JP2004097046A - Culture container - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture container which has a simple structure and can reduce contamination caused by dusts, bacteria, and the like in each treating process. <P>SOLUTION: This culture container 1 comprises a culture container 2 manufactured by connecting at least one main culture container 4, at least one culture medium container 5 having a culture medium sealed therein, and at least one waste culture medium container 6 capable of receiving a liquid in the main culture container 4 to each other through connection pipes 7, 8 capable of limiting the flow of the liquid therebetween in a state closed from the outside, and a vertical interval-adjusting means 3 capable of adjusting the heights of the culture medium container 5 and the waste culture medium container 6 with respect to the main culture container 4. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a culture device for culturing cells.
[0002]
[Prior art]
To culture the cells, an extraction step of extracting cells to be cultured from a body fluid such as a bone marrow fluid extracted from a patient, a medium preparation step of preparing a medium suitable for the cells to be cultured, and A plurality of steps are sequentially performed, such as a primary culturing step in which the medium is put into a culture vessel together with the medium and placed under predetermined culturing conditions, and a secondary culturing step in which the primary cultivated cells are mixed with a living tissue supplement and further cultured. Is
[0003]
Conventionally, it has been considered that such cells are cultured in a clean bench in which the whole is sealed and the amount of dust inside is controlled (for example, see Patent Document 1).
[0004]
[Patent Document 1]
Japanese Patent Publication No. Hei 3-57744 (page 2, column 3, etc.)
[0005]
That is, in the clean bench, for example, an airflow that flows from the ceiling side to the floor side is formed, and when dust and the like are generated in each processing step, the dust and the like flow to the floor side by the airflow. And collected by a dust collector placed under the floor. A robot arm is arranged in the clean bench so that cells can be moved between each step.
[0006]
[Problems to be solved by the invention]
However, when all the processing steps are performed in a relatively large clean bench as described above, since the space where each step is performed is continuous, dust generated in one step is distributed to another step. May be contaminated with cells that have been used. Therefore, when culturing a plurality of cells at the same time, it is necessary to take sufficient care not to cause mixing between the cells or to contaminate the substance to be added.
[0007]
The present invention has been made in view of the above circumstances, and has as its object to provide a culture vessel and a culture apparatus that can reduce contamination by dust, bacteria, and the like in each processing step with a simple configuration. I have.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides the following means.
The invention according to claim 1 is characterized in that at least one main culture container, at least one culture container enclosing a culture medium, and at least one waste medium container capable of containing a fluid in the main culture container, A culture vessel connected in a sealed state to the outside by a connection pipe capable of restricting the flow of a fluid, and a height difference adjusting means capable of adjusting the heights of a culture vessel and a waste culture vessel with respect to the main culture vessel, respectively. A culture device is provided.
[0009]
According to the present invention, the cells are sealed in the main culture container, the restriction on the flow of the fluid in the connection pipe between the culture container and the main culture container is released, and the height difference adjusting means is operated to adjust the culture medium. By arranging the container at a position higher than the main culture container, the medium flows into the main culture container from the medium container through the connecting pipe. In this state, by restricting the flow of the fluid in the connection pipe again, the inside of the main culture vessel is sealed from other vessels. This makes it possible to culture the cells in the main culture vessel sealed from the outside.
[0010]
Further, when it is necessary to replace the medium in the main culture vessel after a predetermined time has elapsed, the flow restriction of the connecting pipe between the main culture vessel and the waste culture vessel is released, and the height difference adjusting means is used. Is operated to arrange the main culture container at a position higher than the waste medium container, whereby the medium flows from the main culture container toward the waste medium container. Thereby, it becomes possible to discharge the waste medium that has become unnecessary in the main culture vessel to the waste culture vessel.
[0011]
Then, the culture of the cells is continued by flowing the culture medium again from the culture vessel toward the main culture vessel to restrict the flow of the fluid in the connection pipe.
As described above, according to the culture container of the present invention, in a completely sealed container, it is possible to perform cultivation of cells and exchange by supply / discarding of a medium required for culturing cells by gravity. .
[0012]
The invention according to claim 2 is the culture apparatus according to claim 1, wherein the height difference adjusting means is provided rotatably around the longitudinal axis with the longitudinal axis arranged horizontally, and the main culture vessel and the culture medium vessel are provided. And a culture apparatus comprising a rotating drum for fixing waste medium containers at different circumferential positions.
According to the present invention, it is possible to easily adjust the height difference between the main culture container, the medium container, and the waste medium container fixed at different circumferential positions by the rotation of the rotary drum. Further, by continuously rotating the rotating drum, it becomes possible to use the drum as a centrifuge.
