Patent Application
20140302597
The present invention relates to a cell culture container, an automated cell subculture device and a cell subculture method which perform subculture of various cells.
Heretofore, subculture of various cells (floating cells, adherent cells) has been performed. In particular, anchorage-dependent cells are adhered to the bottom surface (culture surface) of the culture container and proliferated, and therefore it is necessary to pay attention to always maintaining the cell density within a constant range. If the cell density is too low and the distance between neighboring cells is too large, anchorage-dependent cells may die because of lowered proliferation rate. On the other hand, if the cell density is too high and the distance between neighboring cells is too small, the anchorage-dependent cells may stop proliferating and die in that state. Therefore, when culturing the anchorage-dependent cells, they need to be sequentially subcultured into a culture container.
In a subculture at an early stage of culture, incubation is performed in a culture container with a small culture zone, so that the density of anchorage-dependent cells is not too low. To this end, when the anchorage-dependent cells proliferate to a certain degree, they are subcultured into a culture container with a greater culture zone, or a plurality of culture containers having the same culture zone. This operation is repeated until the anchorage-dependent cells are proliferated to the target number of cells. Heretofore, in the cell culture operation involving such a subculture, the cells in the primary culture container need to be dispensed into a plurality of subculture containers by a skilled operator using a pipette. Heretofore, this operation has been disadvantageously troublesome.
Patent Literature 1 proposes a cell culture device in which a first culture unit and a second culture unit are connected by piping, and a culture medium exchange operation and a subculture operation are performed in a timely manner and automatically by a cell culture program. Moreover, Patent Literature 2 proposes a culture container having culture zones partitioned by a fixed partition piece for primary culture in a subculture container and an automatic subculture device.
However, in the cell culture devices described in Patent Literature 1 and Patent Literature 2, when cells are subcultured, a cell suspension is transferred to a subculture zone using a transfer pump, which has posed the problems of the loss of cells and the stress exerted on the cells associated with transferring, and complication of the structure of the device. Moreover, in the fixed partition structure of Patent Literature 2, uniformly inoculating cells during the subculture is difficult.
An object of the present invention is to provide a cell culture container, an automatic subculture device and a cell subculture method which is capable of performing subculturing of cells without performing a dispensing operation using a pipette, and of preventing microbial contamination in the culture container during cell subculture.
Typical examples of the inventions disclosed in the present application are as follows:
The cell culture container of the present invention includes 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 in a sealable culture container which forms a first culture zone, and has means for raising and lowering the first partition member in a state that the culture container is sealed.
Moreover, the cell culture container of the present invention 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 has means for raising and lowering the second partition member in a state that the culture container is sealed.
Moreover, the cell culture container of the present invention 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.
Another culture container of the present invention includes a fourth partition member, which forms a culture zone in a sealable culture container, and has means for moving the fourth partition member in a state that the culture container is sealed to change the area of the culture zone.
According to the present invention, the subculture efficiency of the cell culture container can be improved, and microbial contamination in the culture container can be prevented during cell subculture.
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 to
In
The flow of the subculture of this example will be described below with reference to
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 in
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 partition 4 stated above, it is desirable to use materials suitable for cell culture such as polycarbon and polystyrene.
The ceiling substrate 2 and the bottom surface substrate 3 of the culture container 1 stated 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 substrate 3 desirably 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 to
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 mechanisms 5, 6 which raise and lower the partition 4 in the culture container 1 are provided in an upper part of the ceiling substrate, but the supporting mechanisms 5, 6 may be provided on the outside of the side face of the culture container 1. In this case, it can be realized by providing a vertical moving mechanism which vertically moves the magnets 5A, 6A in place of the rotation mechanism.
In addition, a hole may be provided in the culture container 1, the supporting member may be connected with the partition 4 through this hole, and the partition 4 may 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 in
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.
In
The culture container of this example includes a movable partition 4 having a wall surface formed to be lower than a wall surface of a culture container 1 which forms a third culture zone (primary culture zone) in the sealable culture container 1 which 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 partition 14 having the wall surface formed to be lower than the wall surface of the culture container 1 which 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 container 1 is 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 to
Herein, the parts other than a movable partition 15 of
The culture container of this example, as shown in
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 to
Herein, the parts other than the movable partition 16, a magnet 16A fitted into the movable partition 16, a second target cell 17, and a culture medium 18 for second target cell in
The culture container of this example, as shown in
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 to
Herein, 19 is a control processor, and 20 is a monitor. 21 is a mixed gas producing device, 22 is a gas pump, 23 is a culture cell suspension tank, 24 is a culture medium tank, 25 is a trypsin tank, 26 is a PBS tank, 23A, 24A, 25A, 26A are electromagnetic valves connected to the corresponding tanks, respectively, and 27 is a liquid pump. It should be noted that the broken lines in
First, in the primary culture of target cells, as shown in
Next, as shown in
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 cm2 and a subculture zone of 1000 cm2 was 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 2×103 cells/cm2.
Primary culture was performed for three days by the procedure of Example 1, and about 2×106 cells were confirmed from the culture surface. In addition, a subculture was performed for three days and about 2×107 cells 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.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/078966 | 12/14/2011 | WO | 00 | 6/13/2014 |
