JP2022123160A - Culture device and culture method - Google Patents

Abstract

To reduce the number of devices for agitating a liquid culture medium in a culture vessel while reducing the risk of contamination in the extended culture of cells.SOLUTION: A culture device is provided with: a culture vessel 12 in which a liquid culture medium is accommodated; a culture vessel driver which drives the culture vessel 12 so as to agitate the liquid culture medium CS in the culture vessel 12; and a liquid culture medium feeder for adding an additional amount of the liquid culture medium CS to the culture vessel 12 in accordance with an increase in the number of cells in the liquid culture medium CS. The culture vessel driver drives the culture vessel 12 in such a manner that the area of a surface part, which comes into contact with the liquid culture medium CS that moves by means of the agitation, of the culture vessel 12 can become smaller with the decrease in the volume of the liquid culture medium CS in the culture vessel 12.SELECTED DRAWING: Figure 7

Description

TECHNICAL FIELD The present invention relates to a culture apparatus and culture method for culturing cells using a culture solution.

Conventionally, in large-scale cell culture, for example, culture medium with a density of 10 ⁴ to 10 ⁷ cells per milliliter is started from a small amount of cells of several milliliters, and then cultured with a large amount of cells of 10 ⁹ to 10 ¹⁰ cells. It is being propagated until First, cells are cultured using several milliliters of culture solution in a Petri dish, for example. When the cell density in the petri dish reaches a predetermined density, the cells in the petri dish are divided and seeded in multiple petri dishes for culturing. It becomes possible to transfer to a large-capacity culture vessel, such as a flask. After being transferred to a large-capacity culture vessel several times in this way, the cells are finally placed in a culture capable of accommodating a large-capacity culture medium, as described in Patent Document 1, for example. transferred to the bag. Cultivation is performed in the culture bag using, for example, 50 liters of culture medium until the required number of cells is reached. Such culture is called expansion culture.

Japanese translation of PCT publication No. 2014-507959

However, in such expansion culture, cells are transferred to a large-capacity culture vessel a plurality of times, so there is a risk of contamination occurring during the transfer. Moreover, for each of the plurality of culture vessels, a device for stirring the culture solution in the culture vessel is required.

Accordingly, it is an object of the present invention to reduce the number of devices for agitating the culture medium in the culture vessel while reducing the risk of contamination in cell expansion culture.

In order to solve the above technical problems, according to one aspect of the present invention,
A culture device for expanding culture of cells in a culture solution,
a culture vessel containing a culture solution;
a culture vessel driving unit that drives the culture vessel so that the culture solution in the culture vessel is agitated;
a culture solution supply unit for adding culture solution to the culture container as the number of cells in the culture solution increases;
The culture vessel driving unit drives the culture vessel such that the smaller the amount of the culture medium in the culture vessel, the smaller the portion of the surface of the culture vessel that is in contact with the culture medium that moves due to stirring. A culture device is provided.

Also, according to another aspect of the present invention,
A method for culturing cells in which cells are expanded and cultured in a culture medium contained in a culture vessel,
driving the culture vessel so that the culture solution in the culture vessel is agitated;
adding a culture medium to the culture vessel as the number of cells in the culture medium increases;
A culturing method is provided in which the culture vessel is driven such that the smaller the amount of culture medium, the smaller the portion of the surface of the culture vessel that is in contact with the culture medium that is moved by agitation.

According to the present invention, it is possible to reduce the number of devices for agitating the culture medium in the culture vessel while reducing the risk of contamination in cell expansion culture.

1 is a block diagram showing the configuration of a culture apparatus according to an embodiment of the present invention; FIG. A schematic perspective view of an example of a culture vessel Schematic partial cross-sectional view of a culture vessel driving part in a culture apparatus Schematic partial cross-sectional view of part of the culture vessel driving unit shown in FIG. 3 as seen from a different direction Schematic partial cross-sectional view of the culture vessel driving unit shown in FIG. 3 with the culture vessel tilted. Sectional view showing the inclination state of the culture vessel when the amount of culture solution is relatively small. The top view which shows the inclination state of a culture container when a culture solution is a relatively small amount. A diagram showing agitation of the culture medium when the culture medium is relatively small. Sectional view showing the state of inclination of the culture vessel when the amount of culture medium is relatively large. A top view showing the tilted state of the culture vessel when the amount of the culture medium is relatively large. A diagram showing agitation of the culture medium when the medium volume is relatively large.

