JP2005348672A - Cell culture device and cell culture method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell-culturing apparatus which can supply a gas necessary for the culture in a large amount in a simple structure without causing the enhancement of cost and without increasing a shear force. <P>SOLUTION: The cell-culturing apparatus is characterized by having a culture tank 2 having a culture region 32 for culturing cells, and an adjusting tank 3 having an adjusting region 12 for adjusting a culture medium concentration of the gas supplied to the culture region 32, wherein the adjusting region 12 is disposed in a non-culturing region where the cells are not cultured. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell culture apparatus and a cell culture method for culturing cells such as animal cells and plant cells.

Cells such as animal cells and plant cells are widely used for the production of useful substances.
Examples of methods for culturing these cells, for example, animal cells, include the following.
(1) Liquid culture method using a petri dish This is a method in which contamination of germs is blocked from the outside by a petri dish, and a culture solution and cells are put into the petri dish, and oxygen necessary for the cells is supplied by diffusion from the water surface.
(2) Agar culture method using a petri dish The culture solution is solidified with agar, etc., and the cells are attached to the solid so that they can proliferate. There is.
(3) Rotating culture method A culture solution and cells are put in a bottle (container) with a lid, and oxygen is supplied while rotating the bottle to attach the cells to the inner wall of the bottle. Can be cultured.
(4) Stirring culture method A culture solution and cells are put into a bottle with a lid and stirred with a magnetic stirrer, etc. It is mainly used for the growth of floating cells and can be cultured in a larger amount than the method using a petri dish. is there.

In these culture methods, oxygen required by the cells is sent by diffusion from the water surface or forced ventilation. However, if a large amount of oxygen is supplied, strong shearing is applied to the cells regardless of whether they are stirred or diffused. There is a risk of damaging cells by applying force. In addition, when oxygen supply is limited to prevent cell destruction, there arises a problem that the cell concentration cannot be increased above a certain level.
Therefore, Patent Document 1 describes a technique for supplying a large amount of oxygen without increasing the shearing force by using an oxygen permeable membrane.

On the other hand, in animal cell culture, when a tissue or organ is formed from cells, it is necessary to keep the cells in a fixed position without moving as much as possible. In this case, it is possible to provide a scaffold and allow cells to adhere to the scaffold and grow, but the three-dimensional culture required for tissues and organs cannot be performed. Therefore, a technology that enables three-dimensional culture using a rotary culture apparatus that rotates a disc-shaped liquid culture vessel has been developed.
JP-A-5-304943

However, the following problems exist in the conventional technology as described above.
When an oxygen permeable membrane is used to supply a large amount of oxygen, there is a problem that the apparatus becomes complicated and causes a cost increase.
Further, in the case of three-dimensional culture using a rotary culture device, since only a very small device has been completed, there arises a problem that a large amount of culture is difficult.

  On the other hand, since animal cells need to be cultured for a long period of time, it is necessary to change the medium during the culture. However, in that case, it is necessary to extract the culture solution without cells in a short time, so a filtration device is required, and complicated operations such as flow rate adjustment to supply a new medium of the same amount as the extraction amount are required. This has the disadvantage of poor workability. Further, continuously performing the extraction of the culture medium and the introduction of the medium has a disadvantage that the efficiency is poor because the input medium may be extracted together with the culture medium.

  The present invention has been made in consideration of the above points, and supplies a large amount of gas necessary for culturing such as oxygen without increasing the shearing force applied to the cells without causing an increase in cost. An object of the present invention is to provide a cell culture device and a cell culture method that can be used.

Another object of the present invention is to provide a cell culture apparatus and a cell culture method that enable a large amount of three-dimensional culture.
Furthermore, another object of the present invention is to provide a cell culture apparatus and a cell culture method that eliminate the complexity of medium exchange and can efficiently perform continuous medium exchange.
In the following description, a cell that has been proliferated into a single mass is referred to as a cell mass.

In order to achieve the above object, the present invention employs the following configuration.
The cell culture device of the present invention has a culture tank having a culture region for culturing cells, and an adjustment tank having an adjustment region for adjusting the gas concentration of the medium supplied to the culture region, It is provided in a non-culture area where the cells are not cultured.
The cell culture method of the present invention is a cell culture method for culturing cells with a medium in a culture region, and the gas concentration of the medium supplied to the culture tank in a non-culture region where the cells are not cultured. And a step of supplying the culture medium with the gas concentration adjusted from the non-culture region to the culture region.

