WO2021002358A1

WO2021002358A1 - Culture apparatus and culture method

Info

Publication number: WO2021002358A1
Application number: PCT/JP2020/025668
Authority: WO
Inventors: 樋口　朗
Original assignee: 株式会社京都製作所
Priority date: 2019-07-04
Filing date: 2020-06-30
Publication date: 2021-01-07
Also published as: JP2022123160A

Abstract

A culture apparatus is provided with: a culture vessel 12 in which a liquid culture medium is accommodated; a culture vessel shaking section which shakes the culture vessel 12 so as to agitate the liquid culture medium CS in the culture vessel 12; and a liquid culture medium feeding section for adding an additional amount of the liquid culture medium CS to the culture vessel 12 in accordance with the increase in the number of cells in the liquid culture medium CS. The culture vessel shaking section shakes the culture vessel 12 in such a manner that the area of a surface part, which comes in 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.

Description

Culture device and culture method

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

Conventionally, in the mass culture of cells, for example, the culture broth from 10 per milliliter ⁴ 10 ⁷ cell density culture initiation from a small amount of cells of a few milliliters, and 10 ⁹ to 10 ¹⁰ quantity of cells It is being propagated until it becomes. First, for example, cells are cultured in a petri dish using a culture solution of several milliliters. When the cell density in the petri dish reaches a predetermined density, the cells in the petri dish are divided into multiple dishes and cultured, and this is repeated to make a total culture solution volume of several tens of milliliters, as compared with the petri dish. It can be transferred to a large capacity culture vessel, for example a flask. After several transfers to such a large volume culture vessel, the cells are finally cultured in a large volume of culture medium, as described in, for example, Patent Document 1. Transferred to a bag. In the culture bag, for example, 50 liters of culture medium is used to culture until the required number of cells is reached. Such a culture is called an expanded culture.

Special Table 2014-507959

However, in such expanded culture, the timing of transfer to a large-volume culture vessel and the rate of increase in volume (ratio of the volume of the culture solution before and after transfer) are the growth curves of the cells (time and cell density). In relation to), it is generally determined by the logarithmic growth phase (the period showing a high cell proliferation rate). Especially when the permissible rate of capacity increase is small, the frequency of transfer is high, the burden on the operator is increased, and the risk of contamination is also high. In addition, since the growth curve may change significantly depending on the cell line, it is essential to monitor the cell density so as not to miss the timing of transfer. This is also a burden on the worker. Further, for each of several culture containers having different capacities, a device for stirring the culture solution in the container is required.

Therefore, the present invention works while realizing continuous expansion culture from a small amount to a large amount in one container with high culture efficiency in a short period of time and reducing the risk of contamination in the expansion culture of cells. The challenge is to enable automation to reduce the burden on the person.

In order to solve the above technical problems, according to one aspect of the present invention,
A culture device that expands and cultures cells in a culture medium.
A culture container that houses the culture solution and
A culture vessel swinging portion that swings the culture vessel so that the culture solution in the culture vessel is agitated,
It has a culture solution supply unit for adding the culture solution to the culture medium as the number of cells in the culture solution increases.
The culture vessel swinging portion swings the culture vessel so that the smaller the amount of the culture solution in the culture vessel, the smaller the surface portion of the culture vessel that comes into contact with the culture solution that moves by stirring. Incubators are provided.

Further, 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.
The culture vessel is shaken so that the culture solution in the culture vessel is agitated.
As the number of cells in the culture solution increased, the culture solution was added to the culture vessel, and the culture solution was added.
A method for culturing cells is provided in which the culture vessel is shaken so that the smaller the amount of the culture solution, the smaller the surface portion of the culture vessel that comes into contact with the culture solution that moves by stirring.

According to the present invention, in the expansion culture of cells, continuous expansion culture from a small amount to a large amount can be realized with high culture efficiency in a short period of time in one container, and the work is performed while reducing the risk of contamination. It is possible to enable automation to reduce the burden on the person.

Schematic block diagram which shows the structure of the culture apparatus which concerns on one Embodiment of this invention. Schematic perspective view of an example of a culture vessel Schematic partial cross-sectional view of the swinging portion of the culture vessel in the culture apparatus Schematic partial cross-sectional view of a part of the swinging portion of the culture vessel shown in FIG. 3 viewed from different directions. Schematic partial cross-sectional view of the swinging portion of the culture vessel shown in FIG. 3 in a state where the culture vessel is tilted. The figure which shows an example of the structure of the culture solution supply part The figure which shows an example of the structure of the cell density measurement part Cross-sectional view showing the tilted state of the culture vessel when the amount of the culture solution is relatively small. Top view showing the tilted state of the culture vessel when the amount of the culture solution is relatively small. The figure which shows the stirring of the culture solution when the culture solution is a relatively small amount. Cross-sectional view showing the state of inclination of the culture vessel when the amount of the culture solution is relatively large. Top view showing the state of inclination of the culture vessel when the amount of the culture solution is relatively large. The figure which shows the stirring of the culture solution when the culture solution is a relatively large amount. Block diagram showing the control system of the culture device The figure for demonstrating an example of the culture solution supply schedule The figure which shows an example of the Gantt chart of the swing pattern of a culture vessel. The figure which shows an example of the graph which shows the change of the culture solution amount in a culture vessel. The figure which shows an example of the graph which shows the change of a cell density

The culture apparatus according to one aspect of the present invention is a culture apparatus for expanding and culturing cells in a culture solution, and the culture is such that the culture container containing the culture solution and the culture solution in the culture container are stirred. The culture vessel swinging portion has a culture vessel swinging portion that swings the container, and a culture solution supply portion that adds a culture solution to the culture vessel as the number of cells in the culture solution increases. The smaller the amount of the culture solution in the culture container, the smaller the surface portion of the culture container that comes into contact with the culture solution that moves by stirring is shaken.

According to this aspect, in the expansion culture of cells, continuous expansion culture from a small amount to a large amount can be realized with high culture efficiency in a short period of time in one container, and the work is performed while reducing the risk of contamination. It is possible to enable automation to reduce the burden on the person.

For example, the culture vessel includes a circular bottom surface and a cylindrical inner peripheral surface erected from the outer peripheral edge of the bottom surface. In this case, the swinging portion of the culture container tilts the culture container so that the culture solution collects in the corner sandwiched between the bottom surface and the inner peripheral surface, and the culture solution reciprocates along the corner. The tilt direction of the culture vessel is changed, and the smaller the amount of the culture solution, the smaller the reciprocating range of the culture solution. As a result, the culture solution can be stirred while suppressing its evaporation.

For example, the swinging portion of the culture vessel tilts the culture vessel more as the amount of the culture solution is smaller. As a result, the surface area of the culture solution is reduced, so that evaporation of the culture solution from the liquid surface can be suppressed.

For example, the culture device 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 tilt angle of the culture container is increased so that the area of the liquid surface of the culture solution becomes smaller. Thereby, the evaporation of the culture solution from the liquid surface can be suppressed.

