JP2013116093A - Culture vessel, automatic culture device and culture medium supplying method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To cause a large-sized culture vessel to bend easily, the amount of medium in each place becomes uneven, the quality of regenerated tissue is not stable, and an error occurs in handling.
SOLUTION: A culture vessel 10 having a structure in which walls 18, 20, and 22 in which cell culture inserts can be arranged in a row is provided in a culture area, and further, the culture vessel can be installed, and a plurality of cell culture inserts 11 can be simultaneously installed. An automatic culture apparatus that can be processed. As a result, the deflection of the enlarged culture vessel 10 can be minimized, the amount of medium supplied at the time of cell seeding and medium replacement by the robot can be made uniform, and the quality of living tissue produced by automatic culture can be stabilized.
[Selection] Figure 1

Description

  The present invention relates to a culture vessel and an automatic culture apparatus for culturing cells, and particularly to a culture technique for efficiently producing tissue from a large number of cells.

  In recent years, development of a technique for reconstructing a biological tissue having a partial function of an internal organ by culturing cells outside the body has been active. The reconstructed living tissue is not only used for treatment of a disease, but also used for a test for evaluating the effect of a drug or the like. For example, cultured skin epithelium has already been put to practical use in drug effect tests such as cosmetics and transdermal drugs, but the production of the cultured skin epithelium is carried out manually by skilled workers under strict processes. ing. Therefore, when producing a large amount of cultured skin epithelium, the burden on the worker is increased and the time and cost required to educate and nurture the worker, human error, and biology by humans who possess bacteria etc. Contamination and the like may occur, and a lot of cost is required for the countermeasures. This is a big barrier in mass production. Therefore, it is expected that these problems can be solved by automating a series of culture operations using an instrument.

  Currently, a culture container for producing cultured skin epithelium mainly uses a 6-well plate in which cell culture inserts are installed in each well. Each well is independent. Supply culture medium to each well and cell culture insert. However, since 6 cultured skin epithelia are produced per 6-well plate, assuming mass production of several hundred to several thousand cultured skin epithelium, the number of culture containers to be processed by the apparatus increases. End up. Therefore, it is required to increase the number of cell culture inserts per culture container and to realize simultaneous processing of the culture solution to the cell culture inserts, which is difficult to do manually.

  In order to solve this problem, for example, Patent Document 1 proposes a configuration in which one well is provided and the culture solution in the well portion can be processed simultaneously.

  Further, for example, Patent Document 2 presents a means for enabling the feeding and discharging of a culture solution to a well portion that is not independent.

JP-T-2004-521645 JP 2005-526514 A

  In the culture container as described in Patent Document 1, when the culture container is enlarged, a difference occurs in the amount of the medium around each cell culture insert due to the deformation of the culture container, and there is a difference in the medium components supplied to the cells. There is a problem that it does not become a cultured skin epithelium with the quality of. In addition, there is a problem that the position of each cell culture insert varies with the deformation of the culture vessel, and the processing of the robot or the like has a reproduction and cannot be performed accurately.

  In a culture vessel such as Patent Document 2, since the feeding port and the discharge port for the culture solution are on the same surface, when the culture vessel is enlarged, stagnation occurs and a cell culture insert in which the surrounding medium is not exchanged is produced. There arises a problem that the new medium supply to the cells is not uniform.

  Currently, the development of a technique for reconstructing a biological tissue having a partial function of an internal organ from a cell is actively developed. For example, cultured skin epithelium is manufactured as a product and is being used for drug evaluation tests for newly developed cosmetics and transdermal drugs. However, its manufacture is done manually by highly skilled engineers.

  For the production of the cultured skin epithelium, a culture vessel in which a cell culture insert is installed in an independent well of the 6-well plate described above is used. Cells are seeded on cell culture inserts. Then, cell culture is performed by supplying and exchanging the culture solution to each cell culture insert and each well at a predetermined time. The maximum number of cultured skin epitheliums that can be produced in one culture container is six. You cannot work on multiple cell culture inserts at the same time manually. Therefore, in order to produce hundreds to thousands of cultured skin epithelium, a large amount of culture containers are required, and enormous work time is required. For this reason, the production of cultured skin epithelium is a small-scale production and the quality is not stable, thereby resulting in high production costs.