  Therefore, in the cell culture device and the cell culture method of the present invention, a medium in which the concentration of gas such as oxygen is adjusted in advance in a non-culture area where cells are not cultured may be supplied to the culture area. When supplying, it is not necessary to apply a shearing force to the cells, and the cells can be prevented from being destroyed. Moreover, in the present invention, since only the adjustment tank for adjusting the gas concentration of the culture medium is provided, it becomes possible to prevent the apparatus from becoming complicated and causing a cost increase as in the case of using the oxygen permeable membrane.

It is preferable that at least a part of the culture medium circulates between the culture region and the adjustment region.
This makes it possible to perform continuous medium exchange efficiently and easily. Further, in the present invention, since at least a part of the medium containing a medium component introduced from the culture area and not consumed in the culture area is used, the amount of the medium to be newly added in the adjustment area can be reduced, resulting in an increase in cost. Can be suppressed.

  When at least a part of the culture medium is circulated between the culture region and the adjustment region, the inside of the adjustment tank is stored in the first storage part for storing the medium introduced from the culture region, and the gas concentration is adjusted. It is preferable that a partition member for partitioning the lower part of the first storage part and the second storage part in communication with the second storage part for storing the culture medium delivered to the culture region is provided.

  As a result, in the present invention, the medium whose gas concentration has been adjusted can be prevented from mixing with most of the old medium introduced from the culture region, and the first reservoir and the second reservoir can be communicated from the culture region. By using at least part of the medium containing medium components that could not be consumed in the culture area introduced into the second reservoir through the section, the amount of medium newly added in the adjustment area can be reduced. The rise can be suppressed.

In the present invention, a discharge part for discharging the overflowed culture medium and an introduction part for introducing the culture medium from the culture tank are provided in the first storage part, and the introduction part is located above the discharge part. A configuration that is spaced apart can also be suitably employed.
Thereby, in this invention, since the height of the culture medium of a 1st storage part is restrained to the height of the discharge | emission part where a culture medium overflows, miscellaneous bacteria can flow back into a culture tank via an introduction part. Can be prevented.
Furthermore, a configuration in which a sterilization filter is provided in the flow path of the culture medium from the adjustment region to the culture region is also preferable in order to prevent contamination with bacteria in the culture region.

Moreover, in this invention, the structure by which a culture tank and an adjustment tank are provided separately, and the structure by which an adjustment tank is arrange | positioned in a culture tank are employable.
When the adjustment tank is disposed in the culture tank, a configuration having a cylindrical body that forms the non-culture area partitioned from the culture area inside can be suitably employed.
In this configuration, since the gas concentration is adjusted in the non-culture area partitioned from the culture area by the cylinder, the gas can be supplied to the culture medium without applying shearing force to the cells, and the cells can be prevented from being destroyed. Further, in this configuration, since the adjustment tank is disposed in the culture tank, the apparatus can be reduced in size and price, the amount of the medium to be used can be reduced, and the cost of the medium can be reduced.

Further, when the adjustment tank is disposed in the culture tank, the cylinder rotates around a substantially vertical axis by driving a rotary drive source between a lower end opening of the cylinder and the bottom of the culture tank. It is also possible to employ a configuration in which a rotator that feeds an internal medium to the culture region is provided.
In this configuration, the medium between the lower end opening of the cylinder and the rotating body is sent to the outside by centrifugal force due to the rotation of the rotating body, and the medium whose gas concentration is adjusted inside the cylinder is accordingly opened at the lower end opening. Is supplied to the culture area.

As the rotator, a configuration that rotates in a non-contact manner with respect to the rotation drive source as in the case of using a magnetic stirrer or the like can be suitably employed.
In this configuration, it is possible to prevent contamination of germs and the like from the connecting portion between the rotating body and the rotation drive source.

On the other hand, the cell culture device of the present invention is a cell culture device having a culture tank for culturing cells with a culture medium, and has an injection part into which the culture medium is injected at the bottom of the culture tank. The horizontal cross-sectional area is formed so as to increase gradually as it goes upward.
The cell culture method of the present invention is a cell culture method for culturing cells with a medium in a culture tank, wherein the inside of the culture tank is formed such that the horizontal cross-sectional area gradually increases upward. And the step of injecting the culture medium from the bottom of the culture tank.

