JPWO2013183121A1 - Culture container and automatic culture device - Google Patents

Abstract

A culture container, a first container, a plurality of second containers stored and held in the first container, a container lid member for sealing each of the first container and the second container, Each of the first container and the second container, and a connection port for connecting the flow path circuit for supplying the cells and the culture medium to the culture container, the first container and the second container, , A container for containing the medium and cells, or only the medium, and as the connection port, the first container has a second supply port for supplying the medium, and supplying and discharging the gas. And a second discharge port for discharging the culture medium, and each of the second containers is supplied with the culture medium, a first supply port for supplying and discharging a gas, and the culture medium And a first discharge port for discharging the Supply port and the second supply port is always functions as a flow path for flowing in one direction of the medium to the culture vessel.

Description

  The present invention relates to a culture container and an automatic culture apparatus for culturing cells or tissues by automatic operation.

  The production of regenerative tissue used for regenerative medical treatment is based on GMP (Good Manufacturing Practice), which is a standard for manufacturing management and quality control of pharmaceuticals and the like. In general, regenerative tissues are manufactured according to SOP (Standard Operational Procedure) by a manufacturing worker with specialized cell culture technology in a CPC (Cell Processing Center) that provides a clean manufacturing environment. The Therefore, a great labor cost, labor, and operation cost are generated. In addition, since all manufacturing processes are performed manually, there is a limit to the amount of regenerative tissue manufactured. Therefore, the manufacturing cost is increased, and as a result, the spread of regenerative medical treatment is hindered.

  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 realized, and mass production becomes possible. In addition, since the operation by the automatic culture apparatus is constant, it is expected to contribute to the constant quality of the regenerated tissue obtained after production.

  Here, the automatic culture apparatus cultures cells as an alternative to manual work, but it is necessary to comply with GMP for manual work contents. In addition, although GMP specialized for automatic culture devices is not stipulated at present, development guidelines on automatic culture devices for clinical use (regenerative medicine field (design guidelines for human cell culture processing equipment [revised], 2009) In accordance with the guidelines provided by the Ministry of Economy, Trade and Industry, it is necessary to comply with this development guideline. As a means for solving these problems, for example, an apparatus for automating the culture process as described in Patent Documents 1 and 2 is required. By performing operations such as medium exchange automatically, the risk of biological contamination is reduced and production efficiency is improved. That.

  When the target organ for regenerative medical treatment is corneal epithelium, esophageal mucosa, epidermis, etc., the cell types to be cultured are epithelial cells such as corneal epithelial cells, oral mucosal epithelial cells, and epidermal cells. In this case, the culture container preferably has a two-layer structure. Epithelial cells co-culture with feeder cells, such as mouse-derived 3T3-J2 cells, and grow with growth factors produced by the feeder cells. However, when epithelial cells and feeder cells are cultured on the same culture plane, after production Feeder cells are mixed in the regenerated tissue. On the other hand, when culturing using a culture container having a two-layer structure, since two types of cells are cultured on different culture surfaces, mixing of mouse-derived cells and the like that are different from humans into regenerated tissues can be avoided. it can. This is more preferred in regenerative medical treatment for humans. Similarly, when culturing epithelial cells using an automatic culture apparatus, a two-layer structure is desirable. As shown in Patent Document 1, development of a closed culture vessel having a two-layer structure has already been promoted.

JP 2008-148602 A JP 2007-31668 A

  As described above, the automatic culture device keeps the quality of the regenerated tissue after production in order to keep the quality of the regenerated tissue constant. It is necessary that the old medium is not mixed. In addition, when monitoring by medium component analysis using an old medium that is the drainage liquid at the time of medium exchange, in order to accurately perform the medium component analysis, a new medium is mixed in the old medium that is the drainage liquid. Things need to be avoided.

  In addition, as described above, the automatic culture apparatus is required to meet the development guidelines for GMP and automatic culture apparatus that are required for manual work. One of the requirements is that the quality of the regenerated tissue produced is constant. There is a need to. There are multiple conditions that make the quality constant, but one of them is to replace the old medium and supply a new medium in the medium replacement process in which the medium is replaced in all cases. It is necessary that the old medium is not mixed with the new medium. The old medium is a medium used for the growth of cells. For example, glucose is consumed and lactic acid is discharged instead. When an old medium is mixed with a new medium, the amount of glucose after the medium exchange is different from the concentration of the new medium, and as a result, the reproducibility of the culture process is lost. Similarly, since lactic acid contained in the old medium changes the pH of the medium, mixing of lactic acid derived from the old medium after the medium exchange also affects the reproducibility of the culture process. From the above, in order to improve the reproducibility of the culture process, it is necessary that the old medium and the new medium are not mixed in the medium replacement process. In addition, in order to grasp the culture state in the culture process, it is desirable to mount a monitoring function by medium component analysis using an old medium as a drainage. In order to accurately perform medium component analysis, it is necessary to avoid mixing a new medium into the old medium that is the drainage. This is because, as described above, the component composition of the old culture medium is different from that of the new culture medium. Therefore, if the new culture medium is mixed with the old culture medium used for the medium component analysis, the results of the medium component analysis are different. As described above, a technique for controlling the mixing of an old medium and a new medium is also necessary in the culture process.

  Finally, when culturing with an automatic culture apparatus, it is necessary to carry out liquid feeding such as a medium necessary for cultivation and gas feeding containing 5% carbon dioxide by a flow channel tube attached to the culture vessel. When 10 culture vessels are cultured as in the above-described esophageal mucosal regeneration, the total length of the flow path tube attached to the closed culture vessel for feeding and feeding is enormous. In addition, the smaller the number of flow channel tubes to be attached per culture vessel, the simpler the flow channel circuit and the better. This is because if the flow path circuit is complicated, it may cause a failure of the automatic culture apparatus and a cost increase.

  In addition, the automatic culture apparatus is required to satisfy the development guidelines for GMP and automatic culture apparatus that are required in manual work. In particular, in order to safely implement regenerative medicine, it is necessary to ascertain that the quality of the remaining regenerated tissue is good by taking out the regenerated tissue aseptically and conducting an inspection on the day before using the regenerated tissue for treatment. . In addition, after removing the regenerated tissue for examination, the remaining regenerated tissue needs to be able to continue culturing while maintaining sterility until the time of treatment.

  In addition, the number of regenerative tissues necessary for regenerative medical treatment differs for each target organ. For example, in the case of esophageal mucosal regeneration, clinical research on regenerative medical treatment by using about 8 regenerated tissues, which are oral mucosa sheets with a diameter of 24 mm, layered to about 3-5 layers, has already been advanced. On the day before the treatment, it is necessary to inspect whether the manufactured regenerated tissue has good quality, and a regenerated tissue for inspection is further required. In the case of esophageal mucosal regeneration, a total of about 10 regeneration tissues are required. Since the regenerated tissue used for examination is taken out on the day before transplantation, it is necessary that the regenerated tissue for transplantation can be cultured while maintaining sterility. Therefore, an automatic culture device for regenerative medicine can produce at least a plurality of regenerative tissues for transplantation and inspection, and culture that maintains sterility after taking out the regenerated tissue for inspection the day before. Must.

  In the invention of Patent Document 1, when replacing the medium, a new medium is supplied in a state where the old medium is present in the culture container, and after the state in which the new medium and the old medium are mixed inside the culture container, an excessive amount of liquid is supplied. The continuous medium exchange method is taken out. The discharged medium has a large proportion of the old medium, but the old medium is mixed into the new medium although the concentration is low. Since the composition of the old medium varies depending on the cell growth stage (number of cells, degree of differentiation), the concentration of each component of the medium after the medium exchange by this method also varies depending on the cell growth stage. Further, when the medium component analysis is performed using the old medium obtained by this method, there is a problem that the monitoring result is not accurate because the collected old medium contains a new medium.

  In the invention of Patent Document 1, automatic culture is performed using a plurality of culture containers having a two-layer structure. The culture vessel and the flow channel tube for feeding liquid into the culture vessel are detachable. It is in a separated state during culturing and in a combined state during liquid feeding. Therefore, the flow path circuit is not a closed system but an open system, and has a risk of biological contamination due to invasion of bacteria or the like from the attachment / detachment portion of the culture vessel. Although it is possible to take out only one piece for testing from a plurality of culture containers that are co-cultured, it is sterile because it has a risk of biological contamination from the attachment / detachment part of the culture container. Extraction has not been realized.

  In the invention of Patent Document 2, automatic culture is performed using a closed culture vessel having a single one-layer structure. The closed culture vessel and the flow tube are always connected, so that a closed flow channel circuit is obtained. This eliminates the risk of biological contamination due to the invasion of bacteria and the like. However, since it has a single-layer structure, when culturing epithelial cells using this culture vessel, feeder cells to be co-cultured are mixed into the regenerated tissue after production. Since feeder cells may use animal-derived cells that are not derived from humans, contamination of feeder cells leads to a decrease in safety for regenerative medical treatment.

  In addition, the device of Patent Document 2 requires a flow channel tube for supplying only air for adjusting the pressure in the culture vessel in addition to a flow channel tube for feeding and discharging a liquid such as a culture medium. If this method is applied to a closed culture vessel having a two-layer structure, the flow tube required for one closed culture vessel is a flow tube that is responsible for feeding and discharging liquid to and from the upper and lower layers. In addition, a flow path tube for supplying air is required, and a total of five tubes are required. In the method of Patent Document 2, one closed culture vessel is automatically cultured. For example, when 10 closed culture vessels are used for regenerative medical treatment for esophageal mucosal regeneration, the total number of channel tubes is It will be 50, which will be quite a lot. As a result, the flow path circuit becomes complicated.

  In addition, since only one closed culture vessel is cultured in one culture, a part of the regenerated tissue cannot be aseptically removed for the purpose of examination or the like the day before the regenerative medical treatment is performed. A regenerative tissue for regenerative medical treatment and a regenerative tissue for examination cannot be extracted separately, and all of them are extracted. In this case, the regenerated tissue is placed in an environment different from the cultured state from the time when the test and the treatment are performed at the same time or after the test until the time of the treatment. In the former case, it is difficult to reflect the test result in the schedule of regenerative medical treatment. In the latter case, there is a risk that the quality of the regenerated tissue will change.

  Based on the above, the object of the present invention is to provide a simple flow path circuit so that the old culture medium and the new culture medium do not mix with each other. It is an object of the present invention to provide a culture container and an automatic culture apparatus that enable this.

  Another object of the present invention is to provide a culture container and an automatic culture apparatus capable of aseptically removing one regenerated tissue from a plurality of cell containers in which a plurality of cells are co-cultured.

  An example of a representative example of the present invention is as follows. A culture container for holding and culturing cells, wherein the first container, a plurality of second containers stored and held in the first container, and each of the first container and the second container are sealed. A container lid member for stopping, each of the first container and the second container, and a connection port for connecting a flow path circuit for supplying the cells and the culture medium to the culture container. And the second container is a container for containing the medium and cells or only the medium, respectively, and a part of the partition between each of the first container and the second container is a liquid and a gas. A gap that allows gas to flow between each other exists between each of the first container and the second container, and the connection port serves as the first port. In the container, the medium is supplied and the gas is supplied and discharged. A second supply port and a second discharge port for discharging the medium are connected, and each of the second containers has a first for supplying the medium and supplying and discharging the gas. A supply port and a first discharge port for discharging the culture medium are connected, and the first supply port and the second supply port are flow paths that always flow the culture medium in one direction with respect to the culture vessel. Function.