  In order to carry out mass production, it is desirable to have a system in which cell culture inserts can be installed in one culture vessel as much as possible, and a plurality of cell culture inserts that are impossible by hand are simultaneously processed by the apparatus. However, when the culture vessel is enlarged, the culture vessel becomes less rigid and deforms. As a result, the amount of medium around each cell culture insert is not uniform, and the supply of the influence of the cultured skin epithelium varies depending on the position, resulting in unstable quality. Furthermore, the position accuracy of the apparatus for exchanging the liquid on the upper part of the cell culture insert is deteriorated, and the processing becomes difficult. Therefore, it is a problem how to suppress the deformation of the large culture container and eliminate the non-uniformity of the culture medium exchange depending on the position of the culture container.

  An object of the present invention is to solve such problems and to provide a culture vessel, an automatic culture apparatus, a medium supply method, and the like that can mass-produce tissues using cells.

  In order to achieve the above object, in the present invention, a culture container capable of holding one or a plurality of cell culture containers having a substance-permeable membrane on the bottom, a medium injection region for injecting a medium, and a cell culture container One or a plurality of culture regions in which the culture medium can be supplied and exchanged from the lower part of the material permeable membrane, the culture medium discharge region for discharging the culture media, and the culture medium being supplied from the culture medium injection region to the culture region There is provided a culture vessel comprising an inlet wall in which an injection hole is formed, and an outlet wall in which one or a plurality of discharge holes are formed for discharging the medium from the culture region to the medium discharge region.

  In order to achieve the above object, according to the present invention, there is provided an automatic culture apparatus for culturing cells using a culture container, the culture container having one or a plurality of cell culture containers having a substance-permeable membrane on the bottom surface. A culture medium injection area for injecting a culture medium, a culture area in which a cell culture container can be held and the culture medium can be supplied and exchanged from the lower part of the substance-permeable membrane, and a culture medium discharge area for discharging the culture medium An inlet wall having one or more injection holes for supplying the culture medium from the culture medium injection area to the culture area, and one or more discharges for discharging the medium from the culture area to the culture medium discharge area. A medium supply mechanism for holding a culture container and supplying and exchanging the medium to the culture container, and an adjustment mechanism for maintaining an environment in which cells in the culture container can be cultured. Having the first A culture environment, a second culture environment that maintains a clean environment and enables a culture vessel to be transported to the first culture environment, and a control system that controls the first culture environment and the second culture environment Provided is an automatic culture apparatus having the above structure.

  Furthermore, in order to achieve the above object, the present invention provides a medium supply method of an automatic culture apparatus for culturing cells, and can hold one or a plurality of cell culture containers having a substance-permeable membrane on the bottom surface. A culture medium injection area for injecting a culture medium, a culture area for holding a cell culture container and supplying and exchanging the culture medium from the lower part of the substance permeable membrane, a culture medium discharge area for discharging the culture medium, and a culture medium injection An inlet wall in which one or a plurality of injection holes are formed for supplying the culture medium from the area to the culture area, and a discharge outlet wall in which one or a plurality of discharge holes are formed for discharging the medium from the culture area The culture container is provided in a first culture environment having a culture medium supply mechanism for supplying and replacing the culture medium to the culture container, and an adjustment mechanism for maintaining an environment in which the cells in the culture container can be cultured. Install The culture medium injection region of the culture container installed in the first culture environment from the culture medium supply system of the second culture environment in which the culture medium supply system for maintaining the clean environment and supplying the culture medium to the cell culture container is installed A medium supply method for injecting a medium is provided.

  This minimizes the deflection of the larger culture vessel, makes it possible to equalize the amount of medium supplied during cell seeding and medium exchange by a robot, and to stabilize the quality of living tissue produced by automatic culture.

