Cell detachment method and cell detachment system

Abstract

To provide a cell detachment method that increases both cell survival rate and detachment rate when detaching cells from a culture substrate.SOLUTION: Provided is a method for detaching adherent cells cultured on a culture substrate, including a vibration applying step of applying ultrasonic vibrations to the culture substrate, and a shear force applying step of applying shear force to the culture surface of the culture substrate, the vibration applying step beginning before the shear force applying step begins, and the shear force being applied by means other than ultrasonic vibrations.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a cell detachment method and a cell detachment system. [Background technology]

[0002] In the fields of cell medicine and regenerative medicine, large amounts of cells are required, and in particular, there is a demand for an efficient and stable supply of adhesive cells, which account for the majority of biological tissues.

[0003] In the culture of adherent cells, the target cells are obtained through the steps of culturing the cells on a culture substrate such as a polystyrene dish, detaching the cells from the culture substrate, recovering the cells, and washing them. To further expand the cells, a portion of the acquired cells is transferred to a new culture substrate and cultured, a process known as passaging. During these cell acquisition and passaging processes, detachment methods have been investigated to recover cells at a high detachment rate without damaging them.

[0004] Meanwhile, cell detachment methods using shear force have been proposed. Patent Document 1 discloses a cell detachment device that applies ultrasonic vibrations to a container holding cells and a culture medium or liquid, thereby detaching at least some of the cells from the container. Patent Document 2 also discloses a method for detaching cells by mechanical stimulation caused by the flow of a culture medium. The present inventors have discovered a problem with the above prior art. Specifically, when handling cells that are difficult to detach, such as cells that have a high adhesive force to the container, detachment under conditions that increase detachment efficiency may result in a decrease in cell viability. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 047368 [Patent Document 2] Japanese Patent Application Publication No. 2013 / 57636 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an object of the present disclosure is to provide a cell detachment method that increases both the survival rate and detachment rate of cells, and a cell detachment system using the same. [Means for solving the problem]

[0007] While investigating methods for detaching cells, the inventors discovered that highly efficient detachment without damaging the cells is possible when ultrasonic vibrations are applied to the culture substrate and then shear force is applied to the culture surface of the culture substrate. That is, the cell detachment method according to the present disclosure includes: A method for detaching adherent cells cultured on a culture substrate, comprising: a vibration applying step of applying ultrasonic vibration to the culture substrate; A shear force application step of applying shear force to the culture surface of the culture substrate. Includes the vibration applying step starts before the shear force applying step starts, The shear force is applied by a means other than ultrasonic vibration. In addition, the cell detachment system according to the present disclosure includes: A cell detachment system having a detachment unit that detaches adherent cells cultured on a culture substrate, and a control unit, The peeling unit has a vibration applying unit that applies vibrations in an ultrasonic band to the culture substrate, and a shear force applying unit that applies shear force to the culture surface of the culture substrate, The control unit controls the vibration applying unit so that the start of driving occurs before the start of driving of the shear force applying unit. [Effects of the Invention]

[0008] According to the present disclosure, it is possible to provide a cell detachment method and a cell detachment system using the same that increase both the cell survival rate and detachment rate. [Brief explanation of the drawings]

[0009] [Figure 1A] FIG. 1 is a diagram illustrating the cell detachment method of the present disclosure. [Figure 1B] FIG. 1 is a diagram illustrating the cell detachment method of the present disclosure. [Figure 2A] FIG. 1 is a diagram illustrating the cell detachment method of the present disclosure. [Figure 2B] FIG. 1 is a diagram illustrating the cell detachment method of the present disclosure. [Figure 3] 1 is a schematic diagram illustrating an example of a piezoelectric body according to the present disclosure. [Figure 4] 1A and 1B are diagrams illustrating a vibration imparting mechanism that imparts vibration in the ultrasonic band in the present disclosure. [Figure 5A] FIG. 1 is a diagram illustrating a bolt-clamped Langevin transducer. [Figure 5B] FIG. 1 is a diagram illustrating a stick-on type vibrator. [Figure 6] FIG. 1 illustrates an example of a cell detachment system according to the present disclosure. [Figure 7] FIG. 1 illustrates an example of a cell detachment system according to the present disclosure. [Figure 8] FIG. 1 is a diagram illustrating an adhesive strength measuring device used in the examples of the present disclosure. [Figure 9] 1 is a diagram illustrating a device 1 having a mechanism for applying vibration in the ultrasonic band that can be used in an embodiment of the present disclosure. [Figure 10] FIG. 2 is a diagram illustrating a device 2 having a mechanism used to apply shear force in the present disclosure. [Figure 11] FIG. 1 is a diagram illustrating a device 3 having a mechanism used to apply ultrasonic vibration and shear force that can be used in an embodiment of the present disclosure. [Figure 12] FIG. 1 is a diagram illustrating a device 4 having a mechanism used to apply shear force in the present disclosure. [Figure 13] FIG. 1 is a diagram illustrating a device 5 having a mechanism used to apply ultrasonic vibration and shear force that can be used in an embodiment of the present disclosure. [Figure 14A]FIG. 1 is a diagram illustrating a device 6 having a mechanism used to apply shear force in the present disclosure. [Figure 14B] FIG. 1 is a diagram illustrating a device 6 having a mechanism used to apply shear force in the present disclosure. [Figure 15] FIG. 1 is a diagram illustrating a device 7 having a mechanism used to apply shear force in the present disclosure. [Figure 16] FIG. 1 is a diagram illustrating a device 8 having a mechanism used to apply shear force in the present disclosure. [Figure 17] FIG. 1 is a diagram illustrating a device 9 having a mechanism used to apply shear force in the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, a cell detachment method according to an embodiment of the present disclosure will be described. (Cell detachment method) The present disclosure provides a method for detaching adherent cells cultured on a culture substrate, comprising: a vibration applying step of applying ultrasonic vibration to the culture substrate; A shear force application step of applying shear force to the culture surface of the culture substrate. Includes the vibration applying step starts before the shear force applying step starts, The present invention provides a method for detaching cells, wherein the shear force is applied by a means other than ultrasonic vibration.

