Method for confirming the proper installation of a culture apparatus and the flow path of the culture apparatus.

Abstract

To provide a culture apparatus that can confirm the proper installation of the flow channels in the culture apparatus, and a method for confirming the proper installation of the flow channels in the culture apparatus. [Solution] A culture apparatus comprising a liquid bottle, a gas supply source for supplying gas to the liquid bottle, a flow path connecting the liquid bottle and the gas supply source, a sterile connector provided in the flow path and in order from the liquid bottle side, a manual valve, at least one automatic valve, and a pressure sensor, characterized in that it comprises a determination unit that determines abnormalities in the manual valve and automatic valve in the flow path, and / or the normality of the attachment of tubes to the manual valve and automatic valve, based on the pressure sensor measurement value when a combination of opening and closing of the automatic valve and manual valve is performed while gas is supplied from the gas supply source.

Description

Technical Field

[0001] The present invention relates to a culture device and a method for confirming the proper installation of the flow path of the culture device.

Background Art

[0002] Regenerative medicine that uses regenerated tissue manufactured from cells as raw materials to restore the functions of organs such as the liver is expected as a radical treatment method for diseases that have not had conventional treatment methods. The treatment targets cover a wide range, including skin, cornea, esophagus, heart, bone, cartilage, etc., and the clinical application examples thereof are also increasing rapidly. In the manufacturing process of regenerated tissue, biological samples collected from the patient himself or others are separated and purified, and processed such as amplification and tissue formation. This process is carried out in a cell processing facility (CPC: Cell Processing Center) according to a standard operating procedure (SOP: Standard Operating Procedure) that meets the Good Manufacturing Practice (GMP), which is the standard for manufacturing management and quality control of pharmaceuticals and the like. Therefore, the operation of CPC requires a large amount of cost and personnel with specialized culture techniques. In addition, since the manufacturing process is mainly manual, there is a limit to the increase in the manufacturing volume. Low productivity and high manufacturing costs have hindered the spread of regenerative medicine, and automation of the culture operation, which particularly requires labor and cost in the manufacturing process, is demanded. Automation of the culture operation enables labor saving, cost reduction, and mass production.

[0003] As an example of an automatic culture device, for example, as shown in Patent Document 1, there is a device that automatically handles a closed system flow path having a closed space. In the closed system flow path, a closed system culture container is constantly connected by a flow path tube or the like, and cells are cultured inside the closed system culture container to manufacture regenerated tissue.

[0004] A closed-loop system allows the movement of liquids and gases held inside through the operation of valves, pumps, etc., installed on its exterior. This enables the automated culture device to automatically perform cell seeding, culture medium exchange, microscopic observation, etc., while maintaining the closed nature of the culture space. Furthermore, Patent Document 1 describes a method for determining the correctness of the installation of a closed-loop system when an operator installs it, by using the gas pressure that normally controls the gas phase component of the culture vessel, and by measuring the rise or fall of the gas pressure value with a pressure sensor.

[0005] On the other hand, the sterile connector disclosed in Patent Document 2 provides a connector that can aseptically connect a liquid bottle to a closed flow path as described above. A typical product name is the AQS17004 from Calder Products' Aseptic Connector Series. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2016-208866 [Patent Document 2] U.S. Patent No. 12042621 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In a closed-loop system, rubber tubing functions as a valve when correctly installed in an automatic valve (pinch valve), controlling the direction of liquid and air flow. Therefore, after the operator visually confirms the installation of the rubber tubing in the automatic valve, the system is equipped with a function to automatically verify that the tubing functions correctly as a valve. There are two types of automatic valves: one that only allows air and liquid to pass through, and another that has a liquid bottle connected upstream. Patent document 1 deals with the former, the automatic valve that only allows air and liquid to pass through, and does not mention a method for verifying the latter, the automatic valve to which a liquid bottle is connected.

[0008] The reason for this is that, in the example of the sterile connection connector mentioned above, a protective film is provided to cover the inner surface which maintains sterility. By closely joining the protective films of the two connecting parts to be connected and fixing them together, the protective films are then pulled out, allowing electrical connection between the two sterile tubes. However, if pressure is applied from one of the tubes to this connecting part alone, there is a risk that the protective film of the sterile connection connector will rupture and be damaged.

[0009] In closed-loop systems, where automatic valves and manual valves (which are also automatic valves but require manual operation during use) are used together, if even one automatic valve among many is not properly installed, unintended pressurization can be applied to the sterile connection connector, potentially damaging the protective membrane. To address this, automatic valves connected to liquid bottles are equipped with a manual valve between them and the sterile connection connector connected by a tube. This manual valve is closed during checks of the flow path's functionality to protect the sterile connection connector from unintended pressurization.

[0010] Thus, in the conventional automated installation and verification process, the manual valve remains closed between the automated valve to which the liquid bottle is connected and the sterile connection connector connected to it by a tube. The manual valve is opened only when the liquid bottle is connected to the closed system flow path at the required time, finally establishing contact with the automated valve and supplying liquid to the automated culture system.

[0011] The object of the present invention is to provide a culture apparatus that can confirm the normal installation of the flow channels of the culture apparatus, and a method for confirming the normal installation of the flow channels of the culture apparatus. [Means for solving the problem]

[0012] The configuration of the present invention for achieving the above objective is as follows. A culture apparatus comprising a first liquid bottle, a first gas supply source for supplying gas to the first liquid bottle, a first flow path connecting the first liquid bottle and the first gas supply source, a first sterile connector provided in the first flow path and arranged in order from the side closest to the first liquid bottle, a first manual valve, at least one first automatic valve, and a first pressure sensor, characterized in that it comprises a first determination unit that determines abnormalities in the first manual valve and the first automatic valve in the first flow path, and / or the normality of the attachment of tubes to the first manual valve and the first automatic valve, based on the measurement value of the first pressure sensor when a combination of opening and closing of the first automatic valve and the first manual valve is performed while gas is supplied from the first gas supply source.

[0013] Furthermore, the culture apparatus comprises a second liquid bottle, a second gas supply source for supplying gas to the second liquid bottle, a second flow path connecting the second liquid bottle and the second gas supply source, a second sterile connector provided in the second flow path and arranged in order from the side closest to the second liquid bottle, a second manual valve, at least one second automatic valve, and a second pressure sensor, and is characterized by comprising a second determination unit that determines abnormalities in the manual valve and automatic valve in the second flow path, and / or the normality of the tube attachment to the second manual valve and the second automatic valve, based on the measurement value of the second pressure sensor when a combination of opening and closing of the second automatic valve and the second manual valve is performed while gas is being supplied from the second liquid bottle by the second gas supply source.

[0014] Furthermore, the present invention relates to a method for confirming the normality of the installation of a flow path in a culture apparatus, comprising a first liquid bottle, a first gas supply source for supplying gas to the first liquid bottle, a first flow path connecting the first liquid bottle and the first gas supply source, and provided in the first flow path, in order from the side closest to the first liquid bottle, a first sterile connector, a first manual valve, at least one first automatic valve, and a first pressure sensor, and is characterized by including a first pressure measurement step of measuring the pressure value at the first pressure sensor when a combination of opening and closing of the first automatic valve and the first manual valve is performed while gas is supplied from the first gas supply source, and a first determination step of determining whether there is an abnormality in the first manual valve and the first automatic valve in the first flow path, and / or the normality of the attachment of tubes to the first manual valve and the first automatic valve, based on the pressure value measured in the pressure measurement step.

