JP4361291B2 - Cell culture vessel - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cell culture container in which seed cells after culturing in a seed culture container are transferred to the main culture container and continuously subjected to main culture to be harvested.
[0002]
[Prior art]
Conventionally, when a considerable amount of cells are harvested by culturing and proliferating cells, a method of culturing by taking a predetermined stage is taken due to its characteristics (scale-up). That is, inoculate a small amount of seed cells into a small seed culture container (seed cell culture container) at the stage of seed culture, increase the inoculated seed cells to a certain amount, and then a considerable number of the same size as the seed cell container. In this method, the cells are transferred to a vessel or a culture vessel that is several to 10 times larger than the seed cell culture vessel, and further, the cells are continuously cultured and passed through a stage of growth (main culture). Such a culture method cannot be performed by the techniques described in, for example, JP-A-6-38734 and JP-A-5-38281.
Therefore, at present, in the case of manual work, inoculate cells in a dish prepared with a medium, and after culturing (seed culture), evenly transferred to a plurality of dishes with the same size medium, cultured and harvested (main culture) Is taken.
In addition, as a culture container when used in an automatic cell culture apparatus, a main culture container that is several to ten times larger than a seed culture container connected by a tube is used.
[0003]
[Problems to be solved by the invention]
However, in the case of manual work, there is a problem that the work of transferring the cells from the individually formed seed culture container to the main culture container becomes complicated and requires a lot of labor.
In addition, in a conventional culture vessel used for an automatic cell culture apparatus capable of mass processing (production), a seed culture vessel and a main culture vessel for performing main culture are connected by a tube, so that two The handling of the seed culture container and the main culture container, which are culture containers, is inconvenient, resulting in a situation where the tube may be cut or detached, which may hinder sterility maintenance, seed cells, culture media, and various drugs. There are various problems in that the mechanism for feeding them becomes complicated and the cost of sterilization of the container increases.
[0004]
The present invention has been devised in view of the above problems, and based on the knowledge obtained by the present inventor, for example, suitable for regenerative medicine of mesenchymal stem cells instead of the conventional cell culture container, is inexpensive. It is an object of the present invention to provide a cell culture container that is easy to handle and safe.
[0005]
[Means for Solving the Problems]
In order to achieve the above object, the present invention adopts the following configuration. That is, the present invention is the outer shell of the upper surface, inoculated port, the medium supply port, and a seed culture vessel agent supply port is provided, is provided at the bottom of this seed culture vessel, as with the seed culture vessel And a main culture vessel provided with an inoculation port, a medium supply port, and a drug supply port in the outer shell on the upper surface, and the main culture vessel is communicated with the seed culture vessel and cultured in the seed culture vessel. The cultured cells are collected so as to flow down into the main culture vessel and are subjected to main culture.
[0006]
The cell culture container according to claim 1 is a cell culture container in which a main culture container is integrally provided in a lower part of a seed culture container, and an inoculation port, a culture medium are provided on an outer shell of the seed culture container. Provided with a supply port and a drug supply port, and disposed inside the seed culture vessel below the inoculation port, the medium supply port, and the drug supply port, and subjected to a seed culture that has been subjected to a surface treatment that increases cell adhesion While forming a compartment, a drainage compartment arranged on one side of the seed culture compartment, and a cell recovery compartment arranged on the other side of the seed culture compartment, the main culture container is similar to the seed culture container The outer shell is provided with an inoculation port, a medium supply port, and a drug supply port, and in the same manner as the seed culture container, inside the main culture container, below the inoculation port, the medium supply port, and the drug supply port. Surface treatment that is placed and increases cell adhesion Forming a main culture compartment, a drainage compartment arranged on one side of the main culture compartment, and a cell recovery compartment arranged on the other side of the main culture compartment, and the drainage compartment of the seed culture vessel It communicates with the drainage compartment of the main culture container disposed below, the cell recovery compartment of the seed culture container communicates with the culture compartment of the main culture container disposed below, and further the cells of the main culture container A cell collection port for taking out the cells to the outside is provided in the collection compartment, and a drainage port for taking out the waste solution to the outside is provided in the drainage compartment of the main culture container.