A culture apparatus according to one aspect of the present invention is a culture apparatus that performs expansion culture of cells in a culture medium, and includes a culture vessel containing a culture medium and the culture medium in the culture vessel so that the culture medium is agitated. a culture vessel driving unit for driving a container; and a culture medium supply unit for adding culture medium to the culture container as the number of cells in the culture medium increases. The culture vessel is driven such that the smaller the amount of the culture medium, the smaller the portion of the surface of the culture vessel that is in contact with the culture medium that is moved by agitation.

According to this aspect, in cell expansion culture, the number of devices for stirring the culture solution in the culture vessel can be reduced while reducing the risk of contamination.

For example, the culture vessel includes a circular bottom surface and a cylindrical inner peripheral surface standing from the outer peripheral edge of the bottom surface. In this case, the culture vessel driving unit tilts the culture vessel so that the culture medium is accumulated in the corner sandwiched between the bottom surface and the inner peripheral surface, and the culture medium reciprocates along the corner. By changing the inclination direction of the culture vessel, the reciprocation range of the culture medium is reduced as the amount of the culture medium is reduced. Thereby, the culture solution can be stirred while suppressing its evaporation.

For example, the culture vessel drive unit tilts the culture vessel more as the amount of culture solution decreases. As a result, since the surface area of the culture solution is reduced, the evaporation of the culture solution from the liquid surface can be suppressed.

For example, the culture apparatus has a humidity sensor that measures the humidity inside the culture container. In this case, when the humidity detected by the humidity sensor decreases, the culture vessel driving section increases the inclination angle of the culture vessel so that the surface area of the culture solution becomes smaller. This makes it possible to suppress the evaporation of the culture solution from the liquid surface.

For example, the culture apparatus has a dissolved oxygen sensor that measures the amount of oxygen dissolved in the culture solution in the culture vessel. In this case, the culture vessel drive unit drives the culture vessel such that at least one of the reciprocation period and the reciprocation range of the culture solution increases when the amount of dissolved oxygen detected by the dissolved oxygen sensor decreases. . As a result, the culture solution is more agitated, sufficient oxygen is supplied to the culture solution and homogenization is performed, and cell damage can be suppressed.

For example, when the amount of culture solution in the culture container exceeds a predetermined threshold amount, the culture container driving section changes the tilt direction of the culture container so that the culture solution circulates along the corner. Thereby, a large amount of culture solution can be sufficiently stirred.

For example, the culture solution supply unit may add culture solution to the culture container at the timing when the cell density approaches the upper limit until the culture solution from less than 1 liter in the culture container reaches 50 liters.

A culture method according to another aspect of the present invention is a cell culture method in which cells are expanded and cultured in a culture solution contained in a culture container, wherein the culture container is agitated so that the culture solution in the culture container is agitated. and add the culture medium to the culture vessel as the number of cells in the culture medium increases. The culture vessel is driven to become smaller.

According to this aspect, in cell expansion culture, the number of devices for stirring the culture solution in the culture vessel can be reduced while reducing the risk of contamination.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a block diagram showing the configuration of a culture device according to one embodiment of the present invention.

As shown in FIG. 1, the culture apparatus 10 includes a culture container 12 containing a culture solution CS containing cells, and a culture container driving unit for driving the culture container 12 to agitate the culture solution CS in the culture container 12. 14 and a culture solution supply unit 16 that supplies the culture solution CS to the culture container 12 .

In addition, in the case of the present embodiment, the culture apparatus 10 includes a humidity sensor 18 that measures the humidity within the culture vessel 12 and a dissolved oxygen sensor 20 that measures the amount of oxygen dissolved in the culture medium CS within the culture vessel 12. have.

The culture apparatus 10 has a gas supply unit 17 that supplies a humidified mixed gas of oxygen, carbon dioxide, and nitrogen to the culture vessel 12 .