It is a schematic block diagram of the culture container based on a 1st Example. It is a top view of the culture container based on a 1st Example. It is the front view which showed the internal structure of the culture container before a cell liquid and culture medium supply based on a 1st Example. It is a front view of the culture container which installed the cell culture insert based on a 1st Example. It is a front view of the state which supplied the culture medium to the substance-permeable membrane lower part of the cell culture insert based on a 1st Example. It is a front view of the state before supplying a culture medium to the substance permeable membrane upper part of a cell culture insert based on a 1st Example. It is a front view of the state which is supplying the culture medium to the substance permeable membrane upper part of the cell culture insert based on a 1st Example. It is a front view of the state after supplying a culture medium to the substance permeable membrane upper part of a cell culture insert based on a 1st Example. It is a figure which shows an example of the whole schematic structure of the automatic culture apparatus based on a 1st Example. It is a block diagram which shows the state of the automatic culture apparatus before installing a culture container based on a 1st Example. It is a side block diagram which shows the state of the automatic culture apparatus before installing the culture container based on a 1st Example. It is a block diagram which shows the state inside an incubator before installing a culture container based on a 1st Example. It is a side block diagram which shows the state inside an incubator before installing the culture container based on a 1st Example. It is a block diagram which shows the state at the time of inserting the culture container into an automatic culture apparatus inside based on a 1st Example. It is a side block diagram which shows the state at the time of inserting the culture container based on a 1st Example into an automatic culture apparatus inside. It is a block diagram which shows the state of the automatic culture apparatus at the time of installing the culture container based on a 1st Example. It is a side block diagram which shows the state of the automatic culture apparatus at the time of installing the culture container based on a 1st Example. It is a block diagram which shows the state at the time of inserting the culture medium supply system based on a 1st Example into the inside of an automatic culture apparatus. It is a side block diagram which shows the state at the time of inserting the culture medium supply system based on a 1st Example into the inside of an automatic culture apparatus. It is a block diagram which shows the structural example of the control system of the whole automatic culture apparatus.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments are merely examples for realizing the present invention, and do not limit the technical scope of the present invention. Should. In each drawing, the same reference numerals are assigned to common components. In the present specification, a container structure capable of holding one or a plurality of cell culture containers such as a cell culture insert is referred to as a culture container.

  The culture vessel used in the automatic culture apparatus of the present invention has a configuration in which a large number of cell culture inserts can be installed, and a medium for supplying nutrients from the lower part of the cell culture insert can be simultaneously supplied and exchanged.

  In a preferred embodiment of the present invention, the culture vessel is composed of three areas: a culture medium injection area (area), a culture area (area), and a culture medium discharge area (area). The culture medium injection area and the culture medium discharge area are located in parallel with one side of the culture container, and there is a culture area having a plurality of walls between the two areas. The culture medium injection area has one or a plurality of injection ports, and the culture medium is injected into the area from the port. The medium discharge area has one or more ports serving as discharge ports, and the medium in the area is discharged from the port.

  The culture area has one or more walls on which a plurality of cell culture inserts can be arranged in a row. There are a plurality of injection holes in the wall between the culture medium injection area and the culture area, and the culture medium is supplied to the culture area through the holes. Moreover, there are a plurality of discharge holes in the wall between the culture area and the medium discharge area, and the medium is sent to the medium discharge area through the holes. The waste liquid in the medium discharge area moves to the outside from the port that is the discharge port. In order to make the flow of the medium in one direction, the injection hole on the medium injection area side is positioned higher than the discharge hole on the medium discharge area side. A plurality of cell culture inserts can be installed in a culture zone sandwiched between a plurality of walls, and a guide is provided at the installation location. The plurality of walls surrounding the culture zone can increase the rigidity of the culture vessel and prevent the occurrence of deflection, whereby the culture medium can be uniformly supplied to all cell culture inserts and the quality can be stabilized.

  In a preferred embodiment of the automatic culture apparatus in which the culture vessel is installed, a safety cabinet called a second culture environment, or an incubator having a clean bench or cell isolator and a culture medium supply mechanism called a first culture environment Has an integrated structure. Place the sterile-packed culture container in the safety cabinet or clean bench or cell isolator, which is the second culture environment, and remove the packaging. Open the door of the incubator, which is the first culture environment in the automatic culture device, and set the culture vessel on the installation stand inside. Subsequently, the medium supply mechanism is set in the culture container. In this automatic culture device, cell culture and medium can be supplied simultaneously to the upper and lower culture surfaces of the cell culture insert by the medium supply mechanism, so that cell seeding, medium supply, and medium can be supplied to a large number of cell culture inserts. Automatic cell culture work such as switching can be performed.

  The culture container, automatic culture apparatus, culture container installation method, medium supply method, and the like of the first embodiment will be described with reference to the drawings.

<Composition of culture vessel>
First, the configuration of the culture vessel 10 and its cell solution supply and medium supply method will be described with reference to FIGS. 1, 2 and 3A to 3F. 1 is a schematic configuration diagram of a culture vessel according to Example 1, and FIG. 2 is a top view of the culture vessel of Example 1. FIG. 3A, 3B, 3C, 3D, 3E, and 3F are front views of the automatic culture apparatus of Example 1, respectively.

  FIG. 3A shows a front view showing the state of the culture vessel 10 before supplying the cell solution / medium, FIG. 3B shows a front view of the state of the culture vessel 10 in which the cell culture insert 11 is installed, and FIG. 3C shows the cell culture insert. 11 shows a front view of the state in which the culture medium 12 is supplied to the lower part, FIG. 3D shows a front view of the state before supplying the culture medium 12 to the upper part of the cell culture insert 11, and FIG. FIG. 3F shows a front view of the state after supplying the culture medium 12 to the upper part of the cell culture insert 11.