[0007]
Further, the present invention is the addition to form the bottom of the culture container and the seed culture vessel with a transparent member even without low, the seed culture vessel, a part of the bottom of the seed culture vessel, protrudes from the main culture vessel In this way, the cells are formed so as to be observable with a microscope by observing cells adhering to the main culture vessel and the protruding bottom portion of the seed culture vessel.
[0008]
Further, the present invention is that the pre-Symbol main culture container and the seed culture vessel, characterized in that it is formed of polystyrene.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
In the following, an embodiment of the present invention will be described in detail with reference to FIGS.
FIG. 1 is an external perspective view of the entire cell culture container in the present embodiment, and FIG. 2 is a side view of the cell culture container as viewed from the direction of arrow II in FIG. 1, and in particular, the arrangement of the seed culture container and the main culture container It is a side sectional view shown with a perspective section for easy understanding of the relationship (in addition, the arrangement of the medium supply port and the drug supply port is different from the other drawings, but is functionally equivalent). FIG. 3 is an external perspective view highlighting the external structure of the cell culture container, and FIG. 4 is a partially broken external perspective view showing the internal structure in an easy-to-understand manner by partially cutting the cell culture container. FIG.
[0010]
The cell culture container according to the present embodiment includes, for example, a box-shaped seed culture container 1 (transparent body) formed of a synthetic resin material such as polystyrene, and a larger capacity (shape) than the seed culture container 1. It consists of a box-shaped main culture vessel 2 (transparent body) molded of a synthetic resin material such as polystyrene. The seed culture container 1 is integrated with the corner of the upper surface of the main culture container 2 in an offset arrangement so that a part of the bottom part protrudes rearward outside, and the seed culture container 1 and the main culture container 2 are integrated. The internalized cell culture container can be observed from the outside.
[0011]
First, the configuration of the seed culture container 1 will be described. That is, the outer shell 1a on the upper surface of the seed culture container 1 contains an inoculation port 1c for inoculating cells (tissue) with an air filter 1b, a medium supply port 1d for supplying a medium, for example, a growth promoting substance. A drug addition port 1e for adding a drug, a drug containing a cleaning agent, a drug containing a cell release agent, or the like is provided. The inside of the seed culture vessel 1 includes a seed culture compartment 1A in the center along the front-rear direction, a drain compartment 1B to the right of the seed culture compartment 1A, and a cell recovery compartment 1C to the left of the seed culture compartment 1A. It is divided into two compartments. That is, the seed culture section 1A is disposed below the inoculation port 1c, the medium supply port 1d, and the drug addition port 1e. The partition walls 1A1 and 1A2 are provided on both the left and right sides, and between the first half portions of the partition walls 1A1 and 1A2. A V-shaped upper wall 1A3 is formed. Further, the bottom of the seed culture section 1A is subjected to surface treatment by, for example, ultraviolet irradiation or corona discharge so as to increase the adhesion of cells. On the other hand, such a surface treatment is not applied to the drainage compartment 1B and the cell recovery compartment 1C so that cells do not adhere and grow. Furthermore, a drainage communication hole 1B1 for discarding the supernatant (old medium) into the main culture container 2 is provided at the front end of the drainage compartment 1B, and cells are stored at the front end of the cell recovery compartment 1C. A cell supply hole 1C1 for transferring (supplying) the culture vessel 2 is provided.
[0012]
In this way, by tilting the seed culture container 1 integrated with the main culture container 2 to the right, the liquid that has become unnecessary for seed culture flows from the seed culture section 1A over the partition wall 1A1 to the drainage section 1B. By tilting leftward, the cells can flow from the seed culture section 1A over the partition wall 1A2 to the cell collection section 1C and transferred to the main culture container 1.
[0013]
Next, the configuration of the main culture container 2 will be described. The main culture vessel 2 has the same structure as the seed culture vessel 1 described above. That is, the outer shell 2a on the upper surface of the main culture vessel 2 contains an inoculation port 2c for inoculating cells (tissue) with an air filter 2b, a medium supply port 2d for supplying a medium, for example, a growth promoting substance. A drug addition port 2e for adding a drug, a drug including a cleaning agent, a drug including a cell peeling agent, and the like is provided. The inside of the main culture vessel 2 includes a main culture section 2A in the center along the front-rear direction, a drainage section 2B to the right of the main culture section 2A, and a cell recovery section 2C to the left of the main culture section 2A. It is divided into two compartments. That is, the main culture section 2A is disposed below the inoculation port 2c, the medium supply port 2d, and the drug addition port 2e, and the partition walls 2A1, 2A2 are provided on both the left and right sides, and between the first half portions of the partition walls 2A1, 2A2. A V-shaped upper wall 2A3 is formed. Further, the bottom of the main culture section 2A is subjected to a surface treatment by, for example, ultraviolet irradiation or corona discharge so as to increase the adhesion of cells.
