Cell detachment device, cell detachment system, cell detachment method, and program

Abstract

To efficiently and stably detach sample cells from a culture substrate.SOLUTION: A cell detachment device for detaching cells adhered to the culture surface of a culture substrate from the culture surface includes an ultrasonic generator that generates ultrasonic vibrations in the culture substrate, a beating unit that strikes the culture substrate, and a shaking unit that shakes the culture substrate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell detachment device, a cell detachment system, a cell detachment method, and a program. [Background technology]

[0002] In the medical field, for treatment and research and development, cells are sometimes cultured on the bottom of a culture substrate, such as a culture vessel such as a culture plate or dish. However, because the cultured cells (sample cells) adhere to the bottom of the culture substrate, it is necessary to detach and remove the sample cells from the substrate. Methods for detaching sample cells from a culture substrate include using a detachment enzyme or a chemical that acts on the cell membrane, using a temperature-responsive polymer, or applying ultrasonic waves to apply vibrational energy to the cells to detach them.

[0003] Patent Document 1 discloses a cell detachment device in which an ultrasonic wave emitting means is disposed on the underside of a culture substrate, and the ultrasonic wave emitting means emits acoustic radiation pressure toward a processing target area on the underside of the substrate, thereby detaching sample cells. In the cell detachment device disclosed in Patent Document 1, ultrasonic waves are applied while the processing target area is moved via an ultrasonic transmitter. This allows ultrasonic waves to be selectively applied to the sample cells, gradually detaching the sample cells from the container. Patent Document 2 discloses a cell detachment device in which a culture vessel held in a holder is moved back and forth and collided with a collision target together with the holder, detaching sample cells by the impact and vibration. These methods do not apply appropriate force to cells or sheet-shaped cell cultures, which have particularly strong adhesive properties, and therefore, when attempting to detach them in a short period of time, the cells or sheet-shaped cell cultures can be damaged, weakening the cells or tearing the sheet, among other problems. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2023-069062 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-113133 Summary of the Invention [Problem to be solved by the invention]

[0005] The adhesive strength of sample cells to the underside of the culture substrate varies depending on their characteristics and culture conditions. In the case of the cell detachment devices disclosed in the above-mentioned patent documents, when attempting to detach particularly highly adhesive sample cells or sheet-shaped cell cultures containing sample cells, detachment may not be possible or may require a long time. Furthermore, simply increasing the energy of the ultrasound or impact to promote detachment in a short period of time may result in damage to the cells, such as injury or partial death, and may also require consideration of damage to the sheet-shaped cell culture, such as wrinkles, tears, or holes.

[0006] In view of the above, one of the objects of the present invention is to provide a cell detachment device, a cell detachment system, a cell detachment method, and a program that can efficiently and stably detach sample cells from a culture substrate. [Means for solving the problem]

[0007] In order to solve the above problems, a cell detachment device according to one aspect of the present invention comprises: A cell detachment device for detaching cells adhered to a culture surface of a culture substrate from the culture surface, comprising: an ultrasonic wave generating unit that generates ultrasonic vibrations in the culture substrate; A beating unit that beats the culture substrate; and a shaking unit that shakes the culture substrate. [Effects of the Invention]

[0008] According to one aspect of the present invention, sample cells can be efficiently and stably detached from a culture substrate. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a diagram showing a schematic configuration of a cell detachment device according to one aspect of an embodiment of the present invention. [Figure 2] FIG. 2 is a diagram illustrating the structure of a vibrator. [Figure 3] 10 is a flowchart illustrating an example of a peeling step according to one aspect of the embodiment. [Figure 4] 1 is a diagram showing a schematic configuration of an example of a cell detachment device according to a first embodiment. [Figure 5] 3A and 3B are diagrams illustrating an example of the structure of a shaking unit in the first embodiment. [Figure 6] 3A to 3C are diagrams illustrating an example of the structure of a mounting section in the first embodiment. [Figure 7] 3A to 3C are diagrams illustrating an example of the structure and driving style of a tapping unit in the first embodiment. [Figure 8] FIG. 2 is a diagram showing a schematic configuration of a modified example of the cell detachment device according to the first embodiment. [Figure 9] FIG. 10 is a diagram showing a schematic configuration of an example of a cell detachment device according to a second embodiment. [Figure 10] FIG. 10 is a diagram showing a schematic configuration of an example of a cell detachment device according to a third embodiment. [Figure 11] FIG. 10 is a diagram showing a schematic configuration of a modified example of the cell detachment device according to the third embodiment. [Figure 12] FIG. 10 is a diagram showing a schematic configuration of an example of a cell detachment device according to a fourth embodiment. [Figure 13] FIG. 10 is a diagram showing a schematic configuration of a modified example of the cell detachment device according to the fourth embodiment. [Figure 14] FIG. 10 is a diagram showing a schematic configuration of an example of a cell detachment device according to a fifth embodiment. [Figure 15] FIG. 13 is a diagram showing a schematic configuration of an example of a cell detachment device according to a sixth embodiment. [Figure 16] FIG. 13 is a diagram showing a schematic configuration of an example of a cell detachment device according to a seventh embodiment. [Figure 17] FIG. 13 is a diagram showing a schematic configuration of an example of a cell detachment device according to an eighth embodiment. [Figure 18] FIG. 1 is a diagram showing a schematic configuration of a cell detachment device used in Comparative Example 1. [Figure 19] FIG. 10 is a diagram showing a schematic configuration of a cell detachment device used in Comparative Example 2. [Figure 20] FIG. 10 is a diagram showing a schematic configuration of a cell detachment device used in Comparative Example 3. [Figure 21] FIG. 10 is a diagram showing a schematic configuration of a cell detachment device used in Comparative Example 4. [Figure 22] FIG. 10 is a diagram showing a schematic configuration of a cell detachment device used in Comparative Example 5. [Figure 23] FIG. 10 is a diagram showing a schematic configuration of a cell detachment device used in Comparative Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of a cell detachment device, a cell detachment system, and a cell detachment method according to one aspect of the present invention will be described with reference to the drawings. The following embodiments do not limit the scope of the present invention. Note that the configurations shown in the following embodiments are merely examples, and the present invention is not limited to the configurations shown in the drawings. Furthermore, although multiple features are described in the embodiments, not all of these multiple features are necessarily essential to the present invention, and multiple features may be combined arbitrarily. Furthermore, in the drawings, the same reference numbers are used for identical or similar components, and redundant explanations are omitted.

[0011] In the following, the concept of a cell detachment device according to one aspect of the present invention will first be described with reference to Fig. 1, followed by a description of the individual components of the cell detachment device. After that, the first to eighth embodiments will be specifically described, and the effects of the present invention will be described with reference to comparative examples.

[0012] (Cell detachment device) A cell detachment device according to one embodiment of the present invention detaches cells or sheet-like cell cultures adhered to the culture surface of a culture substrate from the culture surface. The cell detachment device 1, whose schematic configuration is shown in FIG. 1 , includes an ultrasonic generator 2 that generates ultrasonic vibrations in the culture substrate 9, a beating unit 4 that strikes the culture substrate to impart impact and vibration, and a shaking unit 3 that shakes the culture substrate. The ultrasonic generator 2 can generate ultrasonic vibrations in the culture substrate 9 that act on a time scale on the order of microseconds. The shaking unit 3 can generate a water flow within the culture substrate 9 that acts on a time scale on the order of seconds. In addition to these two types of effects, the cell detachment device according to one embodiment of the present invention also applies an impact on the cells or sheet-like cell culture, generated by the beating unit 4, on a time scale on the order of milliseconds.

[0013] By arranging these components in the cell detachment device 1, it is possible to apply actions on a wide time scale, from seconds to microseconds, to cells or sheet-like cell cultures adhered to the culture surface of the culture substrate. The time required for cells to recover their adhesive strength is generally on the order of minutes to ten minutes. Therefore, the cell detachment device makes it possible to apply forces from the three components in a period shorter than this time scale. The cell detachment device of the present invention and each of the components included therein are described in detail below.

[0014] (ultrasonic generator) The ultrasonic generator described here refers to a component in the cell detachment device that can generate ultrasonic vibrations on the culture substrate. The ultrasonic vibrations used in the present invention can be vibrations with a frequency of, for example, about 10 kHz to 1 MHz. The ultrasonic vibration generating means can be any means that can apply ultrasonic vibrations to cells, and is not particularly limited. One example of the ultrasonic vibration generating means is an ultrasonic oscillator such as lead zirconate titanate (PZT) that can be used as a vibrating body.

[0015] Here, ultrasonic vibrations can be applied to the culture substrate by directly contacting a vibrator with the outer surface of a culture substrate filled with a medium and sealed. Alternatively, instead of contacting the ultrasonic irradiation means directly with the container, a vibration transmitter can be interposed between the ultrasonic irradiation means and the culture substrate, allowing ultrasonic waves to be incident on the cells to be detached.

[0016] (Vibrator used in the ultrasonic generator) The vibrator used in the ultrasonic generator described above is not limited as long as it generates ultrasonic vibrations, but for example, a piezoelectric body bonded to a vibration plate can be used. In this case, if the piezoelectric body is circular, the vibration plate is preferably made of glass, SUS, or quartz. By using glass, SUS, or quartz as the vibration plate, it is possible to output a large amplitude at a relatively high driving frequency (vibration frequency) in the ultrasonic range without damaging the vibrator. The vibrator can also include an ultrasonic vibrator.

[0017] When the piezoelectric body is ring-shaped, it is preferable that the outer diameter of the diaphragm be equal to that of the piezoelectric body. Furthermore, the thickness of the diaphragm in this case is such that the piezoelectric body and the diaphragm are bonded together, and the midpoint of the thickness direction of the deflection during deflection vibration, i.e., the neutral plane where there is neither tension nor compression during deflection, is located on the diaphragm side, in order to efficiently utilize the distortion of the piezoelectric body for deflection. Furthermore, commercially available Langevin vibrators or rectangular vibrators can also be used as the vibrator. Examples of Langevin vibrators include those in which a piezoelectric body is sandwiched between two metal blocks and fastened together with bolts or the like to form an integrated structure, such as those manufactured by Honda Electronics and Fuji Ceramics.

[0018] (Piezoelectric material used in the ultrasonic generator) The piezoelectric element used in the vibrator described above is preferably a ring-shaped or annular piezoelectric element, but the piezoelectric element that deflects the vibrator may also be disk-shaped. However, for the piezoelectric element used in the cell detachment device, a ring-shaped element is preferred, as it ensures a clear view of the area below the culture substrate so that cell information can be directly observed using an observation mechanism. The piezoelectric element exhibits piezoelectric properties when subjected to polarization treatment, and the polarity of polarization can be changed depending on the electrode pattern, as shown in Figure 2. Figure 2 is a top view of a ring-shaped piezoelectric element with electrodes formed thereon, and schematically shows the role of each electrode and the state of polarization.

[0019] In the piezoelectric element shown in Figure 2, the area where the electrodes (A1, A2, A3, B1, B2, B3) are patterned is the driving phase that contributes to deformation, and the area between electrodes A1 and B1 is the sensor phase that detects the degree of deformation. A polarized piezoelectric element can excite either a standing wave mode or a traveling wave mode by controlling the phase of the applied AC voltage for each electrode pattern. Applying an AC voltage with a 180-degree phase difference between the positively polarized electrodes (A1, A3, B1, B3) and the negatively polarized electrodes (A2, B2) generates a standing wave in the piezoelectric element. In contrast, when the electrodes are divided into phase A (electrodes A1, A2, A3) and phase B (electrodes B1, B2, B3) on either side of the dashed line as shown in Figure 2, applying an AC voltage with a 90-degree phase difference between phase A and phase B generates a two-phase traveling wave in the piezoelectric element. In Figure 2, GND refers to ground and is used to ground the electrodes located on the backside.

[0020] The electrode pattern described above can be formed by printing electrodes made of Ag, for example, but other electrode materials such as noble metals Au, Pt, Pd, etc., and base metals Cu, etc., can also be used. Furthermore, the formation method is not limited to printing, and known methods such as plating and sputtering can also be used.

[0021] Furthermore, although a piezoelectric element with a material composition of PbZrTiO3 (PZT) was used as the vibrating body, it is preferable to use a lead-free piezoelectric material that does not substantially contain Pb in consideration of environmental regulations. Examples of lead-free piezoelectric materials include BaTiO3 (BT), NaNbO3, BiNaTiO3, and BiFeO3 as the main components, and combinations of these or metal elements added to the main components are also acceptable. In particular, by using a BaTiO3 (BT)-based material, vibration performance equivalent to that of PbZrTiO3 (PZT) can be obtained. Furthermore, materials other than ceramics, such as single crystal materials and polymer-based piezoelectric materials, can also be used.

[0022] The thickness of the piezoelectric material used in the vibrating body is preferably such that when the laminated piezoelectric material and the diaphragm undergo bending vibration, the midpoint of the bending thickness direction, i.e., the neutral plane that is neither tensile nor compressive during bending, is located on the diaphragm side. This allows the strain of the piezoelectric element to be efficiently utilized for bending. Furthermore, the combination of the thickness of the piezoelectric material and the diaphragm can be determined by the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm.

[0023] The ultrasonic generator according to one embodiment of the present invention may use a single vibrator, or may use a combination of multiple vibrators of the same or different types. Furthermore, by combining a vibrator with a vibration plate, it is possible to freely design the area in which ultrasonic vibrations are generated. When combining a vibrator with a vibration plate, they may be combined via a vibration-transmitting material. They may also be directly bonded with an adhesive or physically fixed with bolts or the like. For example, by combining a vibration plate larger than the vibrator, it is possible to transmit ultrasonic band vibrations to a larger-sized culture substrate, or to transmit vibrations to multiple culture substrates.

[0024] Furthermore, the ultrasonic generator according to one embodiment of the present invention preferably generates vibrations in a direction that includes a component perpendicular to the culture surface of the culture substrate. To transmit vibrations in a direction that includes a component perpendicular to the culture surface of the culture substrate, the most efficient transmission is achieved by transmitting the vibrations from the bottom of the culture substrate. By transmitting vibrations in a direction that includes a component perpendicular to the culture surface of the culture substrate, ultrasonic vibrations can be effectively transmitted to cells adhering to the culture surface, thereby weakening the adhesive force between the cells and the culture surface.

[0025] (vibration transmitter) A vibration transmitter can be disposed between the ultrasonic generator and the culture substrate. This allows ultrasonic vibrations generated by the ultrasonic generator to be transmitted to the culture substrate via the vibration transmitter. Any known material capable of transmitting ultrasonic vibrations can be used as the vibration transmitter. Such a vibration transmitter is used to transmit vibrations without attenuation by matching the ultrasonic generator and the culture substrate. In addition to physical matching such as acoustic impedance, mechanical matching such as adhesion is also important for matching the ultrasonic generator and the culture substrate. Therefore, it is preferable to use a vibration transmitter that has these properties. Specific examples of the vibration transmitter include water and acoustic transmission gel, but more preferably, solid materials with low fluidity such as rubber or gel. In the cell detachment device according to one embodiment of the present invention, the vibration transmitter is preferably made of a material that is at least elastic. Having at least elasticity in the vibration transmitter not only effectively transmits ultrasonic vibrations, but also effectively applies the impact force from the beating unit to the culture substrate placed on the vibration transmitter.