[0011] A specific example of the cell detachment method of the present disclosure is shown in FIGS. 1A and 1B. As shown in FIG. 1A, first, a vibration applying step (step S110) is started in which a culture substrate is placed and vibrations in the ultrasonic band are applied to the culture substrate. Next, the culture substrate is placed and the shear force application step (step S140) begins. Finally, the detached cells are collected (step S150).

[0012] As shown in FIG. 1B, the vibration applying step (step S110) starts before the shear force applying step (step S140). A further embodiment is shown in Figures 2A and 2B.

[0013] As shown in Figure 2A, first, a vibration applying step (step S210) is started, in which a culture substrate is placed and ultrasonic vibrations are applied. Then, an information acquiring step (step S220) is executed, in which cell information on the culture substrate is acquired. Based on the acquired cell information, a shear force applying step start time determining step (step S230) is executed, in which the start timing of the shear force applying step is determined. Next, a culture substrate is placed, and a shear force applying step (step S240) is started. Finally, the detached cells are collected (step S250).

[0014] As shown in FIG. 2B, the vibration applying step (step S210) starts before the shear force applying step (step S240) starts.

[0015] (Vibration application process) In the vibration applying step, vibrations in the ultrasonic band are applied to the culture substrate, thereby facilitating detachment of the cells from the culture substrate. The inventors have discovered that applying ultrasonic vibrations to a culture substrate on which cells are cultured can reduce the adhesive force of the cells before they detach from the culture substrate, and have investigated how to actively utilize this effect.

[0016] In the present disclosure, ultrasonic vibration refers to vibration with a frequency of 10 kHz or more and 1 MHz or less. The duration for which ultrasonic vibration is applied in the vibration application step is not particularly limited, but from the viewpoint of effectively reducing the adhesive force of cells and increasing cell viability, it is, for example, from 1 second to 1 hour, preferably from 10 seconds to 30 minutes, and more preferably from 30 seconds to 15 minutes. Depending on the cellular environment (e.g., type of cell detachment solution, environmental temperature, CO2 concentration, etc.) in the cell detachment method of the present disclosure, applying ultrasonic vibration for a long period of time may not be desirable. Furthermore, if the duration is too short, the adhesive force of cells may not be sufficiently reduced by ultrasonic vibration.

[0017] The maximum amplitude of the vibration surface of the ultrasonic vibration in the vibration application step is not particularly limited, but from the viewpoint of maintaining cell viability, it is desirable to set it to 10 μm or less. This range can suppress the effects of heat generation from the vibrator and transmit an appropriate amount of vibration energy to the cells to reduce their adhesive strength. Furthermore, setting the amplitude to 10 μm or less is preferable because it makes it difficult for cavitation to occur when ultrasonic vibration is applied, making it difficult for cell viability to decrease.

[0018] The direction of ultrasonic vibration preferably includes a component perpendicular to the culture surface. In the present disclosure, the direction of ultrasonic vibration refers to the direction connecting the vibration surface of the vibrator that generates ultrasonic vibration and the vibration transmission member that transmits the ultrasonic vibration.

[0019] In particular, by applying ultrasonic vibrations from the bottom surface of the culture substrate, it becomes possible to apply ultrasonic vibrations having a component perpendicular to the culture surface, and by including a component perpendicular to the culture surface, it becomes easier to reduce the adhesive strength of the cells.

[0020] Various vibration modes can be used for ultrasonic vibration patterns, depending on the vibrator and ultrasonic device used as the vibration imparting means. While various vibration modes can be selected depending on the cell type and detachment conditions, a vibration mode that excites a uniform amplitude throughout the vessel is preferred, such as when using a vibrator with high in-plane uniformity, such as a Langevin vibrator. Furthermore, when using a vibrator that can generate various vibration modes through bending vibration, such as a ring-shaped vibrator with an annular piezoelectric element attached to a glass plate, a mode that imparts a uniform amplitude with a constant period, such as a traveling wave or carrier wave that can be generated by dividing the electrodes into multiple parts, is preferred. By achieving a uniform in-plane amplitude at the bottom of the culture substrate vessel, the amplitude variation between any position in the vessel when ultrasonic vibrations are applied is reduced, thereby reducing the differential effects between cultured cells. Furthermore, depending on the type of vibrator used in the vibration imparting means and the driving method, heat may be generated during operation. This tendency tends to intensify as the device continues to be driven at the resonant frequency, depending on the applied voltage, the frequency band used, and the device environment. To address this issue, the amount of heat generated can be controlled 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 vibration imparting means 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.

