JP2010104299A - Automatic culture apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To sufficiently sterilize a culture space in a cartridge-type closed culture vessel, to control culture conditions, etc., by accurately analyzing medium components, and to efficiently supply a fresh medium to the culture space of the cartridge-type closed culture vessel. <P>SOLUTION: The automatic culture apparatus is provided with a first flow path connected to a vicinity of one end of the cartridge-type closed culture vessel and acting as a supplying or discharging flow path for a gas and/or liquid to the culture space, a second flow path connected to a vicinity of the other end of the cartridge-type closed culture vessel and acting as a supplying or discharging flow path for the gas and/or liquid, a third flow path connected to a vicinity of the connection position of the first flow path to the cartridge-type closed culture vessel and acting as a supplying or discharging flow path for the gas and/or liquid, a medium component analyzing apparatus connected to the third flow path and analyzing components in the culture liquid, and a switching means to connect either one of the first flow path and the third flow path to the culture space of the cartridge-type closed culture vessel. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a culture apparatus capable of culturing cells or tissues using a detachable cartridge type closed culture vessel, for example, a culture apparatus capable of producing a regenerative tissue usable for regenerative medicine.

  The production of regenerative tissue used for transplantation as regenerative medical treatment is based on GMP (Good Manufacturing Practice), which is the standard for manufacturing management and quality control of pharmaceuticals, etc. CPC (Cell Processing) that provides a clean manufacturing environment Manufactured in accordance with SOP (Standard Operational Procedure) by a person with specialized cell culture technology. In Japan, relevant laws and regulations have already been enforced by the Ministry of Health, Labor and Welfare in Japan (for example, Ministry of Health and Welfare Order No. 179, Yakuhin No. 480). Outside Japan, relevant laws and regulations have been issued mainly by Western institutions (eg, US Food and Drug Administration, European Commission).

  Regenerative tissue is manufactured by manufacturing personnel with specialized cell culture technology in an environment where safety and cleanliness are strictly controlled as defined by these laws and regulations. Operational costs are incurred. In addition, since all manufacturing processes are performed manually, there is a limit to the amount of regenerative tissue manufactured. As a result, the manufacturing cost for manufacturing the regenerative tissue is high, which hinders the spread of regenerative medical treatment. Therefore, in order to overcome this situation, there is a need to introduce an automatic culture apparatus that automates part or all of the culture process. By carrying out the culturing process with an automatic culturing device rather than manually, labor saving and cost reduction can be achieved, and mass production becomes possible. In addition, unlike the manual operation, the operation by the automatic culture apparatus is always performed uniformly, so that it is expected to contribute to the constant quality of the regenerated tissue obtained after the production. Here, the automatic culture apparatus cultures cells as an alternative to manual labor, but it is considered essential to satisfy GMP as in the manual manufacturing process. That is, it must be shown that a high-quality regenerated tissue can be produced in a state where the automatic culture apparatus maintains cleanliness based on scientific evidence.

  In order to maintain cleanliness, it is necessary to ensure that the inside of the culture vessel in which cells are directly seeded and cultured in the culture process is not biologically contaminated. Therefore, it is essential to sterilize the culture container in advance and use it after eliminating the possibility of biological contamination. By this sterilization operation, only the seeded cells are cultured in the culture container in the culturing process, and it is possible to avoid the occurrence of biological contamination in which undesirable organisms such as accidentally mixed bacteria and bacteria grow.

  By the way, a culture vessel used for an automatic culture apparatus is an open culture vessel (for example, a culture vessel having a lid) that is easy to open and close and has a large contact area with the outside, and a closed that is not easy to open and close and has a small contact area with the outside. Mainly classified into system culture containers (for example, cartridge-type culture containers (Patent Documents 1, 2, and 3)). Culture techniques using open culture vessels have already been established, and various types are commercially available. It is commonly used for manual culture, from the research and development stage to the production and sales stage of regenerated tissue. However, the culture medium is easily spilled and has a large contact area with the outside world, which causes biological contamination. Therefore, specialized culture techniques are required to culture while maintaining cleanliness. An automatic culture apparatus having a drive device such as a robot arm, a transfer robot and the like for the open culture container has already been reported.

On the other hand, closed culture vessels are positioned as next-generation culture vessels that are assumed to be used in automatic culture apparatuses, and are being developed in various research institutions together with automatic culture apparatuses. Although a culture technique using a closed culture vessel and an automatic culture apparatus has not been sufficiently established, some reports have already been made (Patent Documents 2 and 4). The closed culture container generally has a structure in which the medium is not easily spilled and has a small contact area with the outside world, and thus has an advantage that it is easier to maintain cleanliness than the open culture container. As an example of an automatic culture device using a closed culture vessel, an automatic culture device that uses a cartridge-type closed culture vessel and is connected to the automatic culture device via a crimping joint to perform cell seeding, medium exchange, etc. Have been reported (Patent Documents 1 and 5). This cartridge type closed culture vessel has a valve structure, and exchanges such as inflow and outflow of the culture medium are performed only through this. In addition, the culture tank of the cartridge-type closed culture vessel exchanges gas such as O ₂ and CO ₂ through the gas exchange membrane to the outside, and the efficiency of gas inflow and outflow is lower than that of the open culture vessel. descend. In addition, as with a general culture container, it is essential to perform a sterilization operation before use in the culture process to eliminate the possibility of biological contamination. Furthermore, it is essential to conduct a leak test to confirm that the medium is assembled in a state where airtightness is sufficiently ensured without leaking.

  Conventionally used sterilization methods include a method of sterilization by exposure to high temperature (for example, dry heat sterilization is about 180 ° C. and high temperature steam sterilization is about 120 ° C.). However, in general, the culture vessel is often made of a high molecular compound such as polycarbonate, polystyrene, or polyethylene having cell adhesion. Therefore, when exposed to high temperatures by the conventional methods mentioned here, the culture vessel is deformed and cannot be used for culture. As another method, there is a sterilization method using a sterilization gas (for example, ethylene oxidite gas, hydrogen peroxide gas, ozone gas). Although it is not necessary to expose to high temperature, it is necessary for the sterilization gas to spread throughout the culture vessel. It is effective if the structure allows the gas to flow into and out of the culture vessel efficiently, but otherwise the sterilization efficiency decreases. In the case of a cartridge type closed culture vessel, it is difficult to fill the inside with the sterilization gas even if it is exposed to the sterilization gas from the outside. As another sterilization method for closed culture vessels, a method has been reported in which all components are sterilized in advance and assembled in a clean environment (Patent Document 2). However, the assembly process is generally complicated and requires special equipment. As a result, a clean environment with a large space is required. This leads to an increase in production costs. Further, after the assembly, it is necessary to conduct a leak test to confirm whether the closed culture container is assembled normally and has no defects such as a leak. When equipment for carrying out this leak test is introduced, a larger space is required. As described above, it is necessary to improve the sterilization of the inside of the cartridge-type closed culture vessel using the conventional method and the leakage test in consideration of economy.

  By the way, in order for the automatic culture apparatus to be able to produce a high-quality regenerated tissue, it is necessary to exchange the medium efficiently during the culture process. The medium is a liquid that provides various nutrient components necessary for cell growth, maintains the pH in the solution, and provides an environment necessary for culture. During the culturing process, the medium is periodically changed according to the state of the cells. When exchanging the medium, it is necessary to completely replace the old medium with the new medium. When the old medium is mixed, the concentration of various nutrients contained in the newly supplied medium changes and affects the metabolism of the cells. As a result, the quality of the regenerated tissue obtained after production can be made non-uniform. This means that the culture protocol for producing regenerative tissue is not uniform. Thus, the medium change needs to be replaced completely and efficiently from the old medium to the new medium.