[0013]
The invention according to claim 3 provides the culture device according to claim 2, wherein the culture container is provided so as to be mounted on an outer surface of the rotary drum.
According to the present invention, by allowing the culture vessel to be mounted on the outer surface of the rotating drum, it is possible to easily perform a replacement operation when performing culture by replacing a large number of culture vessels with a common rotating drum. .
[0014]
According to a fourth aspect of the present invention, in the culturing apparatus according to the second or third aspect, a position in which the culture vessel is disposed radially outward in the main culture vessel in a state where the culture vessel is fixed to a rotating drum. And a culture device provided with a take-out port capable of taking out the cells inside.
According to the present invention, when the cultured cells are finally recovered from the main culture vessel, the rotating drum is rotated, and only the cells in the main culture vessel are centrifuged from other fluids. It is possible to collect cells at an outlet disposed radially outward of the main culture vessel.
[0015]
According to a fifth aspect of the present invention, there is provided the culture apparatus according to any one of the first to fourth aspects, wherein the connecting pipe includes a valve, and the valve allows or restricts the flow of the fluid in the connecting pipe. I will provide a.
According to the present invention, it is possible to control the flow of the fluid in the connecting pipe by controlling the valve. Thereby, the connecting pipe can be easily opened and closed, and the steps of inflow of the culture medium from the culture vessel to the main culture vessel, discharge of the culture medium from the main culture vessel to the waste culture vessel, and culture of the cells in the main culture vessel are switched. It becomes possible.
[0016]
According to a sixth aspect of the present invention, in the culture apparatus according to any one of the first to fourth aspects, the connecting pipe is formed of a flexible material, and is formed from a tube in which the flow of an internal fluid is restricted by being crushed. The present invention provides a culture apparatus having a pinch valve that restricts the flow of a fluid by sandwiching the tube in the radial direction from the outside and allows the flow of the fluid by opening the tube.
According to this invention, when the connecting pipe is pushed in the radial direction from the outside by the operation of the pinch valve, the connecting pipe made of a flexible material is crushed, and the internal flow path is closed. This limits the flow of the fluid. Therefore, the configuration of the culture vessel can be simplified without providing a valve in the connecting tube. When the culture container is disposable for each cell, it is preferable because the cost can be reduced.
[0017]
According to a seventh aspect of the present invention, in the culturing apparatus according to any one of the first to sixth aspects, at least one enzyme container enclosing a protease is contained in the main culture container. A culture tube in which the height difference adjusting means is capable of adjusting the height of the enzyme container with respect to the main culture container, by a connection pipe capable of restricting the concentration of the enzyme container.
[0018]
According to the present invention, for example, when culturing adherent cells such as mesenchymal stem cells, the cells adhere to and grow on the inner wall of the main culture vessel after a predetermined culture period has elapsed. Therefore, when such cells are collected, after the medium in the main culture container is discharged into the waste medium container, the flow restriction of the connecting pipe between the enzyme container and the main culture container is released, and the height difference is increased. The adjusting means is operated to arrange the enzyme container at a position higher than the main culture container. Thus, the protease can be supplied into the main culture vessel to detach the cells from the inner wall of the main culture vessel.
[0019]
BEST MODE FOR CARRYING OUT THE INVENTION
Before describing the embodiment of the present invention, a manufacturing process of a bone filling material as a living tissue filling material will be schematically described. In order to produce a bone filling, as shown in FIG. 17, first, bone marrow fluid is collected from a patient's iliac bone or the like. The collected bone marrow fluid is centrifuged and swirled to extract bone marrow cells having a high specific gravity.
[0020]
The extracted bone marrow cells are put into a culture vessel together with a previously prepared medium and mixed. A portion of the medium is removed and sent for infection testing.
After this, the mixed bone marrow fluid and medium are brought to a predetermined temperature (eg, 37 ± 0.5 ° C.), humidity (eg, 100%) and CO 2 ₂ By maintaining the culture conditions such as the concentration (for example, 5%), the cells are primarily cultured under a constant culture condition for a predetermined time. At a predetermined exchange time during the culturing of the cells, the medium is discarded from the inside of the culture vessel. Then, the medium is mixed again, and the culturing step is repeated and continued. A portion of the discarded medium is sent for infection testing.
[0021]
After a predetermined culture period is over, after the medium is discarded from the culture vessel, a protease such as trypsin is charged and mixed into the culture vessel. As a result, the mesenchymal stem cells attached to and grown on the bottom surface of the culture container are detached from the bottom surface of the main culture container. The mesenchymal stem cells thus detached are extracted by being subjected to a centrifugal separator.