[0021] The environmental temperature during the vibration application step is not particularly limited and can be set appropriately depending on the cell type used. From the viewpoint of maintaining cell viability, a temperature of 20°C or higher and 40°C or lower is preferred, and in the case of mammalian cells, for example, a temperature of 30.0°C or higher and 37.5°C or lower is particularly preferred. This range is because the environmental temperature is close to the temperature during culture, which reduces the impact of temperature changes on the cells and allows for maintaining a high viability. When using a known container using, for example, a temperature-responsive membrane as the culture substrate, the environmental temperature during the vibration application step may be set to a temperature range corresponding to the temperature responsiveness.

[0022] The vibration imparting step may be performed in an environment with a CO concentration of 5%, similar to cell culture. The environmental conditions for the vibration imparting step can be set appropriately depending on the type and characteristics of the cells and the method of using the cells after detachment.

[0023] (Shearing force application process) In the shear force application step, a shear force is applied to the culture surface of the culture substrate to detach the cells from the culture substrate.

[0024] The inventors further discovered that when the adhesive strength of cultured adherent cells to the culture substrate is reduced by ultrasonic vibration, the cells are more easily detached by applying shear force. Shear force refers to a force acting laterally on the cells. In other words, by reducing the adhesive strength by ultrasonic vibration and then separately applying shear force to the cells as a force for detaching them from the sides, it became possible to detach the cells without reducing their viability.

[0025] Here, methods of applying shear force include at least beating (beating the culture substrate), shaking (shaking the culture substrate), rocking (rocking the culture substrate), and eccentric rotation (eccentric rotation of the culture substrate), discharging liquid onto the culture substrate, and stirring liquid on the culture substrate. Note that shear force applied by ultrasonic vibration is not included in the shear force. Shear force is considered to be applied by means other than ultrasonic vibration. Specific examples include the following.

[0026] An example of hitting a culture substrate is to impart an impact force to the culture substrate 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 imparting an impact force by colliding a member with the culture substrate is not limited to these. Furthermore, the hitting unit that hits the substrate can impart an impact force by colliding the culture substrate with a flat, arc-shaped, or protruding member, or these methods can be combined. Among these, the hitting unit of the present disclosure preferably uses a means for imparting an impact force to the culture substrate by colliding a member having an appropriate mass with the culture substrate, from the viewpoint of miniaturizing the device configuration.

[0027] Furthermore, the weight and raw material of the colliding member can be selected appropriately depending on the impact force to be applied, and known materials such as inorganic materials such as metals and ceramics, organic materials such as resins and rubber, and even composites of these can be used as raw materials.

[0028] As a power source for moving the member to be collided, a method that can generate force by passing electricity, such as a motor or solenoid, can be used. These power sources may be used directly or via a member that can store energy, such as a spring.

[0029] The tapping unit of the present disclosure can also control the impact force and inertial force applied to the culture substrate by arranging a microdeformation member on the opposite side of the tapping direction. From the viewpoint of minimizing the contact points, the microdeformation member is preferably cylindrical, hemispherical, or a combination thereof, and is preferably made of an elastic material, i.e., rubber.

[0030] An example of shaking the culture substrate is to use a configuration that generates a flow in the liquid in the culture substrate by applying acceleration that includes a component in a direction parallel to the culture surface of the culture substrate. For example, a configuration can be used in which the culture substrate is rotated by a motor and then reciprocated on one axis using a slide rail or the like. Alternatively, a configuration can be used in which the culture substrate is accelerated using a linear motor or the like to apply acceleration including a component in a direction parallel to the culture surface and a component in a direction non-parallel to the culture surface. The axis of shaking can be appropriately designed so that an acceleration including a component in a direction parallel to the culture surface of the culture substrate can be applied by shaking.

[0031] A specific example of shaking according to the present disclosure will now be illustrated. For example, reciprocating shaking, which involves reciprocating motion in the horizontal or vertical direction, can be used. The reciprocating shaking may include a horizontal or vertical component, and shaking may be performed only in the horizontal or vertical direction, or a combination of these may be performed obliquely. Furthermore, the shaking axis is not limited to a single axis, and shaking may be performed on multiple axes, such as two or more axes, in different shaking directions.

[0032] Other shaking methods include, for example, rotary shaking, which involves a circular motion in a direction that includes a horizontal component. Rotary shaking may involve only horizontal shaking, or it may involve diagonal shaking that includes vertical shaking. The circular motion may be a perfect circle, an ellipse, a figure-eight shape, or a combination of these.

[0033] Other shaking methods include, for example, seesaw shaking, which refers to a shaking method in which the culture medium moves back and forth like a seesaw around a single point in the culture substrate as an axis, and examples of such shaking include horizontal and vertical reciprocation, and a combination of these.

[0034] Another example of a shaking method is a swinging motion in which the sample is swung in an arc around a fulcrum like a pendulum. For shaking the culture substrate, these specific examples may be used alone or in combination.

[0035] Examples of eccentric rotation of the culture substrate include rotation with an eccentric axis, such as stirring. Specifically, as mentioned above, examples include the above-mentioned configuration in which the axis of rotation is eccentric, horizontal eccentric vibration, and vortex stirring.