  When using an open culture container, many of them have a structure with a lid, so when replacing the medium, open the lid of the open culture container, collect all the old medium, and wash it (eg PBS (Phosphate Buffered Salts) In general, the solution is washed several times with a solution, and then a new medium is added. For this reason, the ratio of the old medium mixed with the new medium after the medium replacement becomes very small. However, when a closed culture vessel is used, the culture medium must be changed by an operation different from that of the open culture vessel because the lid is not structured. A closed culture vessel generally has a flow path (hereinafter referred to as a supply flow path) through which a new culture medium is supplied from the outside and a flow path (hereinafter referred to as a discharge flow path) through which an old culture medium is discharged simultaneously. It is a connected structure. When the medium is replaced, a new medium is supplied from the supply flow path, and a continuous medium in which excess liquid is discharged from the discharge flow path after the new medium and the old medium are mixed inside the culture vessel. The exchange method is often taken (Patent Documents 5 and 6). Although the ratio of the old culture medium is large in the liquid discharged from the discharge channel, a new culture medium and an old culture medium are mixed in the culture vessel. In order to completely replace the inside of the culture container with the new medium, the proportion of the old medium is reduced according to the amount to be flowed, and therefore it is necessary to flow a sufficient amount of the new medium. In other words, it is inefficient to completely replace the culture medium in the culture vessel with a new culture medium by the method of continuous replacement.

  On the other hand, in order for the automatic culture apparatus to be able to produce a high-quality regenerated tissue, it is also necessary to accurately grasp the cell culture state during the culture process. Cells seeded in the culture vessel have biological quality such as growth rate and metabolism depending on the quality of the collected cells (eg, the state at the time of collection, the nature of the donor who provided the cells (age, history, etc.)). Different. Although the operation can be made uniform by an automatic culture apparatus without human intervention, the growth situation of the cells varies depending on the quality of the collected cells. Therefore, as a culture process control, if the behavior of cells is evaluated at each time point during the culture process, and the culture protocol suitable for the state of the cells at that time is not corrected according to the result, the regenerated tissue obtained after production It is difficult to make the quality constant. The cell state measurement method is preferably a non-invasive method that does not affect cells and tissues when the obtained regenerated tissue is assumed to be used for regenerative medical treatment.

  As a method for non-invasively evaluating cells in culture, for example, evaluation of cell proliferation and cell shape over time by microscopic observation can be mentioned, but medium component analysis is known as another effective method. By monitoring various components of the medium (for example, glucose, lactic acid, and ammonium ions), the growth status of the cells can be grasped sequentially. When cells are undergoing normal metabolic activity, glycolysis consumes glucose and produces lactic acid. As the number of cells increases with the progress of the culture process and the metabolism becomes active, the consumption of glucose and the output of lactic acid increase. When the medium component analysis is performed, for example, in the production of an antibody drug, a medium of about several mL is collected and used for analysis from within a large volume of about several hundred L if necessary. Since the volume of the culture medium being cultured is sufficiently large relative to the amount collected for analysis, the operation of collecting hardly affects the culture process. On the other hand, in the case of production of regenerative tissue in regenerative medicine, the amount of medium used for culturing is about several mL to several tens mL, which is smaller than that of antibody drug production. When a medium of several milliliters is collected for medium component analysis from here, it is an amount that occupies a large proportion of the volume of the medium used for culture, so it is necessary to replenish a new medium with a volume equal to the collected amount. Arise. This is equivalent to a part of the medium being replaced, and the concentration of the medium components changes during the culture process, affecting the metabolism of the cells and consequently preventing the homogenization of the regenerated tissue after culture. .

JP 2006-320304 A JP 2008-113600 A JP 2002-153261 A JP 2004-089138 A JP 2006-149237 A JP 2004-208664 A

  As described above, in the culture apparatus that cultures cells and tissues using a cartridge type closed culture vessel, there is a problem that the culture space in the cartridge type closed culture vessel cannot be sufficiently sterilized. In addition, the culture apparatus has a problem that the medium components cannot be accurately analyzed, and the culture conditions cannot be controlled using the analysis results. Furthermore, the culture apparatus has a problem that a new medium cannot be efficiently supplied to the culture space in the cartridge type closed culture vessel.

  The culture apparatus according to the present invention that achieves the above-described object is provided with a sterilization chamber that can accommodate a cartridge type closed culture vessel having a culture space therein, and is disposed in the sterilization chamber, and is crimped to the cartridge type closed culture vessel. One end connected to a coupling part that can be connected in a state of being connected to the culture space of the cartridge type closed culture vessel, and the cartridge type closed culture vessel and the joint. With the other end connected to the inside of the cartridge type closed culture vessel and a trap for collecting gas and / or liquid supplied to the culture space of the cartridge type closed culture vessel. One end is connected to the collecting device, and the other end faces the inside of the cartridge-type closed culture vessel in a state where the cartridge-type closed culture vessel and the joint are connected. It is those with a decane.

  According to the culture apparatus configured as described above, the inside of the culture space can be sterilized by introducing a sterilization gas through the introduction tube in a state where the cartridge type closed culture vessel and the joint portion are connected. Can do. Further, according to the culture apparatus of the present invention, the sterilization chamber (cartridge type) is introduced by introducing sterilization gas through the introduction pipe in a state where the cartridge type closed culture vessel and the joint part are separated from each other. The outside of the closed culture vessel) can be filled with sterile gas. As described above, in the culture device according to the present invention, prior to culturing cells or tissues using a cartridge-type closed culture vessel, the culture space in the cartridge-type closed culture vessel and the outside of the cartridge-type closed culture vessel Can be reliably sterilized.

  In addition, the culture apparatus according to the present invention that achieves the above-described object can be connected to the vicinity of one end of a cartridge-type closed culture container having a culture space therein, and the culture of the cartridge-type closed culture container A first flow path serving as a flow path for supplying and discharging gas and / or liquid to and from the space can be connected to the vicinity of the other end of the cartridge type closed culture vessel. The cartridge can be connected in the vicinity of the position where the second flow path serving as a flow path for supplying and discharging gas and / or liquid to the culture space and the first flow path in the cartridge-type closed culture vessel are connected. A third flow path serving as a flow path for supplying and discharging gas and / or liquid to and from the culture space of the mold-closed culture vessel, and connected to the third flow path, A media component analyzer capable of analyzing, in which and a switching means for communicating to said first flow path and either the culture space of the cartridge-type closed system culture vessel of the third flow path.

  In the culture device configured as described above, the medium filled in the culture space in the cartridge-type closed culture vessel can be supplied to the component analyzer through the third flow path. According to the culture apparatus according to the present invention, the medium components can be analyzed with high accuracy, and the culture conditions and the like can be controlled using the analysis results.

  Furthermore, the culture apparatus according to the present invention that achieves the above-described object includes a joint portion that can be connected in a crimped state to a cartridge-type closed culture vessel having a culture space therein, the cartridge-type closed culture vessel, and the joint portion. Are connected to one end portion of the cartridge type closed culture vessel, and a flow path for supplying and discharging gas and / or liquid to the culture space of the cartridge type closed culture vessel. In a state where the first flow path, the cartridge-type closed culture vessel, and the joint portion are connected to each other, the cartridge-type closed culture vessel can be connected to the vicinity of the other end of the cartridge-type closed culture vessel. A second flow path serving as a flow path for supplying or discharging gas and / or liquid to / from the culture space of the system culture container, the joint portion, and the cartridge type closed system A driving device that drives the feeding container so as to be able to contact and separate, and that can drive the joint portion from the substantially horizontal direction to the substantially vertical direction connecting the first flow path and the second flow path. It is. In addition, it is preferable that the direction which connects the said 1st flow path and the said 2nd flow path drives the said joint part from a horizontal direction to a perpendicular direction.