  First, the structure of the culture vessel 10 of the present embodiment will be described with reference to FIGS. The culture vessel 10 mainly comprises an inlet area 13, a culture area 14, and a discharge area 15. There are one or more inlets 16 in the inlet area 13. The discharge port area 15 has one or a plurality of discharge ports 17. For the sake of illustration, only one inlet 16 and one outlet 17 are shown, but the number is not limited to one.

  The culture area 14 has a wall 18 that connects the inlet area 13 and the outlet area 15. The cell culture insert 11 is placed on the wall 18 and the cells are cultured. A guide 19 is provided on the upper portion of the wall 18 and functions as a cell culture container holding member together with the wall 18 to determine a position (vertical, horizontal, height, inclination) on which the cell culture insert 11 is placed. The width between the adjacent walls 18 is the amount that one cell culture insert 11 can enter, and the width depends on the size of the cell culture insert 11. In the present specification, a region sandwiched between adjacent walls 18 is called a culture zone.

  There is an injection hole 21 in the inlet wall 20 between the inlet area 13 and the culture area 14, and a discharge hole 23 in the outlet wall 22 between the outlet area 15 and the culture area 14. Note that the wall 18 may be referred to as a partition wall in order to distinguish between the inlet wall 20 and the outlet wall 22. Since the inlet wall 20, the discharge outlet wall 22, and the partition wall 18 are joined to the culture vessel bottom surface 24, these walls 18, 20, and 22 increase the rigidity of the culture vessel 10, so that the entire culture vessel 10 is bent. The occurrence is prevented. A culture zone 25 is a place where one or a plurality of cell culture inserts 11 can be arranged in a row, surrounded by two adjacent partition walls 18, an inlet wall 20, an outlet wall 22, and a culture vessel bottom surface 24. As shown in FIG. 1, one or a plurality of culture zones 25 are formed in the culture area 14 of the culture vessel 10.

  Next, a method for supplying and discharging the culture medium to and from the culture vessel 10 in this embodiment will be described with reference to FIGS. 3A to 3F. The culture vessel 10 is exemplified by a culture method using the cell culture insert 11.

  As shown in FIG. 3A, the inside of the culture vessel 10 includes a partition wall 18, an inlet wall 20, an outlet wall 22, an inlet 16, an outlet 17, an inlet hole 21, an outlet hole 23, a guide 19, and a culture vessel bottom surface 24. Consists of. Only two guides 19 are shown, and the rest are omitted for convenience of illustration. When the cell culture insert 11 is inserted, the positional relationship with the guide 19 is as shown in FIG. 3B.

  As shown in FIG. 3B, a cell permeable membrane 26 is stretched on the cell culture insert 11, and cells are cultured on the upper portion 27 of the material permeable membrane 26. When the culture medium 12 is injected into the lower part 28 of the material permeable membrane 26, the medium 12 passes through the material permeable film 26, supplies the components in the medium to the cells cultured in the upper part of the material permeable film 27, and promotes cell growth. .

As shown in FIG. 3A, the center height of the injection port 16 is h ₁ , the center height of the discharge port 17 is h ₂ , and the height of the injection hole 21 is h ₃ , based on the culture vessel bottom bottom 28. The height of the discharge hole 23 is h ₄ , and the height of the bottom surface of the material permeable membrane 26 is h ₅ . The thickness of the culture vessel bottom 24 is t.

Each positional relationship is made into the following formula | equation (1)-Formula (3).

h ₃ > h ₁ ≧ t …………… Formula (1)
h ₅ ≧ h ₂ ≧ t, h ₃ > h ₅ …………… Formula (2)
h ₃ > h ₄ ............ Formula (3)

With reference to FIG. 3C, a method of supplying the medium to the lower part 28 of the material permeable membrane of the culture vessel 10 of this example will be described. First, the culture medium 12 is supplied from the inlet 16 to the inlet area 13. At that time, the discharge port 17 is closed. When the culture medium 12 accumulates in the inlet area 13 and the water level rises to the injection hole 21 (h ₃ ), the culture medium 12 moves to the culture area 14 according to the equation (1). Thereafter, the culture medium 12 in the culture area 14 enters the discharge port area 15 through the discharge hole 23 according to the equations (2) and (3).

