Culture device and method for checking installation normality of flow path for culture device

Abstract

In order to provide a culture device for which it is possible to check the installation normality of a flow path for the culture device, and a method for checking the installation normality of a flow path for the culture device, the present invention is configured as follows.　This culture device comprises: a liquid bottle; a gas delivery source for delivering a gas to be delivered to the liquid bottle; a flow passage for connecting the liquid bottle and the gas delivery source; and an aseptic connector, a manual valve, at least one automatic valve, and a pressure sensor, which are provided to the flow passage in the stated order from a position closest to the liquid bottle. The culture device is characterized by comprising a determination unit for determining the abnormality of the manual valve and the automatic valve in the flow passage and / or the normality of attachment of a tube to the manual valve and the automatic valve on the basis of a measurement value of the pressure sensor obtained when the combination of opening and closing of the manual valve and the automatic valve is executed during delivery of the gas from the gas delivery source.

Description

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

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

[0002] Regenerative medicine, which uses regenerated tissue manufactured from cells to restore the function of organs and other tissues, is expected to be a curative treatment for diseases for which there were previously no treatments. The treatment targets are diverse, including skin, cornea, esophagus, heart, bone, and cartilage, and the number of clinical applications is rapidly increasing. In the manufacturing process of regenerated tissue, biological samples collected from the patient or another person are separated, purified, amplified, and tissue-formed. This process is carried out in a Cell Processing Center (CPC) in accordance with Standard Operating Procedures (SOPs) that meet Good Manufacturing Practice (GMP), which is the standard for manufacturing management and quality control of pharmaceuticals and other products. Therefore, operating a CPC requires significant costs and personnel with specialized cell culture techniques. In addition, since the manufacturing process is mainly manual, there are limits to increasing the production volume. Low productivity and high manufacturing costs are hindering the widespread adoption of regenerative medicine, and automation of the culture process, which is particularly labor-intensive and costly within the manufacturing process, is needed. Automating the culture process will enable labor savings, cost reduction, and mass production.

[0003] As an example of an automated culture device, there is a device that automatically handles a closed system flow channel having a closed space, as shown in Patent Document 1. In the closed system flow channel, a closed system culture vessel is constantly connected by a flow channel tube or the like, and cells are cultured inside the closed system culture vessel to produce 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 Calder Products Aseptic Connector Series AQS17004.

[0006] Japanese Patent Publication No. 2016-208866, U.S. Patent No. 12042621

[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 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.

[0012] The present invention has the following configuration to achieve the above objective: 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.

[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.

[0017] This figure shows one configuration of the flow path in the automated culture apparatus according to Example 1. This figure shows the configuration of the liquid bottle before connection according to Example 1. This figure shows a time chart of the flow path installation determination protocol in the flow path configuration according to Example 1. This figure shows the relevant flow path circuit diagram in the flow path according to Example 1. This figure shows the relevant flow path circuit diagram in the flow path according to Example 1. This figure shows the relevant flow path circuit diagram in the flow path according to Example 1. This figure shows the relevant flow path circuit diagram in the flow path according to Example 1. This figure shows the relevant flow path circuit diagram in the flow path according to Example 1. This figure shows the pressure measurement results in the liquid delivery device according to Example 1. This is a flow path circuit diagram related to the pressure propagation in the liquid delivery process according to Example 1. This is a flow path circuit diagram related to the pressure propagation in the liquid delivery process according to Example 1. This is a flow path circuit diagram related to the pressure propagation in the ventilation process according to Example 1. This figure shows one configuration of the automated culture apparatus according to Example 2. This figure shows a cell bottle alone before connection to the closed system flow path according to Example 2. This figure shows a time chart of the flow path installation determination protocol in the flow path configuration according to Example 2. This figure shows the pressure measurement results in the liquid delivery device according to Example 2. This figure shows a flowchart of the overall operation of the cell culture process in the automated culture apparatus according to Example 2.