[0026] (Tapping club) A tapping unit according to one embodiment of the present invention applies an impact force to cells by tapping a culture substrate. The tapping unit according to one embodiment of the present invention can apply an impact force to a culture substrate, for example, by colliding a member having an appropriate mass with the culture substrate. The colliding member may be rod-shaped, hammer-shaped, or spherical, but the means for applying an impact force by colliding the member with the culture substrate is not limited to these. Furthermore, the tapping unit according to one embodiment of the present invention can also apply an impact force to the culture substrate by colliding the culture substrate with a flat, arc-shaped, or protruding member, or these methods can be combined. From the perspective of miniaturizing the device configuration, the tapping unit according to one embodiment of the present invention is preferably configured to apply an impact force to the culture substrate by colliding a member having an appropriate mass with the culture substrate.

[0027] The weight and raw material of the colliding member can be selected appropriately depending on the impact force to be applied. In this case, known materials such as inorganic materials such as metals and ceramics, organic materials such as resins and rubber, and composites of these can be used as the raw material. Furthermore, as a power source for moving the colliding member, a method that can generate force by passing electricity, such as a motor or solenoid, can be used. In this case, these power sources can be used directly or via a member that can store energy, such as a spring.

[0028] The tapping unit according to one embodiment of the present invention can also control the impact force and inertial force applied to the culture substrate by arranging a microdeformable member on the opposite side of the tapping direction. The microdeformable member is preferably cylindrical, hemispherical, or a combination thereof, from the viewpoint of minimizing contact points, and is preferably made of an elastic material, i.e., a rubber material.

[0029] The beating unit according to one embodiment of the present invention can strike the culture substrate in a direction that includes a component parallel to the culture surface. To strike the culture substrate in a direction that includes a component parallel to the culture surface, it is preferable to strike the side of the culture substrate. However, since circular containers known as dishes have lids, it is preferable to avoid the lid so that the side of the container body where the culture surface is located can be struck directly. Note that "parallel" in this specification does not only mean parallel in the mathematical sense, but also includes cases where the orientation is slightly deviated from parallel in the mathematical sense, as long as the effects of the present invention can be obtained (the same applies hereinafter).

[0030] (Shaking part) The shaking unit according to one embodiment of the present invention shakes the culture substrate. The shaking described below refers to applying acceleration including a component parallel to the culture surface of the culture substrate to generate a flow in the liquid within the culture substrate. Therefore, the shaking unit can use a configuration that generates such a flow.

[0031] The shaking unit can be configured, for example, to rotate the culture substrate using a motor and then reciprocate the culture substrate along one axis using a slide rail or the like. Alternatively, the shaking unit can be configured to apply acceleration including a component parallel to the culture surface and a component non-parallel to the culture surface by accelerating the culture substrate using a linear motor or the like. That is, the shaking axis of the shaking unit can be appropriately designed so that acceleration including a component parallel to the culture surface and a component non-parallel to the culture surface of the culture substrate can be applied by shaking.

[0032] Here, specific examples of shaking performed on the culture substrate in the shaking unit described above are given. Shaking performed by the shaking unit includes, for example, reciprocating shaking in the horizontal or vertical direction. The reciprocating shaking may include a horizontal or vertical component, and may be performed only in the horizontal or vertical direction, or may be performed diagonally by combining these. Furthermore, the number of shaking axes is not limited to one, and shaking in different shaking directions may be performed on multiple axes, such as two or more axes.

[0033] Shaking also includes, for example, rotational shaking, which is a circular motion in a direction that includes a horizontal component. Rotational shaking only needs to include a horizontal component, and may involve shaking only in the horizontal direction, or may involve shaking diagonally in a manner that also includes the vertical direction. In this case, circular motion includes motion in a perfect circle, an ellipse, a figure-eight shape, or a combination of these.

[0034] Shaking also includes, for example, seesaw shaking. Seesaw shaking refers to a shaking method in which a seesaw-like motion is performed around a single point in the culture substrate as an axis, and examples include horizontal and vertical reciprocating motions, or a combination of these. Shaking also includes, for example, swinging motion in an arc around a fulcrum like a pendulum.

[0035] The shaking unit according to one embodiment of the present invention can be configured to reciprocately shake the culture substrate. Reciprocating shaking refers to a shaking motion that includes a turning motion, and the turbulence generated at the timing of the turning motion has the effect of promoting the detachment of cells that are beginning to detach. The reciprocating shaking described below can include, for example, reciprocating shaking in the horizontal or vertical direction, and rotational shaking in a circular motion, including a turning motion, as well as seesaw shaking and rocking motion. Of these shaking motions, it is more preferable to shake the culture substrate in a direction that includes a component parallel to the culture surface. Shaking the culture substrate in a direction that includes a component parallel to the culture surface can also promote the cell detachment effect.

[0036] Furthermore, the shaking unit preferably shakes the culture substrate in a direction non-parallel to the culture surface. Examples of shaking in a non-parallel direction include seesaw shaking and rocking motion. Shaking the culture substrate in a direction non-parallel to the culture surface can also promote the cell detachment effect. By performing such shaking, shear forces can be applied to the cells from various directions by shaking in a direction non-parallel to the culture surface, thereby promoting the detachment effect.

[0037] (Placement section) A cell detachment device according to one embodiment of the present invention has a mounting section on which a culture substrate, a beating section, and an ultrasonic generator are mounted, and the shaking section can be configured to shake the mounting section. Note that, in the present invention, "mounting" may refer to either a state in which the member is fixed after installation or a state in which the member is simply mounted, and a separate driving mechanism may be provided between the member to be mounted and the mounting section.

[0038] Regarding the placement of the culture substrate, a placement member for the culture substrate can be provided as long as it does not interfere with the transmission of ultrasonic vibrations or the application of impact force by beating. Alternatively, the culture substrate can be placed directly on the ultrasonic wave generating unit or beating unit via a vibration transmitting material. Regarding the method of placing the ultrasonic wave generating unit and the beating unit on the shaking unit, the placing units may be provided so that the ultrasonic wave generating unit and the beating unit each have a place for placing the culture substrate, or the placing units may be provided so that the ultrasonic wave generating unit and the beating unit serve as a place for placing one culture substrate. Furthermore, when there are multiple places for placing the culture substrate, a transfer means such as a robot arm or a transfer arm may be provided in addition to the placing unit so that the culture substrate can be transferred between the places.

[0039] A cell detachment device according to one embodiment of the present invention includes a mounting unit for mounting a culture substrate, a beating unit, and an ultrasonic generator, and the shaking unit can be configured to shake the mounting unit. For example, by mounting the culture substrate, the beating unit, and the ultrasonic generator on the mounting unit and configuring the shaking unit to shake the mounting unit, at least two of the three forces of ultrasonic vibration, beating, and shaking can be simultaneously applied to the culture substrate. As a result, forces over a wide range of time scales can be simultaneously applied, which is expected to enable cell detachment in a shorter time with minimal damage, depending on the cell type.

[0040] (Control unit) Furthermore, a cell detachment device according to one embodiment of the present invention may have a control unit that controls the operation of at least one of the ultrasonic generator, the beating unit, and the shaking unit. Known techniques can be used to control the operation of each unit. The control unit preferably controls each parameter of each component, namely, time, direction, intensity, and period, individually, or in combination with several of these parameters. The control unit may be provided independently of the cell detachment device described below and connected to one or more cell detachment devices via wired or wireless connection to construct a cell detachment system. The control unit may be attached to at least one of the ultrasonic generator, the beating unit, and the shaking unit, and may be provided independently of the cell detachment device or may be included within the cell detachment device.

[0041] (time) The control unit of the cell detachment device preferably controls the driving time of at least one of the beating unit, the shaking unit, and the ultrasonic generator. The total driving time of each unit can be appropriately set depending on the cell characteristics of the cell culture or sheet-shaped cell culture to be detached, the environmental temperature, the type of detachment solution, and the detachment conditions.

[0042] The control unit of the cell detachment device according to one embodiment of the present invention can control the driving of the thumping unit, the shaking unit, and the ultrasonic wave generating unit so that there is a time when all of the units are driven. By allowing time for each unit to be driven, forces of different action scales can be applied simultaneously, thereby improving the effectiveness of cell detachment.

[0043] (direction) Furthermore, it is preferable that the control unit of the cell detachment device controls at least one of the direction of beating, the direction of shaking, and the direction of ultrasonic vibration. Here, the direction of ultrasonic vibration refers to the direction connecting the vibration surface of the vibrator that generates ultrasonic vibration and the vibration transmitter that transmits the ultrasonic vibration. The direction of beating refers to the vector in the direction of the action of beating on the culture substrate. The direction of shaking refers to the vector of the movement imparted to the culture substrate by the shaking action.

[0044] The control unit of the cell detachment device described above can control the direction of ultrasonic vibrations to a direction that includes a component perpendicular to the culture surface of the culture substrate. The tapping direction can also be controlled to a direction that includes a component parallel to the culture surface, and the shaking direction can also be controlled to a direction that includes a component parallel to the culture surface. The control unit can also control the tapping and shaking directions so that the tapping direction and the shaking direction are different from each other. Furthermore, the control unit can also control the shaking direction, the tapping direction, and the ultrasonic vibration direction so that they are different from each other. By varying the direction of action applied by each of the three units, forces can be applied to the cells on the culture surface from different directions, resulting in a more favorable detachment effect. The tapping direction, the shaking direction, and the ultrasonic vibration direction do not need to be constant; for example, each unit can be controlled to change at least one of these over time. Furthermore, in the exemplary configuration shown, the shaking direction and the tapping direction act in different directions (perpendicular directions in the figure) within a plane parallel to the culture surface. However, the plane containing the shaking direction and the beating direction is not limited to a plane parallel to the culture surface.

[0045] (strength) The control unit of a cell detachment device according to one embodiment of the present invention can control at least one of the intensity of ultrasonic vibration, the intensity of beating, and the intensity of shaking. Intensity refers to the amount of energy applied to the culture substrate per unit time, and can be controlled, for example, by controlling the number of applications and the application strength. For example, the number of applications of ultrasonic vibration can be increased by increasing the frequency, and the intensity can be increased by increasing the voltage and the amplitude. Furthermore, the number of applications can be increased by increasing the number of applications per unit time, e.g., per minute, and the intensity can be increased by increasing the mass of the beating unit or the spring constant. Regarding shaking, the number of applications can be increased by increasing the number of applications per unit time, e.g., per minute, or by increasing the shaking speed, and the intensity can be increased by increasing the amplitude.

[0046] The control unit may also control at least one of the ultrasonic vibration intensity, the tapping intensity, and the shaking intensity so as to change over time. Specific examples of changing over time include, but are not limited to, monotonically increasing or decreasing over time, or changing at a constant intensity value over time. Alternatively, both the intensity and direction may be changed simultaneously over time.

[0047] Furthermore, the above-mentioned control unit preferably controls the number of ultrasonic vibrations per minute, the number of tappings per minute, and the number of shakings per minute so that a predetermined relationship is satisfied. Specifically, it is preferable to control the ultrasonic generator, tapping unit, and shaking unit so that the relationship of number of shakings per minute < number of tappings per minute < number of ultrasonic vibrations per minute is satisfied. If this relationship is not satisfied, the detachment effect of the cells or sheet-shaped cell culture may be insufficient, and the detachment time may be prolonged.

[0048] (period) The control unit of the present invention can control the tapping unit, the vibration unit, and the ultrasonic generator so that at least one of the above-mentioned controls regarding time, period, and intensity is periodically performed. The "periodic control" described here refers to a control in which a combination of the control regarding the time, direction, and intensity of the action of each of the exemplified units is repeated at least once. The periodic control also includes, but is not limited to, repeating at least one or more combinations of control of the remaining control targets while subjecting them to non-periodic changes, such as monotonous changes.

[0049] (Maintenance environment for cell detachment device) Furthermore, a cell detachment device according to one embodiment of the present invention can use a known temperature control method to control the environmental temperature around the culture substrate. The environmental temperature is not particularly limited and can be controlled to suit the cell type used. From the perspective of maintaining cell viability, the environmental temperature is preferably 20 to 40°C, and in the case of mammalian cells, for example, a temperature between 30.0°C and 37.5°C is particularly preferable. This ensures that the temperature is close to the temperature during culture, reduces the impact of temperature changes on the cells, and maintains high cell viability. Furthermore, when a known container using a temperature-responsive membrane is used as the culture substrate, the environmental temperature may be set to a temperature range corresponding to the temperature response.

[0050] Furthermore, the cell detachment device described above may have a CO2 concentration adjustment mechanism that maintains a 5% CO2 concentration in the surrounding environment of the culture substrate, similar to general cell culture. These environmental conditions can be appropriately set depending on the type and characteristics of the cells and the method of using the cells after detachment. Furthermore, the cell detachment device may be used in known clean benches or safety cabinets used in cell culture operations, and components that can suppress particle emission may be used in the device. To suppress particles, a known HEPA filter or other suitable filter may be installed in the heat exhaust section within the device. Furthermore, known exterior shapes, textured shapes, and even exterior materials may be used on the interior and exterior surfaces of the device to enhance sterilization and disinfection.

[0051] (Removal method) A cell detachment method according to one aspect of the present invention includes the steps of: (A) an ultrasonic wave generating step of generating ultrasonic vibrations in a culture substrate; (B) a beating step of beating the culture substrate; (C) a shaking step of shaking the culture substrate. These steps can be executed by the above-mentioned control unit, which controls the driving of at least one of the ultrasonic wave generating step, the beating step, and the shaking step, and by controlling these steps with the control unit, the detachment effect of the cells or sheet-shaped cell culture can be improved.

[0052] Specific examples of the control of these processes performed by the control unit include, for example, control of the drive time of at least one of the ultrasonic wave generation process, the tapping process, and the shaking process. This control can also include control of at least one of the direction of ultrasonic vibration, the direction of tapping, and the direction of shaking. This control can also include control so that the number of tappings per minute and the number of shakings per minute satisfy the relationship: number of shakings per minute < number of tappings per minute. Furthermore, this control can also include a step of controlling so that at least one of the above-exemplified controls is performed periodically. These exemplified controls can be performed individually or in combination.

[0053] In the above-described cell detachment method, the cells adhered to the culture surface are preferably a sheet-shaped cell culture. The cell detachment method includes the above-described three steps of ultrasonic generation, beating, and shaking, thereby enabling the cells or sheet-shaped cell culture adhered to the culture surface of the culture substrate to be subjected to effects on a wide time scale, from seconds to microseconds. Therefore, it is possible to detach not only cells adhered to the culture surface, but also sheet-shaped cell cultures formed by cell-cell adhesion without damaging the adhesion between cells.

[0054] Next, a specific example of a cell detachment method according to one embodiment of the present invention will be described with reference to the flowchart in Fig. 3(a). Note that the processes executed in each step of the flowchart described below are executed by the control of each part by the control unit 6. The detachment process using the cell detachment device 1 illustrated in Fig. 1 is started, for example, in response to an instruction input from an operator to the control unit 6. In response, the flow proceeds to step S310, where the culture substrate 9 is placed at a predetermined position on the cell detachment device 1, for example, by the above-mentioned robot arm or the like.

[0055] After the placement of the culture substrate 9 is completed, in step S320, the control unit 6 acquires control parameters previously input by the operator or control parameters set according to the culture substrate 9 and stored in a memory (not shown). The control parameters include the start time of each component, the duration of operation of each component, the application direction, application strength, and cycle. In step S330, the control unit 6 controls each component according to the acquired control parameters to perform the detachment operation. Then, in step S340, the control unit 6 terminates the detachment operation according to the control parameters. After the detachment operation is completed, in step S350, the culture substrate 9 is removed from the placement position, for example, by the robot arm used initially, and the culture substrate 9 is transported to a step in which cells are removed from the culture substrate 9 and recovered.

