Culture apparatus and culture method

Abstract

To provide a culture apparatus and a culture method which can perform extended culture by a single culture vessel, and can reduce physical damage given to cells as much as possible, and a reference value of monitoring can be shared by all culture scales in monitoring a culture state of cells in culture solution by an optical sensor.SOLUTION: A culture apparatus is constituted of: a culture vessel 1 which houses culture solution containing cells; a rotary drive part 2 which rotary-drives the culture vessel 1 about an axis X; a tilting mechanism 3 for tilting the culture vessel 1 along a vertical plane including the axis X; and an optical sensor 4 which measures a state quantity regarding a culture state of cells in the culture solution inside the culture vessel 1, as primary elements. The culture vessel 1 has a translucent part 1a at one end part in a direction along the axis X. When the culture vessel 1 is tilted, the rotary drive part 2 and the optical sensor 4 move together with the culture vessel 1, and a physical relationship between the optical sensor 4 and the translucent part 1a does not change but is constant.SELECTED DRAWING: Figure 1

Description

Technical Field

[0003]

[0001] The present invention relates to a culture apparatus and a culture method used for culturing cells (including animal cells, insect cells, plant cells, bacteria, fungi, viruses, algae, yeast, etc.).

Background Art

[0002] In recent years, in the fields of production of biopharmaceuticals, regenerative medicine, immunotherapy, etc., it has been required to efficiently and massively culture cells in an artificial environment. Cell culture is performed using a culture solution (medium), but as the cell density in the culture solution increases, depletion of components necessary for growth and accumulation of metabolic products (waste products) of the cells themselves occur, the growth rate decreases, and the cell density reaches a saturated state. For this reason, a method (scale-up culture) of gradually increasing the volume of the culture solution and gradually expanding the culture scale (scale-up) is used. In scale-up culture, every time the culture scale is expanded, fresh culture solution is replenished, so that components necessary for growth are replenished, metabolic products are diluted, and an environment in which cells can easily grow is maintained.

[0003] Typically, in large-scale culture, separate culture vessels are provided for each culture scale, and the culture medium containing the proliferated cells is sequentially transferred from small-scale culture vessels to large-scale culture vessels to carry out the culture process. However, the process of transferring the culture medium is cumbersome, and there is a risk of contamination during the transfer, which can lead to the culture medium becoming contaminated. To address these problems, Patent Document 1 proposes a culture system in which cells are cultured in a single culture tank, and fresh culture medium (fresh culture medium) is sequentially (in stages) replenished into the culture tank from a separate culture medium tank, thereby sequentially expanding the culture scale. The culture tank is equipped with a stirrer for agitating the culture medium, an aeration device for supplying gas such as oxygen to the culture medium, a temperature control device for adjusting the temperature inside the culture tank, and sensors for measuring the culture state of the cells inside the culture tank online. The measurement data from the sensors is output to an analyzer, which analyzes the culture state of the cells. Then, based on the analysis data output from the analyzer, it is determined whether the culture process for each culture scale has been completed, and fresh culture medium is replenished from the culture medium tank to the culture vessel according to the determination result. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2020-124169 [Overview of the project] [Problems that the invention aims to solve]

[0005] In the culture apparatus described in Patent Document 1, when the culture medium in the culture tank is stirred by the stirrer, there is a concern that cells near the tip of the stirring blade may suffer physical damage due to the shear force associated with the stirring. Furthermore, while the amount of culture medium in the culture tank differs for each culture scale, the stirrer stirs the culture medium at a fixed position in the culture tank regardless of the culture scale. As a result, the distribution of cells in the culture medium changes for each culture scale, and even if the culture state of the cells in the culture tank is the same, the sensor measurement data will show different characteristics for each culture scale. Therefore, when monitoring the culture state of cells in the culture tank based on the sensor measurement data, it becomes necessary to individually set the monitoring reference value (threshold) for each culture scale.

