JP2018046800A - Cell culturing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent beforehand damage to a load sensor provided in a culture chamber to detect a load received from a placing table by supporting the placing table even if an accident such as leakage of a fluid contained in a culture vessel occurs when culturing cells by placing the culture vessel on top of the placing table stored in the culture chamber in which the environment is managed to suit cell culturing.SOLUTION: In order to detect a load received from a placing table 2, the placing table 2 on which is placed culture vessels K1a, K1b, and K1c, is supported, and shield members 6 which cover the top of a plurality of load sensors 13 provided in the culture chamber 10 are made to correspond to where the load sensors 13 are positioned and are provided on the placing table 2 such that the load sensors 13 support the placing table 2 through the shielding members 6.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture apparatus.

  In recent years, in the fields of pharmacology and biochemistry such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy, cells (including tissues, microorganisms, viruses, etc.) can be efficiently cultured in large quantities in an artificial environment. It has been demanded.

  In order to meet such demands, the present applicant has constructed a closed environment using a bag-shaped culture vessel made of a flexible material, and efficiently cultured cells while reducing the risk of contamination. Studies have been conducted on cell culture techniques that can be performed.

  For example, in Patent Document 1, the present applicant connects a plurality of culture containers via a transfer tube, transfers cells cultured in a small capacity culture container to a larger capacity culture container, We have proposed a cell culture method that allows cells to proliferate efficiently while maintaining a proper level.

  Further, in Patent Document 1, a plurality of culture containers are arranged with a difference in height on the mounting table, and the culture containers arranged at a relatively high position are arranged at a relatively low position by the drop. It has also been proposed to sequentially transfer the contents to the culture vessel.

Japanese Patent Laying-Open No. 2015-188392

  By the way, cell culture is usually performed in a culture chamber controlled in an environment suitable for cell culture. When transferring the content liquid between the culture containers as described above, the culture chamber is selected according to the transfer situation. The timing of operations performed in the system must be properly controlled. For this purpose, it is necessary to monitor the state of transfer of the content liquid between the culture vessels. For example, a plurality of load sensors are provided in the culture chamber, and a mounting table in which the culture vessels are arranged and stored in the culture chamber is provided. It is conceivable to monitor the state of transfer of the content liquid between the culture vessels by detecting the change in the load bias accompanying the transfer of the content liquid supported by these load sensors.

  However, cell culture usually requires a period of several days to several weeks, during which an accident such as leakage of the content liquid from the culture vessel may occur. When such an accident occurs, the leaked content liquid may fall on the load sensor and the load sensor may be damaged. If the load sensor is damaged, not only labor and cost are required for the repair, but also cell culture cannot be performed until the repair is completed.

  The present invention has been made in view of the circumstances as described above, and in performing cell culture by placing a culture vessel on a mounting table stored in a culture chamber managed in an environment suitable for cell culture, Even if an accident such as leakage of the contents liquid from the culture vessel occurs, it is possible to prevent damage to the load sensor arranged in the culture chamber in order to support the mounting table and detect the load received from the mounting table. An object of the present invention is to provide a cell culture device that can be used.

  A cell culture device according to the present invention is a cell culture device that performs cell culture by placing a culture vessel on a mounting table stored in a culture chamber managed in an environment suitable for cell culture, the mounting table described above A plurality of load sensors that support and detect a load received from the mounting table are disposed in the culture chamber, and a shielding member that covers the upper side of the load sensor corresponds to the position where the load sensor is disposed. The mounting table is provided on the mounting table, and the mounting table is supported by the load sensor via the shielding member.

  According to the present invention, even when an accident such as leakage of the content liquid from the culture vessel placed on the mounting table occurs during cell culture, the leaked content liquid is blocked by the shielding member. Thus, it is possible to prevent the load sensor from falling on the load sensor, thereby preventing the load sensor from being damaged.