According to the present invention, the medium always flows in one direction in the cell seeding process and the medium exchange process. In the medium exchange for exchanging the entire amount, the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved. Analysis accuracy of medium component analysis using the collected old medium is improved. Since the flow path tube attached to the closed culture vessel serves both as a liquid feeding and air feeding function, the flow path circuit is simplified.
Further, according to the present invention, one regenerated tissue can be aseptically removed from a cell container in which a plurality of cells are co-cultured. Aseptic culture can be continued for the remaining culture vessels.

FIG. 2 is a plan view of a culture vessel corresponding to the AA ′ cross section of FIG. 1B in which a plurality of upper-layer culture vessel units according to the first embodiment of the present invention are housed in a lower-layer integrated culture vessel box. . It is a longitudinal cross-sectional view of the culture container corresponding to the BB 'cross section of FIG. 1A in a 1st Example. In a 1st Example, it is a longitudinal cross-sectional view which shows the state which accommodated one culture container unit for upper layers in the culture container box of a lower layer integrated type. In a 1st Example, it is a longitudinal cross-sectional view which shows the state which isolate | separated one culture container unit for upper layers from the culture container box of a lower layer integrated type. FIG. 3 is a circuit diagram of an entire flow path when 10 regenerated tissues are cultured in the automatic culture apparatus according to the first embodiment. FIG. 3B is a diagram showing an associated channel circuit extracted from the overall channel circuit diagram of FIG. 3A for one upper layer culture vessel unit in the first example. It is the figure which showed the control mechanism of the automatic culture apparatus which has a culture container which concerns on a 1st Example. In 1st Example, it is the figure which showed a series of protocols which culture | cultivate a cell using the automatic culture apparatus of a present Example. In the 1st Example, it is the figure which showed an example of the table for controlling cell seeding with an automatic culture apparatus. In 1st Example, it is the figure which showed the culture medium and the flow of air at the time of cell seeding | inoculation by the flow path circuit which extracted the relevant part from the whole flow path circuit diagram with respect to one culture container unit for upper layers. . In 1st Example, it is the figure which showed the culture medium and the flow of air at the time of cell seeding | inoculation with respect to the culture container for lower layers. In the 1st Example, it is the figure which showed an example of the table for controlling culture medium exchange by an automatic culture apparatus. In 1st Example, it is the figure which showed the flow of the culture medium and air at the time of the culture medium exchange of an upper layer with a flow path circuit with respect to one culture container unit for upper layers. It is the figure which showed the culture medium and the flow of air at the time of the culture medium exchange of an upper layer. It is the figure which showed the culture medium and the flow of air at the time of the culture medium exchange of an upper layer. It is the figure which showed the culture medium and the flow of air at the time of the culture medium exchange of an upper layer. According to the first embodiment, the flow of the medium and the air during the exchange of the lower layer medium was shown by the flow path circuit that extracted the relevant part from the entire flow path circuit diagram for one upper culture container unit. FIG. It is the figure which showed the culture medium at the time of lower layer culture medium exchange, and the flow of air. It is the figure which showed the culture medium at the time of lower layer culture medium exchange, and the flow of air. It is the figure which showed the culture medium at the time of lower layer culture medium exchange, and the flow of air. It is a top view of the culture container which culture | cultivates the culture container for a test separately from the culture container main body for lower layers based on the 2nd Example of this invention. It is a longitudinal cross-sectional view which shows a part of culture container which comprised the bellows type isolation film between each culture container unit for upper layers and the culture container main body for lower layers based on the 3rd Example of this invention. In the 3rd Example, it is the figure which showed a series of protocols which take out one culture container unit for upper layers from a culture container aseptically. In the 3rd Example, it is the figure which showed a series of protocols which take out one culture container unit for upper layers from a culture container aseptically. In the 3rd Example, it is the figure which showed a series of protocols which take out one culture container unit for upper layers from a culture container aseptically. It is a longitudinal cross-sectional view which shows an example of the manufacturing method of the culture container which comprised the bellows type isolation film between each culture container unit for upper layers and the culture container main body for lower layers based on the 4th Example of this invention. It is a longitudinal cross-sectional view which shows an example of the culture container which comprised the bellows type isolation membrane between each culture container unit for upper layers and the culture container main body for lower layers based on the 5th Example of this invention. It is an accommodation state of the culture container unit for upper layers with respect to the culture container main body for lower layers in a 5th Example. It is a longitudinal cross-sectional view which shows an example of the accommodation method of the culture container unit for upper layers with respect to the culture container main body for lower layers in a 5th Example. It is a longitudinal cross-sectional view which shows an example of the culture container which comprised the bellows type isolation membrane between each culture container unit for upper layers and the culture container main body for lower layers based on the 6th Example of this invention. It is a longitudinal cross-sectional view which shows an example of the culture container which comprised the bellows type isolation membrane between each culture container unit for upper layers and the culture container main body for lower layers based on the 7th Example of this invention. It is the figure in the 7th Example which showed the state which takes out one culture container unit for upper layers aseptically from the culture container.

  In a typical embodiment of the present invention, the culture vessel is a two-layered closed culture vessel having a culture space inside and having a first vessel, a second vessel, and a lid member thereof, A plurality of second containers can be accommodated in the first container. The first container includes a second supply port for supplying a medium, supplying and discharging a gas, and a second container for discharging the medium. Two discharge ports are connected. Each of the second containers is connected to a first supply port for supplying a medium, supplying and discharging a gas, and a first discharge port for discharging the medium. A 1st supply port and a 2nd supply port function as a flow path which always flows a culture medium to one direction with respect to a culture container.

  In another embodiment of the present invention, a closed culture vessel having a two-layer structure having a culture space inside and having a first vessel and a second vessel, wherein a plurality of second vessels are contained in the first vessel. A container can be accommodated, and has a first supply port and a second supply port for supplying a cell suspension or a medium and supplying and discharging air to and from the first container and the second container, respectively. And a first discharge port and a second discharge port for discharging the medium and discharging the air from the first container and the second container, respectively. And the culture container of the structure which connected between the container lid member and the 1st container and had the expansion | extension member (isolation film) which can accommodate the said 2nd container in the inside is provided.

  In order to achieve the above object, in another embodiment of the present invention, in an automatic culture apparatus using a closed culture vessel having a two-layer structure including the first container and the second container, the medium is A flow path circuit that can be configured to always flow in one direction and a control protocol for controlling the flow path circuit are employed. That is, an automatic culture apparatus using a closed culture vessel having a culture space inside, a cell bag in which a cell suspension is stored, a medium bag in which a medium is stored, a refrigerator and a medium in which the medium is refrigerated and stored A heater for preliminarily warming the medium to 37 ° C. at the time of replacement, a culture vessel for culturing cells, a fluid movement control mechanism for feeding / feeding cell suspension, medium and air, and a source of carbon dioxide and the like A gas tank for adjusting the gas concentration, a filter for adjusting the atmospheric pressure with the outside, a two-way valve and a three-way valve for opening and closing the flow path. A flow path circuit including a culture container, a cell bag, a culture medium bag, a fluid movement control mechanism unit, and the like is installed in a thermostat, and the temperature of the entire flow path circuit is controlled. The culture environment of the culture vessel is controlled by a control device. In addition, a temperature sensor is installed in the device to monitor the internal temperature. In addition, a microscope is installed to monitor the state of cell growth optically as appropriate.

  That is, in an automatic culture apparatus using a closed culture vessel having a culture space in the preferred embodiment of the present invention that achieves the above-described object, when seeding cells in the first vessel in the culture vessel, While the cell suspension is supplied from the cell bag to the first container via the first supply port by the movement control mechanism, the air in the culture container is discharged to the outside from the second supply port. At the time of cell seeding in the second container in the culture container, after switching between the first supply and the second supply port, the cell suspension is transferred from the cell bag to the first via the second supply port by the fluid movement control mechanism. While supplying the container, the air in the culture container is discharged to the outside from the first supply port. In addition, the order which seeds a cell to a 1st container and a 2nd container is arbitrary.

  At the time of exchanging the medium in the first container in the culture container, the medium in the culture container is discharged from the second supply port to the outside while the medium is fed from the medium bag to the first supply port by the fluid movement control mechanism. The medium that reached the first supply port was refrigerated at 4 ° C. during storage but passed through a heater that warms to 37 ° C., so the temperature is 37 ° C. at this point. Since the medium reaching the first supply port temporarily stands by in a constant temperature bath maintained at 37 ° C., the temperature of the medium is also maintained at 37 ° C. Next, while the old medium in the first container is discharged from the first discharge port by the fluid movement control mechanism, the air in the culture container is supplied to the inside from the second supply port. Subsequently, while the culture medium waiting at the first supply port is supplied into the first container by the fluid movement control mechanism, the air in the culture container is discharged to the outside from the second supply port. This operation is started immediately after the old medium discharged from the first discharge port is completely discharged from the first container. It is not necessary to complete the complete feeding of the old medium discharged from the first discharge port to the drain bag or the like. Finally, a part of the old medium discharged from the first discharge port in the channel tube is collected from the drainage collection bag, and the rest is discharged to the drainage bag. As described above, the old medium can be discharged in the first container without being mixed with the new medium. The medium in the first container after the medium exchange is only the new medium supplied at the time of the medium exchange.

  When the medium of the second container in the culture container is replaced, the roles of the first supply port and the second supply port are switched compared to when the medium of the first container is replaced. Further, the discharge port is the first discharge port in the first container, whereas it is the second discharge port in the second container. First, while the medium is fed from the medium bag to the second supply port by the fluid movement control mechanism, the air in the culture vessel is discharged from the first port to the outside. Since the temperature of the culture medium that has reached the second port temporarily stands by as in the case of the first container, it is maintained at 37 ° C. at this time. Next, while the old medium in the second container is discharged from the second discharge port by the fluid movement control mechanism, the air in the culture container is supplied to the inside from the first supply port. Subsequently, while the medium that is waiting at the second supply port is supplied to the second container by the fluid movement control mechanism, the air in the culture container is discharged to the outside from the first supply port. This operation starts immediately after the old medium discharged from the second discharge port is completely discharged from the first container. It is not necessary to complete the complete feeding of the old medium discharged from the second discharge port to the drain bag or the like. Finally, a part of the old medium discharged from the second discharge port in the channel tube is collected from the drainage collection bag, and the rest is discharged to the drainage bag. As described above, as in the first container, the old medium can be discharged in the second container without being mixed with the new medium. The medium in the second container after the medium exchange is only a new medium supplied at the time of the medium exchange. It should be noted that the order of exchanging the medium for the first container and the second container is arbitrary.

  Further, during the culture, for example, for the purpose of inspection, by using an elongating member installed in the culture container, only one second container can be removed aseptically. In addition, after aseptically removing only one, the remaining first container and second container can be continuously cultured while maintaining sterility.

  With the above configuration and protocol, the culture medium flows in one direction in the flow path circuit. Old and new media do not mix. As a result, the accuracy of medium component analysis on the collected old medium is also improved. Further, as described above, the first supply port and the second supply port have both the function of feeding the medium and feeding the gas. Therefore, the number of channel tubes to be attached to the closed culture vessel passes as compared with the case where the gas channel tube is provided independently. Therefore, the flow path circuit becomes simpler. Furthermore, it is possible to remove some culture containers aseptically during the culture. Since the remaining culture containers after removal also maintain sterility, the culture can be continued.

According to the culture container and the automatic culture apparatus using the closed culture container according to the present invention, one regenerated tissue can be aseptically taken out from the cell container in which a plurality of cells are co-cultured. . Aseptic culture can be continued for the remaining culture vessels. In addition, in the cell seeding process and the medium exchange process, the medium always flows in one direction. In the medium exchange for exchanging the entire amount, the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved. Analysis accuracy of medium component analysis using the collected old medium is improved. Since the flow path tube attached to the closed culture vessel serves both as a liquid feeding and air feeding function, the flow path circuit is simplified.
Hereinafter, embodiments of an automatic culture apparatus according to the present invention will be described in detail with reference to the drawings.