[0006] The object of the present invention is to provide a culture apparatus and culture method that allows for large-scale culture in a single culture vessel, minimizes physical damage to cells, and enables the use of a common reference value for monitoring the culture state of cells in the culture medium using an optical sensor across all culture scales. [Means for solving the problem]

[0007] To solve the above problems, the present invention provides a culture apparatus comprising: a culture vessel containing a culture medium containing cells, which is rotationally driven about its axis and is tiltable along a vertical plane including the axis, and which has a light-transmitting portion at one end in the direction along the axis; a rotational drive unit for rotating the culture vessel; a tilting mechanism for tilting the culture vessel; and an optical sensor positioned at a predetermined location facing the light-transmitting portion, which measures a state quantity related to the culture state of cells in the culture medium inside the culture vessel from outside the culture vessel via the light-transmitting portion, and which moves while maintaining a constant positional relationship with the light-transmitting portion as the culture vessel tilts, wherein a predetermined amount of fresh culture medium is successively replenished in the culture vessel based on the state quantity measured by the optical sensor, and the culture vessel is successively tilted by the tilting mechanism at a predetermined angle in accordance with the increase in the amount of culture medium inside the culture vessel, thereby culturing cells while successively expanding the culture scale inside the culture vessel.

[0008] Furthermore, in order to solve the above problems, the present invention provides a culture medium containing cells in a culture vessel that is rotationally driven around its axis, tiltable along a vertical plane including the axis, and having a light-transmitting portion at one end in the direction along the axis, and while performing a culture process in which cells are cultured in the culture medium while the culture vessel is rotated with respect to the horizontal plane at a predetermined inclination angle and the liquid surface of the culture medium intersects the light-transmitting portion, a state quantity relating to the culture state of the cells in the culture medium is measured from outside the culture vessel via the light-transmitting portion using an optical sensor, and the state quantity measured by the optical sensor Based on this, the end of the culture step is determined, and thereafter, a predetermined amount of fresh culture medium is replenished in the culture vessel, and while maintaining a constant positional relationship between the light-transmitting part and the optical sensor, the culture vessel is tilted to an angle such that the intersection position of the liquid level of the culture medium in the culture vessel and the light-transmitting part is the same as in the culture step, and the next culture step is performed in the same manner as the previous culture step, and the end of the next culture step is determined based on the state quantity measured by the optical sensor, and thereafter these processes are repeated to culture cells while successively expanding the culture scale in the culture vessel. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a culture apparatus and culture method that allows for large-scale culture in a single culture vessel, minimizes physical damage to cells, and enables the use of a common reference value for monitoring the culture state of cells in the culture medium using an optical sensor across all culture scales. [Brief explanation of the drawing]

[0010] [Figure 1] This is a side view showing the overall configuration of the culture apparatus according to the embodiment. [Figure 2] This is a view from above of the culture apparatus according to the embodiment. [Figure 3] This is a rear view of the culture apparatus according to the embodiment. [Figure 4]This figure shows the culture vessel tilted 90° upwards from the state shown in Figure 1. [Figure 5] This diagram schematically shows the state when the culture process is performed using the culture apparatus according to the embodiment. [Figure 6] This diagram shows the process of gradually expanding the culture scale. [Figure 7] This diagram shows the process of gradually expanding the culture scale. [Modes for carrying out the invention]

[0011] Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[0012] Figures 1 to 4 show the overall configuration of the culture apparatus according to this embodiment. The culture apparatus of this embodiment is mainly composed of a culture vessel 1 that contains a culture medium containing cells, a rotation drive unit 2 that rotates the culture vessel 1 around an axis X, a tilting mechanism 3 that tilts the culture vessel 1 along a vertical plane including the axis X, and an optical sensor 4 that measures state quantities related to the culture state of cells in the culture medium inside the culture vessel 1.

[0013] The culture vessel 1 is formed in a drum shape with a circular or polygonal cross-section perpendicular to the axis X, and has a light-transmitting section 1a at one end along the axis X and an opening 1b at the other end. The light-transmitting section 1a is constructed by attaching a circular light-transmitting plate made of transparent glass or transparent resin to the frame of a circular through-hole provided in the central region of one end of the culture vessel 1. The opening 1b is opened when supplying or replenishing culture medium to the culture vessel 1 or when discharging culture medium from the culture vessel 1, and is airtightly closed with a lid member 1c during the culture process. A connecting section 1d for connecting to a rotary drive unit 2 is attached to one end of the culture vessel 1. A baffle for mixing and stirring may be provided on the inner surface of the culture vessel 1. A temperature control unit for adjusting the temperature inside the culture vessel 1 may also be provided. This temperature control unit can be constructed, for example, by attaching an electric heater or a fluid jacket for the circulation of temperature-controlled fluid to the outer circumference of the culture vessel 1.