If the culture medium 12 in the other culture zone 25 in the culture area 14 is small, the culture medium 12 in the discharge port area 15 supplies the culture medium 12 from the discharge hole 23. Thereby, the culture medium 12 of all the culture zones 25 becomes uniform height. However, in order to supply the culture medium 12 from the substance permeable membrane 26, the liquid level of the culture medium 12 is set to be higher than h ₅ . Here, for example, a position sensor may be installed in the culture vessel 10 to measure the position of the liquid level.

  If necessary, excess medium 12 can be removed from outlet 17 and adjusted. Further, the medium 12 in the inlet area 13 can be removed by sucking the medium 12 through the inlet 16. For example, when it is desired to remove the old culture medium 12 by exchanging the culture medium, the culture medium 14 in the culture area 14 and the discharge port area 15 can be extracted by the formula (2) by sucking the culture medium from the discharge port 17. Thereafter, the medium is replaced with a new medium 12 by the above-described medium supply method.

  With reference to FIG. 3D, a method for supplying a medium to the substance permeable membrane upper portion 27 of the culture vessel 10 in the present embodiment will be described. As shown in FIG. 3D, there are a liquid injection nozzle 46, a tube 48, and a liquid injection nozzle set 47 that can be attached to and detached from the manipulator described in FIG. The liquid injection nozzle set 47 includes one or a plurality of liquid supply nozzles 46. These can be moved by a manipulator 45.

  As shown in FIG. 3E, the tip of the liquid injection nozzle 46 can be moved to the upper portion 27 of the substance permeable membrane 26 by the manipulator 45. Then, a cell suspension, a medium, or the like can be sent through the tube 48 and discharged from the liquid injection nozzle 46 by a peristaltic pump 51, which will be described later, and the medium 12 is sucked from the liquid injection nozzle 46 by the perista pump 51 or the like. can do. After feeding a predetermined amount of the medium 12 from the liquid injection nozzle 46, the liquid injection nozzle 46 is moved by the manipulator 45 to perform culture.

  At the time of cell seeding, FIG. 3D → FIG. 3E → FIG. 3F is repeated. At the time of medium exchange, FIG. 3F → FIG. 3E → FIG. 3D → (replacement of the injection nozzle 46 as necessary) → FIG. 3D → FIG. 3E → FIG. This treatment can be performed simultaneously using a plurality of injection nozzles 46 for cell seeding and medium exchange of a plurality of cell culture inserts 11.

<Configuration of automatic culture device>
An example of the overall configuration of the automatic culture apparatus 40 of the present embodiment will be described with reference to FIG. The automatic culture apparatus 40 includes, as basic components, a safety cabinet, a clean bench 41 including a cell isolator, an incubator 42 that forms a first culture environment, and a control unit 43. In the present specification, the incubator 42 has a first culture environment for culturing cells by holding one or a plurality of culture containers, and the clean bench 41 has a mechanism for maintaining a clean environment, It functions as a second culture environment in which one or a plurality of culture containers can be transported to the first culture environment. Each basic configuration will be described below.

  First, the clean bench 41 that is the second culture environment will be described. The clean bench 41 can maintain the internal cleanliness by a HEPA (High Efficiency Particulate Air) filter or the like. There is an up-and-down sliding door 44 in front of the clean bench 41, and a disinfected hand or glove can be put into the clean bench 41 by opening a part of the door 44.

  Inside the clean bench 41, a culture medium supply system 49 is installed. This has the refrigerator 50 which functions as a refrigeration storage part which stores a culture medium etc. by Peltier etc., for example, and preserve | saves a cell suspension and a culture medium in it. The medium and the like are fed by a peristaltic pump 51 constituting the medium supply system 49 and supplied to the culture vessel 10 installed in the incubator 41 that is the first culture environment. A method of installing the medium supply system 49 in the second culture environment will be described later.

  An incubator 42 as a first culture environment is connected to the back of the clean bench 41. An openable / closable door 52 is installed between the clean bench 41, which is the second culture environment, and the incubator 42, which is the first culture environment. The door 52 is closed during the culture, and the inside of the incubator 42, which is the first culture environment. Incubate the cells at As shown in FIG. 4, the opened door 52 is stored in an intermediate chamber 53 between the clean bench 41 and the incubator 42. It is desirable that the door 52 slides downward and the intermediate chamber 53 has a negative pressure so that dust or the like is not generated.