Furthermore, the culture apparatus 10 controls the culture vessel driving section 14 and the culture medium supply section 16 based on a predetermined culture program, and drives the culture vessel based on the detection results of the humidity sensor 18 and the dissolved oxygen sensor 20, respectively. It has a control unit 22 that controls the unit 14 .

The culture container 12 is a container that accommodates the culture medium CS, and cells are cultured using the culture medium CS therein. In this culture vessel 12, as the number of cells increases, the culture solution CS is gradually added from a small amount (less than 1 liter, for example, 50 ml), and the cells are cultured using the culture solution, that is, expansion culture is performed. will be Therefore, the culture container 12 has a capacity capable of accommodating and stirring the maximum amount (for example, 50 liters) of culture solution used for culture.

FIG. 2 is a perspective view showing the shape of an example of the culture vessel. Although an XYZ orthogonal coordinate system is shown in the drawings, this is for facilitating understanding of the embodiments of the invention and does not limit the invention. Also, the X-axis direction and the Y-axis direction are horizontal directions, and the Z-axis direction is a vertical direction.

As shown in FIG. 2, in the case of the present embodiment, the culture vessel 12 includes a disk-shaped bottom plate portion 12a, a cylindrical side wall portion 12b erected from the outer peripheral edge of the bottom plate portion 12a, and supported by the side wall portion 12b. and a top plate portion 12c. That is, the culture container 12 is in the shape of a so-called basin. The height of the side wall portion 12b is smaller than the radius of the bottom plate portion 12a. Further, the top plate portion 12c is detachable and functions as a lid.

FIG. 3 is a schematic partial cross-sectional view of a culture vessel drive section in the culture apparatus. Moreover, FIG. 4 is a schematic partial cross-sectional view of part of the culture vessel drive unit shown in FIG. 3 as seen from a different direction.

As shown in FIGS. 3 and 4, the culture vessel driving unit (culture vessel driving device) 14 in the culture apparatus 10 includes a stage 30 that holds the culture vessel 12 and a rotation center that extends in the vertical direction (Z-axis direction). and a rotary actuator 34 comprising a rotary table 32 that rotates about an axis C0.

The stage 30 and rotary actuator 34 are drivingly connected via a swing head 36 and a tilting mechanism 38 .

The swing head 36 supports the stage 30, and swings a swing axis C1 extending in the horizontal direction (X-axis direction) and a swing axis C1 extending in the horizontal direction (Y-axis direction) perpendicular to the swing axis C1. It is provided in the culture vessel driving device 14 so as to be swingable around the axis C2. The swing head 36 also has a connecting shaft 40 at its lower portion for drivingly connecting with the rotary actuator 34 via the tilting mechanism 38 . When the stage 30 takes a horizontal posture, the connecting shaft 40 of the swing head 36 extends in the vertical direction (Z-axis direction).

The tilting mechanism 38 is a link mechanism for tilting the stage 30 via the swing head 36, that is, for tilting the culture container 12 on the stage 30 with respect to the horizontal direction. For this purpose, the tilting mechanism 38 includes a base portion 42, a swinging head connecting portion 44 connected to the swinging head 36, and a link arm 46 connecting the base portion 42 and the swinging head connecting portion 44. .

A base portion 42 of the tilting mechanism 38 is attached to the rotary table 32 of the rotary actuator 34 . Therefore, when the rotary actuator 34 is driven, the base portion 42 rotates together with the rotary table 32 around the rotation center axis C0.

The swinging head connecting portion 44 of the tilting mechanism 38 is slidably fitted on the connecting shaft 40 of the swinging head 36 via, for example, a bearing.

A link arm 46 of the tilting mechanism 38 is configured to connect the base portion 42 and the swinging head connecting portion 44 . Specifically, the link arm 46 has one end rotatably fixed to the swing head connecting portion 44 and the other end rotatably fixed to the base portion 42 . The rotation axis C3 at one end of the link arm 46 and the rotation axis C4 at the other end extend horizontally and are parallel to each other.