Hereinafter, an automatic culture apparatus according to a first embodiment of the present invention in which a plurality of upper-layer culture container units are accommodated in one lower-layer culture container will be described in detail with reference to the drawings. In the present specification, gas, liquid, gas and liquid flowing through the flow path of the automatic culture apparatus may be collectively referred to as fluid.
1A is a plan view of a culture vessel corresponding to the AA ′ cross section of FIG. 1B, and FIG. 1B is a longitudinal cross-sectional view of the culture vessel corresponding to the BB ′ cross section of FIG. 1A.
The culture container 100 of the present embodiment includes a plurality of upper-layer culture container units 101 (10 a to j in this case) for culturing a regenerated tissue, and one lower-layer integrated type for culturing feeder cells. It can be accommodated in the culture container box 102. Each upper layer culture vessel unit 101 has an inverted truncated cone shape. Each upper layer culture container unit 101 is housed inside the culture container box 102 through openings 115 provided at a plurality of predetermined positions on the upper surface of the box-shaped culture container box 102, and each upper layer culture container unit 101. A plurality (10 in this case) of container lid members 103 corresponding to the unit 101 are fixed to the upper surface of the lower layer culture container main body 104 with screws or the like so as to close the openings 115. Thus, a closed space 110 surrounded by the container lid member 103 and the lower layer culture container body (second container) 104 is formed in the culture container 100, and each upper layer culture container unit (first container) 101 is It is held in the enclosed space 110. The upper layer culture vessel unit 101 is installed on the vessel lid member 103 by a hook structure or the like, and can be easily taken out as necessary. In the example of FIG. 1A, the plurality of openings 115 are provided in parallel to the upper surface of the culture vessel box 102, but other arrangement methods such as a staggered pattern may be used.

  Next, basic components of the lower layer integrated culture vessel 100 will be described in detail. The material of the lower layer-integrated culture vessel box 102 is a plastic having rigidity as well as plasticity such as polycarbonate, polystyrene, and polypropylene. In this example, the case where the shape of the bottom face of the lower-layer integrated culture container box is square is shown. In addition, an example is shown in which ten upper-layer culture container units 101 are accommodated in one culture container box 102 in a state where two vertical and five horizontal culture container units 101 are arranged side by side. In addition, arrangement | positioning of the culture container unit 101 for upper layers is not limited to this. The container lid member 103 and the lower layer culture container body 104 constituting the culture container box 102 are formed by injection molding, cutting, or the like.

  The upper culture container unit 101 is accommodated in the culture container box 102. For example, a cell culture insert container generally used for cell culture is used as the upper culture container unit. Cell culture insert containers may be commercially available, such as those manufactured by BD, manufactured by Corning, manufactured by Greiner, etc., and usable products are not limited. Moreover, application to a temperature-responsive cell culture insert container manufactured by Cellseed is also possible. The bottom surface 120 of the upper-layer culture container unit 101, which is a cell culture insert container or the like, is a porous film, and has many holes with a diameter of about 0.4 μm, for example. Thereby, a culture medium and a liquid factor can be moved between the upper layer and the lower layer. Since the pore diameter is sufficiently smaller than the cells, the cells in the upper layer do not move through the pores to the lower layer. Moreover, since it is the hole of the magnitude | size, a culture medium and a liquid factor do not move rapidly in a short time of about several minutes, for example. Therefore, even if the upper culture container unit is lifted in a state where the culture medium or the like is contained in the upper culture container unit 101, the inner culture medium is retained within, for example, a few minutes. In addition, between the upper edge part of the culture container unit 101 for upper layers, and the opening part 115 of the culture container main body 104 for lower layers provided in the upper surface of the culture container box 102, mutual gas distribution is enabled. There is a gap 122.

  In the upper layer culture container unit 101 such as a cell culture insert container, the cells are seeded and cultured on the bottom surface of the upper layer culture container unit 101. In the culture container box 102, the cells are seeded and cultured on the bottom surface of the lower layer culture container body 104. The container lid member 103 or the lower layer culture container body 104 is provided with an elastic member 105 such as an O-ring. Thereby, the particle | grains containing gas, a microbe, etc. are not mixed inside from the exterior of the culture container 100 of a cell. Therefore, the inside of the culture vessel 100 can be maintained aseptic during culture. The connection of the container lid member 103 to the lower culture container body 104 is fixed by screwing screws provided on the container lid member 103 and the lower culture container body 104 together. In this case, when removing the container lid member 103 from the lower layer culture container body 104, the container lid member 103 is rotated and removed. Alternatively, the container lid member 103 is fixed by an engagement or fitting engagement portion provided in the container lid member 103 and the lower layer culture vessel main body 104. In this case, the container lid member 103 is lifted and removed in the axial direction without rotating. In addition, the fixing method to the culture container main body 104 for lower layers of the container lid member 103 is not limited to these methods.

  The lower layer culture vessel main body 104 includes a lower layer supply flow path 107 having a lower layer supply connection projection structure (second supply port) 106 at one end thereof for supplying a medium and supplying and discharging air / water vapor. The lower layer discharge passage 109 has a lower layer discharge connection projection structure (second discharge port) 108 at one end thereof for discharging the medium and discharging air / water vapor. The position of the lower layer supply channel 106 in the lower layer culture vessel main body 104 should be changed depending on the amount of the medium 150 to be introduced, but may be any position above the level of the introduced medium liquid. The position of the lower layer discharge channel 109 in the lower layer culture vessel body 104 is used for discharging the medium from the lower layer culture vessel body 104. It is desirable to install the lower part at the same height. By doing so, the discharge efficiency of the culture medium can be improved. The discharging efficiency is further improved by discharging the medium while appropriately tilting the culture container by a rotating mechanism for rotating the main culture container shown in FIG. 3A described later.

  Each container lid member 103 has an upper layer supply connection projection structure (first supply port) 111 for supplying a culture medium and supplying / discharging air / water vapor to each upper layer culture container unit 101 at one end thereof. And an upper layer discharge flow path 114 having an upper layer discharge connection projection structure (first discharge port) 113 for discharging the culture medium at one end thereof. In FIG. 1B corresponding to the BB ′ cross section of FIG. 1A, the upper layer supply flow path 112 and the upper layer discharge flow path 114 should be displayed overlapping each other, but here the configuration is easy to understand. In order to do this, the upper layer supply channel 112 and the upper layer discharge channel 114 are separated from each other in the left-right direction (the same applies to the following drawings).

  The position of the upper layer supply channel 111 in the container lid member 103 should be changed depending on the amount of the medium to be introduced into the container, but may be any position above the introduced medium liquid level. Since the position of the upper layer culture vessel unit 101 on the bottom surface of the upper layer culture vessel unit 101 is used for discharging the medium from the upper layer culture vessel unit 101, the bottom surface of the upper layer culture vessel unit 101 and the upper layer ejection channel 114 are It is desirable to be close. However, if it is too close, in the production of the regenerated tissue, the upper layer discharge flow path 114 comes into contact with the growth of the cells, and this impedes the growth of the cells. In the case of oral mucosal epithelial cells, the cells grow to a height of about several hundred μm when cultured to regenerated tissue. Therefore, the upper layer discharge channel 114 can be close to the bottom surface of the upper layer culture vessel unit 101 up to a position sufficiently higher than that, for example, up to about 500 μm. The distance at which proximity is possible is determined according to the type of cells to be cultured. When the culture solution is discharged, the discharge efficiency can be improved by discharging the medium while being appropriately tilted by the rotation mechanism in FIG.

  The upper layer supply flow path 112 and the upper layer discharge flow path 114 installed in each container lid member 103 are arranged so as not to interfere with cell observation as much as possible. When there is no hindrance in cell observation, for example, when the inside of a cell culture container is observed with a microscope, it means that the cell is provided with a shape that does not inhibit the microscope optical axis and is arranged at a position that does not inhibit the microscope optical axis. The lower layer supply flow path 107, the lower layer discharge flow path 109, the upper layer supply flow path 112, and the upper layer discharge flow path 114 are flows made of an elastic body such as silicon having an inner diameter suitable for the protrusion structure size of the flow path. Road tube is connectable. Thereby, it can connect to the channel circuit which an automatic culture apparatus has.

  At the time of culture, epithelial cells are seeded in the upper culture container unit 101, and feeder cells are seeded in the lower culture container main body 104. However, in some applications, other cells may be seeded or cultured without seeding the cells. As an example of culturing without seeding cells, it is possible to culture without feeder cells by seeding epithelial cells in the upper layer culture vessel unit 101 and not seeding cells in the lower layer culture vessel body 104. It has been reported that.

  With reference to FIG. 2A and FIG. 2B, the configuration of the cell culture container will be described by focusing on one upper layer culture container unit. FIG. 2A shows an enlarged state in which one upper layer culture container unit is accommodated in the culture container box 102, and FIG. 2B shows one upper layer culture container unit from the culture container box. The separated state is shown. The upper culture container unit 101 has an outer peripheral edge portion 1010 at the upper end thereof, and the outer peripheral edge portion 1010 is held on the inward protruding portion 1030 of the container lid member 103. The inward protruding portion 1030 is held on an annular protruding portion 1040 provided around the opening 115 of the lower layer culture vessel body 104. Screw structures 1050A and 1050B are provided on the outer peripheral portion of each container lid member 103 and the outer peripheral portion of the annular projecting portion 1040 of the lower layer culture vessel main body 104, and the container lid member 103 is provided with the lower layer culture vessel by this screw structure. It is fixed to the main body 104. The fixing method is not limited to screwing, and any means such as engagement, fitting, or the like may be used.

  As described above, the culture container 100 for holding and culturing the cells includes the culture container unit 101 for accommodating only the culture medium and the cells, or the culture medium, and the culture medium and cells or the culture medium for accommodating the culture container unit for the upper layer And a container lid member 103 for sealing each upper layer culture container unit and the lower layer culture container, and a flow path circuit and an outer surface of the culture container. A connectable upper layer supply channel 112, a lower layer supply channel 107, an upper layer discharge channel 114, and a lower layer discharge channel 109, and discharge or supply of the medium to each of the upper layer culture container units; Or when discharging | emitting or supplying with respect to the culture container for lower layers, a communication state is switched by the means which controls liquid feeding. As will be described later, in the upper layer supply channel 112 and the lower layer supply channel 107, the cell suspension / medium always flows in one direction with respect to the culture vessel 100, and the gas flows in both directions.

  Next, FIG. 3A is an overall flow path circuit diagram when 10 regenerated tissues are cultured. FIG. 3B shows an associated channel circuit extracted from the entire channel circuit diagram for one upper-layer culture container unit.

  With reference to FIG. 3A, the entire flow path when culturing the lower layer integrated culture container 100 that accommodates the ten upper-layer culture container units 101 will be described. The culture target is epithelial cells such as corneal epithelial cells, oral mucosal epithelial cells, and epidermal cells. As described with reference to FIGS. 1A and 1B, the lower layer integrated culture vessel 100 is composed of two layers including a layer for culturing epithelial cells and a layer for culturing feeder cells that produce growth factors for epithelial cells. Structure. Since two types of cells are used, the cell bags 203 and 204 are of two types. Further, in order to prevent two types of cells from being mixed at the time of cell seeding, the flow path circuit for seeding is divided. In this figure, an epithelial cell is put into the cell bag 202, passes through the first flow path circuit (1) 205 indicated by a broken line, and is sequentially seeded in 10 upper culture container units 101 (A to J). . Feeder cells are put into the cell bag 203, pass through the second flow path circuit 204 indicated by a solid line, and seeded into the lower layer integrated culture container box 102. At the time of cell seeding, each cell suspension is sent from the cell bags 202 and 203 to the culture vessel 100.