  Therefore, in the cell culture apparatus and the cell culture method of the present invention, it is possible to provide a velocity gradient so that the rising flow rate of the medium injected from the bottom of the culture tank and rising increases and decreases toward the bottom. Therefore, by adjusting the flow rate of the medium so that the sedimentation rate of the cells is between the ascending flow rate at the upper part and the ascending flow rate at the lower part, three-dimensional culture can be performed in a state where the cells remain in a certain position in the medium. In this case, a large amount of three-dimensional culture can be realized by increasing the culture tank.

  One end of a fibrous medium for attaching the cells to the bottom or top of the culture tank is fixed, or a granular medium having substantially the same specific gravity as the medium is attached to the culture tank. In the case where the cells have adhesiveness, the cells can be attached to a fibrous medium that remains linear in the medium or a granular medium that floats in the medium.

Moreover, the structure provided with the control apparatus which controls the flow rate of the said culture medium based on the position of the said cell is also employable suitably.
In this case, even if the sedimentation rate of the cells cannot be handled by the above velocity gradient, the cells are controlled by controlling the flow rate of the medium so that the sedimentation rate of the cells is between the ascending flow rate at the upper part and the ascending flow rate at the lower part. It becomes possible to perform three-dimensional culture in a state where it is kept at a fixed position in the medium.

  In the present invention, it is possible to supply a large amount of gas such as oxygen to the culture medium without complicating the apparatus configuration as in the case of using an oxygen permeable membrane, and it is possible to reduce the cost. Further, in the present invention, even when the size of the cell mass changes, it is possible to realize a large amount of three-dimensional culture by holding the cells at a fixed position corresponding to the size of the cell mass. Furthermore, according to the present invention, continuous medium exchange can be performed efficiently and easily.

Hereinafter, embodiments of the cell culture apparatus and cell culture method of the present invention will be described with reference to FIGS.
(First embodiment)
FIG. 1 is a diagram showing a schematic configuration of a cell culture device 1 according to the present invention.
The cell culture device 1 is provided with a culture tank 2 for culturing cells using a liquid medium (culture medium) B, a gas concentration of the medium B provided separately from the culture tank 2 and supplied to the culture tank 2. Adjustment tank 3 for adjustment, supply pipe (flow path) 4 for supplying medium B from adjustment tank 3 to culture tank 2, and introduction of old medium B discharged from culture tank 2 to adjustment tank 3 It is mainly composed of the introduction pipe 5 and the like.

  The culture tank 2 has a culture region 32 inside, and is formed in a substantially truncated cone shape whose horizontal cross-sectional area gradually increases from the bottom toward the upper side, and its upper end is formed in a cylindrical shape. The upper end of the opening is closed by a box-like closing member 6. The top wall 6a of the closing member 6 is provided with an operation port 7 for inoculating and extracting cells to be cultured. The bottom wall 6b located at a position lower than the upper end of the culture tank 2 has a vertical direction. The upper end of the introduction pipe 5 arranged along the side is fixed so as to open inside the closing member 6. Further, a cylindrical injection tube (injection part) 8 is inserted and fixed to the top wall part 6 a of the closing member 6. The injection tube 8 is arranged along the vertical direction, the lower end thereof opens at the bottom inside the culture tank 2, and one end of the supply tube 4 is connected to the upper end.

  The adjustment tank 3 has a rectangular box shape in cross section, and the medium B introduced from the culture tank 2 is separated by a partition wall (partition member) 9 vertically suspended from the top wall portion 3a. The first reservoir 11 for storing and the second reservoir 12 as an adjustment region (non-culture region where cell culture is not performed) for storing the medium B whose gas concentration is adjusted and sent to the culture tank 2 are The first storage part 11 and the second storage part 12 are partitioned in a state where the lower parts are communicated with each other. Connected to the upper side of the first reservoir 11 is one end of an overflow pipe (discharge unit) 13 that overflows and discharges the old medium B introduced from the culture tank 2. The other end of the overflow pipe 13 is connected to a replaceable sterile container 14. The sterile container 14 is provided with an air escape filter 15.