The rotary actuator 34 to which the base portion 42 of the tilting mechanism 38 is attached is moved up and down in the vertical direction (Z-axis direction) by the ball screw mechanism 48 .

The ball screw mechanism 48 includes a screw shaft 50 extending in the vertical direction (Z-axis direction), a nut 52 engaging the screw shaft 50, and a motor (not shown) rotating the screw shaft 50. . The nut 52 is attached to the lift bracket 54 . A rotary actuator 34 is attached to the lifting bracket 54 .

When the ball screw mechanism 48 is driven, the rotary actuator 34 moves up and down together with the lifting bracket 54 via the nut 52 . For example, as shown in FIG. 5 , when the ball screw mechanism 48 raises the rotary actuator 34 , the stage 30 is tilted via the tilting mechanism 38 . Specifically, the base portion 42 of the tilting mechanism 38 attached to the rotary actuator 34 is lifted, causing the link arm 46 to push the swing head connecting portion 44 . As a result, the swing head 36 rotates together with the swing head connecting portion 44 around at least one of the swing axes C1 and C2 (the swing axis C2 in FIG. 13). As a result, the stage 30 is tilted, and the culture vessel 12 on the stage 30 is also tilted.

As shown in FIG. 5, when the rotary actuator 34 is driven to rotate the rotary table 32 with the stage 30 tilted, the tilting mechanism 38 rotates about the rotation center axis C0, thereby tilting the stage 30 in the tilting direction. changes. As a result, the culture medium CS in the culture container 12 is agitated, and the cells in the culture medium CS are cultured.

In the culture vessel driving device 14, even if the rotary actuator 34 rotates the tilting mechanism 38 once, for example, the stage 30 itself does not rotate, and instead the stage 30 tilts only once. be. That is, the lowest portion of the culture vessel 12 on the stage 30 is simply changed to another portion in sequence.

Returning to FIG. 1, in the case of the present embodiment, the culture medium supply unit 16 that supplies the culture medium CS to the culture vessel 12 includes a culture medium tank 60 that stores the culture medium CS and a culture medium in the culture medium tank 60. and a supply pump 62 for sending CS into the culture vessel 12 . The supply pump 62 is controlled by the controller 22 .

In addition to the culture solution CS, a mixed gas is supplied to the culture container 12 by the gas supply unit 17 . For example, the gas supply unit 17 is configured to mix oxygen, carbon dioxide, and nitrogen and humidify the mixed gas with a humidification unit (not shown). The humidified mixed gas is introduced into the culture vessel 12 through a filter (not shown) that prevents contamination of germs and a gas introduction port (not shown) provided in the culture vessel 12 . The culture container 12 is provided with a gas exhaust port for exhausting the gas inside, and the gas that has passed through the gas exhaust port is exhausted to the atmosphere through a filter. Further, the gas supply unit 17 is controlled by the control unit 22 in terms of the supply timing and supply amount of the gas.

The humidity sensor 18 is mounted inside the culture vessel 12, specifically, for example, on the inner circumferential surface 12d so as not to be submerged in the culture solution CS, and measures the humidity inside the culture vessel 12. FIG. Humidity sensor 18 also outputs a signal corresponding to the measured humidity to control unit 22 .

The dissolved oxygen sensor 20 measures the amount of oxygen dissolved in the culture medium CS within the culture vessel 12 . For example, a fluorescent dissolved oxygen sensor is used as the dissolved oxygen sensor 20 . For example, a fluorescent dissolved oxygen sensor includes a chip that is placed on the bottom surface 12e of the culture vessel 12 and is coated with a fluorescent material, a light source that irradiates the chip with ultraviolet rays or the like from outside the culture vessel 12, and a light source that emits light from the chip. a light-receiving element for receiving the emitted fluorescence.

When a fluorescent substance absorbs light energy such as ultraviolet light from a light source, it transitions from the ground state to the excited state. Excited fluorophore molecules typically emit fluorescence and return to the ground state. At this time, however, if oxygen molecules are present around the molecules in the excited state, the excitation energy is taken away by the oxygen molecules, and so-called oxygen quenching occurs, in which the emission intensity of fluorescence decreases. Utilizing this oxygen quenching, that is, utilizing the fact that the emission intensity of fluorescence is inversely proportional to the concentration of oxygen molecules, the fluorescence type dissolved oxygen sensor measures the amount of dissolved oxygen in the culture medium in the culture vessel.