  That is, the cell bag 202 and the culture medium bag 211 are composed of the first flow path circuit 205, the electromagnetic valves 206B and 206D, the fluid movement control mechanism (tube pump) 208A, the first flow path circuit 221 and the first electromagnetic valve 307. It is connected to the flow path (first supply port) 111 of the upper layer culture vessel unit 101 (A to J) via the (two-way valves A to J). The first channel circuit 205 is branched to the first channel circuit 221 corresponding to the ten upper-layer culture container units by the multi-branch portion 209A. The cell bag 203 and the culture medium bag 211 are connected to the culture container box 102 via the second flow path circuit 204, the electromagnetic valves 206C and 206E, the fluid movement control mechanism (tube pump) 208B, and the second flow path circuit 22). The flow path (second supply port) 106 is connected. On the other hand, the flow path (first discharge port) 113 of each upper layer culture vessel unit 101 is connected to the drainage bag 213, the drainage collection via the first flow path circuit 223, the fluid movement control mechanism 208C, and the three-way valve 207A. It is connected to the bag 214A. Ten branch paths (223) constituting the first flow path circuit are combined into one at the multi-branch portion 209B. The flow path (second discharge port) 108 of the culture container box 102 is connected to the drainage bag 213 and the drainage recovery bag 214B via the second flow path circuit 224, the fluid movement control mechanism 208D, and the three-way valve 207B. It is connected to the. In addition, 212 is a heater, 215A and 215B are gas supply units, 216 is a gas concentration adjusting unit, and 217 is a filter.

  Thus, in the first channel circuit (205, 221, 223) and the second channel circuit (204, 222, 224), the two-way valves 206, 307, the fluid movement control mechanism 208, The three-way valve 207 is controlled based on a predetermined sequence by a control protocol given in advance. Thereby, the flow path circuit is controlled so that the culture medium always flows in one direction with respect to the culture container 100 and the new culture medium is supplied after the old culture medium is discharged.

  FIG. 3B shows a portion extracted from the culture vessel 100 shown in FIG. 3A that is related to one upper-layer culture vessel unit and lower-layer culture vessel body.

  The protocol for cell seeding and medium exchange will be described using this figure. In FIG. 3A, the multi-branch portion 209A branches the flow path to ten upper-layer culture container units. FIG. 3B shows a case where the flow path is connected from the multi-branch portion 209A to one upper culture container unit 101. FIG. 3B shows the direction of cell suspension / medium and air flow. In the upper layer supply channel 221 and the lower layer supply channel 222, the cell suspension / medium flows only in the direction from the outside to the inside of the culture vessel 100 (101, 104). On the other hand, air flows in both directions. On the other hand, in the upper layer discharge channel 114 and the lower layer discharge channel 109, both the cell suspension / medium and air flow only in the direction from the inside to the outside of the culture vessel 100.

  At the time of liquid feeding, predetermined two-way valves (206, 307) and three-way valve (207) are opened and closed in advance. Then, the fluid movement control mechanism unit 208 is operated to feed the liquid while controlling the flow rate and the liquid feeding time. In the case of epithelial cells, the sent cell suspension branches to the upper layer culture container unit 101 for cell seeding at the multi-branch portion 209A. In advance, the two-way valve and the three-way valve installed in the flow path circuit connected to the culture container unit for the upper layer to be fed are opened so that the feeding can be performed. On the other hand, the two-way valve and the three-way valve installed in the flow path circuit connected to the culture vessel that is not the liquid feeding target are closed so that the liquid feeding is impossible. Cell seeding is sequentially performed on the ten upper-layer culture container units 101 and the lower-layer integrated culture container box 102. After all seeding is completed, the rotating mechanism 311 attached to the lower part of the lower layer integrated culture container box 102 is operated. Although the culture container is kept horizontal during cell seeding and cell culture, the culture container is tilted immediately after cell seeding and when changing the medium. By continuously rocking at the time of cell seeding, the distribution of cells after seeding is made uniform. Thereafter, the culture vessel is returned to a horizontal state and cultured in that state.

During the culture period, the medium is changed at a predetermined date and time. In the case of epithelial cells, it is generally performed once every 1-3 days. During culturing other than during cell seeding and medium exchange, the temperature in the thermostatic chamber in which the flow path circuit is installed is maintained at 37 ° C. Thereby, the temperature in a culture container is also maintained at 37 degreeC. Further, CO ₂ or the like is supplied from the gas supply unit 215 as necessary. The concentration is performed by a gas concentration adjusting unit. For example, a gas containing 5% CO ₂ is appropriately sent into the culture vessel. The gas composition and the air supply schedule are determined according to the cell type to be cultured and the type of medium used. In addition, when taking in gas from the outside of the flow path circuit or adjusting the pressure inside the flow path circuit, it is performed through a filter. For example, a filter having a quality that does not pass particles of 0.22 μm or more is used.

  Aseptic desorption parts 1103 are attached before and after the upper layer culture container unit 101 in the flow path circuit. During culturing, liquid can be fed in the same manner as the channel tube. When the culture is continued while maintaining the sterility of the remaining culture containers, the culture container unit for the upper layer is separated from the flow path circuit by using the aseptic detachment part 1103 when the culture is continued while maintaining the sterility. Subsequently, the upper layer culture container unit is aseptically removed from the lower layer culture container body by the method described above. The flow path circuit after being removed by the aseptic desorption section maintains the closed state of the flow path tube by the aseptic desorption section 1103 left at the place where the culture container was removed. Thereby, even if it removes a culture container for a test | inspection on the previous day, culture | cultivation can be continued with respect to the remaining culture container.

  FIG. 4 is a block diagram showing a control mechanism of an automatic culture apparatus having a closed culture vessel 100. Each component controlled by the control device 1402 is connected to a culture vessel 1401 arranged inside the thermostatic device 1403. Needless to say, what is placed in the thermostat 1403 is a culture vessel such as the closed culture vessels 101 and 104 described in each embodiment, or the culture vessel installed in an automatic culture apparatus.

In FIG. 4, the control device 1402 includes a temperature adjustment unit 1404 for controlling the temperature of the thermostatic device 1403, a temperature sensor 1406, and a gas supply unit 1405, and a gas for controlling the gas concentration in the culture vessel. Concentration adjustment unit 1411, fluid movement control 1407 having a channel tube connected to each channel circuit component for automatically exchanging the culture medium in the culture vessel, and controlling the operation of each component For this purpose, a microscope 1408 for cell observation and a CO ₂ / O ₂ sensor (not shown) are connected.

The control device 1402, the display screen 1410, and the database 1412 are processed by a normal computer including a processing unit including a CPU (CentrAl Processing Unit), a storage unit, a display device, an input / output unit including a keyboard, and the like. And the display unit of the display device. The control device 14022 operates various programs stored in the storage unit on the CPU as a processing unit. Accordingly, the temperature adjustment unit 1404, the gas supply unit 1405, the fluid movement control mechanism unit 1407, the microscope 1408, the CO ₂ / O ₂ sensor, the gas concentration adjustment unit 1411, the cell / medium / drainage / drainage collection bag 1409, The culture environment in the thermostat 1403 is controlled, and a predetermined culture process in the culture container 1401 can be performed.

The gas concentration adjusting unit 1411 does not need to be directly connected to the culture vessel 1401. The temperature controller 1404, the gas concentration controller 1411, and the CO ₂ / O ₂ sensor may be connected to the thermostat 1403. In the case of this configuration, since it is necessary to supply gas to the cell culture container 1401 from outside the container, a part of the lid of the cell culture container 1401 has gas permeability such as polycarbonate, polystyrene, polymethylpentene and the like. Cell culture can be performed by depositing a transparent thin film to enable gas exchange inside the cell culture vessel 1401.

  A series of culture procedures when culturing cells using the automatic culture apparatus of the present embodiment having the above configuration will be described. FIG. 5 is a flowchart for explaining the operation of the automatic culture apparatus. Hereinafter, the operation of the automatic culture apparatus will be described. In addition, what is necessary is just to arrange | position a culture container in parallel in the multi-branch part of a flow path, when increasing the number of culture containers to be used. Moreover, the culture | cultivation procedure in that case should just perform operation shown below with respect to each culture container in order.

  First, the automatic culture apparatus is activated (step S1), and the schedule is determined (step S2). Further, after opening and closing the appropriate two-way valve and three-way valve, the fluid movement control mechanism is operated, seeding the culture vessel (step S3), culturing the cells in the culture vessel (step S4), and Observation with a microscope is performed (step S5). It is determined whether the cell is in a normal state (step S6). If it is normal, the culture medium in the culture vessel is replaced (step S7). Thereafter, culture and medium exchange just before transplantation are performed (steps S8 to S10). Further, the transplanted tissue is collected (step S11), and the series of processes is terminated (step S12).

  Next, the control protocol of the automatic culture apparatus will be described with respect to cell culture (step S4), culture medium exchange (step S7), culture immediately before transplantation, and culture medium exchange (step S10). Note that the collection of the test tissue (step S9) is preferably performed in the culture vessel described in Example 2 and later, and therefore the description thereof is omitted here.

First, seeding in the closed culture vessel 100 will be described.
FIG. 6 is a diagram showing an example of a table 600 for controlling seeding in the culture vessel 100 held in the database 1412. In the figure, black circles indicate the opening (ON) of the solenoid valves 206, 207, and 307 and the operating state of the pump 208, and X indicates the closing (OFF) of the solenoid valve and the stopped state of the pump (hereinafter the same).
First, two-way valves (solenoid valves) 206 and 307, three-way valves (solenoid valves) 207, a fluid movement control mechanism unit (see FIG. 1) for seeding each of the ten upper-layer culture container units 101 (101A to 101J). (Tube pump) 208 and the like are controlled according to a predetermined sequence given by table 600. For example, when the volume of each upper layer culture container is 2 ml, each valve and pump are controlled for 30 seconds for seeding the upper layer culture container 101A, and then seeding to the upper layer culture container 101B. Therefore, a series of processing is performed over 30 seconds, and thereafter, processing for seeding is performed in the same manner up to the upper layer culture vessel 101J.

  Finally, two-way valves (solenoid valves) 206 and 307, three-way valves (solenoid valves) 207, fluid movement control mechanisms (tube pumps) 208, etc. for seeding in the lower layer, that is, the integrated culture vessel box 102 Are controlled in accordance with a predetermined sequence given in the table 600. As an example, when the volume of the culture container main body 104 for the lower layer is 30 ml, a series of processing is performed over 450 seconds, and when it is 50 ml, a series of processing is performed over 750 seconds.

  FIG. 7A shows a protocol for seeding cells into the upper layer. As shown in the figure, the solution is fed from the cell bag 202 into one upper layer culture vessel 101 via the upper layer supply channel 221. At this time, the two-way valve on the flow path through which the culture medium and air flow is previously opened. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding. That is, the cell bag 202 is cultured in the upper layer via the first flow path circuit (205), the electromagnetic valve 206B, the fluid movement control mechanism 208A, the first flow path circuit (221), and the second electromagnetic valve 307. It is connected to the flow path (first supply port) 111 of the container 101, and cell seeding is performed on the upper layer culture container 101. At the same time, air in the lower layer culture vessel body 104 is discharged from the lower layer supply channel 107 to the outside of the culture vessel. The air is finally discharged out of the flow path from the filter 217A. That is, in the lower layer culture vessel body 104, the flow path (second supply port) 106 has a second flow path circuit (222), a fluid movement control mechanism 208B, a second flow path circuit (204), an electromagnetic valve. It is connected to the filter 217A via 206A. Since there is a gap between the upper layer culture vessel 101 and the lower layer culture vessel body 104 that allows gas to flow between each other, the gas in the upper layer culture vessel is supplied to the second channel circuit and the filter 217A. It is exhausted through. When the predetermined amount has been delivered, the process ends. Cell seeding is carried out in the same manner for the other upper-layer culture vessels 101. Here, cell seeding is performed in the order of the upper layer and the lower layer, but the order is arbitrary.