[0014]
On the other hand, such a surface treatment is not performed on the drainage compartment 2B and the cell collection compartment 2C so that cells do not adhere and grow. Furthermore, at the front end of the drainage compartment 2B, there is a nozzle-shaped drainage port 2B1 for taking out (discarding) the supernatant (old medium) to the outside, and at the front end of the cell recovery compartment 2C, A nozzle-shaped cell recovery port 2C1 for taking out the cells to the outside is provided.
[0015]
In this way, by tilting to the right of the main culture vessel 2, the liquid that has become unnecessary for the culture passes over the partition wall 2A1 from the main culture section 2A, flows out into the drainage section 2B, and passes through the drainage port 2B1. Further, by tilting to the left, the main cultured cells can pass over the partition wall 2A2 from the main culture section 2A, flow out to the cell recovery section 2C, and be taken out to the outside via the cell recovery port 2C1. It becomes possible.
[0016]
In addition, a communication pipe 1D bent in an L shape is attached to the cell supply hole 1C1 of the cell collection section 1C of the seed culture container 1, and an outlet thereof opens above the main culture section 2A. As a result, as shown in FIG. 5 (5), the seed culture vessel 1 is tilted so that the cells in the cell collection section 1C are once poured into the communication pipe 1D and received in the state shown in FIG. 5 (6). It can be made.
[0017]
During culture, the medium supply ports 1d and 2d and the drug supply ports 1e and 2e are respectively plug members (not shown) for blocking communication with the outside in order to maintain an aseptic condition as an optimum culture condition. ) Is provided, and only when a culture medium or a medicine is supplied, the stopper member is removed and a supply tube having a pinch valve is connected. Similarly, the cell recovery port 2C1 and the drainage port 2B1 are normally plugged with a plug member (not shown).
[0018]
Next, the operation of the present embodiment will be described along the cell culture procedure with reference to FIG. Since the arrangement of the medium supply ports 1d and 2d and the drug supply ports 1e and 2e shown in FIG. 5 is schematically shown, it is functionally different from those shown in FIGS. Are equivalent.
(Disposal of chemicals and medium during seed culture)
The seed cells are inoculated into the seed culture section 1A of the sterilized seed culture container 1. Next, an appropriate amount of a medium is filled from the medium supply port 1d and a drug is filled from the drug supply port 1e, and the seed cells are cultured (see FIG. 5 (1)). The depth of the culture medium in the seed culture section 1A is, for example, about 5 mm from the bottom (the same applies to the main culture section described later). After a predetermined time has elapsed, as shown in FIG. 5 (2), the seed culture container 1, that is, the cell culture container is tilted to the right as a whole, and when tilted 90 degrees as shown in FIG. The (supernatant) flows over the partition wall 1A2 of the seed culture section 1A and flows into the drainage section 1B of the seed culture container 1. Further, when the cell culture container is returned to a horizontal state as a whole (see FIG. 5 (4)), the used medicine and the old medium are discharged from the drainage compartment 2B of the main culture container 2 via the drainage communication hole 1B1. Through the drainage port 2B1.
[0019]
A new medium and an appropriate amount of a drug such as a growth promoting substance are supplied to the seed culture section 1A and the culture is continued. After a certain period of time, the used medicine and the old medium are again discharged to the outside by the above method. Such an operation is repeated several times.
[0020]
(Transfer of seed cells to main culture section)
After treatment with a drug such as a cell release agent, this time, when the cell culture container is tilted to the left as shown in FIG. 5 (5), the medium containing the cells crosses the partition wall 1A1 of the seed culture compartment 1A and becomes a cell. When it flows into the collection compartment 1C and the cell culture container is in a vertical state (see FIG. 5 (6)), it temporarily accumulates in the communication pipe 1D via the cell supply hole 1C1. Then, when the posture of the cell culture container is again returned to the horizontal state as shown in FIG. 5 (7), the medium containing the seed cells is supplied to the main culture section 2A of the main culture container 2 by flowing down. It becomes.