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 shaking portion shakes the culture vessel so that when the amount of dissolved oxygen detected by the dissolved oxygen sensor decreases, at least one of the reciprocating cycle and the reciprocating range of the culture solution increases. Move. As a result, the culture solution is more agitated, sufficient oxygen is supplied and homogenized to the culture solution, and cell damage can be suppressed.

For example, when the amount of the culture solution in the culture container exceeds a predetermined threshold, the swinging portion of the culture container changes the tilt direction of the culture container so that the culture solution orbits along the corner. .. As a result, a large amount of the culture solution can be sufficiently stirred.

For example, the culture apparatus sets the culture time based on the culture condition acquisition unit that acquires information on the culture conditions including at least the initial number of cells, the target number of cells, the target cell density, and the doubling time, and the culture condition information. It has a culture time calculation unit to be calculated, a supply schedule creation unit that creates a culture solution supply schedule based on the culture condition information and the culture time, and the culture solution supply unit is set to the supply schedule. Based on this, the amount of culture medium supplied per unit time is changed. This makes it possible to automate the expansion culture.

For example, in the culture apparatus, the difference between the cell density measuring unit that measures the cell density of the culture solution in the culture vessel and the cell density measured by the cell density measuring unit and the target cell density is equal to or less than a predetermined value. It has a supply schedule correction unit that corrects the supply schedule so as to be, and the culture solution supply unit changes the culture solution supply amount per unit time based on the corrected supply schedule. As a result, the cell density can be maintained at the target cell density while the expansion culture is automatically performed.

For example, the cell density measuring unit is configured to measure the cell density by inputting a user's measurement instruction at an arbitrary timing. This allows the user to check the cell density at any time.

For example, the supply schedule correction unit is configured to correct the supply schedule by inputting a correction instruction from the user. This makes it possible to create a supply schedule desired by the user.

For example, the cell density measuring unit includes a tube, a roller pump provided in the tube, and a cell density sensor for measuring the cell density of the culture solution in the tube, and the tube has one end thereof in the culture vessel. The other end is arranged so as to be located above the liquid level of the culture solution while being located in the culture solution of the above, and the roller pump is rotated in the normal direction to fill the tube with the culture solution via the one end. , The culture solution in the tube is returned to the culture vessel by reversing. As a result, the entire culture broth is used for culturing without a part of the culture broth staying in the tube.

For example, the cell density sensor measures the culture solution in the portion of the tube arranged so as to have an inclination angle of 30 ° or less with respect to the vertical direction. As a result, the precipitation of cells in the tube is suppressed, and the cell density sensor can accurately measure the cell density.

For example, the culture apparatus has a swing schedule creation unit that creates a swing schedule showing a change in the swing pattern of the culture container swing section with respect to the culture container based on the supply schedule. The rocking portion swings the culture vessel based on the rocking schedule. As a result, the culture vessel can be shaken according to the amount of the culture solution in the culture vessel.

For example, the culture apparatus displays a Gantt chart creation unit that creates a Gantt chart showing at least one change in a swing pattern, cell density, culture temperature, gas concentration, and gas flow rate of the culture vessel, and the Gantt chart. It has a display unit and a display unit. This allows the user to check the culture status.

For example, the culture apparatus has a graph creation unit that creates a graph showing changes in at least one of the amount of culture solution and the cell density in the culture vessel, and the display unit together with the Gantt chart have the graph creation unit. Display the graph created by. This allows the user to check the status of at least one of the amount of culture medium and the cell density in the culture vessel.

For example, the culture apparatus has a culture end condition acquisition unit for acquiring information on the culture end condition including at least one of the number of cells reached, the amount of the reached culture solution, and the reached culture time, and the culture end condition is satisfied. After that, the culture solution supply unit stops the supply of the culture solution, and the culture vessel swinging unit stops the shaking of the culture vessel. As a result, the expanding culture that is being executed can be automatically terminated.

For example, the culture apparatus has a notification unit for notifying the user when the culture end condition is satisfied, and a culture end input unit for the user to end the culture, and the user input to the culture end input unit can be performed. After the execution, the culture solution supply unit stops the supply of the culture solution, and the culture vessel swinging unit stops the shaking of the culture vessel. As a result, it is suppressed that the cells die after being left unattended after the completion of the culture.

For example, the culture solution supply unit is provided in the culture solution storage container for storing the culture solution, a tube having one end connected to the culture solution storage container and the other end connected to the culture solution container, and the tube. A portion of the tube is disposed on the outer surface of the culture vessel, including a roller pump. As a result, the temperature of the culture solution to be supplied to the culture container from now on becomes about the same as the temperature of the culture solution in the culture container, and as a result, the temperature change of the culture solution in the culture container is suppressed.

When the temperature is controlled before the culture solution is supplied to the culture container as described above, for example, the culture apparatus includes a refrigerator for refrigerating and accommodating the culture solution storage container and a heater for heating the culture container. You may.

Another aspect of the culture method of the present invention is a method for culturing cells in which cells are expanded and cultured in a culture solution contained in the culture container, and the culture container is such that the culture solution in the culture container is stirred. The culture solution is added to the culture container as the number of cells in the culture solution increases, and the smaller the amount of the culture solution, the more the surface portion of the culture container that comes into contact with the culture solution that moves by stirring. The culture vessel is shaken so that

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a schematic configuration diagram showing a configuration of a culture apparatus according to an embodiment of the present invention.

As shown in FIG. 1, the culture apparatus 10 shakes the culture container 12 that houses the culture solution CS containing cells and the culture container 12 that shakes the culture solution CS in the culture container 12 to stir. It has a moving unit 14 and a culture solution supply unit 16 that supplies the culture solution CS to the culture container 12.

Further, in the case of the present embodiment, the culture apparatus 10 includes a humidity sensor 18 for measuring the humidity in the culture vessel 12, a dissolved oxygen sensor 20 for measuring the amount of oxygen dissolved in the culture solution CS in the culture vessel 12. It has a cell density measuring unit 22 for measuring the cell density in the culture solution CS in the culture vessel 12.

The culture device 10 has a gas supply unit 24 that supplies a mixed gas of humidified oxygen, carbon dioxide, and nitrogen to the culture container 12.

Further, the culture apparatus 10 controls the culture vessel shaking unit 14, the culture solution supply unit 16, and the gas supply unit 24 based on the detection results of the humidity sensor 18, the dissolved oxygen sensor 20, and the cell density measurement unit 22, respectively. It has a control unit 26.

The culture container 12 is a container for accommodating the culture solution CS, and cells are cultured inside the culture container 12 using the culture solution CS. In this culture vessel 12, cells are cultured using the culture solution CS, that is, expanded culture, while gradually adding the culture solution CS from a small amount (less than 1 liter, for example, 50 ml) as the number of cells increases. Will be done. Therefore, the culture vessel 12 has a capacity capable of accommodating and stirring the maximum amount (for example, 50 liters) of the culture solution used for culturing.

FIG. 2 is a perspective view showing the shape of an example of the culture vessel 12. Although the XYZ Cartesian coordinate system is shown in the drawings, this is for facilitating the understanding of the embodiment of the invention and does not limit the invention. Further, 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 is supported by 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 a side wall portion 12b. It is provided with a top plate portion 12c to be formed. That is, the culture vessel 12 has a so-called basin shape. The height of the side wall portion 12b is made smaller than the radius of the bottom plate portion 12a. Further, the top plate portion 12c is removable and functions as a lid.

