for the cells and allow them to stand (S05). Depending on the progress of cell culture, a decision is made as to whether to start replacing the liquid medium (S06). When replacing the liquid medium, the gas bag 29 is filled with gas (S07), the old medium is discharged from the culture vessel (S08), and new liquid medium is supplied from the medium bottle (S09). Humidified gas is then supplied and allowed to stand (S10), and depending on the progress of the cell culture, a decision is made as to whether the culture should continue (S11), and the culture medium is replaced again. At the end of cell culture, the automatic culture is stopped, the cultured cells are manually removed (S12), the drainage bag 50 is collected to check for bacterial growth (S13), and the used flow path is removed from the thermostatic bath 63, completing the process.

[0049] Fig. 4 shows the configuration of a cell culture vessel 1 according to an embodiment of the present invention. Fig. 4(A) is a top view of the cell culture vessel 1 of this embodiment, and Fig. 4(B) is a cross-sectional view taken along line BB' in Fig. 4(A).

[0050] A drainage port 6, a liquid / gas supply port 5, and an exhaust port 12 are welded to the top surface of a sealed container (also called a "bag") 2, and an open culture vessel (also called a "culture dish" or "dish") 3 is placed inside the sealed container 2. The drainage pipe connected to the drainage port 6 has a length that reaches close to the bottom of the open culture vessel 3. The liquid supply port and the air supply port are shared as the liquid / gas supply port 5, but the liquid supply port and the air supply port may be provided separately. By providing the liquid supply port above within the opening surface of the open culture vessel 3, loss of culture medium can be reduced.

[0051] The drainage port 6 can be provided with the tip of the drainage pipe connected to the drainage port 6 located near the bottom of the culture dish, thereby improving the efficiency of medium exchange. On the other hand, there is no problem if the air supply port and the exhaust port are located at a position not above the opening of the open culture vessel 3.

[0052] With this configuration, the open-system culture vessel 3 previously used for manual culture (open system) can be easily converted into a closed-system cell culture vessel. In particular, in the case of adherent culture, cells are cultured by adhering to the open-system culture vessel 3, so changing the culture vessel is a major change that changes the scaffolding of the cells and can affect the culture. Therefore, being able to transition from manual culture (open system) to automated culture (closed system) without changing the culture vessel is effective from the perspective of increasing the success rate of cell culture.

[0053] In the closed culture vessel of this embodiment, it is possible to use a different open culture vessel 3 without changing the positions of the drainage port 6, the liquid and air supply port 5, and the exhaust port 12 welded to the top surface of the sealed vessel 2. This will be explained using Figures 5(A) and 5(B).

[0054] 5(A) and 5(B), the drainage port 6, the liquid / gas supply port 5, and the exhaust port 12 are arranged in a line at the top of the drawing of the sealed vessel 2. As described above, even if the liquid supply port is positioned at the top within the open surface of the vessel of the open culture vessel 3 and the tip of the drainage tube connected to the drainage port 6 is positioned near the bottom of the culture dish, it is possible to use an open culture vessel 3 with a relatively small diameter (FIG. 5(A)) or a large inner diameter approximately the same as the inner diameter of the bag (FIG. 5(B)). In other words, various commercially available open culture dishes can be used as they are.

[0055] The open culture dishes that have been used in clean rooms for cell culture, known as Cell Processing Centers (CPCs), can be continued to be used as they are, and the medium exchange required for cell culture, as well as gas exchange and humidification within the culture vessels, can be performed in a biologically closed space outside the clean room, which will greatly contribute to reducing the cost of cell culture.

[0056] The reason for arranging them in a straight line is so that they can be hooked onto the edge of the open culture vessel 3 and fixed in position, and by hooking them onto the edge and fixing them, the liquid drainage port 6, liquid supply / gas supply port 5, and exhaust port 12 can be stably fixed to the open culture vessel 3 even if the size of the open culture vessel 3 is different. Furthermore, arranging them in a straight line is not essential, and the exhaust port 12 can be located anywhere as long as it can exhaust gas from inside the sealed vessel 2.

