Culture device

Abstract

When culturing cells using a culture medium in a culture vessel, the evaporation of the culture medium is suppressed. [Solution] The culture apparatus comprises a culture vessel containing a culture medium for culturing cells, a gas supply device for supplying gas to the culture vessel, and a humidifier for humidifying the gas flowing from the gas supply device to the culture vessel. The humidifier includes a hollow fiber membrane filter comprising a hollow fiber membrane through which gas from the gas supply device passes and a casing housing the hollow fiber membrane, a water supply device for filling the casing of the hollow fiber membrane filter with water, and a first heater for heating the hollow fiber membrane filter.

Description

Technical Field

[0001] The present invention relates to a culture apparatus for culturing cells using a culture solution in a culture vessel.

Background Art

[0002] For example, Patent Document 1 discloses a shaking type culture apparatus for culturing cells using a culture solution in a culture bag (culture vessel). This culture apparatus performs cell culture in the culture vessel by heating the culture vessel with a rubber heater or the like and shaking the culture vessel. Further, this culture apparatus performs culture while supplying a mixed gas into the culture vessel.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] When culturing cells using a culture solution in a culture vessel as in the culture apparatus described in Patent Document 1, evaporation of the culture solution may be a problem. When the culture solution evaporates, the osmotic pressure of the culture solution increases, and water may move out of the cells, causing damage to the cells. In particular, when culturing cells using a small amount of culture solution, the evaporation rate of the culture solution becomes high, and the osmotic pressure also becomes high, which can cause significant damage to the cells.

[0005] Therefore, an object of the present invention is to suppress evaporation of the culture solution when culturing cells using the culture solution in a culture vessel.

Means for Solving the Problems

[0006] To solve the above technical problems, according to one aspect of the present invention, a culture vessel for containing a culture solution for culturing cells, A gas supply device that supplies gas to the culture vessel, The system includes a humidifier that humidifies the gas flowing from the gas supply device to the culture vessel, The humidifier, A hollow fiber membrane filter comprising a hollow fiber membrane through which gas from the gas supply device passes, and a casing that houses the hollow fiber membrane, A water supply device for filling the casing of the hollow fiber membrane filter with water, A culture apparatus is provided, which includes a first heater for heating the hollow fiber membrane filter. [Effects of the Invention]

[0007] According to the present invention, when culturing cells using a culture medium in a culture vessel, the evaporation of the culture medium can be suppressed. [Brief explanation of the drawing]

[0008] [Figure 1] A schematic diagram showing the configuration of a culture apparatus according to one embodiment of the present invention. [Figure 2] A schematic perspective view of an example of a culture vessel. [Figure 3] Schematic partial cross-sectional view of the culture vessel rocking mechanism in a culture apparatus. [Figure 4] Schematic partial cross-sectional view of a portion of the culture vessel's rocking mechanism shown in Figure 3, viewed from a different direction. [Figure 5] Schematic partial cross-sectional view of the culture vessel rocking mechanism shown in Figure 3, where the culture vessel is tilted. [Figure 6A] Cross-sectional view showing the tilt of the culture vessel when the amount of culture medium is relatively small. [Figure 6B] Top view showing the tilt of the culture vessel when the amount of culture medium is relatively small. [Figure 7] A diagram showing the stirring of the culture medium when the amount of culture medium is relatively small. [Figure 8A] Cross-sectional view showing the tilt of the culture vessel when the amount of culture medium is relatively large. [Figure 8B] Top view showing the tilt of the culture vessel when the culture medium is in a relatively large volume. [Figure 9] Figure showing agitation of the culture solution when the amount of the culture solution is relatively large [Figure 10] Schematic configuration diagram of the gas supply unit [Figure 11] Schematic internal structure diagram of the hollow fiber membrane filter [Figure 12] Figure showing the dilution ratio and osmotic pressure of the culture solution [Figure 13] Figure showing the relationship between the supply gas flow rate and the evaporation rate of the culture solution [Figure 14] Figure showing the amount of the culture solution and the evaporation rate of the culture solution with respect to the culture elapsed time [Figure 15] Schematic configuration diagram of the gas supply unit in the culture apparatus according to another embodiment

Mode for Carrying Out the Invention

[0009] The culture apparatus according to one aspect of the present invention includes a culture container that stores a culture solution for culturing cells, a gas supply device that supplies gas to the culture container, and a humidifying device that humidifies the gas flowing from the gas supply device toward the culture container. The humidifying device includes a hollow fiber membrane filter including a hollow fiber membrane through which the gas from the gas supply device passes and a casing that houses the hollow fiber membrane, a water supply device that fills water into the casing of the hollow fiber membrane filter, and a first heater that heats the hollow fiber membrane filter.

[0010] According to this aspect, when culturing cells using a culture solution in a culture container, evaporation of the culture solution can be suppressed.

[0011] For example, the culture apparatus is disposed below the culture container and has a second heater that heats the culture solution in the culture solution. In this case, the heating temperature of the first heater is higher than the heating temperature of the second heater. Thereby, it is possible to suppress condensation and reduction of water vapor in the gas flowing out from the humidifying device before reaching the culture container.

[0012] For example, the culture apparatus has a first membrane filter provided in the gas supply path between the humidifier and the culture vessel, and positioned such that the normal of the filter surface is inclined with respect to the vertical. This first membrane filter suppresses contamination of the culture medium in the culture vessel. Furthermore, the inclination prevents condensation from spreading uniformly across the entire filter surface of the first membrane filter, which would increase flow resistance.

[0013] For example, the culture apparatus has a third heater that heats the first membrane filter. In this case, the heating temperature of the third heater is higher than that of the first heater. As a result, water vapor in the gas can pass through the first membrane filter without condensation forming on the first membrane filter.

[0014] For example, the portion of the gas supply channel between the first membrane filter and the culture vessel extends horizontally. This prevents condensation water generated in the gas supply channel from dripping into the culture vessel.

[0015] For example, the first membrane filter is positioned lower than the connection point of the culture vessel. This prevents condensation water generated in the gas supply path from dripping into the culture vessel.

[0016] For example, the culture apparatus has a second membrane filter installed in a gas discharge channel connecting the inside of the culture vessel to the outside air, and the filter surface normal is positioned at an angle to the vertical. This suppresses contamination of the culture medium inside the culture vessel by the second membrane filter. Furthermore, the angle prevents condensation from spreading uniformly across the entire filter surface of the second membrane filter, which would increase flow resistance.

[0017] For example, the culture apparatus has a fourth heater that heats the second membrane filter. In this case, the heating temperature of the fourth heater is higher than that of the first heater. This allows water vapor in the gas to pass through the second membrane filter without condensation forming on the second membrane filter.

[0018] For example, the portion of the gas discharge channel between the second membrane filter and the culture vessel extends horizontally. This prevents condensation water generated in the gas discharge channel from dripping into the culture vessel.

[0019] For example, the second membrane filter is positioned lower than the connection point of the culture vessel. This prevents condensation water generated in the gas discharge channel from dripping into the culture vessel.

[0020] For example, the culture vessel is cylindrical in shape, comprising a bottom plate, a top plate, and side walls. In this case, the culture apparatus has a fifth heater for heating the top plate and a sixth heater for heating the side walls, and the heating temperatures of the fifth and sixth heaters are higher than those of the second heater. This suppresses the occurrence of condensation on the top plate and side walls.