[0014] The rotary drive unit 2 comprises a drive motor 2a whose rotational speed can be adjusted variably, and a power transmission mechanism 2b that transmits the rotational power of the drive motor 2a, and is attached to the tilting frame 3a of the tilting mechanism 3, which will be described later, via a mounting plate 2c. The culture vessel 1 is connected to the power transmission mechanism 2b via a connecting part 1d attached to one end thereof, and rotates at a predetermined speed around the axis X in accordance with the rotation of the drive motor 2a.

[0015] As shown in Figure 2, the tilting mechanism 3 comprises a tilting frame 3a and a tilting support section 3b that supports the tilting frame 3a so that it can tilt along a vertical plane containing the axis X. The tilting frame 3a consists of left and right side sections 3a1 supported by the tilting support section 3b and a rear section 3a2 connecting the rear ends of the left and right side sections 3a1. A pivot shaft 3a11 having a center line Y perpendicular to the axis X is connected to each of the left and right side sections 3a1, and a rotational drive unit 2 is attached to the rear section 3a2 via a mounting plate 2c. The tilting support section 3b is erected on a base 3b1 and comprises left and right support sections 3b2 that support the pivot shaft 3a11 of the tilting frame 3a so that it can rotate around the center line Y, and also has a function to lock the rotation of the pivot shaft 3a at any position.

[0016] The culture vessel 1 can be tilted to any position between a position where the axis X is aligned with the horizontal plane (Figure 1: tilt angle θ=0°) and a position where the axis X is perpendicular to the horizontal plane (Figure 4: tilt angle θ=90°) by operating the tilting mechanism 3.

[0017] The optical sensor 4 is a spectroscopic sensor (reflective or transmissive type) such as a near-infrared spectroscopic sensor, a mid-infrared spectroscopic sensor, or a Raman spectroscopic sensor. The optical sensor 4 (sensor probe) is attached to the tip of the sensor bracket 4a. The rear end of the sensor bracket 4a is attached to the mounting plate 2c in an adjustable position, and the tip of the sensor bracket 4a extends through the central hole of the mounting plate 2c, the hollow part of the power transmission mechanism 2b, and the central hole of the connecting part 1d to a predetermined value near the light-transmitting part 1a. In this way, the optical sensor 4, attached to the tilting frame 3a via the sensor bracket 4a and the mounting plate 2c, faces the light-transmitting part 1a at a predetermined position close to the light-transmitting part 1a.

[0018] In this embodiment, the optical sensor 4 is a reflection-type near-infrared spectroscopy sensor (NIR). Near-infrared light is irradiated onto the culture solution in the culture vessel 1 through the light-transmitting portion 1a, and the reflected light reflected by the cells in the culture solution is received through the light-transmitting portion 1a to obtain its spectrum (NIR spectrum). The spectrum of the reflected light acquired by the optical sensor 4 correlates with the state quantity related to the culture state of the cells in the culture solution. By analyzing the spectrum of the reflected light, the state quantity related to the culture state of the cells in the culture solution can be measured (detected). Examples of the state quantity related to the culture state of the cells include the cell density in the culture solution, the specific growth rate, the amount of cell metabolites, the cell survival rate, etc. In this embodiment, the optical sensor 4 measures the cell density in the culture solution.