  In the culture apparatus according to the present invention configured as described above, the joint portion is connected to the first flow channel and the second flow channel in a state where the joint portion and the cartridge type closed culture vessel are connected. The direction is a substantially vertical direction, and the first channel is at a first position where the first channel is downward. At the first position, the washing solution or the culture solution is passed from the first channel in the culture space of the cartridge type closed culture vessel. In addition, the washing solution or the culture solution filled in the culture space of the cartridge type closed culture vessel can be discharged from the first channel. The culture apparatus according to the present invention can efficiently supply a liquid such as a new culture medium or a washing solution to the culture space in the cartridge type closed culture vessel.

  The culture apparatus according to the present invention can reliably sterilize the inside of the culture space of the cartridge type closed culture container prior to culturing cells or tissues using the cartridge type closed culture container. Therefore, the culture apparatus according to the present invention can provide a culture environment suitable for culturing cells and tissues that require extremely high cleanliness.

  Moreover, according to the culture apparatus which concerns on this invention, a component analysis can be carried out with high precision about the culture medium with which the culture space of the cartridge type closed system culture container was filled. Therefore, the culture apparatus according to the present invention can optimally control the medium conditions and the like based on the result of the component analysis of the medium, and can provide an optimal culture environment for the cells and tissues to be cultured.

  Furthermore, according to the culture apparatus according to the present invention, liquids such as a culture medium and a washing liquid can be reliably supplied to the culture space of the cartridge type closed culture vessel, and a process such as medium exchange and washing can be performed efficiently. . Therefore, with the culture device according to the present invention, the culture process of cells and tissues can be performed more easily and efficiently.

  Hereinafter, the culture apparatus according to the present invention will be described in detail with reference to the drawings. FIG. 1 is an overall view of an element part for sterilizing a cartridge type closed culture vessel inside an automatic culture apparatus before a culture process and further performing a leak test. FIG. 2 is an exploded perspective view showing a state in which the cartridge type closed culture vessel and the joint are connected in the sterilization chamber. FIG. 3 shows the first stage and the second stage when sterilizing gas or clean air is introduced in the process of sterilizing the cartridge type closed culture vessel. The first stage also corresponds to a state when performing a leak test. FIG. 4 is an overall view of a flow path for performing medium exchange and medium component analysis inside the automatic culture apparatus. FIG. 5 is an exploded perspective view showing a state where the joint portion and each flow path are connected when performing medium exchange and medium component analysis. FIG. 6 shows a process of performing medium exchange and medium component analysis using a state in which the cartridge type closed culture vessel is connected to the joint portion and each flow path. FIG. 7 is a diagram for evaluating the efficiency of medium exchange and the accuracy in medium component analysis using glucose and lactic acid as indices for the conventional method of continuously exchanging the medium. FIG. 8 is a diagram for evaluating the efficiency of medium exchange and the accuracy in medium component analysis using glucose and lactic acid as an index in the method for stepwise medium exchange according to the present invention. FIG. 9 shows the results of culturing rabbit-derived corneal epithelial cells for 10 days as an example, and conducting medium component analysis for glucose and lactic acid. FIG. 10 shows a phase contrast microscopic image and a histological evaluation result of a corneal epithelial cell sheet which is a regenerated tissue finally obtained after 10 days of culture for the same sample as FIG. is there. FIG. 11 is a flowchart showing the instruction contents for correcting the culture process based on the results of medium component analysis. FIG. 12 shows a screen example of the information terminal that gives the instruction content according to the flowchart of FIG. FIG. 13 is a top view showing an entire automatic culture apparatus including an element unit for sterilizing a cartridge-type closed culture vessel in advance and an element unit for performing medium exchange and medium component analysis.

<< First Embodiment >>
An example of a culture apparatus to which the present invention is applied and which sterilizes a cartridge type closed culture container before the culture process and enables a leak test will be described with reference to FIGS. FIG. 1 schematically shows the configuration of the culture apparatus, FIG. 2 schematically shows an exploded perspective view of the cartridge type closed culture vessel and the joint in the culture apparatus, and FIG. 3 shows the culture apparatus. The sterilization process in is shown.

  In the culture apparatus according to the present invention, a cartridge-type closed culture vessel 2 is disposed inside the sterilization chamber 1. In this example, the entire cartridge-type culture vessel has a substantially rectangular parallelepiped shape, but may have any shape such as a substantially cylindrical shape. In the cartridge type closed culture vessel 2, the culture tank 3 as a culture space has a substantially cylindrical shape, and the upper surface and the bottom surface of the cylindrical shape are formed by gas permeable membranes 4 and 5 through which gas can permeate from the outside. ing. The gas permeable membranes 4 and 5 are fixed to the cartridge type closed culture container member 10 with an adhesive or the like. The material of the gas permeable membranes 4 and 5 is, for example, polycarbonate or silicone polycarbonate. Although the gas permeable membranes 4 and 5 have a lower gas exchangeability than a general open culture vessel, oxygen and carbon dioxide can flow in and out through the outside and inside during culturing. .

  In this example, the culture tank 3 has a substantially cylindrical culture space. In addition, the shape of the culture tank used as culture | cultivation space is not limited to a substantially cylindrical shape, What kind of shapes, such as a substantially rectangular parallelepiped shape, may be sufficient. The culture tank 3 is provided with a flow path 6 and a flow path 7 that allow introduction and discharge of gas and / or liquid from the outside. These flow paths 6 and 7 are formed at positions facing each other, and communicate with one end face of the cartridge type closed culture container member 10. One of these flow paths 8 and 9 is used for introducing gas and / or liquid from the outside, and the other is used for outflow of gas and / or liquid from the outside. Further, the cartridge type closed culture vessel is provided with a valve 8 and a valve 9 that can block or communicate the culture tank 3 and the outside at positions where the flow paths 6 and 7 communicate with the outside, respectively. The valves 8 and 9 are provided with cross cuts for penetrating the tubular member. These valves 8 and 9 are fitted into holes 11 and 12 formed in the cartridge type closed culture container member 10 and fixed by means such as an adhesive. The material of these valves 8 and 9 is a viscoelastic member such as silicone resin. The depth of the holes 11 and 12 and the height of the valves 8 and 9 are designed to be equal. Therefore, after incorporating the valves 8 and 9 into the holes 11 and 12, one end surface of the cartridge type closed culture container member 10 is a flat surface without any irregularities.

  The joint portion 13 includes an elastic seal 14 and a rectangular parallelepiped joint member 15. The material of the elastic seal 14 is a viscoelastic member such as silicone resin, for example, and is fixed to one end face of the joint member 15 by means such as an adhesive. The elastic seal 14 is formed with ring-shaped fine protrusions 16 and 17 at positions facing the valves 8 and 9 of the cartridge type closed culture vessel 2. When connecting the cartridge type closed culture vessel 2 and the joint portion 13, the pressure is particularly strongly pressed between the valves 8 and 9 and the ring-shaped fine protrusions 16 and 17. Further, in a state where the cartridge type closed culture vessel 2 and the joint portion 13 are connected, the position of the pressure contact surface by the valves 8 and 9 and the ring-shaped fine protrusions 16 and 17 is from one end surface of the cartridge type closed culture vessel member 10. It will be a place that gets inside. Therefore, one end surface of the cartridge type closed culture vessel member 10 and the surface excluding the ring-shaped fine protrusions 16 and 17 of the elastic seal 14 are efficiently and strongly pressure-bonded, resulting in leakage of gas or liquid. Can be suppressed.