  Next, the incubator 42 as the first culture environment will be described. The inside of the incubator 42 maintains an environment close to a temperature of 37 ° C., a humidity of 100%, and 5% carbon dioxide by an adjustment mechanism (not shown). There is also a port for supplying a predetermined gas from the outside, which is also not shown. There is a culture vessel table 55 that slides forward when the culture vessel 10 is installed and has a guide 54 for positioning the culture vessel 10 on the upper surface. After placing the culture vessel 10 on the culture vessel stand 55, it is pushed in and installed in the incubator 42. Inside the incubator 42 is a manipulator 45 capable of holding a liquid injection nozzle set 47 in which a plurality of liquid injection nozzles 46 for supplying and discharging the medium 12 and the like to the cell culture insert 11 of the culture vessel 10 are set.

  The manipulator 45 installed in the first culture environment can move the liquid injection nozzle set 47 described above in the X-Y-Z direction. Further, there are a waste liquid pad 56 for collecting the waste liquid and a humidity pad 57 capable of removing water to maintain the humidity. These are all made of a material that can withstand high humidity and, if necessary, have a mechanism that can be decontaminated with hydrogen peroxide mist or peracetic acid.

  The control unit 43 is integrated with the clean bench 41 as the second culture environment and the incubator 42 as the first culture environment, and controls them. That is, environmental monitoring in the incubator 42 and storage of various sensing data, control of the manipulator 45, and control of the medium supply system 49 in the clean bench 41 are integrated. Data display and operation of the control unit 43 are performed by, for example, a touch panel type display / operation unit 58 in front of the clean bench 41. Details of the control unit 43 will be described later with reference to FIG.

<A series of operations such as culture vessel installation>
Next, an example of the operation of installing the culture container of the present example in the automatic culture apparatus will be described with reference to FIGS. 5A, 5B to 9A, and 9B. 5A, FIG. 6A, FIG. 7A, FIG. 8A, and FIG. 9A are schematic diagrams regarding the installation of the culture vessel in the automatic culture apparatus of the present example, respectively, and FIG. 5B, FIG. 6B, FIG. 7B, FIG. 9B is a schematic side view thereof.

  First, FIG. 5A, FIG. 5B, FIG. 6A, and FIG. 6B show the configuration of the automatic culture apparatus 40 before the culture container 10 is installed. As shown in FIGS. 5A and 5B, the inside of the clean bench 41, which is the second culture environment, maintains cleanliness by decontamination with a HEPA filter, a germicidal lamp, and hydrogen peroxide as necessary. 6A and 6B, when the incubator door 52 that is the first culture environment is opened, the inside of the incubator 42 and the inside of the clean bench 41 are connected. Inside the incubator 42, a manipulator 45 and a culture vessel table 55 are installed. The culture vessel table 55 has a guide 54 for correcting the position when the culture vessel 10 is installed.

  Next, the set of the culture vessel 10 will be described with reference to FIGS. 7A, 7B, 8A, and 8B. The culture container 10 is packaged 29, and the inside of the package 29 is in a sterilized state. In the case of the clean bench 41, it is sterilized with ethanol or the like before entering, and the clean bench door 44 is opened a little. For example, it may be decontaminated with a pass box like an isolator and put inside. As shown in FIGS. 8A and 8B, the culture vessel table 55 in the incubator 42 is pulled out, and the culture vessel 10 with the packaging 29 removed is placed on the culture vessel table 55. When installing, pay attention to the position of the guide 54.

  Finally, installation on the clean bench 10 which is the second culture environment such as the culture medium supply system 49 will be described with reference to FIGS. 9A and 9B. The medium supply system 49 and the like are all covered with the package 30, and the inside is sterilized. When the culture vessel 10 is put into the clean bench 41, the medium supply system 49 and the like are also put into the clean bench 10 and the packaging 30 is removed. As shown in FIG. 4, the culture medium supply system 49 is installed in the clean bench 41, and a connector is connected to supply necessary power. The liquid injection nozzle set 47 is installed on the fixture 59 of the manipulator 45. The liquid injection nozzle set 47 is connected to the peristaltic pump 51 of the culture medium supply system 49 and can supply the culture medium 12 and the like in the refrigerator 50 and collect the waste liquid.

  The inlet 16 of the culture vessel 10 and the tube 48 can be connected and connected to the peristaltic pump 51. A waste liquid pad 56 and a humidity pad 57 can be installed. The waste liquid pad 56 can be connected to the discharge port 17 of the culture vessel 10 by a tube 48 and connected to the peristaltic pump 51. A notch 60 in the shape of a tube 48 is provided in a part of the incubator door 52 so that the tube 48 can be installed between the clean bench 41 and the incubator 42. FIG. 4 shows a state in which these are installed, and then the incubator door 52 is closed, and an automatic culture process can be performed.