[0022]
The extracted mesenchymal stem cells are mixed in a culture vessel in which a bone filling material and an appropriate medium have been charged after the cell number is adjusted. In practice, mesenchymal stem cells are attached to a bone substitute and injected into a medium. Then, in the same manner as above, the mixed mesenchymal stem cells and the medium are brought to a predetermined temperature (for example, 37 ± 0.5 ° C.), humidity (for example, 100%) and CO 2. ₂ By maintaining the culture conditions such as the concentration (for example, 5%), the cells are subcultured under a constant culture condition for a predetermined time.
[0023]
In the secondary culture step as well, as in the primary culture step, the medium is changed periodically, and a part of the medium to be supplied and a part of the medium to be discarded are each sent for an infection test. Then, when a predetermined culture period has elapsed, a sample is extracted for quality inspection and infection inspection for shipping, and the manufactured bone filling material is sealed and provided as a product.
[0024]
Next, a culture device according to a first embodiment of the present invention will be described below with reference to the drawings.
The culture apparatus 1 according to the present embodiment is used for the primary culture step, among the culture steps described above.
[0025]
As shown in FIGS. 1 and 2, the culture apparatus 1 according to the present embodiment includes a culture vessel 2 and a rotating drum (level difference adjusting means) 3 for attaching the culture vessel 2 to an outer peripheral surface. . As shown in FIG. 3, the culture vessel 2 includes one main culture vessel 4, one culture vessel 5, one waste culture vessel 6, and a first culture vessel 4 connecting the main culture vessel 4 and the culture vessel 5. And a second connecting pipe 8 for connecting the main culture container 4 and the waste medium container 6 to each other.
[0026]
The main culture container 4, the medium container 5, and the waste medium container 6 are each made of a material such as hard polystyrene. Further, the first and second connecting pipes 7 and 8 are constituted by flexible tubes such as vinyl chloride. Thereby, it becomes possible to bend each connecting pipe 7, 8 and to arrange each container 4, 5, 6 at an arbitrary position, and to set the intermediate position in the longitudinal direction of the connecting pipe 7, 8 in the radial direction. By being crushed by a clip or a pinch valve, the flow of the fluid inside can be restricted. Before the first and second connecting pipes 7 and 8 are attached to the rotary drum 3, the first and second connecting pipes 7 and 8 are closed by, for example, a detachable clip (not shown) in order to restrict the flow of the fluid. I have.
[0027]
The medium container 5 is disposed on the main culture container 4 side, the main culture container 4 is disposed on the waste medium container 6 side, and the fluid in the medium container 5 is supplied to the main culture container 4 and the fluid in the main culture container 4 is disposed in the waste medium container. Included are inclined surfaces 5a and 4a for facilitating the flow into the container 6. In FIG. 3, reference numeral 9 denotes a mounting hole for mounting the containers 4, 5, and 6 to the rotating drum 3.
[0028]
Further, in the main culture container 4 and the medium container 5, for example, MEM (Minimal Essential Medium: minimum essential medium), FBS (Fetal Bovine Serum: fetal bovine serum), antibiotics, etc., at a predetermined mixing ratio, for example, The medium adjusted at 84: 15: 1 is enclosed. Human serum may be used instead of FBS. As the antibiotic, any antibiotic such as a cephem, a macrolide, a tetracycline, a fosfomycin, an aminoglycoside, and a new quinolone can be used in addition to the penicillin antibiotic.
The waste medium container 6 contains aseptic gas, for example, 5% CO. ₂ Gas is enclosed.
[0029]
The main culture container 4 includes, for example, an injection part 10 that can penetrate an injection needle and is closed by elasticity after the injection needle is pulled out. In this way, a bone marrow fluid is injected into the main culture container 4 by piercing the injection needle of a syringe from which a bodily fluid, for example, a bone marrow fluid has been collected, into the main culture container 4, and the main culture is drawn by pulling out the injection needle from the injection unit 10. The inside of the container 4 can be shut off from the outside.
[0030]
The rotary drum 3 has a cylindrical surface centered on a horizontal rotation axis, and is connected to a rotary drive source such as a motor (not shown). Container mounting portions 11, 12, and 13 for mounting the containers 4, 5, and 6 are provided on the outer surface of the cylindrical surface that is the outer peripheral surface of the rotary drum 3. The container mounting part 11 of the main culture container 4 is arranged between the container mounting part 12 of the culture medium container 5 and the container mounting part 13 of the waste medium container 6, and is spaced apart in the circumferential direction of the rotating drum 3 respectively. Is located at For example, in the example shown in FIGS. 1 and 2, the rotary drums 3 are arranged at intervals of 90 ° in the circumferential direction.
The mounting of the containers 4, 5, 6 on the rotating drum 3 is performed using, for example, bolts 14. Instead of the bolt 14, any fixing means such as a thumb screw, belt, hook, etc. may be used.
