CELL CULTURE CONTAINER, AND AUTOMATED CELL SUBCULTURE DEVICE AND CELL SUBCULTURE METHOD USING SAME

Provided is a cell culture container that makes it possible to subculture cells without a dispensing operation using a pipette and also to prevent microbial contamination in the culture container during cell subculture. The cell culture container is provided, within a culture container (1) forming a sealable first culture zone, with a partition member (4) having a wall surface formed lower than the wall surface of the culture container and forming a second culture zone; and has means for raising and lowering the partition member while the culture container is in a sealed state. Magnets (4A, 5A, 6A) are used as the means for raising and lowering the partition member.

DESCRIPTION OF EMBODIMENTS

The embodiments of the present invention will be described with reference to drawings. In each of the drawings, the identical components are given the identical numbers, and repeated explanation is omitted.

One constitutional example of the cell culture container of Example 1 of the present invention will be described with reference toFIG. 1.

InFIG. 1,1is a cell culture container,2is a ceiling substrate of the culture container1, and3is a bottom surface substrate of the culture container1. The area of the bottom surface substrate3partitioned by the frame of the culture container1is a first culture zone (subculture zone).4is a movable partition, and4A is a magnet fitted into the movable partition4. The movable partition4is lower than the wall surface of the culture container1, and the region the bottom surface substrate3of the partitioned culture container1is a second culture zone (primary culture zone).5and6are supporting mechanisms which are located outside the ceiling substrate of the culture container1,5A and6A are magnets which are fitted into the supporting mechanisms5,6, respectively, and5B and6B are rotation mechanisms which rotate the supporting mechanisms5,6, respectively.7is a first target cell,8is a culture medium which provides nutrient compositions of cells.9is the inlet and outlet of the culture medium8,10is the inlet of mixed gas (air including 5% of CO2, and at humidity of 90% or higher), and11is the outlet for mixed gas.12is a drive mechanism for shaking the culture container1for uniform inoculation and cell peeling as well as for inclining the culture container1for discharging culture medium and the like.13is an observation mechanism for observing cells.

The flow of the subculture of this example will be described below with reference toFIGS. 1 and 2. First, in the primary culture of target cells, as shown inFIG. 1, after the culture medium8is provided from a culture medium inlet outlet9to the primary culture zone in the movable partition4, the suspension of the target cell is poured into the container. At this time, the magnets5A,6A in the supporting mechanisms5,6are in such a state that they face the magnet4A in the movable partition4with the same poles facing each other (N-pole against N-pole), and the movable partition4is closely fitted to the bottom surface substrate3. In this state, the entire culture container1can be shaken by the drive mechanism12to uniformly inoculate the cells. In addition, a mixed gas (air containing 5% of CO2, and at humidity of 90% or higher) required for cell culture is supplied from a mixed gas inlet10into the culture container1. Since the movable partition4is lower than the wall surface of the culture container1, the mixed gas can be supplied to the primary culture zone. The culture process of cells are observed and measured by the observation mechanism13.

Around the time when the cell proliferation limit is reached on the primary culture surface, the culture medium of the primary culture zone is discharged, and the cells are washed with PBS (physiological saline). Trypsin is then poured into the primary culture zone to remove the cells off from the culture surface, and the culture medium is then poured to cause the cells to float in the culture medium. Next, as shown inFIG. 2, the supporting mechanisms5,6are rotated 180° by the rotation mechanisms5B,6B, so that the magnets5A,6A in the supporting mechanisms5,6face the magnet4A in the movable partition4with their different poles facing each other (S-pole against N-pole). As a result, by the attractive force of the magnets, the movable partition4is detached from the bottom surface substrate3by the supporting mechanisms5,6located on the outside of the culture container, and is raised to the ceiling substrate2of the culture container. The partition4detaches from the bottom surface substrate3, whereby a gap is produced between the partition4and the bottom surface substrate3, and the cell suspension which is in the primary culture zone is diffused in the subculture zone of the cell culture container1having a culture surface greater than the primary culture zone. In addition, a culture medium which is suitable for the culture zone is poured into the subculture zone from the culture medium inlet outlet9, and the cells are uniformly dispersed into the subculture zone by shaking with the drive mechanism12. After the cells are adhered to the culture surface, the culture medium is exchanged to remove trypsin and subculturing is then performed. After the subculturing, a cell suspension containing more cells can be collected by an operation similar to the process described above.