[0021] For example, the culture apparatus has a culture medium supply unit that supplies culture medium to the culture vessel. In this case, as the amount of culture medium in the culture vessel supplied by the culture medium supply unit increases, the amount of gas supplied per unit time by the gas supply unit is changed. When there is a large amount of culture medium in the culture vessel and evaporation of the culture medium does not significantly affect the cells, the supply of an excess amount of gas to the culture vessel is suppressed.

[0022] For example, the amount of gas supplied by the gas supply device is changed so that the evaporation rate of the culture medium in the culture vessel and the osmotic pressure of the culture medium, which is determined from the evaporation rate, become predetermined values. This makes it possible to suppress cell deformation due to the osmotic pressure of the culture medium.

[0023] For example, the predetermined value of the osmotic pressure of the culture medium may be in the range of 260 to 315 mOsm / kg.

[0024] For example, the heating temperature of the first heater is changed as the amount of culture medium supplied to the culture vessel by the culture medium supply unit increases. This prevents an excess amount of water vapor from being supplied to the culture vessel when there is a large amount of culture medium in the culture vessel and evaporation of the culture medium does not significantly affect the cells.

[0025] For example, before the culture medium supply unit supplies the culture medium to the culture vessel, the gas supplied from the gas supply device and humidified by the humidifier is supplied to the culture vessel. This suppresses the evaporation of the small amount of culture medium immediately after it is supplied to the culture vessel.

[0026] Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[0027] Figure 1 is a schematic diagram showing the configuration of a culture apparatus according to one embodiment of the present invention.

[0028] As shown in Figure 1, the culture apparatus 10 includes a culture vessel 12 that contains a culture medium CS containing cells, a culture vessel shaking unit 14 that shakes the culture vessel 12 to agitate the culture medium CS inside the culture vessel 12, and a culture medium supply unit 16 that supplies the culture medium CS to the culture vessel 12.

[0029] In this embodiment, the culture apparatus 10 includes a humidity sensor 18 for measuring the humidity inside the culture vessel 12, a dissolved oxygen sensor 20 for measuring the amount of oxygen dissolved in the culture medium CS inside the culture vessel 12, and a cell density measuring unit 22 for measuring the cell density in the culture medium CS inside the culture vessel 12.

[0030] Furthermore, the culture apparatus 10 has a gas supply unit 24 that supplies a mixed gas of humidified oxygen, carbon dioxide, and nitrogen to the culture container 12.

[0031] Furthermore, the culture apparatus 10 includes a control unit 26 that controls the culture vessel rocking unit 14, the culture medium 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.

[0032] The culture vessel 12 is a container for holding the culture medium CS, and cell culture using the culture medium CS is performed inside it. In this culture vessel 12, as the number of cells increases, cell culture using the culture medium CS is performed, i.e., expansion culture, by gradually adding the culture medium CS starting from a small amount (less than 1 liter, for example 50 milliliters). Therefore, the culture vessel 12 has a capacity that can hold and stir the maximum amount of culture medium used for culture (for example 50 liters).

[0033] Figure 2 is a perspective view showing an example of the shape of a culture vessel 12. Note that the XYZ Cartesian coordinate system is shown in the drawing, but this is for the purpose of facilitating the understanding of the embodiments of the invention and does not limit the invention. Also, the X and Y axes are horizontal, and the Z axis is vertical.

[0034] As shown in Figure 2, in this embodiment, the culture vessel 12 comprises a disc-shaped bottom plate portion 12a, a cylindrical side wall portion 12b that rises from the outer edge of the bottom plate portion 12a, and a top plate portion 12c supported by the side wall portion 12b. In other words, the culture vessel 12 is a so-called cylindrical shape. The height of the side wall portion 12b is smaller than the radius of the bottom plate portion 12a. The top plate portion 12c is detachable and functions as a lid.

[0035] Figure 3 is a schematic partial cross-sectional view of the culture vessel rocking section 14 in the culture apparatus 10. Figure 4 is a schematic partial cross-sectional view of a part of the culture vessel rocking section 14 shown in Figure 3, viewed from a different direction.

[0036] As shown in Figures 3 and 4, the culture vessel rocking section 14 in the culture apparatus 10 comprises a stage 30 that holds the culture vessel 12 and a rotary actuator 34 that has a rotary table 32 that rotates around a rotational center axis C0 extending in the vertical direction (Z-axis direction).

[0037] The stage 30 and the rotary actuator 34 are driven and connected via the oscillating head 36 and the tilting mechanism 38.

[0038] The rocking head 36 supports the stage 30 and is mounted on the culture vessel rocking section 14 so as to be able to rock about a rocking axis C1 extending horizontally (X-axis direction) and a rocking axis C2 extending horizontally (Y-axis direction) and perpendicular to the rocking axis C1. The rocking head 36 also has a connecting shaft 40 at its lower part for drive connection to the rotary actuator 34 via a tilting mechanism 38. When the stage 30 is in a horizontal position, the connecting shaft 40 of the rocking head 36 extends vertically (Z-axis direction).

[0039] The tilting mechanism 38 is a link mechanism for tilting the stage 30 via the rocking head 36, that is, for tilting the culture vessel 12 on the stage 30 with respect to the horizontal direction. To this end, the tilting mechanism 38 includes a base portion 42, a rocking head connecting portion 44 connected to the rocking head 36, and a link arm 46 connecting the base portion 42 and the rocking head connecting portion 44.

[0040] 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 together with the rotary table 32 around the rotational axis C0.

[0041] The pivoting head connecting portion 44 of the tilting mechanism 38 is slidably attached to the connecting shaft 40 of the pivoting head 36, for example, via a bearing.

[0042] The link arm 46 of the tilting mechanism 38 is configured to connect the base portion 42 and the oscillating head connecting portion 44. Specifically, the link arm 46 has one end that is rotatably fixed to the oscillating head connecting portion 44 and the other end that is rotatably fixed to the base portion 42. The pivot axis C3 of one end of the link arm 46 and the rotation axis C4 of the other end each extend horizontally and are parallel to each other.

[0043] The rotary actuator 34, to which the base portion 42 of the tilting mechanism 38 is attached, is raised and lowered vertically (in the Z-axis direction) by the ball screw mechanism 48.

[0044] The ball screw mechanism 48 includes a screw shaft 50 extending vertically (in the Z-axis direction), a nut 52 that engages with the screw shaft 50, and a motor (not shown) that rotates the screw shaft 50. The nut 52 is attached to a lifting bracket 54. A rotary actuator 34 is attached to the lifting bracket 54.

[0045] 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 Figure 5, when the rotary actuator 34 is raised by the ball screw mechanism 48, the stage 30 tilts via the tilting mechanism 38. Specifically, the base portion 42 of the tilting mechanism 38 attached to the rotary actuator 34 rises, causing the link arm 46 to push the oscillating head coupling portion 44. As a result, the oscillating head 36, together with the oscillating head coupling portion 44, rotates about at least one of the oscillating axes C1 and C2 (oscillating axis C2 in Figure 13). As a result, the stage 30 tilts, and the culture vessel 12 on the stage 30 also tilts.