[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.

[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, referred to as the inlet valve 15, gas inlet valve 17, liquid bottle on / off valve 19, and air supply valve 21, respectively. Hereinafter, these will be collectively referred to as "automatic valves." Pinch valves are preferred, which open and close the rubber tube that constitutes the flow path using external force. When the rubber tube, which is clamped in a drive valve actuated by spring force, is closed, the automatic valve can operate when power is applied to control the rubber tube to an open state. Here, a valve that closes when not in operation and not powered is 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, which will be 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 main purpose of the 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, so it has the same effect as closing a valve. The aforementioned air supply valve 21 can be opened when the pump 22 is not operating, allowing gas to be delivered 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 further introduce CO into the gas phase. ２Cell culture can be performed by ventilating the area to add gas. 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 of 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 measured by the first pressure sensor 13. The flow path installation determination protocol in this embodiment is based on the above phenomena.

[0028] In this embodiment, Figure 3 shows the time chart of the flow path installation determination protocol in the flow path configuration shown in Figure 1. 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 through the inlet valve 15 to the gas inlet valve 17. The relevant flow path circuit diagram in the flow path shown in Figure 1 is shown in Figure 4(A). 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 not in operation, 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 in operation, and the gas inlet valve 17 downstream of it is also in operation, 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 the flow control unit 12 starts supplying air. 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 the flow control unit 12 starts supplying air. 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 during operation and deactivation are obtained only when the installation of the inlet valve 15 and the gas inlet valve 17 downstream of it 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 operated, 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 operation and deactivation of the air supply valve 21.

[0031] Next, the proper installation of the tube to the pump 22 is confirmed. During this confirmation process, pressure is propagated, 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 process 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 system checks the proper attachment of the tube to the air supply valve 21 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, 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 properly installed. 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 correctly installed.

[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 increases, 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 equal to the density ρ (m³) of the liquid. ３ / kg), gravitational acceleration G (m / s ２ ), and the liquid height (m) can be expressed by the following formula: P = ρ × G × h In particular, in the case of water, it can be simply expressed as P = 10000 × h, and at a depth of 1 m, it is 10 kPa. The lower limit of the regulated pressure P' is, for example, the bottom area of ​​a liquid bottle with a diameter of 8 cm and a bottom area of ​​approximately 50 cm². ２It is such that with 500 ml of liquid, the water depth becomes 10 cm, and a water pressure of 1 kPa is applied to the opening of the supply pipe near the bottom surface of the bottle. Therefore, if a pressure higher than this is not applied to the liquid, the liquid inside the supply pipe cannot be moved, so it is necessary to set this as the lower limit value P'. Strictly speaking, since a pressure loss as the fluid for moving the supply pipe is added, when the inner diameter of the pipe is extremely small or when the length of the pipe itself is longer than the water depth, a pressure setting considering this is necessary.

[0037] On the other hand, the upper limit value P'' of the adjustment pressure is within the upper limit value of the pressure range indicated by the specifications of the automatic valve. As the reason, in FIG. 1, when the automatic valves 15, 17, 19, and 21 are not operating, if a higher pressure is applied upstream by the pressure controller 11, the automatic valve will be forcibly opened and the pressure will propagate downstream, damaging components with low pressure resistance performance. As a guideline for P'', for example, when using an SMC model LPV21 as an automatic valve that can open and close a rubber tube with an outer diameter of 1 / 4 inch and an inner diameter of 1 / 8 inch, the operating pressure range is 0 - 0.2 MPa. Since a supply pressure exceeding this cannot be closed by the automatic valve, a value of about 10 kPa to 100 kPa should be selected.