[0015] Furthermore, the present invention relates to a method for confirming the normality of the installation of a flow path in a culture apparatus, comprising a second liquid bottle, a second gas supply source for supplying gas to the second liquid bottle, a second flow path connecting the second liquid bottle and the second gas supply source, and provided in the second flow path, in order from the side closest to the second liquid bottle, a second sterile connector, a second manual valve, at least one second automatic valve, and a second pressure sensor, and is characterized by including a second pressure measurement step of measuring the pressure value at the second pressure sensor when a combination of opening and closing of the second automatic valve and the second manual valve is performed while gas is being supplied from the second gas supply source to the second liquid bottle, and a second determination step of determining whether there is an abnormality in the second manual valve and the second automatic valve in the second flow path, and / or the normality of the attachment of tubes to the second manual valve and the second automatic valve, based on the pressure value measured in the second pressure measurement step. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a culture apparatus that can confirm the normal installation of the flow channels of the culture apparatus, and a method for confirming the normal installation of the flow channels of the culture apparatus. [Brief explanation of the drawing]

[0017] [Figure 1]It is a diagram showing a configuration of a flow path in the automatic culture device according to Example 1. [Figure 2] It is a diagram showing the configuration of the liquid bottle before connection according to Example 1. [Figure 3] It is a diagram showing a time chart of the flow path installation determination protocol in the flow path configuration according to Example 1. [Figure 4(A)] It is a diagram showing a related flow path circuit diagram in the flow path according to Example 1. [Figure 4(B)] It is a diagram showing a related flow path circuit diagram in the flow path according to Example 1. [Figure 4(C)] It is a diagram showing a related flow path circuit diagram in the flow path according to Example 1. [Figure 4(D)] It is a diagram showing a related flow path circuit diagram in the flow path according to Example 1. [Figure 4(E)] It is a diagram showing a related flow path circuit diagram in the flow path according to Example 1. [Figure 5] It is a diagram showing the pressure measurement result in the liquid feeding device according to Example 1. [Figure 6(A)] In the liquid feeding process according to Example 1, it is a flow path circuit diagram related to the propagation of pressure. [Figure 6(B)] In the liquid feeding process according to Example 1, it is a flow path circuit diagram related to the propagation of pressure. [Figure 7] In the ventilation process according to Example 1, it is a flow path circuit diagram related to the propagation of pressure. [Figure 8] It is a diagram showing a configuration of the automatic culture device according to Example 2. [Figure 9] It is a diagram showing a single cell bottle before connection to the closed system flow path according to Example 2. [Figure 10] It is a diagram showing a time chart of the flow path installation determination protocol in the flow path configuration according to Example 2. [Figure 11] It is a diagram showing the pressure measurement result in the liquid feeding device according to Example 2. [Figure 12] It is a diagram showing a flowchart of the overall operation of the cell culture process in the automatic culture device according to Example 2. [Modes for carrying out the invention]

[0018] Hereinafter, Embodiment 1 of the present invention will be described with reference to the drawings and other drawings. The following description illustrates a specific example of the content of the present invention, and the present invention is not limited to this description. Various changes and modifications are possible by those skilled in the art within the scope of the technical ideas disclosed herein.

[0019] Furthermore, in all the figures used to illustrate the present invention, components having the same function are denoted by the same reference numerals, and repeated explanations may be omitted. [Examples]

[0020] An example of a liquid delivery device consisting of a closed-system flow path for supplying liquid or gas to a culture vessel will be described using Figure 1. Liquid bottle 1 is a liquid bottle connected to the closed-system flow path. Figure 2 shows liquid bottle 1 alone before connection to the closed-system flow path. Container 2 can hold any amount of liquid inside, and the inside is sealed by closing the screw cap 4 through which the suction tube 3 passes. A sterile connection connector 5 is connected to the extension of the suction tube 3, and the contact parts located on the inner surface of the sterile connection connector 5 are covered with a protective film 6 made of high-density polyethylene fiber nonwoven fabric or the like, so that gas can pass through the protective film 6 but bacteria and viruses cannot enter the container. In addition, a vent filter 7 is connected to the screw cap 4 by a tube, so that gas can pass through but bacteria and viruses cannot enter the bottle side.

[0021] The sterile connection connector 5 on the liquid bottle 1 side is connected to the sterile connection connector 5 on the closed system flow path side according to a defined procedure, and then the protective film 6 is removed to install it in the closed system flow path, integrating it as a liquid delivery device that delivers liquid to the culture vessel 8 shown in Figure 1. In Figure 1, 9 is the gas supply source (gas supply source) and is connected to the pressure controller 11 via the gas supply pipe 10. The pressure controller 11 is connected to the flow control unit 12, and the pressure between the pressure controller 11 and the flow control unit 12 is measured by the first pressure sensor 13. The flow control unit 12 can supply gas downstream at any flow rate. 14 is the second pressure sensor, which can detect the gas pressure downstream from the flow control unit 12.

[0022] Valves 15, 17, 19, and 21 are automatic valves, designated as inlet valve 15, gas inlet valve 17, liquid bottle on / off valve 19, and air supply valve 21, respectively. Hereafter, these will be collectively referred to as "automatic valves." Pinch valves are preferred, which open and close the rubber tubes that constitute the flow path using external force. When the rubber tubes, which are clamped in a drive valve actuated by spring force, are closed, the automatic valves can operate when power is applied to control the rubber tubes to an open state. Here, valves that are closed when not in operation and not powered are used. Automatic valve 15 is an inlet valve and is connected to the upstream flow control unit 12 and the downstream branching section 16. Automatic valve 17 is a gas inlet valve, with one end connected to the branching section 16 and the other end connected to the vent filter 18 and exposed to the outside air. Automatic valve 19 is a liquid bottle on / off valve, with one end connected to the branching section 16 of the air supply pipe 10 and the other end connected to the sterile connection connector 5 via a manual valve 20. The automatic valve 21 is an air supply valve and is connected to the liquid supply pipe 26 so as to bypass the pump 22 described below.

[0023] 22 is a pump, one end of which is connected to the liquid delivery pipe 26, and the other end of which is connected to the culture vessel 8 by a tube. The primary purpose of pump 22 is to deliver a desired amount of liquid from the liquid bottle 1 to the culture vessel 8. A suitable pump is a tube pump that generates pressure by squeezing a rubber tube with a rotating roller, and when the liquid delivery is stopped, the rubber tube is compressed by the roller, thus acting in the same way as closing a valve. The aforementioned air supply valve 21 can be opened when pump 22 is not operating, allowing gas to be supplied from the air supply pipe 10 to the culture vessel 8.

[0024] The culture vessel 8 is an airtight container consisting of a main body and a lid. In the configuration shown in this figure, a liquid supply pipe connected to the pump 22 is connected to the top, and a pressure adjustment pipe 23 is provided on the lid, with the other end connected to a vent filter 24 and exposed to the outside air. The main purpose of the culture vessel 8 is to hold cells at the bottom, suspend them with the liquid culture medium necessary for growth, maintain the growth temperature, and enable cell culture by ventilating and adding CO2 gas to the gas phase. 25 is a control unit that controls the operation of the pressure controller 11, flow control unit 12, first pressure sensor 13, second pressure sensor 14, automatic valves 15, 17, 19, 21 and pump 22.

[0025] The following describes the principle of the protocol for confirming the normal installation of the closed-loop flow path through the control of the control unit 25 in the configuration of this embodiment. As shown in Figure 1, two pressure sensors (first pressure sensor 13 and second pressure sensor 14) are installed in the closed-loop flow path. The normal installation is determined by confirming, using the pressure sensor values, that the manual installation of the flow path tube to the automatic valve and pump was performed correctly.

[0026] When the automatic valve is not in operation, i.e., when the automatic valve is closed or the pump is stopped and acting like a shut-off valve, the upper limit of the pressure that can be sealed inside the flow tube is determined by the specifications of the automatic valve and the pump. Normally, the pressure used for air supply is operated at or below the pressure that the automatic valve can seal, and as a guideline, the supply pressure is set to about half of the sealing pressure. The supply pressure is adjusted by the control of the pressure controller 11 and can be constantly monitored by the first pressure sensor 13. In addition, a second pressure sensor 14 connected between the flow control unit 12 and the automatic valve 15 can measure the pressure value at this position.

[0027] When air is supplied through this flow path to a closed automatic valve connected downstream, the gas does not pass through that section. However, the flow control unit 12 attempts to maintain a predetermined amount of air supplied by supplying air downstream, causing the pressure value at the second pressure sensor 14 to rise. After a certain period of time, the pressure sensed by the second pressure sensor 14 reaches approximately the same level as that at the first pressure sensor 13. On the other hand, when the automatic valve and pump are operating, the flow path is open and the flow path tube is not sealed, allowing gas to pass through the inside of the flow path tube. Therefore, the second pressure sensor 14 measures a lower pressure value than that at the first pressure sensor 13. The flow path installation determination protocol in this embodiment is based on the above phenomena.