[0046] As shown in Figure 5, when the rotary actuator 34 is driven and the rotary table 32 rotates with the stage 30 tilted, the tilting mechanism 38 rotates around the rotational axis C0, thereby changing the tilt direction of the stage 30. As a result, the culture medium CS in the culture vessel 12 is agitated, and the cells in the culture medium CS are cultured.

[0047] In this culture vessel rocking unit 14, even if the rotary actuator 34 rotates the tilting mechanism 38 by, for example, one full rotation, the stage 30 itself does not rotate; instead, only the tilting direction of the stage 30 rotates by one full rotation. In other words, the lowest part of the culture vessel 12 on the stage 30 is sequentially changed to a different part.

[0048] Returning to Figure 1, in this embodiment, the culture medium supply unit 16 that supplies the culture medium CS to the culture vessel 12 is controlled by the control unit 26. The supply of the culture medium CS to the culture vessel 12 will be described later.

[0049] In addition to the culture medium CS, a mixed gas is supplied to the culture vessel 12 by the gas supply unit 24. The gas supply to the culture vessel 12 will be described later.

[0050] The humidity sensor 18 is mounted inside the culture vessel 12, specifically on the inner surface 12d, for example, so as not to be submerged in the culture medium CS, and measures the humidity inside the culture vessel 12. The humidity sensor 18 also outputs a signal corresponding to the measured humidity to the control unit 26.

[0051] The dissolved oxygen sensor 20 measures the amount of oxygen dissolved in the culture medium CS in 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 comprises a chip coated with a fluorescent substance and placed on the bottom surface 12e of the culture vessel 12, a light source that irradiates the chip with ultraviolet light or the like from outside the culture vessel 12, and a light-receiving element that receives the fluorescence emitted from the chip.

[0052] When a fluorescent substance absorbs light energy, such as ultraviolet light, from a light source, it transitions from its ground state to an excited state. The excited molecules of the fluorescent substance usually return to the ground state while emitting fluorescence. However, if oxygen molecules are present around the excited molecules at this time, the excitation energy is absorbed by the oxygen molecules, and the fluorescence emission intensity decreases, a phenomenon known as oxygen quenching. By utilizing this oxygen quenching, that is, by utilizing the fact that the fluorescence emission intensity is inversely proportional to the oxygen molecule concentration, fluorescent dissolved oxygen sensors measure the amount of dissolved oxygen in the culture medium in a culture vessel.

[0053] Furthermore, the dissolved oxygen sensor 20 outputs a signal corresponding to the measured amount of dissolved oxygen to the control unit 26.

[0054] The cell density measurement unit 22 measures the cell density of the culture medium CS in the culture vessel 12. The measured cell density is output to the control unit 26. The cell density during culture is monitored by the periodic measurements of this cell density measurement unit 22.

[0055] The control unit 26 consists of, for example, a control board equipped with a memory device and a CPU. By operating according to a program stored in the memory device, the CPU performs operations related to cell culture, which will be described later.

[0056] First, the control unit 26 controls the culture medium supply unit 16.

[0057] Controlled by the control unit 26, the culture medium supply unit 16 supplies additional culture medium CS to the culture vessel 12 as the number of cells in the culture medium CS increases. For example, in a single culture vessel 12, culture medium CS is supplied to the culture vessel 12 in stages, starting from less than 1 liter (e.g., 200 milliliters) of culture medium CS until it reaches 50 liters.

[0058] Furthermore, the control unit 26 controls the culture vessel oscillating unit 14 (its rotary actuator 34 and ball screw mechanism 48) based on the amount of culture medium CS in the culture vessel 12.

[0059] Controlled by the control unit 26, the culture vessel oscillating unit 14 oscillates the culture vessel 12 in such a way that evaporation of the culture medium CS within the culture vessel 12 is suppressed while the culture medium CS is agitated. Specifically, the culture vessel oscillating unit 14 oscillates the culture vessel 12 in such a way that the smaller the amount of culture medium CS in the culture vessel 12, the smaller the surface area of ​​the culture vessel 12 that comes into contact with the agitated culture medium. The oscillating of the culture vessel 12, i.e., the agitation of the culture medium CS, will now be explained.

[0060] Figure 6A is a cross-sectional view showing the tilt of the culture vessel 12 when the amount of culture medium is relatively small. Figure 6B is a top view showing the tilt of the culture vessel 12 when the amount of culture medium is relatively small.

[0061] As shown in Figures 6A and 6B, the culture medium is stirred while the culture vessel 12 is tilted. The tilt angle θ of the culture vessel 12 (angle relative to the culture vessel 12 in a horizontal position) is increased as the amount of culture medium CS in the culture vessel 12 decreases.

[0062] Thus, the smaller the amount of culture medium CS, the smaller the surface area of ​​the culture medium CS by tilting the culture vessel 12 more. By reducing the surface area of ​​the culture medium CS, evaporation of the culture medium CS from the surface LS can be suppressed.

[0063] Here, we will explain "evaporation of the culture medium." When the culture medium CS evaporates, the cell density within the culture medium CS increases. When the amount of culture medium CS is large (e.g., 1 liter or more), the increase in cell density due to evaporation is relatively small, and the impact on cells due to this increase in density is small. On the other hand, when the amount of culture medium CS is small (e.g., less than 1 liter), the increase in cell density due to evaporation is relatively large, and the impact on cells due to this increase in density is large. The smaller the amount of culture medium CS, the greater the impact on cells due to its evaporation, and in some cases, some cells may die or be damaged.

[0064] Therefore, the smaller the amount of culture medium CS, the greater the tilt of the culture vessel 12 (by increasing the tilt angle θ), the less the effect of evaporation of the culture medium CS on the cells.

[0065] Furthermore, if the amount of culture medium CS in the culture vessel 12 is such that the effect on the cells due to the evaporation of the culture medium CS is sufficiently small, the tilt angle θ of the culture vessel 12 may remain constant.

[0066] As the culture vessel 12 tilts, the culture medium CS accumulates in the corner 12f between the circular bottom surface 12e of the culture vessel 12 and the cylindrical inner surface 12d that rises from the outer edge of the bottom surface 12e, as shown in Figure 6B. In this state, the direction of tilt of the culture vessel 12 is changed.

[0067] Figure 7 shows the stirring of the culture medium when the amount of culture medium is relatively small. Figure 7 shows the culture vessel 12 being stirred, viewed from above (view along the Z axis).

[0068] As shown in Figure 7, a relatively small amount of culture medium CS (e.g., less than 1 liter) is reciprocated along the corner 12f sandwiched between the bottom surface 12e and the inner surface 12d of the culture vessel 12. For example, the rotary actuator 34 repeatedly rotates the tilting mechanism 38 forward and backward within a 90-degree angular range, causing the tilt direction of the culture vessel 12 to change within a 90-degree angular range. As a result, the culture medium CS is reciprocated within a 90-degree angular range. Consequently, the culture medium CS is agitated. Note that, as shown in Figure 7, if the Y-axis positive direction is set to 0 degrees with respect to the Z-axis, for example, the culture medium CS reciprocates between the position of -45 degrees (315 degrees) and the position of +45 degrees, centered on the 0-degree position.