[0036] Examples of discharging liquid onto a culture substrate include a method in which liquid is discharged from a needle or the like and directed onto the culture surface to spread and apply the liquid along the culture surface; a method shown in Figures 15A and 15B and described later in which liquid is discharged and sucked up from a pipetter or the like to apply a flow of liquid from different directions along the culture surface; and a method shown in Figure 16 and described later in which a flow path is created along the culture surface and a pump or syringe is used to send the liquid in one direction or in two directions to apply the liquid.

[0037] An example of agitating the liquid is a method of applying the shear force by creating a flow parallel to the culture surface using a stirrer 9 with stirring blades as shown in Figure 17. As shown in Figure 17, the device 9 is a device with a shear force applying mechanism, and has a holding mechanism 11 that holds the culture substrate 10 and a stirring mechanism 137 for applying the shear force. In addition to these, known methods such as a method using a stirring bar can also be used to agitate the liquid.

[0038] Of these methods for applying shear force, beating, shaking, eccentric rotation, and liquid ejection are preferred. The shear force application methods exemplified here may be used alone or in combination.

[0039] The magnitude of the shear force applied to the culture surface is preferably varied over time during the shear force application process. Examples of variations over time include monotonically, at a constant rate, or by an arbitrary amount of change in the shear force, or by periodic, multiple, or random increases and decreases. Furthermore, the magnitude of the shear force may be varied periodically, for example, at a rate of 5% or more, sinusoidally, or in a pulsed manner.

[0040] These changes over time may be caused by a change in the strength of the shearing force. For example, the strength of the shearing force itself may be changed, or the strength of the shearing force may be changed as the impulse of the shearing force per unit time.

[0041] The magnitude of the shear force applied in the shear force applying step of the present disclosure is preferably changed periodically. By changing the shear force at a constant period, it is possible to apply a shear force of a constant strength range uniformly per unit time, which is preferable from the viewpoint of improving peeling efficiency.

[0042] In the shear force application step of the present disclosure, it is desirable to change the direction of the shear force. The change in the direction of the shear force can be changed over time or can be achieved by simultaneously applying shear forces from different directions. Specific examples of the types and directions of shear force exemplified in the present disclosure include shear forces from one direction, such as when discharging a liquid, shear forces acting from two different directions, such as when reciprocating shaking is performed on one axis or when beating from two directions, and shear forces from multiple directions, such as when rotating a two-axis stage or beating a culture substrate while rotating it.

[0043] It is also desirable to change the direction of shear force and the magnitude of the shear force applied to the culture surface during the shear force application process. This is because there are variations in the growth and adhesion states of individual cells, so by changing the magnitude and direction of the shear force, it is possible to apply multifaceted forces to the cells and apply shear force that is appropriate for each individual cell.

[0044] The direction in which the shear force acts preferably includes a component parallel to the culture surface. By including a component parallel to the culture surface, cells whose adhesive strength has decreased in the vibration application step can be more effectively detached.

[0045] In the shear force application step, the time for applying shear force, i.e., the time for performing the shear force application step, is not particularly limited, but from the viewpoint of effectively detaching cells and increasing cell viability, it is, for example, from 1 second to 1 hour, preferably from 5 seconds to 30 minutes, and more preferably from 10 seconds to 10 minutes. If the time for applying shear force is too long, excessive damage to the cells may be caused by the shear force, which is undesirable. On the other hand, if the time is too short, sufficient shear force may not be applied to the cells, resulting in a reduced detachment effect.

[0046] The environmental temperature during the shear force application step is not particularly limited, but from the viewpoint of maintaining cell viability, a temperature of 20°C or higher and 40°C or lower is preferred, and a temperature of 30.0°C or higher and 37.5°C or lower is particularly preferred. Within this range, the environmental temperature is close to the temperature during culture, which reduces the effect of temperature changes on the cells and allows the viability to be maintained at a high level. When a known container using, for example, a temperature-responsive membrane is used as the culture substrate, the environmental temperature during the shear force application step may be set to a temperature range corresponding to the temperature responsiveness.

[0047] The shear force application step may be performed in an environment with a CO concentration of 5%, similar to cell culture. The environmental conditions for the shear force application step can be appropriately set depending on the type and characteristics of the cells and the method of using the cells after detachment.

[0048] Furthermore, the cell detachment method of the present disclosure may include a step other than the vibration applying step or the shear force applying step. The step may be included between the vibration applying step and the shear force applying step, during the shear force applying step, after the shear force applying step has ended, or before the vibration applying step begins.

[0049] Other processes include a detached cell recovery process, an information acquisition process, a process for determining the start time of the shear force application process, and further, a process for replacing the liquid in the culture substrate with a cell detachment solution, a process for rinsing the cells with the cell detachment solution, and a process for diluting or homogenizing the detachment solution.