[0028] Figure 3 shows the time chart of the flow path installation determination protocol in the flow path configuration shown in Figure 1 of this embodiment. The automatic control timings of the gas inlet valve 17, inlet valve 15, liquid bottle on / off valve 19, air supply valve 21, flow control unit 12, and pump 22 are shown, with the automatic valves and pumps being ON when operating and OFF when not operating. The manual valve 20, which is operated manually, and the pressure values ​​of the second pressure sensor 14, which change in conjunction with these, are also shown. First, the normality of the tube attachment to the inlet valve 15 is confirmed in the path from the flow control unit 12 to the gas inlet valve 17 via the inlet valve 15. Figure 4(A) shows the relevant flow path circuit diagram in the flow path shown in Figure 1. The gas inlet valve 17 is operated (the valve is opened) and the inlet valve 15 is deactivated (the valve is closed), and air supply is started by the flow control unit 12. From here on, valves and pumps are deactivated unless otherwise specified. In particular, the manual valve 20 connected to the liquid bottle on / off valve 19 is manually closed, protecting the connected sterile connection connector 5 from external pressure. At this time, the inlet valve 15 is inactive, so if it is properly installed, the second pressure sensor 14 will show a pressure increase. Next, the inlet valve 15 is activated (the valve is opened). In this state, the inlet valve 15 is active, and the gas inlet valve 17 downstream of it is also active, so if it is properly installed, the pressure shown by the second pressure sensor 14 will decrease. At this point, it cannot be determined that the inlet valve 15 is properly installed. This is because there is a possibility that the gas inlet valve 17 downstream is not properly installed.

[0029] Next, the normality of the tube attachment to the gas inlet valve 17 is checked in the path from the flow control unit 12 through the inlet valve 15 to the gas inlet valve 17. The inlet valve 15 is activated, the gas inlet valve 17 is deactivated, and air supply is started by the flow control unit 12. At this time, since the gas inlet valve 17 is deactivated, the second pressure sensor 14 shows a pressure increase when the installation is normal. Next, the gas inlet valve 17 is activated, and air supply is started by the flow control unit 12. At this time, the inlet valve 15 is activated, and the gas inlet valve 17 downstream of it is also activated, so the pressure shown by the second pressure sensor 14 decreases when the installation is normal. At this point, it can be determined that both the gas inlet valve 17 and the inlet valve 15 are installed normally. This is because the pressure value response results expected when the inlet valve 15 and the gas inlet valve 17 downstream are installed are obtained only when the installation is correct.

[0030] Next, the proper installation of the tube to the air supply valve 21 is confirmed. In this confirmation step, pressure is propagated, and the related flow path circuit diagram is shown in Figure 4(B). The inlet valve 15 is activated, and the air supply valve 21 is deactivated, and air supply is started by the flow control unit 12. At this time, since the air supply valve 21 is deactivated, the second pressure sensor 14 will show a pressure increase if the installation is normal. Next, the air supply valve 21 is activated. If the installation is normal, the pressure shown by the second pressure sensor 14 will decrease. At this point, it can be determined that the air supply valve 21 is properly installed. This is because the installation of the inlet valve 15 and the gas inlet valve 17 downstream of it has been determined to be correct, and furthermore, the expected pressure value response results have been obtained for both the activated and deactivated states of the air supply valve 21.

[0031] Next, the proper installation of the tube to the pump 22 is confirmed. Pressure is propagated during this confirmation process, and the related flow path circuit diagram is shown in Figure 4(C). The inlet valve 15 is operated and the supply valve 21 is deactivated, and the flow control unit 12 starts supplying air. At this time, since the pump 22 is deactivated, if the installation is normal, the second pressure sensor 14 will show a pressure increase. Subsequently, when the air supply is stopped by the flow control unit 12, the high pressure is temporarily maintained. When the pump 22 is operated in the next step, if the installation is normal, the gas is supplied downstream and the second pressure sensor 14 will gradually show a decrease in pressure from high pressure. Once a constant target pressure is reached without becoming negative pressure, the pump 22 is stopped. At this point, it can be determined that the pump 22 is properly installed. This is because the installation of the inlet valve 15 and the gas inlet valve 17 and supply valve 21 downstream has been determined to be correct, and furthermore, the expected pressure value response results have been obtained for when only the pump 22 is operating and when it is not operating.

[0032] At this point, the basic installation of the closed-system flow path into the device is complete. Next, we will explain the process of installing liquid bottle 1 into the closed-system flow path. The reason for installing liquid bottle 1 at this stage is that if an abnormality is detected in the flow path installation judgment protocol up to this point, and the problem cannot be resolved by redoing the installation assuming an installation error, it will be necessary to remove the abnormal flow path and replace it with a new closed-system flow path and perform automated culture operation under a normal flow path. Liquid bottle 1 connected to the abnormal flow path may contain valuable samples, and redoing the preparation carries risks. In addition, the volume of liquid bottle 1 needs to be changed according to the amount of liquid required for culture, and installing liquid bottle 1 into the flow path in a later step rather than operating it as an integrated part of the closed-system flow path broadens the applicability of automated cell culture.

[0033] Figure 4(D) is an explanatory diagram showing the liquid bottle 1 installed in a closed flow path. After connecting the sterile connection connector 5 connected to the liquid bottle 1 with the sterile connection connector 5 on the flow path side, the manual valve 20, which was closed up to this point, is manually opened.

[0034] Next, the normality of the tube attachment to the air supply valve 21 is checked in the path from the flow control unit 12 through the inlet valve 15 to the air supply valve 21. Pressure is propagated during this check process, and the related flow path circuit diagram is shown in Figure 4(E). The inlet valve 15 is activated to activate the liquid bottle shut-off valve 19, and the flow control unit 12 starts supplying air. At this time, since the liquid bottle shut-off valve 19 is activated and the manual valve 20 is open, the second pressure sensor 14 shows a low pressure when the installation is normal. Next, when the liquid bottle shut-off valve 19 is deactivated, the second pressure sensor 14 shows a high pressure when the installation is normal. At this point, it can be determined that the liquid bottle shut-off valve 19 is installed correctly. This is because the expected pressure values ​​for both the activated and deactivated states are obtained only when the liquid bottle shut-off valve 19 is installed correctly.

[0035] When liquid bottle 1 is placed in a closed flow path, if the manual valve 20 is closed due to a work error, this flow path placement determination protocol indicates that the flow control unit 12 starts supplying air and the pressure rises, thus indicating that it was a human error caused by forgetting to open the manual valve 20.

[0036] The adjustment pressure of the first pressure sensor 13 used for pressure determination is adjusted according to the following guidelines. The lower limit P' is greater than or equal to the water pressure applied to the suction port of the supply pipe, depending on the amount of liquid held in the liquid bottle 1, and the upper limit P'' is within the upper limit of the pressure range indicated in the specifications of the automatic valve. Specifically, the pressure P at the bottom of the container is expressed by the following formula, using the liquid density ρ (m³ / kg), gravitational acceleration G (m / s²), and liquid height (m). P = ρ × G × h In the case of water in particular, it can be simply expressed as P = 10000 × h, which is 10 kPa at a depth of 1 m. The lower limit of the regulating pressure P' is, for example, that a liquid bottle with a diameter of 8 cm and a bottom area of ​​approximately 50 cm² has a bottom area of ​​500 ml of liquid, a water depth of 10 cm, and a water pressure of 1 kPa is acting on the opening of the supply pipe near the bottom of the bottle. Therefore, unless a pressure greater than this is applied to the liquid, the liquid inside the supply pipe cannot be moved, so this must be set as the lower limit of P'. Strictly speaking, pressure loss as the fluid moves through the supply pipe is added, so if the inner diameter of the pipe is extremely small or the length of the pipe itself is longer than the water depth, it is necessary to set the pressure taking this into consideration.

[0037] On the other hand, the upper limit of the regulated pressure P'' is within the upper limit of the pressure range indicated in the specifications of the automatic valve. The reason for this is that if a higher pressure is applied upstream by the pressure controller 11 when automatic valves 15, 17, 19, and 21 are not operating in Figure 1, the automatic valves will be forcibly opened, the pressure will propagate downstream, and components with low pressure resistance will be damaged. As a guideline for P'', for example, if an SMC model LPV21 automatic valve capable of opening and closing a rubber tube with an outer diameter of 1 / 4 inch and an inner diameter of 1 / 8 inch is used, the operating pressure range is 0-0.2 MPa, and supply pressures exceeding this cannot be closed by the automatic valve, so a range of approximately 10 kPa to 100 kPa should be selected.