It is explanatory drawing which shows the outline of the cell culture apparatus which concerns on embodiment of this invention. It is explanatory drawing which shows the outline of an example of the cell culture kit used for embodiment of this invention. It is a top view which shows the outline of an example of the mounting base in embodiment of this invention. It is a perspective view which shows the outline of the flame | frame of the mounting base shown in FIG. It is AA sectional drawing of FIG. It is a perspective view which shows the outline of the shielding member provided in the front end side of a mounting base. It is a perspective view which shows the outline of the shielding member provided in the rear end side of a mounting base. It is explanatory drawing which shows the state which the front-end | tip part of the support pin with which a load sensor is provided engaged with the engagement recessed part provided in the shielding member provided in the front-end side of a mounting base. It is explanatory drawing which shows the example comprised so that a mounting base could advance / retreat on the guide rail attached to the culture chamber. It is explanatory drawing which shows the example comprised so that a mounting base could advance / retreat on the guide rail attached to the culture chamber. It is explanatory drawing which shows the example comprised so that a mounting base could advance / retreat on the guide rail attached to the culture chamber. It is explanatory drawing which shows the example comprised so that a mounting base could advance / retreat on the guide rail attached to the culture chamber.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

  The cell culture apparatus 1 shown in FIG. 1 has a culture vessel K1a, a culture vessel K1a, a storage container 2 stored in a culture chamber 10 controlled in an environment suitable for cell culture (for example, 37 ° C., 5% carbon dioxide concentration). An apparatus for culturing cells by arranging K1b and K1c. When performing cell culture, for example, a cell culture kit K as shown in FIG. 2 is used.

  In the cell culture kit K shown in FIG. 2, the first culture container K1a, the second culture container K1b, the third culture container K1c, and the medium supply container K2 are transferred to the transfer tubes K4a, K4b, K4c via the three-way stopcocks K3a, K3b. , K4d, K4e.

  More specifically, the first culture container K1a and the medium supply container K2 are connected by transfer tubes K4a and K4e via a three-way stopcock K3a, and the second culture container K1b and three-way stopcock K3b are connected by a transfer tube K4b. The third culture vessel K1c and the three-way stopcock K3b are connected by a transfer tube K4c, and the three-way stopcock K3a and the three-way stopcock K3b are connected by a transfer tube K4d. A transfer tube K4e is attached to a liquid feed pump P such as a tube pump.

  The culture containers K1a, K1b, K1c, and the culture medium supply container K2 are provided with a transfer port for transferring the content liquid to and from other containers, and a transfer tube K4a is connected to the transfer port. , K4b, K4c, K4e are connected.

  The culture vessels K1a, K1b, and K1c preferably have gas permeability (oxygen permeability and carbon dioxide permeability) necessary for cell culture so that cell culture can be performed in a closed system. Furthermore, in order to realize high cell growth efficiency, it is preferable that it has low cytotoxicity, low elution property, and radiation sterilization suitability, so that the progress of cell culture and the state of cells can be observed. It is preferable to have transparency to the extent that it can be seen through.

  On the other hand, the medium supply container K2 has gas barrier properties against oxygen and carbon dioxide in order to suppress changes in pH due to oxidation of the stored medium and dissipation of carbon dioxide contained in the medium at a high concentration. Is preferred.

  The culture containers K1a, K1b, and K1c are preferably formed into a bag shape using a soft wrapping material having desired physical properties. For example, the peripheral portion is sealed by heat fusion, and a desired shape such as a rectangular shape is used. However, the specific form is not limited as long as the cells and culture medium to be cultured can be accommodated without hindrance to the culture. The same applies to the medium supply container K2.

  On the mounting table 2, culture containers K1a, K1b, K1c provided in the cell culture kit K described above are arranged. In the example shown in FIG. 1, the first culture container K1a is relatively relative to the second culture container K1b by appropriately adjusting the height of the trays 3a, 3b, 3c on which the culture containers K1a, K1b, K1c are installed. The culture vessels K1a, K1b, K1c are elevated on the mounting table 2 so that the second culture vessel K1b is placed at a relatively high position relative to the third culture vessel K1c. It can be arranged with a difference.

  In FIG. 1, the state in which the mounting table 2 is stored in the culture chamber 10 is viewed from the front, and the culture vessels K1a, K1b, and K1c arranged on the mounting table 2 with a difference in height are indicated by a one-dot chain line. Show. Although not particularly illustrated, the cell culture device 1 can be provided with a cold storage room managed at a temperature suitable for storage of the culture medium (for example, 4 ° C.) below the culture room 10. The stored medium supply container K2 is stored.