  Further, the lower end portion (introduction portion) 5 a of the introduction pipe 5 described above is provided on the upper side of the first storage portion 11. The lower end portion 5a of the introduction pipe 5 is disposed so as to be spaced above the height direction position (overflow line) of the overflow pipe 13.

  On the other hand, the second reservoir 12 is mixed with a medium supply pipe 16 for supplying a new medium B and about 5% of the medium B in the gas phase for oxygen and pH adjustment necessary for cell culture. A gas supply pipe 17 for supplying a gas containing carbon dioxide and a gas exhaust pipe 21 are connected. The tip of the culture medium supply pipe 16 is connected so as to be positioned above the liquid surface position in the adjustment tank 3 defined by the overflow line. The tip of the gas supply pipe 17 is connected so as to be positioned below the liquid level in the adjustment tank 3 described above so that the mixing property of the supplied gas and the culture medium B is improved. Further, a sterilization filter 18 for sterilizing the gas supplied to the adjustment tank 3 is interposed in the middle of the gas supply pipe 17.

  The supply pipe 4 is connected to the lower part of the second reservoir 12 in the adjustment tank 3. The supply pipe 4 is provided with a pump 19 serving as a drive source for supplying the culture medium B to the culture tank 2 and two sterilization filters 20 for sterilizing the culture medium B supplied to the culture tank 2. It is heavy and intervened.

Next, the operation of the cell culture device 1 having the above configuration will be described below.
First, in the adjustment tank 3, the new medium B is supplied from the medium supply pipe 16 and the sterilized gas (oxygen, carbon dioxide) is supplied from the gas supply pipe 17, whereby the second reservoir 12. In this case, a medium (culture solution) having an oxygen concentration and pH suitable for cell culture is stored. At this time, since the second reservoir 12 is partitioned from the first reservoir 11 by the partition wall 9, the new medium whose gas concentration has been adjusted is stored without being mixed with the old medium in the first reservoir 11. Is done.

  The medium B with the adjusted gas concentration is sent from the second reservoir 12 by driving the pump 19 and sterilized by the sterilization filter 20, and then the culture medium 2 is supplied from the supply pipe 4 to the culture tank 2 through the injection pipe 8. It is injected into the bottom of (culture region 32). The medium B supplied to the bottom of the culture tank 2 rises, but the horizontal cross-sectional area inside the culture tank 2 gradually increases as it goes upward, so that the ascending flow rate of the culture medium B increases toward the bottom and decreases toward the top. A velocity gradient is applied so that Here, the culture solution was sampled at a plurality of points in different height directions, and the relationship between the ascending flow rate at that point and the average particle size of the cells was measured. As shown in FIG. A relationship proportional to the square of the particle size was obtained.

  Therefore, the flow rate of the medium B (the supply amount per unit time to the culture tank 2) is adjusted so that the sedimentation rate of the cells is between the upward flow rate of the culture medium B in the upper part of the culture tank 2 and the upward flow rate in the lower part. By this, the cells in the medium B can be kept at a fixed position in the medium. Therefore, it becomes possible to culture the cells three-dimensionally.

  In addition, when cell culture progresses and the cell becomes a larger lump (cell lump), the sedimentation rate of the cell lump also increases and descends in the culture tank 2, but as the descent proceeds, the ascending flow rate of the medium B also increases. Since it becomes large, the cells can be kept at a fixed position at a height where the sedimentation rate of the cell mass and the ascending flow rate of the medium B are balanced. In other words, even if the sedimentation speed varies depending on the size of the cell mass, the cell mass of each particle size can be kept at the height at which the rising flow velocity according to the sedimentation speed is generated. 3D culture of cell mass is possible.

  Then, the culture medium B that has risen in the culture tank 2 overflows from the upper end of the culture tank 2 and leaks to the closing member 6, falls from the lower end 5 a of the introduction pipe 5, and falls into the first reservoir 11 of the adjustment tank 3. Introduced into The old medium B consumed in the culture tank 2 and introduced into the first reservoir 11 of the adjustment tank 3 is then discharged from the overflow pipe 13 to the sterile container 14 by overflowing the first reservoir 11. .