[0056] Note that the ultrasonic wave generating unit 2, the beating unit 4, and the shaking unit 3 can all be operated simultaneously for the detachment operation. However, various operation methods can be envisioned depending on, for example, the cells or sheet-shaped cell culture to be detached. Examples of envisioned operation methods are described below. FIGS. 3(b) to 3(d) are flowcharts showing specific examples of the operation method. In the example shown in FIG. 3(b), first, in step S3301, only the ultrasonic wave generating unit 2 and the shaking unit 3 are operated according to predetermined parameters. The detachment operation using these operations is performed for a predetermined time (step S3302). Then, in step S3303, the detachment operation is performed using the beating unit 4 and the shaking unit 3. Alternatively, in the example shown in FIG. 3(c), first, in step S3311, only the ultrasonic wave generating unit 2 and the shaking unit 3 are operated, and then, in addition to these operations, the beating unit 4 is operated for a predetermined time, intensity, or cycle (step S3312). In step S3313, the detachment operation started in step S3312 is continued for a predetermined time. In another example shown in FIG. 3(d), in step S3321, only the ultrasonic generator 2 is operated, and operation by the ultrasonic generator 2 alone is continued for a predetermined time (step S3322). Then, while this operation is continuing, the beating unit 4 and the shaking unit 3 are started (step S3323). Note that the process exemplified here can be modified as appropriate depending on the adhesive strength of cells, cell characteristics, etc., as described above. For example, the shaking unit 3 can be configured to shake the culture substrate 9 simultaneously with the vibration by the ultrasonic generator 2 and the beating by the shaking unit 4. Alternatively, the shaking unit 3 can be configured to shake the culture substrate 9 simultaneously with the vibration by the ultrasonic generator 2, or to shake the culture substrate 9 simultaneously with the beating by the shaking unit 4.

[0057] Before proceeding to a detailed description of the embodiments of the cell detachment device according to the present invention, the cells to be detached by the present invention, the related sheet-shaped cell culture, and the culture substrate will be described below. Also, buffer solutions, culture media, serum, antibiotics, and cell detachment solutions that are generally used in actual cell detachment processes and that may be used when carrying out the detachment process in the following embodiments will be described below.

[0058] (cell) In the present invention, the cells to be detached are not particularly limited as long as they can be cultured in vitro as an adherent to a culture substrate. Examples include Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), normal diploid fibroblasts derived from human fetal lung (TIG-3 cells), and human fetal kidney-derived cells (HEK293 cells). Other examples include various cultured cell lines such as A549 cells derived from human alveolar basal epithelial adenocarcinoma, mouse macrophage-like cells (RAW264.7), and HeLa cells derived from human cervical cancer. Further examples include epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, cardiac myocytes, and neuronal cells that constitute the nervous system. Other examples include glial cells, fibroblasts, hepatic parenchymal cells involved in the metabolism of the body, non-parenchymal hepatic cells, and adipocytes. Further examples of cells with differentiation potential include induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, and pancreatic stem cells. Other examples include various stem cells such as skin stem cells, muscle stem cells, and germline stem cells, or progenitor cells of various tissues, as well as cells induced to differentiate therefrom. The present invention may also be applied to sheet-shaped cells. Among these, the cell detachment method of the present invention is suitable for cells with strong intercellular bonds, cells with high adhesive strength to substrates, and cells with high trypsin sensitivity. Furthermore, given the need for large-scale cell culture, the method is particularly suitable for, for example, CHO cells used for protein production and mesenchymal stem cells that can be used in cell therapy.

[0059] (Sheet-shaped cell culture) In the present invention, a sheet-shaped cell culture (cell sheet) refers to a thin membrane in which cells are interconnected to form a sheet. The cells constituting the sheet-shaped cell culture are not particularly limited as long as they can form a sheet-shaped cell culture. Examples include adhesive cells such as adhesive somatic cells. Examples of somatic cells include myoblasts (e.g., skeletal myoblasts), muscle satellite cells, and mesenchymal stem cells (e.g., those derived from bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue, endometrium, placenta, and umbilical cord blood). Further examples include tissue stem cells such as cardiomyocytes, fibroblasts, and cardiac stem cells, pluripotent stem cells such as embryonic stem cells and iPS cells, synovial cells, chondrocytes, and epithelial cells (e.g., oral mucosal epithelial cells, retinal pigment epithelial cells, and nasal mucosal epithelial cells). Other examples include endothelial cells (e.g., vascular endothelial cells), hepatic cells (e.g., hepatic parenchymal cells), pancreatic cells (e.g., pancreatic islet cells), kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, and skin cells. Somatic cells may be differentiated from iPS cells (iPS cell-derived cells). Other examples include iPS cell-derived cardiomyocytes, fibroblasts, myoblasts, epithelial cells, endothelial cells, hepatic cells, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, and chondrocytes. The sheet-shaped cell culture of this embodiment may be obtained by culturing cells into a sheet, or by culturing cells into a sheet and then cutting the edges of the sheet-shaped cells to a desired size.

[0060] (Culture substrate) The culture substrate used in one embodiment of the present invention refers to a culture vessel used for cell culture. The culture substrate is not particularly limited as long as it is a cell-adherent culture substrate. For example, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, multi-well plates, multi-plates, Petri dishes, culture bags, bottles, etc. can be used.

[0061] The material of the culture substrate in this embodiment may be any material that is chemically stable and capable of culturing the desired cells. For example, culture substrates made of polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, and polyglycolic acid can be used. Culture substrates made of polyvinyl alcohol, polyvinyl acetate, or poly(meth)acrylic acid can also be used. Culture substrates made of poly(meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, and polysulfone can also be used. Culture substrates made of cellulose, cellulose derivatives, polysilicone, polymethylpentene, glass, metal, etc. can also be used. Among these, culture substrates made of polystyrene are more preferred.

[0062] In one embodiment of the present invention, the culture substrate may be a stimuli-responsive culture substrate, such as a temperature-responsive culture substrate whose culture surface changes in hydrophilicity in response to an external stimulus such as temperature. To obtain a sheet-shaped cell culture, it is necessary to detach the cells while maintaining adhesion between them, and in such cases, a temperature-responsive culture substrate can be suitably used.

[0063] (buffer) In one embodiment of the present invention, any buffer solution can be used without limitation as long as it can maintain a neutral pH. For example, Tris buffer solutions such as Tris-HCl buffer solutions, phosphate buffer solutions, HEPES buffer solutions, HBSS buffer solutions, citrate-phosphate buffer solutions, glycylglycine-sodium hydroxide buffer solutions, Britton-Robinson buffer solutions, and GTA buffer solutions can be used. Among these, phosphate buffer solutions that closely resemble the in vivo environment are preferred, and phosphate-buffered saline (PBS), which has been adjusted to be isotonic with intracellular fluid, is more preferably used.

[0064] (Culture medium) The type of culture medium used in one embodiment of the present invention is not particularly limited. For example, Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, or McCoy's 5A medium can be used. Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM), or RPMI 1640 medium can also be used. Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemSpan H3000 (Stem Cell Technology), or StemSpan SFEM (Stem Cell Technology) can also be used. Stemline II (Sigma-Aldrich), Endothelial Cell Growth Medium 2 Kit (Promocell), or Mesenchymal Stem Cell Growth Medium 2 (Promocell) can also be used. MSCGM Bullet Kit (Lonza) and mTeSR1 or 2 medium (Stem Cell Technology) can also be used. Repro FF or Repro FF2 (Reprocell), NutriStem medium (Biological Industries), MF-Medium mesenchymal stem cell growth medium (Toyobo Co., Ltd.), etc. can also be used. It is preferable to use the actual medium from these that is suitable for culturing the respective cells.

[0065] (serum) The above-mentioned media may be supplemented with serum or antibiotics. Examples of serum include fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum, with FBS being commonly used due to its ease of availability. Alternatively, serum-free media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) without containing raw or unpurified serum may also be used.

[0066] (antibiotics) Examples of antibiotics that may be added to the above-mentioned medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, and amphotericin B.

[0067] (Cell detachment solution) In the cell detachment method according to the present invention, the cell detachment solution described below can also be used as a solution for detaching cells. In the embodiments of the present invention described below, the pH of the cell detachment solution is preferably in the neutral range. This neutral range is suitable for cell culture and can maintain a stable high cell viability. The pH of the cell detachment solution can be adjusted appropriately with hydrochloric acid, sodium hydroxide, or the like. Various buffer solutions are also suitable for maintaining a stable pH.

[0068] In the embodiments described below, the viscosity of the cell detachment solution is preferably 1.80 mPa·s or less. Maintaining this viscosity prevents the flow of the detachment solution generated by ultrasonic vibrations from being impeded, thereby maintaining high detachment efficiency. The viscosity of the cell detachment solution can be adjusted as needed by adding polymers or sugars, for example.

[0069] The cell detachment solution may also contain protease. However, since protease degrades a portion of cells, while it can improve detachment efficiency, it may also reduce the quality of the cells. For this reason, the amount of protease relative to the total mass of the cell detachment solution is preferably 0.0005% by mass or less, and more preferably, the cell detachment solution does not contain protease. Examples of protease that can be used include those that degrade a portion of cells and make them easier to detach from the substrate. Examples include trypsin, accutase, collagenase, natural protease, chymotrypsin, elastase, papain, pronase, or recombinant forms thereof.

[0070] Furthermore, a solution containing a metal ion chelating agent (hereinafter, sometimes referred to as a chelating agent) can be more preferably used as the cell detachment solution. By using a cell detachment solution containing a chelating agent, cells can be effectively detached by ultrasonic vibration. The chelating agent in the embodiments described below is not particularly limited. For example, ethylenediaminetetraacetic acid (hereinafter, sometimes referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycoletherdiaminetetraacetic acid, nitrilotriacetic acid, or diethylenetriaminepentaacetic acid can be used. In addition, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediaminedisuccinic acid, etidronic acid, citric acid, gluconic acid, phosphonobutanetriacetic acid, etc. can also be used. Among these, chelating agents that form chelates with divalent cations are preferred, and Ca 2+ and Mg 2+Chelating agents that form a chelate with ethylenediaminetetraacetic acid are particularly preferred, with ethylenediaminetetraacetic acid being most preferred. When ethylenediaminetetraacetic acid is used as the chelating agent, the pH of the cell detachment solution is preferably 7.0 or higher and 8.0 or lower. By maintaining the pH of the cell detachment solution at a higher pH within the neutral range that can maintain a high cell viability, the chelating ability of ethylenediaminetetraacetic acid can be enhanced, thereby making it possible to further increase the detachment efficiency. Note that the chelating agent may be used alone or in combination with two or more types. The content of the chelating agent is preferably 0.01 mM or higher and 5.0 mM or lower. This range ensures a reliable chelating effect and suppresses a decrease in activity due to the presence of an excess chelating agent.

[0071] The cell detachment solution of this embodiment may contain a hydrophilic polymer containing a polyalkylene glycol structure. Polyalkylene glycol can increase the cell viability in cell detachment methods using ultrasound. An example of a hydrophilic polymer containing a polyalkylene glycol structure is polyethylene glycol. The hydrophilic polymer preferably has a peak molecular weight Mp measured by gel permeation chromatography of 800 to 50,000, more preferably 1,200 to 20,000. By setting the peak molecular weight in this manner, the polymer has little effect on cells and can suppress the thickening effect of the culture medium caused by the polymer.

[0072] When detaching a sheet-shaped cell culture using the detachment method according to one embodiment of the present invention, it is preferable to use the medium used for culture as is or to replace it with fresh medium before the detachment procedure. This is because, in the case of a sheet-shaped cell culture, the sheet shape is maintained by adhesion between cells, and the use of a medium allows the cells to be detached while maintaining high cell activity, making the sheet-shaped cell culture less susceptible to damage such as tearing or holes. Furthermore, when detaching a sheet-shaped cell culture, the medium can be appropriately diluted with a buffer solution or the like, or the cell detachment solution can be diluted with the buffer solution or the like to an extent that does not weaken the adhesion between cells.

[0073] (Cell culture conditions) The cell culture conditions can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to a dish, and 1.0 × 10 1 ~5.0×10 4 cells / cm 2 The cells are seeded in an amount of about 1000 cells per well and cultured in an environment at 37°C and a CO2 concentration of 5%. At this time, it is preferable to culture the cells until the cell occupancy rate in the substrate is about 70 to 80%, that is, until the substrate becomes subconfluent.

[0074] (Culture conditions for sheet-shaped cell culture) The culturing conditions for sheet-shaped cells can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to a dish, and 1.0 × 10 1 ~1.0×10 6 cells / cm 2 The cells are seeded in an amount of about 100 ml and cultured at 37°C in a CO2 atmosphere. Preferably, the cells are cultured until the cell occupancy rate in the substrate reaches about 100% (i.e., the substrate reaches a confluent state).

[0075] Next, specific embodiments of the cell detachment device according to one aspect of the present invention described above will be described below with reference to the drawings.

[0076] First Embodiment A first embodiment of a cell detachment device according to one aspect of the present invention will be described below with reference to Figures 4 to 7C. Figure 4 is a schematic perspective view of the cell detachment device 100 according to the first embodiment, and Figures 5(a) and 5(b) are diagrams illustrating the drive system of the shaking unit of the cell detachment device 100. Figures 6(a) and 6(b) are diagrams illustrating the placement unit, and Figures 7(a) to 7(c) are diagrams illustrating the structure of the beating unit, etc.

[0077] As shown in FIG. 4 , the cell detachment device 100 according to this embodiment includes an ultrasonic generator 2 that generates ultrasonic vibrations in a culture substrate 9, a beating unit 4 that strikes the culture substrate 9, and a shaking unit 3 that shakes the culture substrate 9. More specifically, the cell detachment device 100 also includes a base plate 8, a mounting unit 5, a holder 27, and rubber feet 26. The shaking unit 3 is mounted on the base plate 8, to which the rubber feet 26 are attached. A mounting unit 5 is provided on the shaking unit 3, and an ultrasonic generator 2 that generates ultrasonic vibrations and a beating unit 4 are mounted on the mounting unit 5. A holder 27 for holding the culture substrate 9 is further mounted on the ultrasonic generator 2, and holds the culture substrate 9 so that it does not move significantly even when subjected to forces such as shaking, beating, and ultrasonic waves. The vibration transmitter 10 described above is provided between the ultrasonic generator 2 and the culture substrate 9.

[0078] By configuring each part in this manner, the culture substrate 9 placed on the ultrasonic generator 2 can receive ultrasonic vibrations while simultaneously being struck by the beating unit 4. Furthermore, by placing the mounting unit 5 on the shaking unit 3, the ultrasonic generator 2 and the beating unit 4 can be shaken simultaneously with the culture substrate 9, and the three forces of ultrasonic vibration, beating, and shaking can be applied to the culture substrate 9 at any desired timing.

[0079] In this embodiment, the ultrasonic generator 2 uses a vibrator with a piezoelectric element fixed to an elastic body or sandwiched between elastic bodies, and the vibrator vibrates when an alternating voltage is applied to the piezoelectric element of the vibrator. As described above, the ultrasonic generator 2 holds a culture substrate 9, such as a cell culture vessel, by a holder 27, and the culture substrate 9 is placed on the holder 27 while being appropriately held so as not to move significantly due to shaking, beating, ultrasonic vibration, or the like. This allows the ultrasonic irradiation and beating operations to be controlled in parallel or independently, even during the shaking operation of the culture substrate 9. The holder 27 is also provided with a weight 39, which can apply weight to the culture substrate 9 to prevent it from floating due to the ultrasonic waves. The base plate 8 is placed on, for example, a table or workbench via rubber feet 26.