[0050] The vibration applying step and the shear force applying step may be repeated at regular intervals or at random intervals. The ratio of the total time for which the vibration applying step is performed to the total time for which the shear force applying step is preferably 0.01 or more and 100 or less. In other words, when the time for which the vibration applying step is performed is a and the time for which the shear force applying step is b, it is preferable that the relationship between a and b satisfy Formula 5. 0.01≦a / b≦100 (formula 5)

[0051] The time difference between the start of the vibration applying step and the start of the shear force applying step is not particularly limited, as long as the start of the vibration applying step precedes the start of the shear force applying step. From the viewpoint of increasing cell viability, the time difference is, for example, within 30 minutes, preferably within 10 minutes, and more preferably within 5 minutes. Furthermore, the shear force applying step may be started before the end of the vibration applying step, simultaneously with the end of the vibration applying step, or after the end of the vibration applying step. By starting the shear force applying step before the end of the vibration applying step, shear force can be applied while weakening the adhesive force of the cells, allowing for more effective cell detachment. Furthermore, information about the cells adhered to the culture substrate may be measured and the measurement information may be used to determine whether to transition from the vibration applying step to the shear force applying step.

[0052] Furthermore, the time difference between the start of the vibration applying step and the end of the shear force applying step is not particularly limited, but from the viewpoint of increasing cell viability, it is preferably, for example, 10 seconds or more and 30 minutes or less. Furthermore, the shear force applying step may end after the vibration applying step, or may end before the vibration applying step, or the vibration applying step and the shear force applying step may end simultaneously. Furthermore, information on the cells adhered to the culture substrate may be measured and the measurement information may be used to determine whether to end the vibration applying step or the shear force applying step.

[0053] When the adhesion force between the cells and the culture substrate at the start of the vibration application step is A1, the adhesion force between the cells and the culture substrate at the start of the shear force application step is A2, and the adhesion force between the cells and the culture substrate when both the vibration application step and the shear force application step are completed is A3, it is preferable that the relationship among A1, A2, and A3 satisfies Formula 1. A1 > A2 > A3 (Formula 1)

[0054] When the adhesion area between the cells and the culture substrate at the start time of the vibration application step is B1, the adhesion area between the cells and the culture substrate at the start time of the shear force application step is B2, and the adhesion area at the time when both the vibration application step and the shear force application step are completed is B3, it is preferable that the relationship among B1, B2, and B3 satisfies Formula 2 and Formula 3. B2 < 0.5 × B1 (Formula 2) 0 ≤ B3 < B2 (Formula 3)

[0055] [[ID=I3]] By satisfying the relationship between B1 and B2 where B2 < 0.5 × B1 (Formula 2), the adhesion force of the cells at the start of the shear force application step is sufficiently reduced by the vibration application step, and even when a shear force is applied, the cells are likely to be detached in a state with a high survival rate. Also, by satisfying (Formula 3), the detachment rate of the cells becomes high.

[0056] Also, when the luminance information obtained by optically measuring the cells at the start time of the vibration application step is C1, and the luminance information obtained by optically measuring the cells at the start time of the shear force application step is C2, it is preferable that the relationship between C1 and C2 satisfies Formula 4. C1 < C2 (Formula 4) By satisfying (Formula 4), the detachment rate of the cells becomes high.

[0057] (Information acquisition step) The method for detaching cells of the present disclosure may further include an information acquisition step, which is a step of acquiring cell information. And further, it may include a shear force application start time determination step of determining the start timing of the shear force application step based on the acquired cell information.

[0058] The cell information disclosed herein includes cell type, passage number, culture time, as well as cell measurement information. Examples of measurement information in the present disclosure include cell adhesion strength, cell area, the area of ​​a halo region in a phase contrast microscope image (also called a phase contrast image), brightness values ​​in a phase contrast image, and brightness values ​​when observing the cell culture surface of a culture substrate using oblique incidence illumination.

[0059] An example of measurement information on cell adhesion force is a method in which a water stream with a known shear force is sprayed onto a cell culture surface to apply shear force to detach the cells, and the applied shear force is estimated from the size of the area where the cells have detached, and this is considered to be adhesion force. By evaluating the changes in adhesive strength during the vibration application process and the shear force application process, it is possible to optimize the control of changes in the ultrasonic intensity during the vibration application process, the start time of the shear force application process, the magnitude and direction of the shear force during the shear force application process, and even the time.

[0060] An example of measurement information of the cell area is, for example, obtaining a phase contrast image of the adhered cells and measuring the area occupied by one cell through image processing, thereby quantifying the cell area.

[0061] It is known that adhered cells appear larger in area in phase contrast images, and cells whose adhesive strength has decreased due to the vibration and shear stress application steps assume a spherical shape due to surface tension, and therefore appear smaller in area in phase contrast images. This fact may be used to determine the timing for starting the shear stress application step. Other examples of cellular information include the brightness value in a phase contrast image or the area of ​​a halo region.

[0062] In a phase-contrast image of cells in an adherent state, only regions with a small phase difference (when the cells are flat) are detected. On the other hand, when a detachment solution is added, cell adhesion weakens and the cells lift off the culture substrate, the phase difference increases in the lifted areas, and a halo region with a higher image brightness value is observed in the phase-contrast image. In other words, as cell adhesion weakens, the area of ​​the halo region increases and its value approaches a constant value. This can be used to determine the timing of the start of the shear force application process, and further, the control of changes in the magnitude, direction, and even time of the shear force application process can be determined based on the amount of change in cell information.