[0031]
In addition, when the containers 4, 5, 6 are mounted on the container mounting portions 11, 12, 13, the connecting pipes 7, 8 between the containers 4, 5, 6 are provided with connecting pipe paths. Pinch valves 15 and 16 are provided for radially sandwiching intermediate positions of the connecting pipes 7 and 8 in the longitudinal direction. Each of the pinch valves 15, 16 has two pins 15a, 16a extending radially outward from the inside of the rotary drum 3 toward the outside of the cylindrical surface, and a connecting pipe 7 is provided between the pins 15a, 16a. , 8 can be opened and closed by switching the distance between the pins 15a, 16a. Each of the pinch valves 15 and 16 is controlled to be opened and closed by a controller (not shown).
[0032]
The operation of the culturing apparatus 1 according to the present embodiment thus configured will be described below.
In order to culture cells using the culture apparatus 1 according to the present embodiment, first, as described above, the injection needle of a syringe from which bone marrow fluid has been collected is passed through the injection section 10 and the bone marrow fluid is injected into the main culture container 4. Inject.
[0033]
Then, the rotating drum 3 is rotated by a predetermined angle, and the rotating drum 3 is fixed at a position where the main culture vessel 4 is disposed horizontally at the uppermost position. In this state, the main culture vessel 4 is placed under predetermined culture conditions, for example, temperature (for example, 37 ± 0.5 ° C.), humidity (for example, 100%) and CO 2. ₂ Culture is performed by arranging under conditions such as concentration (for example, 5%). And CO ₂ The concentration conditions are as follows: ₂ Is dissolved or part or all of the main culture vessel 4 is CO 2 ₂ This can be achieved by using a transmission filter.
[0034]
Thereafter, when a predetermined medium exchange time has been reached, as shown in FIG. 4, the rotating drum 3 is rotated by a predetermined angle so that the main culture container 4 is arranged higher than the waste medium container 6. . In the case of the present embodiment, since the main culture vessel 4 is disposed at the top during the culture, the rotating drum 3 does not need to be rotated. However, in order to move the culture medium in the main culture container 4 to the waste culture medium container 6 more smoothly, for example, as shown in FIG. 4, the main culture container 4 is tilted upward and the waste culture container 6 is tilted downward. It is preferable that the arrangement is made as follows.
[0035]
In this state, the pinch valve 16 that has closed the second connection pipe 8 is opened. Thereby, the unnecessary medium is discharged from the main culture container 4 into the waste medium container 6 via the second connecting pipe 8 by gravity. When a predetermined amount of the medium in the main culture vessel 4 is discharged, the pinch valve 16 is operated to close the second connection pipe 8 again.
[0036]
Next, the rotating drum 3 is rotated by a predetermined angle so that the main culture container 4 is arranged lower than the medium container 5 as shown in FIG. That is, the main culture vessel 4 is arranged obliquely downward and the culture vessel 5 is arranged obliquely upward. Then, the pinch valve 15 that has closed the first connection pipe 7 is opened. Thereby, a new culture medium is supplied from the culture medium container 5 to the main culture container 4 via the first connection pipe 7. As a result, the medium exchange step is completed, so that the rotating drum 3 is rotated by a predetermined angle, the main culture vessel 4 is returned to the highest position, and the culture is continued.
[0037]
By continuing the culture while repeating such a medium exchange step a plurality of times, the mesenchymal stem cells grow sufficiently on the bottom surface of the main culture vessel 4. The main culture vessel 4 containing the grown mesenchymal stem cells can be transported alone by sealing and cutting all the connecting tubes 7, 8 connected to the main culture vessel 4 with heat.
[0038]
As described above, according to the culture apparatus 1 of the present embodiment, the culture medium and the like necessary for the culture are sealed in advance, and the inside of the culture vessel 2 is sealed from the outside during the culture period of the primary culture step. Can be shut off. This can prevent external dust and the like from being mixed. In addition, the medium exchange operation in which the medium is easily scattered can be performed simply by rotating the rotary drum 3 and arranging the containers 4, 5, 6 at predetermined positions. The medium can be moved between the containers 4, 5, and 6 by gravity while keeping the medium sealed inside 8. Therefore, there is no influence such as contamination on other external cells.
[0039]
Although the culture vessel 2 in the present embodiment has been described as being applicable to the primary culture step of growing mesenchymal stem cells while changing the medium a plurality of times at a predetermined time, the main culture vessel 4 For example, a bone filling material such as a β-tricalcium phosphate porous body is sealed, and the mesenchymal stem cells cultured in the primary culture step can be put therein, so that it can be applied to the secondary culture step. May be.
Alternatively, a blood collection tube (not shown) may be connected to the main culture container 4, and bone marrow fluid collected from a patient may be directly injected into the main culture container 4 by the blood collection tube.