  In the present embodiment, the cell culture device 1 is preferably configured to efficiently culture cells that have been damaged and have lost their functions, such as cells collected from patients and cells thawed after cryopreservation. . Curing culture for culturing such cells so that their original functions are restored, activating culture for culturing while activating the cells by antibody stimulation, and amplification culture for proliferating the activated cells in large quantities, A procedure for performing cell culture by the cell culture apparatus 1 will be described below by giving an example of performing the above in order.

  First, curing culture is performed in the first culture container K1a, and a culture solution containing cells to be cultured and a medium is poured into the first culture container K1a in a sterile environment in advance. Then, the culture vessels K1a, K1b, K1c are arranged on the mounting table 2 with a height difference as shown in FIG. 1, and after the mounting table 2 is stored in the culture chamber 10, the culture chamber 10 is isolated from the outside. It is managed in an environment suitable for cell culture.

When performing curing culture, a medium is added from the medium supply container K2 to the first culture container K1a as necessary. In order to add the medium, the three-way stopcock K3a is operated to enable the flow between the transfer tubes K4e and K4a, the liquid feed pump P is driven, and the medium supply container K2 passes through the transfer tubes K4e and K4a. What is necessary is just to transfer a culture medium to one culture container K1a. After adding the culture medium, the three-way stopcock K3a is operated to shut off the transfer tube K4a, and the culturing is performed for a predetermined period while avoiding the outflow of the content liquid in the first culture container K1a.
In addition, in performing curing culture, the method and apparatus which this applicant proposed by Unexamined-Japanese-Patent No. 2016-140295 can be utilized.

  When the curing culture in the first culture container K1a is completed, the content liquid in the first culture container K1a is transferred to the second culture container K1b and a medium is added to perform activation culture in the second culture container K1b. An activated substance such as an anti-CD3 antibody that activates cells by antibody stimulation is immobilized on the inner bottom surface of the second culture container K1b, so that the cells cultured in the second culture container K1b Activated by binding to.

  In order to transfer the contents of the first culture container K1a to the second culture container K1b, the three-way stopcocks K3a, K3b can be operated to allow flow between the transfer tubes K4a, K4d, K4b, and between the culture containers K1a, K1b. What is necessary is just to send the contents liquid of the 1st culture container K1a to the 2nd culture container K1b via the transfer tubes K4a, K4d, and K4b by the drop. At this time, the tray 3a on which the first culture vessel K1a is installed is pivotally supported at one end so that the first culture vessel K1a can be tilted so that the transfer port is on the lower side. It is preferable, and the content liquid of the 1st culture container K1a can be efficiently sent by this.

  On the other hand, in order to add the medium to the second culture container K1b, the three-way stopcocks K3a, K3b are operated to enable the flow between the transfer tubes K4e, K4d, K4b, the pump P is driven, and the medium is supplied from the medium supply container K2. The medium may be transferred to the second culture container K1b via the transfer tubes K4e, K4d, and K4b. However, if the medium that has been stored in the cold storage room at a low temperature is added to the second culture container K1b as it is, the cell growth efficiency is improved. We are anxious about it falling. For this reason, when adding a culture medium to the second culture container K1b, the culture medium is once transferred from the culture medium supply container K2 to the first culture container K1a, and the culture medium is heated to be equal to the temperature in the culture chamber 10. Then, it may be transferred to the second culture container K1b.

  Further, by transferring the medium to the first culture container K1a and then to the second culture container K1b, the cells remaining in the first culture container K1a can be transferred together with the medium to the second culture container K1b. it can. This also makes it possible to reduce the remaining cells in the first culture container K1a. By repeating such a transfer operation a plurality of times, the remaining cells in the first culture container K1a can be further reduced.

  The transfer from the medium supply container K2 to the first culture container K1a and the transfer from the first culture container K1a to the second culture container K1b may be performed as described above, and after the medium is added to the second culture container K1b. Operates the three-way stopcock K3b, shuts off the transfer tube K4b, and performs activated culture for a predetermined period while avoiding the outflow of the content liquid in the second culture vessel K1b.