The joint 13 is formed with a hole through which the introduction pipe 18 and the discharge pipe 19 pass. One end of the introduction tube 18 is connected to the introduction device 20. A pump 21 is disposed in the middle of the introduction pipe 18. The introduction device 20 includes a sterilization gas (for example, ethylene oxidite gas, hydrogen peroxide gas, ozone gas), clean air (for example, 3500 particles / m ³ or less having a diameter of 0.5 μm or more passed through a HEPA filter, a diameter A device capable of introducing clean air having a particle number of 5 μm or more of less than 1 / m ³ ) and sterilized water (for example, clean water previously sterilized by an autoclave and free of impurities by an ion exchange membrane) It is. Further, one end of the discharge pipe 19 is connected to the collection device 22. A pump 23 is disposed in the middle of the discharge pipe 19. The collection device 22 can collect the gas and liquid discharged through the discharge pipe 19, and can measure the volume particularly in collecting the gas. The introduction pipe 18 and the discharge pipe 19 pass through the joint member 15, the elastic seal 14, and the valves 8 and 9 of the cartridge type closed culture container, respectively, in a state where the cartridge type closed culture container 2 and the joint part 13 are connected. The tip reaches the flow paths 6 and 7.

  Further, the joint portion 13 is connected to an operating shaft 25 via an arm 24. With this as a reference, the joint portion 13 can be driven to rotate and vertically. Therefore, the cartridge type closed culture vessel 2 and the joint portion 13 can be connected or disconnected.

  In the culture apparatus of this example, the culture tank 3 is configured as a single space, but the culture tank 3 may have a two-layer structure, for example, a cell culture insert that is a kind of an open culture container. . In this case, for example, cell types that are cultured by co-culture with feeder cells that play a role of providing nutrients during culture, such as corneal epithelial cells and oral mucosal cells, can be cultured while being spatially separated. When such cells are targeted, a two-layer structure is preferable, and in that case, it is necessary to prepare a flow path, a valve, an introduction pipe, a discharge pipe, and the like for another tank.

  A process of sterilizing the cartridge type closed culture vessel 2 in the culture apparatus configured as described above will be described. In FIG. 3, the shaded area indicates a portion where sterilization gas is introduced and filled. As shown in FIG. 3A, first, sterilized gas is introduced into the culture tank 3 from the introduction device 20 through the introduction tube 18 in a state where the cartridge type closed culture vessel 2 and the joint portion 13 are connected. . The sterilizing gas supplies an excess amount (for example, twice the volume ratio) than the total volume of the culture tank 3 and the volumes of the flow paths 6 and 7. Thereby, the inside of the culture tank 3 and the flow paths 6 and 7 in the cartridge type closed culture vessel can be almost completely filled with the sterilization gas. Excess sterilized gas passes through the discharge pipe 23 from the flow path 7 and is collected in the collection container 22. As described above, after maintaining the state in which the sterilization gas is almost completely filled in the culture tank 3 and the flow paths 6 and 7 in the cartridge type closed culture vessel for a predetermined time, the joint 13 is connected to the cartridge type with the operation shaft 25 as a reference. Separated from the closed culture vessel 2. Next, as shown in FIG. 3B, sterilization gas is supplied again from the introduction device 20 through the introduction tube 18 in a state where the cartridge type closed culture vessel 2 and the joint portion 13 are separated from each other. The chamber 1 can be filled with sterilization gas. That is, according to this process, the outside of the cartridge type closed culture vessel 2 can be filled with sterilization gas. By introducing the above two-stage sterilization gas, the sterilization gas is filled into the inside of the sterilization chamber 1 inside the culture tank 3, the flow paths 6 and 7, and the outside of the cartridge type closed culture vessel 2 for a predetermined time. The sterilization process is completed by maintaining the state.

  Next, when removing the sterilizing gas, the cartridge type closed culture vessel 2 and the joint part 13 as shown in FIG. 3A are connected to each other, and the introduction device 20 passes through the introduction tube 18. Clean air is introduced into the culture tank 3. Then, clean air is introduced in the same manner with the cartridge type closed culture vessel 2 and the joint 13 as shown in FIG. By repeating the introduction of the washing air a plurality of times, the sterilization gas filled in the sterilization chamber 1 inside the culture tank 3, the flow paths 6 and 7, and the outside of the cartridge type closed culture vessel 2 is removed. Can do.

  Thereafter, a leak test is performed. In the leak test, the cartridge-type closed culture vessel 2 and the joint portion 13 as shown in FIG. 3 (A) are connected to each other, and sterilized water is introduced in the same manner as sterilization gas and cleaning air. The cartridge type closed culture vessel 2 is visually checked for leakage to the outside when the inside is filled with sterilized water. When it is confirmed by a leak test that there is no problem in the performance of the cartridge type closed culture vessel, sterilized water is discharged from the discharge pipe 19 by introducing clean air heated to a high temperature of 30 to 100 ° C. Air-dry until sterilized water does not remain inside or outside the mold-closed culture vessel 2.

  Through the above steps, the inside of the cartridge-type closed culture vessel 2 and the sterilization chamber 1 can be sterilized and airtightness without leakage of gas or liquid from the culture tank 3 can be confirmed. Judge that it can be used in the process. If the leak test reveals that the cartridge type closed culture vessel leaks and there is a problem with performance, discard the cartridge type closed culture vessel that had the problem, prepare a new one, and start again from the sterilization process. It is desirable to carry out again.

  According to the present culture apparatus, the above-described sterilization process ensures that organisms that cause biological contamination such as fungi and bacteria that are supposed to adhere to the outer periphery of the cartridge type closed culture vessel 2 and the inside of the culture tank 3 are reliably obtained. Can be removed. As described above, the whole can be efficiently sterilized with respect to the cartridge type closed culture vessel in which the sterilization gas is difficult to enter into the inside by the sterilization method normally used because of the closed system structure.

<< Second Embodiment >>
An example of a culture apparatus to which the present invention is applied and which enables medium exchange and medium component analysis will be described with reference to FIGS. FIG. 4 schematically shows a configuration around a cartridge type closed culture vessel, FIG. 5 schematically shows an exploded perspective view of a joint portion in the culture apparatus, and FIG. 6 shows a culture medium in the culture apparatus. The exchange process is shown. In the culture apparatus shown in this example, the same components as those described in the first embodiment described above may be denoted by the same reference numerals and detailed description thereof may be omitted.

  The culture apparatus shown in this example includes a first flow path 30, a second flow path 31, and a third flow path 32 as shown in FIGS. The tip of the second channel 31 faces the inside of the channel 6 in a state where the cartridge type closed culture vessel 2 and the joint 13 are connected. The first flow path 30 and the third flow path 32 are connected via a flow path switching unit 33 and face the inside of the flow path 7 in a state where the cartridge type closed culture vessel 2 and the joint portion 13 are connected.

Supply pumps 34, 35, and 36 are connected to midway portions of the first flow path 30, the second flow path 31, and the third flow path 32, respectively. These supply pumps 34, 35, and 36 can flow gas and liquid in either direction through the first flow path 30, the second flow path 31, and the third flow path 32, respectively. The starting end of the first flow path 30 is connected to the culture medium tank 38, the cleaning liquid tank 39, the clean air tank 40, and the drainage tank 41 via the flow path switching unit 37. The clean air tank 40 is a tank that can supply clean air that has passed from the outside through a high-efficiency particulate air (HEPA) filter from the outside, and can be heated to about 30 to 100 ° C. as necessary. Further, this clean air has a cleanness such that the number of particles having a diameter of 0.5 μm or more is 3500 / m ³ or less and the number of particles having a diameter of 5 μm or more is less than 1 / m ³ . On the other hand, the second flow path 31 is connected to the clean air tank 43 and the drainage tank 44 via the flow path switching unit 42. As with the clean air tank 40, the clean air tank 43 can supply clean air heated to about 30 to 100 ° C. Further, the third flow path 32 is connected to the culture medium component analyzer 45.