[0080] In this embodiment, a control unit 6 including drive circuits for each operation is connected to the cell detachment device 100 via an electric cable 41. This allows each part of the cell detachment device 100 to operate according to operation commands from the control unit 6 and with various parameters set. Furthermore, in this embodiment, the control unit 6 is connected to a graphic controller (touch panel) 7 via the electric cable 41, allowing an operator to easily operate the device via the graphic controller 7. Note that while FIG. 4 illustrates the electric cable 41 as being wired, it may also be connected wirelessly, for example, via radio waves.

[0081] The graphic controller 7 is detachable, and in this embodiment, switches are provided on the panel of the control unit 6 so that the operator can perform the peeling operation with minimal operations on the control unit 6. In this case, examples of the switches include a power switch, a switch for selecting a beating mode, a shaking mode, and an ultrasonic vibration mode, and a button switch for the shot operation. Another example of a component that may be provided other than the switches is a rotary dial, which can also be used to adjust the periodic control of each operation.

[0082] In addition, the control unit 6 may be expanded to include other devices such as a microscope in addition to the graphic controller 7. Furthermore, functions can be enhanced by using a commercially available PC, memory, etc. The control unit 6 in this embodiment is provided with a connection terminal such as a USB for this purpose. Furthermore, the control unit 6 may be provided independently of the cell detachment device 100, as exemplified in FIG. 4, or it may be provided within the housing of the cell detachment device 100. Furthermore, as in this embodiment, the control unit 6 alone may perform ultrasonic band vibration control, beating control, and shaking control, or separate control units may be provided for each control. Furthermore, the control unit may be provided with a computer or the like and run by software, or it may be provided with an operation panel for operation by switching, etc.

[0083] Furthermore, as described above, the control unit 6 can independently control the ultrasonic irradiation, tapping, and shaking operations, and can variably command the intensity, movement amount, direction, time, period (frequency), number of times, speed, etc. In this embodiment, the ultrasound generator 2 applies ultrasonic vibrations to the adhesion site and the sample cells via the vibration transmission element (not shown). The required vibration intensity varies depending on the cell type. Therefore, it is necessary to control the parameters for generating ultrasonic vibrations in the ultrasonic band according to each case. Examples of parameters of the ultrasound generator 2 for generating ultrasonic vibrations include the voltage, frequency, and waveform applied to the piezoelectric element. Furthermore, depending on the type and driving method of the vibrator used in the ultrasound generator 2, heat may be generated during driving. Although this tendency depends on the applied voltage, frequency band used, and device environment, continued driving at the resonant frequency tends to intensify this tendency. In response to this, it is possible to control the amount of heat generated by repeating drive and pause at regular intervals (burst drive), or by repeatedly and continuously changing the drive frequency within a certain frequency range (sweep drive), either alone or in combination. Furthermore, to suppress heat generation, a cooling mechanism can be provided around the ultrasonic generator 2 to control the vibrator directly or indirectly via the ambient temperature. Furthermore, the resonant frequency may change due to heat generated by the vibrator during operation. In such cases, known methods, such as resonant frequency tracking control using current detection, can be used.

[0084] Next, an example of the structure for reciprocating shaking by the shaking unit 3 shown in Fig. 4 will be described with reference to Fig. 5(a) and 5(b). Fig. 5(a) is a schematic diagram in which part of the structure is omitted for the purpose of explaining the shaking unit 3, and Fig. 5(b) is a diagram that schematically shows an outline of the shaking operation.

[0085] In the cell detachment device 100 according to this embodiment, the agitation unit 3 and other components supported by the base plate 8 shown in FIG. 4 are driven in the agitation direction shown in FIG. 4. A guide rail 58 is fastened to the base plate 8 so as to agitate the agitation unit 3 and other components back and forth. A base-side spring post 60 is fixed to the base plate 8, and one end of a pressure spring 59 is connected to the base-side spring post 60. The opposite end of the pressure spring 59 is fixed to a vibration-side spring post 61 provided in the agitation unit 3. The pressure spring 59 can be provided as needed, and the agitation unit 3 may be driven by the vibration slide piece 49 alone.

[0086] An overview of the shaking operation will now be described with reference to Figure 5B. The shaking unit 3 is shaken by converting the rotational movement of the shaking motor 55 into movement of the shaking rod 53 in the extension direction of the guide rail 58, which is then converted into movement of the shaking slide top 49. Specific details of the drive operation will now be described. The rotation of the shaking motor 55 is transmitted to the shaking rod 53 via a gear 56. A rotating disk 44 is fixed to the rotating shaft of the shaking motor 55, and as the shaking motor 55 rotates, the rotating disk 44 rotates in the direction indicated by the arrow in the figure. Note that the rotating direction of the rotating disk 44 may be reversed.

[0087] The rotating disc 44 supports a linear bushing holder 45. The linear bushing holder 45 is rotatably installed at a position offset from the rotation axis of the rotating disc 44, and a linear bushing 46 is incorporated into the linear bushing 46. One end of a vibration rod 53 is further incorporated into the linear bushing 46 so that it can move in a linear manner. The other end of the vibration rod 53 is fixed to a rotatable vibration fulcrum shaft 48. With the above configuration, the rotational movement of the vibration motor 55 is converted into the reciprocating movement of the vibration rod 53 around the vibration fulcrum shaft 48.

[0088] In this embodiment, a connecting rod 54 is supported at one end by a connecting rod fixture 47 fixed to the linear bush holder 45 so as to be arranged in parallel with the shaking rod 53. The other end of the connecting rod 54 is fixed to the shaking fulcrum shaft 48. With the above configuration, the rotational movement of the shaking motor 55 is converted into a reciprocating movement of the connecting rod 54, which is arranged in parallel with the shaking rod 53, in the extension direction of the guide rail 58. A shaking slide top 49 slidably abuts on the connecting rod 54, and the reciprocating movement of the connecting rod 54 is converted into a reciprocating movement of the shaking slide top 49 in the extension direction of the guide rail 58. The shaking movement of the shaking unit 3 can be performed by the reciprocating movement of the shaking slide top 49.

[0089] In this embodiment, the oscillation slide top 49 is supported by an oscillation top slide shaft 51 arranged parallel to the oscillation adjustment screw rod 50. With this configuration, the oscillation stroke of the oscillation unit 3 can be freely adjusted by rotating the oscillation adjustment screw rod 50. In this embodiment, as shown in FIG. 5(a), a stroke knob 63 is provided at the end of the oscillation adjustment screw rod 50. An operator can adjust the oscillation stroke by manually turning this stroke knob 63. In addition, this stroke knob 63 may be electrically rotated by a motor or the like connected to the above-mentioned control unit 6, for example, so that it can be automatically controlled.

[0090] As another example, a stroke indicator 62 (see FIG. 4) may be provided on the vibration slide top 49 so that the vibration stroke can be determined. In this case, a position detector such as an encoder may be provided for the stroke indicator 62, and the position of the vibration slide top 49 may be displayed by the control unit 6 or the graphic controller 7. In addition, in this embodiment, a vibration detector 52 is provided so that the vibration period and an arbitrary origin can be detected. By providing the vibration detector 52, it becomes possible to detect the vibration period and an arbitrary origin, enabling more accurate vibration operation.

[0091] 4 and 5B, the shaking direction is shown as a reciprocating motion in one direction. However, the shaking direction is not limited to the illustrated direction, and may be, for example, multiple directions, a mode in which the direction changes intermittently, or a mode in which multiple axes are combined, and is not limited to shaking along the illustrated single axis. Also, in this embodiment, the ultrasonic wave generating unit 2 and the tapping unit 4 are mounted on top of the shaking unit 3, but their arrangement is not limited to the illustrated mode, and they may, for example, be embedded inside the shaking unit 3. Even in such a case, the same effect can be obtained by configuring the shaking unit 2 and the tapping unit 4 to perform shaking operations in conjunction with the shaking operation of the shaking unit 3.

[0092] As described above, the reciprocating distance of the shaking unit 3 in this embodiment in a horizontal plane can be freely adjusted using the stroke knob 63. In this embodiment, the reciprocating movement of the shaking unit 3 is performed by rotating the rotating disk 44 using the rotational force of the actively moving shaking motor 55, accumulating inertial force. In this embodiment, the rotating disk 44 is provided to offset the linear bushing 46 and obtain a stable rotation period. By offsetting the center of gravity of the linear bushing 46, the shaking operation is stabilized. In addition, the shaking fulcrum shaft 48 and the rotating disk 44 are connected by the shaking rod 53 via the linear bushing 46, so that the shaking fulcrum shaft 48 is configured to swing and rotate in response to the rotation. In this embodiment, by operating the shaking fulcrum shaft 48 so that it does not complete a full rotation, the shaking unit 3 can be made thinner and more compact while still providing space for arranging the detector 52.

[0093] Next, the mounting unit 5 of the cell detachment device 100 according to this embodiment will be described with reference to Figures 6(a) and 6(b). Figure 6(a) is a schematic perspective view of the main parts of the cell detachment device 100, showing the state in which the culture substrate 9 is mounted on the device, and Figure 6(b) is a schematic perspective view of the same configuration with the culture substrate 9 removed.

[0094] In this embodiment, the ultrasonic wave generating unit 2 and the tapping unit 4 are placed on the mounting unit 5, and the culture substrate 9 is placed on the ultrasonic wave generating unit 2. A vibration transmitting material (not shown) with high ultrasonic transmittance, such as water, rubber, or gel, is placed, coated, or dripped on the ultrasonic wave generating unit 2. The culture substrate 9 is placed on the ultrasonic wave generating unit 2 via such a vibration transmitting material. Note that the vibration transmitting material is not limited to a single material or member, but can also be made up of multiple materials, or multiple members can be used in combination. For example, silicone rubber with water dripped thereon can be used as the vibration transmitting material.

[0095] In this embodiment, a holder 27 is disposed on the upper surface of the mounting unit 5 so as to hold the culture substrate 9 at an appropriate position on the ultrasound generating unit 2. The holder 27 may be configured to minimize the inhibition of deformation of the culture substrate 9 by providing, for example, three protrusions to prevent misalignment of the outer diameter of the culture substrate 9. The structure of the holder 27 is not limited to this example. For example, if there are no protrusions, the holder 27 may have a two-sided support such as a V-groove or a circumferential support. Even with such a shape, an effect similar to that achieved by the configuration with the protrusions can be achieved. The holder 27 may be detachable so as to be replaceable depending on the size of the culture substrate 9. The holder 27 in this embodiment may also include a detachable screw 42 for fixing the holder 27 to the mounting unit 5 to position and fasten the holder 27 itself.

[0096] Next, the tapping unit 4 will be described with reference to FIGS. 6(a) and 6(b) again. The beating unit 4 is mounted and fixed on the upper surface of the mounting unit 5, and is appropriately positioned to strike the culture substrate 9. For example, a moderate stroke is required for beating, and the larger the stroke, the stronger the beating force. Various sizes of culture substrates 9 can be used, for example, general culture vessels with diameters of 35 to 100 mm. To be able to uniformly accommodate vessels of these sizes, a stroke of 65 mm or more is required. However, this can also be achieved by, for example, making the position of the beating unit 4 changeable in the stroke direction as needed. In this case, a stroke of approximately 0.1 mm to 10 mm is sufficient.

[0097] In this embodiment, the force (thumping force) of the tapping unit 4 that taps the culture substrate 9 is variable. The required strength of the tapping force differs depending on the cell type, so it must be controlled according to each case. Therefore, in this embodiment, the tapping force is made variable. As shown in FIG. 6A or 6B, the tapping unit 4 is provided with an index line 43 that indicates the tapping force, and the tapping force can be adjusted by changing the position of the spring pressure indicator 22 on the index line 43. In this embodiment, the tapping force can be adjusted, for example, from 0 N to 20 N.

[0098] Next, details of the beating unit 4 in the cell detachment device 100 according to this embodiment will be described with reference to Figures 7(a), 7(b), 7(c), and 7(d). Figure 7(a) is a schematic cross-sectional view of the beating unit 4, Figure 7(b) is a timing chart showing an example of the beating operation of the beating unit 4, and Figures 7(c) and 7(d) are diagrams showing modified examples of the beating unit 4 in the same style as Figure 7(a).

[0099] In the tapping unit 4, the tapping member 12 is configured to tap the culture substrate 9, and tapping pressure is generated by charging a spring 19 with force. As described above, the tapping pressure can be increased or decreased by moving the spring pressure adjustment unit, which is composed of a screw or the like and serves as the spring pressure indicator 22. A rotating shaft 14 to which a motor or the like is connected is provided within the tapping unit 4. By rotating the rotating shaft 14 in the direction of the arrow in the figure, the cam 15 pushes the transmission rod 18 to the right of the page in the figure. This compresses the spring 19 and charges the tapping pressure. When the cliff of the cam 15 passes the abutment position with the transmission rod 18, the spring pressure is released, and the tapping motion by the tapping member 12 is performed.

[0100] 7(b), the light shielding plate 16 provided on the cam 15 blocks the light from the detector 17, which allows the detector 17 to detect and count the tapping cycle and the number of taps, thereby obtaining the cumulative number of taps. The cumulative number of taps may be displayed on the graphic controller 7 (see FIG. 4), for example.

[0101] It is also possible to variably control the period by using the count of the detected output. A modified example of the tapping unit 4 having such a configuration is shown in FIG. 7(c). As shown in FIG. 7(c), the tapping unit 4 is provided with a motor that can variably control the pressure applied to the spring 19 and a control circuit that controls the motor. By appropriately controlling these components, it is possible to automatically adjust the strength of the tapping by the tapping member 12. The tapping unit 4 can also be configured to tap the culture substrate 9 by rotating the tapping member 12. Another embodiment of the tapping unit of the present invention is shown in FIG. 7(d). In the embodiment shown in FIG. 7(d), a cam 15 is rotated by a motor (not shown), which rotates the tapping member 12, which is biased by the spring 19, thereby tapping the culture substrate 9.

[0102] As described above, in the first embodiment, the beating direction of the beating unit 4 and the shaking direction of the shaking unit 3 are different. More specifically, the beating direction and the shaking direction can be made perpendicular so that the forces applied by each unit are effectively applied in different directions. However, the beating action and the shaking action may be superimposed. Figure 8 shows a schematic perspective view of the main parts of a cell detachment device 1a configured in this way. For example, for cell detachment where applying a force in a specific direction to the culture medium is effective, it may be effective to configure the device so that the shaking direction of the shaking unit 3a matches the beating direction.

[0103] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. It also prevents the sheet-shaped cell culture from tearing in a short time and can be expected to be detached cleanly from the culture substrate.

[0104] <Second embodiment> A second embodiment of the present invention will be described below with reference to Figures 9(a) and 9(b). Figure 9(a) is a diagram showing the schematic configuration of a cell detachment device according to this embodiment in the same format as Figure 1, and Figure 9(b) is a top view of the cell detachment device shown in Figure 9(a). In the drawings shown below, components that exhibit the same effects as those described in the first embodiment will be given the same reference numerals, and detailed description thereof will be omitted here.