[0038] Figure 5 shows the operational data for the liquid delivery device described in Example 1, obtained by executing the flow path installation 0 determination protocol shown in Figure 4(E). The horizontal axis represents the measurement time, and the vertical axis represents the measurement value of the second pressure sensor 14. Liquid bottle 1 has a capacity of 1 L and holds 500 ml of water, and 5% CO2 gas controlled to 15 kPa by the pressure controller 11 was prepared. The flow control unit 12 was set to supply 50 sccm of air when in operation. The figures are the average of three trials each for the scenario where the manual valve 20 was closed due to forgetting to open it, and when it was opened correctly, and are shown from the time when air supply started.

[0039] As a result, when the manual valve 20 was closed, the pressure reached 10 kPa in 6 seconds, and thereafter approached 15 kPa, which was adjusted by the pressure controller 11. On the other hand, when the manual valve 20 was opened by operating it, the pressure rise was low, with a maximum of 1.8 kPa. Based on these results, the automatic judgment can be operated to determine that if the value of the second pressure sensor 14 is between 7 kPa and 15 kPa 6 seconds after air supply, the manual valve 20 is closed or another abnormality has occurred, resulting in improper installation of the liquid bottle 1.

[0040] When the inlet valve 15 is activated and the liquid bottle on / off valve 19 is activated, and the flow control unit 12 starts supplying air, the gas enters the liquid inside through the suction pipe 3 and is released from the vent filter 7. Depending on the type of liquid, there is a risk that bubbles may form and cover the liquid surface, so it is desirable to minimize the amount of air supplied necessary to determine whether the connection is possible. Up to this point, the method involves the flow control unit 12 continuing to supply air until the target pressure of the second pressure sensor 14 is approximately the same as the target pressure indicated by the first pressure sensor 13, and it is necessary to consider making the determination in a short time to minimize bubble generation. Alternatively, the normality of the automatic valve installation can be determined in a relatively short time by predicting the target pressure from the amount of pressure change per unit time from the measurement value of the second pressure sensor 14 from the start of air supply.

[0041] Furthermore, when connecting liquid bottles, even if the connection between sterile connectors appears correct, if there is a gap between the parts, the pressure pushing down on the liquid surface inside liquid bottle 1 will weaken, causing the pressure detected by the second pressure sensor 14 to change, thus detecting a connection abnormality. At this time, the gas used for connection determination is supplied under positive pressure. This means that even if there is a connection defect such as a gap between the parts as described above, the gas that acts during connection determination will only leak out of the flow path, thus avoiding the risk of outside air containing bacteria entering the flow path.

[0042] Next, when an abnormality is detected during the liquid bottle connection check, the operator performs a check of the flow path to determine whether it is due to forgetting to open the manual valve 20 as described above, improper installation of the sterile connection connector 5, improper attachment of the tube to the basic automatic valve, or some other abnormality. At this time, the operation of each automatic valve, both active and inactive, which is undergoing connection checks, continues, while the air supply is stopped, and the control shifts to preparing for the operator's check. The operator can start the air supply at any time and check the flow path by differentiating between normal and faulty sections while checking the reading of the second pressure sensor 14. In this way, when an abnormality is detected during the liquid bottle connection check, the state at the time of the check in the flow path configuration to be checked is reproduced, and the timing of air supply is arbitrarily controlled, so that the abnormal installation state of the flow path can be quickly restored to a normal state, and the system can transition to automatic culture operation.

[0043] The method of supplying liquid to the culture vessel 8 using this liquid supply device will now be explained. The liquid supply process in the time chart shown in Figure 3 and the related flow path circuit diagrams where pressure is propagated during this liquid supply process are shown in Figures 6(A) and 6(B). In Figure 6(A), the liquid bottle on / off valve 19 is operated, and the other valves are not operated, while the pump 22 is operated. Once a predetermined amount of liquid has reached the culture vessel through the suction tube from the liquid bottle 1 and the liquid is held, the pump is stopped. Next, when the gas introduction valve 17 is operated, air enters through the vent filter 18, and the liquid in the liquid supply pipe 26 moves to the liquid bottle 1 due to gravity, and the flow path upstream from the branch section 16 becomes empty. Next, in Figure 6(B), when the liquid bottle on / off valve 19 is deactivated and the pump 22 is operated, the liquid held in the liquid supply pipe 26 before and after the pump 22 moves to the culture vessel, and air enters through the vent filter 18 and pushes the liquid, so the liquid supply pipe 26 to the culture vessel 8 becomes empty. The liquid transfer process is terminated by deactivating the pump and the gas introduction valve 17.

[0044] The ventilation method for the culture vessel 8 using this liquid delivery device is described below. The ventilation process in the time chart shown in Figure 3 and the related flow path circuit diagram where pressure is propagated during this ventilation process are shown in Figure 7. The inlet valve 15 and the supply valve 21 are operated, and the flow control unit 12 is operated with the other valves inactive. As a result, the pipeline is opened from the supply source to the culture vessel 8, gas can be supplied to the culture vessel 8 at a predetermined flow rate, and the ventilated gas is discharged from the vent filter 24, achieving ventilation inside the culture vessel 8. As described above, the gas necessary for ventilating the culture vessel 8 is used as the gas for determining the installation of the flow path, so the device configuration is simple. [Examples]

[0045] Hereinafter, Embodiment 2 of the present invention will be described with reference to drawings and other illustrations.

[0046] Using Figure 8, the components of the automated culture apparatus used in this embodiment, which performs culture using a closed-system culture vessel, will be explained.

[0047] The automated culture apparatus 100 comprises a culture vessel 101, a ventilation adapter 107, a gas supply unit 109, pumps 116 and 117 for supplying liquid or gas, flow paths connecting these, an automatic valve 119 for opening and closing the flow paths, a control unit 138 for controlling the flow control unit 111, pumps 116 and 117, and the automatic valve, a rocking mechanism 130 for rocking the culture vessel 101, and an incubator 135 as a temperature holding mechanism that houses the culture vessel 101, ventilation adapter 107, rocking mechanism 130, etc., and controls the temperature. The culture vessel 101 is a culture vessel with a ventilation surface (also called a gas exchange membrane) 104 with a gas-permeable membrane placed on its bottom surface, and an open bottom surface 171 is in contact with its lower surface. The bottom surface 171 of the opening has the function of horizontally supporting the ventilation surface 104, which is a thin membrane, and has multiple openings for ventilation, as well as a function to prevent liquid leakage downward by making airtight contact with the outer circumference of the ventilation surface 104.

[0048] The gas space 108 has an upper end that contacts the lower surface of the opening bottom surface 171 so as to indirectly support the ventilation surface, and a lower end that contacts the upper surface (inner surface) of the ventilation adapter 107, forming a gas space 108 that can airtightly maintain a desired gas concentration. Here, "airtight" means that the gas supplied from the gas supply unit does not leak out of the gas space 108, and does not mean that the gas is contained within the gas space 108 and that the contained gas stagnates (does not flow).

[0049] Inside the culture vessel 101, cells 102 are held and cultured together with the culture medium 103. The culture vessel 101 is connected to a pressure regulating tube 105 and a vent filter 106, which allow gas to enter and exit the culture vessel while preventing the entry of bacteria and viruses from the outside.

[0050] Furthermore, the culture vessel 101 is attached to the ventilation adapter 107, enabling the supply of gas and humidification necessary for culture to the culture vessel 101. The gas supply unit 109 consists of a gas cylinder 110 maintaining a predetermined gas concentration, a gas flow control unit (mass flow controller: MF) 111, a second pressure sensor 112, and a humidification bottle 113, which is the humidification unit, and is connected upstream of the ventilation adapter 107. Gas supply and humidification are controlled by the gas flow control unit to a predetermined amount of air, which is supplied, humidified by passing through the water inside the humidification bottle 113, and then supplied to the ventilation adapter 107. Downstream of the ventilation adapter 107, a gas flow meter (mass flow meter: MFM) 145, a CO2 sensor 114, and a CO2 gas vent filter 115 are connected, and the gas is released into the atmosphere outside the device. The gas flow meter 145 and CO2 sensor 114 allow monitoring of whether gas exchange in the ventilation adapter 107 is being performed appropriately by measuring the gas flow rate and CO2 levels. Furthermore, changes in CO2 concentration can be used to predict the culture state.

