Cell culture system

An operation isolator forms an aseptic space. An incubator is connected to the operation isolator, in which cells are stored and cultured. A storage chamber stores articles used in the operation isolator. In order to carry articles from the outside into the storage chamber, a decontamination pass-box is provided. The storage chamber and the operation isolator are directly or indirectly connected to each other.

TECHNICAL FIELD

The present invention relates to a cell culture system used in a regenerative medicine, in which damaged biofunctions are rejuvenated using stem cells, and particularly relates to a cell culture system, in which an incubator for storing and culturing cells is connected to an operation isolator for preparing cells.

BACKGROUND ART

As disclosed in PATENT DOCUMENT 1, an organ cell obtained from a multifunctional stem cell such as an iPS cell, a vascular endothelial cell forming a vascular endothelium, or a mesenchymal cell forming a support structure for a cell functioning in tissue is cultured. A co-culture is then performed with the optimum mixture ratio so that a steric organ bud having a microvascular structure can be induced in a test tube and eventually transplanted into an organism to produce an organ.

In order to culture such cells or tissue, there is known a system, which comprises an isolator forming an aseptic space, and an incubator detachably connected to the isolator to store and culture cells, to perform necessary operations for culturing in the isolator. In a system disclosed in PATENT DOCUMENT 2, for example, two isolators connected to incubators are connected to each other through a sterilized chamber, so that different kinds of cells can be cultured in each of the incubators.

SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

When culturing different cells and performing a co-culture, it is necessary to prepare each cell separately so that a cultured cell can be mixed with the other cell. This preparation is required to be promptly performed in a short amount of time. In a conventional system, however, many articles cannot be stored in the isolator, and thus for each cell, articles needed for culturing such as material, an apparatus, culture vessel and so on have to be subjected to sterilization as necessary, and transported from outside. Because of this, an operation necessary for culturing a cell cannot be performed effectively.

In the system disclosed in PATENT DOCUMENT 2, since two isolators are connected through the sterilized chamber, a preparation for different cells, which have been simultaneously cultured at the same time, can be performed. In this case, when the cells are transferred between the isolators, contamination by various germs from outside should not occur. The cells delivered from one isolator are transferred to the other isolator through the sterilized chamber, which should be sterilized for each transfer since the sterilized chamber has a portion exposed to the outside. Thus, it is difficult to perform the preparatory operation in the isolator within a short amount of time.

The object of the present invention is to provide a cell culture system, by which an operation needed to culture cells in the operation isolator can be performed effectively and promptly in a short amount of time while cells being treated are not contaminated by various germs.

Means for Solving the Problems

A cell culture system of the present invention, which has an operation isolator forming an aseptic space and an incubator connected to the operation isolator for storing and culturing cells, comprises a storage chamber for storing an article, which is used in the operation isolator, and a pass-box through the article is transferred from the outside into the storage chamber, the storage chamber and the operation isolator being directly or indirectly connected to each other.

The cell culture system may be provided with a transportation isolator, which is disposed with a transporting mechanism in an aseptic space therein, and a plurality of operation isolators and incubators may also be provided. In this case, it is preferable that the plurality of operation isolators is arranged along the transportation path and connected to the transportation isolator, and the storage chamber and the operation isolator are connected to each other through the transportation isolator. In such a structure, the cell culture system may further comprise a plurality of containers containing the article, the article being stored in the storage chamber and transported by the transporting mechanism in a state in which the article is contained in the container. The transportation path of the transportation isolator may be formed in a straight line, and the transporting mechanism may transport the container in a reciprocal manner.

Preferably, the storage chamber is detachably provided.

Further, different kinds of cells may be cultured in the plurality of incubators, and the cells cultured in the different incubators may be mixed in any one of the operation isolators. Alternately, the same kind of cells may be cultured in the plurality of incubators, and the cells cultured in the different incubators may be mixed in any one of the operation isolators.

Effects of the Invention

According to the present invention, an operation needed to culture cells in the operation isolator can be performed effectively and promptly in a short amount of time while treated cells are not contaminated by various germs.