In the culture apparatus of this example, the flow path switching unit 33 is incorporated in the joint member 15 at a lower position when the joint portion 13 is set up in the vertical direction when performing medium exchange and medium component analysis. Yes. The flow path switching unit 33 includes an input port 50, a disk-shaped valve 51, and an output port 52. The input port 50 has two flow path coupling portions 53, one connected to the first flow path 30 and the other connected to the third flow path 32. The disk-like valve 51 is formed with only one channel selection channel 54. The disk-like valve 51 can be rotated in the circumferential direction by remote operation from the outside. The output port 52 includes a flow channel 55 having a size that covers a moving region of the flow channel selection flow channel 54 when the disk-like valve 51 rotates, and a tube-shaped flow channel 56 connected to the flow channel 55. Is formed. The tubular flow path 56 has a length that penetrates the joint member 15 and the elastic seal 14 when the flow path switching section 33 is incorporated in the joint section 13. Therefore, when the cartridge type closed culture vessel 2 and the joint part 13 are connected, the tip of the tubular flow channel 56 in the output port 52 reaches the flow channel 7 inside the cartridge type closed culture vessel. According to the flow path switching unit 33, when the disk-like valve 51 rotates, one of the first flow path 30 and the third flow path 32 coupled to the two flow path coupling sections 53 is connected to the flow path. It can be connected to the tubular flow path 56 in the output port 52 via the selection flow path 54.
Therefore, the medium or the like can flow between the first flow path or the third flow path and the cartridge type closed culture vessel 2 by the flow path switching unit 33. Note that a hole 57 for incorporating the flow path switching unit 33 is formed in the joint member 15.

  In the culture apparatus of the present example configured as described above, when performing medium exchange and medium component analysis, as shown in FIG. 6, the cartridge type closed culture vessel 2 and the joint part 13 are similar to the sterilization process. It is made in the state which connected. Further, at that time, the cartridge type closed culture vessel 2 and the joint portion 13 are fixed in a state where they are erected in the vertical direction.

  In FIGS. 6A, 6B, 6C, and 6D, the cartridge type closed culture vessel 2, the joint portion 13, and the respective flow paths are stationary at the same position. In addition, the direction indicated by the arrow in the figure indicates the direction in which the medium or the like flows. First, as shown in FIG. 6 (A), in medium replacement and medium component analysis, clean air is sent from the second flow path 31 to the inside of the culture tank 3 of the cartridge type closed culture vessel 2 to collect the old medium. Meanwhile, all the old medium is aspirated from the third flow path 32. This old medium is collected by the medium analyzer 45 and the medium component analysis is performed by the medium component analyzer 45. Subsequently, as shown in FIG. 6 (B), as the cleaning process in the cartridge type closed culture vessel 2, the second liquid is supplied to the inside of the cartridge type closed culture vessel 2 from the first flow path 30 while the second liquid is supplied. Clean air is sucked from the flow path 31 and is continued until the cleaning liquid is filled in the cartridge type closed culture vessel 2 (that is, the culture tank 3, the flow paths 6 and 7). Thereafter, as shown in FIG. 6C, all the cleaning liquid is collected from the first flow path 30 and sent to the drainage tank while supplying clean air from the second flow path 31 to the inside of the cartridge type closed culture vessel 2. Deliver liquid. The cleaning process consisting of FIGS. 6B and 6C is preferably repeated a plurality of times as necessary. Finally, as shown in FIG. 6D, as a step of supplying a new medium to the cartridge type closed culture vessel 2, a new medium is supplied from the first flow path 30 into the culture tank 3 of the cartridge type closed culture vessel. While supplying the liquid, clean air is sucked from the second flow path 31 and continued until the culture medium 3 of the cartridge type closed culture vessel 2 is filled with a new medium. The cartridge-type closed culture container 2 filled with a new medium is left in the culture chamber to culture the cells. Therefore, the cartridge-type closed culture container 2 is returned to the horizontal direction, removed from the joint portion 13, and cultured. Bring it to the culture section and leave it as it is to culture the cells. Meanwhile, as the cleaning of the flow path used for the medium exchange and the medium component analysis, the first flow path 30 and the second flow path 31 are directly connected by a separately prepared cleaning flow path (not shown). Clean air is sucked in from the second flow path 31 while sending the cleaning liquid, and then the clean air warmed to 30 to 100 ° C. is sent from the first flow path 30 to discharge the cleaning liquid and air-dry the inside. To do. Similarly, the second flow path 31 and the third flow path 32 are directly connected to each other by a cleaning flow path (not shown), and clean air is sucked from the second flow path 31 while sending the cleaning liquid from the third flow path 32. Subsequently, clean air heated to 30 to 100 ° C. is sent from the third flow path 32 to discharge the cleaning liquid and the like, and the inside is air-dried.

  The culture apparatus of the present example described above can remove the old medium and supply a new medium in a discontinuous manner. Therefore, when performing a medium component analysis using the collected old medium, a new medium is used. It is possible to measure a more accurate value without being influenced by. On the other hand, as in the prior art, for the purpose of medium component analysis at the same time as medium exchange in a closed culture vessel, when the new medium and the old medium are continuously replaced, the old medium is mixed with the new medium outside the culture container. It is discharged from the discharge channel. In this case, even if the medium component analysis is performed using the liquid collected from the discharge flow path, a concentration different from the original concentration of only the old medium is measured because the new medium is mixed. In addition, the concentration will change as new media infusion in the media change proceeds. This means that the analysis accuracy by medium component analysis is lowered.

  In addition, the culture apparatus of this example can use almost the entire amount of the old medium for medium component analysis. Therefore, in the culture apparatus of this example, since various kinds of medium components can be analyzed in the medium component analyzer 45, for example, a cell or tissue culture for the purpose of producing a regenerative tissue for regenerative medicine It can be said that it is particularly preferable.