[0057] On the other hand, when culturing cells using a closed-system culture vessel with the bag of this embodiment, it is important to manage the pressure inside the bag. If the pressure inside the bag becomes too high relative to the ambient air pressure, depending on the relative positions of the liquid / gas supply port 5, the liquid drainage port 6, and the open-system culture vessel 3, as shown in FIG. 6, there is a non-zero possibility that the liquid supplied from the liquid / gas supply port 5 may not be properly supplied to the open-system culture vessel 3, or that the tip of the drainage tube connected to the liquid drainage port 6 may rise above the bottom of the open-system culture vessel 3, making it difficult to properly aspirate the drainage liquid. For this reason, as shown in FIG. 7, it is effective to control the pressure and suppress deformation of the bag. Furthermore, as mentioned above, instead of managing the pressure, deformation of the closed-system culture vessel may be suppressed by controlling the amount of gas supplied from the liquid / gas supply port 5 so that it is approximately equal to the amount of exhaust gas exhausted from the closed-system culture vessel. Note that the components shown in FIG. 7 are the same as those in FIG. 1, and the same reference numerals are used for each component, so a description of each component will be omitted.

[0058] Furthermore, in order to suppress deformation of the sealed vessel 2, it may be effective to use a clip as shown in Fig. 8. If there is excess space in the bag around the open culture vessel 3, clips are used to fasten the periphery of the open culture vessel 3 so as to reduce that space. When sealing the open culture vessel 3 with a bag, it is possible to address this issue by sealing so as to minimize such excess space, but such clips are useful in cases where sealing does not go well.

[0059] Any clip can be used as long as it can fasten the bag so that the extra space is small. For example, a clip consisting of a pair of opposing strip-shaped members as shown in Figure 8(C) (commercially available for fastening the opening of a plastic bag) can be used.

[0060] As a measure to prevent misalignment of the positional relationship between the liquid / gas supply port 5, the liquid / drain port 6 and the open culture vessel 3, there is also a method of fixing the liquid / gas supply port 5 and the liquid / drain port 6 to a lid-like member that covers the open culture vessel 3, as shown in Figure 9. Alternatively, the lid may be pressed down from the outside of the bag with a belt or the like to prevent it from lifting. The optimal lid-like member is one that has a groove that fits into the opening of the open culture vessel 3, but even a member that does not have a groove and simply covers the open culture vessel 3 can still be effective in preventing misalignment of the positional relationship between the liquid / gas supply port 5, the liquid / drain port 6 and the open culture vessel 3.

[0061] 10, the fluid supply / gas supply port 5 and the fluid discharge port 6 may be provided with a support member 10 that can be fitted and fixed to the outside of the open culture vessel 3 to prevent the ports from coming off the culture dish. The support member 10 is formed of a material with appropriate rigidity, such as plastic, and may have any shape as long as it can contact the outer surface of the culture dish 3 and prevent the ports from coming off.

[0062] 11, a connection port 14 may be provided that penetrates the inside and outside of the bag (sealed container), and the inside of the connection port 14 may be connected to the drainage port 6 and the fluid supply port 5 by flexible piping 11 such as rubber tubing. The piping 11 absorbs deformation of the bag and prevents the ports from shifting position. The drainage port 6 and the fluid / gas supply port 5 are fixed in position relative to the open culture vessel 3 by providing a support member positioned on the outside of the open culture vessel 3, as shown in FIG. 10. A lid such as that shown in FIG. 9 may be used instead of the support member.

[0063] An example of the procedure for culturing using the vessel of this embodiment is shown in Figure 12. The vessel of this embodiment can be provided in two ways.

[0064] (1) Only the bag with the port is packaged, sterilized, and provided. In this case, the bag is packaged with one side open. Users can use the culture dishes they have traditionally used for manual culture by sealing them into the bag themselves. Users can freely choose the type of culture dish, but sealing work is required.

[0065] (2) The culture dish to be used by the user is sealed in a bag with a port and provided in a sterilized state. The user can omit the task of sealing the culture dish, but must choose the culture dish from the options provided by the provider.

[0066] (1) involves steps 1 to 8. (2) involves steps 4 to 8. Figure 12 shows the procedure for (1). In both cases (1) and (2), a tube with a sterile connector is fitted to the port installed on the bag, allowing it to be aseptically connected to the flow path of an automatic culture device or the like outside the clean bench.