[0063] Another example of cell information is the brightness value observed when observing the cell culture surface of a culture substrate using oblique illumination. Cells whose adhesion has been weakened by the action of a cell detachment solution shrink their pseudopodia, reducing the microscopic adhesion surface with the culture substrate, which makes light more likely to scatter at the interface between the culture substrate and the cells. Therefore, when observed using oblique illumination, which increases the proportion of scattered light, the brightness value of the cell culture surface of the culture substrate increases as the cell adhesion weakens, and this value approaches a constant value. This can be used to determine the timing of the end of the vibration application process and the start of the shear force application process, or to determine at least one of the ultrasonic vibration application conditions of the vibration application process, such as vibration intensity and vibration application time, and the shear force application conditions of the shear force application process, such as shear force application intensity, shear force application time, and control of shear force application changes.

[0064] The cell detachment method of the present disclosure may be equipped with a control unit that pre-sets the conditions for the vibration application process and shear force application process (ultrasonic intensity, application time of ultrasonic vibration, vibration mode, type, intensity, and application time of shear force, etc.) according to the cell type, or may be controlled by combining the cell type with information such as observations.

[0065] (Ultrasonic vibration) In the present disclosure, ultrasonic vibrations can be exemplified by vibrations having a frequency of 10 kHz to 1 MHz. The vibration generating means can be any means capable of applying ultrasonic vibrations to cells, and is not particularly limited. One example is the use of an ultrasonic oscillator such as lead zirconate titanate (PZT) as the vibrating body.

[0066] Here, the vibrator can be brought into direct contact with the outer surface of the culture substrate, which has been filled with a culture medium and sealed, to impart vibration to the culture substrate. Alternatively, instead of bringing the vibration imparting means into direct contact with the container, a vibration transmitting member described as an ultrasound transmitting substance can be interposed between the vibration imparting means and the treatment target area, and ultrasound can be imparted to the cells to be ablated.

[0067] (vibrator) In the present disclosure, the vibrator may be any vibrator that generates vibrations in the ultrasonic band, and examples include a piezoelectric body bonded to a vibration plate. When 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, a large amplitude can be output at a relatively high driving frequency (vibration frequency) in the ultrasonic band without being damaged as a vibrator. The vibrator may include an ultrasonic vibrator. In the case of a ring-shaped piezoelectric body, it is preferable that the outer diameter of the diaphragm is equal to the outer diameter of the piezoelectric body.

[0068] The thickness of the vibration plate is preferably such that when the piezoelectric body and the vibration plate are bonded together and the vibration occurs, the midpoint of the thickness direction of the bending, i.e., the neutral plane that is neither tensile nor compressed when the piezoelectric body bends, is on the vibration plate side, in order to efficiently utilize the distortion of the piezoelectric body for bending.

[0069] The vibrator of the present disclosure can also be a commercially available Langevin vibrator or rectangular vibrator. Langevin vibrators have a piezoelectric body sandwiched between two metal blocks and fastened together with bolts or the like to form an integrated structure, and are available from Honda Electronics and Fuji Ceramics, for example.

[0070] As the vibrator of the present disclosure, a ring device in which a ring-shaped piezoelectric body is bonded to a glass plate or a metal plate, or a Langevin vibrator (Langevin device), which allows easy amplitude control on the order of micrometers, is preferably used.

[0071] When the ring device is used in standing wave mode, it is possible to transmit vibrations with the strongest amplitude to a position corresponding to the vibration mode, such as the center, depending on the size and resonant frequency of the piezoelectric body and diaphragm. When the ring device is used in traveling wave mode or carrier wave mode, it is possible to periodically move the positions of the antinodes and nodes of the bending vibration, making it possible to apply more uniform vibrations to the culture substrate. In addition, the Langevin device is capable of applying uniform vibrations to the culture substrate.

[0072] (piezoelectric material) In the present disclosure, the piezoelectric body used in the vibrator is preferably a ring-shaped or annular piezoelectric body, but the shape of the piezoelectric body that deflects the vibrator may be a disk-shaped piezoelectric body. When cell information is directly observed in the information acquisition step, the piezoelectric body is preferably annular in shape so that the field of view below the culture substrate can be secured.

[0073] Piezoelectric materials exhibit piezoelectric properties when polarized, and the polarity of polarization can be changed depending on the electrode pattern, as shown in Figure 3. In Figure 3, the area patterned with electrodes α1, α2, α3, β1, β2, and β3 is the driving phase 301 that contributes to deformation, and the area sandwiched between electrodes α1 and β1 is the sensor phase 302 that detects the degree of deformation. A polarized piezoelectric material can excite standing wave and traveling wave vibration modes by controlling the phase of the input AC voltage for each electrode pattern. A standing wave is generated by applying an AC voltage with a 180-degree phase difference to negatively polarized electrodes (α2 and β2) relative to positively polarized electrodes (α1, α3, β1 and β3). In contrast, as shown in Figure 3, when phase A 303 (electrodes α1, α2, α3) and phase B 304 (electrodes β1, β2, β3) are separated to the left and right of the dashed line, applying an AC voltage with a 90-degree phase difference between phase A and phase B generates a traveling wave with two-phase drive. In Figure 3, GND refers to ground, and is used to ground the electrodes placed on the backside.