EXPLANATION OF REFERENCES

EMBODIMENT OF THE INVENTION

In the following, the present invention will be described with reference to embodiments shown in the drawings.

FIG. 1shows the general structure of a cell culture system to which a first embodiment of the present invention is applied. A transportation isolator11is an elongated box-shaped casing extending directly upward and downward inFIG. 1. The inside of the transportation isolator11is maintained at a positive pressure, and is not in communication with the outside of the cell culture system to form an aseptic space. A belt conveyor (transporting mechanism)12is disposed in the transportation isolator11. The belt conveyor12, which is configured such that the transportation path is formed in a straight line along the longitudinal direction of the transportation isolator11, reciprocally transports a cage C, which is a container containing an article. Articles such as material, an apparatus, culture vessel and so on, which are needed for culturing and are transported from outside of the cell culture system, are contained in the cage C. Thus, the first through third operation isolators21-23and a storage chamber52, which are described later, are arranged along and connected to the linearly formed transportation path, and the belt conveyor12is moveable forwards and backwards, so that the cage C is promptly transported and the transportation speed is easily controlled when the transportation is automated. Further, since the transportation isolator11provided to cover the transportation path is formed in a simple elongated box-shape, decontamination of the inside space can be performed easily and with certainty without any blind spots. Note that the cage C may be directly mounted on the belt conveyor12, or may be housed in a tray or the like.

InFIG. 1, first through third operation isolators21-23are arranged along the transportation path and connected to the left side of the transportation isolator11. The first through third operation isolators21-23extend parallel to each other in a vertical direction with respect to the longitudinal direction. The inside of each of these operation isolators21-23is maintained at a positive pressure, and is not in communication with the outside of the cell culture system to form an aseptic space.

The first operation isolator21is provided with gloves24for opening and closing doors32and35described later, and for carrying out various operations. The first operation isolator21is connected to the transportation isolator11through an opening31, which is opened and closed by a door32. When the door32is closed, the first operation isolator21is shut off airtight from the transportation isolator11. When the door32is open, a cage C can be transferred between the inside of the first operation isolator21and the inside of the transportation isolator11.

An incubator33for storing and culturing cells is connected to the first operation isolator21through a connecting device34. An opening provided on the connecting device34side of the first operation isolator21is opened and closed by a door35, and the connecting device34is provided on an outer wall of the first operation isolator21to enclose the door35. The first operation isolator21is shut off airtight from the inside space of the connecting device34by the door35. An opening provided on the connecting device34side of the incubator33is opened and closed by a door36, and the connecting device34is provided to enclose the periphery of the door35. The inside of the incubator33is sealed airtight by the door36. The incubator33can be separated from the first operation isolator21under the condition in which the doors35and36are closed.

A decontamination gas supply device37, which supplies decontamination gas such as hydrogen peroxide vapor, for example, is connected to the first operation isolator21and the connecting device34. That is, while the door32is closed and the doors35and36are open, the insides of the first operation isolator21and the incubator33are decontaminated. On the other hand, when the incubator33is attached or detached, the inside of the connecting device34is supplied with decontamination gas while the doors35and36are closed to perform a decontaminating operation for the exposed portions of the doors35and36.

The second and third operation isolators22and23have similar structures as those of the first operation isolator21. That is, an incubator41is connected to the second operation isolator22through a connecting device42, and a decontamination gas supply device43is connected to the second operation isolator22and the connecting device42. Similarly, an incubator44is connected to the third operation isolator23through a connecting device45, and a decontamination gas supply device46is connected to the third operation isolator23and the connecting device45. InFIG. 1, common references to the first operation isolator21are shown regarding the opening31, doors32,35, and36, and gloves24.

A decontamination pass-box51is provided on the right side of the transportation isolator11inFIG. 1. The decontamination pass-box51is provided for transferring articles such as material, an apparatus, culture vessel and so on, which are needed for culturing cells, from the outside into the transportation isolator11. That is, in the decontamination pass-box51, the articles and the cage C are decontaminated, and the articles are then transferred into the cage C. Further, cells to be cultured are transferred through the decontamination pass-box51.