The above-mentioned non-contact subculture operation in a closed system culture container is realized by using the magnet mechanism, and microbial contamination in the culture container during subculturing can be prevented. In addition, during the subculturing operation, since a transfer pump is not required, no loss of cells or stress on cells associated with transferring by the pump occurs.

For the components of the movable partition4stated above, it is desirable to use materials suitable for cell culture such as polycarbon and polystyrene.

The ceiling substrate2and the bottom surface substrate3of the culture container1stated above can be formed from base materials of solid substrates such as glass, silicon halides, quartz, or plastics and polymers. More desirably, these substrates have such optical transparency that can be observed by an optical microscope and other means, and further the bottom surface substrate3desirably has a material on which cleaning can be performed before depositing cells onto the surface and surface reforming of the substrate by preprocessing.

In this example, the magnets are used as means for raising and lowering the partition member. The principle of transfer using the magnets will be described with reference toFIG. 3. A magnet is a substance which has two magnetic poles (N-pole, S-pole) and serves as a source of production of a bipolar magnetic field (on the left hand ofFIG. 3). The magnetic poles of the magnet do not exist solely, but both poles always constitute a magnet together. When two magnets are approached to each other, a force which attracts each other (attractive force) acts between different poles (on the right hand ofFIG. 3), while a force which repels each other (repulsive force) acts between the same poles (at the center ofFIG. 3). In addition to permanent magnets (ferrite magnets, neodymium magnets, etc.), electromagnets which temporarily produce a magnetic force by energized coils are also available.

It should be noted that in the above example, the configurations of the outer frame and partition of the culture container being square have been described, but it goes without saying that they may be circular, polygonal or in other shapes.

Moreover, in this example, it has been described that the supporting mechanisms5,6which raise and lower the partition4in the culture container1are provided in an upper part of the ceiling substrate, but the supporting mechanisms5,6may be provided on the outside of the side face of the culture container1. In this case, it can be realized by providing a vertical moving mechanism which vertically moves the magnets5A,6A in place of the rotation mechanism.

In addition, a hole may be provided in the culture container1, the supporting member may be connected with the partition4through this hole, and the partition4may be moved by moving this supporting member. In this case, the sealing property between the hole and supporting member needs to be ensured.

In addition, in this example, the subculture by means of the culture container by using the subculture of adherent cells has been explained, but the culture container of this example can also be applied to the subculture of floating cells.

Moreover, in this example, it has been stated that the operation from the primary culture to subculture is automatically performed, but as shown inFIG. 4, the culture container of this example can also be applied to subculture by manual operation.

The cell subculture method of Example 1 is a cell subculture method which uses a cell culture container including a partition member having a wall surface formed to be lower than a wall surface of the culture container which forms the second culture zone in a sealable culture container which forms a first culture zone, and having means for raising and lowering the partition member in a state that the culture container is sealed, the method including the steps of culturing cells in the second culture zone in a state that the partition member is lowered, raising the partition member, and dispersing the cells cultured in the second culture zone into the first culture zone, and culturing cells in the first culture zone and the second culture zone.

According to this example, the subculture efficiency of the cell culture container can be improved, and microbial contamination in the culture container can be prevented during cell subculture.

FIG. 5shows a constitutional example of the cell culture container of Example 2 of the present invention. Example 2 is a cell culture container with a two-passage structure.

InFIG. 5, the parts other than a movable partition14and a magnet14A fitted into the movable partition14are similar to those in Example 1.

The culture container of this example includes a movable partition4having a wall surface formed to be lower than a wall surface of a culture container1which forms a third culture zone (primary culture zone) in the sealable culture container1which forms a first culture zone (second-passage subculture zone), has means for raising and lowering the partition in a sealing state, and includes the movable partition14having the wall surface formed to be lower than the wall surface of the culture container1which forms a second culture zone (first passage subculture zone) in the first culture zone, and has means for raising and lowering the partition in a state that the culture container1is sealed.