[0074] The electrode pattern may be formed by printing Ag, but the electrode material may be other than Ag, such as noble metals as Au, Pt, Pd, etc., or base metals such as Cu, etc. The electrode may be formed by printing, plating, or sputtering.

[0075] The piezoelectric material used for the vibrating body was PbZrTiO3 (PZT), but considering environmental regulations, a lead-free piezoelectric material that does not contain substantial Pb is preferable. Examples of lead-free piezoelectric materials include BaTiO3 (BT), NaNbO3, BiNaTiO3, and BiFeO3 as their main components, and combinations of these materials or addition of metal elements to the main components are also acceptable. In particular, BaTiO3 (BT)-based materials have been confirmed to have vibration performance equivalent to that of PbZrTiO3 (PZT). Materials other than ceramics, such as single crystal materials and polymer-based piezoelectric materials, are also acceptable.

[0076] The thickness of the piezoelectric material in the vibrating body is such that when the bonded and stacked piezoelectric material and the diaphragm flex and vibrate, the midpoint of the thickness direction of the flexure, that is, the neutral plane that is neither tensile nor compressed when flexing, is located on the diaphragm side, in order to efficiently utilize the distortion of the piezoelectric element for flexure.Therefore, the combination of the thickness of the piezoelectric material and the diaphragm was calculated based on the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm.

[0077] Fig. 4 shows an example of applying ultrasonic vibrations to a culture substrate using a piezoelectric material. In Fig. 4, a vibration applying mechanism 4102 comprises a vibrating body 4101, a vibration transmission member 413, and a cover 415, and the vibrating body 4101 comprises a diaphragm 411 and a piezoelectric material 412. A culture substrate 421 is placed on the vibration transmission member 413. In this example, the culture substrate 421 is covered with a culture substrate lid 422, and a weight 414 is placed on top of the culture substrate lid 422. By covering the culture substrate with the culture substrate lid 422 and further placing a weight 414 in this way, ultrasonic vibrations may be applied efficiently.

[0078] (Vibration source amplitude evaluation) The amplitude of the vibration source of the ultrasonic vibration generator can be measured using a laser Doppler vibrometer (Graphtec, AT7200). The measurement method involves driving the ultrasonic vibration generator at the desired resonant frequency and voltage, scanning the transducer surface two-dimensionally in the X and Y directions with a laser, obtaining the vibration velocity at each point, and then calculating the amplitude in the Z direction. This allows the vibration waveform of the transducer to be measured three-dimensionally, and the maximum amplitude of the vibration source when driven can be determined. For a bolt-tightened Langevin transducer, as shown in Figure 5A, two annular piezoelectric elements P1 and P2 are arranged on either side of electrodes E1 and E2, respectively, and are further sandwiched between two metal blocks M1 and M2, and fastened axially with bolt 500. The amplitude of the vibration source is the amplitude on the surfaces of M1 and M2. 5B, in a flat vibrator (attached vibrator) in which a piezoelectric element is attached to a glass plate or a metal plate with adhesive or the like, the amplitude is the amplitude of the glass plate 501 or the metal plate 502.

[0079] (cell) The cells in the present disclosure are not particularly limited as long as they can be cultured in vitro on a culture vessel. For example, various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), human alveolar basal epithelial adenocarcinoma-derived A549 cells, mouse macrophage-like cells (RAW264.7), and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile cells, and the like, can be used. Examples of cells that can be detached include skeletal muscle cells, smooth muscle cells, cardiac muscle cells, neuronal cells that constitute the nervous system, glial cells, and fibroblasts; hepatic parenchymal cells involved in the metabolism of living organisms; non-hepatic parenchymal cells and adipocytes; and cells with differentiation potential, such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, as well as progenitor cells of various tissues, and cells induced to differentiate therefrom. The cell detachment method disclosed herein may also be applied to sheet-like cells. Among these, the cell detachment method disclosed herein is suitable for cells with strong intercellular bonds, cells with high adhesion to culture substrates, and cells with high trypsin sensitivity. 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.

[0080] (Culture substrate) The culture substrate in the present disclosure refers to a culture substrate used for cell culture. The culture substrate is not particularly limited as long as it is a cell-adherent culture substrate, and examples thereof include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, multi-well plates, multi-plates, Petri dishes, culture bags, bottles, etc.

[0081] The material of the culture substrate in the present disclosure may be any material that is chemically stable and capable of culturing the desired cells, and examples thereof include polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(meth)acrylic acid, poly(meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, glass, metal, etc. Among these, polystyrene is preferred.

[0082] (Cell detachment solution) In the present disclosure, a cell detachment solution is a solution used in the cell detachment method of the present disclosure for detaching cells.

[0083] The pH of the cell detachment solution of the present disclosure is preferably in the neutral range. This is because the neutral range is suitable for cell culture and can maintain a stable high cell viability. The pH can be adjusted appropriately with hydrochloric acid, sodium hydroxide, or the like. In addition, various buffer solutions are preferably used to maintain a stable pH.

[0084] The viscosity of the cell detachment solution in the present disclosure is preferably 1.80 mPa·s or less. This is because a viscosity in this range does not impede the flow of the detachment solution generated by ultrasonic vibrations, thereby maintaining high detachment efficiency. The viscosity of the cell detachment solution can be adjusted as appropriate by adding polymers or sugars, for example.