The decontamination pass-box51is connected to the transportation isolator11through the storage chamber52. That is, the storage chamber52is disposed between the decontamination pass-box51and the transportation isolator11, and a cage C, in which an article is contained, is temporarily stored in the storage chamber52. Gloves61for opening and closing a door56described later, and for carrying out various operations are provided for the decontamination pass-box51.

The decontamination pass-box51and the storage chamber52are positioned facing the first operation isolator21, and extend in a direction opposite to the first operation isolator21. The storage chamber52is connected to the transportation isolator11through an opening53, so that the cage C can be transferred between the inside of the storage chamber52and the inside of the transportation isolator11. Due to this construction, the inside of the storage chamber52is maintained at a positive pressure to form an aseptic space similarly to the inside of the transportation isolator11, and the storage chamber52and the first operation isolator21are indirectly connected to each other through the transportation isolator11.

The decontamination pass-box51is connected to the storage chamber52through an opening55, which is open and closed by the door56. When the door56is closed, the decontamination pass-box51is shut off airtight from the storage chamber52, and when the door56is open, the cage C can be transferred between the inside of the decontamination pass-box51and the inside of the storage chamber52.

A transportation opening57is formed in a portion of the decontamination isolator51opposite to the storage chamber52, and is opened and closed by a door58. The door58is exposed to the outside of the cell culture system, and an article can be transferred in and out of the decontamination pass-box51by opening the door58. When the door58is open, the door56is closed, and thus the storage chamber52and the transportation isolator11are not in communication with the outside. That is, the storage chamber52and the transportation isolator11form an aseptic space.

A decontamination device59is connected to the decontamination pass-box51and the transportation isolator11. While the doors56and58are closed, the inside of the decontamination pass-box51and articles such as material, an apparatus, culture vessel and so on, which are transferred from the outside, are decontaminated. A decontaminating operation of the inside of the transportation isolator11is performed under the condition in which the door32and so on, which are provided between the transportation isolator11and the first through third operation isolators21-23, are closed while the inside of the storage chamber52is decontaminated.

With reference toFIGS. 2 and 3, the construction of the storage chamber52is described.

The storage chamber52is provided with a motor roller conveyor71and three belt conveyors81. The motor roller conveyor71is arranged on the side of a front window63to transport a cage C storing articles such as material, an apparatus, culture vessel and so on, which are needed for culturing cells, between the openings53and55, i.e., right and left directions inFIG. 2. Each of the belt conveyors81transports the cage C in up and down directions perpendicular to the transporting direction of the motor roller conveyor71, and transfers the cage C from the motor roller conveyor71to a storage area A, or transfers it from the storage area A to the motor roller conveyor71.

The example ofFIG. 2is shown such that three cages C are mounted on the motor roller conveyor71and three cages C are mounted on each of the belt conveyors81, but a maximum of six cages C can be mounted in the storage area A. That is, when six cages C are stored in the storage area A, there is no cage C on the motor roller conveyor71. Note that the number of cages C which can be stored in the storage area A is merely one example, and the number of belt conveyors81is also merely one example. The numbers can be freely determined in accordance with the object.

The motor roller conveyor71has a plurality (nine in the drawing) of rollers72, which are arranged in parallel to each other. Each of the rollers72is rotationally supported about the axis by a support mechanism73, and is rotated in forward and reverse directions periodically by a drive mechanism not shown, and thus, a cage C is transported from the opening55to the opening53or in the opposite direction. The support mechanism73is driven up and down by a piston of an air cylinder74, and each of the rollers72is set to either an up-position, at which the roller is projected above the conveying surface of the belt conveyors81, or a down-position, at which the roller is depressed into the conveying surface of the belt conveyors81. That is, the conveying surface of the motor roller conveyor71is set to the up-position when transporting the cage C, and is set to the down-position when not transporting the cage C.

The nine rollers72of the motor roller conveyor71can be driven or stopped in groups of three rollers. That is, inFIG. 2, the three rollers72positioned at the opening55side, the three rollers72positioned in the center, and the three rollers72positioned at the other opening53side are independently controlled from each other. For example, under the condition in which only the three rollers72on the opening55side are stopped, the six rollers72in the center and other opening53side can be driven.