Note that the volume of the cell collection compartment 1c is set to be about 1.5 times larger than the amount (volume) of the culture solution, so that the cell culture container can be made as shown in FIGS. 5 (5) to 5 (1). Even if the state is brought down (returned), it is possible to avoid returning the culture medium to the original place.
[0021]
Further, in the main culture container 2, the same culture operation as that performed in the seed culture container 1 is performed. The used medicine and the old medium are moved to the right by tilting the cell culture container over the partition wall 2A2 and the drainage compartment 2B. Can be discharged to the outside through the drainage port 2B1. In addition, after discharging the waste liquid to the outside, a new medium, a growth promoter, or the like is introduced from the medium supply port 2d or the drug supply port 2e, and the culture is continued. When the main culture is completed, the main culture vessel 2, that is, the cell culture vessel as a whole is tilted to the left and poured into the cell recovery section 2C so as to get over the partition wall 2A1, and a medium containing cells from the cell recovery port 2C1. Is taken out to the outside.
[0022]
Further, in the seed culture and main culture processes, as shown in FIG. 2, the bottoms of the containers 1 and 2 are brought into contact with the bottoms of the seed culture container 1 and the main culture container 2 that are arranged in an offset manner. It is possible to observe cells that are attached to and proliferate, and culture can be performed while observing.
[0023]
According to this embodiment, mesenchymal stem cells and the like inoculated from a single patient are cultured, and in order to obtain a considerable amount, while changing the medium and adding various drugs at regular intervals, Although the culture is performed, the integrated cell culture container performs these operations by a method using a gravitational action when transferring a substance. For this reason,
It is possible to discharge a used drug or an old medium without connecting a tube to the medium supply ports 1d and 2d and the drug supply ports 1e and 2e, or to transfer cells from the seed culture container 1 to the main culture container 2. Therefore, handling of the cell culture container is simplified, sterility maintenance can be secured, and the cost required for sterilization can be reduced.
[0024]
In addition, although the said embodiment demonstrated the case where the material of the cell culture container was made into polystyrene, you may form with synthetic resin materials other than polystyrene, or a transparent glass material. In this case, the seed culture section and the main culture section are appropriately subjected to a surface treatment that enhances cell adhesion.
In the above embodiment, the partition walls 1A1, 1A2, 2A1, and 2A2 are formed so as to be substantially vertical from the bottom, but they can be inclined so as to spread outward. In addition, the air filters 1b and 2b are integrally provided at the inoculation ports 1c and 2c, but instead, the air filters 1b and 2b may be formed separately from the inoculation ports 1c and 2c.
[0025]
【The invention's effect】
As described above, according to the present invention, since the seed culture container is provided on the main culture container, the cells cultured in the seed culture container are tilted into the main culture container disposed immediately below the seed culture container. Can be moved by using the action of gravity to flow down, so that it is possible to prevent the conventional tube from being cut off or detached and hindering the maintenance of sterility. In addition, the cell culture container can be easily handled.
[0026]
According to the invention relating to the cell culture container of claim 1 , the seed culture container is provided with a seed culture compartment, a drainage compartment, and a cell recovery compartment, and the main culture container provided at the lower part of the seed culture vessel is also provided. Similarly, the main culture compartment, drainage compartment, and cell collection compartment are provided, while the cell collection compartment of the seed culture vessel communicates with the main culture compartment of the main culture vessel. Until the seed cells are proliferated, the waste liquid can be used to remove used medicine and old media from the drainage compartment of the seed culture container by tilting the entire cell culture container in an appropriate direction. It can be discharged from the drainage port through the drainage compartment of the culture vessel.
[0027]
When a predetermined amount of cells is seed-cultured in the culture compartment of the seed culture container, the seed cells cultured in the seed culture compartment of the seed culture container are treated with a drug such as a cell release agent, and then the cell culture container is By tilting, it is allowed to flow down from the cell collection compartment of the seed culture vessel to the culture compartment of the main culture vessel. The seed cells after being transferred to the main culture container are subjected to main culture in the main culture section of the main culture container. In the main culturing process, the main culturing is performed by adding a drug or the like added from a drug supply port or the like in the outer shell of the main culture container, or exchanging a medium. In the process until the main culture is completed, the used waste liquid is discharged to the outside through the drainage port by tilting the entire cell culture container and flowing to the drainage compartment. When the main culture is completed in the main culture section, the main cultured cells are treated with a drug such as a cell release agent, and then the entire cell culture container is tilted and transferred to the cell recovery section of the main culture container. It comes out from the mouth.