[0069] The smaller the amount of culture medium CS, the smaller the reciprocating range (angle range) of the culture medium CS. This is to suppress the evaporation of the culture medium CS.

[0070] Specifically, when the culture medium CS moves across the surface of the culture vessel 12 due to agitation, a small amount of culture medium CS remains on the surface after the majority (clumpy) of culture medium CS has passed. For example, as shown in Figure 7, after the majority (clumpy) of culture medium CS has moved to the 45-degree position, a small amount of culture medium CS remains at the 0-degree position. This remaining small amount of culture medium CS is prone to evaporation. Therefore, before this small amount of culture medium CS evaporates, the clumpy culture medium CS returns and absorbs it. Also, the smaller the amount of culture medium CS, the greater the impact on cells due to evaporation, so the range of travel of the culture medium CS is reduced. This makes it possible to suppress the evaporation of culture medium CS when the amount of culture medium CS is relatively small.

[0071] As the number of cells increases, culture medium CS is added to the culture vessel 12, increasing the amount of culture medium CS in the culture vessel 12. This increase expands the range over which the culture medium CS can be stirred. This is because, while the increased amount of culture medium CS reduces the impact on cells due to evaporation, it necessitates greater agitation of the culture medium CS.

[0072] When the amount of culture medium CS is relatively small (e.g., less than 1 liter), as described above, the culture medium CS is moved back and forth within the culture vessel 12 to suppress evaporation. In contrast, when the amount of culture medium CS is relatively large (e.g., 1 liter or more) as the number of cells increases, the culture medium CS is circulated within the culture vessel 12.

[0073] Figure 8A is a cross-sectional view showing the tilt of the culture vessel 12 when the culture medium is relatively large. Figure 8B is a top view showing the tilt of the culture vessel 12 when the culture medium is relatively large.

[0074] As shown in Figures 8A and 8B, and also referring to Figures 6A and 6B, when the amount of culture medium CS is relatively large, the tilt angle θ of the culture vessel 12 is smaller than when the amount of culture medium CS is relatively small. This is because it reduces the depth of the culture medium CS, allowing gases such as oxygen to circulate throughout the entire culture medium CS.

[0075] The greater the depth of the culture medium (CS), the less effectively gases such as oxygen, absorbed through the surface of the culture medium by agitation, can spread throughout the entire medium. Specifically, gases have difficulty reaching the deeper parts of the culture medium. As a result, the amount of dissolved oxygen in the deeper parts of the culture medium may become insufficient, potentially damaging the cells.

[0076] As the culture vessel 12 tilts, the culture medium CS accumulates in the corner 12f between the bottom surface 12e and the inner surface 12d of the culture vessel 12, as shown in Figure 8B. In this state, the direction of tilt of the culture vessel 12 is changed.

[0077] Figure 9 shows the stirring of the culture medium CS when the amount of culture medium CS is relatively large. Figure 9 shows the culture vessel 12 being stirred, viewed from above (view along the Z axis).

[0078] A relatively large amount (e.g., more than 1 liter) of culture medium CS is circulated along the corner 12f sandwiched between the bottom surface 12e and the inner surface 12d of the culture vessel 12. For example, as the rotary actuator 34 continuously rotates the tilting mechanism 38 in one direction, the tilt direction of the culture vessel 12 continues to rotate in one direction. As a result, the culture medium CS is circulated. Consequently, the culture medium CS is agitated.

[0079] As described above, the control unit 26 changes the stirring mode (oscillation pattern) based on the amount of culture medium CS in the culture vessel 12. For example, if the amount of culture medium CS in the culture vessel 12 is small compared to a predetermined threshold amount (e.g., 1 liter), the culture medium CS is stirred by reciprocating it, as shown in Figure 7. Furthermore, the smaller the amount of culture medium CS, the smaller the reciprocating range of the culture medium CS is made. On the other hand, if the amount of culture medium CS in the culture vessel 12 exceeds a predetermined threshold amount, the culture medium CS is stirred by circulating it, as shown in Figure 9. The amount of culture medium CS in the culture vessel 12 may be calculated, for example, from the weight of the culture medium CS in the culture vessel 12 measured by a weight sensor (not shown).

[0080] In addition, in this embodiment, the control unit 26 is configured to control the culture vessel oscillating unit 14 based on the measurement results of the humidity sensor 18 and the dissolved oxygen sensor 20 while the culture medium CS is being stirred.

[0081] Specifically, when the humidity in the culture medium CS detected by the humidity sensor 18 decreases, for example, when the humidity decreases below a predetermined lower limit of the appropriate range, the culture vessel rocking unit 14 controlled by the control unit 26 increases the tilt angle of the culture vessel 12 (i.e., the stage 30) so that the area of ​​the liquid surface LS of the culture medium CS decreases.

[0082] When the humidity inside the culture vessel 12 decreases, the culture medium CS is more likely to evaporate from its liquid surface LS. Therefore, evaporation can be suppressed by reducing the surface area of ​​the culture medium CS.

[0083] Furthermore, if the amount of dissolved oxygen detected by the dissolved oxygen sensor decreases, for example, if it falls below the lower limit of a predetermined appropriate range, the culture vessel rocking unit 14 controlled by the control unit 26 rocks the culture vessel 12 so that at least one of the period and range of the reciprocating motion of the culture medium CS increases. The dissolved oxygen sensor 20 (its tip) is positioned on the culture vessel 12 so that it can contact the culture medium CS and detect the amount of dissolved oxygen, regardless of the amount of culture medium CS in the culture vessel 12. In this embodiment, the dissolved oxygen sensor 20 is provided on the outer peripheral edge of the bottom surface 12e of the culture vessel 12. Also, when the dissolved oxygen sensor 20 measures the amount of dissolved oxygen, the culture vessel 12 is rocked by the culture vessel rocking unit 14 so that the culture medium CS comes into contact with the dissolved oxygen sensor 20. In this case, in order to bring the culture medium CS into contact with the dissolved oxygen sensor 20 and accurately detect the amount of dissolved oxygen, the oscillation speed or oscillation pattern of the culture vessel 12 may be temporarily changed, or the oscillation of the culture vessel 12 may be temporarily stopped.

[0084] When the amount of dissolved oxygen in the culture medium CS within the culture vessel 12 decreases, the cells in the culture medium CS are damaged. Therefore, by increasing at least one of the reciprocating period and reciprocating range of the culture medium CS, the culture medium CS is agitated more, thereby allowing more oxygen to be taken into the culture medium CS. As a result, cell damage can be suppressed.

[0085] Furthermore, if the culture medium CS in the culture vessel 12 is relatively large and being circulated, increasing the circulation speed will further agitate the culture medium CS, thereby allowing more oxygen to be incorporated into the culture medium CS.

[0086] Furthermore, in this embodiment, the control unit 26 controls the amount of gas supplied to the culture vessel 12 by the gas supply unit 24 based on the amount of culture medium in the culture vessel 12. The configuration of the gas supply unit 24 will be explained first.

[0087] Figure 10 is a schematic diagram of the gas supply unit.

[0088] As shown in Figure 10, the gas supply unit 24 includes a gas supply device 60 that supplies a mixed gas of oxygen, carbon dioxide, and nitrogen to the culture vessel 12, and a humidifier 62 that humidifies the gas flowing from the gas supply device 60 to the culture vessel 12.