  Next, a protocol for seeding cells on the lower layer culture vessel body 104 is shown. As shown in FIG. 6, the two-way valve on the flow path through which the culture medium and air flow is opened in advance. Keep everything else closed. In this state, the fluid movement control mechanisms 209A and 209B are operated to carry out liquid feeding and air feeding. That is, as shown in FIG. 7B, from the cell bag 203, the second flow path circuit (204), the electromagnetic valve 206C, the fluid movement control mechanism 208B, and the second flow path circuit (222) are passed through the second flow path circuit (204). The liquid is fed from the supply port 106 to the culture container main body 104 for the lower layer. At the same time, the air in the culture container is supplied from the supply flow path (first supply port) 111 of the upper layer culture container 101 to the first flow path circuit (221), the fluid movement control mechanism 208A, and the electromagnetic valve 206F. Through the culture vessel. The air is finally discharged out of the flow path from the filter 217B. The process ends when the amount of liquid is completely delivered. In this way, cell seeding from the cell bag to the culture container is smoothly performed.

Next, medium exchange will be described.
The medium exchange is sequentially performed on the ten upper-layer culture container units 101 and one lower-layer integrated culture container box 102.
FIG. 8 is a diagram illustrating an example of a table 800 for controlling medium replacement in a closed culture container. With this table 800, two-way valves (solenoid valves) 206 and 307, three-way valves (solenoid valves) 207, fluid movement control mechanisms (tube pumps) 208, and the like are exchanged for predetermined medium given by the table 800. It is controlled according to the sequence.

  A protocol for exchanging the medium of the upper layer culture vessel 101 will be described with reference to FIGS. 9A to 9D show a flow path circuit formed in accordance with the table 800 for one upper layer culture vessel 101, and the flow of the medium and air when the upper layer medium is exchanged thereby.

  First, as shown in FIG. 9A, the medium is fed from the medium bag 202 to the first supply port 111 of the culture container unit for the upper layer through the first flow path circuit (221), and is set in a standby state. At the same time, the air in the culture vessel is discharged from the flow channel (second supply port) 106 to the outside of the culture vessel 100 via the second flow channel circuit (222). The air is finally discharged out of the flow path from the filter 217A. At this time, according to the table 800, the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding. In addition, since the culture medium bag 211 is installed in a refrigerator at 4 ° C., the culture medium immediately after being fed from the culture medium bag 211 is 4 ° C., but is heated to 37 ° C. by the heater 212, and a culture container and the like are installed. Maintain at 37 ° C. in a thermostat.

  Thus, in the state of FIG. 9A, the tip of the culture medium in the first flow path circuit is maintained at the first supply port 111 or in the vicinity thereof. In addition, what is necessary is just to change this stop position suitably according to a use.

  Next, as shown in FIG. 9B, the old medium used for the culture in the upper layer culture vessel unit 101 is used for upper layer discharge while maintaining the standby state of the new medium in the first flow path circuit (221). The liquid is discharged from the flow path 114. That is, the medium in the culture container is discharged to the drainage bag 213 by the fluid movement control mechanism 208C via the first discharge port 113 and the first flow path circuit (223). At that time, a part of the drainage liquid required for the medium component analysis is collected in the drainage collection bag 214A. When discharging the old medium from the culture container, the culture container is tilted by the rotation mechanism 311 so that the old medium can be easily discharged from the discharge port side in the culture container. At the same time, air is supplied from the second supply port 106 into the culture vessel 100 by the fluid movement control mechanism 208A via the lower layer supply channel, that is, the second channel circuit (222). The air is finally supplied from the filter 217A into the flow path. At this time, according to the table 800, the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed. In this state, the fluid movement control mechanisms 208A, 208B and 208C are operated to discharge and supply air. The old medium is finally discharged into the drainage bag 213 or the drainage collection bag 214A. The drainage ends when the entire amount of the old medium in the upper layer is discharged from the upper layer. However, it is not necessary to complete the whole quantity at this point.

  Subsequently, as shown in FIG. 9C, liquid is fed from the culture medium bag 211 to the first supply port 111 of the culture container unit 101 for upper layer or the vicinity thereof through the first flow path circuit (221), and the tip is this portion. Then, the new medium that has been kept at 37 ° C. is supplied into the upper layer culture vessel unit 101. At the same time, the air in the culture vessel 100 is discharged out of the culture vessel via the second supply port 106 and the lower layer supply channel (second channel circuit 222). The air is finally discharged out of the flow path from the filter 217A. At this time, according to the table 800, the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208B are operated to perform liquid feeding and air feeding.

  Finally, as shown in FIG. 9D, the old medium remaining in the first flow path circuit (223) between the upper layer discharge flow path 114 and the drainage bag 213 or the drainage recovery bag 214A is drained. The liquid is discharged into the liquid bag 213 or the drainage recovery bag 214A. At the same time, air is supplied from the second supply port 106 into the culture vessel 100 via the lower layer supply channel, that is, the second channel circuit (222). The air is finally supplied from the filter 217A into the flow path. At this time, according to the table 800, the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed. In this state, the fluid movement control mechanisms 208B and 208C are operated to drain and supply air. The drainage ends when the entire old medium in the upper layer is discharged to the drainage bag 213 or the drainage collection bag 214A.

Next, a protocol for exchanging the medium to the lower layer integrated culture container box 102 will be described.
10A to 10D show a flow path circuit formed according to the table 800 for the lower culture container box 102 and the flow of the culture medium and air when the lower culture medium is changed.

  First, as shown in FIG. 10A, the medium is fed from the medium bag 211 to the second supply port 106 of the culture vessel 100 or the vicinity thereof through the lower layer supply channel, that is, the second channel circuit (222). Here, the standby state is set. At the same time, the air in the culture vessel 100 is discharged from the first supply port 111 to the outside of the culture vessel via the first flow path circuit (221). The air is finally discharged out of the flow path from the filter 217B. At this time, according to the table 800, the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding. In addition, since the culture medium bag 211 is installed in the refrigerator, the culture medium immediately after being fed from the culture medium bag 211 is 4 ° C., but is heated to 37 ° C. by the heater 212, and a thermostatic device 1403 in which a culture container and the like are installed. Maintain at 37 ° C.

  Next, as shown in FIG. 10B, the old medium used for culture in the lower layer integrated culture container box 102 is removed from the lower supply channel while maintaining the standby state of the new medium in the second supply port 106. The liquid is discharged from 109 through the second flow path circuit (224). At the same time, air is supplied from the first supply port 111 into the culture vessel 100 through the first flow path circuit (221). The air is finally supplied from the filter 217B into the flow path. At this time, according to the table 800, the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208D are operated to drain and supply air. The drainage ends when the entire amount of the old medium in the lower layer is discharged from the lower layer. The old medium is finally discharged to the drainage bag 213 or the drainage collection bag 214B, but it is not necessary to complete the total amount at this point.

  Subsequently, as shown in FIG. 10C, the solution is fed from the culture medium bag 211 to the second supply port 106 of the culture container box 102 of the lower layer integrated type or the vicinity thereof through the second flow path circuit (224). A new medium kept at a temperature of 0 ° C. is supplied into the lower culture container box 102. At the same time, the air in the culture vessel 100 is discharged from the first supply port 111 to the outside of the culture vessel via the first flow path circuit (221). The air is finally discharged out of the flow path from the filter 217B. At this time, according to the table 800, the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding.

  Finally, as shown in FIG. 10D, the old medium remaining in the second channel circuit (224) between the lower layer discharge channel 109 and the drainage bag 213 or the drainage recovery bag 214B is drained. The liquid is discharged into the liquid bag 213 or the drainage recovery bag 214B. At the same time, air is supplied into the culture vessel 100 from the first supply port 111 via the first flow path circuit (221). The air is finally supplied from the filter 217B into the flow path. At this time, according to the table 800, the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208D are operated to drain and supply air. The drainage ends when the old medium in the lower layer is entirely discharged into the drainage bag 213 or the drainage collection bag 214B.

  According to the present embodiment, a part of the flow path tube attached to the closed culture vessel serves both as a liquid feeding function and an air feeding function, so that the entire flow path circuit is simplified. For example, when the lower layer culture container main body 104 is individually provided in each of the ten upper layer culture container units 101 (101A to 101J), the first supply port, the first discharge port, the second supply port, the second Since it is necessary to connect a flow tube to each of the discharge ports, a total of 40 flow tubes are required. On the other hand, according to the present embodiment, since only two channel tubes are connected to the lower layer culture vessel body 104 as a whole, a total of 22 channel tubes are sufficient. Also, the number of solenoid valves that control the flow path is reduced. Furthermore, since the seeding to the lower layer culture container main body is performed only once, the treatment time is shortened, and the culture can be promptly transferred.

  The details of a series of processes in the automatic culture apparatus of the present embodiment described above will be described together with reference to the control flow of FIG. In addition, what is necessary is just to arrange | position a culture container in parallel in the multi-branch part of a flow path, when increasing the number of culture containers to be used. Moreover, the culture | cultivation procedure in that case should just perform operation of S1-S8 and S10-S12 shown below with respect to each culture container in order.

<Step S1: Start>
First, as shown in FIG. 5, the automatic culture apparatus is activated. The operation is started when the operator presses the start switch of the operation unit in the control device. At this time, the channel circuit and the like are installed in the automatic culture apparatus in advance. On the operation screen of the control unit display, confirm that the value is appropriate for the internal environment of the automatic culture apparatus. For example, it is confirmed that the temperature of the thermostat is 37 ° C. These numerical values are not limited. For example, the temperature can be selected from the range of 0 ° C to 45 ° C. Further, the inside of the apparatus is sterilized with a sterilizing gas or sterilized with ethanol by an appropriate prior operation, and is in a clean state. In addition, sterilization is performed in advance on the flow path portion used for culture.

<Step S2: Schedule determination>
An automatic culture schedule to be performed by an automatic culture apparatus is determined according to the type and amount of cells to be cultured. Conditions such as date, frequency, fluid volume, etc. for performing operations such as cell seeding, medium exchange, microscopic observation, drainage collection, examination tissue collection, and transplantation tissue collection are input from the operation unit of the control unit.

<Step S3: Cell seeding>
After opening and closing the appropriate two-way valve and three-way valve, the fluid movement control mechanism is operated to suck the cell suspension from the cell bag. In order to culture oral mucosal epithelial cells in the case of esophageal mucosal regeneration, the cell suspension is composed of oral mucosal epithelial cells suspended in KCM medium (keratinocyte culture medium) and 3T3-J2 cells also suspended in KCM medium. is there. By driving the fluid movement control mechanism, the cell suspension is aspirated while discharging the air in the flow path to the outside of the flow path through the air filter. And it seeds to a culture container. Cell seeding is sequentially performed on each upper layer and lower layer of each culture vessel. After sowing, the culture vessel is rocked a plurality of times by a rotating mechanism so that the cell distribution on the culture surface becomes uniform.