[0105] The cell detachment device 200 according to this embodiment differs from the first embodiment in that it includes an ultrasonic wave generating unit 202, a shaking unit 203, and a beating unit 204, which will be described in detail below. In FIG. 9(a), the arrows indicate the direction of force applied to the culture substrate 9 by each unit. As shown in the figure, the ultrasonic wave generating unit 202, the shaking unit 203, and the beating unit 204 according to this embodiment are configured to be able to change the force applied to the culture substrate 9 by each unit at various angles. The direction of each force is changed based on a command from the control unit 206.

[0106] For example, the shaking unit 203 in this embodiment is movable in the XY shaking directions shown in the figure via guide rails, linear guides (not shown), etc., provided on the base plate 8. Note that a known XY drive system can be applied to the configuration for driving the shaking unit 203 in the XY directions of a Cartesian coordinate system, and therefore a description thereof will be omitted here. As in the first embodiment, the ultrasonic generator 202 and the tapping unit 204 are mounted on the shaking unit 203. However, unlike the first embodiment, the ultrasonic generator 202 is configured to be able to change the direction of ultrasonic vibration application, as shown in FIG. 9(a). Furthermore, the tapping unit 204 is also configured to be able to change the tapping direction, as shown in FIGS. 9(a) and 9(b). The culture substrate 9 is mounted on the ultrasonic generator 202 via a holder 27 and a vibration transmitter (not shown), and ultrasonic vibrations can be applied via the vibration transmitter in the direction indicated by the arrow in the figure.

[0107] Next, referring to FIG. 9C , an example of a configuration in which the ultrasonic wave generating unit 202 changes the angle of ultrasonic irradiation in the cell detachment device 200 according to this embodiment will be described. In the cell detachment device 200 according to this embodiment, a shaking unit 203 is installed on the base plate 8, and an ultrasonic wave generating unit 202 in the ultrasonic band, which is capable of changing the vibration angle, is mounted on a mounting unit 205 provided on the shaking unit 203. The ultrasonic wave generating unit 202 is supported on the shaking unit 203 via a pan-tilt mechanism 210 provided in the mounting unit 205 so as to be capable of pan-tilt rotation. The ultrasonic wave generating unit 202 generates vibrations by controlling the direction of vibration application in the ultrasonic band so that vibrations are generated in a direction including a component perpendicular to the bottom surface of the culture substrate 9. In this embodiment, supporting the ultrasonic wave generating unit 202 via the pan-tilt mechanism 210 allows the ultrasonic wave generating unit 202 to rotate about two axes.

[0108] By providing such a mechanism, the ultrasonic generator 2 can freely change the angle and position at which ultrasonic vibrations are applied. Furthermore, since the ultrasonic generator 2 moves up and down when applying vibrations, it is possible to press or release the vibration transmitter on the vibrator against any location on the culture substrate. In this embodiment, the vibration application angle is set to 0° in the vertical direction and 90° in the horizontal direction.

[0109] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. It is also possible to prevent tearing of the sheet-shaped cell culture in a short time and to expect that the cell culture will be detached cleanly from the culture substrate. Furthermore, according to this embodiment, it is possible to apply vibration, beating, and shaking actions from directions suitable for cell detachment, enabling more efficient cell detachment.

[0110] <Third embodiment> A third embodiment of the present invention will be described below with reference to Figures 10(a) to 10(c). Figure 10(a) is a diagram showing the general configuration of a cell detachment device according to this embodiment in the same format as Figure 1, and Figures 10(b) and 10(c) are diagrams showing the cell detachment device in the respective states of applying ultrasonic vibrations and beating. In the drawings shown below, components that exhibit the same effects as those described in the first embodiment are given the same reference numerals, and detailed descriptions thereof will be omitted.

[0111] In the cell detachment device 300 according to this embodiment, the ultrasonic wave generating unit 302 and the beating unit 304 are disposed separately on the shaking unit 303 via the mounting unit 305, and the application of ultrasonic vibrations and the execution of beating are performed separately. That is, in this embodiment, simultaneous application of ultrasonic vibrations and shaking, and simultaneous application of beating and shaking can be performed independently.

[0112] Here, an example of a process performed by the cell detachment device 300 when actually detaching cells will be described. During cell detachment, for example, a culture substrate 9 is first placed on the ultrasonic generator 302. Under the control of the controller 306, the ultrasonic generator 302 applies ultrasonic vibrations to the culture substrate 9 and the shaker 303 shakes the culture substrate 9. FIG. 10(b) shows the state after a predetermined processing time has elapsed. The culture substrate 9 is then transported to and placed on the beating unit 304. Then, under the control of the controller 306, the beating unit 304 beats the culture substrate 9 and the shaker 303 shakes the culture substrate 9. FIG. 10(c) shows the state after a further predetermined time has elapsed. The culture substrate 9 is then transported again to the ultrasonic generator 302. These processes may be performed once or repeatedly.

[0113] In this embodiment, when applying force by ultrasonic vibration and tapping, the culture substrate 9 needs to be transported. This transport and movement of the culture substrate 9 does not necessarily have to be performed by human hands, but can be performed using a work robot such as a cylinder or transport robot or a gripper robot. Furthermore, the control at that time can be performed by a work robot provided in association with the control unit 306.

[0114] Next, a modification of the third embodiment will be described with reference to FIGS. 11(a) to 11(c). FIG. 11(a) is a diagram showing the general configuration of a cell detachment device 300a according to this modification in the same format as FIG. 10(a), and FIGS. 11(b) and 11(c) are diagrams showing the cell detachment device in the respective states of applying ultrasonic vibrations and performing beating. In the embodiment illustrated in FIGS. 10(a) to 10(c), the beating direction was set parallel to the culture surface of the culture substrate 9. In contrast, in this modification, the beating direction is set perpendicular to the culture surface. That is, in this modification, only the beating unit 304a differs from the third embodiment.

[0115] In this modification, during cell detachment processing, for example, the culture substrate 9 is first placed on the ultrasonic generator 302, and under the control of the controller 306, the ultrasonic generator 302 applies ultrasonic vibrations to the culture substrate 9 and the shaker 303 shakes the culture substrate 9. FIG. 11(b) shows the state after a predetermined processing time has elapsed, and the culture substrate 9 is then transported to and placed on the beating unit 304a. Then, under the control of the controller 306, the beating unit 304a beats the culture substrate 9 and the shaker 303 shakes the culture substrate 9. FIG. 11(c) shows the state after a further predetermined time has elapsed, and the culture substrate 9 is then transported again to the ultrasonic generator 302. These processes may be performed only once or repeatedly.

[0116] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. It can also prevent tearing of the sheet-shaped cell culture in a short time and can be expected to detach the cell culture from the culture substrate in a clean state. Furthermore, according to this embodiment, the effects of beating and vibration can be separated depending on the adhesion characteristics of the cells, making it possible to detach cells according to their characteristics.

[0117] <Fourth embodiment> A fourth embodiment of the present invention will be described below with reference to Figures 12(a) and 12(b). Figure 12(a) is a diagram showing the schematic configuration of a cell detachment device according to this embodiment in the same format as Figure 1, and Figure 12(b) is a diagram showing a part of the internal structure of the cell detachment device shown in Figure 12(a) in a swinging state. In the drawings shown below, components that exhibit the same effects as those described in the first embodiment will be given the same reference numerals, and detailed description thereof will be omitted.

[0118] The cell detachment device 400 according to this embodiment differs from the cell detachment device 1 according to the first embodiment in that the agitation unit 403 performs a rocking motion. Specifically, in this embodiment, the agitation unit 403 is fastened with screws or the like to a linear guide 57 that slides along a guide rail 58, and rocks along the shape of the guide rail 58. At this time, the heights from the base plate 8 of the first rail fixtures 64 that secure the rails, which are installed at both ends of the guide rail 58, and the second rail fixture 65 installed in the center of the guide rail 58, are different. This allows the guide rail 58 to be fixed in a curved state, and allows the agitation unit 403 to rock at an angle.

[0119] 12(c) shows a schematic diagram of the relationship between the culture substrate 9 and the stripping solution 38 retained therein when the shaking unit 403 is at the end of the rocking motion. As described above, in this embodiment, by performing a rocking motion with an inclination angle, the culture substrate 9 is tilted so that both ends are higher along the shape of the rail, as shown in FIG. 12(c). As a result, even if the level of the stripping solution 38 rises when it hits the wall of the culture substrate 9, the side surface of the culture substrate 9 also becomes higher and tilted, which prevents the stripping solution 38 from spilling out of the culture substrate 9.

[0120] While the description herein concerns a case in which the guide rail 58 is oscillated so that its end is elevated relative to the base plate 8, the oscillating motion may be reversed. Such a modification will be described with reference to FIGS. 13(a) and 13(b). In a modified cell detachment device 400a, as shown in FIG. 13(a), the heights of the first rail fixture 64 and the second rail fixture 65 from the base plate 8 are reversed compared to the example shown in FIG. 12(b). In this case, the culture substrate 9 can be tilted upside down relative to the cell detachment device 400 according to the fourth embodiment. As shown in FIG. 13(b), the culture substrate 9 is tilted downward at both ends, following the rail shape. This results in greater vertical fluctuation of the detachment solution 38 near the wall surface of the culture substrate 9, enabling greater shear force to be applied to the bottom surface of the culture substrate 9. While this enhances the detachment effect, depending on the volume of the detachment solution, the shaking action may cause the detachment solution to easily spill outside the culture substrate.

[0121] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. Furthermore, it is possible to prevent tearing of the sheet-shaped cell culture in a short time and to expect that the cell culture will be detached cleanly from the culture substrate. Furthermore, it is possible to generate turbulence in the detachment solution 38 by creating an up-and-down flow in the detachment solution 38 during shaking, thereby enabling more effective detachment.

[0122] <Fifth embodiment> A fifth embodiment of the present invention will be described below with reference to Fig. 14. Fig. 14 is a diagram showing the schematic configuration of a cell detachment device according to this embodiment in the same format as Fig. 1. In the drawings shown below, components that exhibit the same effects as those described in the first embodiment will be given the same reference numerals, and detailed description thereof will be omitted here.

[0123] The cell detachment device 500 according to this embodiment differs from the cell detachment device 1 according to the first embodiment in that the shaking unit 503 performs a revolving motion. Specifically, the shaking unit 503 according to this embodiment has the same function as the shaking unit 203 described in the second embodiment, and is configured using an automatic stage that moves independently in the X and Y directions. To operate this stage in a revolving motion, the revolving motion can be achieved by changing the vibration phase difference in each of the X and Y directions. For example, by setting the amplitude in the X and Y directions to the same distance and the phase difference to 90°, a perfect circular revolving motion can be achieved. Furthermore, by periodically switching the phase difference between 90° and 270°, revolving motion and back-and-forth motion can also be achieved.

[0124] It is possible to set various operating conditions, including the amplitude and phase difference described above, and even periodic changes in the phase difference, and accordingly, it is possible to apply various forces to the culture substrate.

[0125] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. It also prevents tearing of the sheet-shaped cell culture in a short time and can be expected to be detached cleanly from the culture substrate. Furthermore, these can be appropriately set depending on the cell type and detachment method.

[0126] Sixth Embodiment A sixth embodiment of the present invention will be described below with reference to Fig. 15. Fig. 15 is a diagram showing the schematic configuration of a cell detachment device according to this embodiment in the same format as Fig. 1. In the drawings shown below, components that exhibit the same effects as those described in the first embodiment will be given the same reference numerals, and detailed description thereof will be omitted here.

[0127] The cell detachment device 600 according to this embodiment differs from the cell detachment device 1 according to the first embodiment in that the agitation unit 603 performs a rotational switching motion. Specifically, the agitation unit 603 according to this embodiment is configured to have a drive system including a motor or the like, which performs the rotational motion. By controlling the drive system including the motor, the agitation unit 603 can perform shaking with a rotational switching motion, such as rotation in only one direction or periodic reverse rotation, in addition to the rotational speed.

[0128] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. Furthermore, it is possible to prevent tearing of the sheet-shaped cell culture in a short time and to expect that it will be detached cleanly from the culture substrate. Furthermore, for example, by including reverse rotation during shaking, it is possible to generate turbulence in the detachment solution, resulting in more effective detachment.

[0129] Seventh Embodiment A seventh embodiment of the present invention will be described below with reference to Fig. 16. Fig. 16 is a diagram showing the schematic configuration of a cell detachment device according to this embodiment in the same format as Fig. 12(a) or Fig. 13(b). In the drawings shown below, components that exhibit the same effects as those described in the first embodiment will be given the same reference numerals, and detailed description thereof will be omitted here.

[0130] The cell detachment device 700 according to this embodiment differs from the first embodiment in the characteristics of the rubber feet 726 that are disposed below the base plate 8 and support the cell detachment device 700 on a table or the like. In this embodiment, a soft viscoelastic material is used for the rubber feet 726, which actively expand and contract during shaking, making the shaking direction of the shaking unit 3 non-parallel. As shown in the figure, when shaking is reversed, a moment due to the force generated during reversal is generated in the shaking unit 3. This moment causes the rubber feet 726, made of a viscoelastic material and located at a position corresponding to the end being reversed, to contract, while the rubber feet 726 at the opposite end expand, tilting the shaking unit 3. This makes it possible to apply additional force acting in the vertical direction to, for example, the detachment solution during shaking.

[0131] As an effect of using a soft viscoelastic material for the rubber feet 726, for example, the viscoelastic material generates a moment when the device is inverted, which has the added effect of absorbing the force acting on the base plate 8 when the device is inverted and preventing the cell detachment device 700 from shifting sideways.

[0132] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. Furthermore, it is possible to prevent tearing of the sheet-shaped cell culture in a short time and to expect that it will be detached cleanly from the culture substrate. Furthermore, it is possible to generate turbulence in the detachment solution by creating an up-and-down flow in the detachment solution during shaking, thereby enabling more effective detachment.

[0133] Eighth Embodiment An eighth embodiment of the present invention will be described below with reference to Fig. 17. Fig. 17 is a diagram showing the schematic configuration of a cell detachment device according to this embodiment in the same format as Fig. 1. In the drawings shown below, components that exhibit the same effects as those described in the first embodiment will be given the same reference numerals, and detailed description thereof will be omitted.

[0134] The cell detachment device 800 according to this embodiment differs from the other embodiments described above in that it does not include the mounting unit 5 described in the first embodiment and the like. In the cell detachment device 800, an ultrasonic wave generating unit 2 in the ultrasonic range is installed on a shaking unit 803 installed on a base plate 8. Meanwhile, the beating unit 4 is installed on a holding member 66 installed on the base plate 8, independent of the shaking unit 3. In this embodiment, ultrasonic vibration and shaking can be applied simultaneously, but beating is applied separately. When applying ultrasonic vibration and shaking and beating force, the culture substrate 9 must be transported. As in the third embodiment, the culture substrate 9 can be transported not by hand, but by a work robot such as a cylinder, a transport robot, or a gripper robot. Furthermore, as a variation of this embodiment, the beating unit 4 may be installed on the shaking unit, and only the ultrasonic wave generating unit 2 may be used separately.

[0135] The cell detachment device according to the present embodiment described above can increase both the cell survival rate and detachment rate. It can also prevent tearing of the sheet-shaped cell culture in a short time and can be expected to detach the cell culture from the culture substrate in a clean state. Furthermore, according to this embodiment, the effects of beating and vibration can be separated depending on the adhesion characteristics of the cells, making it possible to detach cells according to their characteristics.