[0089] The gas supply device 60 is configured to supply a mixed gas contained in a gas tank (not shown) at a predetermined timing and amount based on instructions (control signals) from the control unit 26. For example, the gas supply device 60 is a flow control valve located between the gas tank and the humidifier 62.

[0090] The humidifier 62 is positioned between the gas supply device 60 and the culture vessel 12. The humidifier 62 also includes a hollow fiber membrane filter 64 through which the mixed gas Gd from the gas supply device 60 passes, a plurality of water supply devices 66 that fill the hollow fiber membrane filter 64 with water, and a first heater 68 that heats the hollow fiber membrane filter 64. In this embodiment, the water supply devices 66 are two water supply containers 66.

[0091] Figure 11 is a schematic diagram of the internal structure of a hollow fiber membrane filter.

[0092] The hollow fiber membrane filter 64 comprises a plurality of hollow fiber membranes 70 through which the mixed gas Gd from the gas supply device 60 passes, and a casing 72 that houses the plurality of hollow fiber membranes 70. The casing 72 is provided with ports 72a that connect to two water supply containers 66. Water W from the water supply containers 66 fills the casing 72 via the ports 72a. The filling of the casing 72 with water W begins when one of the water supply containers 66 is filled with water W and the other is empty. When water from the water supply container 66 that is filled with water W is supplied to the casing 72, air inside the casing 72 moves into the empty water supply container 66. Finally, when the water levels in the two water supply containers 66 reach the same level, the casing 72 is filled with water.

[0093] In this embodiment, two water supply containers 66 are provided, but if multiple water supply containers 66 are to be provided, there may be three or more. Furthermore, the water supply containers 66 are located above the hollow fiber membrane filter 64, and water is stored in all of the water supply containers 66 so that the water level in each container is at the same level.

[0094] Furthermore, the water supply container 66 may be configured to have a variable internal volume. That is, the water supply container 66 may be formed in a bellows shape, for example, by being composed of a piston such as a syringe, or at least a part of it may be made of an elastic material.

[0095] In contrast, if the water supply container 66 has a fixed internal volume and is sealed, there is a possibility that sufficient water cannot be supplied into the casing 72. Furthermore, there is a possibility that it will not be able to absorb the volume changes caused by vaporized gas from the supply water due to temperature changes, etc., and by the permeable gas that passes through the hollow fiber membrane.

[0096] In addition to the configuration in which the internal volume of the water supply container 66 is variable, a detection unit (not shown) may be provided to detect the amount of movement of the piston representing the change in internal volume, the amount of deformation of the bellows, or the amount of deformation of the elastic part of the elastic body. This makes it possible to suppress damage to the water supply container 66, leakage, etc., that may occur due to the volume change of the water supply container 66 caused by gas. Furthermore, it is also possible to detect the amount of water supplied from the water supply container 66 to the casing 72 via these detection units. Based on the detection result of the amount of water supplied, the timing of replenishing the water supply can be notified to the operator, thereby preventing a decrease in humidification function due to insufficient water supply.

[0097] When the mixed gas Gd passes through the hollow fiber membrane 70 with the casing 72 filled with water, the mixed gas Gd passing through the hollow fiber membrane 70 is humidified by the water vapor that permeates through the hollow fiber membrane 70. As a result, the humidified mixed gas Gw flows out from the casing 72. The pressure around the hollow fiber membrane 70 (water pressure) is set to be higher than the pressure inside the hollow fiber membrane 70 (mixed gas pressure) in order to allow water vapor to move into the hollow fiber membrane 70.

[0098] The amount of water vapor per unit volume contained in the humidified mixed gas Gw is determined by the heating temperature T1 [°C] of the first heater 68 that heats the hollow fiber membrane filter 64. The higher the heating temperature T1, the greater the amount of water vapor contained in the humidified mixed gas Gw. The first heater 68 is, for example, a heater that is waterproofed by coating the heating element with silicone rubber (hereinafter referred to as the "silicone rubber heater").

[0099] In the case of the culture apparatus 10 of this embodiment, a second heater 74 is provided below the culture vessel 12 to heat the culture medium CS inside in order to maintain the culture medium CS in the culture vessel 12 at a predetermined temperature (for example, about 37°C). The second heater 74 is, for example, a silicone rubber heater provided on the stage 30 of the culture vessel rocking section 14. The heating temperature T1 of the first heater 68 that heats the hollow fiber membrane filter 64 is set higher than the heating temperature T2 of the second heater 74. This takes into account that the water vapor in the mixed gas Gw that flows out from the humidifier 62 condenses and decreases before reaching the culture vessel 12. In other words, the heating temperature T1 of the first heater 68 is set higher than the heating temperature T2 of the second heater 74 in order to contain a larger amount of water vapor in the mixed gas Gd inside the hollow fiber membrane filter 64 than the amount of water vapor required for the culture vessel 12. For example, heating temperature T1 is set to a temperature 10 to 15°C higher than heating temperature T2.

[0100] The humidified mixed gas Gw that flows out from the humidifier 62 is supplied to the culture vessel 12 via the gas supply path Pin. The gas supply path Pin consists of a first filter unit 76, an L-shaped tube joint 78 provided on the top plate portion 12c of the culture vessel 12, a flexible insulated tube 80 connecting the humidifier 62 and the first filter unit 76, and a flexible insulated tube 82 connecting the first filter unit 76 and the L-shaped tube joint 78.

[0101] The first filter unit 76 is a unit for suppressing contamination of the culture medium CS in the culture vessel 12, and includes a first membrane filter 86 through which a humidified mixed gas Gw passes within its housing 84. In this embodiment, the first membrane filter 86 is positioned such that the normal of the filter surface 86a through which the mixed gas Gw passes is inclined with respect to the vertical direction (Z-axis direction) (in this embodiment, inclined at 90 degrees with respect to the vertical direction). The reason for positioning the first membrane filter 86 in this orientation will now be explained.

[0102] Condensation water generated in the gas supply path Pin from the humidifier 62 to the first membrane filter 86 is captured by the first membrane filter 86. However, if the normal to the filter surface 86a extends vertically, as in this embodiment, the captured condensation water spreads uniformly across the entire filter surface 86a of the first membrane filter 86. As a result, the flow resistance of the first membrane filter 86 increases, and the required amount of mixed gas or water vapor does not reach the culture vessel 12.

[0103] To address this, the first membrane filter 86 is positioned such that the normal to its filter surface 86a is inclined with respect to the vertical direction (Z-axis direction). This causes the captured condensation water to move to the lower part of the first membrane filter 86. As a result, the entire filter surface 86a is prevented from being covered with condensation water.

[0104] In the culture apparatus 10 of this embodiment, a third heater 88 is provided to heat the first filter unit 76, i.e., the first membrane filter 86. The third heater 88 is, for example, a silicone rubber heater. This third heater 88 heats the first membrane filter 86 at a heating temperature T3 that is higher than the heating temperature T1 of the first heater 68 that heats the hollow fiber membrane filter 64. For example, the heating temperature T3 is set 3 to 5°C higher than the heating temperature T1. As a result, water vapor in the mixed gas Gw can pass through the first membrane filter 86 without condensation forming on the first membrane filter 86.