<Step S4: Cell Culture>
Culturing is performed for a predetermined time in a state where the culture vessel is left still horizontally. For example, in the case of oral mucosal epithelial cells, the stationary period is about 5 days after sowing. During the culture, the ambient environment of the culture vessel is maintained at 37 ° C. by a thermostat. Moreover, the gas of a predetermined component is sent into the inside of a culture container as needed. In the case of oral mucosal epithelial cell culture, the CO2 concentration is maintained at 5% and the humidity at 100%. The air inside the automatic culture apparatus is constantly stirred by a fan so that the temperature distribution is always uniform.

<Step S5: Observation with a microscope>
Cell images are acquired using a microscope installed in an automatic culture apparatus. The light source installed in the automatic culture apparatus is appropriately illuminated, and the cells are focused and imaged by a microscope. If necessary, set a fixed point on the culture surface and photograph. The acquired cell image is stored in a database so that it can be viewed as necessary on a display installed outside the automatic culture apparatus.

<Step S6: Cell State Determination>
Judging from the information regarding the growth state of the cells obtained by microscopic observation, the frequency and timing of medium replacement are adjusted. For example, when the cell adhesion is insufficient, the medium exchange in the next step is not performed, and the cell culture in step S4 is continued.

<Step S7: Medium replacement>
The medium exchange is generally performed once every few days. The frequency is adjusted according to the state of cell growth. After opening and closing the appropriate electromagnetic valve, the fluid movement control mechanism is operated, and the fluid movement control mechanism is driven to suck the medium from the medium bag. At the same time, the air in the flow path is discharged out of the flow path through the filter. The medium immediately after being fed from the medium bag is 4 ° C., but the process proceeds to the next step with the temperature of the medium maintained at 37 ° C. due to the gas phase in the heater and thermostatic chamber.

  Subsequently, the old medium is discharged from the culture vessel. At this time, the culture vessel is tilted by the rotating mechanism so that the entire amount of the old medium is discharged. Immediately after discharging, a new medium maintained at 37 ° C. is supplied into the culture vessel. This avoids drying of the cells on the culture surface and a temperature drop on the culture surface.

  Part of the old medium discharged from the culture vessel is sent to the drainage collection bag and the rest is sent to the drainage bag. The collected old medium is evaluated for cell growth using medium component analysis by a separately prepared medium component analyzer. For example, the amount of glucose used during cell growth and the amount of lactic acid excreted is measured to grasp the cell growth state. In addition, a mycoplasma test or the like is performed to determine whether the medium is contaminated. When there is contamination, the culture is immediately terminated, and the cells are aseptically discarded by appropriate operations so that the place where the automatic culture apparatus is installed is not contaminated.

<Step S8: Determination immediately before transplantation>
A determination is made as to whether or not the transplant is suitable.

<Step S10: Culture and medium exchange just before transplantation>
Immediately before the transplantation is performed, the medium is exchanged by the same operation as steps S4 to S7.

<Step S11: Collection of transplanted tissue>
As a result of the evaluation in step S7, when it is determined that a regenerated tissue suitable for transplantation has been cultured, the tissue is collected for transplantation and used for regenerative medical treatment. After removing the culture container, it is transported to an operating room where regenerative medical treatment is performed while maintaining sterility and biological quality, and used for treatment.

<Step S12: End>
Remove the channel used for culture. Subsequently, sterilization with a sterilization gas or disinfection with ethanol is performed by an appropriate operation inside the apparatus to obtain a clean state. The various software of the automatic culture apparatus is terminated, and the operation of the automatic culture apparatus is terminated.

  As mentioned above, although an example of embodiment of this invention was demonstrated according to drawing, this invention is not limited to these Examples. For example, in the embodiment, a peristaltic pump is assumed as a fluid movement control mechanism for moving fluid, but it goes without saying that other drive mechanisms such as a syringe pump may be used.

  According to a preferred embodiment of the automatic culture apparatus configured as described above, it is possible to take out a regenerated tissue for examination or the like in advance while maintaining sterility. Since the remaining regenerated tissue after removal maintains the sterility in the same manner, the culture can be continued. In the cell seeding step and the medium exchange step, the medium always flows in one direction. In the medium exchange for exchanging the entire amount, the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved. Analysis accuracy of medium component analysis using the collected old medium is improved. After collecting the old medium, a new medium warmed to 37 ° C. in advance is supplied immediately. A part of the flow path tube attached to the closed culture vessel serves both as a liquid feeding function and an air feeding function, so that the entire flow path circuit is simplified.

  As mentioned above, although an example of embodiment of this invention was demonstrated according to drawing, this invention is not limited to these Examples. For example, in the embodiment, a peristaltic pump is assumed as a fluid movement control mechanism for moving fluid, but it goes without saying that other drive mechanisms such as a syringe pump may be used.

  As mentioned above, although an example of embodiment of this invention was demonstrated according to drawing, this invention is not limited to these Examples. For example, in the embodiment, a peristaltic pump is assumed as a fluid movement control mechanism for moving fluid, but it goes without saying that other drive mechanisms such as a syringe pump may be used.

  According to this example, in the cell seeding step and the medium exchange step, the medium always flows in one direction, so the old medium does not mix with the new medium, and the reproducibility of the culture is improved. A new medium is quickly supplied after all the old medium is discharged at the time of medium exchange.

  In addition, a part of the flow path tube attached to the closed culture vessel serves both as a liquid feeding function and an air feeding function, so that the entire flow path circuit is simplified. In particular, since the lower layer is an integrated culture vessel box, it is only necessary to connect two channel tubes in total to the lower layer culture vessel body, the channel circuit is simplified, and an electromagnetic for controlling the channel is provided. There are fewer valves.

  When the regenerative tissue in the culture container is subjected to regenerative medical treatment, it is evaluated whether it has a quality suitable for the regenerative medical treatment. Therefore, there is a need to take out one of the cell containers that are co-cultured in the same environment aseptically in advance and use them for inspection. That is, there is a case where it is desired to collect the inspection tissue (step S9) in FIG.

  The present embodiment provides a culture vessel 100 that meets such needs.

The culture container 100 of Example 2 shown in FIG. 11 has a cell bag, a culture medium bag, a drainage bag, a drainage collection bag, etc. in parallel with the culture container unit 101 for the upper layer and the culture container box 102 integrated with the lower layer. And an upper culture container unit 1101 for test and a test container box 1102 for test. The upper culture container unit for inspection 1101 and the test culture container box 1102 are co-cultured in the same environment as the culture container 100 of the first embodiment.
That is, the test upper layer culture container unit 1101 has an upper layer supply connection projection structure 1111 and an upper layer discharge connection projection structure 1113 corresponding to the upper layer supply connection projection structure 111 and the upper layer discharge connection projection structure 113. The culture container box for examination 1102 includes a culture container body corresponding to the lower layer culture container body 104 of Example 1, a lower layer supply connection projection structure 1106 corresponding to the lower layer supply connection projection structure 106, and a lower layer discharge connection. A lower-layer discharge connecting projection structure 1108 corresponding to the projection structure 108 and an opening corresponding to the opening 115 are provided. In this example, a state in which the lower layer integrated culture container box 102 is connected to the test culture container box 1102 by a second flow path circuit including the flow path tube 222 is shown. The connection relationship between the upper culture container units 1101 for testing is not shown in the figure, but one test is performed in parallel with the plurality of upper culture container units 101 having the same configuration as in Example 1. The upper layer culture container unit 1101 is placed on the culture container box 1102 for inspection, and the first flow path circuit including the flow path tube is used as a cell bag, a medium bag, a drainage bag, a drainage collection bag, etc. Just connect.

  A sterile desorption part 1103 is installed in the second flow path circuit before and after the culture container box 1102 for inspection. The aseptic desorption part 1103 can supply and supply air in the same manner as the channel tube, and can also cut the channel. The flow path after cutting maintains sterility. Further, even after the culture container box for examination 1102 is removed by the aseptic desorption part 1103, in order to continue the culture of the lower layer integrated culture container box 102, the three-branch part 1104 is provided in the second channel circuit. A bypass channel tube 1105 that bypasses the culture vessel box 1102 for inspection is attached in advance. Similarly, a bypass channel tube that bypasses the upper-layer culture vessel unit for testing 1101 is also attached in advance to the first channel circuit in the same manner by the three-branch portion.

  After the inspection culture container box 1102 is removed, liquid feeding and air supply are performed to the lower layer integrated culture container box 102 by the detour channel tube 1105. Similarly, when the test upper layer culture container unit 1101 is removed, liquid supply and air supply are performed to each upper layer culture container unit 101 by the detour channel tube 1105.

  According to the present embodiment, it is possible to perform a process including the collection of the examination tissue (step S9) in FIG. 5 as follows (the description of the same process as that of the first embodiment is omitted).

<Step S9: Collection of examination tissue>
One day out of the culture containers being cultured is collected for examination on the day before the scheduled transplant date. First, open the door of the automatic culture device. In order to avoid a temperature drop in the apparatus, only the flow path may be taken out and the apparatus door may be closed. Subsequently, the flow path tubes of the test culture container box 1102 and the test upper culture container unit 1101 are removed by the aseptic desorption part 1103. Next, the test upper culture container unit 1101 is removed from the test culture container box 1102. The removed upper layer culture container unit is promptly inspected. Similarly, the flow path after removing the upper culture container unit for testing is immediately returned into the apparatus, and the apparatus door is closed.

  During the operation up to this point, the flow path is exposed to room temperature and the temperature is lowered. Therefore, the air conditioner and the heater in the apparatus are controlled and adjusted so that the temperature quickly returns to the culture temperature of 37 ° C.

  It is evaluated whether the regenerated tissue in the culture container unit for upper layer 1101 taken out for examination has a quality suitable for regenerative medical treatment. For example, in the case of a regenerated tissue by oral mucosal epithelial cells, it has a layered structure of about 3 layers by histological evaluation, or oral mucosal stem cells are present in the basal layer of regenerated tissue by immunohistochemical staining evaluation, or oral mucosa Evaluate whether or not an epithelial cell-specific protein is expressed.

  According to the culture container of Example 2 configured as described above, it is possible to take out a regenerated tissue for testing or the like in advance while maintaining sterility. Since the remaining regenerated tissue after removal maintains the sterility in the same manner, the culture can be continued. In the cell seeding step and the medium exchange step, the medium always flows in one direction. In the medium exchange for exchanging the entire amount, the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved. Analysis accuracy of medium component analysis using the collected old medium is improved. After collecting the old medium, a new medium warmed to 37 ° C. in advance is supplied immediately. A part of the channel tube attached to the closed culture vessel serves both as a liquid feeding and a gas feeding function, so that the entire channel is simplified.

  The present embodiment is another example of the culture container 100 that meets the need to take out one of the cell containers that are co-cultured in the same environment aseptically in advance and test them. It is to provide.

  Example 3 will be described with reference to FIGS. 12A to 12D. In the culture container 100 of this example, as in Example 1, for example, ten upper-layer culture container units 101 (A to J) for culturing a regenerated tissue are integrated with a lower layer for culturing feeder cells. It can be accommodated in the culture container box 102.

  In this example, a separation membrane 403 for aseptically removing one upper layer culture container unit 101 from the lower layer integrated culture container box 102 is provided. That is, instead of the screw structure shown in FIG. 2A and FIG. 2B, an isolation film 403 is provided between each container lid member 103 and the periphery of the opening 115 of the lower layer culture container main body 104.