  FIG. 7 shows the efficiency of medium exchange using the glucose and lactic acid concentrations as indices for the conventional method in which the medium is continuously exchanged as a comparative control of the method in which the collection of the old medium and the supply of the new medium are performed discontinuously. It is the figure which evaluated the precision by a culture-medium component analysis. The internal volume of the cartridge type closed culture vessel used for the experiment is 2 mL. The inside of the cartridge type closed culture vessel is preliminarily filled with solution A (lactic acid 30 mM, glucose 0 mM). Solution B (lactic acid 0 mM, glucose 25 mM) is continuously injected from the introduction channel connected to the cartridge type closed culture vessel as continuous medium exchange, and discharged on the opposite side of the cartridge type closed culture vessel. The liquid discharged from the flow path was collected in units of 0.5 mL. A total of 4 mL of solution B was injected. Thereafter, medium components were analyzed for all the collected fractions, and the concentrations of glucose and lactic acid were measured. This experiment simulates the process of exchanging medium by an automatic culture apparatus using a cartridge type closed culture vessel. Solution A assumes an old medium and Solution B assumes a new medium. Since lactic acid is contained in solution A at a concentration of 30 mM and solution B at a concentration of 0 mM, when the concentration of lactic acid becomes 0 mM by medium exchange, the liquid in the cartridge-type closed culture vessel is used with lactic acid as an index. Can all be replaced by solution B. Similarly, since glucose is contained in solution A at a concentration of 0 mM and solution B at a concentration of 25 mM, when the concentration of glucose reaches 25 mM due to medium replacement, glucose is used as an index for the cartridge-type closed culture vessel. It can be assumed that all of the liquid inside has been replaced with solution B. The concentration contained in solution A for lactic acid and the concentration contained in solution B for glucose were used as controls, respectively, and the amount contained in each fraction was calculated as a relative concentration and plotted. The number on the x-axis of the graph indicates the total amount of collected liquid. For example, in the case of the point plotted at x = 2.0, the result of analyzing the fraction of 0.5 mL discharged after completing the recovery to 1.5 mL from the discharge pipe is shown. From the graph, in the fraction from 0 mL to 0.5 mL first recovered from the discharge pipe, the deviation from the concentration of lactic acid in Solution A was 1.9%, but the deviation was small, but from 1.5 mL to 2.0 mL As for the fractions collected so far, a large shift of 75.7% occurred. When glucose was used as an index, a deviation of 79.3% occurred in the fraction collected from 1.5 mL to 2.0 mL. As described above, in the conventional method in which the medium is continuously exchanged, the liquid recovered from the discharge pipe has a deviation of 75% or more from the original concentration of the solution A assuming an old medium. It was found that the accuracy of component analysis was reduced. Further, from the graph, 5.7% of the lactic acid contained only in the solution A remains in the fraction collected from 2.5 mL to 3.0 mL, and 4% of the glucose contained only in the solution B. A deviation of 3% occurred. Lactic acid remained in 1.8% even in the fraction collected from 3.0 mL to 3.5 mL. As described above, in the conventional method in which the medium is continuously exchanged, even if a volume of 1.5 times or more of the internal volume is injected from the introduction tube, the solution A assuming the old medium remains and the solution B is completely present. It was found that the efficiency of the medium exchange decreased.

  On the other hand, FIG. 8 shows a method for performing the recovery of an old medium and the supply of a new medium in a discontinuous (stepwise) manner according to the present invention, using the glucose and lactic acid concentrations as indices as in FIG. It is the figure which evaluated the precision by a culture medium component analysis. Similar to the experiment shown in FIG. 7, the cartridge-type closed culture vessel used had an internal volume of 2 mL. The inside of the cartridge type closed culture vessel is preliminarily filled with solution A (lactic acid 30 mM, glucose 0 mM). From the introduction channel connected to the cartridge type closed culture vessel, all the liquid inside was first recovered as continuous medium exchange. Subsequently, a washing solution (lactic acid 0 mM, glucose 0 mM) was injected to fill the cartridge type closed culture vessel, and then all the liquid inside was collected. This operation was repeated again. Subsequently, solution B (lactic acid 0 mM, glucose 25 mM) was injected to fill the cartridge type closed culture vessel, and then all the liquid inside was collected. Medium component analysis was performed on each of the fractions obtained as described above. As in the experiment shown in FIG. 7, the concentration contained in solution A for lactic acid and the concentration contained in solution B for glucose were used as controls, and the amount contained in each fraction was calculated as a relative concentration, which was graphed. The same applies to the x axis. From the graph, the fraction collected from 0 mL to 2.0 mL initially collected showed a small deviation of 1.5% from the lactic acid concentration of Solution A. Regarding glucose, it was 0.0%. As described above, in the method of performing medium exchange in a stepwise manner according to the present invention, the liquid recovered from the discharge pipe has a deviation of only about several percent from the original concentration of the solution A assuming an old medium. Further, the recovered amount was 2.0 mL. Compared with the result of the method in which the medium is continuously changed as shown in FIG. 7 described above, the difference between the concentration of the solution A and the original concentration of only the fraction from 0 mL to 0.5 mL was not large. A fraction of 2.0 mL, which is 4 times, can be collected accurately. If the amount that can be recovered is large, it is possible to perform a plurality of analyzes in the medium component analysis or to further analyze the medium components other than glucose and lactic acid, and as a result, the medium component analysis can be performed with high accuracy. Further, regarding the fraction from 6.0 mL to 8.0 mL collected at the end after two washings, the deviation from the concentration of the solution B is 2.4% for glucose and 0.2% for lactic acid. 0%. As described above, in the method in which the medium is changed in stages, the solution A assuming the old medium hardly remains, and the solution B is almost completely replaced. That is, the efficiency as medium replacement was improved.

9 and 10 show the results of actually using the culture apparatus of this example, inoculating rabbit-derived corneal epithelial cells at a density of 1.0 × 10 ⁴ cells / cm ² and culturing them for 10 days. It was shown to. FIG. 9 is a graph showing the results of measuring glucose and lactic acid concentrations as a medium component analysis every day during the culturing process. FIG. 10 is a photograph showing the phase contrast microscopic image of the cells on the 10th day after culturing in the sample of FIG. 9 and the histological evaluation results by HE staining. In the experiment shown in FIGS. 9 and 10, sample A is a sample in which a good regenerated tissue was finally obtained. In the sample B, a good regenerated tissue was not finally obtained. In Sample A, the amount of glucose decreased as the number of culture days increased, that is, as the number of culture days increased, the overall metabolic rate increased and the glucose consumption increased. In addition, the amount of lactic acid in Sample A increased. Like glucose, as the number of days of culture increased, the overall metabolic amount increased and the amount of lactic acid produced increased. In addition, as shown in FIG. 10, the sample A has grown until the cells are sufficiently dense from the phase contrast microscopic image of the finally obtained regenerated tissue. According to the graph, it can be seen that the structure is layered to about three layers. On the other hand, both the amounts of glucose and lactic acid for sample B were not significantly reduced compared to sample A. Further, as shown in FIG. 10, in the sample B, the cells were not proliferated from the phase contrast microscopic image of the finally obtained regenerated tissue until the cells became sufficiently dense. Was about 0 to 1 layer. Sample A is a model as data from which a good regenerated tissue was finally obtained. On the other hand, the sample B is a model as data in which a good regenerated tissue was not finally obtained. In FIG. 9, the dotted line portion shows the eighth day of culture. At this point, Sample B consumed less glucose and produced less lactic acid compared to the data of Sample A, which finally yielded a good regenerated tissue. Actually, as shown in FIG. 10, the cell density of sample B was small when the culture was completed, and the thickness of the obtained regenerated tissue was thin, and a good regenerated tissue was not obtained. From the above results, by culturing cells using the culture apparatus of this example, medium component analysis can be performed with high accuracy at a point in the middle of the culturing process. By comparing with the obtained data, it was proved that it was possible to determine whether or not a sample in the middle of culture could be finally obtained as a good regenerated tissue.

  By the way, in the culture apparatus of this example as described above, it has become clear that the medium component analysis can be performed very accurately, and the state of cells and tissues during culture can be determined based on the analysis results. An instruction for correcting the culture protocol can be output to the manufacturer using the result of analyzing the old medium collected from the flow path 32 by the medium component analyzer 45. FIG. 11 shows this processing flow, and FIG. 12 shows a sample screen for instructions for correcting the culture protocol output according to the processing flow.