The dissolved oxygen sensor 20 also outputs a signal corresponding to the measured dissolved oxygen amount to the control unit 22 .

The control unit 22 is composed of, for example, a control board on which a memory and a CPU are mounted. By operating according to the program stored in the memory, the CPU executes operations related to cell culture, which will be described later.

First, the control unit 22 controls the supply pump 62 of the culture medium supply unit 16 according to a predetermined culture program.

Under the control of the controller 22 , the supply pump 62 adds the culture medium CS to the culture vessel 12 as the number of cells in the culture medium CS of the culture vessel 12 increases. For example, the supply pump 62 adds the culture medium CS to the culture vessel 12 in stages until less than 1 liter (eg, 200 milliliters) of the culture medium CS in the culture vessel 12 becomes 50 liters. Specifically, the control unit 22 controls the supply pump 62 so as to add the culture solution CS at the timing when the cell density of the culture solution CS in the culture vessel 12 approaches a predetermined upper limit. The predetermined upper limit is the cell density at which cells can be damaged. Also, the supply timing of the culture solution is determined in advance as information of the culture program based on the cells and the culture solution to be used.

Also, the control unit 22 controls the culture vessel driving device 14 (its rotary actuator 34 and ball screw mechanism 48) based on the amount of the culture medium CS in the culture vessel 12. FIG.

Under the control of the controller 22 , the culture vessel driving device 14 drives the culture vessel 12 so that the culture medium CS is stirred while the evaporation of the culture medium CS in the culture vessel 12 is suppressed. Specifically, the culture vessel driving device 14 drives the culture vessel 12 such that the smaller the amount of the culture medium CS in the culture vessel 12, the smaller the portion of the surface of the culture vessel 12 that is in contact with the culture medium that moves due to stirring. Drive container 12 . The driving of the culture container 12, that is, the agitation of the culture solution CS will be described.

FIG. 6A is a cross-sectional view showing the tilted state of the culture vessel when the amount of culture solution is relatively small. Also, FIG. 6B is a top view showing the tilted state of the culture vessel when the amount of the culture solution is relatively small.

As shown in FIGS. 6A and 6B, agitation of the culture solution is performed with the culture vessel 12 tilted. The inclination angle θ of the culture vessel 12 (the angle with respect to the culture vessel 12 in the horizontal state) is increased as the amount of the culture medium CS in the culture vessel 12 is decreased.

Thus, the smaller the amount of the culture medium CS, the smaller the size of the area of the liquid surface LS of the culture medium CS by tilting the culture container 12 more. Evaporation of the culture solution CS from the liquid surface LS can be suppressed by reducing the size of the area of the liquid surface LS.

Here, "evaporation of the culture solution" will be explained. When the culture medium CS evaporates, the cell density in the culture medium CS increases. When the amount of the culture solution CS is large (for example, 1 liter or more), the amount of increase in cell density due to evaporation of the culture solution CS is relatively small, and the effect of the increase in density on the cells is small. On the other hand, when the amount of the culture solution CS is small (for example, less than 1 liter), the increase in cell density due to evaporation of the culture solution CS is relatively large, and the effect of the increase in density on the cells is large. The smaller the amount of the culture medium CS, the greater the effect of its evaporation on the cells, and in some cases, some of the cells die or are damaged.

Therefore, the smaller the amount of the culture medium CS, the more the culture container 12 is tilted (by increasing the tilt angle θ), thereby reducing the influence of evaporation of the culture medium CS on the cells.

If the amount of the culture medium CS in the culture vessel 12 is greater than or equal to the amount that the evaporation of the culture medium CS has a sufficiently small effect on the cells, the inclination angle θ of the culture vessel 12 may be constant. .