[0040]
In addition, although the connecting pipes 7 and 8 are opened and closed by the pinch valves 15 and 16 provided on the rotating drum 3, instead of this, the connecting pipes 7 and 8 themselves may be provided with openable valves. .
[0041]
In the culture apparatus 1 according to the embodiment, the culture container 2 in which the main culture container 4, the culture container 5, and the waste culture container 6 are interconnected by the connection pipes 7, 8 has been described as an example. As shown in FIG. 6, the various containers 4, 5, 6 and the connecting pipes 7, 8 may be integrally attached to a flexible sheet 17 that can be bent. The seat 17 is provided with a bolt mounting hole 18 for mounting to the rotary drum 3 and a through hole 19 through which pins of the pinch valves 15 and 16 pass.
[0042]
With this configuration, when attaching the culture container 2 to the rotating drum 3, the sheet 17 in which the various containers 4, 5, 6 and the connection pipes 7, 8 are integrated is formed into a cylindrical shape as shown in FIG. It only has to be curved and attached to the outer surface of the rotating drum 3. Therefore, the positioning of each of the containers 4, 5, 6 and the connecting pipes 7, 8 can be omitted, and the mounting operation can be facilitated.
[0043]
Next, a culture apparatus according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the culture device 20 according to the present embodiment, portions having the same configuration as those of the culture device 1 according to the above-described first embodiment will be denoted by the same reference numerals, and description thereof will be simplified.
[0044]
The culture vessel 21 of the culture apparatus 20 according to the present embodiment is provided with an enzyme vessel 24 connected to a main culture vessel 22 via a third connection pipe 23 as shown in FIG. The difference from the culture container 2 in the first embodiment is that the cell collection tube 25 is provided in the container 22.
For example, a proteolytic enzyme such as trypsin is sealed in the enzyme container 24, and the third connecting tube 23 is closed by a removable clip. The enzyme container 24 is, for example, mounted on the rotating drum 26 at the same circumferential position as the medium container 5 and in a line in the longitudinal direction.
[0045]
The rotary drum 26 is provided with a container mounting part 27 for mounting the enzyme container 24, and when the enzyme container 24 is mounted, a pinch valve 28 that closes the third connecting pipe 23 in the radial direction when the enzyme container 24 is mounted. Is provided.
[0046]
The cell collection tube 25 is provided at the center of a surface that is to be arranged radially outward when the main culture container 22 is attached to the rotating drum 25. As shown in FIG. 9, the surface of the main culture vessel 22 provided with the cell collection tube 25 is formed in a funnel shape that gradually narrows from the periphery toward the central cell collection tube 25. The cell collection tube 25 is a closed tube made of a flexible tube such as vinyl chloride similar to the connection tubes 7, 8, and 23, and having a closed end.
[0047]
The operation of the culturing apparatus 20 according to the present embodiment thus configured will be described below.
The operation until the cells are cultured while performing the medium exchange periodically is the same as that of the culture apparatus 1 according to the first embodiment.
[0048]
According to the culture device 20 according to the present embodiment, after the mesenchymal stem cells have grown in the attached state in the main culture container 22, the rotating drum 26 is rotated by a predetermined angle, thereby disposing the main culture container 22 obliquely upward. The culture medium container 6 is arranged so as to be obliquely downward. Then, the pinch valve 16 that has closed the second connection pipe 8 is operated to open the second connection pipe 8, and a predetermined amount of the medium to be discarded in the main culture vessel 22 is discarded by gravity. Discharge to medium container 6.
[0049]
After that, the rotary drum 26 is again rotated by a predetermined angle, so that the enzyme container 24 is arranged higher than the main culture container 22. Then, the pinch valve 28 that has closed the third connection pipe 23 is operated to open the third connection pipe 23, and the trypsin stored in the enzyme container 24 is removed from the third connection pipe 23. To the main culture vessel 22 via gravity.
[0050]
Then, the main culture container 22 is vibrated by operating the pinch valve 28 and closing the third connection pipe 23 again for a predetermined time, or by oscillating the rotating drum 26 to apply vibration to the main culture vessel 22. The mesenchymal stem cells adhering to the bottom surface of the culture vessel 22 are peeled off. As a result, the detached mesenchymal stem cells are mixed in a fluid comprising trypsin and a culture medium in a state where the adhesion between the cells has been cut.
[0051]
Thereafter, by continuously rotating the rotating drum 26 in one direction, the mesenchymal stem cells in the main culture vessel 22 and the medium containing trypsin are centrifuged. Since the mesenchymal stem cells have a higher specific gravity than substances such as trypsin, they are moved radially outward by centrifugation.