[0136] <Example> The following describes in detail the application of the present invention to cell detachment treatment, with reference to examples and corresponding comparative examples. The examples and comparative examples described below are merely examples for verifying the effects of the present invention and are not intended to limit the present invention. Furthermore, for evaluation, the driving conditions described below were continued until the sheet-shaped cell culture was detached, thereby obtaining a sheet-shaped cell culture. The detachment time was calculated as a ratio to the detachment time of a comparative example detached at the same time, and was evaluated as follows: A: 50% or more reduction; B: 25% to less than 50% reduction; C: 10% to less than 25% reduction; and D: the same or more reduction but less than 10% reduction. Furthermore, sheet quality was evaluated as follows: A: small holes less than 10 μm; B: slight tears; C: major tears; and D: tears resulting in no sheet shape.

[0137] (Cell culture) The cells used in the examples described below were cultured under the following conditions.

[0138] (Culture of A549 cells on a substrate) A549 cell (human alveolar basal epithelial adenocarcinoma) cell culture and sheet-shaped cell culture were obtained as follows. A549 cells were cultured in a φ35 polystyrene dish (Corning) at 15,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium used was DMEM (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 80%.

[0139] The sheet-shaped cell culture was grown in a φ35 temperature-responsive dish (Upcell (registered trademark), manufactured by CellSeed) at 65,000 cells / cm. 2After seeding at a density of 100, the cells were cultured for 6 days, with the medium being changed every 48 hours. The cells were observed under a phase-contrast microscope and the formation of a cell sheet was confirmed. The cell occupancy rate of the dish was approximately 100%.

[0140] (CHO cell culture on a substrate) A cell culture of CHO cells (Chinese hamster ovary cells) was obtained as follows: CHO-K1 cells were cultured in a φ35 polystyrene dish (Corning) at 15,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium used was Ham's F12 (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell coverage of the dish was approximately 80%.

[0141] (C2C12 cell culture on a substrate) A sheet-shaped cell culture of C2C12 cells (mouse striated muscle cells) was obtained as follows: C2C12 cells were cultured in a φ35 temperature-responsive dish (Upcell®, CellSeed) at a density of 65,000 cells / cm. 2 The cells were seeded at a density of 100 μg / ml and cultured at 37°C in a 5% CO2 environment. The culture medium used was DMEM / F12 (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell occupation area of ​​the dish was approximately 100%.

[0142] (Culture of MDCK cells on a substrate) A sheet-shaped cell culture of MDCK cells (canine renal tubular epithelial cells) was obtained as follows: MDCK cells were cultured in a φ35 temperature-responsive dish (Upcell®). 、 CellSeed) at 65,000 cells / cm 2 The cells were seeded at a density of 100 μg / ml and cultured at 37°C in a 5% CO2 environment. The medium used was Eagle's MEM medium (Fujifilm, Wako Pure Chemical Industries, Ltd.) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The medium was changed every 48 hours for 8 days, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage of the dish was approximately 100%.

[0143] (Culture of HEK293 cells on a substrate) A sheet-shaped cell culture of HEK293 cells (human embryonic kidney cells) was obtained as follows: HEK293 cells were cultured in a φ35 temperature-responsive dish (Upcell®). 、 CellSeed) at 65,000 cells / cm 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The medium used was Eagle's MEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The medium was changed every 48 hours for 9 days, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell occupation rate on the dish was approximately 100%.

[0144] (Culture of BAEC cells on a substrate) A sheet-shaped cell culture of BAEC cells (bovine aortic endothelial cells) was obtained as follows: BAEC cells were cultured in a φ35 polystyrene dish (manufactured by Corning) at a density of 20,000 cells / cm. 2The cells were seeded at a density of 100 μg / ml and cultured at 37°C in a 5% CO2 environment. The culture medium used was DMEM (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The culture medium was changed every 48 hours for 7 days, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage area of ​​the dish was approximately 100%.

[0145] (HMSC cell culture on a substrate) The cell culture and sheet-shaped cell culture of human mesenchymal stem cells (hMSCs) were obtained as follows. For the cell culture, hMSCs were grown in a φ35 polystyrene dish (Corning) at 4000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium used was Mesenchymal Stem Cell Growth Medium 2 (Promocell) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The culture medium was changed every 48 hours for 7 days, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell coverage of the dish was approximately 80%.

[0146] The sheet-shaped cell culture was grown in a φ35 temperature-responsive dish (Upcell (registered trademark), manufactured by CellSeed) at 30,000 cells / cm. 2 The cells were seeded at a density of 100x100x100mm. After that, the culture medium was changed every 48 hours for 8 days, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell occupancy rate of the dish was about 100%.

[0147] Example 1 The A549 cell sheet-shaped cell culture obtained under the above conditions was subjected to a detachment study using the following method. Four hours before the detachment study, the culture medium in the dish in which the sheet-shaped cell culture had been cultured was replaced with fresh medium, and the culture was then performed in a 37°C incubator. The dish was then removed from the 37°C incubator, and the dish was placed in the cell detachment device 100 according to the first embodiment illustrated in FIG. 4 and operated under detachment condition 1 shown in Table 1.

[0148] The ultrasonic wave generating unit 2 for the ultrasonic wave band in the cell detachment device 100 shown in FIG. 4 can be used by replacing the transducer. In this example, a Langevin transducer was used, with a resonant frequency of 34.5 kHz and an applied voltage of approximately 20 V. The voltage was adjusted so that the amplitude of the vibration on the bottom surface of the culture substrate 9 placed on the transducer was 5.0 μm. The driving frequency was 32 to 37 kHz, and the sweep time was 1 second. Silicone rubber with a thickness of 1.0 mm and a hardness of 10 was used as the vibration transmitter, and was attached to the bottom surface of the culture substrate 9.

[0149] The hammering unit 4 used consisted of the mechanism shown in Figure 7(a), and the hammering force was adjusted by adjusting the spring constant and length of the spring 19 used, resulting in a spring force range of 0.2 to 4.0 N. The drive frequency was in the range of 0.1 to 10 Hz, adjustable in 0.1 Hz increments. The shaking unit 3 used consisted of the mechanism shown in Figure 5A, with an amplitude of 1 to 50 mm and a drive frequency in the range of 0.1 to 10 Hz, adjustable in 0.1 Hz increments.

[0150] In this example, the ultrasonic generator 2, beating unit 4, and shaking unit 3 were driven in the operating directions shown in Figure 4. The control conditions were as follows: the ultrasonic generator 2 was driven from the start of detachment, the beating unit 4 was driven for 30 seconds, followed by a 30-second pause, and this cycle was repeated; and the shaking unit 3 was driven continuously from the start of detachment. In other words, vibration and shaking were continuously applied, and beating was added during this period. Operation according to these drive conditions was continued until the sheet-shaped cell culture was detached, resulting in a detached sheet-shaped cell culture. In this example, detachment of the sheet-shaped cell culture refers to a state in which the entire sheet-shaped cell culture is lifted from the culture surface of the culture vessel.

[0151] The detachment time was calculated as a ratio to the detachment time of the cell detachment device of Comparative Example 1, which was performed during the same period. Evaluation was performed as follows: AA: 30% or more reduction; A: 20% or more but less than 30% reduction; B: 10% or more but less than 20% reduction; C: 1% or more but less than 10% reduction; and D: less than 1% reduction, indicating a time equivalent to or longer than that of the Comparative Example. The sheet quality was evaluated as follows: A: no holes or tears, or at least one of small holes and tears less than 500 μm occurred; B: at least one of holes and tears greater than 500 μm but less than 1 mm occurred; C: at least one of holes and tears greater than 1 mm but less than 2 mm occurred; and D: at least one of holes and tears greater than 2 mm occurred. The sheet-shaped cell culture obtained in this example was evaluated, and the detachment time was A and the sheet quality was A. The evaluation results are shown in Table 2. A grade of C or higher was considered good for both detachment time and sheet quality.

[0152] (Examples 2 to 4, 10, 16 to 20) In these examples, the combination of cell type and detachment method was changed as shown in Table 2, and a dish in which a sheet-shaped cell culture had been cultured was set in the cell detachment device 100 shown in Figure 4. Then, control conditions were set according to each of the detachment conditions shown in Table 1, and detachment of the sheet-shaped cell culture was investigated in the same manner as in Example 1. In this example, the forces acting on the culture substrate 9 from the ultrasonic wave generating unit 2 in the ultrasonic band, the beating unit 4, and the shaking unit 3 each act in the direction shown in Figure 4. The peeling results are shown in Table 2.

[0153] Example 5 In this example, the cell detachment test for a sheet-shaped cell culture was conducted in the same manner as in Example 1, except that the combination of cell type and detachment method was changed as shown in Table 2 and the cell detachment device 200 according to the second embodiment illustrated in FIG. 9(a) was used. The detachment conditions were as shown in Table 1. Regarding the actual operating conditions of the cell detachment device 200, the vibration angle was set to 20° at the start of detachment, and vibration was applied by adjusting the position so that the strongest vibration was transmitted to the peripheral bottom surface of the culture substrate 9. Subsequently, as detachment progressed, the vibration angle was gradually reduced to match the position of the edge of the detached sheet-shaped cell culture, while the center of the vibration was moved closer to the center of the culture substrate 9. Then, when 30% of the radial area of ​​the culture substrate 9 had been detached, the vibration angle was set to 0° (vertical), and vibration application was continued with the center of the vibrator aligned with the center of the culture substrate. That is, the operating direction of the ultrasonic wave generating unit 2 in this example was controlled to change from 20° to 0°. The operating directions of the beating unit 4 and the shaking unit 3 were the same as those illustrated in FIG. 4. The peeling results are shown in Table 2.

[0154] Example 6 In this example, similar to Example 5, the combination of cell type and detachment method was changed as shown in Table 2, and the cell detachment device 200 according to the second embodiment illustrated in FIG. 9(a) was used, with the vibration angle of the ultrasonic generator 2 set to 20°. In this example, the detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1, except that the vibration position was aligned with the center of the culture substrate. The detachment conditions were as shown in Table 1. In this example, the direction of action of the ultrasonic generator 2 was set to 20°, and the directions of action of the beating unit 4 and shaking unit 3 were the same as those illustrated in FIG. 4. The detachment results are shown in Table 2.

[0155] Example 7 In this example, the combination of cell type and detachment method was changed as shown in Table 2, and detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1, except that a cell detachment device 1a according to a modified example of the first embodiment shown in Figure 8 was used. The detachment conditions were as shown in Table 1. The shaking unit 3a of the cell detachment device 1a according to this example is a two-axis moving stage, and the driving direction can be freely changed. Here, the operating direction was set so that shaking occurs in the same direction as the beating direction. That is, in this example, the operating direction of the shaking unit 3a was controlled to be the same as the operating direction of the beating unit 4, and the operating directions of the ultrasonic generator 2 and beating unit 4 were the same as the operating directions shown in Figure 4. The detachment results are shown in Table 2.

[0156] Example 8 In this example, the detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1, except that the combination of cell type and detachment method was changed as shown in Table 2 and the cell detachment device 300 according to the third embodiment illustrated in FIG. 10(a) was used. The detachment conditions were as shown in Table 1. The placement unit 305 of the cell detachment device 300 according to this example is divided into an ultrasonic generator 302 and a beating unit 304, and the culture substrate 9 is transported as shown in FIGS. 10(b) and 10(c) to apply force from each unit. The time required for transporting the culture substrate 9 between the ultrasonic generator 2 and the beating unit 4 and setting the culture substrate 9 was 10 seconds. The directions of force application by the ultrasonic generator 302, beating unit 304, and shaking unit 303 in this example were the same as those shown in FIG. 10A, i.e., FIG. 4. The detachment results are shown in Table 2.

[0157] Example 9 In this example, the cell detachment test for a sheet-shaped cell culture was performed in the same manner as in Example 1, except that the combination of cell type and detachment method was changed as shown in Table 2 and the cell detachment device 400 according to the fourth embodiment illustrated in Figures 12(a) and 12(b) was used. The detachment conditions were as shown in Table 1. In the cell detachment device 400 according to this example, the ultrasonic generator 2 and the beating unit 4 had the same mechanisms as those in the first embodiment illustrated in Figure 4. The shaking unit 403 used a curved rail (58) as shown in Figure 12B as the rail for the axial movement stage. During shaking, the inclination angle was 5° at an amplitude of 25 mm. The direction of force exerted by the ultrasonic generator 2, beating unit 4, and shaking unit 403 in this example was the same as that shown in Figure 12B, but the shaking direction was different from that shown in Figure 4 and included a component non-parallel to the culture surface. The detachment results are shown in Table 2.

[0158] Example 11 In this example, the cell detachment test for a sheet-shaped cell culture was conducted in the same manner as in Example 1, except that the combination of cell type and detachment method was changed as shown in Table 2 and the cell detachment device 700 according to the seventh embodiment, illustrated in Figure 16, was used. The detachment conditions were as shown in Table 1. The cell detachment device 700 according to this example has a configuration similar to that of the cell detachment device 100 according to the first embodiment (Figure 4), except that the rubber feet 726 are made of butyl rubber with a rubber hardness of 30, making them more easily deformable. When changing to rubber feet 726, the mounting unit 5 was adjusted so that the sinking amount of the rubber feet 726 was 1 mm for a vibration amplitude of 10 mm of the vibration unit 3. The direction of force exerted by the ultrasonic generator 2, tapping unit 4, and vibration unit 3 in this example was the same as that shown in Figure 16, but the vibration direction was different from that shown in Figure 4 and included a component non-parallel to the culture surface. The detachment results are shown in Table 2.

[0159] Example 12 In this example, the detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1, except that the combination of cell type and detachment method was changed as shown in Table 2, and a cell detachment device 300a according to a modified example of the third embodiment shown in FIG. 11(a) was used. The detachment conditions were as shown in Table 1. The mounting unit 305 of the cell detachment device 300a according to this example is configured separately as an ultrasonic generator 2 and a beating unit 304a. The beating direction of the beating unit 304a is perpendicular to the bottom surface of the culture substrate 9, which is the same as the direction of application of ultrasonic vibrations. As shown in FIGS. 11B and 11C, in this example, the culture substrate 9 is moved to apply force. The time required for transferring the culture substrate 9 between the ultrasonic generator 2 and the beating unit 304a and setting the culture substrate 9 was 10 seconds. The directions of force exerted by the ultrasonic generator 2, tapping unit 304a, and shaking unit 3 in this example are the same as those shown in Fig. 11A, with the tapping unit 304a acting in the same direction as the direction of vibration in the ultrasonic band, and the other directions being the same as those shown in Fig. 4. Peeling results are shown in Table 2.

[0160] Example 13 In this example, the cell detachment test for a sheet-shaped cell culture was conducted in the same manner as in Example 1, except that the combination of cell type and detachment method was changed as shown in Table 2, and the cell detachment device 800 according to the eighth embodiment shown in FIG. 17 was used. The detachment conditions were as shown in Table 1. The cell detachment device 800 according to this example does not have the mounting unit 5 of the first embodiment, but has a shaking unit 803 installed on the base plate 8, and an ultrasonic generator 2 installed on the shaking unit 803. Meanwhile, the beating unit 4 is installed on a holding member 66 attached to the base plate 8. In this example, ultrasonic vibration and shaking can be applied simultaneously, and beating is applied separately, so the time required for transferring the culture substrate 9 and setting the culture substrate 9 during this period was 10 seconds. The force acting directions of the ultrasonic generator 2, beating unit 4, and shaking unit 803 in this example are the same as those shown in FIG. 17, i.e., the same as those shown in FIG. 4. The detachment results are shown in Table 2.