FIG. 3 is a schematic partial cross-sectional view of the culture vessel swinging portion 14 in the culture apparatus 10. Further, FIG. 4 is a schematic partial cross-sectional view of a part of the culture vessel swinging portion 14 shown in FIG. 3 viewed from different directions.

As shown in FIGS. 3 and 4, the culture vessel swinging portion (culture vessel swinging device) 14 in the culture apparatus 10 extends in the vertical direction (Z-axis direction) with the stage 30 holding the culture vessel 12. A rotary actuator 34 including a rotary table 32 that rotates around a rotation center axis C0 is provided.

The stage 30 and the rotary actuator 34 are drive-connected via the swing head 36 and the tilting mechanism 38.

The swing head 36 supports the stage 30 and swings extending in the horizontal direction (X-axis direction) and extending in the horizontal direction (Y-axis direction) and orthogonal to the swing axis C1. The culture vessel swinging portion 14 is provided so as to swing around the shaft C2. Further, the swing head 36 is provided with a connecting shaft 40 for driving and connecting with the rotary actuator 34 via a tilting mechanism 38 below the swing head 36. 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 swinging head 36, that is, tilting the culture vessel 12 on the stage 30 in the horizontal direction. Therefore, the tilting mechanism 38 includes a base portion 42, a swing head connecting portion 44 connected to the swing head 36, and a link arm 46 connecting the base portion 42 and the swing head connecting portion 44. ..

The 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 around the rotation center axis C0 together with the rotary table 32.

The swing head connecting portion 44 of the tilting mechanism 38 is extrapolated to the connecting shaft 40 of the swing head 36 so as to be slidable, for example, via a bearing.

The link arm 46 of the tilting mechanism 38 is configured to connect the base portion 42 and the swing head connecting portion 44. Specifically, the link arm 46 includes one end rotatably fixed to the swing head connecting portion 44 and the other end rotatably fixed to the base portion 42. The rotation shaft C3 at one end and the rotation shaft C4 at the other end of the link arm 46 extend in the horizontal direction 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 with the screw shaft 50, and a motor (not shown) for rotating the screw shaft 50. .. The nut 52 is attached to the elevating bracket 54. A rotary actuator 34 is attached to the elevating bracket 54.

When the ball screw mechanism 48 is driven, the rotary actuator 34 moves up and down together with the elevating bracket 54 via the nut 52. For example, as shown in FIG. 5, when the rotary actuator 34 is raised by the ball screw mechanism 48, 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 rises, whereby the link arm 46 pushes the swing head connecting portion 44. As a result, the swing head 36 and the swing head connecting portion 44 rotate about at least one of the swing shafts C1 and C2 (the swing shaft 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 in a state where the stage 30 is tilted, the tilting mechanism 38 rotates around the rotation center axis C0, whereby the tilting direction of the stage 30 Changes. As a result, the culture solution CS in the culture container 12 is stirred, and the cells in the culture solution CS are cultured.

In such a culture vessel swinging portion 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 tilting direction of the stage 30 only rotates once. Is. That is, the lowest portion of the culture vessel 12 on the stage 30 is only sequentially changed to another portion.

Returning to FIG. 1, in the case of the present embodiment, the culture solution supply unit 16 that supplies the culture solution CS to the culture container 12 is controlled by the control unit 26.

FIG. 6 is a diagram showing an example of the configuration of the culture solution supply unit 16.

As shown in FIG. 6, in the case of the present embodiment, the culture solution supply unit 16 is attached to the culture solution storage container 60 for storing the culture solution CS, one end 62a connected to the culture solution storage container 60, and the culture solution container 12. It includes a tube 62 having a connected other end 62b and a roller pump 64 provided on the tube 62. The control unit 26 controls the supply amount of the culture solution CS to the culture container 12 by controlling the roller pump 64 in the culture solution supply unit 16. The details of controlling the supply amount of the culture solution CS by the control unit 26 will be described later.

In the case of the present embodiment, as shown in FIG. 6, a part of the tube 62 is arranged on the outer surface of the culture vessel 12. For example, a part of the tube 62 is arranged in a helical or mianda shape on the outer surface of the top plate portion 12c of the culture vessel 12. The reason why the tube 62 is arranged on the outer surface of the culture vessel 12 in this way is that the temperature of the culture solution CS in the tube 62 is brought close to the temperature of the culture solution CS in the culture vessel 12. For example, as shown in FIG. 6, when the culture container 12 is kept warm by the heater 66 and / or when the culture solution storage container 60 is refrigerated in the refrigerator 68, the culture solution CS in the culture container 12 The difference between the temperature and the temperature in the culture solution CS in the tube 62 is large. By disposing the tube 62 on the outer surface of the culture container 12, the culture solution CS flowing in the tube 62 is warmed to the temperature of the culture solution CS in the culture container 12. As a result, the temperature change of the culture solution CS in the culture vessel 12 caused by the inflow of the culture solution CS from the tube 62 is suppressed, and the damage to the cells inside the culture solution CS is suppressed.

Further, in the case of the present embodiment, the culture vessel 12 is shaken by the culture vessel swinging portion 14 as described above. Therefore, as the tube 62, a silicon tube made of a material having high flexibility, for example, silicone rubber, is preferable. Further, the roller pump 64 may be provided in the culture vessel 12, and the portion of the tube 62 from the roller pump 64 to the culture vessel 12 may be arranged on the outer surface of the culture vessel 12.

Further, in the case of the present embodiment, the other end 62b of the tube 62 is provided along the inner peripheral surface 12d of the culture vessel 12, specifically, the culture flowing out from the opening of the other end 62b of the tube 62. The other end 62b of the tube 62 is provided along the inner peripheral surface 12d so that the liquid CS flows along the inner peripheral surface 12d. As a result, the culture solution CS flowing out from the other end 62b of the tube 62 is warmed by the inner peripheral surface 12d. In this case, it is preferable that the other end 62b is cut diagonally so that the opening of the other end 62b faces the inner peripheral surface 12d.

By arranging the tube 62 on the outer surface of the culture vessel 12 in this way, it is not necessary to separately provide a heating device such as a heater for heating the culture solution in the tube 62.

Further, in addition to the culture solution CS, the mixed gas is supplied to the culture container 12 by the gas supply unit 24. For example, the gas supply unit 24 is configured to mix oxygen, carbon dioxide, and nitrogen, and humidify the mixed gas by a humidifying unit (not shown). The humidified mixed gas is introduced into the culture vessel 12 through a filter (not shown) for preventing contamination of various germs and a gas introduction port (not shown) provided in the culture vessel 12. The culture vessel 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 discharged to the atmosphere through a filter. Further, in the gas supply unit 24, the supply timing and the supply amount of the gas are controlled by the control unit 26.

The humidity sensor 18 is attached to the inner peripheral surface 12d of the culture vessel 12 so as not to be immersed in the culture solution CS, and measures the humidity in the culture vessel 12. Further, the humidity sensor 18 outputs a signal corresponding to the measured humidity to the control unit 26.