[0019] FIG. 5 schematically shows the state when the culturing process is executed by the culturing apparatus of this embodiment. A predetermined amount of culture solution C (containing cells) is accommodated inside the culture vessel 1. The culture vessel 1 is inclined so that the axis X makes a predetermined inclination angle θ with respect to the horizontal plane, and is rotationally driven around the axis X by the rotation driving unit 2 in a state where the liquid surface L of the culture solution C intersects the inner surface of the light-transmitting portion 1a at a predetermined position S. At the same time, gases such as oxygen, nitrogen, carbon dioxide, etc., for example, oxygen O, are supplied into the culture solution C in the culture vessel 1 from the gas aeration unit 5 in a manner such as bubbling. The cells in the culture solution C grow in the culture solution C under the mixing and stirring accompanying the rotation of the culture vessel 1 and the oxygen supply from the gas aeration unit 5. The optical sensor 4 constantly monitors the culture state of the cells in the culture solution C through the light-transmitting portion 1a and measures the cell density in the culture solution C in real time. Incidentally, the intersection position S between the liquid surface L of the culture solution C and the light-transmitting portion 1a can be set at an arbitrary position on the inner surface of the light-transmitting portion 1a by adjusting the inclination angle θ of the culture vessel 1. In this embodiment, the intersection position S is set at the position where the inner surface of the light-transmitting portion 1a intersects the axis X.

[0020] When the cell density measured by the optical sensor 4 reaches a predetermined reference value, it is determined that the culturing process has ended, and the rotation of the culture vessel 1 is temporarily stopped, and a predetermined amount of fresh culture solution C is replenished from the opening 1b into the culture vessel 1. By replenishing the culture solution C, the amount of the culture solution C in the culture vessel 1 increases, and the intersection position S between the liquid surface L of the culture solution C and the light-transmitting portion 1a fluctuates. Therefore, the culture vessel 1 is tilted in a direction to cancel the fluctuation of the intersection position S of the liquid surface L (the direction in which the tilt angle θ decreases). That is, the culture vessel 1 is tilted until the tilt angle θ is reached at which the intersection position S between the liquid surface L of the culture solution C and the light-transmitting portion 1a is at the same position as before the replenishment of the culture solution C (the position where the inner surface of the light-transmitting portion 1a intersects the axis X). By tilting the culture vessel 1 in this way, the position (intersection position S) of the liquid surface L of the culture solution C visible through the light-transmitting portion 1a becomes the same before and after the replenishment of the culture solution C. Further, when the culture vessel 1 is tilted, since the rotation drive unit 2 and the optical sensor 4 attached to the tilt frame 3a move along with the culture vessel 1, the positional relationship between the optical sensor 4 and the light-transmitting portion 1a does not change. Therefore, the position of the measurement point T of the optical sensor 4 with respect to the light-transmitting portion 1a (a region near the inner surface of the light-transmitting portion 1a and centered on the optical axis of the irradiation light of the optical sensor 4) is the same before and after the replenishment of the culture solution C. Therefore, the distance between the position (intersection position S) of the liquid surface L of the culture solution C visible through the light-transmitting portion 1a and the measurement point T of the optical sensor 4 (hereinafter, the distance between the intersection position S and the measurement point T is referred to as the "measurement point depth") is the same before and after the replenishment of the culture solution C. That is, the measurement point T of the optical sensor 4 and the measurement point depth are constant without fluctuating before and after the replenishment of the culture solution C. In this way, after replenishing a predetermined amount of the culture solution C into the culture vessel 1 and tilting the culture vessel 1 to a predetermined tilt angle θ, the rotation of the culture vessel 1 is restarted to execute the next culturing process.

[0021] Generally, spectroscopic sensors such as near-infrared spectrometers pick up the surrounding environment of the measurement point as noise. Therefore, if the surrounding environment changes, the spectrum acquired during measurement of the target object will fluctuate, making it difficult to correctly detect the target object. In this embodiment, as described above, the measurement point T and measurement point depth of the optical sensor 4 relative to the light-transmitting section 1a remain constant before and after the replenishment of culture medium C, and the surrounding environment of the measurement point T of the optical sensor 4 does not change. Therefore, the measurement accuracy of the optical sensor 4 in the culture process before the replenishment of culture medium C can be reproduced in the culture process after the replenishment of culture medium C. For this reason, when determining the end of the culture process based on the state quantity (cell density in this embodiment) related to the culture state of the cells measured by the optical sensor 4, the end of the culture process after the replenishment of culture medium C can be correctly determined (with the same accuracy as the culture process before replenishment) using the reference value (threshold) of the above state quantity set for the culture process before the replenishment of culture medium C. For example, in the culture process before replenishing culture medium C (the culture process before scaling up), spectral data from the optical sensor 4 corresponding to the cell density at which the culture process should be terminated can be acquired, and a calibration curve can be created from this spectral data. Using this calibration curve, it is possible to correctly determine when the culture process after replenishing culture medium C (the culture process after scaling up) should be terminated.