As described above, in the present embodiment, the culture tank 2 for culturing cells and the adjustment tank 3 for adjusting the gas concentration of the culture medium B are separated, and the gas of the culture medium B in the second reservoir 12 which is a non-culture area. The simple configuration of adjusting the concentration can prevent cell destruction due to shear force applied to the cells during gas supply, so that the device configuration is not complicated as in the case of using an oxygen permeable membrane. A large amount of gas can be supplied to the culture medium B. Therefore, in this embodiment, there exists an effect that cost reduction is realizable.
Further, in the present embodiment, since a part of the medium B is circulated between the culture tank 2 (the culture region 32 thereof) and the adjustment tank 3, continuous medium exchange is performed efficiently and easily. In addition, since the medium B introduced from the culture tank 2 is added to the adjustment tank 3, the amount of the medium B to be newly added can be reduced, and an increase in cost can be further suppressed.

  And in this Embodiment, since the adjustment tank 3 is partitioned into the 1st storage part 11 and the 2nd storage part 12 by the partition wall 9, it introduce | transduces from the culture tank 2 to the new culture medium B in which the gas concentration was adjusted. The old medium B can be discharged into the sterile container 14 without mixing, and the new medium B can be continuously supplied to the culture tank 2. Furthermore, in this embodiment, since the lower end portion 5a of the introduction pipe 5 for introducing the medium B from the culture tank 2 to the adjustment tank 3 is provided above the overflow pipe 13, miscellaneous bacteria are the first. It is possible to prevent contamination from flowing backward from the reservoir 11 to the culture tank 2 through the introduction pipe 5 (the lower end 5a thereof). In addition, in this embodiment, since the sterilization filter 20 is provided in the supply pipe 4 that supplies the culture medium B from the adjustment tank 3 to the culture tank 2, it is possible to more reliably prevent contamination of the culture tank 2 with bacteria. It becomes possible to do.

  Moreover, in this Embodiment, since the horizontal direction cross section in the culture tank 2 becomes large gradually as it goes upwards, it becomes possible to provide a velocity gradient to the flow velocity of the culture medium B inject | poured from the bottom part. Therefore, even when the size of the cell mass changes, it is possible to retain the cell mass at a fixed position corresponding to the size of the cell mass and perform three-dimensional culture. Depending on the size of 2, a large amount of three-dimensional culture can be realized. Further, since the height (position) that remains in accordance with the size of the cell mass is determined in this way, it becomes easy to select and collect cells of a specific size.

(Second Embodiment)
Next, a second embodiment of the cell culture device according to the present invention will be described with reference to FIG. In this figure, the same reference numerals are given to the same elements as those of the first embodiment shown in FIG. 1, and the description thereof is omitted.
The main difference between the second embodiment and the first embodiment described above is that the culture medium 2 is provided with a fibrous medium, and the culture tank 2 is provided with an injection portion for the medium B. That is.

  As shown in FIG. 3, one end of a fibrous medium 30 such as a thread having a length equivalent to the height of the culture tank 2 and a specific gravity equal to or lower than that of the medium B is fixed to the bottom wall 2 a of the culture tank 2. ing. In addition, near the bottom of the side wall 2b of the culture tank 2, an inlet (injection part) 22 connected to the supply pipe 4 and into which the medium B is injected intersects with the vertical axis along the side wall 2b. It is provided so as to open obliquely upward.

In the present embodiment, in addition to the same operations and effects as in the first embodiment, since the fibrous medium 30 functions as a scaffold, even when adherent cells are cultured, the cells are allowed to adhere. 3D culture can be carried out.
Moreover, since the culture medium B injected into the inside of the culture tank 2 from the inlet 22 has a component around the vertical axis, it rises in a spiral shape, and a swirling flow is generated in the culture medium B. Therefore, the pressure of the outer culture medium B is increased by the centrifugal force, and the pressure on the center side is reduced. As a result, the cells in the culture solution are easily collected on the center side and attached to the fibrous medium 30. Further, since the fibrous medium 30 has a length equivalent to the height of the culture tank 2, even if the size of the cell mass varies, the cell mass can be attached at a position corresponding to the size.
Also in this embodiment, the flow rate of the medium B injected into the culture tank 2 is controlled to a flow rate (flow rate) preferable for cell culture.

  In this embodiment, since the fibrous medium 30 has a specific gravity equal to or lower than that of the culture medium B, one end of the fibrous medium 30 is fixed to the bottom wall 2a of the culture tank 2, but the fibrous medium When 30 has a specific gravity equal to or higher than that of the culture medium B, one end of the fibrous medium 30 may be fixed to the top wall (top) 6a of the closing member 6 and suspended.