By tilting the culture container 12, as shown in FIG. 6B, the culture solution CS is sandwiched between the circular bottom surface 12e of the culture container 12 and the cylindrical inner peripheral surface 12d standing from the outer peripheral edge of the bottom surface 12e. It collects in the corner 12f which was closed. In this state, the inclination direction of the culture container 12 is changed.

FIG. 7 is a diagram showing agitation of the culture medium when the amount of culture medium is relatively small. FIG. 7 shows the culture vessel 12 being stirred from above (viewed from the Z-axis direction).

As shown in FIG. 7, a relatively small amount (for example, less than 1 liter) of culture medium CS is reciprocated along corner 12f sandwiched between bottom surface 12e and inner peripheral surface 12d of culture vessel 12. As shown in FIG. For example, when the rotary actuator 34 repeats forward and reverse rotation of the tilting mechanism 38 within an angular range of 90 degrees, the tilt direction of the culture vessel 12 changes within an angular range of 90 degrees. Thereby, the culture medium CS is reciprocated within an angle range of 90 degrees. As a result, the culture medium CS is agitated. As shown in FIG. 7, when the positive direction of the Y-axis is set to be the direction of 0 degrees with respect to the Z-axis, for example, from the position of -45 degrees (315 degrees) around the 0-degree position to the position of +45 degrees. , the culture medium CS is reciprocated.

The smaller the amount of the culture medium CS, the smaller the reciprocation range (angular range) of the culture medium CS. The reason is to suppress the evaporation of the culture solution CS.

Specifically, when the culture medium CS moves on the surface of the culture vessel 12 due to stirring, a very small amount of the culture medium CS remains on the surface after the majority (lump) of the culture medium CS has passed. For example, as shown in FIG. 7, after the majority (lump) of the culture medium CS moves to the position of 45 degrees, a very small amount of the culture medium CS remains at the position of 0 degrees. This small amount of the remaining culture medium CS is easy to evaporate. Therefore, before the very small amount of culture medium CS evaporates, the bulky medium CS returns and absorbs the very small amount of culture medium CS. In addition, since the smaller the amount of the culture medium CS, the greater the effect of evaporation on the cells, the reciprocating range of the culture medium CS is made smaller. As a result, evaporation of the culture solution CS can be suppressed when the amount of the culture solution CS is relatively small.

As the number of cells increases, the culture medium CS is added to the culture vessel 12, and the amount of the culture medium CS in the culture vessel 12 increases. The reciprocation range of the culture solution CS is expanded according to the increase. This is because, while increasing the culture medium CS reduces the effect of its evaporation on the cells, it is necessary to agitate the medium CS more.

When the culture medium CS is relatively small (eg, less than 1 liter), the culture medium CS is reciprocated within the culture vessel 12 to prevent evaporation, as described above. On the other hand, when the culture solution CS is added as the number of cells increases and the amount of the culture solution CS is relatively large (for example, 1 liter or more), the culture solution CS is circulated in the culture container 12 .

FIG. 8A is a cross-sectional view showing the tilted state of the culture vessel when the amount of culture solution is relatively large. FIG. 8B is a top view showing the tilted state of the culture vessel when the amount of culture solution is relatively large.

As shown in FIGS. 8A and 8B, and with reference to FIGS. 6A and 6B , when the culture medium CS is relatively large, the culture vessel 12 is more stable than when the culture medium CS is relatively small. is small. This is to reduce the depth of the culture solution CS and spread gas such as oxygen throughout the culture solution CS.

As the depth of the culture solution increases, gas such as oxygen taken in through the liquid surface of the culture solution by agitation becomes more difficult to spread throughout the culture solution. Specifically, it is difficult for the gas to reach the deep part of the culture solution. As a result, there is a possibility that the amount of dissolved oxygen in the deep part of the culture medium will be insufficient, and the cells will be damaged.

By tilting the culture container 12, as shown in FIG. 8B, the culture solution CS accumulates in the corner 12f sandwiched between the bottom surface 12e and the inner peripheral surface 12d of the culture container 12. As shown in FIG. In this state, the inclination direction of the culture container 12 is changed.

FIG. 9 is a diagram showing agitation of the medium when the medium is relatively large. FIG. 9 shows the culture vessel 12 being stirred from above (viewed from the Z-axis direction).