  When the activation culture in the second culture container K1b is completed, the content liquid in the second culture container K1b is transferred to the third culture container K1c and a medium is added, and amplification culture is performed in the third culture container K1c.

  In order to transfer the contents of the second culture container K1b to the third culture container K1c, the three-way stopcock K3b can be operated to allow flow between the transfer tubes K4b and K4c, and the drop between the culture containers K1b and K1c The content liquid in the second culture container K1b may be sent to the third culture container K1c via the transfer tubes K4b and K4c. At this time, the tray 3b on which the second culture vessel K1b is installed is pivotally supported at one end so that the second culture vessel K1b can be tilted so that the transfer port is on the lower side. Is preferable as in the case of transferring the contents of the first culture vessel K1a to the second culture vessel K1b.

  On the other hand, in order to add the medium to the third culture container K1c, the three-way stopcocks K3a, K3b are operated to enable the flow between the transfer tubes K4e, K4d, K4c, the pump P is driven, and the medium supply container K2 is operated. What is necessary is just to transfer a culture medium to the 3rd culture container K1c via transfer tube K4e, K4d, K4c. When adding the medium to the third culture container K1c, the medium is once transferred from the medium supply container K2 to the first culture container K1a or the second culture container K1b in the same manner as when adding the medium to the second culture container K1b. Then, after sufficiently warming, it may be transferred to the third culture vessel K1c. After the medium is added to the third culture vessel K1c, the three-way stopcock K3b is operated to shut off the transfer tube K4c, and the amplification culture is performed for a predetermined period while avoiding the outflow of the content liquid in the third culture vessel K1c. The addition of the medium to the third culture container K1c may be divided into a plurality of times depending on the progress of the amplification culture.

  As described above, when cell culture is performed while transferring the content liquid between the culture containers K1a, K1b, K1c and the medium supply container K2, various operations as described above are performed from the outside. It is performed in a culture chamber 10 that is isolated and managed in an environment suitable for cell culture. For this reason, it is required to monitor the transfer state of the content liquid and appropriately control the operation timing according to the transfer state, but in this embodiment, the support unit 2 is supported and received from the mount unit 2. A plurality of load sensors 13 for detecting a load are arranged in the culture chamber 10 to monitor the transfer state of the content liquid by detecting a change in the load bias accompanying the transfer of the content liquid, and according to the transfer state. The operation timing can be appropriately controlled.

For example, if the load sensor receives from the mounting table 2 as a reference value when nothing is accommodated in any of the culture containers K1a, K1b, K1c arranged on the mounting table 2, the first culture container The load that the load sensor receives from the mounting table 2 during the curing culture in K1a is biased toward the first culture container K1a with respect to the reference value due to the weight of the content liquid in the first culture container K1a. Become. When the curing culture in the first culture container K1a is finished and the content liquid in the first culture container K1a is transferred to the second culture container K1b, the load received by the load sensor from the mounting table 2 is the first culture container K1a. The bias changes from the side to the second culture container K1b side.
Furthermore, when a culture medium is added to the second culture container K1b, the load that the load sensor receives from the mounting table 2 changes more toward the second culture container K1b due to the weight of the added culture medium.

Therefore, by setting in advance how the bias of the load received by the load sensor from the mounting table 2 changes depending on the increase / decrease of the contents in the culture vessels K1a, K1b, K1c, the loading detected by the load sensor is set. From the change in the bias of the load received from the pedestal 2, the state of transfer of the content liquid can be monitored. Then, for example, when the content liquid in the first culture container K1a is transferred to the second culture container K1b, if the load sensor detects a deviation of the load received from the mounting table 2 when the transfer is completed in advance, it is determined that the transfer is completed. Then, the operation timing can be controlled so that the operation of adding the medium to the second culture container K1b is performed.
It is also possible to measure the amount of medium added to the culture vessels K1a, K1b, K1c from the amount of change in load received from the mounting table 2.