(Third embodiment)
Next, a third embodiment in which the form of a medium for attaching adherent cells is different will be described with reference to FIG.
Also in this figure, the same reference numerals are given to the same elements as those of the second embodiment shown in FIG. 3, and the description thereof is omitted.
As shown in FIG. 4, in the third embodiment, a granular medium 31 made of beads or the like is placed inside the culture tank 2. Since the granular medium 31 has substantially the same specific gravity as the medium B, the granular medium 31 has a floating property with respect to the medium B.

Also in this embodiment, the same operation and effect as in the second embodiment can be obtained.
In addition, in order to cope with the variation in the size of the cell mass, the granular medium is in the height direction of the culture tank 2 by using a plurality of types of granular media having buoyancy with respect to the medium B and having different specific gravities. The cell mass can be attached as a scaffold at a position corresponding to the size of the cell mass.

(Fourth embodiment)
Next, a fourth embodiment of the cell culture device according to the present invention will be described with reference to FIG. In this figure, the same reference numerals are given to the same elements as those of the first embodiment shown in FIG. 1, and the description thereof is omitted.
The main difference between the fourth embodiment and the first embodiment is that the adjustment tank 3 is disposed in the culture tank 2.

  The cell culture apparatus 1 shown in FIG. 5 has a configuration in which a cylindrical body 33 as the adjustment tank 3 is disposed in the culture tank 2 along the vertical direction. The inside of the adjustment tank 3 is divided into a culture region 32 by a cylindrical body 33, thereby forming a non-culture region 34 in which cell culture is not performed. In addition, the injection tube 8 is disposed in the cylinder 33 so that the lower end thereof is located above the lower end opening 33 a of the cylinder 33. A gas supply pipe 17 with a sterilization filter 18 is connected to the injection pipe 8, and a culture medium supply pipe 4 with a sterilization filter 20 is connected outside the culture tank 2.

  Further, a stirring bar (rotating body) 35 is provided between the lower end opening 33 a of the cylinder 33 and the bottom 2 a of the culture tank 2. The stirrer 35 is a permanent magnet coated with plastic, and is driven between a cylindrical body 33 and the bottom 2a of the culture tank 2 by driving a magnetic stirrer (rotation drive source) 36 installed below the culture tank 2. Thus, the magnetic stirrer 36 is configured to rotate about a substantially vertical axis without contact with the magnetic stirrer 36.

  Further, a sterile container 14 connected by a discharge pipe 37 is installed in the vicinity of the culture tank 2. The end of the discharge pipe 37 facing the culture region 32 in the culture tank 2 is opened above the culture region 32 at a position higher than the upper end opening 33b of the cylinder 33, and the discharge pipe facing the inside of the sterile container 14 The end of 37 is opened at a position lower than the end facing the culture region 32. An exhaust pipe 39 in which a sterilizing filter 38 is interposed is connected to the top of the sterile container 14.

In the cell culture apparatus 1 having the above-described configuration, the sterilized gas and the new medium are mixed and supplied into the adjustment region 34 in the cylinder 33. In the vicinity of the lower end opening 33a of the cylindrical body 33, the magnetic stirrer 36 is rotated by driving the magnetic stirrer 36, so that the medium between the stirrer 35 and the lower end opening 33a is moved outside by the centrifugal force. Accordingly, the medium whose gas concentration is adjusted in the cylinder 33 is opened at the lower end (according to the pressure drop due to the decrease of the medium between the stirrer 35 and the lower end opening 33a). It is introduced into the culture region 32 from the part 33a and rises with a velocity gradient corresponding to the horizontal cross-sectional area of the culture tank 2.
Thereby, as described above, it is possible to perform three-dimensional culture by retaining the cell clusters of each particle size at the height (position) according to the size of the cell clusters.

  At this time, the adjustment region 34 in the cylinder 33 is partitioned from the culture region 32 by the cylinder 33, and the upper end opening 33b of the cylinder 33 is higher than the inclined portion of the culture tank 2, that is, the culture tank. Since the horizontal cross-sectional area of 2 is located at a constant height, the flow rate of the medium is slow and the cells are settled, and the cells do not flow into the cylinder 33 and only the old medium is It will flow from the top. Therefore, the culture medium can be circulated while adjusting the gas concentration without applying shearing force to the cells.