[0105] In this embodiment, the portion of the gas supply path Pin between the first membrane filter 86 and the culture vessel 12, i.e., the insulating tube 80, extends horizontally (in the X-axis direction). This suppresses condensation of water vapor in the mixed gas Gw that has passed through the first membrane filter 86, and prevents the condensed water from dripping into the culture vessel 12 (compared to the case where this portion extends vertically (in the Z-axis direction)). As a result, a localized and rapid decrease in the concentration of the culture medium CS due to the dripping of condensed water is suppressed. Such a decrease in the concentration of the culture medium CS can change the osmotic pressure of the cells, potentially damaging them. To further suppress the dripping of condensed water, if possible, the first membrane filter 86 may be positioned lower than the connection between the gas supply path Pin and the culture vessel 12, i.e., the connection between the insulating tube 80 and the L-shaped tube joint 78.

[0106] As shown in Figure 10, a second filter unit 90 is also provided on the gas discharge channel Pout that connects the inside of the culture vessel 12 to the outside air. The gas discharge channel Pout consists of the second filter unit 90, an L-shaped tube joint 92 provided on the top plate portion 12c of the culture vessel 12, and a flexible heat-insulating tube 94 that connects the second filter unit 90 and the L-shaped tube joint 92.

[0107] The second filter unit 90 is a unit for suppressing contamination of the culture medium CS in the culture vessel 12, and includes a second membrane filter 98 through which exhaust gas Ge passes within its housing 96. In this embodiment, the second membrane filter 98 is positioned such that the normal to the filter surface 98a through which the exhaust gas Ge passes is inclined with respect to the vertical direction (Z-axis direction) (in this embodiment, inclined at 90 degrees with respect to the vertical direction). This suppresses the increase in flow resistance caused by condensation covering the entire filter surface 98a, similar to the first membrane filter 86. As a result, an excessive increase in pressure inside the culture vessel 12 is suppressed.

[0108] In the culture apparatus 10 of this embodiment, a fourth heater 100 is provided to heat the second filter unit 90, i.e., the second membrane filter 98, similar to the first filter unit 76 (first membrane filter 86). The fourth heater 100 is a silicone rubber heater, similar to the third heater 88. This fourth heater 100 heats the second membrane filter 98 at a heating temperature T4 that is higher than the heating temperature T1 of the first heater 68 that heats the hollow fiber membrane filter 64, for example, at the same heating temperature T3 as the third heater 88. As a result, water vapor in the exhaust gas Ge can pass through the second membrane filter 98 without condensation forming on the second membrane filter 98.

[0109] In this embodiment, the portion of the gas discharge channel Pout between the second membrane filter 98 and the culture container 12, i.e., the insulating tube 94, extends horizontally (in the X-axis direction). This suppresses condensation of water vapor in the exhaust gas Ge before it passes through the second membrane filter 98, and prevents the condensed water from dripping into the culture container 12 (compared to the case where this portion extends vertically (in the Z-axis direction)). As a result, a localized and rapid decrease in the concentration of the culture medium CS due to the dripping of condensed water is suppressed. Furthermore, to further suppress the dripping of condensed water, if possible, the second membrane filter 98 may be positioned lower than the connection between the gas discharge channel Pout and the culture container 12, i.e., the connection between the insulating tube 94 and the L-shaped tube joint 92.

[0110] In the case of the culture apparatus 10 of this embodiment, it further includes a fifth heater 102 for heating the top plate portion 12c of the cylindrical culture container 12 and a sixth heater 104 for heating the side wall portion 12b. The fifth and sixth heaters 102 and 104 are, for example, film heaters attached to the outer surface of the culture container 12, and are transparent heaters using ITO electrodes or the like so that the culture medium CS inside the culture container 12 can be seen.

[0111] The fifth and sixth heaters 102 and 104 heat the top plate 12c and side wall 12b of the culture vessel 12 so that water vapor inside the culture vessel 12 does not condense on the inner surfaces of the top plate 12c and side wall 12b of the culture vessel 12. For this purpose, the heating temperatures T5 and T6 of the fifth and sixth heaters 102 and 104 are set higher than the heating temperature T2 of the second heater 74 located below the culture vessel 12. For example, the heating temperatures T5 and T6 are set 0 to 5°C higher than the heating temperature T2.

[0112] The gas supply device 60, the first heater 68, the second heater 74, the third heater 88, the fourth heater 100, the fifth heater 102, and the sixth heater 104 in the gas supply unit 24 are controlled by the control unit 26.

[0113] First, the control unit 26 controls the first heater 68, the second heater 74, the third heater 88, the fourth heater 100, the fifth heater 102, and the sixth heater 104 to heat at the corresponding heating temperatures T1 to T6 as described above. While maintaining control over these heaters, the control unit 26 controls the amount of gas supplied per unit time from the gas supply device 60.

[0114] Specifically, the control unit 26 gradually or linearly reduces the amount of gas supplied per unit time by the gas supply device 60 as the amount of culture medium CS supplied by the culture medium supply unit 16 increases. In other words, the less culture medium CS there is in the culture vessel 12, the more gas is supplied.

[0115] As shown in Figures 6A and 6B, when the amount of culture medium CS in the culture vessel 12 is small, the evaporation of the culture medium CS causes significant damage to the cells in the culture medium CS. On the other hand, as shown in Figures 8A and 8B, when the amount of culture medium CS in the culture vessel 12 is large, the evaporation of the culture medium CS has almost no effect on the cells in the culture medium CS.

[0116] Therefore, when the amount of culture medium CS in the culture vessel 12 is small, a large amount of humidified mixed gas Gw, i.e., a large amount of water vapor, is supplied to the culture vessel 12 to create a humidity environment of 95% RH or higher inside the culture vessel 12. This makes it difficult for the culture medium CS to evaporate, and damage to the cells in the culture medium CS is suppressed. On the other hand, when the amount of culture medium CS in the culture vessel 12 is large, adding an excessive amount of mixed gas Gw in that state will increase the pressure inside the culture vessel 12, so the amount of gas supplied should be reduced. However, the amount of gas supplied necessary for pH adjustment of the culture medium CS should be maintained.

[0117] This section discusses the effects of evaporation of culture medium on cells. Evaporation of the culture medium changes its osmotic pressure, affecting the cells. Figure 12 shows the dilution ratio and osmotic pressure when Iskov-modified Dulbecco's medium (IMDM), an example of a culture medium, is diluted with distilled water. As shown in Figure 12, increasing the dilution ratio decreases the osmotic pressure of the culture medium. When cells are placed in a hypertonic solution with high osmotic pressure, the volume of the cells decreases as water is drawn out of the cells. On the other hand, when placed in a hypotonic solution with low osmotic pressure, the cells expand as water is drawn into their interiors. In this way, cells are deformed and damaged by the osmotic pressure of the culture medium.

[0118] Generally, the optimal osmotic pressure for a culture medium in cell culture is 265-315 mOsm / kg. Cells are damaged by water movement in both hypertonic and hypotonic solutions that fall outside this range. Specifically, in the Iskov-modified Dulbecco medium shown in Figure 12, these relationships are adjusted so that the optimal osmotic pressure is achieved at a dilution ratio of 1.00. As the dilution ratio increases, the osmotic pressure decreases, and as the culture medium evaporates and becomes more concentrated, the osmotic pressure increases.