[0052]
According to the culture device 20 according to the present embodiment, the main culture container 22 is formed in a funnel shape, so that the mesenchymal stem cells moved outward in the radial direction are moved toward the center by the funnel-shaped container wall. Collected. Since the cell collection tube 25 is provided at the center, the collected mesenchymal stem cells are collected in the cell collection tube 25.
[0053]
Further, since the cell collection tube 25 is made of vinyl chloride which can be sealed or welded by heat, as shown in FIG. As shown in FIG. 5, the cutting end 25a can be closed while maintaining the inside in a sterile and sealed state as shown in FIG. I have. Hereinafter, this cutting method is referred to as aseptic tube cutting.
[0054]
Also, as shown in FIG. 11A, after the cell collection tube 25 and the other connection tube 30 arranged in parallel are cut at the same time, as shown in FIG. After the tube 30 is shifted so as to coincide with the tube 30, the heating plate 29 is removed as shown in FIG. 3C so that the cell collection tube 25 can be connected to another connection tube 30. Has become. Although the connecting portion is closed at the time of connection, the internal flow path can be communicated with the tube walls being connected by external force. That is, the cell collection tube 25 and the other connection tube 30 can be connected while the inside is kept in a sterile state. Hereinafter, this connection method will be referred to as aseptic tube connection.
[0055]
That is, by connecting the cell collection tube 25 from which the mesenchymal stem cells have been collected to the connecting tube 30 connected to another main culture vessel (not shown) in which the living tissue filling material is enclosed by aseptic tube connection. In addition, it becomes possible to transfer the recovered mesenchymal stem cells to the secondary culture step while keeping them in a sterile state.
Since the mesenchymal stem cells are arranged inside the cell collection tube 25, there is a possibility that the mesenchymal stem cells may be damaged by the heating plate 29. Therefore, as shown in FIG. A preliminary space 31 for cutting may be formed at the tip of the. That is, by cutting along the cutting line A passing through the preliminary space 31, the heating plate 29 is prevented from directly touching the mesenchymal stem cells in the cell collection tube 25, and the integrity of the mesenchymal stem cells is maintained. In addition, aseptic tube connection can be performed.
[0056]
In the present embodiment, the enzyme container 24 is provided, and the mesenchymal stem cells are detached by trypsin. However, instead of this, the inner wall of the main culture container 22 becomes hydrophobic at a predetermined temperature as a boundary. A temperature responsive treatment that switches between hydrophilicity may be performed.
The temperature-responsive treatment is performed by immobilizing a temperature-responsive polymer poly (N-isopropylacrylamide) with a covalent bond. The region subjected to the temperature responsive treatment has a boundary temperature of 32 ° C., above which a weak hydrophobicity similar to that of a commercially available cell culture vessel is exhibited, but a high hydrophilicity is obtained by cooling the temperature below the boundary temperature. . Therefore, for example, by culturing at 37 ° C. and then cooling to 32 ° C. or less, it is possible to change the inner surface of the main culture vessel 22 to exhibit high hydrophilicity and to exfoliate the mesenchymal stem cells noninvasively. Become.
[0057]
Next, a culture apparatus 40 according to a third embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 13 and 14, the culture device 40 according to the present embodiment includes a main culture container 41, a medium container 44 connected to the main culture container 41 via connecting pipes 42 and 43, and a waste medium container. A culture vessel 46 having a 45 and a mounting table (elevation difference adjusting means) 47 on which the culture vessel 46 is mounted and whose inclination angle is changed are provided.
[0058]
The main culture vessel 41 is formed in a flat thin box shape and has a relatively large bottom area. The medium container 44 and the waste medium container 45 are formed in a box shape having a height higher than that of the main culture container 41, and have an internal volume several times larger than the internal volume of the main culture container 41. are doing. The medium container 44 and the waste medium container 45 are arranged on the same side of the main culture container 41.
Further, the same culture medium as described above is sealed in the main culture container 41 and the medium container 44, and the waste medium container 45 is empty.
[0059]
The mounting table 47 includes a tray portion 48 for fixing the culture vessel 46 and a rocking device 49 for rocking the tray portion 48 around a horizontal axis. The tray portion 48 is provided with the same pinch valves 50 and 51 as those in the first embodiment, and opens and closes the connecting tubes 42 and 43 so as to radially interpose the intermediate positions of the connecting tubes 42 and 43 in the longitudinal direction. You can do it. The swinging device 49 includes a base 52 and a motor 53 that swings the tray portion 48 with respect to the base 52.
[0060]
The operation of the culturing apparatus 40 according to the present embodiment thus configured will be described below.