[0028]
Thus, in the invention according to claim 2, as in the invention according to claim 1, the mechanism of feeding of seed cells, culture media, various drugs, etc. is formed using the weight action of the substance. Since the structure can be formed simply, the cell can be easily cultured, and the cell can be cultured without contact with external air during the process of seed culture to main culture. The sterilization cost and the separation and collection of the waste liquid and the cells can be efficiently performed. As a result, the cost required for the cell culture can be greatly reduced as a whole.
Then, by performing an operation of tilting the seed culture container, the seed cells can be easily transferred to the main cell compartment of the main cell container while maintaining aseptic conditions, and effects such as efficient cell culture can be achieved.
[0029]
Further, according to the present invention, since the culture state of the cells can be observed with a microscope in the seed culture and the culture process by the main culture, there is an effect that the target cells can be obtained smoothly.
[0030]
According to the invention described in claim 4, since the seed culture vessel and the main culture vessel are formed of polystyrene (transparent synthetic resin material), the mirror surface is attached to the cell by, for example, ultraviolet irradiation or corona discharge. Since the surface treatment can be performed so as to enhance the adhesion, when the cell culture container is tilted and the waste liquid is discharged, it is possible to prevent the cells in the culture compartment from peeling off and falling off.
[Brief description of the drawings]
FIG. 1 is an external perspective view of an entire cell culture container according to an embodiment of the present invention.
FIG. 2 is a side view of the cell culture container as viewed from the direction of arrow II in FIG. 1, and is a side cross-sectional view particularly shown in a transparent cross section so that the arrangement relationship between the seed culture container and the main culture container can be easily understood.
FIG. 3 is an external perspective view showing the appearance structure of a cell culture container with emphasis.
FIG. 4 is a partially cutaway external perspective view showing a part of a cell culture container in a broken view so that the internal structure can be easily understood.
FIG. 5 is an operation explanatory diagram for explaining the mechanism of cell transfer and drug disposal in the cell culture container according to the present embodiment.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Seed culture container 1A ... Seed culture compartment 1B ... Drainage compartment 1C ... Cell collection | recovery compartment 1B1 ... Drainage communication hole 1C1 ... Cell supply port 1a, 2a ... Outer shell 1A1, 1A2 on upper surface ... Partition wall 1D ... Communication pipe 2 ... main culture vessel 2A ... main culture compartment 2B is drainage compartment 2C ... cell collection compartments 2A1, 2A2 ... partition wall 2B1 ... drainage port 2C1 ... cell collection ports 3A, 3B ... microscope

Claims (1)

A cell culture container in which a main culture container is integrally provided at a lower portion of a seed culture container, and an inoculation port, a medium supply port, and a drug supply port are provided in an outer shell of the seed culture container, and the seed culture container A seed culture compartment disposed below the inoculation port, the medium supply port, and the drug supply port and subjected to a surface treatment for increasing cell adhesion, and a drainage solution disposed on one side of the seed culture compartment While forming the compartment and the cell collection compartment arranged on the other side of the seed culture compartment, the inoculation port, the medium supply port, and the drug supply are provided in the outer shell of the main culture container in the same manner as the seed culture vessel A book which is provided with a mouth and is subjected to a surface treatment to increase cell adhesion, which is arranged inside the main culture container below the inoculation port, the medium supply port, and the drug supply port in the same manner as the seed culture container. Culture compartment, on one side of the main culture compartment A drainage compartment placed on the other side of the main culture compartment, and a drainage compartment of the seed culture vessel communicated with a drainage compartment of the main culture vessel placed below The cell collection compartment of the seed culture container is communicated with the culture compartment of the main culture container disposed below, and further, a cell collection port for taking out the cells to the cell collection compartment of the main culture container, A cell culture vessel, wherein a drainage port for taking out the waste solution from the outside is provided in the drainage compartment of the main culture vessel.

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