[0051] The supply of liquid or gas is performed by pumps 116 and 117. Pump 116 is connected by a tube to the inlet tube 218 of the culture vessel 101, and the other end is connected to the suction tube of the culture medium bottle 120 via a vent filter 143 that allows outside air to be introduced, an automatic valve 119, a manual valve 142, and a sterile connection connector 5. When the automatic valve 119 is opened and the flow path is opened, pump 116 is configured to draw liquid culture medium held in the culture medium bottle 120 and deliver it to the culture vessel 101.

[0052] Pump 117 performs the following three additional steps. First, the cell suspension held in the cell bottle 122 is transferred to the culture vessel 101 via the suction tube (also called the "drainage tube") 121 by pressurization to perform cell seeding. Next, the culture medium 103 can be discharged from the culture vessel 101 and transferred to the supernatant collection bag 123 and the supernatant analysis bag 124. In addition, the cells that have grown in the culture vessel 101 are collected by suction into the cell collection bottle 126 via the cell collection tube 125.

[0053] The cell bottle 122 can be installed in the closed system flow path by joining the sterile connection connector 33 and the sterile connection connector 154 on the closed system flow path side according to a prescribed procedure, and removing the protective membrane 163 from each, thereby integrating as a liquid delivery device for delivering liquid in the automated culture apparatus 100 shown in Figure 8. Regarding the cell seeding configuration, the pump 117 is connected to two automatic valves 119 and a vent filter 143 upstream. Downstream of the pump 117, automatic valves 147, 148, and 149 are connected, of which the vent filter 150 is connected to automatic valve 147, and the sterile connection connector 154 is connected downstream of automatic valve 149 via a manual valve 155.

[0054] Figure 9 shows the cell bottle 122 alone before being connected to the closed-loop system. The container 159 can hold any amount of liquid inside, and the inside is sealed by closing the screw cap 161 through which the liquid delivery tube 160 passes. A sterile connection connector 162 is connected to the extension of the liquid delivery tube 160, and the contact parts located on the inner surface of the connector are covered with a protective film 163, so that gases can pass through the protective film 163 but bacterial cells and viruses cannot enter the container. In addition, a vent filter 164 is connected to the screw cap 161 by a tube, so that gases can pass through but bacterial cells and viruses cannot enter the bottle side.

[0055] 127 is a weight sensor that measures the weight of the culture medium bottle 120 connected to the flow path, and the supernatant collection bag 123 is also measured when the bottle is replaced. 128 is also a weight sensor that measures the weight of the cell bottle 122 connected to the flow path, and the cell collection bottle 126 when the bottle is replaced. This allows the pump to operate while measuring the weight of the bottle or bag during fluid delivery, and the pump to stop the fluid delivery in response to changes in weight, thereby recording and storing the change in weight as the fluid volume is delivered.

[0056] As described above, the culture medium bottle 120, cell bottle 122, cell recovery bottle 126, supernatant recovery bag 123, supernatant analysis bag 124, the channels connecting each component, pumps 116 and 117, automatic valve 119, and weight sensors 127 and 128 are located in the fluid control unit 129, which is the main body of the device outside the incubator 135.

[0057] The rocking mechanism 130 consists of a rocking stage 131 that holds the ventilation adapter 107, a link mechanism 132 that supports the rocking stage 131 from three directions, rocking shafts 133 connected to the link mechanism 132, and a rocking stage 134 fixed inside the chamber of the incubator 135. To rock the culture vessel, if the movement is in the left-right direction of the paper, the right rocking shaft 133 is lowered and the left rocking shaft 133 is raised simultaneously, and the movement of the central rocking shaft is controlled without moving the central rocking shaft. This causes the rocking stage to move with an inclination, allowing the culture vessel to be tilted. Then, by reversing the movement of the left and right axes, the culture vessel 101 can be tilted in the opposite direction. By performing these operations continuously, and by moving the rocking shaft in the depth direction of the paper, the culture vessel can be tilted back and forth. By repeatedly performing the left-right and back-forward movements alternately, the cells 102 and culture medium 103 inside the culture vessel 101 on the rocking stage 134 can be stirred.

[0058] Incubator 135 is an example of a temperature maintenance mechanism and is a so-called dry incubator consisting of a constant temperature section 136 and an opening / closing door 137. Incubator 135 can house culture vessels 101, a rocking mechanism 130, and a humidifying bottle 113, and can maintain the temperature inside the incubator at a temperature suitable for cell culture. By adopting a small dry incubator instead of a large CO2 incubator and having a configuration that can supply the gas necessary for culture, the device can be miniaturized, making it possible to operate multiple devices simultaneously in a space-saving manner.

[0059] The control unit 138 can control the operation of the gas supply unit 109, pumps 116 and 117, and automatic valve 119, as well as the operation of the oscillating mechanism 130. By recording the detection values ​​of the gas supply unit 109, weight sensors 127 and 128, and pressure sensors 112 and 144, and automatically controlling these mechanized elements at predetermined timings, a predetermined amount of cell suspension can be delivered from the cell bottle 122 to the culture vessel 101 during cell seeding; a predetermined amount of humidified gas from the humidification bottle 113 can be supplied to the ventilation adapter 107 for a predetermined amount and time during gas exchange; a predetermined amount of culture medium can be delivered from the culture medium bottle 120 to the culture vessel 101 during culture medium addition; a predetermined amount of culture medium can be supplied to the culture vessel 101 after the culture medium 103 in the culture vessel 101 has been discharged into the supernatant recovery bag 123 during culture medium exchange; and a portion of the culture medium in the culture vessel 101 can be delivered to the supernatant analysis bag 124 during supernatant sampling. During cell retrieval, the culture medium in the culture vessel 101 is discharged into the supernatant retrieval bag 123, the cell suspension is agitated using the agitation mechanism 130, and then the liquid can be transferred to the cell retrieval bottle 126.

[0060] The following is a time chart of the flow path installation determination protocol in the flow path configuration shown in Figure 8 of this embodiment, as shown in Figure 10. The automatic valves and pumps are set to ON when in operation and OFF when not in operation. The time chart shows the operation timing of the automatic control of the automatic valves 157, 158, 118, 119, 151, 147, 148, 149, the flow control unit 111, and the pump 117, as well as the manual valves 142 and 155 that are operated manually, and the pressure values ​​of the second pressure sensor 112 that change in conjunction with them. Initially, the operator installs the flow path in the automatic device, and the automatic confirmation of the automatic valves 119 and pumps 116 and 117, other than the connection of the liquid-containing bottle, is assumed to have been performed separately. In the "culture medium bottle connection" step in Figure 10, the culture medium bottle 120 is filled with liquid culture medium in a clean environment, and after connecting the sterile connection connector 141 to the sterile connection connector 141 of the flow path, the manual valve 20, which had been closed up to this point, is manually opened.

[0061] The "culture medium bottle connection confirmation" step verifies the normality of tube attachment and culture medium bottle connection along the path from the flow control unit 111 through automatic valves 157 and 118 to automatic valve 119 and the downstream manual valve 142. Automatic valves 157 and 118 are activated to activate automatic valve 119, and air supply is started by the flow control unit 12. At this time, manual valve 20 is open, and if the installation is normal, the second pressure sensor 14 will show low pressure. Next, automatic valve 119 is deactivated, and air supply is started by the flow control unit 12. At this time, automatic valve 119 is deactivated, and manual valve 20 is open, so if the installation is normal, the second pressure sensor 14 will show high pressure. At this point, it can be determined that automatic valve 119 is installed correctly. This is because the expected pressure value response results for operation and non-operation are obtained only when automatic valve 119 is installed correctly.

[0062] When the culture medium bottle 120 is placed in a closed flow path, if the manual valve 142 is closed due to a work error, this flow path placement determination method indicates that the pressure will start rising as soon as the flow control unit 111 starts supplying air, thus indicating that it is a human error caused by forgetting to open the manual valve 142.