  Here, FIG. 3 shows an outline of an example of the mounting table 2 in plan view. In the mounting table 2 shown in FIG. 3, trays 3a, 3b, and 3c on which the culture vessels K1a, K1b, and K1c are installed are mounted on a frame 4 that is framed in a square shape as shown in FIG. On the rear end side of the left and right side frames 4 a of the frame 4, traveling rollers 5 that travel on guide rails 11 that are horizontally attached to both side walls of the culture chamber 10 are provided. And the said side frame 4a is supported by the fixed roller 12 provided before the guide rail 11 of the culture chamber 10, and the mounting base 2 can be moved forward and backward on the guide rail 11 attached to the culture chamber 10. (See FIGS. 9 to 12).

  With this configuration, when the culture vessels K1a, K1b, and K1c are installed on the mounting table 2 or removed from the mounting table 2, the mounting table 2 is pulled out to the front of the culture chamber 10, and the work Can be easily performed. After the culture containers K1a, K1b, and K1c are installed, the mounting table 2 can be stored in the culture chamber 10 to perform cell culture.

  Further, as described above, the plurality of load sensors 13 that support the mounting table 2 and detect the load received from the mounting table 2 are disposed in the culture chamber 10, but the position at which the load sensor 13 is disposed is as follows. The mounting table 2 is appropriately set so as to support the mounting table 2 at at least three points and to support the mounting table 2 in a balanced manner. In this embodiment, two load sensors 13 are arranged on each of the front side and the back side of the culture chamber 10 so as to support the four corners of the mounting table 2. The load sensor 13 includes, for example, a support pin 13a held so as to protrude (preferably vertically protrude) from the floor surface of the culture chamber 10, and the load received by the support pin 13a is applied to the load detection element (load cell). It can be configured to be transmitted.

  When cell culture is performed, if an accident occurs in which the content liquid leaks from the culture containers K1a, K1b, and K1c arranged on the mounting table 2, the leaked content liquid is transferred to the load sensor 13. If the content liquid enters the load sensor 13 through the gap between the support pin 13a and the holding member that holds the support pin 13a, the load sensor 13 may be damaged.

  For this reason, the mounting table 2 is provided with a shielding member 6 that covers the upper side of the load sensor 13 in correspondence with the position where the load sensor 13 is disposed. 2 is supported by the load sensor 13. As a result, even when an accident such as leakage of the content liquid from the culture vessels K1a, K1b, K1c arranged on the mounting table 2 occurs during cell culture, the leaked content liquid is shielded. It is possible to prevent the load 6 from falling on the load sensor 13 by being blocked by the member 6, thereby preventing the load sensor 13 from being damaged.

5 is a cross-sectional view taken along the line AA of FIG. 4, FIG. 6 is a perspective view schematically showing a shielding member 6a provided on the front end side of the mounting table 2, and FIG. 7 is a rear end side of the mounting table 2. It is a perspective view which shows the outline of the shielding member 6b provided.
In these figures, the shielding members 6 a and 6 b formed in a columnar shape are shown attached to the frame 4 by bolts 7. As long as the intended purpose can be achieved by covering the upper side of the load sensor 13, the specific mode is not particularly limited, but at the four corners of the mounting table 2 corresponding to the position where the load sensor 13 is disposed. Among the provided shielding members 6, it is preferable that the shielding member 6 a provided on the front end side of the mounting table 2 is provided with an engaging recess 6 c that engages with the tip of the support pin 13 a included in the load sensor 13 ( (See FIG. 8).

  As described above, when the mounting table 2 is configured to be able to move back and forth on the guide rail 11 attached to the culture chamber 10, the mounting table 2 moves back on the guide rail 11 (see FIG. 9), It can be configured that the traveling roller 5 is separated from the rear end of the guide rail 11 and the front end of the side frame 4 a of the frame 4 is separated from the fixed roller 12 when reaching the position. Thereby, the mounting table 2 can be separated from the guide rail 11 at the retreat limit and supported by the load sensor 13 (see FIG. 10).

  At this time, the tip of the support pin 13a included in the load sensor 13 is engaged with the engagement recess 6c provided in the shielding member 6a, so that the load sensor 13 and the mounting table 2 are aligned. Thus, each time the mounting table 2 is supported by the load sensor 13, the mounting table 2 can be supported by the load sensor 13 in the same posture. As a result, the detection accuracy can be increased without variation in the bias of the load detected by the load sensor 13.