To culture cells using the culture device 40 according to the present embodiment, first, bone marrow cells are supplied into the main culture container 41, the tray section 48 is set horizontal, and the bottom surface of the main culture container 41 is set horizontal. While keeping the cells, the cells are cultured under the same predetermined culture conditions as described in the above embodiment.
Then, when a predetermined medium exchange time comes, the motor 53 of the mounting table 47 is operated to swing the tray portion 48 with respect to the base 52, so that the main culture container 41 is moved as shown in FIG. It is arranged at a position higher than the waste medium container 45.
[0061]
In this state, among the pinch valves 50 and 51 provided in the tray portion 48, the pinch valve 51 that has closed the connection pipe 43 connecting the main culture container 41 and the waste medium container 45 is opened. As a result, the medium to be discarded in the main culture container 41 is discharged into the waste medium container 45 by gravity. Since the adherent mesenchymal stem cells remain attached to the bottom surface in the main culture container 41, the connection tube 43 is closed again by operating the pinch valve 51.
[0062]
Thereafter, the motor 53 of the mounting table 47 is operated to arrange the medium container 44 at a position higher than the main culture container 41 as shown in FIG. Then, the pinch valve 50 that has closed the connection pipe 42 connecting the main culture vessel 41 and the culture vessel 44 is opened. Thereby, the new culture medium sealed in the culture medium container 44 is supplied into the main culture container 41 by gravity. As a result, the medium in the main culture vessel 41 is replaced with a new medium, and the medium exchange is completed by closing the pinch valve 50 again.
[0063]
By continuing the culture while repeating this medium exchange step a plurality of times, mesenchymal stem cells can be grown to the required number along the bottom surface of the main culture vessel 41. By cutting the connecting tubes 42 and 43 by aseptic tube cutting, the main culture vessel 41 after the culture can be independently transported with the mesenchymal stem cells sealed inside.
[0064]
As described above, according to the culture apparatus 40 according to the present embodiment, the culture medium in the main culture vessel 41 can be exchanged only by swinging the tray section 48, and the cells can be grown to a required number in a sterile state. it can. In addition, since the cells are cultured in a state where the inside of the culture container 46 is sealed from the outside, it is possible to prevent the outside from being mixed with other cells and the like. Furthermore, since a simple configuration that only swings the tray portion 48 can be adopted, the cost can be reduced and the operation is simple.
[0065]
In addition, since the main culture container 41 is formed in a thin box shape to achieve the required minimum medium depth, it is possible to reduce the amount of the medium used.
Further, since the various containers 41, 44, 45 and the connecting pipes 42, 43 are mounted on the flat tray portion 48, the mounting operation is easy.
[0066]
In the present embodiment, the medium container 44 and the waste medium container 45 are connected to the same side surface of the main culture container 41, but may be connected to different side surfaces or upper and lower surfaces instead. In this case, it is necessary to provide a mechanism for swinging the tray portion 48 so as to generate a height difference between the main culture container 41 and the medium container 44 or the waste medium container 45 attached thereto. Further, an enzyme container or other containers may be connected to the main culture container 41.
[0067]
In addition, by providing a rotation mechanism for rotating the tray unit 48 about its normal line, the mesenchymal stem cells cultured in the main culture container 41 may be centrifuged. Further, a closed connecting tube for collecting the centrifuged mesenchymal stem cells or transferring the cells to another culture vessel may be provided on one side of the main culture vessel 41.
[0068]
In addition, since the containers 41, 44, and 45 can be separated by aseptically cutting the connecting tubes 42 and 43, the relationship between the cultured mesenchymal stem cells and the discarded medium is unclear. In order to prevent this from happening, each container 41, 44, 45 may be provided with a symbol indicating that it is a related container, in particular, a bar code. The barcodes may be the same barcode or an interconnected barcode.
[0069]
Further, a part or the whole of the main culture container 41 may be configured to be transparent, and an observation window (not shown) may be formed in the tray portion 48 on which the main culture container 41 is mounted. According to this, it is possible to observe the culture state through an observation window, for example, with an inverted microscope during the culture period.
[0070]
Further, in each of the above embodiments, the mesenchymal stem cells have been described as an example of cells to be cultured. Alternatively, other cells such as ES cells, somatic stem cells, bone cells, chondrocytes, and nerve cells may be used. It may be applied when culturing somatic cells.
The body fluid supplied to the main culture vessels 4, 22, 41 may be peripheral blood or umbilical cord blood in addition to bone marrow fluid. Alternatively, only the bone marrow cells obtained by centrifuging the collected bone marrow fluid may be supplied into the main culture vessels 4, 22, 41.
In addition, as a living tissue replacement material, any biocompatible material such as other ceramics, collagen, and polylactic acid may be used in addition to a bone replacement material made of β-tricalcium phosphate.