  The component analyzer 45 that performs the processing measures the amounts of glucose and lactic acid in the old medium collected in the third flow path 32. The component analyzer 45 compares the measurement result with data in which a good regenerated tissue was finally obtained in the past. When glucose consumption or lactic acid production is clearly small, it is thought that cell metabolism is low and the number of cell growth is small. Therefore, in order to improve the growth status of the cells, addition of growth factors (eg, EGF, insulin, cytokine, BMP, TGF-β, IGF, PDGF, VEGF, HGF) and replacement with a medium containing high-concentration serum (For example, if the normal medium has a serum concentration of 5%, the medium is replaced with a medium having a serum concentration of 10%.) Increase in the frequency of medium replacement (for example, it is usually changed once every two days for one day. The manufacturer is instructed to modify the culture protocol. When the amount of glucose consumed or the amount of lactic acid produced is clearly large compared with the data obtained in the past in which good regenerated tissues were finally obtained, it is considered that cell metabolism is active and the number of cells grown is large. If it is possible to change the date on which surgery is performed on patients scheduled to receive regenerative medicine treatment, change the date on which surgery is performed on patients scheduled to receive regenerative medical treatment, by accelerating the date and time for collection as regenerative tissue. Give instructions to the manufacturer. In addition, if it is not possible to change the date of surgery for patients scheduled for regenerative medicine treatment, the growth factor content is higher than that of the normal medium in order to suppress cell metabolism and reduce the cell growth rate. Change to medium that is low or removed, change to medium containing low-concentration serum (for example, if the medium has a serum concentration of 5%, change to medium with 1% serum), decrease medium replacement frequency ( For example, the manufacturer is instructed to modify the culture protocol, such as, for example, it is normally changed once every two days to be changed once every three days. Instructions to continue culturing under the same conditions without changing the culturing protocol when glucose consumption and lactic acid production are comparable compared to the data obtained in the last good regenerated tissue To the manufacturing workers. In accordance with the obtained instructions, the manufacturer makes modifications to the culture protocol such as changing the medium composition.

  Specifically, the sample screen shown in FIG. 12 is taken when the measurement results of glucose and lactic acid are obtained as a result of the medium component analysis performed on the eighth day of culture in the experiment shown in FIG. In the upper part of the sample screen, the time-dependent values of glucose and lactic acid obtained by the 8th day in this case are shown on the graph by the name of in-culture data. Since the data on the 8th day is the latest data, it is highlighted on the graph. Furthermore, all the data when the culture was obtained in the past and finally a good regenerated tissue was obtained is shown by the name of the reference data on the same graph for comparison. The lower part of the graph shows the relative value of the measurement results in this case with respect to the data on the 8th day of culture and the data for the same number of days of culture when the cultures were obtained in the past and finally a good regenerated tissue was obtained. Has been. The lower part of the sample screen shows instructions for the manufacturer to modify the culture protocol based on the results of comparison with the data obtained in the past when a good regenerated tissue was finally obtained. . The manufacturer will modify the culture protocol as necessary, referring to the data obtained from the measurement and the instructed contents.

  As described above, according to the culture apparatus of the present example, even if cells are purified, seeded and cultured in the same manner, cell growth such as cell growth tendency and degree of differentiation due to the difference in cell source quality Appropriate culture conditions can be set even when the states are variously different, and very high quality cells and tissues can be produced. For example, when corneal epithelial cells are collected from the corneal limbus and cultured, and a corneal epithelial cell sheet is produced as a regenerated tissue, the proportion of corneal epithelial stem cells contained in the corneal epithelial cells collected as a cell source, cell viability, cell There are differences in the cell sources collected in terms of numbers. It is very difficult to seed all the same conditions for various items to evaluate the state of the cell source. Even under the same conditions for some items, differences gradually occur during the culturing process, resulting in differences in the quality of the finally obtained regenerated tissue. According to the culturing apparatus of this example, the state of the cells can be sequentially grasped during the culturing process, and the appropriate culturing for the cells at the time when the regenerated tissue is finally predicted is being predicted. Since the protocol can be modified, a good and uniform regenerative tissue is finally obtained.

  The component analyzer 45 is not limited to one that measures the amounts of glucose and lactic acid contained in the medium, and may be one that measures the amounts of glutamic acid and ammonia. Further, the culture apparatus of this example may be applied to an analysis apparatus that measures not only the component analysis but also the number of cells, dissolved oxygen concentration, dissolved carbon dioxide concentration, and the like.

<< Third Embodiment >>
FIG. 13 shows an overall schematic configuration of the automatic culture apparatus to which the present invention is applied in combination with the first embodiment and the second embodiment described above. The automatic culture apparatus includes a culture container loading unit 60 for loading the cartridge type closed culture container 2 from the outside, a sterilization chamber 1 for sterilizing the cartridge type closed culture container 2, and a medium exchange chamber 61 for exchanging the medium. And a culture chamber 62 for cell culture.

  The sterilization chamber 1 is connected to an external introduction device 20 and a collection device 22, and the cartridge-type closed culture vessel 2 introduced from the outside is subjected to sterilization and leakage tests before being used in the culture process (No. 1). 1 embodiment). Thereafter, the cells are input from the cell input unit 63 as a single cell suspension in the medium. A pipette manipulator 64 for handling the cell suspension is arranged in the apparatus, and the cell suspension is loaded into the cell while the tip stored and stored in the tip storage unit 65 is attached to the tip. Aspirate from part 63. On the other hand, the cartridge type closed culture vessel 2 after the sterilization process is carried to the medium exchange chamber 61 by the handling manipulator 66. Here, the cell suspension collected in advance by the pipette manipulator 64 is seeded into the cartridge-type closed culture vessel 2 and then carried to the culture chamber 62. A turntable 67 is provided in the culture chamber 62, and a base 68 on which the cartridge type closed culture vessel 2 is allowed to stand at the time of culture is disposed thereon. In this embodiment, there are three bases, and three cartridge type closed culture volumes 2 can be cultured simultaneously in one culture. The entire culture chamber 62 is maintained in an environment suitable for culture (for example, a temperature of 37 ° C. and a humidity of 99.9%). When the medium needs to be exchanged, it is conveyed to the culture chamber 62 by the handling manipulator 66, and the medium exchange and the medium component analysis are simultaneously performed (see the second embodiment). The culture medium tank 38, the cleaning liquid tank 39, the clean air tank 40, the drainage tank 41, and the culture medium component analysis apparatus 45 necessary for the culture medium component analysis, which are necessary for the medium exchange, are arranged outside the automatic culture apparatus.

The whole block diagram of the element which performs one operation which sterilizes a cartridge type closed system culture container, showing one example of the present invention. FIG. 4 is an exploded perspective view showing a cartridge type closed culture container and a joint part in an element part for performing an operation of sterilizing the cartridge type closed culture container according to an embodiment of the present invention. 1 shows an embodiment of the present invention, and shows the state of the first stage and the second stage when introducing sterilized gas, clean air or sterilized water in a process of sterilizing a cartridge type closed culture vessel and conducting a leak test. The figure shown. The whole figure regarding the flow path which shows one Example of this invention and performs a culture medium exchange and a culture medium component analysis inside an automatic culture apparatus. The exploded perspective view which shows one Example of this invention and shows a mode that the coupling | joint part and each flow path are connected when performing culture medium exchange and culture medium component analysis. The figure which shows one Example of this invention, and showed the process of culture medium exchange and culture medium component analysis in the state which the cartridge type closed system culture container connected with the joint part and each flow path. The figure which shows one Example of this invention and evaluated the content rate of glucose and lactic acid of the solution collect | recovered when the culture medium was continuously replaced | exchanged with respect to the cartridge type closed system culture container. The figure which shows one Example of this invention and evaluated the content rate of glucose and lactic acid of the solution collect | recovered when the culture medium was replaced | exchanged in steps with respect to the cartridge type closed system culture container. The figure which shows one Example of this invention and evaluated the change of content with time of glucose and lactic acid with respect to the corneal epithelial cell which culture | cultivation ended. The figure which shows one Example of this invention and showed the phase-contrast microscope image of the regenerated tissue finally obtained with respect to the corneal epithelial cell which culture | cultivation ended, and the histological evaluation result. The figure which shows one Example of this invention, and showed the flowchart for evaluating the growth condition of a cell during a culture | cultivation process, and correcting a culture | cultivation protocol based on the evaluation result. The figure which shows one Example of this invention and which showed the example of a screen of the information terminal which gives instruction content to the manufacturing worker according to the flowchart for correcting a culture | cultivation protocol. BRIEF DESCRIPTION OF THE DRAWINGS The top view which showed one Example of this invention and showed the whole automatic culture apparatus using the cartridge type closed system culture container.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Sterilization chamber, 2 ... Cartridge type closed culture vessel, 3 ... Culture tank, 4 and 5 ... Gas permeable membrane, 6 and 7 ... Channel, 8 and 9 ... Valves, 10 ... cartridge type closed culture vessel members, 13 ... joints, 14 ... elastic seals, 15 ... joint members, 16 and 17 ... ring-shaped microprojections, 18 ... Introducing pipe, 19 ... discharge pipe, 25 ... operating axis, 30 ... first channel, 31 ... second channel, 32 ... third channel, 33 ... channel Switching unit, 50 ... input port, 51 ... disc-shaped valve, 52 ... output port, 53 ... channel connection unit, 54 ... channel for channel selection, 30 ... culture Container input unit, 61 ... medium exchange chamber, 62 ... culture chamber, 63 ... cell input unit, 64 ... pipette manipulator, 65 ... chip storage unit 66 ... handling manipulator, 67 ... turn table, 68 ... base