[0063] Next, the cell bottle connection process and installation verification method will be explained. In the "cell bottle connection" process shown in Figure 10, the cell bottle 122 is filled with a liquid cell suspension in a clean environment, and the sterile connection connector 154 is connected to the sterile connection connector 154 of the flow path. Then, the manual valve 155, which had been closed up to this point, is manually opened. In addition, the flow path side vent filters 152 and 153 are manually connected with a tube or the like to allow gas to pass through, and the process of connecting the cell bottle 122 to the closed flow path is carried out.

[0064] The "cell bottle connection confirmation" step verifies the normality of the tube attachment and connection of the cell bottle 122 along the path from the flow control unit 111 to the automatic valves 147, 148, 149 and manual valve 155 and sterile connection connector 154, via automatic valves 157, 118, and 151. Automatic valves 157 and 118 are activated to activate automatic valves 147 and 148, and the flow control unit 111 starts supplying air. At this time, air is supplied through automatic valve 147, and the gas pressure propagates across the liquid surface in the cell bottle 122, causing the cell suspension to begin moving through the supply pipe towards the closed system flow path. When manual valve 155 is open and properly installed, the second pressure sensor 14 shows a pressure lower than the supply pressure.

[0065] Next, automatic valves 147 and 149 are deactivated, and air supply is started by the flow control unit 111. At this time, automatic valve 149 is deactivated and manual valve 20 is open, so if the installation is normal, the second pressure sensor 14 will show high pressure. At this point, it can be determined that automatic valve 147 is installed correctly. This is because the expected pressure value response results for operation and non-operation are obtained only when the tubing to automatic valve 147 is installed correctly.

[0066] At this point, the pressure inside the cell bottle has increased and needs to be released. After stopping the air supply with the flow control unit 111, the automatic valve 158 is activated, followed by the automatic valve 147. At this point, the gas that was pressurizing the inside of the cell bottle 122 is released to the outside air through the vent filter 143, and the pressurization is released. Meanwhile, there is a possibility that pressure and liquid from inside the bottle are moving in the tube downstream of the cell bottle, so if automatic valves 148 and 149 are activated simultaneously, the pressure inside the tubes at automatic valve 147 and automatic valves 148 and 149 will become equal, and at this point, the normalization of the pressure around the cell bottle and the confirmation of the proper installation of the closed system flow path are completed.

[0067] In summary, when the cell bottle 122 is placed in a closed flow path, if the manual valve 155 is closed due to a work error, this installation confirmation method indicates that the pressure will start to rise as the flow control unit 111 starts supplying air, thus confirming that the error was due to human error in forgetting to open the manual valve 155.

[0068] The adjustment pressure of the first pressure sensor 13 used for pressure determination is adjusted according to the following guidelines: The lower limit P' is greater than or equal to the water pressure considered depending on the potential energy of the liquid volume held in the liquid bottle 1 (P1), the pressure loss of the vent filter (P2), the loss resistance of the supply pipe (P3), and the potential energy of the container receiving the liquid (P4); and the upper limit P'' is within the upper limit of the pressure range indicated in the specifications of the automatic valve.

[0069] Specifically, the pressure P1 at the bottom of the container is expressed by the following formula, using the density ρ (m³ / kg) of the liquid, the acceleration due to gravity G (m / s²), and the liquid height (m). P1 = ρ × G × h The following is the same as described in Example 1. Furthermore, the pressure loss due to the vent filter is about 0.1 kPa, and if these are combined, a pressure of 1 kPa or more must be applied to the liquid in order to move the liquid inside the supply pipe, so this must be set as the lower limit P'. Strictly speaking, since the pressure loss of the fluid moving through the supply pipe is added, if the inner diameter of the pipe is extremely small, if the length of the pipe itself is longer than the water depth, or if there is a head of several meters between the pipe and the container to which the liquid is being delivered, it is necessary to set the pressure taking this into consideration. On the other hand, the upper limit P'' of the adjustment pressure is the same as described in Example 1, and from the above, the supply pressure should be selected to be between 10 kPa and 100 kPa.

[0070] Figure 11 shows the operational data for the fluid delivery device described in Example 2, obtained by performing the installation confirmation method shown in Figures 8 and 10. The horizontal axis represents the measurement time, and the vertical axis represents the measurement value of the second pressure sensor 112. The cell bottle 122 has a capacity of 0.6 L and was filled with 500 ml of water.

[0071] In the gas supply unit 109, 5% CO2 gas with a pre-controlled pressure of 15 kPa was prepared. The flow rate control unit 111 was set to supply 50 sccm of gas during operation. The values ​​shown are the average of three trials each, assuming that the manual valve 155 was closed instead of opened, and when it was opened correctly. The values ​​represent the period from the start of gas supply to the end of gas supply (13 seconds).

[0072] As a result, when the manual valve 155 was closed, the pressure reached 1.8 kPa in 4 seconds for all liquid volumes, and continued to rise gradually thereafter, reaching 5.6 kPa after 11 seconds. On the other hand, when the manual valve 155 was opened by operation, the pressure rise reached 1.8 kPa in 4 seconds, and continued to rise gradually thereafter, reaching 3.8 kPa after 11 seconds.

[0073] Based on these results, regardless of the liquid volume in the bottle, an automatic flow path installation determination process can be implemented that determines if the value of the second pressure sensor 112 is between 5 kPa and 10 kPa 10 seconds after air supply, indicating that the manual valve 155 is closed or that the cell bottle 122 is improperly installed due to another malfunction. This tendency can be appropriately applied by maintaining a constant ratio between the liquid bottle capacity and liquid volume, and adjusting the determination time and air supply conditions.

[0074] Figure 12 is a flowchart showing the overall operation of the cell culture process in the automated culture apparatus 100 shown in Figure 8. Following "START", the flow channel is installed in the automated culture apparatus 100 (S01). Next, the process for confirming the installation of the flow channel described above is automatically executed (S02). If the determination is an error at this stage, the flow channel installation is repeated, and the flow channel installation process is repeated until it is determined that the installation is correct. If the error is still not resolved, it is assumed that the flow channel itself is defective, and a new flow channel is installed to address the issue.

[0075] Next, the liquid bottles, one containing culture medium in the culture medium bottle and the other containing cell suspension in the cell bottle, are connected to the flow path (S03). Then, the liquid bottle placement confirmation process described above is automatically executed (S04). If the judgment is found to be an error, the connection and placement of the liquid bottles are redone, and the placement of the liquid bottles is repeated until it is determined that the placement is correct. If the error is still not resolved, it is assumed that the liquid bottle itself is defective, and a new liquid bottle is installed as a solution.

[0076] Cell seeding into the container in Figure 12 (S05) is performed as follows. In Figure 8, in the initial state, the pump is stopped and the roller is stopped by pinching the rubber tube, so pumps 116 and 117 are closed as valves. The automatic valve is closed by pinching the rubber tube. A predetermined amount of cell suspension is held in the cell bottle 122 and placed on the weight sensor 128, and the culture vessel 101 is empty and placed horizontally on the ventilation adapter 107. At the start of cell seeding, automatic valves 147 and 149 and pump 117 are opened to open automatic valves 119 and 158, and the drain pipe 121 of the culture vessel 101 and the piping to the CO2 gas vent filter 115 are opened.

[0077] Next, the pump 117 is activated, and air is supplied through the vent filter 153 to pressurize the cell suspension inside the cell bottle 122. The cell suspension then passes through the conduit and is delivered to the culture vessel 101 via the drain pipe 121 (S05).

[0078] Next, when automatic valves 147 and 149 are closed, automatic valve 148 is opened, and pump 117 is activated, the cell suspension in the tubing closest to the culture vessel is transferred to the culture vessel 101. After that, when pump 117 is stopped and automatic valves 147, 148, 149, and 151 are opened, the gas that pressurized the cell bottle 122 is discharged to the vent filter 143 which is open to the outside, and the cell suspension in the tubing returns to the cell bottle 122 due to gravity, so that there is no liquid left in the tubing, and all automatic valves are closed and the cell seeding process is completed (S05).