A relatively large amount (for example, 1 liter or more) of the culture medium CS is circulated along the corner 12f sandwiched between the bottom surface 12e and the inner peripheral surface 12d of the culture container 12 . For example, when the rotary actuator 34 continues to rotate the tilting mechanism 38 in one direction, the tilt direction of the culture container 12 continues to rotate in one direction. Thereby, the culture medium CS is circulated. As a result, the culture medium CS is agitated.

Thus, the controller 22 changes the stirring mode based on the amount of the culture solution CS in the culture container 12 . For example, when the amount of the culture medium CS in the culture container 12 is smaller than a predetermined threshold amount (for example, 1 liter), the culture medium CS is reciprocated as shown in FIG. Stir the culture medium CS. Also, the smaller the amount of the culture medium CS, the smaller the reciprocating range of the culture medium CS. On the other hand, when the amount of the culture medium CS in the culture vessel 12 exceeds a predetermined threshold amount, the culture medium CS is agitated by circulating it as shown in FIG. The amount of the culture medium CS in the culture vessel 12 may be calculated from the weight of the culture medium CS in the culture vessel 12 measured by a weight sensor (not shown), for example.

In addition, in the case of the present embodiment, the control unit 22 is configured to control the culture vessel driving device 14 based on the measurement results of the humidity sensor 18 and the dissolved oxygen sensor 20 during stirring of the culture solution CS. there is

Specifically, when the humidity in the culture medium CS detected by the humidity sensor 18 decreases, for example, when the humidity decreases beyond the lower limit of a predetermined appropriate range, the culture vessel driving device 14 controlled by the control unit 22 increases the inclination angle of the culture container 12 (that is, the stage 30) so that the area of the liquid surface LS of the culture solution CS is reduced.

When the humidity inside the culture container 12 is lowered, the culture solution CS is easily evaporated from the liquid surface LS. Therefore, by reducing the area of the liquid surface LS of the culture solution CS, the evaporation can be suppressed.

Further, when the amount of dissolved oxygen detected by the dissolved oxygen sensor decreases, for example, when it decreases beyond the lower limit of a predetermined appropriate range, the culture vessel driving device 14 controlled by the control unit 22 causes the culture medium CS to reciprocate. The culture vessel 12 is driven such that at least one of the period of motion and the range of reciprocation is increased. The dissolved oxygen sensor 20 (its chip) is provided on the culture container 12 at a position where it can detect the amount of dissolved oxygen by coming into contact with the culture solution CS regardless of the amount of the culture solution CS in the culture container 12 . In the case of this embodiment, the dissolved oxygen sensor 20 is provided on the outer peripheral edge of the bottom surface 12 e of the culture vessel 12 . Further, when the dissolved oxygen sensor 20 measures the dissolved oxygen amount, the culture vessel 12 is driven by the culture vessel driving device 14 so that the culture solution CS contacts the dissolved oxygen sensor 20 . In this case, in order to bring the culture solution CS into contact with the dissolved oxygen sensor 20 and accurately detect the amount of dissolved oxygen, the drive speed or drive mode of the culture container 12 may be temporarily changed, or the culture container 12 may be may temporarily stop driving.

When the amount of dissolved oxygen in the culture solution CS in the culture container 12 decreases, the cells in the culture solution CS are damaged. Therefore, by increasing at least one of the reciprocation period and the reciprocation range of the culture solution CS, the culture solution CS is more agitated, and thereby more oxygen is taken into the culture solution CS. As a result, cell damage can be suppressed.

In addition, when the culture medium CS in the culture container 12 is relatively large and is circulated, the culture medium CS is stirred more by increasing the circulating speed, thereby increasing the amount of the culture medium CS in the culture medium CS. Can take in oxygen.

As described above, according to such an embodiment, it is possible to reduce the number of devices for stirring the culture solution in the culture vessel while reducing the risk of contamination in cell expansion culture.

That is, by using one culture container and changing the stirring mode based on the amount of the culture solution in the culture container, expansion culture can be performed with reduced risk of contamination. In addition, such expansion culture can be realized by a single device (culture vessel driving device).