[0119] Figure 13 shows the results of investigating the relationship between the gas flow rate of the mixed gas supplied to the culture vessel and the evaporation rate of the culture medium in an example of this embodiment, under certain conditions (50 ml) of culture medium volume. As shown in Figure 13, the evaporation rate increases with increasing gas flow rate. Therefore, when the culture medium volume is large, the gradient showing the evaporation rate with respect to gas flow rate becomes gentler, and when the culture medium volume is small, the gradient showing the evaporation rate with respect to gas flow rate becomes steeper. From this, it is necessary to control the gas flow rate of the humidified mixed gas in order to suppress the evaporation of the culture medium.

[0120] Figure 14 shows an example of this embodiment, illustrating the volume of culture medium and the evaporation rate of the culture medium as a function of the elapsed culture time. Starting with an initial culture medium volume of 50 ml, the estimated volume of culture medium after 97 hours (initial volume plus added volume) is shown as the estimated volume per hour (solid line). The volume change taking evaporation rate into account (dashed line) is also shown. Furthermore, the figure also shows the change in the evaporation rate (ratio of evaporated volume to culture medium volume) (dotted line) determined from the evaporation rate at the gas flow rate in Figure 13.

[0121] As can be seen from the evaporation rate progression in Figure 14, in this embodiment, the evaporation rate reaches its maximum value at 26 hours of culture time, which is approximately 3.5%. In other words, when the evaporation rate is above zero, the culture medium becomes more concentrated, the osmotic pressure increases, and it acts to draw water out of the cells, damaging them. As shown in Figure 14, the evaporation rate naturally decreases as the volume of culture medium increases. In this embodiment, the gas flow rate, agitation conditions, humidity conditions, etc., are controlled so that the maximum evaporation rate is 3.5%. On the other hand, the evaporation rate of 3.5% in Figure 14 is the same as the dilution ratio of 0.965 on the evaporation side in the diagram showing the relationship between the dilution ratio of the culture medium and osmotic pressure in Figure 12, so at this value, the osmotic pressure of the culture medium can be controlled to the optimal value of approximately 290 mOsm / kg.

[0122] In this embodiment, in expansion culture where the amount of cultured cells is increased while increasing the volume of culture medium, the evaporation rate of the culture medium is controlled by controlling the flow rate of the supply gas supplied to the culture vessel. This controls the osmotic pressure of the culture medium within an optimal range, thereby reducing damage to the cells. Specifically, the amount of evaporation when the volume of culture medium is small is controlled so that the osmotic pressure is approximately 260-315 mOsm / kg.

[0123] In this embodiment, the control unit 26 lowers the heating temperature T1 of the first heater 68 that heats the hollow fiber membrane filter 64 as the amount of culture medium CS supplied by the culture medium supply unit 16 increases. This reduces the amount of water vapor contained in the mixed gas Gd in the hollow fiber membrane filter 64. As a result, it is possible to reduce the amount of water vapor contained while maintaining the required amount of mixed gas. By reducing the amount of water vapor, dilution of the culture medium is suppressed. In addition, the power consumption of the first heater 68 can be reduced.

[0124] Furthermore, the heating temperature T1 of the first heater 68 may be lowered, and the heating temperatures T3 of the third heater 88, T4 of the fourth heater 100, T5 of the fifth heater 102, and T6 of the sixth heater 104, which are involved in suppressing condensation, may also be lowered while maintaining the aforementioned correspondence. However, the heating temperature T2 of the second heater 74, which heats the culture medium CS in the culture vessel 12, is maintained at the required temperature regardless of the heating temperatures of the other heaters.

[0125] Furthermore, before the culture medium supply unit 16 supplies the culture medium CS to the culture vessel 12, the gas supply unit 24 may supply humidified mixed gas Gw to the culture vessel 12. This ensures that the culture vessel 12 is sufficiently humidified, i.e., filled with water vapor, before the culture medium CS is supplied to it. As a result, evaporation of the small amount of culture medium CS immediately after it is supplied to the culture vessel 12 is suppressed. It is preferable that the maximum amount of humidified mixed gas Gw is supplied to the culture vessel 12 from the gas supply unit 24 in order to immediately begin supplying the culture medium CS to the culture vessel 12.

[0126] According to this embodiment, when culturing cells using a culture medium in a culture vessel, the evaporation of the culture medium can be suppressed.

[0127] The present invention has been described above with reference to the embodiments described above, but the embodiments of the present invention are not limited to these.

[0128] For example, in the above-described embodiment, as shown in Figure 10, the gas supply passage Pin for supplying the humidified mixed gas Gw and the gas discharge passage Pout for discharging the exhaust gas Ge are connected to the top plate portion 12c of the culture container 12. However, the embodiments of the present invention are not limited to this.

[0129] Figure 15 is a schematic diagram of the gas supply section in a culture apparatus according to another embodiment.

[0130] As shown in Figure 15, in the gas supply unit 224 of the culture apparatus according to another embodiment, the gas supply channel Pin that supplies the humidified mixed gas Gw is connected to the side wall portion 212b of the cylindrical culture container 212 via a straight tube joint 278. As a result, the gas supply channel Pin, which is composed of the first filter unit 76, the insulated tubes 80 and 82, and the tube joint 278, extends horizontally (in the X-axis direction). Consequently, the dripping of condensation water from the gas supply channel Pin onto the culture container 212 is suppressed.

[0131] Similarly, the gas discharge channel Pout, which discharges exhaust gas Ge, is also connected to the side wall portion 212b of the culture vessel 212 via a straight tube joint 292. As a result, the gas discharge channel Pout, which consists of the second filter unit 90, the insulated tube 94, and the tube joint 292, extends horizontally (in the X-axis direction). Consequently, the dripping of condensation water from the gas discharge channel Pout onto the culture vessel 212 is suppressed.

[0132] Furthermore, in the above-described embodiment, the humidifier 62 humidifies the mixed gas in the gas supply unit 24, but the embodiments of the present invention are not limited to this. The humidifier may humidify a single gas necessary for cultivation, such as oxygen or carbon dioxide.

[0133] Furthermore, in the embodiment described above, the culture vessel is cylindrical, as shown in Figure 2. However, the embodiments of the present invention are not limited to this. The culture vessel may be, for example, a large Erlenmeyer flask. Alternatively, the culture vessel may be a flexible culture bag.

[0134] Furthermore, in the above-described embodiment, the culture apparatus 10 is configured to supply additional culture medium CS to the culture container 12 as the culture progresses, i.e., to perform expanded culture. However, the embodiments of the present invention are not limited to this. A culture apparatus that cultures cells using a fixed amount of culture medium may also be used.

[0135] In other words, the culture apparatus according to an embodiment of the present invention broadly comprises a culture vessel containing a culture medium for culturing cells, a gas supply device for supplying gas to the culture vessel, and a humidifier for humidifying the gas flowing from the gas supply device to the culture vessel, wherein the humidifier includes a hollow fiber membrane filter comprising a hollow fiber membrane through which the gas from the gas supply device passes and a casing containing the hollow fiber membrane, a water supply device for filling the casing of the hollow fiber membrane filter with water, and a first heater for heating the hollow fiber membrane filter. [Industrial applicability]

[0136] This invention is applicable to a device for culturing cells using a culture medium in a culture vessel.