[0038] FIG. 5 shows the operation data of the liquid delivery device described in Example 1 and is the result of executing the flow path installation 0 determination protocol in FIG. 4(E). The horizontal axis represents the measurement time, and the vertical axis represents the measured value of the second pressure sensor 14. The capacity of the liquid bottle 1 is 1 L, and 500 ml of water is held in it, and 5% CO ２ gas is prepared by controlling it to 15 kPa with the pressure controller 11. During operation in the flow rate control unit 12, the air supply volume is set to 50 sccm. It is the average value of the results of three trials each for the assumption when the manual valve 20 is closed by forgetting to open it and when the opening operation is correctly performed, and is the notation from the time when the air supply is started.

[0039] As a result, when the manual valve 20 is closed, it reaches 10 kPa in 6 seconds and then approaches 15 kPa adjusted by the pressure controller 11. On the other hand, when the manual valve 20 is operated and opened, the pressure rise value is at most 1.8 kPa and proceeds at a low pressure. Based on this result, in the automatic determination, when the value of the second pressure sensor 14 is 7 kPa or more and less than 15 kPa 6 seconds after the air supply, it can be determined by automatic determination that the manual valve 20 is closed or the installation of the liquid bottle 1 is defective due to the occurrence of another abnormality.

[0040] When the introduction valve 15 is operated and the liquid bottle opening / closing valve 19 is in operation, and the air supply is started by the flow rate control unit 12, the gas penetrates into the internal liquid through the suction pipe 3 and is discharged from the vent filter 7. Depending on the type of liquid, there is a risk of forming bubbles and covering the liquid surface, so it is desirable to minimize the air supply amount required for the connection determination. So far, it is a method of continuing the air supply by the flow rate control unit 12 until the reaching pressure of the second pressure sensor 14 becomes approximately the same as the reaching pressure indicated by the first pressure sensor 13, and it is necessary to consider determining in a short time so as to minimize the generation of bubbles. As another method, it is possible to determine the normal installation of the automatic valve in a relatively short time by predicting the reaching pressure from the pressure change amount per unit time based on the measured value of the second pressure sensor 14 from the start of the air supply.

[0041] Also, when connecting the liquid bottles, even if the connection between the aseptic connectors appears to be correct, if there is a gap between the parts and it is defective, the pressure pushing the liquid surface inside the liquid bottle 1 weakens, so the pressure detection value at the second pressure sensor 14 changes, and it is possible to detect that there is an abnormality in the connection. At this time, the air supply of the gas used for the connection determination is operated at a positive pressure. This means that even if there is a connection defect such as a gap between the above-mentioned parts, the gas acting at the time of connection determination only leaks out from the flow path, so the risk of outside air containing bacteria and the like entering the flow path can be avoided.

[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 distinguishing between normal and faulty areas 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 recovered to a normal state, and the automatic culture operation can be started.

[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 pipe 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 pump and gas introduction valve 17 are deactivated, and the liquid transfer process is terminated.

[0044] The ventilation method for the culture vessel 8 using this liquid delivery device will now be explained. 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, and gas can be supplied to the culture vessel 8 at a predetermined flow rate. The ventilated gas is discharged from the vent filter 24, and ventilation of the inside of the culture vessel 8 is achieved. As described above, the gas necessary for the ventilation of the culture vessel 8 is used as the gas for determining the installation of the flow path, so the device configuration is simple.

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

[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 includes 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 portion 108 has its upper end in contact with the lower surface of the opening bottom surface 171 so as to indirectly support the ventilation surface, and its lower end in contact with the upper surface (inner surface) of the ventilation adapter 107, forming a gas space portion 108 that can be hermetically maintained at a desired gas concentration. Here, "hermetically" means that the gas supplied from the gas supply unit does not leak outside the gas space portion 108, and it does not mean that the gas is confined within the gas space portion 108 and the confined gas stagnates (does not flow).