  Further, as the supply of new medium and gas proceeds, the internal pressure of the culture tank 2 increases, and the old medium and gas above the culture tank 2 are discharged from the discharge pipe 37 to the sterile container 14. Then, the gas in the sterile container 14 is exhausted from the exhaust pipe 39.

  Thus, in this embodiment, in addition to obtaining the same operation and effect as the first embodiment, the adjustment tank 3 (tubular body 33) is disposed in the culture tank 2, Miniaturization of the device is realized. In addition, since the amount of medium to be used can be reduced, the cost of the medium can be reduced. Moreover, since the circulation of the culture medium is realized with a simple configuration of rotating the stirring bar 35, it can contribute to further cost reduction. Furthermore, in the present embodiment, since the stirrer 35 rotates in a non-contact manner with respect to the magnetic stirrer 36, it is possible to prevent contamination of bacteria from the connecting portion as in the case of the connected configuration.

(Fifth embodiment)
Next, a fifth embodiment of the cell culture device according to the present invention will be described with reference to FIG. In this figure, the same components as those of the fourth embodiment shown in FIG. 5 are denoted by the same reference numerals, and the description thereof is omitted.
The main difference between the fifth embodiment and the fourth embodiment described above is that gas and a new culture medium are individually introduced into the cylinder 33.

  That is, the gas supply pipe 17 and the culture medium supply pipe 4 are individually introduced into the cylindrical body 33. In addition, a diffuser plate 40 is provided at the end of the gas supply pipe 17 to make the gas bubbles supplied to the adjustment region 34 fine, so that the introduced gas can be effectively dissolved in the culture medium. It has become.

  In the fourth embodiment, gas and a new medium are mixed and supplied to the adjustment region 34. Therefore, even if a diffuser plate is provided at the end of the injection tube 8, the medium becomes a resistance and effective diffusion is achieved. In this embodiment, since the gas supply pipe 17 is introduced into the cylindrical body 33 separately from the culture medium supply pipe 4, the gas concentration is reduced and effective gas concentration adjustment is realized. be able to.

  The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but it goes without saying that the present invention is not limited to such examples. Various shapes, combinations, and the like of the constituent members shown in the above-described examples are examples, and various modifications can be made based on design requirements and the like without departing from the gist of the present invention.

For example, in the above-described embodiment, the cell culture device 1 has been described as a configuration including the culture tank 2 and the adjustment tank 3. However, the present invention is not limited to this, and in order to perform a large amount of three-dimensional culture, The adjustment tank 3 is not necessarily required, and a gas containing oxygen or the like may be supplied to the culture tank 2 without providing the adjustment tank 3.
Moreover, in the said embodiment, although it was set as the structure which circulates the culture medium B between the culture tank 2 and the adjustment tank 3, it is not restricted to this, It can also be set as the structure which supplies the new culture medium B to the culture tank 2 entirely. Is possible.
5 and 6 seem to rise up from the surface of the culture medium and reach the aseptic container 14, but it emerges horizontally from the side wall of the culture tank 2 and bends downward to be sterile. It may be connected to the container 14. Thereby, it can drive | operate with the pressure inside the culture tank 2 being a normal pressure.

In the above embodiment, the flow rate (flow rate) of the medium B is set so that the sedimentation rate of the cell mass is between the ascending flow rate of the medium B in the upper part of the culture tank 2 and the ascending flow rate in the lower part. For example, a sensor for detecting the position of the cell mass is provided in the culture tank 2, and the flow rate (flow velocity) of the medium B to be sent to the culture tank 2 is controlled by the pump 19 based on the detection result (that is, the position of the cell mass). It is good also as a structure which provides the control apparatus which performs.
In this case, when the size of the cell mass cannot be accommodated by the speed gradient of the medium B formed by the culture tank 2, the sedimentation speed of the cell mass is between the ascending flow rate at the upper part of the culture tank 2 and the ascending flow rate at the lower part. By controlling the flow rate of the medium B so as to become, it becomes possible to perform three-dimensional culture in a state where the cell mass is kept at a fixed position in the medium.