  The isolation film 403 shown in FIGS. 12A to 12D has a bellows shape and is welded to the container lid member 103 and the culture container box 102 integrated with the lower layer. The position is inside the O-ring 105 installed in the container lid member 103 or the lower layer integrated culture container box 102. A plurality of forms can be considered for the configuration of the isolation film 403. As a welding method, the separator is fixed by thermal welding that gives high heat during production, but the method is not limited to this method. Other welding methods for the separator include mechanical pressure, ultrasonic welding, adhesive, light irradiation using a photo-curing resin, and the like. The inside and outside of the isolation film 403 are isolated by the welded isolation film 403. At least, it is impossible to move bacteria that can cause biological contamination. Examples of the material include an elastic member such as a silicon material used for a film used for a high-pressure steam sterilization, a film used for a clave bag, or a flow tube, but is not limited to these materials. Any material can be used as long as it can be welded, cannot move bacteria, and can be quickly welded and cut when the upper-layer culture container unit is taken out.

  FIG. 12A shows a culture state in which the container lid member 103 and the lower layer integrated culture container box 102 are integrated. A culture medium 150 is contained inside the upper layer culture container unit 101 and the lower layer integrated culture container box 102. The isolation membrane 403 is accommodated between the container lid member 103 and the lower layer culture container body 104 and in the vicinity of the outer periphery of the container lid member 103. For example, the intermediate portion of the isolation film 403 is folded or rounded on the lower layer culture vessel main body 104 immediately next to the outer periphery of the vessel lid member 103, and the state is fixed with an adhesive or the like.

  On the other hand, FIG. 12B shows a state in which the bellows type isolation membrane 403 is extended to separate the container lid member 103 and the lower layer integrated culture container box 102. That is, the upper culture container unit 101 and the container lid member 103 are lifted. Since the bottom surface portion of the upper layer culture vessel unit 101 is a porous membrane, the medium 150 can be held in the upper layer culture vessel unit for a short time, for example, about several minutes. Since the isolation membrane 403 is welded to the container lid member 103 and the lower layer culture vessel main body 104 in advance, the inside of the isolation membrane 403 maintains sterility.

  As means for connecting the container lid member 103 and the lower layer integrated culture container box 102, a plurality of locking pieces 123 are provided at equal intervals along the circumferential direction on the bottom surface of the container lid member 103. The stop piece 123 is configured to be locked in a lock hole 124 provided outside the annular protrusion 1040 of the lower layer culture vessel main body 104. When both are integrated, the isolation membrane 403 is accommodated between the vessel lid member 103 and the lower layer integrated culture vessel box 102.

  Next, an arbitrary one of the upper culture container units 101 (A to J) in the upper culture state shown in FIG. 12A is replaced with a lower-layer integrated culture container box. A series of protocols for aseptic removal from the body 102 will be described.

  First, as shown in FIG. 12B, the container lid member 103 of the upper layer culture container unit 101 to be taken out and the lower layer integrated culture container box 102 are separated. Next, as illustrated in FIG. 12C, the cutting jig 407 is used to squeeze the intermediate portion of the isolation film 403 and press the isolation film 403. In this state, the part of the isolation film 403 sandwiched between the cutting jigs 407 is welded. As a method for welding the isolation film 304 at the portion sandwiched between the cutting jigs 307, heat welding is given as an example. However, there are also methods such as ultrasonic welding and mechanical high pressure welding. It is not limited.

  After the isolation film 304 is welded, the welded portion is cut. FIG. 12D shows the state after cutting the welded isolation film 403. That is, as shown in FIG. 12D, one closed space 110 surrounded by the container lid member 103, the lower layer culture container body 104, and the isolation film 403 by welding / cutting is removed from the upper layer culture container unit 101. One closed space surrounded by the container lid member 103 and the upper layer side isolation film 4031, the lower layer culture container body 104, the lower layer side isolation film 4032, and the container cover members 103 of the remaining nine upper layer culture container units 101, It is separated into another enclosed space surrounded by Since the extracted upper culture container unit 101 and the lower culture container main body 104 after being taken out are surrounded by the upper isolation film 4031 and the lower isolation film 4032, respectively, each closed space is kept closed. As a result, the internal sterility is maintained.

  According to the present embodiment, the processing including the collection of the inspection tissue (step S9) in FIG. 5 can be performed as follows. (The description of the same processing as in the first embodiment is omitted).

<Step S9: Collection of examination tissue>
One day out of the culture containers being cultured is collected for examination on the day before the scheduled transplant date. The channel tube of the upper culture container unit 101 used for the inspection is removed by the aseptic desorption part 1103. Next, the upper layer culture container unit is detached from the lower layer culture container main body 104. The isolation membrane 403 installed between the upper layer culture container unit and the lower layer culture container main body removes the upper layer culture container unit from the lower layer culture container main body. The removed upper layer culture container unit is promptly inspected. Similarly, the flow path of the culture container 100 after removing the upper culture container unit for testing is immediately returned to the apparatus, and the apparatus door is closed. During the operation up to this point, the flow path of the culture vessel 100 is exposed to room temperature and the temperature is lowered. Therefore, the air conditioner and heater in the apparatus are controlled and adjusted so that the temperature quickly returns to the culture temperature of 37 ° C. To do. It is evaluated whether the regenerated tissue in the culture container unit for the upper layer taken out for examination has a quality suitable for regenerative medical treatment. For example, in the case of a regenerated tissue by oral mucosal epithelial cells, it has a layered structure of about 3 layers by histological evaluation, or oral mucosal stem cells are present in the basal layer of regenerated tissue by immunohistochemical staining evaluation, or oral mucosa Evaluate whether or not an epithelial cell-specific protein is expressed.

<Step S11: Collection of transplanted tissue>
As a result of the evaluation, if it is determined that a regenerated tissue suitable for transplantation has been cultured, the tissue is collected for transplantation and used for regenerative medical treatment. First, the culture container is removed, and then transported to an operating room where regenerative medical treatment is performed while maintaining sterility and biological quality, and used for treatment.

  In order to maintain the sterility of the regenerated tissue as long as it is accommodated in the isolation membrane, the timing for taking out the regenerative tissue from the isolation membrane is controlled in accordance with the progress of the regenerative medical treatment. For example, it is possible to wait, transport, etc. in the operating room in a state of being accommodated in the isolation membrane.

  Thus, according to this example, it is possible to aseptically remove any one regenerated tissue from a cell container in which a plurality of cells are co-cultured. Aseptic culture can be continued for the remaining culture vessels.

  Next, as Example 4, a method for forming the isolation film 403 of Example 3 will be described. That is, in Example 4, with reference to FIG. 13, the separation membrane 403 is provided between each container lid member 103 holding the upper layer culture container unit 101 and the periphery of the opening 115 of the lower layer culture container main body 104. A manufacturing method to be provided will be described.

  First, as shown in FIG. 13, the upper layer culture vessel unit 101 is installed on the vessel lid member 103 by a hook structure or the like. Next, the upper end surface 4033 of the bellows-type isolation film 403 is welded to the lower surface of the inward projecting portion 1030 of the container lid member 103 by means such as thermal welding. In addition, the welding method can also select another method, and is not limited to thermal welding. Subsequently, the other end 4034 of the isolation membrane 403 is welded around the opening 115 of the lower layer culture vessel body 104 by means such as heat welding. The welding method is not limited to heat welding.

  Finally, the container lid member 103 is installed on the lower layer culture container body 104.

  After manufacturing the lower layer integrated culture container, sterilization is performed. When using only materials applicable to γ-ray sterilization, γ-ray sterilization is performed in a state where the entire closed system flow path connected with a flow path tube or the like is placed in a sterilization bag. At the time of use, the flow path is taken out from the sterilization bag and installed in an automatic culture apparatus to perform automatic culture. When a material that is not applicable to γ-ray sterilization is used, γ-ray sterilization is performed only on the applicable material, and the rest is performed by a method other than γ-ray sterilization, such as gas sterilization. Both materials are integrated in a safety cabinet or the like.

The isolation film 403 may have a configuration other than the bellows type described in the third embodiment.
As Example 5, with reference to FIGS. 14A to 14C, a flat type separation membrane for aseptically removing one upper layer culture container unit from the layer culture container and a method for manufacturing the same will be described.
Similar to the bellows-type isolation membrane described in Example 3, the upper and lower end surfaces of the isolation membrane 403 are welded to the vessel lid member 103 and the lower-layer culture vessel body 104, respectively. Unlike the isolation membrane described in FIG. 12, the example shown in Example 5 accommodates the isolation membrane outside the container lid member 103 and the lower culture vessel body 104. FIG. 14A holds the upper culture vessel unit 101. The state which removed the container lid | cover member 103 from the culture container main body 104 for lower layers is shown. The upper and lower ends of the isolation membrane 403 are welded to the container lid member 103 and the lower layer culture vessel body 104, respectively.

  FIG. 14B shows a state in which the container lid member 103 holding the upper layer culture container unit 101 is held in the lower layer culture container main body 104. In this state, the isolation film 403 is positioned in a state where the middle portion thereof is bent and extends to the outside of the upper layer container lid member 101 and the lower layer culture vessel main body 104.

  A portion extending outside the isolation film 403 is installed, for example, by being rounded right next to the outer periphery of the container lid member 103. Or you may make it the state which folded the part extended outside.

  FIG. 14C shows a method of accommodating the container lid member 103 and the isolation membrane 403 having portions extending outside the lower layer culture vessel main body 104 in a folded state. An intermediate portion of the isolation film 403, that is, a portion extending outward, is held between concentric position fixing jigs 801 and 802, and is bent by being pressed by a compression jig 803 from above. In this way, the isolation film 403 is compactly installed by being folded to the side of the outer periphery of the container lid member 103.

  12 (12A to 12D) and the isolation membrane of FIG. 14 (14A to 14C) are compared. In the method of accommodating the isolation membrane shown in FIG. 12, the inside of the container lid member 103 and the lower layer culture vessel body 104 Since the isolation membrane 403 is installed on the bottom, the thickness of the isolation membrane accommodated by folding is increased, and the culture container body 104 for the lower layer is increased in the vertical direction. During culture, it is necessary to observe the cells cultured in the lower culture vessel body with a microscope. However, if the lower culture vessel body becomes larger in the vertical direction, the light required for microscopic observation is attenuated or clear cells are observed. It may be difficult to adjust the depth of focus for obtaining an image. On the other hand, since no separation membrane is accommodated on the outer side of the container lid member, each of the upper-layer culture container units 101 can be brought close to the lower-layer culture container main body. The unit 101 can be arranged.

  On the other hand, in the method of accommodating the isolation membrane shown in FIG. 14, the isolation membrane is accommodated in the outer lateral portion of the vessel lid member, and accordingly, each of the upper culture container units is separated in the lower culture vessel body. There is a limitation on the number of upper-layer culture container units that can be disposed in the culture container 100. On the other hand, since the folded isolation membrane is not accommodated inside the container lid member and the lower layer culture container main body, the lower layer culture container main body does not increase in the vertical direction. Therefore, the microscopic observation is not affected.

Another configuration example of the isolation film 403 will be described in a sixth embodiment.
FIG. 15 shows an example of a lid upper surface type separation membrane for aseptically removing one upper layer culture container unit from the lower layer culture container main body.

  In Example 6, a separation membrane 403 for taking out one upper layer culture container unit from the lower layer culture container body is provided on the outer periphery of the container lid member 103 and the lower layer culture container body 104. Unlike the isolation membranes described in Example 3 and Example 5, the isolation membrane 403 is welded to the outer periphery of the container lid member 103 and the lower layer culture vessel body 104. The upper end surface of the isolation film 403 is welded to the container lid member 103 by the container lid member bonding portion 504 at the outer peripheral upper end surface of the container lid member 103. Further, the lower end surface of the isolation film 403 is welded to the upper surface of the lower layer culture vessel main body 104 by the lower layer lid bonding portion 505. In the sixth embodiment, as in the fifth embodiment, the separation membrane 403 is accommodated outside the vessel lid member 103 and the lower layer culture vessel main body 104. The upper end surface of the isolation film 403 may be welded to the container lid member 103 by the container lid member bonding portion 504 on the outer peripheral side surface of the container lid member 103.