[0022] Figures 6 and 7 show an example of a process (expansion culture) in which the culture scale is sequentially expanded using the culture apparatus of this embodiment. In this example, the initial scale is set to a state in which a standard amount of culture medium C (containing cells) is contained in the culture vessel 1 {Figure 6(a)}, and the culture scale is expanded by sequentially replenishing the culture medium C (containing cells) in the culture vessel 1 so that the amount of culture medium C (containing cells) in the culture vessel 1 becomes, for example, 3 times the standard amount {Figure 6(b)}, 5 times the standard amount {Figure 6(c)}, 8 times the standard amount {Figure 7(a)}, 12 times the standard amount {Figure 7(b)}, and 20 times the standard amount {Figure 7(c)}.

[0023] First, before proceeding to the expansion culture process, the culture process is experimentally performed on an initial scale as shown in Figure 6(a) to create a reference value (calibration curve) for determining the end of the culture process. In the initial scale shown in Figure 6(a), a standard amount of culture medium C (containing cells) is placed in the culture vessel 1, and the culture vessel 1 is tilted at a predetermined angle θ (θ=65° in this example) so that the intersection point S between the liquid surface L of the culture medium C and the translucent section 1a coincides with the axis X, and the cells are cultured while the culture vessel 1 is rotated at a predetermined speed. Then, spectral data from the optical sensor 4 corresponding to the cell density at which the culture process should be terminated is acquired in the initial scale culture process, and a calibration curve is created from this spectral data. The calibration curve created for the initial scale culture process is used as a reference value (threshold) for determining the end of each process in all culture processes.

[0024] Next, the first culture step is performed using the initial scale shown in Figure 6(a). When cell proliferation progresses during the first culture step and the spectral data measured by the optical sensor 4 reaches the level of the calibration curve described above, the first culture step is terminated. Subsequently, fresh culture medium C is added to the culture vessel 1 until the amount of culture medium C in the culture vessel 1 is three times the standard amount, and the culture vessel 1 is tilted to a predetermined inclination angle θ (θ=55° in this example) so that the intersection point S between the liquid surface L of the culture medium C and the translucent section 1a coincides with the axis X. Then, the second culture step is performed while rotating the culture vessel 1 at a predetermined speed {Figure 6(b)}.

[0025] In the second culture step, cell proliferation progresses, and when the spectral data measured by the optical sensor 4 reaches the level of the calibration curve described above, the second culture step is terminated. Subsequently, fresh culture medium C is added to the culture vessel 1 until the amount of culture medium C in the culture vessel 1 is five times the standard amount, and the culture vessel 1 is tilted to a position with an inclination angle θ (θ=45° in this example) so that the intersection point S between the liquid surface L of the culture medium C and the light-transmitting section 1a coincides with the axis X. Then, the third culture step is performed while rotating the culture vessel 1 at a predetermined speed in this state {Figure 6(c)}.

[0026] Subsequently, the same process as described above is repeated to sequentially perform the fourth culture step shown in Figure 7(a) (scale up the culture medium C to 8 times the volume: tilt angle θ=35°), the fifth culture step shown in Figure 7(b) (scale up the culture medium C to 12 times the volume: tilt angle θ=25°), and the sixth culture step shown in Figure 7(c) (scale up the culture medium C to 20 times the volume: tilt angle θ=15°) to proliferate the cells. After the final sixth culture step is completed, the culture medium C (containing the proliferated cells) is drained from the culture vessel 1.

[0027] The culture apparatus of this embodiment allows for large-scale cell culture in a single culture vessel 1, and since the cells in the culture medium C are mixed and stirred by the rotation of the culture vessel 1, physical damage to the cells can be reduced as much as possible. Furthermore, when monitoring the culture state of the cells in the culture medium C with an optical sensor 4, the monitoring reference value (calibration curve, etc.) can be shared across all culture scales.