The dissolved oxygen sensor 20 measures the amount of oxygen dissolved in the culture solution CS in the culture vessel 12. For example, as the dissolved oxygen sensor 20, a fluorescent dissolved oxygen sensor is used. For example, the fluorescent dissolved oxygen sensor is arranged on the bottom surface 12e of the culture vessel 12 and coated with a fluorescent substance, a light source that irradiates the chip with ultraviolet rays or the like from the outside of the culture vessel 12, and radiates from the chip. It is provided with a light receiving element that receives the received fluorescence.

When the fluorescent substance absorbs light energy such as ultraviolet rays from the light source, it transitions from the ground state to the excited state. Excited molecules of fluorescent material usually radiate fluorescence and return to the ground state. However, at this time, if oxygen molecules are present around the molecules in the excited state, the excitation energy is deprived by the oxygen molecules, and so-called oxygen quenching occurs in which the radiant intensity of fluorescence decreases. Utilizing this oxygen quenching, that is, the fact that the radiant intensity of fluorescence is inversely proportional to the oxygen molecule concentration, the fluorescent dissolved oxygen sensor measures the dissolved oxygen amount of the culture solution in the culture vessel.

Further, the dissolved oxygen sensor 20 outputs a signal corresponding to the measured dissolved oxygen amount to the control unit 26.

The cell density measuring unit 22 measures the cell density of the culture solution CS in the culture vessel 12. The measured cell density is output to the control unit 26. The cell density during culturing is monitored by the periodic measurement of the cell density measuring unit 22. Further, although the details will be described later, the control contents of the culture vessel swinging unit 14 and the culture solution supply unit 16 are changed (corrected) based on the measurement results of the cell density measuring unit 22.

FIG. 7 is a diagram showing an example of the configuration of the cell density measuring unit.

As shown in FIG. 7, in the case of the present embodiment, the cell density measuring unit 22 measures the cell density of the tube 70, the roller pump 72 provided in the tube 70, and the culture solution CS in the tube 70. Includes a density sensor 74.

The tube 70 is arranged so that one end 70a thereof is located in the culture solution CS in the culture vessel 12 and the other end 70b is located above the liquid level LS of the culture solution CS. When the roller pump 72 rotates in the normal direction (rotates in the direction of the arrow R), a flow of the culture solution CS from one end 70a of the tube 70 to the other end 70b is generated in the tube 70. That is, the culture solution CS that has flowed into the tube 70 via one end 70a returns to the culture vessel 12 again via the other end 70b.

When the tube 70 is filled with the culture solution CS, the cell density sensor 74 measures the cell density of the culture solution CS in the tube 70. Examples of the cell density sensor 74 include a sensor for measuring the turbidity of the culture solution CS, a sensor for measuring the degree of laser scattering by cells, and a sensor for measuring the dielectric constant of the culture solution CS.

In the case of the present embodiment, the cell density sensor 74 is provided in the portion of the tube 70 between the one end 70a into which the culture solution CS flows into the culture vessel 12 and the roller pump 72. Alternatively, the cell density sensor 74 may be provided in the portion of the tube 70 between the roller pump 72 and the other end 70b where the culture solution CS flows out toward the culture vessel 12.

Further, in the case of the present embodiment, when the measurement of the cell density by the cell density sensor 74 is completed, the roller pump 72 reverses (rotates in the opposite direction of the arrow R). By this reversal, the culture solution CS in the tube 70 is returned to the culture vessel 12 via one end 70a. At this time, since the other end 70b of the tube 70 is located above the liquid level LS of the culture solution CS, the culture solution CS in the culture container 12 does not flow into the tube 70 via the other end 70b. As a result, a part of the culture solution CS is suppressed from staying in the tube 70, and as a result, the entire culture solution CS is used for culturing.

Depending on the type of cells and cell fluid, the cell density sensor 74 may not be able to accurately measure the cell density due to the precipitation of cells in the tube 70. As a countermeasure, in the case of the present embodiment, the tube 70 includes an inclined portion 70c arranged so as to have an inclination angle of a predetermined angle θ (for example, 30 °) or less with respect to the vertical direction (Z-axis direction). .. The cell density sensor 74 measures the cell density in the inclined portion 70c. Since such an inclined portion 70c is less likely to cause cell precipitation than the portion of the tube 70 having a predetermined angle θ or more, the cell density sensor 74 can accurately measure the cell density.

Returning to FIG. 1, the control unit 26 is composed of, for example, a control board on which a storage device and a CPU are mounted. By operating according to the program stored in the storage device, the CPU executes an operation related to cell culture described later.

First, the control unit 26 controls the culture solution supply unit 16 (in the case of the present embodiment, the roller pump 64).

By being controlled by the control unit 26, the culture solution supply unit 16 (roller pump 64) adds the culture solution CS to the culture solution CS as the number of cells in the culture solution CS of the culture container 12 increases. For example, the roller pump 64 gradually adds the culture solution CS to the culture container 12 until the culture solution CS of less than 1 liter (for example, 200 ml) becomes 50 liters in one culture container 12.

Further, the control unit 26 controls the culture vessel swinging portion 14 (its rotary actuator 34 and ball screw mechanism 48) based on the amount of the culture solution CS in the culture vessel 12.

By being controlled by the control unit 26, the culture vessel swinging unit 14 swings the culture vessel 12 so that the culture solution CS is agitated while suppressing the evaporation of the culture solution CS in the culture vessel 12. To do. Specifically, in the culture vessel swinging portion 14, the smaller the amount of the culture solution CS in the culture vessel 12, the smaller the surface portion of the culture vessel 12 that comes into contact with the culture solution that moves by stirring. Shake the culture vessel 12. The shaking of the culture vessel 12, that is, the stirring of the culture solution CS will be described.

FIG. 8A is a cross-sectional view showing a tilted state of the culture vessel 12 when the amount of the culture solution is relatively small. Further, FIG. 8B is a top view showing a tilted state of the culture vessel 12 when the amount of the culture solution is relatively small.

As shown in FIGS. 8A and 8B, the culture solution is agitated with the culture vessel 12 tilted. The inclination angle θ (angle with respect to the culture container 12 in the horizontal state) of the culture container 12 is increased as the amount of the culture solution CS in the culture container 12 is small.

As the amount of the culture solution CS is small in this way, the size of the area of the liquid level LS of the culture solution CS becomes smaller by tilting the culture container 12 greatly. By reducing the size of the area of the liquid level LS, it is possible to suppress the evaporation of the culture solution CS from the liquid level LS.

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

Therefore, as the amount of the culture solution CS is smaller, the influence of the evaporation of the culture solution CS on the cells is reduced by tilting the culture vessel 12 more (by increasing the inclination angle θ).

When the amount of the culture solution CS in the culture container 12 is equal to or greater than the amount in which the effect of evaporation of the culture solution CS on the cells is sufficiently small, the inclination angle θ of the culture container 12 may be constant. ..