The cell subculture method of Example 2 is a cell subculture method which uses a cell culture container which includes, in a sealable culture container which forms a first culture zone, a first partition member having a wall surface formed to be lower than a wall surface of the culture container which forms a second culture zone, has means for raising and lowering the first partition member in a state that the culture container is sealed, and further includes a second partition member having a wall surface formed to be lower than a wall surface of the culture container which forms a third culture zone in the second culture zone, and having means for raising and lowering the second partition member in a state that the culture container is sealed, the method including a step of culturing cells in the third culture zone in a state that the second partition member is lowered, a step of raising the second partition member in a state that the first partition member is lowered and a dispersing the cells cultured in the third culture zone into the second culture zone, a step of culturing cells in the third culture zone and the second culture zone in a state that the first partition member is lowered, a step of raising the first partition member and dispersing the cells cultured in the third culture zone and the second culture zone into the first culture zone, and a step of culturing cells in the first culture zone and the second culture zone and the third culture zone.

The culture container of this example is capable of performing a primary culture and a two-passage subculture in a single closed section, and of preventing microbial contamination in the culture container during subculturing. In addition, during the subculturing operation, since a transfer pump is not required, no loss of cells or stress on cells associated with transferring by the pump occurs.

With reference toFIGS. 6 and 7, the cell culture container of Example 3 of the present invention will be described. Example 3 includes a partition with a straight-line partition structure.

Herein, the parts other than a movable partition15ofFIGS. 6 and 7and a magnet15A fitted into the movable partition15are similar to those in Example 1.

The culture container of this example, as shown inFIG. 6, after the primary culture is performed in the primary culture zone with a small area in the frame of the culture container1, depending on the cell subculture, as shown inFIG. 7, the movable partition15is moved to an appropriate position to ensure a cell subculture zone. The moving of the movable partition15may be performed by the magnets provided on the outside of the culture container as shown in Example 1, or a hole may be provided on the side face of the culture container, a supporting member may be connected with the movable partition15through this hole, and the movable partition15may be moved by moving this supporting member.

According to this structure, a culture zone for the primary culture and subcultures (several passages allowed) can be freely provided depending on cell types and inoculation concentrations, and the non-contact operation in the closed system culture container can be realized, so that microbial contamination in the culture container during subculturing is prevented. In addition, during the subculturing operation, since a transfer pump is not required, no loss of cells or stress on cells associated with transferring by the pump occurs.

The cell subculture method of Example 3 is a cell subculture method which includes a partition member which forms a culture zone into the sealable culture container, and uses a cell culture container having means for moving the partition member in a state that the culture container is sealed and changing the area of the culture zone, the method including a step of locating the partition member in an initial position to culture cells in the culture zone, a step of moving the partition member to expand the area of the culture zone, and a step of culturing cells in the culture zone with the area thereof expanded.

With reference toFIGS. 8 and 9, the cell culture container of Example 4 of the present invention will be described. Example 4 relates to a cell culture container of with a co-culture structure.

Herein, the parts other than the movable partition16, a magnet16A fitted into the movable partition16, a second target cell17, and a culture medium18for second target cell inFIGS. 8 and 9are similar to those in Example 1.

The culture container of this example, as shown inFIG. 8, using the movable partition4and the movable partition16, the first target cell and second target cell are cultured in the respective primary culture zones in the frame of the culture container1, and when subculturing, as shown inFIG. 9, the movable partition4and the movable partition16are opened to perform co-culturing, so that cell incubation in which both cells can promote the proliferation of each other can be realized.

The cell subculture method of Example 4 is a cell subculture method which uses a cell culture container which includes, in a sealable culture container which forms a first culture zone, a first partition member having a wall surface formed to be lower than a wall surface of the culture container which forms a second culture zone, has means for raising and lowering the first partition member in a state that the culture container is sealed, and further includes a third partition member having a wall surface formed to be lower than a wall surface of the culture container which forms a third culture zone which does not overlap the second culture zone, and has means for raising and lowering the third partition member in a state that the culture container is sealed, the method including a step of culturing cells in the second culture zone and the third culture zone in a state that the first partition member and the third are lowered, a step of raising the first partition member and the third partition member and dispersing the cells cultured in the second culture zone and the third culture zone into the first culture zone, and a step of culturing cells in the first culture zone and the second culture zone and the third culture zone.