  FIG. 8 is an explanatory view showing a state in which the distal end portion of the support pin 13a included in the load sensor 13 is engaged with the engagement recess 6c provided in the shielding member 6a provided on the front end side of the mounting table 2. is there. In the present embodiment, the tip of the support pin 13a included in the load sensor 13 is formed in a hemispherical shape, and the engaging recess 6c is formed in a spherical shape with a slightly larger radius of curvature than that, so that both engage with each other. To make it easier.

  In order to pull out the mounting table 2 supported by the load sensor 13 to the front of the culture chamber 10, as shown in FIG. 11, the front end side of the mounting table 2 is lifted and the support pin 13a and the engagement recess 6c are engaged. And then pulling the mounting table 2 forward. As a result, the traveling roller 5 rides on the guide rail 11, and in this state, the mounting table 2 is returned to the horizontal position, and the side frame 4 a of the frame 4 is supported by the fixed roller 12 and pulled forward. 11 is moved forward and pulled out in front of the culture chamber 10 (see FIG. 12).

  In this embodiment, the engaging recess 6c is formed in the shielding member 6a provided on the front end side of the mounting table 2, and is engaged with the support pin 13a provided in the load sensor 13 disposed on the front side of the culture chamber 10. The reason for doing this is to make it easier to release the engagement between the support pin 13a and the engagement recess 6c when the mounting table 2 is pulled out as described above.

  9 to 12, only the frame 4 is shown as the mounting table 2. Although not particularly illustrated, the frame 4 is held for this purpose so that when the mounting table 2 is pulled out, the rear end side is not lifted up and can be pulled out in front of the culture chamber 10 while being kept horizontal. A mechanism can be provided.

  While the present invention has been described with reference to the preferred embodiment, it is needless to say that the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the present invention.

  For example, in the above-described embodiment, the load sensor 13 includes the support pin 13a, and the engaging recess 6c with which the tip end of the support pin 13a engages is provided in the shielding member 6 provided on the front end side of the mounting table 2. However, if the load sensor 13 can support the mounting table 2 and detect the load received from the mounting table 2, the support pin 13a may be omitted, and instead of the support pin 13a. Other support members may be provided.

  In short, in the present invention, when the culture vessels K1a, K1b, and K1c are placed on the mounting table 2 stored in the culture chamber 10 managed in an environment suitable for cell culture, A plurality of load sensors 13 that support and detect the load received from the mounting table 2 are disposed in the culture chamber 10, and the shielding member 6 that covers the upper side of the load sensor 13 corresponds to the position where the load sensor 13 is disposed. If the mounting table 2 is supported by the load sensor 13 via the shielding member 6, the other detailed configuration is appropriately changed without being limited to the above-described embodiment. can do. Further, the detailed configurations described in the above-described embodiments can be appropriately selected and combined.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used as an apparatus for culturing cells in the fields of pharmacology / biochemistry such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy.

DESCRIPTION OF SYMBOLS 1 Cell culture apparatus 2 Mounting stand 6 (6a, 6b) Shielding member 6c Engagement recessed part 10 Culture chamber 11 Guide rail 13 Load sensor 13a Support pin K1a, K1b, K1c Culture container

Claims (4)

A cell culture device for culturing cells by placing a culture vessel on a mounting table stored in a culture chamber controlled in an environment suitable for cell culture,
A plurality of load sensors that support the mounting table and detect a load received from the mounting table are disposed in the culture chamber,
A shielding member that covers the load sensor is provided on the mounting table in correspondence with a position where the load sensor is disposed, and the mounting table is supported by the load sensor via the shielding member. A cell culture device.   2. The device according to claim 1, wherein the mounting table can be moved forward and backward on a guide rail attached to the culture chamber, and the mounting table is separated from the guide rail in a retreat limit and supported by the load sensor. Cell culture device.   The cell culture device according to claim 1, wherein the load sensor includes a support pin that is held so as to protrude from a floor surface of the culture chamber.   The load sensor is disposed so as to support the mounting table at at least three points, and among the shielding members provided on the mounting table, the load sensor is provided on the shielding member provided on the front end side of the mounting table. The cell culture device according to claim 3, wherein an engagement concave portion with which a distal end portion of a support pin is provided is provided.

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