  According to the method for housing the isolation membrane, the isolation membrane is accommodated on the outer side of the container lid member in the same manner as in Example 5, and therefore the number of upper-layer culture container units that can be placed in the culture container 100 is limited. On the other hand, since the isolation membrane is not accommodated inside the container lid member and the lower layer culture container main body, the microscopic observation is not affected.

  Note that the method of cutting the isolation film 403 by the cutting jig 307 described in the third embodiment can also be applied to cutting the isolation films of the fifth and sixth embodiments.

The isolation membrane 403 described in each of the above examples fixes the container lid member and the lower layer culture container main body by a fixing method that requires a rotating operation such as a screw as described in the first example. It can also be applied to cases. This will be described as Example 7.
FIG. 16A shows a case in which a container lid member 103 and a lower layer are formed in a culture vessel 100 in which a lid upper surface type isolation membrane 403 is installed between a container lid member 103 holding an upper layer culture container unit and a lower layer culture container main body 104. The culture vessel main body 104 is fixed by screwing screws 105 (A, B).

  However, the method of cutting the isolation film 403 by the cutting jig 307 described in Example 3 is performed when a fixing method that requires a rotating operation such as a screw for the container lid member and the lower layer culture vessel main body is employed. Is not applicable.

  A method of removing the container lid member in the case where a fixing method requiring such a rotation operation is employed will be described with reference to FIG. 16B.

  FIG. 16B shows the case of using the method of fixing the container lid member 103 and the lower layer culture vessel body 104 with screws 105 (A, B) when using the isolation membrane of each of the above embodiments. The state where 101 is removed is shown. Since the container lid member 103 is fixed to the lower layer culture container body 104 by the screws 105 (A, B), when removing from the lower layer culture container body 104, the container lid member 103 is removed while rotating. . Therefore, the isolation film 403 is twisted, and a twisted structure 1004 is generated.

In this embodiment, the upper layer culture container unit 101 is aseptically isolated by welding and cutting the twisted structure portion. As a result, when the container lid member 103 and the lower layer culture vessel body 104 are fixed by screws, the twisted structure 1004 corresponding to the narrowing is formed in the isolation film 403 when the container lid member 101 is removed while rotating. can get. That is, by separating the upper and lower sides by the twisted structure 1004, one closed space 110 surrounded by the container lid member 103, the lower layer culture container body 104, and the isolation film 403 is removed from the upper layer culture container unit 101. One closed space surrounded by the lid member 103 and the upper-layer side isolation membrane, and the other lower-layer culture container body 104 and the lower-layer side isolation membrane and the container lid member 103 of the remaining upper-layer culture container unit 101 Separated into a closed space. Since the extracted upper culture container unit 101 and the extracted lower culture container main body 104 are surrounded by the upper isolation film and the lower isolation film, respectively, each closed space is kept closed. As for each, the inside sterility is maintained.
In this embodiment, it is possible to reduce the removal protocol.

  As described above, the present invention is useful as an automatic culture apparatus for culturing cells or tissues by automatic operation using a culture vessel, particularly as an automatic culture apparatus capable of producing a regenerated tissue that can be used for regenerative medicine. .

DESCRIPTION OF SYMBOLS 100 ... Culture container 101 ... Upper layer culture container unit 102 ... Lower layer integrated culture container box 103 ... Container lid member 104 ... Lower layer culture container main body 105 ... Elastic member (O-ring)
106 ... Connection protrusion structure for lower layer supply (second supply port)
107: Lower layer supply flow path 108: Lower layer discharge connection projection structure (second discharge port)
109 ... lower layer discharge flow path 110 ... closed space 111 ... upper layer supply connection projection structure (first supply port)
112 ... Upper layer supply channel 113 ... Upper layer discharge connection projection structure (first discharge port)
114 ... upper layer discharge channel 115 ... opening 120 ... bottom surface 122 of nutrient container unit ... gap 150 ... culture basin 1010 ... outer peripheral edge 1030 ... inward projection 1040 ... annular projection 1050A, 1050B ... screw structure 202 ... cell Bag 203 ... Cell bag 204 ... Second flow path circuit 205 ... First flow path circuit 206 ... Two-way valve (solenoid valve)
207 ... Three-way valve (solenoid valve)
208 ... Fluid movement control mechanism 209 ... Multi-branch part 211 ... Medium bag 212 ... Heater 213 ... Drainage bag 214 ... Drainage recovery bag 217 ... Filter 221 ... First flow path circuit 222 ... Second flow path circuit 223 ... 1st channel circuit 224 ... 2nd channel circuit 307 ... Two-way valve (solenoid valve)
311 ... Rotating mechanism 319 ... Aseptic removal part 403 ... Isolation film 407 ... Cutting jig 600 ... Table 800 ... Table 801 ... Position fixing jig 802 ... Position fixing jig 803 ... Compression jig 1004 ... Twist structure 1102 ... For inspection Culture vessel 1104 ... Three-branch part 1105 ... Detour channel tube 1401 ... Culture container 1402 ... Control device 1403 ... Thermostat 1404 ... Temperature controller 1405 ... Gas supply part 1406 ... Temperature sensor 1407 ... Fluid movement control mechanism part 1408 ... Microscope 1410 Display screen 1411 Gas concentration adjusting unit 1412 Database.

Claims (15)

A culture vessel for holding and culturing cells,
A first container;
A plurality of second containers stored and held in the first container;
A container lid member for sealing each of the first container and the second container;
A connection port for connecting each of the first container and the second container, and a flow path circuit for supplying the cells and culture medium to the culture container;
The first container and the second container are containers for containing a medium and cells, respectively, or only the medium, respectively, and a part of the partition between each of the first container and the second container is a liquid. And gas are configured to be movable relative to each other,
Between each of the first container and the second container, there is a gap that allows gas to flow between each other,
As the connection port,
The first container is connected to the supply of the culture medium, a second supply port for supplying and discharging the gas, and a second discharge port for discharging the culture medium,
Each of the second containers is connected with a supply of the medium, a first supply port for supplying and discharging the gas, and a first discharge port for discharging the medium,
The culture container, wherein the first supply port and the second supply port function as a flow path that always allows the culture medium to flow in one direction with respect to the culture container. In claim 1,
The first supply port and the second supply port function as a flow path for flowing the culture medium only in the direction from the outside to the inside of the culture container, and a flow path for flowing the gas in both directions,
The first discharge port and the second discharge port function as a flow path for flowing the culture medium only in the direction from the inside to the outside of the culture container. In claim 2,
The first container is a box-shaped container, and has a plurality of openings provided in the container,
The container lid member has the first supply port and the first discharge port;
Each of the second containers is housed in the first container through the opening, and the container lid member corresponding to each of the second containers closes the opening. Each is fixed to one container, and thereby, a closed space surrounded by the container lid member and the first container is formed in the culture container, and each of the second containers is held in the closed space. A culture vessel characterized by. In claim 3,
It is connected between the container lid member and the first container, and has an isolation film that can accommodate the second container therein,
The isolation container has a function of maintaining the closed space even when the container lid member is removed from the opening. In claim 4,
The isolation membrane can be accommodated in the container lid member and the first container during culture,
The culture container, wherein the isolation membrane is extendable between the container lid member and the first container when the second container is taken out. In claim 4,
The separator can be separated from the second container in a state where the first container is sealed with the separator and the container lid member by welding and further cutting the separator.
The culture container, wherein the first container after the first container is separated from the second container is sealed by the isolation film and maintains the closed space. In claim 4,
The isolation membrane can be accommodated outside the container lid member and the first container during culture,
The culture container, wherein the isolation membrane is extendable between the container lid member and the first container when the second container is taken out. In claim 7,
A culture container, wherein the container lid member and the second container are connected to each other by welding the upper surface or the side surface of the container lid member to the second container. In claim 3,
The culture container, wherein the container lid member is fixed to the first container by any one of screwing, engaging, and fitting fixing means. In claim 9,
The container lid member is fixed to the first container with a screw,
When taking out the second container, the second container is isolated from the first container by welding and cutting a twisted structure portion generated by rotating the container lid member and twisting the isolation film. A culture vessel characterized in that A culture vessel for holding and culturing cells,
A first container;
A plurality of second containers stored and held in the first container;
A container lid member for sealing each of the first container and the second container;
A first container for inspection;
A second container for inspection stored and held in the first container for inspection;
An inspection container lid member;
A connection port for connecting the first container and the first container for inspection, each of the second containers and the second container for inspection, and a flow path circuit for supplying the cells and the culture medium to the culture container; With
The first container, the second container, the first test container, and the second test container are containers for containing a medium and cells, respectively, or a medium only,
Each of the first container and the second container, and the first container for inspection and the second container for inspection are configured such that a liquid and a gas can move to each other,
Between each of the first container and the second container, and between the first container for inspection and the second container for inspection, there is a gap that allows gas to flow between each other,
As the connection port,
In each of the second containers and the second container for inspection, supply of the medium, a first supply port for supplying and discharging the gas, and a first discharge port for discharging the medium Is connected,
In the first container and the first container for inspection, respectively, a supply of the medium, a second supply port for supplying and discharging the gas, and a second discharge port for discharging the medium Is connected,
The channel circuit is provided with a bypass channel tube that bypasses the first container for inspection and the second container for inspection,
The culture container, wherein the first supply port and the second supply port function as a flow path that always allows the culture medium to flow in one direction with respect to the culture container. An automatic culture apparatus for performing cell seeding and medium exchange in a culture container, and culturing cells in the culture container,
At least one closed culture vessel;
A first flow path circuit for connecting one cell bag, a medium bag, a first filter, and a drainage collection bag to the supply side of the closed culture container;
A second flow path circuit for connecting another cell bag, a medium bag, a second filter and a drainage collection bag to the supply side of the closed culture container;
A fluid movement control mechanism and an electromagnetic valve provided in the first flow path circuit and the second flow path circuit;
A control device,
The closed culture vessel is
A first container;
A plurality of second containers stored and held in the first container;
A container lid member for sealing each of the first container and the second container;
A connection port for connecting each of the first container and the second container, and a flow path circuit for supplying the cells and culture medium to the culture container;
The first container and the second container are containers for containing a medium and cells, respectively, or only the medium, respectively, and a part of the partition between each of the first container and the second container is a liquid. And gas are configured to be movable relative to each other,
Between each of the first container and the second container, there is a gap that allows gas to flow between each other,
In the first container, the second flow path circuit includes a supply of the medium, a second supply port for supplying and discharging the gas, and a second discharge port for discharging the medium. Is connected,
In the second container, the first flow path circuit includes a first supply port for supplying the medium, a first supply port for supplying and discharging the gas, and a first discharge port for discharging the medium. Is connected,
The culture medium is always supplied in one direction to the culture vessel via the first flow path circuit, the second flow path circuit, the first supply port, and the second supply port. An automatic culture device. In claim 12,
The control device temporarily maintains the leading end of the medium in the first flow path circuit in the vicinity of the first supply port, and temporarily sets the leading end of the medium in the second flow path circuit. An automatic culture apparatus characterized by having a function of maintaining a standby state in the vicinity of the second supply port. In claim 13,
It is connected between the container lid member and the first container, and has an isolation film that can accommodate the second container therein,
The isolation membrane has a function of maintaining a closed space surrounded by the container lid member and the first container in the culture container even when the container lid member is removed from the opening. An automatic culture device. In claim 14,
The isolation membrane can be housed inside or outside the container lid member and the first container during culture,
The automatic culture apparatus, wherein the isolation membrane can be extended between the container lid member and the first container when the second container is taken out.

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