[0079] The supply of humidifying gas to the container (S06) in Figure 12 is carried out as follows. In Figure 8, water is held in the humidifying bottle 113, and the opening of a long tube is provided at the bottom of the container. The flow control unit 111 and the gas cylinder 110 are connected to one end of this tube via an automatic valve.

[0080] A short tube opening in the humidifying bottle 113 is located at the top of the bottle container, and the ventilation adapter 107 is connected to one end of this opening. When the flow control unit 111 is activated, gas controlled at a predetermined air supply rate is supplied to the inside of the humidifying bottle, passes through water to be humidified, and then supplied from the humidifying bottle. Subsequently, the gas concentration in the gas space 108 of the ventilation adapter 107 increases and is maintained at a predetermined gas concentration, so that gas exchange to the culture vessel 101 continues. Cell culture is left in a heated environment for a long time to allow the cells to grow.

[0081] The transfer of liquid culture medium from the container (S07) in Figure 12 is performed as follows. In Figure 8, culture medium is held in the culture medium bottle 120 and is installed on the weight sensor 127. First, the automatic valves 118 and 119 are opened, opening the pipeline from the inlet valve 118 of the culture vessel 101 to the culture medium bottle 120. Next, when the pump 116 is activated, the culture medium passes through the pipeline and is transferred to the culture vessel 101 from the inlet valve 118.

[0082] Next, when a predetermined amount of culture medium has moved, the automatic valve 158 is opened and the pump 116 is stopped at the same time. At this time, the flow of culture medium is temporarily paused in the pipeline when the pump 116 stops, and the culture medium in the pipeline closest to the culture medium bottle 120 is returned to the inside of the culture medium bottle 120 by gravity as outside air enters through the vent filter 143 which is open to the outside.

[0083] Next, by closing the automatic valve 119 and operating the pump 116, the culture medium in the pipeline near the culture vessel is delivered to the culture vessel 101. After that, stopping the pump 116 will remove all liquid from the pipeline, and all automatic valves will close, completing the culture medium delivery process. If a culture medium change is necessary as shown in Figure 12, proceed to the next liquid culture medium draining step; otherwise, proceed to the culture supernatant sampling step.

[0084] Sampling of the culture supernatant from the container in Figure 12 (S09) is performed as follows. In Figure 8, the culture vessel 101 holds the culture medium 103, and the supernatant analysis bag 124 is placed empty. The supernatant recovery bag 123 and the supernatant analysis bag 124 are switched to either bag by a manual switching valve (not shown). First, by opening the automatic valve 148 and the automatic valve connected to the supernatant analysis bag 124, the drain pipe 121 of the culture vessel 101 and the pipeline to the supernatant analysis bag 124 are opened. Next, when the pump 117 is activated, the culture medium 103 is pumped through the drain pipe 121 and reaches the supernatant recovery bag 123.

[0085] Next, when a predetermined amount of culture medium has moved, automatic valves 118 and 158 are opened, and pump 116 is stopped at the same time. At this time, the flow of culture medium is temporarily paused in the pipeline when pump 116 stops, and the culture medium in the pipeline closest to the supernatant analysis bag 124 is allowed to move into the supernatant analysis bag 124 by gravity as outside air enters through the vent filter 143, which is open to the outside from the branching point.

[0086] Next, the automatic valve connected to the supernatant analysis bag 124 is closed, and at the same time, the pump 116 starts supplying liquid to the culture vessel. The flow of culture medium, which had been temporarily paused in the tubing when the pump 116 stopped, is then supplied with outside air introduced through the vent filter 143, so the culture medium in the tubing closest to the culture vessel 101 returns to the inside of the culture vessel 101 via the drain pipe 121. When the process is complete, the inside of the pipe is empty, so the culture supernatant inside the culture vessel can always be aspirated and discharged when the culture medium is discharged next time.

[0087] If cell culture is to be continued, the process proceeds to the gas supply step; if the culture is to be terminated, the process proceeds to the cell harvesting step.

[0088] Cell retrieval from the container in Figure 12 (S10) is performed as follows. In Figure 8, culture vessel 101 contains culture medium 103, and the cell retrieval bottle 126 is installed empty. First, by opening the automatic valves connected to the cell retrieval tube 125 and the cell retrieval bottle 126, and the automatic valves connected to the cell retrieval bottle 126 and the pump 117, the tubing between the cell retrieval tube 125 and the pump 117 in the culture vessel 101 is opened.

[0089] Furthermore, opening the automatic valves 118 and 158 opens the pipeline to the pump 117 and the outside air. Next, when the pump 117 is operated by the air supply operation from the culture vessel, the culture medium and cell suspension containing cells are delivered from the cell recovery tube 125 and reach the cell recovery bottle 126. The liquid volume in the culture vessel can be easily estimated from the recorded value by the automatic liquid delivery device, and the liquid delivery process can be performed while monitoring the expected liquid volume with the weight sensor 128, allowing for preventative measures such as excessive gas suction after liquid suction.

[0090] Next, when the cell suspension moves, the rocking mechanism 130 is operated so that the open end of the cell recovery tube 125 moves to the lowest position on the bottom of the container, thereby allowing the cell suspension to concentrate more and increasing the recovery rate. When the liquid delivery is finished, the automatic valve is closed and the pump 116 is stopped at the same time.

[0091] After the cells have been collected, the supernatant collection bag is removed from the flow channel (S11), and then the flow channel is removed from the automated culture device (S12), thus completing all steps of the automated culture process. [Explanation of symbols]

[0092] 1 Liquid bottle, 2 Container, 3 Suction tube, 4 Screw cap, 5 Sterile connection connector, 6 Protective membrane, 7 Vent filter, 8 Culture vessel, 9 Gas supply source, 10 Air supply tube, 11 Pressure controller, 12 Flow control unit, 13 First pressure sensor, 14 Second pressure sensor, 15 Inlet valve, 16 Branch section, 17 Gas inlet valve, 18 Vent filter, 19 Liquid bottle on / off valve, 20 Manual valve, 21 Air supply valve, 22 Pump, 23 Pressure adjustment tube, 24 Vent filter, 25 Control unit, 101 Culture vessel, 102 Cells, 103 Culture medium, 104 Ventilation surface, 105 Pressure adjustment tube, 106 Vent filter, 107 Ventilation adapter, 108 Gas space section, 109 Gas supply section, 110 Gas cylinder, 111 Flow control unit, 144 First pressure sensor, 112 Second pressure sensor, 113 Humidification bottle, 114 CO2 sensor, 115 CO2 gas vent filter, 116, 117 Pump, 218 Inlet tube, 119 Automatic valve, 120 Culture medium bottle, 121 Suction tube, 122 Cell bottle, 123 Supernatant recovery bag, 124 Supernatant analysis bag, 125 Cell recovery tube, 126 Cell recovery bottle, 127, 128 Weight sensor, 129 Fluid control unit, 130 Oscillating mechanism, 135 Incubator, 141 Aseptic connection connector, 142 Manual valve, 154 Aseptic connection connector, 163 Protective membrane, 147-149 Automatic valve, 150 Vent filter, 152 Aseptic connection connector, 153 Manual valve

Claims

1. A culture apparatus comprising: a first liquid bottle; a first gas supply source for supplying gas to the first liquid bottle; a first flow path connecting the first liquid bottle and the first gas supply source; a first sterile connector provided in the first flow path and in order from the side closest to the first liquid bottle; a first manual valve; at least one first automatic valve; and a first pressure sensor, A culture apparatus characterized by comprising: a first determination unit that determines, based on the measurement value of the first pressure sensor when a combination of opening and closing of the first automatic valve and the first manual valve is performed while the gas is being supplied from the first gas supply source, whether there is an abnormality in the first manual valve and the first automatic valve in the first flow path, and / or whether the attachment of the tube to the first manual valve and the first automatic valve is normal.

2. In the culture apparatus according to claim 1, The culture apparatus is characterized in that the first determination unit determines whether the first manual valve and the first automatic valve in the first flow path are abnormal, and / or whether the tubes attached to the first manual valve and the first automatic valve are normal, when the first liquid bottle is not connected to the first flow path via the first sterile connector, and then determines whether the first manual valve and the first automatic valve in the first flow path are abnormal, and / or whether the tubes attached to the first manual valve and the first automatic valve are normal, when the first liquid bottle is connected to the first flow path via the first sterile connector.