Claims

1. A culture vessel containing a culture medium for culturing cells, A gas supply device that supplies gas to the culture vessel, The system includes a humidifier that humidifies the gas flowing from the gas supply device to the culture vessel, The humidifier, A hollow fiber membrane filter comprising a hollow fiber membrane through which gas from the gas supply device passes, and a casing that houses the hollow fiber membrane, A water supply device having a water supply container connected to the casing of the hollow fiber membrane filter, located above the casing, and filling the casing with water, The set includes a first heater for heating the hollow fiber membrane filter, The water supply device has a plurality of water supply containers connected to the hollow fiber membrane filter, A culture apparatus characterized in that water from the water supply container is supplied into the casing, and air from inside the casing moves into the water supply container.

2. The culture apparatus according to claim 1, characterized in that the plurality of water supply containers are located above the casing, water from a first water supply container included in the plurality of water supply containers is supplied into the casing, and air inside the casing moves to a second water supply container included in the plurality of water supply containers.

3. The culture apparatus according to claim 1, wherein the plurality of water supply containers are configured to have a variable internal volume.

4. The culture apparatus according to claim 1, further comprising a detection unit for detecting the amount of change in the internal volume of the water supply container.

5. The culture apparatus according to claim 4, wherein the detection unit detects the amount of water supplied from the water supply container to the casing.

6. The culture apparatus according to any one of claims 1 to 5, wherein the plurality of water supply containers store water such that the water level in all of the plurality of water supply containers is at the same level.

7. A second heater is positioned below the culture vessel and heats the culture medium inside the culture vessel. The culture apparatus according to any one of claims 1 to 6, wherein the heating temperature of the first heater is higher than the heating temperature of the second heater.

8. The culture apparatus according to any one of claims 1 to 7, further comprising a first membrane filter provided in a gas supply path between the humidifier and the culture vessel, wherein the normal of the filter surface is inclined with respect to the vertical direction.

9. The first membrane filter has a third heater for heating it, The culture apparatus according to claim 8, wherein the heating temperature of the third heater is higher than the heating temperature of the first heater.

10. The culture apparatus according to claim 8 or 9, wherein the portion of the gas supply path between the first membrane filter and the culture vessel extends horizontally.

11. The culture apparatus according to claim 8 or 9, wherein the first membrane filter is located at a lower position than the connection between the gas supply passage and the culture vessel.

12. The culture apparatus according to any one of claims 1 to 11, further comprising a second membrane filter provided in a gas discharge passage connecting the inside of the culture vessel to the outside air, the second membrane filter being positioned such that the normal to the filter surface is inclined with respect to the vertical direction.

13. The second membrane filter has a fourth heater for heating it, The culture apparatus according to claim 12, wherein the heating temperature of the fourth heater is higher than the heating temperature of the first heater.

14. The culture apparatus according to claim 12 or 13, wherein the portion of the gas discharge passage between the second membrane filter and the culture vessel extends horizontally.

15. The culture apparatus according to claim 12 or 13, wherein the second membrane filter is located at a lower position than the connection between the gas discharge passage and the culture vessel.

16. The culture vessel is cylindrical in shape, comprising a bottom plate, a top plate, and side walls. It has a fifth heater for heating the top plate portion and a sixth heater for heating the side wall portion, The culture apparatus according to claim 7, wherein the heating temperatures of the fifth and sixth heaters are higher than the heating temperature of the second heater.

17. A culture medium supply unit that supplies culture medium to the culture vessel, The system includes a control unit that controls the gas supply device and the culture medium supply unit, The culture apparatus according to any one of claims 1 to 16, wherein the control unit changes the amount of gas supplied per unit time from the gas supply device as the amount of culture medium in the culture vessel supplied by the culture medium supply unit increases.

18. The culture apparatus according to claim 17, wherein the control unit changes the amount of gas supplied by the gas supply device so that the evaporation rate of the culture medium in the culture vessel and the osmotic pressure of the culture medium determined from the evaporation rate are at predetermined values.

19. The culture apparatus according to claim 18, wherein the predetermined value of the osmotic pressure of the culture medium is in the range of 260 to 315 mOsm / kg.

20. The culture apparatus according to any one of claims 17 to 19, wherein the control unit controls the heating temperature of the first heater and changes the heating temperature of the first heater as the amount of culture medium in the culture vessel supplied by the culture medium supply unit increases.

21. The culture apparatus according to claim 18 or 20, wherein, before the culture medium supply unit supplies the culture medium to the culture vessel, the gas supplied from the gas supply device and humidified by the humidifier is supplied to the culture vessel.

22. A culture vessel containing a culture medium for culturing cells, A gas supply device that supplies gas to the culture vessel, The system includes a humidifier that humidifies the gas flowing from the gas supply device to the culture vessel, The humidifier, A hollow fiber membrane filter comprising a hollow fiber membrane through which gas from the gas supply device passes, and a casing that houses the hollow fiber membrane, A water supply device for filling the casing of the hollow fiber membrane filter with water, A first heater for heating the hollow fiber membrane filter, A culture apparatus comprising: a first membrane filter provided in a gas supply path between the humidifier and the culture vessel, the first membrane filter being positioned such that the normal of its filter surface is inclined with respect to the vertical direction.

23. A culture vessel containing a culture medium for culturing cells, A gas supply device that supplies gas to the culture vessel, The system includes a humidifier that humidifies the gas flowing from the gas supply device to the culture vessel, The humidifier, A hollow fiber membrane filter comprising a hollow fiber membrane through which gas from the gas supply device passes, and a casing that houses the hollow fiber membrane, A water supply device for filling the casing of the hollow fiber membrane filter with water, A first heater for heating the hollow fiber membrane filter, A culture apparatus comprising: a second membrane filter provided in a gas discharge passage connecting the inside of the culture vessel to the outside air, and positioned such that the normal of the filter surface is inclined with respect to the vertical direction.

24. A culture vessel containing a culture medium for culturing cells, A gas supply device that supplies gas to the culture vessel, The system includes a humidifier that humidifies the gas flowing from the gas supply device to the culture vessel, The humidifier, A hollow fiber membrane filter comprising a hollow fiber membrane through which gas from the gas supply device passes, and a casing that houses the hollow fiber membrane, A water supply device for filling the casing of the hollow fiber membrane filter with water, A first heater for heating the hollow fiber membrane filter, A first membrane filter is placed in the gas supply path between the humidifier and the culture vessel, A culture apparatus comprising a second membrane filter positioned in a gas discharge channel connecting the inside of the culture vessel to the outside air.

25. The portion of the gas supply path between the first membrane filter and the culture vessel extends horizontally, The culture apparatus according to claim 24, wherein the portion of the gas discharge passage between the second membrane filter and the culture vessel extends in the horizontal direction.

26. The first membrane filter is located at a lower position than the connection between the gas supply path and the culture vessel. The culture apparatus according to claim 24, wherein the second membrane filter is located at a lower position than the connection between the gas discharge passage and the culture vessel.

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