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

[0050] Further, the culture vessel 101 is attached to the ventilation adapter 107, enabling gas supply and humidification necessary for culturing to be performed on the culture vessel 101. The gas supply unit 109 consists of a gas cylinder 110 holding a predetermined gas concentration, a gas flow control unit (mass flow controller: MF) 111, a second pressure sensor 112, and a humidification bottle 113 as a humidification unit, and is connected upstream of the ventilation adapter 107. Gas supply and humidification are controlled to a predetermined air supply volume by the gas flow control unit and sent, passing through the water inside the humidification bottle 113 to be humidified and then supplied to the ventilation adapter 107. Downstream of the ventilation adapter 107, a gas flow meter (mass flow meter: MFM) 145 and a CO ２ sensor 114 and a CO ２ gas vent filter 115 are connected, and the gas is configured to be discharged to the atmosphere outside the device. The gas flow meter 145 and the CO ２ sensor 114 can monitor whether the gas exchange at the ventilation adapter 107 is properly performed, and monitor it with the measured values of the gas flow rate and CO ２ Further, it can be used for predicting the culture state by utilizing the change in the concentration of CO ２ .

[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 can introduce outside air, 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 configuration for cell seeding, 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 bacteria 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 bacteria 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 it 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 it 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 delivery in response to changes in weight, thereby recording and storing the change in weight as the amount of fluid 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 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. ２ By using a small dry incubator instead of a conventional incubator, and by incorporating a configuration that allows for the supply of the necessary gases for cultivation, 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 by 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 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 the automatic valve 119 and the downstream manual valve 142. Automatic valves 157 and 118 are activated to activate automatic valve 119, and the flow control unit 12 starts supplying air. At this time, the manual valve 20 is open, and if the installation is normal, the second pressure sensor 14 shows a low pressure. Next, automatic valve 119 is deactivated, and the flow control unit 12 starts supplying air. 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 shows a 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 the 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 confirmation 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 after connecting the sterile connection connector 154 to the sterile connection connector 154 of the flow path, 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 create a configuration that allows 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 the connection of the cell bottle 122 along the path from the flow control unit 111 to the automatic valves 157, 118, and 151, then to the automatic valves 147, 148, 149, the manual valve 155, and the sterile connection connector 154. The automatic valves 157 and 118 are activated to activate the automatic valves 147 and 148, and the flow control unit 111 starts supplying air. At this time, air is supplied through the 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 the manual valve 155 is open and properly installed, the second pressure sensor 14 shows a pressure lower than the supply pressure.

[0065] Next, the automatic valves 147 and 149 are deactivated, and the flow control unit 111 starts supplying air. At this time, the automatic valve 149 is deactivated and the manual valve 20 is open, so if the setup is normal, the second pressure sensor 14 will show high pressure. At this point, it can be determined that the automatic valve 147 is properly installed. This is because the expected pressure values ​​for both the activated and deactivated states are obtained only when the tubing to the automatic valve 147 is correctly installed.

[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 in 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 equal to the density ρ (m³) of the liquid. ３ / kg), gravitational acceleration G (m / s ２ ), and the liquid height (m) are expressed by the following formula: P1 = ρ × G × h The rest 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, or if the length of the pipe itself is longer than the water depth, or if the head between the container to which the liquid is being delivered is several meters, 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 liquid 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 is supplied with a pressure pre-controlled to 15 kPa. ２ The gas was prepared. The flow 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 ​​are shown 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 increase 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 work 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 the culture medium in the culture medium bottle and the other containing the 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 determination 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 with the rubber tube pinched, so pumps 116 and 117 are closed as valves. The automatic valve is closed with the rubber tube pinched. A predetermined amount of cell suspension is held in the cell bottle 122 and placed on the weight sensor 128, and the culture container 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 used to open automatic valves 119 and 158, and the drain pipe 121 of the culture container 101 and CO2 are opened. ２ The pipeline leading to the gas vent filter 115 is 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 airflow 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, the 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 sent from the drain pipe 121 and reaches the supernatant recovery bag 123.