<Control system configuration of automatic culture device>
FIG. 10 is a block diagram showing an example of the configuration of a control system for controlling the internal devices in the automatic culture apparatus 40.

  The control system of the automatic culture apparatus 40 includes the control unit 43, the manipulator 45, the display / operation unit 58, etc. shown in FIG. In the figure, a display unit 80 that indicates the control status to the user and an input unit 81 such as a touch panel, a keyboard, and a mouse for inputting data and instructions correspond to the display / operation unit 58.

  A control processing unit 82 such as a central processing unit (CPU) for controlling each operation of the automatic culture apparatus 40, a ROM 85 for storing programs and parameters, and a RAM 86 for temporarily storing data and processing results. Further, a memory 83 for performing an operation such as a cache, and an environment holding device 87 that performs processing such as a heater and carbon dioxide supply and performs control based on various sensor outputs correspond to the control unit 43. The culture medium supply system 49 includes a refrigerator control 88 that controls the environment in the refrigerator 50 shown in FIG. 4 and a pump control 89 that controls the verista pump 51. The medium supply system 49 is controlled by the refrigerator control 88 and the pump control 89. Note that the refrigerator control 88 and the pump control 89 can be program controlled by the CPU in the control unit 43.

  When the user instructs the culture process to be processed from the input unit 81 or the communication unit 84, the CPU of the control processing unit 82 proceeds with the culture environment holding process according to the culture preparation program stored in the ROM 85, and the temperature is 37 ° C., 5%. Carbon dioxide concentration, humidity 100%, clean environment field. The refrigerator control 88 is performed after the culture medium supply system 49 is installed.

  Then, the CPU of the control processing unit 82 performs cell culture processing in the culture vessel 10 by the manipulator 45 and the peristaltic pump 51 of the medium supply system 49 according to the automatic culture program stored in the ROM 85. At any time, the display unit 80 and the communication unit 84 can indicate the processing status to the user. When the cell culture process is completed, the display unit 80 and the communication unit 84 indicate the completion to the user, sense that the culture vessel 10 has been removed or the incubator door 52 has been opened, and the control processing unit 82 terminates the process stored in the ROM 85. Perform termination processing according to the program. As described above, a series of cell culture processes by the automatic culture apparatus 40 of the present embodiment can be realized.

  In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail in order to explain the present invention in an easy-to-understand manner, and are not necessarily limited to those having all the configurations described.

  Each of the above-described configurations, functions, processing units, processing means, and the like may be realized by hardware by designing a part or all of them with, for example, an integrated circuit. In addition, each configuration, function, and the like have been described by exemplifying a case where they are realized by software by executing a program that realizes each function. However, information on programs, tables, files, and the like that realize each function It can be stored not only in memory, but also in recording devices such as hard disks and SSDs (Solid State Drives), or recording media such as IC cards, SD cards, and DVDs. It is also possible to do.

10 Culture vessel 11 Cell culture insert 12 Medium 13 Inlet area (area)
14 Culture area (area)
15 Discharge area (area)
16 Inlet 17 Outlet 18 Partition wall 19 Guide 20 Inlet wall 21 Inlet hole 22 Outlet wall 23 Outlet hole 24 Culture vessel bottom 25 Culture zone 26 Substance permeable membrane 27 Substance permeable membrane upper part 28 Substance permeable membrane lower part 29 Culture container packaging 30 Medium supply system packaging 40 Automatic culture apparatus 41 Clean bench 42 Incubator 43 Control unit 44 Clean bench door 45 Manipulator 46 Injection nozzle 47 Injection nozzle set 48 Tube 49 Medium supply system 50 Refrigerator 51 Perista pump 52 Incubator door 53 Intermediate chamber 54 Guide 55 Culture container base 56 Waste liquid pad 57 Humidity pad 58 Display / operation part 59 Fixing tool 60 Notch 80 Display part 81 Input part 82 Control processing part 83 Memory 84 Communication part 85 ROM
86 RAM
87 Environmental maintenance device 88 Refrigerator control 89 Pump control

Claims (15)