[0071]
Further, the shapes of the rotating drums 3, 26, the mounting table 47, and the various containers 4, 5, 6, 22, 24, 41, 44, 45 are not limited to those described in the above embodiments. In addition, although each of the containers 4, 5, 6, 22, 24, 41, 44, and 45 has been described as an example, one container is provided as an example. A plurality of medium containers 6 and 45 may be provided. In addition, in addition to these containers 4, 5, 6, 22, 24, 41, 44, and 45, for example, in the case of the secondary culture step, a container or the like in which a differentiation inducing factor such as dexamethasone is enclosed is provided in advance. They may be connected in advance, or may be connected by aseptic tube connection.
[0072]
【The invention's effect】
As described above, according to the culture apparatus of the present invention, the culture medium can be exchanged by gravity only by giving a height difference to the main culture vessel, the culture vessel, and the waste culture vessel connected by the openable and closable connection pipe. it can. Therefore, the medium can be exchanged while maintaining the sealed state in the culture container, and it is possible to prevent dust and the like from being mixed into the cultured cells from the outside.
[Brief description of the drawings]
FIG. 1 is a front view showing a culture device according to an embodiment of the present invention.
FIG. 2 is a side view showing the culture apparatus of FIG.
FIG. 3 is a front view showing a culture container used in the culture device of FIG. 1;
FIG. 4 is a front view showing a step of discharging a culture medium from a main culture vessel to a waste culture vessel in the culture apparatus of FIG. 1;
FIG. 5 is a front view showing a step of supplying a culture medium from a culture vessel to a main culture vessel in the culture apparatus of FIG. 1;
FIG. 6 is a front view showing a modification of the culture vessel in the culture apparatus of FIG.
FIG. 7 is a perspective view showing the culture container of FIG.
FIG. 8 is a side view showing a culture device according to a second embodiment of the present invention.
FIG. 9 is a front view showing the culture device of FIG. 8;
FIG. 10 is an explanatory diagram illustrating aseptic tube cutting.
FIG. 11 is an explanatory diagram illustrating aseptic tube connection.
FIG. 12 is a longitudinal sectional view showing an end structure of a cell collection tube.
FIG. 13 is a plan view showing a culture device according to a third embodiment of the present invention.
FIG. 14 is a front view showing the culture device of FIG.
FIG. 15 is a front view showing a step of discharging a culture medium from a main culture vessel to a waste culture vessel in the culture apparatus of FIG. 13;
FIG. 16 is a front view showing a step of supplying a culture medium from a culture vessel to a main culture vessel in the culture apparatus of FIG.
FIG. 17 is an explanatory diagram illustrating a culture step to which the present invention is applied.
[Explanation of symbols]
1,20,40 Culture device
2,21,46 culture vessel
3,26 rotating drum (level difference adjusting means)
4,22,41 Main culture vessel
5,44 medium container
6,45 Waste medium container
7, 8, 23, 30, 42, 43 Connecting pipe
15, 16, 28, 50, 51 Pinch valve
24 Enzyme container
25 Cell collection tube (extraction port)

Claims (7)

At least one main culture container, at least one medium container enclosing a culture medium, and at least one waste medium container capable of containing a fluid in the main culture container, a connection capable of restricting a flow of a fluid therebetween. A culture vessel connected in a sealed state to the outside by a tube,
A culture apparatus comprising a height difference adjusting means capable of adjusting heights of a medium container and a waste medium container with respect to the main culture container, respectively. The height difference adjusting means is provided rotatably around the longitudinal axis in a state where the longitudinal axis is horizontally disposed, from the rotating drum that fixes the main culture container, the medium container and the waste medium container at different circumferential positions. The culture device according to claim 1. The culture device according to claim 2, wherein the culture container is provided so as to be mounted on an outer surface of the rotary drum. The removal port which can take out the cell in the said main culture container in the position arrange | positioned radially outward in the state where the said culture container was fixed to the rotating drum, or Claim 2 or Claim. 4. The culture device according to 3. The culture device according to any one of claims 1 to 4, wherein the connection pipe includes a valve, and the flow of the fluid in the connection pipe is permitted or restricted by the valve. The connection pipe is made of a flexible material, and is formed of a tube in which the flow of an internal fluid is restricted by being crushed,
The culture apparatus according to any one of claims 1 to 4, further comprising a pinch valve that restricts the flow of the fluid by sandwiching the tube in the radial direction from the outside and allows the flow of the fluid by opening the tube. In the main culture container, at least one enzyme container enclosing the protease is connected in a hermetically sealed state to the outside by a connection pipe capable of restricting the flow of a fluid between each other,
The culture apparatus according to any one of claims 1 to 6, wherein the height difference adjusting means can also adjust the height of the enzyme container with respect to the main culture container.

Images