Each of the belt conveyors81has the same structure with a pair of belts82and83. These belts82and83are extended in the longitudinal direction of the rollers72, and positioned between two adjacent rollers72. One end of the belts82and83is wound around pulleys85and86, which are provided at both ends of a support shaft84, and the other end of the belts is wound around pulleys88and89, which are provided at both ends of a support shaft87. Between the support shafts84and87, the belts82and83are wound around a drive shaft92of a drive motor91and idler pulleys93and94, which are provided below the support shafts84and87, and are rotated forwards and backwards by the drive motor91, so that the cage C can be transported between the motor roller conveyor71and the storage area A.

A stopper95is provided on the opposite side of the belt conveyor81with respect to the motor roller conveyor71, and on the outside of the pair of belts82and83. The stopper95is driven to move up and down by a piston of an air cylinder96, and rises and falls between an up-position, which is higher than the conveying surface of the belt conveyors81, and a down-position, which is lower than the conveying surface. When the stopper95is in the up-position, the cage C supported by the stopper95is released upward from the belt conveyor81and the cage C, which is transported from the motor roller conveyor71side to the storage area A, comes into contact with the stopper95and stops.

As shown inFIG. 3, a first guide member64is provided at the end portion of the belt conveyor81closest to the front window63side of the motor roller conveyor71. The first guide member64is arranged along the transporting direction of the motor roller conveyor71, and extends from a portion close to the opening53to a portion close to the other opening55. A second guide member65, which extends in parallel to the first guide member64, is provided at the end portion of the belt conveyor81opposite to the motor roller conveyor71. A cage C can be engaged with the first and second guide members64and65. The first guide member64guides the cage C when the cage C is transported by the motor roller conveyor71, and functions as a stopper for the cage C at the end of the belt conveyor81, which also applies to the second guide member65.

The belt conveyor12of the transportation isolator11, and the motor roller conveyor71and the belt conveyor81of the storage chamber52are driven by a manual operation of an operator turning on and off the electric power supply. The up and down movements of the motor roller conveyor71and stopper95are driven by a manual operation of an operator activating the air cylinders74and96.

An operation of the embodiment is described below.

Prior to introducing a culture of cells, the insides of the decontamination pass-box51, the storage chamber52, the transportation isolator11, the operation isolators21,22, and23, and the incubators33,41, and44are decontaminated. Articles such as material, an apparatus, culture vessel and so on, which are needed for culturing cells, are carried in the decontamination pass-box51, where the articles are decontaminated and stored in cages C. The motor roller conveyor71is set to the up-position, so that the cages C are carried into the storage chamber52one by one, and the maximum number of cages C that are mounted on the motor roller conveyors71is three.

In this state, if additional cages C are introduced from the decontamination pass-box51, a cage C, which is not required to be immediately transferred to the operation isolator, is moved from the motor roller conveyor71to the storage area A. In this case, the motor roller conveyor71is lowered, and the cage C is moved by the belt conveyor81. At this time, if there is no cage C at the position of the stopper95in the storage area A, the stopper95is set to the down-position, and the cage C is transported above the stopper95until it stops by coming into contact with the second guide member65. Conversely, when the cage C is transported onto the stopper95, as shown inFIG. 3, the stopper95is set to the up-position, so that the next cage C transported by the belt conveyor81stops when it comes into contact with the stopper95.

When the cage C stored in the storage area A is transferred to the transportation isolator11, the cage C is moved by the belt conveyor81until it stops when it comes into contact with the first guide member64. The motor roller conveyor71is then moved upward to operate, and the cage C is carried to the transportation isolator11side. When the cage C positioned at the stopper95is moved to the motor roller conveyor71, the stopper95is first set to the down-position, by which the cage C is mounted on the belt conveyor81and moved above the motor roller conveyor71.