[0161] Example 14 In this example, detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1, except that the vibration transmitting substance was changed to water. The directions of force application by the ultrasonic generator 2, beating unit 4, and shaking unit 3 in this example were the same as those shown in Figure 4. The detachment results are shown in Table 2.

[0162] Example 15 In this example, the combination of cell type and detachment method was changed as shown in Table 2, and a dish on which a sheet-shaped cell culture had been cultured was set in the cell detachment device 100 according to the first embodiment illustrated in Figure 4. The operating conditions of the cell detachment device 100 were then set to each of the detachment conditions shown in Table 1, and detachment of the sheet-shaped cell culture was investigated in the same manner as in Example 1. A ring device in which a ring-shaped piezoelectric element was adhered to a glass plate was used as the ultrasonic wave generating unit 2 in this example, and it was driven in a traveling wave mode. The directions of force acting by the ultrasonic wave generating unit 2 in the ultrasonic band, the tapping unit 4, and the shaking unit 3 in this example were the same as those shown in Figure 4. The detachment results are shown in Table 2.

[0163] Example 21 In this example, the cell culture of A549 cells obtained under the above conditions was examined for detachment using the following method. The culture medium in the dish in which the cells were cultured was replaced with PBS(-), a detachment solution, and the cells were incubated at 37°C for 2 minutes. Next, the dish was placed in the cell detachment device 100 according to the first embodiment illustrated in Figure 4, and the device was operated as follows using detachment condition 12 shown in Table 1.

[0164] First, the ultrasonic wave generator 2 in the ultrasonic band was driven continuously for 6 minutes from the start of detachment. Next, 4 minutes after the start of detachment, the beating unit 4 and the shaking unit 3 were driven and continued for 2 minutes. Then, 6 minutes after the start of detachment, all drives were stopped and the cells were detached. The detached cells were collected and counted using a hemocytometer, and the viability was calculated using a viability test using trypan blue staining. In addition, all cells that were not detached by ultrasound were also detached from the dish after ultrasonic detachment using a cell scraper, and the cell count was measured using a hemocytometer. The total number of detached cells was calculated by adding the number of cells detached by ultrasound and the number of cells subsequently detached with the cell scraper. The detachment rate was defined as the ratio of the number of ultrasonically detached cells to the total number of detached cells. The viability was 97.5%, and the detachment rate was 98.9%. The evaluation results are shown in Table 2. A viability and detachment rate of 90% or higher was considered to be a good detachment. Furthermore, the collected cells were subcultured to confirm that there were no problems with adhesion or proliferation.

[0165] (Examples 22 and 23) In these examples, the combination of cell type and detachment method was changed as shown in Table 2, and the cell culture was detached under the same conditions as in Example 21 above. After detachment, evaluation was performed using the same method as in Example 21. Since both the survival rate and detachment rate were 90% or higher, it was determined that the detachment was performed successfully. The results are shown in Table 2.

[0166] (Examples 24 and 25) In these examples, the combination of cell type and detachment method was changed as shown in Table 1, and a dish on which a sheet-shaped cell culture had been cultured was set in the cell detachment device 100 illustrated in Figure 4. Control conditions were then set according to each of the detachment conditions shown in Table 1, and detachment of the sheet-shaped cell culture was investigated in the same manner as in Example 1. A ring device in which a ring-shaped piezoelectric element was adhered to a glass plate was used as the ultrasonic wave generating unit 2 in this example, and it was driven in a traveling wave mode. The forces acting on the culture substrate 9 from the ultrasonic wave generating unit 2 in the ultrasonic band, the tapping unit 4, and the shaking unit 3 in this example each act in the direction shown in Figure 4. The results are shown in Table 2.

[0167] Example 26 In this example, a detachment study was performed on a cell culture of hMSC cells obtained under the above conditions using the following method. First, the culture medium in the cell culture dish was replaced with PBS(-), a detachment solution. Next, the dish was placed in the cell detachment device 100 according to the first embodiment illustrated in Figure 4, and a detachment study was performed. In this example, a ring device consisting of a ring-shaped piezoelectric element attached to a glass plate was used as the ultrasonic generator 2, and it was driven in traveling wave mode. In this example, the control conditions were set according to the detachment conditions 29 listed in Table 1, and the devices were driven as follows: The ultrasonic generator 2 in the ultrasonic band was driven for 90 seconds from the start of detachment. The beating unit 4 was driven for 90 seconds starting 90 seconds after the start of detachment. The shaking unit 3 was driven from the start of detachment and continued for 3 minutes. In this example, all drives were stopped 3 minutes after the start of detachment, and the cells were detached. After detachment, evaluation was performed using the same method as in Example 21. Since both the viability and detachment rate were 90% or higher, it was determined that detachment was performed successfully. The results are shown in Table 2.

[0168] (Comparative Examples 1 to 6) In this comparative example, the combination of cell type and detachment method was changed as shown in Table 2, and detachment of sheet-shaped cell cultures was investigated using the cell detachment device 1000 shown in Figure 18. A549 cells, hMSC cells, HEK293 cells, MDCK cells, and C2C12 cells cultured in a φ35 temperature-responsive dish (Upcell (registered trademark, manufactured by CellSeed)) were used as the sheet-shaped cell cultures. BAEC cells cultured in a φ35 polystyrene dish (manufactured by Corning) were also used. The cell detachment device 900 was modified from the cell detachment device 100 shown in Figure 4 according to the first embodiment, except that the ultrasonic generator 2 was not driven, and forces were applied by the beating unit 4 and the shaking unit 3. All other detachment conditions were the same as in Example 1. The corresponding detachment conditions are shown in Table 2. In this comparative example, the ultrasonic generator 2 did not apply force in any direction, and the forces from the beating unit 4 and the shaking unit 3 were applied in the directions shown in Figure 18.

[0169] The peeling time and sheet quality in this comparative example were evaluated as follows. The peeling times were 34 minutes for comparative example 1, 29 minutes for comparative example 2, 27 minutes for comparative example 3, 70 minutes for comparative example 4, 7 minutes for comparative example 5, and 85 minutes for comparative example 6. For all cell types in comparative examples 1 to 6, multiple holes or tears of 2 mm or more were confirmed, and the sheet quality was therefore rated D. The peeling time obtained in this comparative example was used as the standard to evaluate the peeling time of each example and other comparative examples.

[0170] (Comparative Example 7) In this comparative example, the combination of cell type and detachment method was changed as shown in Table 2, and detachment of a sheet-shaped cell culture was examined using the cell detachment device 1000 shown in FIG. 19. Note that the cell detachment device 1000 is the same as the cell detachment device 300 of the third embodiment shown in FIG. 10A, except that the shaking unit 303 is not driven and instead forces are applied by the ultrasonic generator 2 and the beating unit 4. All other detachment conditions are the same as in Example 8. The corresponding cell types and detachment conditions are shown in Table 2. In this comparative example, the force from the shaking unit 303 is not applied in any direction, and the forces from the ultrasonic generator 2 and the beating unit 4 are applied in the directions shown in FIG. 19. The detachment time and sheet quality in this comparative example 7 were evaluated as follows. The detachment time was 38 minutes and the sheet was evaluated as D. The sheet quality was evaluated as B. The detachment results are shown in Table 2.

[0171] (Comparative Example 8) In this comparative example, the combination of cell type and detachment method was changed as shown in Table 2, and detachment of a sheet-shaped cell culture was examined using the cell detachment device 1100 shown in Figure 20. The cell detachment device 1100 was the same as in Example 1, except that the configuration of the cell detachment device 100 of the first embodiment shown in Figure 4 was changed so that the beating unit 4 was not driven and instead forces were applied by the ultrasonic generator 2 and the shaking unit 3. The corresponding cell types and detachment conditions are shown in Table 2. In this comparative example, the force from the beating unit 4 was not applied in any direction, and the forces from the ultrasonic generator 2 and the shaking unit 3 were applied in the directions shown in Figure 20. The detachment time and sheet quality in this comparative example 8 were evaluated as follows. As no detachment occurred after 60 minutes of detachment, no evaluation results were given. Similarly, as no detachment occurred in the sheet quality evaluation, no evaluation results were given. The detachment results are shown in Table 2.

[0172] Comparative Example 9 In this comparative example, the combination of cell type and detachment method was changed as shown in Table 2, and detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1, except that the cell detachment device 1200 shown in FIG. 21 was used. In this comparative example, the shaking unit 3 of the cell detachment device 100 shown in FIG. 4 according to the first embodiment was replaced with a discharge mechanism. As shown in FIG. 21, the cell detachment device 1200 used in this comparative example has the same mechanisms as the cell detachment device 100 of FIG. 4, with the ultrasonic generator 2 and beating unit 4 mounted on the base plate 8. In the cell detachment device 1200, an XYZ stage 29 was mounted on the base plate 8, and an electric pipetter was mounted on the Z-axis plate of the stage 29 as the discharge mechanism 30.

[0173] The discharge mechanism 30 controls the discharge position on the culture surface of the culture substrate 9 on an XY stage, and the height of the discharge mechanism is controlled using a Z stage so as not to contact the liquid surface in the culture substrate 9. The discharge operation was performed by repeating aspiration and dispensing (pipetting) at a suction speed of 1 mL / s and a discharge speed of 2 mL / s with a pipette volume set to 1 mL. The discharge operation was controlled so as to be repeated while moving the position relative to the boundary of the peeled area of ​​the sheet. The corresponding cell types and peeling conditions are shown in Table 2. The force acting direction in this comparative example is shown in Figure 21. That is, the force acting direction from the ultrasonic generator 2 and the beating unit 4 was the same as the acting direction shown in Figure 4. The force acting direction from the discharge mechanism 30 was approximately perpendicular to the bottom surface of the culture substrate 9. The peeling time and sheet quality in Comparative Example 9 were evaluated as follows: The peeling time was 40 minutes, and the sheet was evaluated as D. The sheet quality was evaluated as C. The peeling results are shown in Table 2.

[0174] (Comparative Example 10) In this comparative example, the combination of cell type and detachment method was changed as shown in Table 2, and detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1, except that the cell detachment device 1300 shown in FIG. 22 was used. In this comparative example, the beating unit 4 of the cell detachment device 100 shown in FIG. 4 according to the first embodiment was replaced with a scraping mechanism 31. The ultrasonic generator 2 and shaking unit 3 are provided on the base plate 8 and use the same mechanisms as those of the cell detachment device 1 in FIG. 4. In the cell detachment device 1300, an XYZ stage 29 is installed on the base plate 8, and a sterilized polystyrene scraper is attached to the mounting part 32 as the scraping mechanism 31 on the Z-axis plate of the stage 29. The mounting part 32 is rotatable.

[0175] The gripping mechanism 31 controls the gripping position on the culture surface of the culture substrate 9 using an XY stage, and its vertical height is controlled using a Z stage so as not to damage the sheet-shaped cell culture during movement. The scratching operation was controlled by moving the position relative to the boundary of the sheet's peeling area and rotating it appropriately to repeatedly press the gripping part at the tip of the gripping mechanism 31 against the culture surface to peel off the sheet. The corresponding cell types and peeling conditions are shown in Table 2. The force acting direction in this comparative example is shown in Figure 22. That is, the force acting direction from the ultrasound generating unit 2 and the shaking unit 3 is the same as the acting direction shown in Figure 4. The peeling time and sheet quality in Comparative Example 10 were evaluated as follows. The peeling time was 42 minutes, and the sheet was evaluated as D. The sheet quality was evaluated as C. The peeling results are shown in Table 2.

[0176] (Comparative Example 11) In this comparative example, the cell type and detachment method combination was changed as shown in Table 2, and the cell detachment device 1400 shown in Figure 23 was used. The detachment of a sheet-shaped cell culture was investigated in the same manner as in Example 1. In this comparative example, the ultrasonic wave generating unit 2 in the ultrasonic band of the cell detachment device 100 shown in Figure 4 according to the first embodiment was replaced with a vibration mechanism. The beating unit 4 used was a lightweight unit with the same mechanism as the cell detachment device 1 shown in Figure 4, and the shaking unit 3 was mounted on a base plate 8 and used a uniaxial moving stage. A vibrator was installed as the vibration mechanism 33 on the shaking unit 3 on the base plate 8. The vibrator was installed and adjusted to an amplitude of 1 mm and a frequency of 500 Hz while checking the amplitude and frequency with a displacement meter. The corresponding cell type and detachment conditions are shown in Table 2. The force application direction in this comparative example is shown in Figure 23. That is, the application direction of the beating unit 4 and shaking unit 3 was the same as that shown in Figure 4. The detachment time and sheet quality in Comparative Example 11 were evaluated as follows. The detachment time was 45 minutes, and the sheet was rated D. The sheet quality was evaluated as D because the sheet shape was not maintained due to the tearing, and at least one of holes of 2 mm or more and tears occurred. The peeling results are shown in Table 2.

[0177] (Comparative Example 12) In this comparative example, the combination of cell type and detachment method was changed as shown in Table 2, and the cell culture was detached in the same manner as in Example 21. After detachment, evaluation was performed in the same manner as in Example 21. As a result, the survival rate was 86.3% and the detachment rate was 44.2%. Since both the survival rate and detachment rate were less than 90%, they were determined to be unsatisfactory. The detachment results are shown in Table 2.

[0178] [Table 1]

[0179] [Table 2]

[0180] With reference to the above examples and comparative examples, when three types of force (ultrasonic vibration, beating, and shaking) are applied as in the examples, the time required for detachment is relatively short, even if these forces are not all applied simultaneously. Furthermore, the quality of the detached sheet-shaped cell culture is also good. In particular, by varying the area of ​​application of ultrasonic vibration, or by applying vertical forces during shaking, the effect of shortening the time required for detachment is remarkable. That is, by implementing one aspect of the present invention, it was confirmed that sample cells can be efficiently and stably detached from a culture substrate, and the quality of the detached sheet-shaped cell culture can also be improved. That is, in the method for detaching cells or sheet-shaped cell cultures of the present invention, a combination of ultrasonic vibration, impact by beating, and water flow by shaking is applied to the culture substrate. This enables detachment in a short time without damaging the cells or sheet-shaped cell cultures.