As the culture vessel 12 is tilted, as shown in FIG. 8B, the culture solution CS is sandwiched between the circular bottom surface 12e of the culture vessel 12 and the cylindrical inner peripheral surface 12d erected from the outer peripheral edge of the bottom surface 12e. It collects in the corner 12f. In this state, the tilting direction of the culture vessel 12 is changed.

FIG. 9 is a diagram showing stirring of the culture solution when the amount of the culture solution is relatively small. FIG. 9 shows a state in which the culture vessel 12 being stirred is viewed from above (viewed in the Z-axis direction).

As shown in FIG. 9, a relatively small amount (for example, less than 1 liter) of the culture solution CS is reciprocated 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 repeats the forward rotation and the reverse rotation of the tilting mechanism 38 in an angle range of 90 degrees, the tilting direction of the culture vessel 12 changes in an angle range of 90 degrees. As a result, the culture solution CS is reciprocated in an angle range of 90 degrees. As a result, the culture solution CS is stirred. As shown in FIG. 9, when the Y-axis plus direction is set to the 0-degree direction with respect to the Z-axis, for example, the position of +45 degrees from the position of -45 degrees (315 degrees) with the position of 0 degrees as the center. The culture solution CS is reciprocated between the two.

The smaller the amount of the culture solution CS, the smaller the reciprocating range (angle range) of the culture solution CS. The reason is to suppress the evaporation of the culture solution CS.

Specifically, when the culture solution CS moves on the surface of the culture vessel 12 by stirring, a very small amount of the culture solution CS remains on the surface after most (lumpy) culture solution CS has passed. For example, as shown in FIG. 9, after most (lumps) of the culture solution CS move to the position of 45 degrees, a very small amount of the culture solution CS remains at the position of 0 degrees. This remaining minute amount of culture solution CS is easy to evaporate. Therefore, before the minute amount of the culture solution CS evaporates, the massive culture solution CS returns and absorbs the minute amount of the culture solution CS. Further, the smaller the amount of the culture solution CS, the greater the influence of evaporation on the cells, so that the reciprocating range of the culture solution CS is reduced. As a result, when the amount of the culture solution CS is relatively small, the evaporation of the culture solution CS can be suppressed.

As the number of cells increases, the culture solution CS is added to the culture container 12, and the amount of the culture solution CS in the culture container 12 increases. The reciprocating range of the culture solution CS is expanded as the amount increases. This is because the influence of the evaporation on the cells is reduced by increasing the culture solution CS, but it is necessary to stir the culture solution CS more.

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

FIG. 10A is a cross-sectional view showing a tilted state of the culture vessel 12 when the amount of the culture solution is relatively large. Further, FIG. 10B is a top view showing a tilted state of the culture vessel 12 when the amount of the culture solution is relatively large.

As shown in FIGS. 10A and 10B, and with reference to FIGS. 8A and 8B, when the culture solution CS is relatively large, the culture container 12 is compared with the case where the culture solution CS is relatively small. The tilt angle θ of is small. This is because the depth of the culture solution CS is reduced and a gas such as oxygen is distributed throughout the culture solution CS.

The greater the depth of the culture solution CS, the more difficult it is for gas such as oxygen taken in through the liquid surface of the culture solution CS by stirring to spread throughout the culture solution CS. Specifically, it is difficult for the gas to reach the deep part of the culture solution CS. As a result, the amount of dissolved oxygen in the deep part of the culture solution CS may be insufficient, and the cells may be damaged.

When the culture vessel 12 is tilted, the culture solution CS is accumulated in the corner 12f sandwiched between the bottom surface 12e and the inner peripheral surface 12d of the culture vessel 12, as shown in FIG. 10B. In this state, the tilting direction of the culture vessel 12 is changed.

FIG. 11 is a diagram showing stirring of the culture solution CS when the amount of the culture solution CS is relatively large. FIG. 11 shows a state in which the culture vessel 12 being stirred is viewed from above (viewed in the Z-axis direction).

A relatively large amount (for example, 1 liter or more) of the culture solution 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, the rotary actuator 34 keeps rotating the tilting mechanism 38 in one direction, so that the tilting direction of the culture vessel 12 keeps rotating in one direction. As a result, the culture solution CS is circulated. As a result, the culture solution CS is stirred.

In this way, the control unit 26 changes the stirring mode (oscillation pattern) based on the amount of the culture solution CS in the culture vessel 12. For example, when the amount of the culture solution CS in the culture container 12 is smaller than the predetermined threshold amount (for example, 1 liter), the culture solution CS is reciprocated by reciprocating the culture solution CS as shown in FIG. The culture solution CS is stirred. Further, the smaller the amount of the culture solution CS, the smaller the reciprocating range of the culture solution CS. On the other hand, when the amount of the culture solution CS in the culture vessel 12 exceeds a predetermined threshold amount, the culture solution CS is stirred by circulating the culture solution CS as shown in FIG. The amount of the culture solution CS in the culture container 12 may be calculated from, for example, the weight of the culture solution CS in the culture container 12 measured by a weight sensor (not shown).

In addition, in the case of the present embodiment, the control unit 26 is configured to control the culture vessel rocking device 14 based on the measurement results of the humidity sensor 18 and the dissolved oxygen sensor 20 while stirring the culture solution CS. ing.

Specifically, when the humidity in the culture solution 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 swinging unit controlled by the control unit 26 In No. 14, the inclination angle of the culture vessel 12 (that is, the stage 30) is increased so that the area of the liquid level LS of the culture solution CS is small.

When the humidity in the culture vessel 12 decreases, the culture solution CS tends to evaporate from the liquid level LS. Therefore, by reducing the area of the liquid level LS of the culture solution CS, its evaporation can be suppressed.

Further, when the amount of dissolved oxygen detected by the dissolved oxygen sensor decreases, for example, when the amount of dissolved oxygen decreases beyond the lower limit of a predetermined appropriate range, the culture vessel swinging unit 14 controlled by the control unit 26 moves the culture solution CS. The culture vessel 12 is swung so that at least one of the reciprocating cycle and the reciprocating range is increased. The dissolved oxygen sensor 20 (the chip thereof) is provided at a position on the culture vessel 12 that can come into contact with the culture solution CS and detect the dissolved oxygen amount regardless of the amount of the culture solution CS in the culture vessel 12. In the case of the present embodiment, the dissolved oxygen sensor 20 is provided on the outer peripheral edge of the bottom surface 12e of the culture vessel 12. Further, when the dissolved oxygen sensor 20 measures the dissolved oxygen amount, the culture vessel 12 is shaken by the culture vessel swinging portion 14 so that the culture solution CS comes into contact with 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 dissolved oxygen amount, the shaking speed and shaking pattern of the culture vessel 12 may be temporarily changed, or the culture may be performed. The swing of the container 12 may be temporarily stopped.

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 reciprocating cycle and the reciprocating range of the culture solution CS, the culture solution CS is more agitated, whereby a large amount of oxygen is taken into the culture solution CS. As a result, cell damage can be suppressed.

When the culture solution CS in the culture vessel 12 is relatively large and orbits, the culture solution CS is more agitated by increasing the orbiting speed, whereby a large amount of the culture solution CS is contained in the culture solution CS. Can take in oxygen.