Claims (15)

A sterilization chamber in which a cartridge type closed culture vessel having a culture space inside can be stored;
A joint that is disposed in the sterilization chamber and that can be connected in a pressure-bonded state to the cartridge-type closed culture vessel;
One end is connected to a gas and / or liquid introduction device for supplying to the culture space of the cartridge type closed culture vessel, and the other end is connected to the cartridge type closed culture vessel and the joint. An introduction tube facing the inside of the cartridge type closed culture vessel,
A state in which one end is connected to a collection device for collecting gas and / or liquid supplied to the culture space of the cartridge type closed culture vessel, and the cartridge type closed culture vessel and the joint are connected And the other end of the cartridge-type closed culture vessel provided with a discharge pipe facing the inside,
A culture apparatus capable of culturing cells and / or tissues using the cartridge-type closed culture vessel attached in the sterilization chamber.   The culture apparatus according to claim 1, wherein the joint portion can be crimped to the cartridge type closed culture vessel.   The culture apparatus according to claim 1, wherein the introduction apparatus supplies sterilization gas, clean air, and sterilized water individually through the introduction pipe.   The culture apparatus according to claim 1, further comprising a drive device that drives the joint portion and the cartridge-type closed culture vessel so as to be able to contact and separate. Further comprising a control device for controlling the amount of gas and / or liquid supplied from the introduction device,
The control device supplies gas and / or liquid from the introduction device based on the volume of the sealed chamber, the volume of the culture space in the cartridge-type closed culture vessel and the volume in the introduction pipe and the discharge pipe. The culture apparatus according to claim 1, wherein the amount is controlled. The collection device measures the volume of the collected gas and / or liquid, and outputs the measurement value to the control device,
In addition to the volume of the sealed chamber, the volume of the culture space in the cartridge-type closed culture vessel and the volume in the tube of the introduction pipe and the discharge pipe, the control device includes the gas collected by the collection device and 6. The culture apparatus according to claim 5, wherein a supply amount of gas and / or liquid from the introduction apparatus is controlled using a volume value of the liquid.   4. The culture apparatus according to claim 3, further comprising a temperature control device for controlling the temperature of the cleaning air supplied to the culture space of the cartridge type closed culture vessel. The cartridge-type closed culture container having a culture space inside can be connected to the vicinity of one end, and serves as a flow path for supplying and discharging gas and / or liquid to the culture space of the cartridge-type closed culture container. A first flow path;
A second flow path that can be connected to the vicinity of the other end of the cartridge-type closed culture container, and serves as a flow path for supplying and discharging gas and / or liquid to the culture space of the cartridge-type closed culture container; ,
The cartridge type closed culture vessel can be connected in the vicinity of the position where the first flow channel is connected, and serves as a gas and / or liquid supply / discharge channel for the culture space of the cartridge type closed culture vessel. A third flow path;
A medium component analyzer connected to the third flow path and capable of analyzing the components in the culture solution;
Switching means for communicating any one of the first flow path and the third flow path to the culture space of the cartridge type closed culture vessel,
When the culture medium to be analyzed for components is discharged from the culture space of the cartridge type closed culture vessel, the switching means switches between the third flow path and the culture space of the cartridge type closed culture vessel. A culture device that can communicate.   The first flow path is connected via a flow path switching unit to a culture medium tank filled with a culture medium, a cleaning liquid tank filled with a cleaning liquid, a cleaning air tank filled with cleaning air, and a drainage tank capable of filling drainage. The culture apparatus according to claim 8, wherein   9. The culture according to claim 8, wherein the second flow path is connected to a clean air tank filled with clean air and a drainage tank capable of filling drainage via a flow path switching unit. apparatus. A sterilization chamber capable of storing the cartridge type closed culture vessel;
A fitting portion that is disposed in the sterilization chamber and that can be connected in a crimped state to the cartridge type closed culture vessel;
The culture apparatus according to claim 8, wherein the first flow path, the second flow path, and the third flow path are connected to the cartridge-type closed culture vessel via the joint portion.   The medium component analyzer measures the concentration of a component that serves as an index for determining the progress of cell or tissue culture, and the concentration measured for a specific component deviates from a preset reference range for the component. The culture apparatus according to claim 8, wherein a culture instruction of a modified protocol is output when A joint that can be connected in a pressure-bonded state to a cartridge-type closed culture vessel having a culture space inside;
In the state where the cartridge type closed culture vessel and the joint portion are connected, the cartridge type closed culture vessel can be connected to the vicinity of one end of the cartridge type closed culture vessel, and the gas to the culture space of the cartridge type closed culture vessel And / or a first flow path serving as a liquid supply or discharge flow path;
In a state where the cartridge type closed culture vessel and the joint are connected, the cartridge type closed culture vessel can be connected to the vicinity of the other end of the cartridge type closed culture vessel, and the gas to the culture space of the cartridge type closed culture vessel And / or a second flow path serving as a liquid supply or discharge flow path;
The joint portion and the cartridge-type closed culture vessel are driven so as to be able to contact and separate, and the direction connecting the first passage and the second passage is driven from a substantially horizontal direction to a substantially vertical direction. A drive device capable of
In a state where the joint portion and the cartridge type closed culture vessel are connected, a direction connecting the joint portion to the first flow path and the second flow path is a substantially vertical direction, and the first flow path is downward In the first position, a cleaning solution or a culture solution is supplied from the first flow path into the culture space of the cartridge type closed culture vessel and the culture space of the cartridge type closed culture vessel is set at the first position. A culture apparatus capable of discharging the cleaning liquid or culture liquid filled therein from the first flow path. The cartridge type closed culture vessel can be connected in the vicinity of the position where the first flow channel is connected, and serves as a gas and / or liquid supply / discharge channel for the culture space of the cartridge type closed culture vessel. A third flow path;
A medium component analyzer connected to the third flow path and capable of analyzing the components in the culture solution;
14. The culture apparatus according to claim 13, further comprising switching means for communicating any one of the first flow path and the third flow path to the culture space of the cartridge type closed culture vessel. In the state where the joint part and the cartridge-type closed culture vessel are separated from each other, a washing channel for connecting the first channel and the second channel is further provided,
The culture apparatus according to claim 12, wherein the cleaning liquid and the cleaning air are supplied in a state where the first flow path and the second flow path are connected by the cleaning flow path.

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