[0181] The above-mentioned invention includes the following configurations, methods, and programs. (Configuration 1) A cell detachment device for detaching cells adhered to a culture surface of a culture substrate from the culture surface, comprising: an ultrasonic wave generating unit that generates ultrasonic vibrations in the culture substrate; A beating unit that beats the culture substrate; A shaking unit that shakes the culture substrate; A cell detachment device comprising: (Configuration 2) The ultrasonic wave generating device further includes a mounting portion on which the tapping portion and the ultrasonic wave generating portion are mounted. 2. The cell detachment device according to claim 1, wherein the shaking unit is configured to shake the placement unit. (Configuration 3) 3. The cell detachment device according to claim 1, wherein the shaking unit is configured to shake the culture substrate simultaneously with the vibration by the ultrasonic generating unit and the beating by the beating unit. (Configuration 4) 3. The cell detachment device according to claim 1, wherein the shaking unit is configured to shake the culture substrate simultaneously with the vibrations caused by the ultrasonic wave generating unit. (Configuration 5) 3. The cell detachment device according to claim 1, wherein the shaking unit is configured to shake the culture substrate simultaneously with the beating by the beating unit. (Configuration 6) 6. The cell detachment device according to any one of configurations 1 to 5, wherein the shaking unit is configured to shake the culture substrate back and forth. (Configuration 7) 7. The cell detachment device according to any one of configurations 1 to 6, wherein the shaking unit is configured to shake the culture substrate in a direction parallel to the culture surface. (Configuration 8) 7. The cell detachment device according to any one of configurations 1 to 6, wherein the shaking unit is configured to shake the culture substrate in directions including directions non-parallel to the culture surface. (Configuration 9) 9. The cell detachment device according to any one of configurations 1 to 8, wherein the shaking unit is configured to change the shaking direction within a plane parallel to the culture surface. (Configuration 10) 10. The cell detachment device according to any one of configurations 1 to 9, wherein the beating unit is configured to bend the culture substrate in a direction parallel to the culture surface. (Configuration 11) 11. The cell detachment device according to any one of configurations 1 to 10, wherein the beating unit is configured to change the beating direction within a plane parallel to the culture surface. (Configuration 12) A cell detachment device according to any one of configurations 1 to 11, wherein a vibration transmitting material can be disposed between the ultrasonic generating unit and the culture substrate, the vibration transmitting material having elasticity, and the ultrasonic band vibrations generated by the ultrasonic generating unit can be configured to be transmitted to the culture substrate via the vibration transmitting material. (Configuration 13) 13. The cell detachment device according to any one of configurations 1 to 12, wherein the ultrasonic generating unit is configured to generate vibrations in the ultrasonic band so as to generate vibrations in the culture substrate in a direction including a component perpendicular to the culture surface. (Configuration 14) 14. The cell detachment device according to any one of configurations 1 to 13, wherein the ultrasonic wave generating unit is configured to generate vibrations in the ultrasonic band by changing the direction of the vibrations with respect to the culture surface. (Configuration 15) 15. The cell detachment device according to any one of configurations 1 to 14, wherein the shaking direction of the shaking unit, the beating direction of the beating unit, and the direction of ultrasonic vibration generated by the ultrasonic generator are different from each other. (Configuration 16) 16. The cell detachment device according to claim 15, wherein the shaking unit and the beating unit are configured so that the shaking direction and the beating direction are different directions within a parallel plane. (Configuration 17) 17. The cell detachment device according to any one of configurations 1 to 16, further comprising a control unit that controls the driving of at least one of the beating unit, the shaking unit, and the ultrasonic wave generating unit. (Configuration 18) a control unit that controls the driving of at least one of the tapping unit, the shaking unit, and the ultrasonic wave generating unit; A cell detachment device according to any one of configurations 1 to 16, A cell detachment system comprising: (Configuration 19) 2. The cell detachment device according to claim 1, wherein the driving of at least one of the beating unit, the shaking unit, and the ultrasonic wave generating unit is controlled by a control unit. (Configuration 20) 20. The cell detachment device according to claim 19, wherein the control unit controls the time for driving at least one of the beating unit, the shaking unit, and the ultrasonic wave generating unit. (Configuration 21) The cell detachment device of Configuration 19, wherein the control unit controls the driving of the beating unit, the shaking unit, and the ultrasonic wave generating unit so that there is a time when the beating unit, the shaking unit, and the ultrasonic wave generating unit are driven simultaneously. (Configuration 22) The cell detachment device according to configuration 19 or 20, wherein the control unit controls at least one of the beating direction of the beating unit, the shaking direction of the shaking, and the direction of vibration in the ultrasonic band of the ultrasonic generating unit. (Configuration 23) 23. The cell detachment device according to claim 22, wherein the control unit controls the beating unit and the shaking unit so that the beating direction of the beating unit and the shaking direction of the shaking unit differ from each other. (Configuration 24) 24. The cell detachment device according to any one of configurations 19 to 23, wherein the control unit controls the ultrasonic generating unit, the beating unit, and the shaking unit so that the number of times per minute of the ultrasonic band vibrations, the number of times per minute of the beating, and the number of times per minute of the shaking satisfy the relationship: number of times per minute of the shaking < number of times per minute of the beating < number of times per minute of the ultrasonic band vibrations. (Configuration 25) The cell detachment device of any one of configurations 19 to 24, wherein the control unit is configured to control the ultrasonic wave generating unit, the beating unit, and the shaking unit according to predetermined parameters of time, direction, intensity, and period related to the generation of the vibration, parameters of time, direction, intensity, and period related to the beating, and parameters of time, direction, intensity, and period related to the shaking. (Configuration 26) 26. The cell detachment device of claim 25, wherein the control unit controls the ultrasonic wave generating unit, the beating unit, and the shaking unit so that control of at least one of the ultrasonic wave generating unit, the beating unit, and the shaking unit is performed periodically. (Configuration 27) The control unit; A cell detachment system comprising the cell detachment device according to any one of aspects 19 to 26. (Configuration 28) 28. The cell detachment device according to any one of configurations 1 to 27, wherein the cells adhered to the culture surface are a sheet-shaped cell culture. (Method 1) A cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, comprising: generating ultrasonic vibrations in the culture substrate; beating the culture substrate; Agitating the culture substrate; A cell detachment method comprising: (Method 2) The cell detachment method described in Method 1, wherein after performing at least two combinations of generating vibration, beating, and shaking, at least two other combinations of generating vibration, beating, and shaking that are different from the two combinations are performed. (Method 3) The cell detachment method described in Method 1, wherein the beating is started periodically in synchronization with the vibrating and shaking while the vibrating and shaking are continued. (Method 4) The cell detachment method described in Method 1, wherein the generating of the vibration is carried out for a predetermined period of time, and then the generating of the vibration, the beating, and the shaking are carried out. (Method 5) 5. The cell detachment method according to any one of Methods 1 to 4, wherein the duration of at least one of the generating of vibration, the beating, and the shaking is controlled. (Method 6) A cell detachment method described in any one of methods 1 to 5, wherein at least one of the vibration direction in generating the vibration, the beating direction in beating, and the shaking direction in shaking is controlled. (Method 7) The cell detachment method according to any one of Methods 1 to 6, wherein the number of vibrations per minute in the vibrating, the number of beatings per minute in the beating, and the number of shakings per minute in the shaking satisfy the relationship: number of shakings per minute < number of beatings per minute < number of vibrations per minute. (Method 8) The cell detachment method described in any one of Methods 1 to 7, wherein at least one of generating vibration, beating, and shaking is performed periodically. (Method 9) 9. The cell detachment method according to any one of Methods 1 to 8, wherein the cells adhered to the culture surface are a sheet-shaped cell culture. (program) A program for causing a computer to execute the cell detachment method according to any one of Methods 1 to 9.

[0182] <Other embodiments> Although the exemplary embodiments have been described above in detail, the present invention can be embodied as, for example, a system, an apparatus, a method, a program, a recording medium (storage medium), etc. Specifically, the present invention may be applied to a system made up of multiple devices (for example, a host computer, an interface device, an imaging device, a Web application, etc.), or may be applied to an apparatus made up of a single device.

[0183] Needless to say, the object of the present disclosure can be achieved by the following: A recording medium (or storage medium) on which software program code (computer program) that realizes the functions of the above-described embodiments is recorded is supplied to a system or device. The storage medium is, of course, a computer-readable storage medium. The computer (or CPU or MPU) of the system or device then reads and executes the program code stored on the recording medium. In this case, the program code itself read from the recording medium realizes the functions of the above-described embodiments, and the recording medium on which the program code is recorded constitutes the present disclosure.

[0184] Although the present disclosure has been described above with reference to embodiments, modifications, and examples, the present invention is not limited to these embodiments. Inventions modified within the scope of the present invention and inventions equivalent to the present invention are also included in the present disclosure. Furthermore, the above-described embodiments, modifications, and examples can be combined as appropriate within the scope of the present invention. [Explanation of symbols]

[0185] 1, 1a, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300: Cell detachment device 2, 202, 302: Ultrasonic generator 3, 3a, 203, 303, 403, 503, 603, 803: Shaking section 4, 204, 304, 304a: Hitting part 5: Placement section 6: Control unit 7: Graphics controller 8: Base plate 9:Culture substrate 10: Vibration transmitter 12: hammering member 14: Rotation axis 15: Cam 16: Blackout version 17: Detector 18: Transmission rod 19: Spring 22: Spring pressure indicator 26, 726: Rubber feet 27: Holder 29: XYZ Stage 30:Discharge part 31: Grasping part 32: Vibration unit 38: Stripping solution 39: Weight 41: Electrical wire and cable 42: Detachable screw 43: Indicator line 44: Rotating disc 45: Linear bush holder 46: Linear bushing 47: Connecting rod fixture 48: Shaking fulcrum axis 49: Shaking slide piece 50: Shaking adjustment screw rod 51: Shaking piece slide axis 52: Shaking detector 53: Shaking rod 54: Connecting rod 55: Shaking motor 56: Gear 57: Linear guide 58: Guide rail 59:Pressure spring 60: Base side spring post 61: Shaking side spring post 62: Stroke indicator 63: Stroke knob 64: First rail fixture 65: Second rail fixture

Claims

1. A cell detachment device for detaching cells adhered to a culture surface of a culture substrate from the culture surface, comprising: an ultrasonic wave generating unit that generates ultrasonic vibrations in the culture substrate; A beating unit that beats the culture substrate; A shaking unit that shakes the culture substrate; A cell detachment device comprising:

2. The ultrasonic wave generating device further includes a mounting portion on which the tapping portion and the ultrasonic wave generating portion are mounted. The cell detachment device according to claim 1 , wherein the shaking unit is configured to shake the placement unit.

3. The cell detachment device according to claim 1 , wherein the shaking unit is configured to shake the culture substrate simultaneously with the vibration by the ultrasonic generator and the beating by the beating unit.

4. The cell detachment device according to claim 1 , wherein the agitation unit is configured to agitate the culture substrate simultaneously with the vibrations caused by the ultrasonic wave generation unit.

5. The cell detachment device according to claim 1 , wherein the shaking unit is configured to shake the culture substrate simultaneously with the beating by the beating unit.

6. The cell detachment device according to claim 1 , wherein the shaking unit is configured to shake the culture substrate back and forth.

7. The cell detachment device according to claim 1 , wherein the shaking unit is configured to shake the culture substrate in a direction parallel to the culture surface.

8. The cell detachment device according to claim 1 , wherein the shaking unit is configured to shake the culture substrate in directions including directions non-parallel to the culture surface.

9. The cell detachment device according to claim 1 , wherein the shaking unit is configured to change the shaking direction within a plane parallel to the culture surface.

10. The cell detachment device according to claim 1 , wherein the beating unit is configured to strike the culture substrate in a direction parallel to the culture surface.

11. The cell detachment device according to claim 1 , wherein the beating unit is configured to change the beating direction within a plane parallel to the culture surface.

12. The cell detachment device of claim 1, wherein a vibration transmitting material can be disposed between the ultrasonic generating unit and the culture substrate, the vibration transmitting material having elasticity, and the ultrasonic band vibrations generated by the ultrasonic generating unit can be configured to be transmitted to the culture substrate via the vibration transmitting material.

13. The cell detachment device according to claim 1 , wherein the ultrasonic generator is configured to generate vibrations in an ultrasonic band so as to generate vibrations in the culture substrate in a direction including a component perpendicular to the culture surface.

14. The cell detachment device according to claim 1 , wherein the ultrasonic wave generating unit is configured to generate vibrations in the ultrasonic band by changing the direction of the vibrations relative to the culture surface.

15. The cell detachment device according to claim 1 , wherein the shaking direction of the shaking unit, the beating direction of the beating unit, and the direction of vibration in the ultrasonic band generated by the ultrasonic generator are different from each other.

16. The cell detachment device according to claim 15 , wherein the shaking unit and the beating unit are configured so that the shaking direction and the beating direction are different directions within a parallel plane.

17. The cell detachment device according to claim 1 , further comprising a control unit that controls driving of at least one of the beating unit, the shaking unit, and the ultrasonic wave generating unit.

18. a control unit that controls the driving of at least one of the tapping unit, the shaking unit, and the ultrasonic wave generating unit; The cell detachment device according to any one of claims 1 to 16, A cell detachment system comprising:

19. The cell detachment device according to claim 1 , wherein driving of at least one of the beating unit, the shaking unit, and the ultrasonic wave generating unit is controlled by a control unit.

20. The cell detachment device according to claim 19 , wherein the control unit controls the time for driving at least one of the beating unit, the shaking unit, and the ultrasonic wave generating unit.

21. 20. The cell detachment device according to claim 19, wherein the control unit controls the driving of the pounding unit, the shaking unit, and the ultrasonic wave generating unit so that there is a time when the pounding unit, the shaking unit, and the ultrasonic wave generating unit are driven simultaneously.

22. The cell detachment device according to claim 19 , wherein the control unit controls at least one of the beating direction of the beating unit, the shaking direction of the shaking, and the direction of vibration in an ultrasonic band of the ultrasonic wave generating unit.

23. The cell detachment device according to claim 22 , wherein the control unit controls the pounding unit and the shaking unit so that the pounding direction of the pounding unit and the shaking direction of the shaking unit differ from each other.

24. 20. The cell detachment device according to claim 19, wherein the control unit controls the ultrasonic generator, the beating unit, and the shaking unit so that the number of times per minute of the ultrasonic band vibrations, the number of times per minute of the beatings, and the number of times per minute of the shaking satisfy the relationship: number of times per minute of the shaking < number of times per minute of the beatings < number of times per minute of the ultrasonic band vibrations.

25. 20. The cell detachment device according to claim 19, wherein the control unit is configured to control the ultrasonic wave generating unit, the beating unit, and the shaking unit according to predetermined parameters of time, direction, intensity, and period related to the generation of the vibration, parameters of time, direction, intensity, and period related to the beating, and parameters of time, direction, intensity, and period related to the shaking.

26. The cell detachment device according to claim 25, wherein the control unit controls the ultrasonic wave generating unit, the beating unit, and the shaking unit so that control of at least one of the ultrasonic wave generating unit, the beating unit, and the shaking unit is performed periodically.

27. the control unit; A cell detachment system comprising the cell detachment device according to any one of claims 19 to 26.

28. 27. The cell detachment device according to claim 1, wherein the cells adhered to the culture surface are a sheet-shaped cell culture.

29. A cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, comprising: generating ultrasonic vibrations in the culture substrate; beating the culture substrate; Agitating the culture substrate; A cell detachment method comprising:

30. 30. The cell detachment method of claim 29, wherein after performing at least two combinations of generating vibration, beating, and shaking, at least two other combinations of generating vibration, beating, and shaking that are different from the two combinations are performed.

31. The cell detachment method according to claim 29, wherein the beating is started periodically in synchronization with the vibrating and shaking while the vibrating and shaking are continued.

32. The cell detachment method according to claim 29, wherein the generating of vibration is performed for a predetermined period of time, and then the generating of vibration, the beating, and the shaking are performed.

33. The cell detachment method according to claim 29, wherein the duration of at least one of the generating of the vibration, the beating, and the shaking is controlled.

34. The cell detachment method according to claim 29, wherein at least one of a vibration direction in generating the vibration, a beating direction in beating, and a shaking direction in shaking is controlled.

35. 30. The cell detachment method according to claim 29, wherein the number of vibrations per minute in the vibrating, the number of beatings per minute in the beating, and the number of shakings per minute in the shaking satisfy the relationship: number of shakings per minute < number of beatings per minute < number of vibrations per minute.

36. The cell detachment method according to claim 29, wherein at least one of the generating of vibration, the beating, and the shaking is performed periodically.

37. 37. The cell detachment method according to any one of claims 29 to 36, wherein the cells adhered to the culture surface are a sheet-shaped cell culture.

38. A program for causing a computer to execute the cell detachment method according to any one of claims 29 to 36.

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