The three cages C on the motor roller conveyor71are moved to the transportation isolator11and are transported to the first, second, and third operation isolators21,22, and23by the belt conveyor12of the transportation isolator11. That is, although the first cage C is initially placed on the belt conveyor12, the first cage C is immediately carried into the first operation isolator21and the second cage C is moved to the second operation isolator22by the belt conveyor12and carried into the second operation isolator22. The third cage C is transported to the third operation isolator23by the belt conveyor12and carried into the third operation isolator23. At this time, cages C, which store simultaneously cultured cells, can be carried into each of the operation isolators21,22, and23.

In each of the operation isolators21,22, and23, packaging for material, an apparatus and a culture vessel stored in the cages C is removed, and the cells are seeded in the culture vessel. In this embodiment, for example, organ cells obtained from human iPS cells are seeded in the operation isolator21, vascular endothelial cells are seeded in the operation isolator22, and mesenchymal cells are seeded in the operation isolator23. These culture vessels are transferred into the incubators33,41, and44, which are then separated from the operation isolators, and culturing is performed for a predetermined period of time. During the culturing period, the incubators33,41, and44connected to the operation isolators21,22, and23as necessary, to carry out a medium replacement or passage.

When the culturing of each of the cells is completed, the incubators33,41, and44are connected to the operation isolators21,22, and23, and the culture vessels, in which these three kinds of cells are stored, are removed from the operation isolators21,22, and23, using predetermined treatments for picking up the cells from the culture vessels. The picked-up cells are stored in a special container, and then stored in cages C, which are transferred to the transportation isolator11and collected in either one of the different operation isolators21,22, and23by the belt conveyor12. The three kinds of cells are mixed with each other at a predetermined mixture ratio in the operation isolator where the cells are collected. The culture vessel storing the mixed cells is transferred to the incubator to which the operation isolator is connected, and for a predetermined period of time, a co-culture is performed in the incubator which is separated from the operation isolator, such that a primordium of human liver is created. The cells, for which the co-culture is completed, are taken out of the incubator and placed in the operation isolator connected to the incubator. In the operation isolator, the cells are then subjected to a predetermined treatment, in which the cells are picked up from the culture vessel and stored in a special container. The container is then transported in the transportation isolator11and the storage chamber52to the outside through the decontamination pass-box51. Note that the mixed cells are not necessarily different kinds of cells, but may be the same kind of cells separately cultured and then mixed together. Further, it is not necessary to co-culture cells after mixing, because the cells may be transported outside in the mixed state.

In the embodiment as described above, the cells cultured in the incubators33,41, and44are transferred between the first through third operation isolators21,22, and23through the transportation isolator11. The transportation isolator11forms an aseptic space, which is not required to be decontaminated every time the transfer of cells between the operation isolators21,22, and23is performed. Therefore, operations using a plurality of incubators, which are required for a culture of cells, can be promptly carried out in a short amount of time, without mixing various germs in treated cells.

In the embodiment described above, although the first through third operation isolators21,22, and23and the incubators33,41, and44are provided, these components can be added and connected to the transportation isolator11, as indicated by a broken line B inFIG. 1. In preparation for such addition, the opening31and the door32may be provided beforehand as shown by a solid line. Further, the storage chamber52and the decontamination pass-box51may be connected to the opening31as shown in a broken line D, and the decontamination pass-box51may be provided for a specific use such as carrying an article out of the system. Furthermore, the operation isolator, the storage chamber, and the decontamination pass-box may be detachably provided, so that they can be increased or decreased as necessary.

Although the storage chamber52in the embodiment is configured to dispose six cages C on a plane for storing them in the storage area A, it is possible to provide the storage spaces vertically in a multistage structure, such that a plurality of cages C is sterically arranged and stored. In this case, an elevator, which moves a cage C up and down in the storage spaces, is provided between the openings53and55, and a moving mechanism composed of a motor roller conveyor, belt conveyor, and so on, which moves the cage C between the elevator and the openings53and55, and moves the cages C between the elevator and each storage space, is provided. Further, the belt conveyor12is not necessarily formed along a straight line, but may have a curved portion or may be formed in a circular shape, in accordance with the layout of the installation site. Furthermore, keeping the cage C on the belt conveyor12, articles such as material, an apparatus, culture vessel and so on, which are needed for culturing cells, can be temporarily stored in the transportation isolator11. As the moving mechanism, a device other than the belt conveyor12can be adopted, which transports a cage C or tray while mounted on the mechanism, such as a rod-less cylinder, an electric linear actuator, or a linear motor car or vehicle moving along a guide rail. A device that transports a cage C or tray while holding it, such as a robot and so on, can also be adopted. Note that the structure of the moving mechanism should be as simple as possible, since decontamination becomes difficult if the structure of the moving mechanism is complex.