[0067] When removing the culture dish from the bag in step 7, the bag is opened. This is done in a highly sterile area, such as a clean bench in a clean room, to prevent bacteria or foreign matter from getting into the culture dish (contamination). Since workers wear double gloves, delicate work is difficult. The bag can be easily cut open using scissors. Alternatively, if a slit (not shown) is made in the bag so that it can be opened by tearing it apart, it can be opened without using any tools.

[0068] In this way, if the top surface of the culture dish can be made open, not only cells but also organized cells in a sheet-like state can be easily removed.

[0069] 1...cell culture vessel, 2...sealed vessel, 3...open culture vessel (culture dish), 4...sealing portion, 5...liquid / air supply port, 6...discharge port, 7...liquid / air supply tube, 8...culture fluid, 12...exhaust port, 80...valve, 201...cylinder, 301...gas tube, 501...first filter, 601...humidification bottle.

Claims

1. A closed system culture vessel having, above the sealed container, an open system culture vessel; an airtight container that airtightly seals the open system culture vessel; a liquid supply port that supplies at least liquid to the open system culture vessel; and a liquid discharge port that discharges liquid from the open system culture vessel, the liquid supply port being located above the open system culture vessel and within the opening surface of the open system culture vessel; and a tip of a drainage pipe connected to the drainage port being positioned near the bottom surface of the open system culture vessel.

2. A closed culture vessel according to claim 1, wherein at least the upper surface of the sealed vessel is made of a flexible material.

3. A closed culture vessel according to claim 2, wherein either the liquid supply port or the liquid discharge port is welded to the top surface of the sealed vessel.

4. A closed culture vessel according to claim 1, wherein the liquid supply port also serves as an air supply port for supplying gas into the sealed vessel.

5. A closed culture vessel according to claim 1, characterized in that the closed vessel further comprises an exhaust port for discharging gas from within the closed vessel.

6. A closed culture vessel according to claim 1, wherein either the liquid supply port or the liquid discharge port has a support member that fits into the opening of the open culture vessel.

7. A closed culture vessel according to claim 6, comprising: a connection port penetrating the inside and outside of the sealed vessel; and flexible piping connecting the liquid supply port and / or the liquid discharge port fitted to the open culture vessel by the support member to the connection port.

8. A closed culture vessel according to claim 1, characterized in that it has a pressure-resistant lid that covers the open culture vessel, and either the liquid supply port or the liquid discharge port is fixed to the lid.

9. A closed culture vessel according to claim 1, characterized in that the sealed vessel has a notch that allows it to be easily opened.

10. A cell culture device equipped with a closed culture vessel according to any one of claims 1 to 9, characterized in that it comprises: a pressure sensor that measures the pressure inside the closed culture vessel; and a control unit that controls the amount of gas supplied through the liquid supply port or the amount of gas exhausted from the closed culture vessel so that the pressure measured by the pressure sensor becomes a predetermined pressure.

11. A cell culture device equipped with a closed culture vessel according to any one of claims 1 to 9, characterized in that the cell culture device is equipped with a control unit that controls the amount of gas supplied through the liquid supply port so that the amount of gas exhausted from the closed culture vessel is approximately equal.

12. A cell culture method using a sealed container made of a heat-weldable material, in which at least a liquid supply port for supplying liquid to an open culture vessel housed therein and a liquid discharge port for discharging liquid from the open culture vessel can be positioned above the open culture vessel, comprising the steps of: housing the open culture vessel in the sealed container from the open end of the sealed container in a highly sterile area; positioning the liquid supply port above the open surface of the open culture vessel and positioning the tip of a drainage tube connected to the liquid discharge port near the bottom of the open culture vessel; heat-welding the open end of the sealed container to seal the sealed container; connecting the liquid supply port and the liquid discharge port to predetermined piping of a cell culture device; supplying predetermined medium and cells from the piping of the cell culture device to the liquid supply port of the open culture vessel and culturing the cells; and, after cell culture in the open culture vessel is completed, disconnecting the piping from the liquid supply port and the liquid discharge port. and a step of cutting open the sealed container and removing the open culture vessel from the sealed container.

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