Although the present invention has been described with reference to the above-described embodiments, the embodiments of the present invention are not limited to these.

For example, in the embodiment described above, the culture vessel is basin-shaped, as shown in FIG. However, embodiments of the present invention are not limited to this. The culture vessel may be, for example, a large Erlenmeyer flask.

That is, in a broad sense, the culture apparatus according to the embodiment of the present invention is a culture apparatus that performs expansion culture of cells in a culture medium, and includes a culture vessel containing a culture medium and a culture medium in the culture vessel. and a culture medium supply unit for adding culture medium to the culture container as the number of cells in the culture medium increases, wherein the culture container The driving unit drives the culture vessel such that the smaller the amount of the culture medium in the culture vessel, the smaller the portion of the surface of the culture vessel that comes into contact with the culture medium that moves due to stirring.

Further, in a broad sense, the culture method according to the embodiment of the present invention is a cell culture method in which cells are expanded and cultured in a culture medium contained in a culture vessel, wherein the culture medium in the culture vessel is The culture vessel is driven to be stirred, and the culture medium is added to the culture vessel as the number of cells in the culture medium increases. The culture vessel is driven so that the portion of the surface of the culture vessel becomes smaller.

INDUSTRIAL APPLICABILITY The present invention is applicable to cell culture in which the culture solution is stirred.

12 culture vessel CS culture solution

Claims (8)

A culture device for expanding culture of cells in a culture solution,
a culture vessel containing a culture solution;
a culture vessel driving unit that drives the culture vessel so that the culture solution in the culture vessel is agitated;
a culture solution supply unit for adding culture solution to the culture container as the number of cells in the culture solution increases;
The culture vessel drive unit drives the culture vessel such that the smaller the amount of culture solution in the culture vessel, the smaller the surface portion of the culture vessel that is in contact with the culture solution that moves due to stirring. culture equipment.
The culture vessel includes a circular bottom surface and a cylindrical inner peripheral surface erected from the outer peripheral edge of the bottom surface,
The culture vessel drive unit tilts the culture vessel so that the culture medium is accumulated in the corner sandwiched between the bottom surface and the inner peripheral surface, and the culture vessel reciprocates along the corner. by changing the tilt direction of
2. The culture apparatus according to claim 1, wherein the smaller the amount of the culture solution, the smaller the reciprocation range of the culture solution.
The culturing apparatus according to claim 2, wherein the culturing vessel drive unit tilts the culturing vessel more as the amount of the culture solution decreases.
Having a humidity sensor that measures the humidity in the culture vessel,
4. The culture according to claim 2 or 3, wherein when the humidity detected by the humidity sensor decreases, the culture vessel drive unit increases the inclination angle of the culture vessel so that the liquid surface area of the culture solution becomes smaller. Device.
Having a dissolved oxygen sensor that measures the amount of oxygen dissolved in the culture solution in the culture vessel,
The culture vessel drive unit drives the culture vessel such that at least one of a reciprocation period and a reciprocation range of the culture solution increases when the amount of dissolved oxygen detected by the dissolved oxygen sensor decreases. 5. The culture device according to any one of 2 to 4.
4. The culture vessel drive unit changes the inclination direction of the culture vessel so that the culture medium circulates along the corner when the amount of the culture medium in the culture vessel exceeds a predetermined threshold amount. 6. The culture device according to any one of 2 to 5.
7. The method according to any one of claims 1 to 6, wherein the culture medium supply unit adds culture medium to the culture vessel at the timing when the cell density approaches the upper limit until the culture medium in the culture vessel from less than 1 liter reaches 50 liters. The culture device according to any one of claims 1 to 3.
A method for culturing cells in which cells are expanded and cultured in a culture medium contained in a culture vessel,
driving the culture vessel so that the culture solution in the culture vessel is agitated;
adding a culture medium to the culture vessel as the number of cells in the culture medium increases;
A method of culturing cells, wherein the culture vessel is driven such that the smaller the volume of the culture medium, the smaller the portion of the surface of the culture vessel that is in contact with the culture medium that is moved by stirring.

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