Further, in the case of the present embodiment, the control unit 26 controls the culture vessel swinging unit 14 and the culture solution supply unit 16 according to the culture schedule. This will be described.

FIG. 12 is a block diagram showing a control system of the culture apparatus 10.

The control unit 26 of the culture apparatus 10 acquires information on the culture from the user, creates a culture schedule based on the acquired information, and based on the created culture schedule, the culture container swing unit 14 and the culture as described above. The liquid supply unit 16 is controlled.

Therefore, as shown in FIG. 12, the control unit 26 includes a culture condition acquisition unit 100 that acquires culture condition information from the user, and a culture end condition acquisition unit 102 that acquires culture end condition information from the user. The culture time calculation unit 104 that calculates the culture time based on the acquired culture conditions, the supply schedule creation / correction unit 106 that creates and corrects the supply schedule of the culture solution CS to be supplied to the culture vessel 12, and the culture vessel 12 shake. A swing schedule creation unit 108 for creating a dynamic schedule, a Gantt chart creation unit 110 for creating a Gantt chart based on a swing schedule, a graph creation unit 112 for creating a graph of culture medium volume and cell density, and a culture It includes a culture end determination unit 114 for determining the end, and an output unit 116 for outputting information about the culture. As described above, when the control unit 26 is composed of a storage device and a control board on which the CPU is mounted, the CPU operates according to the program stored in the storage device, so that the CPU acquires these culture conditions. It functions as a unit 100 or the like.

Further, an input device 80 such as a keyboard, a mouse, and a touch panel is connected to the control unit 26 in order to acquire various instruction inputs from the user. Further, a display device 82 such as a monitor is connected to the control unit 26 so that the output unit 116 outputs information about the culture to the user. The input device 80 and the display device 82 may be integrated as a touch screen.

The culture condition acquisition unit 100 in the control unit 26 acquires culture condition information including at least the initial number of cells, the target cell number, the target cell density, and the doubling time from the user via the input device 80 as the culture condition information. To do. That is, the culture condition acquisition unit 100 acquires the minimum information necessary for creating the culture schedule (supply schedule and swing schedule). The initial number of cells is the number of cells at the start of culturing, the target number of cells is the final number of cells obtained by culturing, and the target cell density is the cell density to be maintained during culturing.

The culture end condition acquisition unit 102 in the control unit 26 inputs information on the culture end condition including at least one of the number of cells reached, the amount of the reached culture solution, and the reached culture time as the culture end condition information from the user to the device 80. Get through.

The culture time calculation unit 104 in the control unit 26 calculates the culture time required for culture based on the culture condition information acquired by the culture condition acquisition unit 100 from the user. Specifically, the culture time calculation unit 104 calculates the time required for the number of cells to increase from the initial number of cells to the target number of cells while maintaining the target cell density as the culture time, based on the doubling time. ..

The supply schedule creation / correction unit 106 in the control unit 26 creates a culture solution supply schedule as a culture schedule based on the culture condition information and the culture time. Specifically, the change in the number of cells during the increase from the initial number of cells to the target number of cells in the culture time calculated by the culture time calculation unit 104 is calculated based on the doubling time, and based on the change in the number of cells. The change in the amount of culture solution CS in the culture vessel 12 required to maintain the target cell density is calculated. Based on the calculation result, the supply schedule creation / correction unit 106 uses the temporal change of the flow rate (supply amount per unit time) of the culture solution CS supplied by the culture solution supply unit 16 to the culture container 12 as the supply schedule. create. The control unit 26 changes the flow rate of the culture solution supply unit 16 (in the case of the present embodiment, the roller pump 64) based on this supply schedule.

It is preferable that the supply schedule is created so that the cell density does not become higher than the target cell density.

FIG. 13 is a diagram for explaining an example of the culture solution supply schedule.

FIG. 13 shows the change in the target flow rate required to maintain the target cell density. A supply schedule is created so that the flow rate (controlled flow rate) of the culture solution supply unit 16 does not fall below this target flow rate. For example, as a supply schedule, a schedule is created in which the flow rate of the culture solution supply unit 16 is gradually increased by the minimum necessary increase amount. According to such a supply schedule, the cell density in the culture medium CS in the culture vessel 12 is lower than the target cell density, but the cell density greatly exceeds the target cell density (cells become overcrowded). That is suppressed. That is, in the present embodiment, the cell density is controlled and the supply of the culture solution is scheduled so as to maintain the so-called logarithmic growth phase, thereby shortening the culture period and increasing the culture efficiency.

Further, the supply schedule may be created as a change in the ON / OFF ratio of the culture solution supply unit 16. That is, the culture solution supply unit 16 may intermittently supply the culture solution CS to the culture container 12. For example, when the roller pump 64 of the culture solution supply unit 16 needs to supply the culture solution CS at a flow rate of 10 ml / min, the solution is supplied at a flow rate of 60 ml / min for 2 seconds and the solution is stopped for 10 seconds. And may be repeated alternately. By such intermittent liquid feeding, when the flow rate of the culture solution supply unit 16 is increased stepwise as shown in FIG. 13, the amount of increase to the next flow rate can be reduced. As a result, the excessive supply of the culture solution CS to the culture vessel 12, that is, the excessive decrease in cell density is suppressed.

Further, the supply schedule creation / correction unit 106 corrects the supply schedule so that the difference between the cell density measured by the cell density measurement unit 22 and the target cell density is equal to or less than a predetermined value during culturing.

Specifically, when the difference (absolute value) between the cell density periodically measured by the cell density measuring unit 22 during culturing and the target cell density exceeds a predetermined threshold value, the supply schedule creation / correction unit 106 Corrects the supply schedule in use. For example, when the measured cell density is higher than the target cell density by more than a predetermined threshold value, the supply schedule creation / correction unit 106 increases the supply amount of the culture solution CS to the culture vessel 12. Correct the supply schedule. Based on the corrected supply schedule, the control unit 26 changes the flow rate of the culture solution supply unit 16 (roller pump 64 in the case of the present embodiment), so that the cells of the culture solution CS in the culture container 12 are changed. The difference between the density and the target cell density is made smaller than the predetermined value.

In addition to periodically measuring the cell density during culturing, the cell density measuring unit 22 may be configured to measure the cell density according to a user's instruction at an arbitrary timing during culturing. Good. For example, the cell density measuring unit 22 measures the cell density during culturing by receiving a user's measurement instruction input to the input device 80. The measured cell density is output to the user by the output unit 116 via the display device 82. This allows the user to check the cell density at any time.

In connection with the cell density measurement unit 22 performing cell measurement based on the user's instruction, the supply schedule creation / correction unit 106 may be configured to correct the supply schedule by inputting the user's correction instruction. For example, after confirming the cell density, the user inputs an increase / decrease value of the supply amount of the culture solution CS to the input device 80 as a correction instruction input. The supply schedule creation / correction unit 106 corrects the supply schedule so that the supply amount of the culture solution CS increases or decreases based on the input increase / decrease value. As a result, the supply schedule of the culture solution CS desired by the user can be created.