FIG. 4shows a general structure of a cell culture system to which a second embodiment of the present invention is applied. The difference from the first embodiment is the direct connection between the storage chamber52and the first operation isolator21. In the second embodiment, the storage chamber52and the first operation isolator21are connected through the opening31, which is opened and closed by the door32, so that a cage C can be transferred between the inside of the storage chamber52and the inside of the first isolator21. The decontamination pass-box51is connected to the storage chamber52through the opening55, which is similar to the first embodiment, and the decontamination gas supply device59is connected to the decontamination pass-box51and the storage chamber52. The other structures are identical to the first embodiment, and the same references are assigned.

In the second embodiment described above, a plurality of incubators33are prepared, and can be connected to or detached from the first operation isolator21, so that a preparation for cells can be performed in the first operation isolator21, and the same or different kind of cells can be cultured in each of the incubators33. The cultured cells are sequentially prepared and mixed with each other in the first operation isolator21, and stored and co-cultured in any one of the incubators33connected thereto. In this case, articles such as material, an apparatus, culture vessel and so on, which are needed for the culturing of cells performed in the first operation isolator21, have already been carried into the storage chamber52and stored there by the time they are to be used, and thus they can be carried in and used in the first operation isolator21as necessary. Especially, since the decontamination pass box51is connected to the storage chamber52and the decontamination pass box51and the storage chamber52can be isolated by the door32, articles such as material, an apparatus, culture vessel and so on can be sequentially carried into the decontamination pass-box51from the outside, decontaminated therein, and contained in cages C and stored in the storage chamber52, while a preparation is performed in the first operation isolator21under the condition in which the first operation isolator21and the storage chamber52are isolated. In the second embodiment, similar to the first embodiment, an operation required for culturing cells can be effectively performed.

In a third embodiment shown inFIG. 5, the first operation isolator21connected to the storage chamber52in the second embodiment is connected to the transportation isolator11, as a fourth operation isolator25. The structure of the fourth operation isolator25is similar to those of the first through third operation isolators21-23. That is, an incubator47is connected to the fourth operation isolator25through a connecting device48, and a decontamination gas supply device49is connected to the fourth operation isolator25and the connecting device48. InFIG. 5, regarding the opening31, the doors32,35, and36, and gloves24, the common references to the first through third operation isolators21-23are assigned, and the fourth operation isolator25is connected to the transportation isolator11through the opening31.

The storage chamber52is connected to the fourth operation isolator25through the opening53, which is similar to the connection to the transportation isolator11, so that a cage C can be transferred between the inside of the storage chamber52and the inside of the fourth operation isolator25. Further, the decontamination pass-box51is connected to the storage chamber52through the opening55and the decontamination gas supply device59is connected to the decontamination pass-box51and the transportation isolator11, which is similar to the first embodiment.

According to such a structure, the fourth operation isolator25is directly connected to the storage chamber52, and the first through third operation isolators21-23are indirectly connected to the storage chamber52through the fourth operation isolator25and the transportation isolator11.

In the third embodiment described above, in a similar way as the first embodiment, articles such as material, an apparatus, culture vessel and so on, which are needed for culturing cells, are carried into and decontaminated in the decontamination pass-box, contained in a cage C and stored in the storage chamber52. For carrying the cages C in the first through third operation isolators21-23, the cages C are transferred from the storage chamber52to the fourth operation chamber25, and transported to each of the operation isolators21,22, and23through the transportation isolator11. Further, it is possible that, in the fourth operation isolator25, the cells are seeded in a culture vessel using material, an apparatus, culture vessel and so on housed in the cage C, and stored in the incubator47to culture. According to the third embodiment described above, effects similar to those of the first embodiment can be obtained.