[0085] Next, when a predetermined amount of culture medium has moved, the automatic valves 118 and 158 are 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 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 the 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] The cell retrieval from the container in Figure 12 (S10) is performed as follows. In Figure 8, the culture vessel 101 holds the culture medium 103, and the cell retrieval bottle 126 is installed empty. First, by opening the automatic valve connected to the cell retrieval tube 125 and the cell retrieval bottle 126, and the automatic valve 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 supplying air 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 amount of liquid 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 amount of liquid with the weight sensor 128, allowing for preventative measures such as excessive gas intake 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.

[0092] 1. Liquid bottle, 2. Container, 3. Suction tube, 4. Screw cap, 5. Aseptic connector, 6. Protective membrane, 7. Vent filter, 8. Culture vessel, 9. Gas supply source, 10. Gas 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. Gas 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 Humidifying bottle, 114 CO ２ Sensor, 115 CO ２Gas 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 connector, 142 Manual valve, 154 Aseptic connector, 163 Protective membrane, 147-149 Automatic valve, 150 Vent filter, 152 Aseptic 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, and provided in the first flow path and 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 the culture apparatus further 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 the gas is supplied from the first gas supply source.

2. The culture apparatus according to claim 1, characterized in that the first determination unit determines whether the first manual valve, the first automatic valve in the first flow path is 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, the first automatic valve in the first flow path is 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. A culture apparatus according to claim 1 or 2, wherein 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 measured 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, and provided in the second flow path and 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 the culture apparatus further comprises a second determination unit that determines abnormalities in the second manual valve and the second 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 the gas is being supplied from the second liquid bottle by the second gas supply source.

5. The culture apparatus according to claim 4, 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. A culture apparatus according to claim 4 or 5, characterized in that the second determination unit determines whether there is an abnormality in the second manual valve, the second automatic valve in the second flow path, 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.

7. A culture apparatus according to claim 1 or 2, 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, wherein 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 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 to the second liquid bottle by the second gas supply source.

8. The culture apparatus according to claim 7, wherein the second determination unit determines whether the measurement 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 supplied from the second gas supply source, and whether the measurement 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 supplied from the second liquid bottle by the second gas supply source, and / or whether the attachment of the tube to the second manual valve and the second automatic valve is normal, and 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, the method comprising: 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 the gas is supplied from the first gas supply source; and a first determination step of determining, based on the pressure value measured in the first pressure measurement step, 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.

10. A method for confirming the normal installation of a flow path in a culture apparatus according to claim 9, 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. A method for confirming the normal installation of a flow path in a culture apparatus according to claim 9 or 10, characterized in that in the first determination step, the abnormality of 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 is determined based on whether or not the measured value of the first pressure sensor rises to a predetermined threshold within a predetermined time.

12. A method for confirming the normal 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, the method comprising: 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 the gas is being supplied from the second liquid bottle by the second gas supply source; and a second determination step of determining, based on the pressure value measured in the second pressure measurement step, 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.

13. A method for confirming the normal installation of a flow path in a culture apparatus according to claim 12, characterized in that, in the second determination step, the abnormality of the second manual valve, the second automatic valve in the second flow path, and / or the normality of the attachment of tubes to the second manual valve, the second automatic valve is 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, the second automatic valve in the second flow path, and / or the normality of the attachment of tubes to the second manual valve, the second automatic valve is determined while the second liquid bottle is connected to the second flow path via the second sterile connector.

14. A method for confirming the normal installation of a flow path in a culture apparatus according to claim 12 or 13, characterized in that in the second determination step, the abnormality of the second manual valve, the second automatic valve in the second flow path, and / or the normality of the attachment of the tube to the second manual valve, the second automatic valve is determined based on whether or not the measured value of the second pressure sensor rises to a predetermined threshold within a predetermined time.

15. A method for confirming the normal installation of a flow path in a culture apparatus according to claim 9 or 10, wherein 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 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 a second pressure measurement step of 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 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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