The swing schedule creation unit 108 in the control unit 26 creates a swing schedule for the culture vessel 12 as a culture schedule based on the supply schedule of the culture solution CS created / corrected by the supply schedule creation / correction unit 106. Specifically, the swing schedule creation unit 108 creates a swing schedule indicating a change in the swing pattern of the culture vessel 12 based on a supply schedule, that is, a change in the amount of the culture solution CS in the culture vessel 12. .. As described above, the swing pattern (tilt angle θ, the cycle of reciprocating motion of the culture solution CS and the reciprocating range) of the culture container 12 differs depending on the amount of the culture solution CS in the culture container 12. The control unit 26 stores in the storage device a swing pattern determination table showing the correspondence between the swing pattern and the amount of the culture solution CS. The swing schedule creation unit 108 creates a swing schedule based on the swing pattern determination table and the supply schedule. The control unit 26 controls the swing of the culture vessel 12 by the culture vessel swing section 14 based on this swing schedule.

The Gantt chart creation unit 110 in the control unit 26 creates a Gantt chart showing changes in the swing pattern based on the swing schedule created by the swing schedule creation unit 108. The created Gantt chart is output to the user by the output unit 116 via the display device 82.

FIG. 14 shows an example of a Gantt chart of a swing pattern of the culture vessel 12.

FIG. 14 shows a Gantt chart 120 of five types of swing patterns A to E performed in a culture time of about 15 days. DX on the horizontal axis represents the Xth day after the start of culturing.

In the Gantt chart 120, the execution period of each of the swing patterns A to E is indicated by the time bars 122A to 122E. Specifically, the length of the time bar indicates the duration of the swing pattern, the left end indicates the start timing of the swing pattern, and the right end indicates the end timing of the swing pattern. The seek slider 124 also indicates the current timing of the culture time. That is, when the culture is started, the seek slider 124 starts to move to the right side of the Gantt chart 120.

By displaying such a Gantt chart 120 on the display device 82, the user can confirm the culture status.

The culture temperature (temperature in the culture vessel), gas concentration (concentration of oxygen and carbon dioxide in the culture vessel), and the gas supply unit 22 are on the time bars 122A to 122E of the swing patterns A to E of the Gantt chart. At least one measurement of gas flow rate may be displayed. This allows the user to visually confirm the relationship between the change in the swing pattern and the related change in parameters such as the culture temperature.

Returning to FIG. 12, the graph creation unit 112 in the control unit 26 creates a graph showing changes in at least one of the culture fluid volume and the cell density in the culture vessel 12. The output unit 116 outputs the created graph to the user via the display device 82 together with the Gantt chart created by the Gantt chart creation unit 110.

FIG. 15 is an example of a graph showing a change in the amount of culture solution in the culture vessel 12. Further, FIG. 16 is an example of a graph showing changes in cell density.

Graph 130 shown in FIG. 15 shows changes in the amount of culture solution in the two types of culture containers 12. The dotted line shows the change in the amount of the culture solution (predicted value) based on the supply schedule of the culture solution CS (before correction) first created by the supply schedule creation / correction unit 106. Further, the solid line shows the change in the amount of the culture solution (actual value) based on the supply schedule corrected based on the result of the periodic measurement of the cell density of the cell density measuring unit 22. The tip of the solid line shows the current value of the culture medium volume. By referring to such a graph 130, the user can confirm the change in the amount of the culture solution in the culture vessel 12. Further, such a graph 130 can be used as a reference when the user corrects the supply schedule as described above.

Graph 140 shown in FIG. 16 shows two types of changes in cell density. The dotted line shows the change in cell density (predicted value) based on the supply schedule of the culture medium CS (before correction) first created by the supply schedule creation / correction unit 106. The solid line shows the change in cell density (actual value) measured by the cell density measuring unit 22. The tip of the solid line shows the current value of the culture medium volume. Further, the graph 140 shows a line showing the target cell density TC, a line showing the upper limit value UC of the cell density, and a line showing the lower limit value LC of the cell density. The upper limit value UC and the lower limit value LC of the cell density are acquired from the user by the culture condition acquisition unit 100. By referring to such a graph 140, the user can confirm the change in cell density. Further, such a graph 140 can be used as a reference when the user corrects the supply schedule as described above.

Returning to FIG. 12, the culture end determination unit 114 in the control unit 26 determines whether or not the culture end condition acquired by the culture end condition acquisition unit 102 from the user is satisfied. When the culture end condition is satisfied, the control unit 26 stops the shaking of the culture container 12 of the culture container shaking unit 14 and stops the supply of the culture solution CS of the culture solution supply unit 16. The control unit 26 also stops the gas supply of the gas supply unit 24. Then, the output unit 116 of the control unit 26 notifies the user of the end of the culture via the display device 82. The end of the culture may be notified by voice via a speaker or the like. Further, when the culture end condition acquisition unit 102 acquires a plurality of culture end conditions from the user, the culture may be terminated when at least one culture end condition is satisfied.

In addition, there is a possibility that the user leaves the culture solution CS after culturing as it is. In this case, since the culture vessel 12 has stopped swinging, the cells in the culture solution CS may die due to lack of oxygen or the like depending on the type of cells and the leaving time. As a countermeasure, when the culture end condition is satisfied, the control unit 26 satisfies the culture end condition while keeping the culture container swinging unit 14, the culture solution supply unit 16, and the gas supply unit 24 operating without stopping. Notify the user via the display device 82, the speaker, or the like. After that, when the user executes an instruction input for the end of culture to the input device 80, the control unit 26 stops the culture container swing unit 14, the culture solution supply unit 16, and the gas supply unit 24.

The output unit 116 of the control unit 26 outputs information about the culture to the user via the display device 82. As described above, the output unit 116 is the Gantt chart created by the Gantt chart creation unit 110, the graph created by the graph creation unit 112, the culture conditions acquired by the culture condition acquisition unit 100, and the culture end condition acquisition unit 102. The display device 82 is displayed with the culture end conditions and the like acquired by. Further, as other information, the output unit 116 may display information such as the elapsed time from the start of culture, the current number of cells, the current amount of culture solution, and the current cell density on the display device 82 in real time. The control unit 26 may be configured so that the information output by the output unit 116, that is, the information displayed by the display device 82 can be selected by the user via the input device 80.

In this way, the control unit 26 having the configuration shown in FIG. 12 controls the culture vessel shaking unit 14 and the culture solution supply unit 16 based on the culture schedule (culture solution supply schedule and culture vessel shaking schedule). , Expansion culture can be automated.

As described above, according to such an embodiment, in the expansion culture of cells, continuous expansion culture from a small amount to a large amount can be realized with high culture efficiency in a short period of time in one container, and there is a risk of contamination. It is possible to enable automation to reduce the burden on the operator while reducing the problem.

That is, by using one culture vessel and changing the stirring mode based on the amount of the culture solution in the culture vessel, it is possible to perform expanded culture in which the risk of contamination is reduced. Moreover, such an expanded culture can be realized by one device (culture container swinging device).