3. In the culture apparatus according to claim 1 or 2, The culture apparatus is characterized in that the first determination unit determines whether there is an abnormality in the first manual valve, the first automatic valve in the first flow path, and / or the normality of the attachment of the tube to the first manual valve, the first automatic valve, based on whether the measurement value of the first pressure sensor rises to a predetermined threshold within a predetermined time.

4. A culture apparatus comprising: a second liquid bottle; a second gas supply source for supplying gas to the second liquid bottle; a second flow path connecting the second liquid bottle and the second gas supply source; a second sterile connector provided in the second flow path and in order from the side closest to the second liquid bottle; a second manual valve; at least one second automatic valve; and a second pressure sensor, A culture apparatus characterized by comprising a second determination unit that determines, based on the measurement value of the second pressure sensor when a combination of opening and closing of the second automatic valve and the second manual valve is performed while the gas is being supplied from the second liquid bottle by the second gas supply source, whether there is an abnormality in the second manual valve and the second automatic valve in the second flow path, and / or whether the tubes attached to the second manual valve and the second automatic valve are normal.

5. In the culture apparatus according to claim 4, The culture apparatus is characterized in that the second determination unit determines whether the second manual valve and the second automatic valve in the second flow path are abnormal, and / or whether the tubes attached to the second manual valve and the second automatic valve are normal, when the second liquid bottle is not connected to the second flow path via the second sterile connector, and then determines whether the second manual valve and the second automatic valve in the second flow path are abnormal, and / or whether the tubes attached to the second manual valve and the second automatic valve are normal, when the second liquid bottle is connected to the second flow path via the second sterile connector.

6. In the culture apparatus according to claim 4 or 5, The culture apparatus is characterized in that the second determination unit determines whether there is an abnormality in the second manual valve, the second automatic valve, and / or the normality of the attachment of the tube to the second manual valve, the second automatic valve, based on whether the measurement value of the second pressure sensor rises to a predetermined threshold within a predetermined time.

7. In the culture apparatus according to claim 1 or 2, The system comprises a second liquid bottle, a second gas supply source for supplying gas to the second liquid bottle, a second flow path connecting the second liquid bottle and the second gas supply source, a second sterile connector provided in the second flow path and arranged in order from the side closest to the second liquid bottle, a second manual valve, at least one second automatic valve, and a second pressure sensor. A culture apparatus characterized by comprising a second determination unit that determines, based on the measurement value of the second pressure sensor when the opening and closing combination of the second automatic valve and the second manual valve is performed while the gas is being supplied to the second liquid bottle by the second gas supply source, whether there is an abnormality in the second manual valve and the second automatic valve in the second flow path, and / or whether the tubes attached to the second manual valve and the second automatic valve are normal.

8. In the culture apparatus according to claim 7, The second determination unit determines whether there is an abnormality in the second manual valve and the second automatic valve in the second flow path, and / or whether the tubes attached to the second manual valve and the second automatic valve are normal, based on whether the measured value of the second pressure sensor rises to a predetermined first threshold within a predetermined time when a combination of opening and closing of the second automatic valve and the second manual valve is performed while gas is being supplied from the gas supply source, and whether the measured value of the second pressure sensor rises to a predetermined second threshold within a predetermined time when a combination of opening and closing of the second automatic valve and the second manual valve is performed while gas is being supplied from the second liquid bottle by the second gas supply source. A culture apparatus characterized in that the second threshold is greater than the first threshold.

9. A method for confirming the normal installation of a flow path in a culture apparatus, comprising: a first liquid bottle; a first gas supply source for supplying gas to the first liquid bottle; a first flow path connecting the first liquid bottle and the first gas supply source; and provided in the first flow path, in order from the side closest to the first liquid bottle, a first sterile connector, a first manual valve, at least one first automatic valve, and a first pressure sensor, wherein A first pressure measurement step involves measuring the pressure value at the first pressure sensor when a combination of opening and closing of the first automatic valve and the first manual valve is performed while the gas is being supplied from the first gas supply source, A method for confirming the normal installation of a flow path in a culture apparatus, comprising: a first determination step of determining whether there is an abnormality in the first manual valve, the first automatic valve, and / or the normality of the attachment of the tube to the first manual valve, the first automatic valve, based on the pressure value measured in the first pressure measurement step.

10. In the method for confirming the normal installation of the flow path of a culture apparatus according to claim 9, A method for confirming the normal installation of a flow path in a culture apparatus, characterized in that, in the first determination step, the abnormality of the first manual valve and the first automatic valve in the first flow path and the normality of the attachment of tubes to the first manual valve and the first automatic valve are determined while the first liquid bottle is not connected to the first flow path via the first sterile connector, and then the abnormality of the first manual valve and the first automatic valve in the first flow path and / or the normality of the attachment of tubes to the first manual valve and the first automatic valve are determined while the first liquid bottle is connected to the first flow path via the first sterile connector.

11. In the method for confirming the normal installation of the flow path of a culture apparatus according to claim 9 or 10, A method for confirming the normality of the installation of a flow path in a culture apparatus, characterized in that the first determination step determines whether there is an abnormality in the first manual valve, the first automatic valve in the first flow path, and / or the normality of the attachment of the tube to the first manual valve, the first automatic valve, based on whether the measured value of the first pressure sensor rises to a predetermined threshold within a predetermined time.

12. A method for confirming the proper installation of a flow path in a culture apparatus, comprising: a second liquid bottle; a second gas supply source for supplying gas to the second liquid bottle; a second flow path connecting the second liquid bottle and the second gas supply source; and provided in the second flow path, in order from the side closest to the second liquid bottle, a second sterile connector, a second manual valve, at least one second automatic valve, and a second pressure sensor, wherein A second pressure measurement step involves measuring the pressure value at the second pressure sensor when a combination of opening and closing of the second automatic valve and the second manual valve is performed while the gas is being supplied from the second liquid bottle by the second gas supply source, A method for confirming the normal installation of a flow path in a culture apparatus, comprising: a second determination step of determining whether there is an abnormality in the second manual valve, the second automatic valve, and / or the normality of the attachment of the tube to the second manual valve, the second automatic valve, based on the pressure value measured in the second pressure measurement step.

13. In the method for confirming the normal installation of the flow path of a culture apparatus according to claim 12, A method for confirming the normal installation of a flow path in a culture apparatus, characterized in that, in the second determination step, the abnormality of the second manual valve and the second automatic valve in the second flow path, and / or the normality of the attachment of the tubes to the second manual valve and the second automatic valve are determined while the second liquid bottle is not connected to the second flow path via the second sterile connector, and then the abnormality of the second manual valve and the second automatic valve in the second flow path, and / or the normality of the attachment of the tubes to the second manual valve and the second automatic valve are determined while the second liquid bottle is connected to the second flow path via the second sterile connector.

14. In the method for confirming the normal installation of the flow path of a culture apparatus according to claim 12 or 13, A method for confirming the normality of the installation of a flow path in a culture apparatus, characterized in that the second determination step determines whether there is an abnormality in the second manual valve, the second automatic valve, and / or the normality of the attachment of the tube to the second manual valve, the second automatic valve, based on whether the measured value of the second pressure sensor rises to a predetermined threshold within a predetermined time.

15. In the method for confirming the normal installation of the flow path of a culture apparatus according to claim 9 or 10, The culture apparatus comprises a second liquid bottle, a second gas supply source for supplying gas to the second liquid bottle, a second flow path connecting the second liquid bottle and the second gas supply source, a second sterile connector provided in the second flow path and arranged in order from the side closest to the second liquid bottle, a second manual valve, at least one second automatic valve, and a second pressure sensor. A second pressure measurement step involves measuring the pressure value of the second pressure sensor when a combination of opening and closing of the second automatic valve and the second manual valve is performed while the gas is being supplied from the second liquid bottle by the second gas supply source, A method for confirming the normal installation of a flow path in a culture apparatus, comprising: a second determination step of determining whether there is an abnormality in the second manual valve, the second automatic valve, and / or the normality of the attachment of the tube to the second manual valve, the second automatic valve, based on the pressure value measured in the second pressure measurement step.

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