It is a figure which shows embodiment of this invention, Comprising: It is a figure which shows schematic structure of the cell culture apparatus which concerns on 1st Embodiment. It is a figure which shows the relationship between the average particle diameter of a cell lump, and a raise flow rate. It is a figure which shows schematic structure of the cell culture apparatus which concerns on 2nd Embodiment. It is a figure which shows schematic structure of the cell culture apparatus which concerns on 3rd Embodiment. It is a figure which shows schematic structure of the cell culture apparatus which concerns on 4th Embodiment. It is a figure which shows schematic structure of the cell culture apparatus which concerns on 5th Embodiment.

Explanation of symbols

B Medium (culture medium)
DESCRIPTION OF SYMBOLS 1 Cell culture apparatus 2 Culture tank 2a Bottom part 3 Adjustment tank 4 Supply pipe (flow path)
5a Lower end (introduction part)
6a Top wall (top)
8 Injection pipe (injection part)
9 Partition wall (partition member)
11 1st storage part 12 2nd storage part (adjustment area)
13 Overflow pipe (discharge section)
20 Sanitization filter 22 Inlet (injection part)
30 Fibrous medium 31 Granular medium 32 Culture area 33 Tube 33a Lower end opening 34 Non-culture area (adjustment area)
35 Stirrer (Rotating body)
36 Magnetic Stirrer (Rotary drive source)

Claims (15)

A culture vessel having a culture region for culturing cells;
An adjustment tank having an adjustment region for adjusting the gas concentration of the medium supplied to the culture region;
The cell culture apparatus, wherein the adjustment area is provided in a non-culture area where the cells are not cultured. The cell culture apparatus according to claim 1, wherein
At least a part of the culture medium circulates between the culture region and the adjustment region. The cell culture device according to claim 2,
Inside the adjustment tank, a first reservoir for storing the medium introduced from the culture region, and a second reservoir for storing the medium to be sent to the culture region with the gas concentration adjusted, A cell culture device, comprising: a partition member that partitions the lower part of the first storage part and the second storage part in communication with each other. The cell culture device according to claim 3,
The first storage part is provided with a discharge part for discharging the overflowed medium and an introduction part for introducing the medium from the culture tank,
The cell culture device, wherein the introduction part is spaced apart from the discharge part. In the cell culture device according to any one of claims 1 to 4,
A cell culture apparatus, wherein a sterilization filter is provided in a flow path of the medium from the adjustment region to the culture region. In the cell culture device according to any one of claims 1 to 5,
The cell culture apparatus, wherein the culture tank and the adjustment tank are provided separately. The cell culture apparatus according to claim 1 or 2,
The cell culture apparatus, wherein the adjustment tank has a cylindrical body that is disposed in the culture tank and forms the non-culture area partitioned from the culture area. The cell culture device according to claim 7,
Between the lower end opening of the cylinder and the bottom of the culture tank, there is provided a rotating body that rotates around a substantially vertical axis by driving a rotational drive source and feeds the medium inside the cylinder to the culture region. A cell culture device. The cell culture device according to claim 8,
The cell culture device, wherein the rotating body rotates in a non-contact manner with respect to the rotation driving source. A cell culture device having a culture tank for culturing cells in a medium,
Having an injection part into which the culture medium is injected at the bottom of the culture tank;
The cell culture apparatus, wherein the culture tank is formed so that an internal horizontal cross-sectional area gradually increases as it goes upward. The cell culture device according to claim 10, wherein
One end of a fibrous medium to which the cells are attached is fixed to the bottom or top of the culture tank. The cell culture device according to claim 10, wherein
A cell culture apparatus, wherein a granular medium having substantially the same specific gravity as that of the culture medium and allowing the cells to adhere thereto is placed in the culture tank. The cell culture device according to any one of claims 10 to 12,
A cell culture device comprising a control device for controlling a flow rate of the culture medium based on a position of the cell. A cell culture method for culturing cells with a medium in a culture region,
Adjusting the gas concentration of the medium supplied to the culture tank in a non-culture area where the cells are not cultured;
Supplying the culture medium with the gas concentration adjusted from the non-culture area to the culture area. A cell culture method for culturing cells with a medium in a culture tank,
A cell culture method comprising the step of injecting the medium from the bottom of the culture tank into the culture tank formed such that the cross section in the horizontal direction gradually increases as it goes upward.

Images