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

For example, in the case of the above-described embodiment, the culture vessel has a basin shape as shown in FIG. However, the 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 for expanding and culturing cells in a culture solution, and is a culture container containing the culture solution and a culture solution in the culture solution. The culture vessel swinging portion that swings the culture vessel so that the culture medium is agitated, and the culture medium supply portion that adds the culture solution to the culture medium as the number of cells in the culture medium increases. The culture vessel swinging portion swings the culture vessel so that the smaller the amount of the culture solution in the culture vessel, the smaller the surface portion of the culture vessel that comes into contact with the culture solution that moves by stirring. It is a thing.

Further, the culture method according to the embodiment of the present invention is, in a broad sense, a method for culturing cells in which cells are expanded and cultured in a culture solution contained in a culture container, and the culture solution in the culture container is used. The culture vessel is shaken so as to be agitated, the culture solution is added to the culture solution as the number of cells in the culture solution increases, and the smaller the amount of the culture solution, the more contact with the culture solution that moves by stirring. This is a method of rocking the culture vessel so that the surface portion of the culture vessel becomes smaller.

The present invention is applicable to cell culture performed while stirring the culture solution.

12 Culture container CS culture solution

Claims

A culture device that expands and cultures cells in a culture medium.
A culture container that houses the culture solution and
A culture vessel swinging portion that swings the culture vessel so that the culture solution in the culture vessel is agitated,
It has a culture solution supply unit for adding the culture solution to the culture medium as the number of cells in the culture solution increases.
The culture vessel swinging portion swings the culture vessel so that the smaller the amount of the culture solution in the culture vessel, the smaller the surface portion of the culture vessel that comes into contact with the culture solution that moves by stirring. Incubator.
The culture vessel comprises a circular bottom surface and a cylindrical inner peripheral surface erected from the outer peripheral edge of the bottom surface.
The culture is tilted so that the culture solution is collected in a corner sandwiched between the bottom surface and the inner peripheral surface of the culture container swinging portion, and the culture solution is reciprocated along the corner. Change the tilt direction of the container,
The culture apparatus according to claim 1, wherein the smaller the amount of the culture solution, the smaller the reciprocating range of the culture solution.
The culture apparatus according to claim 2, wherein the culture container swinging portion tilts the culture container more as the amount of the culture solution is smaller.
It has a humidity sensor that measures the humidity inside the culture vessel.
The second or third claim, wherein when the humidity detected by the humidity sensor of the culture vessel swinging portion decreases, the inclination angle of the culture vessel is increased so that the area of the liquid surface of the culture solution becomes smaller. Incubator.
It has a dissolved oxygen sensor that measures the amount of oxygen dissolved in the culture solution in the culture vessel.
When the amount of dissolved oxygen detected by the dissolved oxygen sensor decreases, the culture vessel swinging portion swings the culture vessel so that at least one of the reciprocating cycle and the reciprocating range of the culture solution increases. The culture apparatus according to any one of claims 2 to 4.
When the amount of the culture solution in the culture container exceeds a predetermined threshold, the swinging portion of the culture container changes the tilt direction of the culture container so that the culture solution orbits along the corner. Item 2. The culture apparatus according to any one of Items 2 to 5.
A culture condition acquisition unit that acquires information on culture conditions including at least the initial number of cells, the target number of cells, the target cell density, and the doubling time.
A culture time calculation unit that calculates the culture time based on the culture condition information,
It has a supply schedule creation unit that creates a supply schedule of the culture solution based on the culture condition information and the culture time.
The culture apparatus according to any one of claims 1 to 6, wherein the culture solution supply unit changes the culture solution supply amount per unit time based on the supply schedule.
A cell density measuring unit that measures the cell density of the culture solution in the culture vessel,
It has a supply schedule correction unit that corrects the supply schedule so that the difference between the cell density measured by the cell density measurement unit and the target cell density is equal to or less than a predetermined value.
The culture apparatus according to claim 7, wherein the culture solution supply unit changes the culture solution supply amount per unit time based on the corrected supply schedule.
The culture apparatus according to claim 8, wherein the cell density measuring unit is configured to measure the cell density by inputting a measurement instruction of a user at an arbitrary timing.
The culture apparatus according to claim 8 or 9, wherein the supply schedule correction unit is configured to correct the supply schedule by inputting a correction instruction from a user.
The cell density measuring unit includes a tube, a roller pump provided in the tube, and a cell density sensor for measuring the cell density of the culture medium in the tube.
The tube is arranged so that one end thereof is located in the culture solution in the culture vessel and the other end is located above the liquid level of the culture solution.
Any of claims 8 to 10, wherein the roller pump rotates forward to fill the tube with the culture solution through one end, and reverses the rotation to return the culture solution in the tube to the culture vessel. The culture apparatus according to one item.
The culture apparatus according to claim 11, wherein the cell density sensor measures a culture solution in a portion of the tube arranged so as to have an inclination angle of 30 ° or less with respect to the vertical direction.
It has a swing schedule creation unit that creates a swing schedule that shows changes in the swing pattern of the culture container swing section with respect to the culture container based on the supply schedule.
The culture apparatus according to any one of claims 7 to 12, wherein the culture vessel swinging portion swings the culture vessel based on the shaking schedule.
A Gantt chart creation unit that creates a Gantt chart showing changes in the shaking pattern of the culture vessel, and
The culture apparatus according to claim 13, further comprising a display unit for displaying the Gantt chart.
The Gantt chart creation unit creates the Gantt chart so that at least one measured value of culture temperature, gas concentration, and gas flow rate is displayed on a time bar indicating the length of duration of the swing pattern. The culture apparatus according to claim 14.
It has a graph creating unit that creates a graph showing changes in at least one of the amount of culture medium and the cell density in the culture vessel.
The culture apparatus according to claim 14 or 15, wherein the display unit displays a graph created by the graph creation unit together with the Gantt chart.
It has a culture end condition acquisition unit for acquiring information on culture end conditions including at least one of the number of cells reached, the amount of reached culture solution, and the reached culture time.
Any one of claims 7 to 16, wherein after the culture termination condition is satisfied, the culture solution supply unit stops the supply of the culture solution, and the culture container swinging unit stops the shaking of the culture vessel. The incubator according to the section.
A notification unit that notifies the user when the culture end condition is satisfied, and
The user has a culture end input unit for terminating the culture.
17 of claim 17, after the user input to the culture end input unit is executed, the culture solution supply unit stops the supply of the culture solution, and the culture container swinging unit stops the shaking of the culture container. The incubator described.
A culture solution storage container for storing the culture solution, a tube having one end connected to the culture solution storage container and the other end connected to the culture solution container, and a roller provided in the tube. Including pump
The culture apparatus according to any one of claims 1 to 18, wherein a part of the tube is arranged on the outer surface of the culture container.
The culture apparatus according to claim 19, further comprising a refrigerator for refrigerating and accommodating the culture solution storage container and a heater for heating the culture container.
A method for culturing cells in which cells are expanded and cultured in a culture medium contained in a culture vessel.
The culture vessel is shaken so that the culture solution in the culture vessel is agitated.
As the number of cells in the culture solution increased, the culture solution was added to the culture vessel, and the culture solution was added.
A method for culturing cells, in which the culture vessel is shaken so that the smaller the amount of the culture solution, the smaller the surface portion of the culture vessel that comes into contact with the culture solution that moves by stirring.