[0028] In this embodiment, the rotation speed of the culture vessel 1 in the first to sixth culture steps may be the same speed between each culture step, or it may be different speeds. For example, the rotation speed of the culture vessel 1 may be gradually increased in accordance with the increase in the amount of culture medium C in the culture vessel 1. Also, when replenishing the culture medium C after the completion of each culture step from the first to fifth culture steps, the culture medium C may be replenished while continuing to rotate the culture vessel 1.

[0029] Furthermore, the tilting of the culture vessel 1 may be performed by an operator manually operating the tilting mechanism 3, or a tilting operation unit that automatically operates the tilting mechanism 3 may be provided (for example, the tilting frame 3a of the tilting mechanism 3 may be operated by a rotary actuator such as an electric motor or a linear actuator such as a hydraulic cylinder). For example, when the spectral data measured by the optical sensor 4 reaches the level of the calibration curve described above, an operation signal may be output to the tilting operation unit described above, causing the culture vessel 1 to be automatically tilted to a predetermined tilt angle θ.

[0030] Furthermore, the supply (including replenishment) of culture medium C to the culture vessel 1 may be performed manually by an operator, or a culture medium supply unit may be provided to automatically supply culture medium C to the culture vessel 1 (for example, a predetermined amount of culture medium C is delivered to the culture vessel 1 from a culture medium tank that stores culture medium C by a liquid delivery means). For example, when the spectral data measured by the optical sensor 4 reaches the level of the calibration curve described above, an operation signal may be output to the culture medium supply unit to automatically replenish a predetermined amount of culture medium C from the culture medium tank to the culture vessel 1. [Explanation of symbols]

[0031] 1 Culture vessel 1a Translucent part 2 Rotary drive unit 3 Tilt mechanism 3a Tilting frame 4 Optical sensors θ Tilt angle C Culture solution L liquid level S intersection position T Measurement point

Claims

1. A culture vessel containing a culture medium containing cells, which is driven to rotate around its axis and is tiltable along a vertical plane including the axis, and which has a light-transmitting portion at one end in the direction along the axis, A rotational drive unit that rotates the culture vessel, A tilting mechanism for tilting the culture vessel, The system includes an optical sensor positioned at a predetermined location facing the light-transmitting portion, which measures the cell density or specific growth rate of cells in the culture medium within the culture vessel from outside the culture vessel via the light-transmitting portion, and which moves while maintaining a constant positional relationship with the light-transmitting portion as the culture vessel tilts. A culture apparatus that sequentially replenishes a predetermined amount of fresh culture medium to the culture vessel based on the cell density or specific growth rate measured by the optical sensor, and sequentially tilts the culture vessel by a predetermined angle using the tilting mechanism in accordance with the increase in the amount of culture medium in the culture vessel, thereby culturing cells while sequentially expanding the culture scale within the culture vessel.

2. The culture apparatus according to claim 1, wherein the tilting mechanism comprises a tilting frame that can be tilted along the vertical plane, the rotary drive unit and the optical sensor are attached to the tilting frame, and the culture vessel is attached to the tilting frame via the rotary drive unit.

3. The culture apparatus according to claim 1 or 2, wherein the optical sensor is a spectroscopic sensor.

4. A culture vessel containing cells is housed in a culture vessel that is rotationally driven around its axis, can tilt along a vertical plane including the axis, and has a light-transmitting portion at one end in the direction along the axis. With the culture vessel tilted at a predetermined angle with respect to the horizontal plane and the liquid surface of the culture medium intersects the light-transmitting section, a culture process is performed in which cells are cultured in the culture medium while the culture vessel is rotated, and the cell density or specific growth rate of the cells in the culture medium is measured from outside the culture vessel via the light-transmitting section using an optical sensor. A culture method in which cells are cultured while successively expanding the culture scale in the culture vessel, with the following steps: determining the end of the culture step based on the cell density or specific growth rate measured by the optical sensor; replenishing the culture vessel with a predetermined amount of fresh culture medium; and tilting the culture vessel to an angle such that the intersection point of the liquid surface of the culture medium in the culture vessel and the optical sensor is the same as in the culture step, while maintaining a constant positional relationship between the light-transmitting part and the optical sensor; and repeating these processes thereafter.

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