A culture vessel capable of holding one or a plurality of cell culture vessels having a substance-permeable membrane on the bottom surface,
A medium injection region for injecting the medium;
A culture region that holds the cell culture vessel and is capable of supplying and exchanging a medium from the lower part of the substance-permeable membrane;
A medium discharge area for discharging the medium;
An inlet wall in which one or a plurality of injection holes are formed to supply a culture medium from the culture medium injection area to the culture area;
A culture vessel comprising a discharge port wall having one or a plurality of discharge holes for discharging a medium from the culture region to the medium discharge region. The culture container according to claim 1,
The medium injection region has a medium injection port for injecting a medium from the outside,
A culture vessel characterized in that the medium injected from the medium injection port can be supplied to one or a plurality of the injection holes. A culture container according to claim 2,
The medium discharge area has a medium discharge port for discharging the medium to the outside,
A cell culture container capable of discharging the medium discharged from one or a plurality of the discharge holes. The culture container according to claim 1,
The cell culture vessel, wherein the culture region has a plurality of culture zones divided by a plurality of partition walls arranged in parallel from the inlet wall to the outlet wall. A culture container according to claim 2,
A cell culture container, wherein the height of the center of the injection hole is higher than the height of the center of the medium injection port. A culture vessel according to claim 3,
The height of the center of the injection hole is higher than the height of the bottom surface of the substance-permeable membrane, and the height of the bottom surface of the substance-permeable film is higher than or equal to the height of the center of the medium outlet. The cell culture container is characterized in that the height of the center of is higher than the height of the discharge hole. The culture container according to claim 4,
The partition wall has a guide for adjusting and holding an installation position of one or a plurality of the cell culture containers. The culture container according to claim 4,
Each of the plurality of culture zones includes one injection hole and one discharge hole on each of the injection wall and the discharge wall,
A cell culture container characterized by being capable of holding a plurality of the cell culture containers in a row. The culture container according to claim 7,
Each of the plurality of culture zones includes one injection hole and one discharge hole on each of the injection wall and the discharge wall,
A plurality of the cell culture containers can be held in a row by the guide. An automatic culture apparatus for culturing cells using a culture vessel,
The culture vessel can hold one or a plurality of cell culture vessels having a substance-permeable membrane on the bottom surface, a medium injection region for injecting a medium, and the cell culture vessel, the lower part of the substance-permeable membrane A culture region capable of supplying and exchanging the medium, a medium discharge region for discharging the medium, and one or a plurality of injection holes for supplying the culture medium from the medium injection region to the culture region A structure comprising an inlet wall and a discharge wall formed with one or a plurality of discharge holes for discharging the medium from the culture region to the medium discharge region,
A culture medium supply mechanism for holding the culture container and supplying and replacing the culture medium to the culture container; and a first culture environment having an adjustment mechanism for maintaining an environment in which cells of the culture container can be cultured;
A second culture environment that maintains a clean environment and allows the culture vessel to be transported to the first culture environment;
An automatic culture apparatus comprising the first culture environment and a control system for controlling the second culture environment. The automatic culture apparatus according to claim 10, wherein
The culture medium supply mechanism of the first culture environment includes a manipulator capable of supplying and exchanging the culture medium to the upper part of the substance permeable membrane of one or a plurality of the cell culture containers. apparatus. The automatic culture apparatus according to claim 10, wherein
An automatic culture apparatus further comprising a door between the first culture environment and the second culture environment and an intermediate chamber for storing the door when the door is opened. The automatic culture apparatus according to claim 11, wherein
The second culture environment is:
An automatic culture apparatus, characterized in that a culture supply system comprising a refrigerated storage unit for storing the culture medium to be supplied to the cell culture container and a culture medium feeding mechanism for feeding the culture medium can be installed. A medium supply method for an automatic culture apparatus for culturing cells,
One or a plurality of cell culture vessels having a substance permeable membrane on its bottom surface can be held, a medium injection region for injecting a medium, the cell culture vessel being held, and a medium being supplied from the lower part of the substance permeable membrane And a culture area that can be exchanged, a culture medium discharge area for discharging the culture medium, and an inlet wall in which one or a plurality of injection holes are formed for supplying the culture medium from the culture medium injection area to the culture area; , Using a culture vessel configured to discharge the medium from the culture region and having a discharge port wall formed with one or a plurality of discharge holes,
The culture container is installed in a first culture environment having a culture medium supply mechanism for supplying and replacing the culture medium to the culture container, and an adjustment mechanism for maintaining an environment in which cells in the culture container can be cultured,
From the culture medium supply system of the second culture environment in which a culture medium supply system that supplies a culture medium to the cell culture container is maintained, the culture container installed in the first culture environment A medium supply method comprising injecting the medium into a medium injection region. The medium supply method according to claim 14, wherein
The medium is supplied from the medium supply system in the second culture environment to the medium supply mechanism in the first culture environment, and the top of the substance permeable membrane of the cell culture container is supplied by the recognition supply mechanism, and A medium supply method comprising supplying the medium to a lower part of the substance permeable membrane.

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