An automatic cell subculture system of Example 5 of the present invention will be described with reference toFIG. 10. The same parts as in the above examples will be referred to by the same numerals below, and their explanation will be omitted, while only different parts will be described.

Herein,19is a control processor, and20is a monitor.21is a mixed gas producing device,22is a gas pump,23is a culture cell suspension tank,24is a culture medium tank,25is a trypsin tank,26is a PBS tank,23A,24A,25A,26A are electromagnetic valves connected to the corresponding tanks, respectively, and27is a liquid pump. It should be noted that the broken lines inFIG. 10are electric signal lines connected to the control processor19and the respective electric control parts. The control flowchart of the same is shown inFIG. 11.

First, in the primary culture of target cells, as shown inFIG. 1, after the culture medium8is provided from a culture medium inlet outlet9to the primary culture zone in the movable partition4(ST1), the suspension of the target cells is poured from the culture cell suspension tank (ST2). At this time, the magnets5A,6A in the supporting mechanisms5,6are in such a state that they face the magnet4A in the movable partition4with the same poles facing each other (N-pole against N-pole). The entire culture container is shaken by the drive mechanism12to uniformly inoculate cells (ST3). In addition, a mixed gas (air containing 5% of CO2, and at humidity of 90% or higher) required for cell culture is supplied from a mixed gas inlet10into the culture container (ST4), to perform primary culture (ST5). Since the movable partition4is lower than the wall surface of the culture container1, the mixed gas can be supplied to the primary culture zone. The culture process of the cells is observed and measured by the observation mechanism13. Around the time when the cell proliferation limit is reach on the primary culture surface, the culture medium of the primary culture zone is discharged (ST6), and PBS (physiological saline) is poured from the PBS tank26to wash the cells (ST7). Trypsin is then poured into the primary culture zone from the trypsin tank25to remove the cells off from the culture surface (ST8), and the culture medium is then poured to cause the cells to float in the culture medium (ST9).

Next, as shown inFIG. 2, the supporting mechanisms5,6are rotated 180° by the rotation mechanisms5B,6B, so that the magnets5A,6A in the supporting mechanisms5,6face the magnet4A in the movable partition4with their different poles facing each other (S-pole against N-pole). As a result, by the attractive force of the magnets, the movable partition4is detached from the bottom surface substrate3by the supporting mechanisms5,6located on the outside of the culture container and is raised to the ceiling substrate2of the culture container. The partition4is detached from the bottom surface substrate3, whereby a gap is produced between the partition4and the bottom surface substrate3, and the cell suspension which is in the primary culture zone is diffused in the subculture zone of the cell culture container1having a culture surface greater than the primary culture zone (ST10). In addition, a culture medium which is suitable for the culture zone is poured into the subculture zone from the culture medium inlet outlet9, and the cells are uniformly dispersed into the subculture zone by shaking with the drive mechanism12(ST11). After the cells are adhered to the culture surface, the culture medium is exchanged (ST12) to perform subculturing (ST13) so as to remove trypsin. After the subculturing, a cell suspension containing more cells can be collected by an operation similar to the process described above (ST17).

An example of cell culture using Example 1 will be described. A culture container having a similar structure to that in Example 1 with the area of a primary culture zone of 100 cm2and a subculture zone of 1000 cm2was used. Moreover, the cells used were 3T3 cells (fibroblast culture cell strain derived from mouse skin), and the culture medium used was DMEM with calf serum and an antibiotic added to it. It should be noted that the inoculation density of the 3T3 cells is 2×103cells/cm2.

Primary culture was performed for three days by the procedure of Example 1, and about 2×106cells were confirmed from the culture surface. In addition, a subculture was performed for three days and about 2×107cells could be collected from the culture surface.

The present invention is not limited to the above-described Examples unless the features of the present invention are damaged, and other forms which are conceivable within the scope the technical ideas of the present invention are also included in the scope of the present invention.

REFERENCE SIGNS LIST