[0085] The cell detachment solution of the present disclosure may contain a protease, but preferably the amount of the protease relative to the total mass of the cell detachment solution is 0.0005% by mass or less, and more preferably the cell detachment solution is protease-free. This is because protease partially degrades cells, thereby increasing detachment efficiency, but may also reduce the quality of the cells.

[0086] In the present disclosure, proteolytic enzymes include, for example, enzymes that degrade parts of cells and make it easier to detach the cells from the culture substrate, such as trypsin, accutase, collagenase, natural proteases, chymotrypsin, elastase, papain, pronase, or recombinant forms thereof.

[0087] As the cell detachment solution in the present disclosure, a solution containing a metal ion chelating agent (hereinafter, sometimes referred to as a chelating agent) is particularly preferred, because by using a cell detachment solution containing a chelating agent, cells can be effectively detached by vibration in the ultrasonic band.

[0088] The chelating agent in the present disclosure is not particularly limited, and examples thereof include ethylenediaminetetraacetic acid (hereinafter sometimes referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycoletherdiaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediaminedisuccinic acid, etidronic acid, citric acid, gluconic acid, phosphonobutanetriacetic acid, etc. 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 the 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. This is because a higher pH within the neutral range, which can maintain high cell viability, can enhance the chelating ability of ethylenediaminetetraacetic acid and make it possible to further increase the detachment efficiency. Chelating agents may be used alone or in combination of two or more types.

[0089] The content of the chelating agent is preferably 0.01 mM to 5.0 mM, inclusive, which ensures a chelating effect and prevents a decrease in activity due to the presence of an excess chelating agent.

[0090] The cell detachment solution of the present disclosure 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. This is because the polymer has little effect on cells and can suppress the thickening effect of the culture medium caused by the polymer.

[0091] (buffer) The buffer solution in the present disclosure can be used without limitation as long as it can maintain a neutral pH range. Examples include Tris buffer solutions such as Tris-HCl buffer solutions, phosphate buffer solutions, HEPES buffer solutions, citrate-phosphate buffer solutions, glycylglycine-sodium hydroxide buffer solutions, Britton-Robinson buffer solutions, and GTA buffer solutions. Among these, phosphate buffer solutions that are close to 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.

[0092] (Culture medium) The type of culture medium is not particularly limited, and examples thereof include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemSpan H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma-Aldrich), Endothelial Cell Growth Examples include Medium 2 Kit (Promocell), Mesenchymal Stem Cell Growth Medium 2 (Promocell), MSCGM Bullet Kit (Lonza), mTeSR1 or 2 medium (Stem Cell Technology), Repro FF or Repro FF2 (ReproCell), NutriStem medium (Biological Industries), and MF-Medium mesenchymal stem cell growth medium (Toyobo Co., Ltd.). Among these, it is preferable to use a medium suitable for culturing each cell.

[0093] (serum) The above-mentioned medium may contain serum or antibiotics. Examples of serum include fetal bovine serum (FBS), baby 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, the medium may contain neither raw nor unpurified serum, but may contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0094] (antibiotics) Examples of antibiotics added to the medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, amphotericin B, and the like.

[0095] (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 the culture substrate, 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 occupies about 70 to 80% of the surface area of ​​the culture substrate, that is, until the cells reach a subconfluent state.

[0096] (Cell detachment system) In a further embodiment, the present disclosure provides a cell detachment system. The cell detachment system of the present disclosure comprises: A cell detachment system having a detachment unit that detaches adherent cells cultured on a culture substrate, and a control unit, The peeling unit has a vibration applying unit that applies vibrations in an ultrasonic band to the culture substrate, and a shear force applying unit that applies shear force to the culture surface of the culture substrate, The cell detachment system is characterized in that the control unit controls the start of driving of the vibration applying unit to occur before the start of driving of the shear force applying unit. In addition, the cell detachment system of the present disclosure includes: The shear force applying unit applies shear force to the culture surface by at least one means selected from the group consisting of beating, shaking, rocking, and eccentric rotation applied to the culture substrate.

[0097] The control unit of the cell detachment system disclosed herein has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit so as to change the magnitude of the shear force over time.

[0098] The control unit of the cell detachment system of the present disclosure has a shear force control unit that controls the shear force application unit, and the shear force control unit is characterized by controlling the shear force application unit so as to periodically change the magnitude of the shear force.

[0099] The control unit of the cell detachment system disclosed herein has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit to change the direction of the applied shear force based on at least one selected from the group consisting of the size, shape, adhesion area, and brightness value of the cell.

[0100] The control unit may be integrated with the shear force control unit, i.e., may control the vibration applying unit so that the start of drive of the vibration applying unit occurs before the start of drive of the shear force applying unit, and may also control the shear force applying unit. Alternatively, the control unit may be separated into a part that controls the shear force control unit so that the start of drive of the vibration applying unit occurs before the start of drive of the shear force applying unit, and a part that controls the shear force control unit.

[0101] For example, the shear force applying unit may be provided with a shear force control unit separate from an information processing device that controls the start of driving of the vibration applying unit to occur before the start of driving of the shear force applying unit.

[0102] 6 and 7 show examples of the cell detachment system of the present disclosure. The cell detachment system 61000 illustrated in FIG. 6 includes a detachment unit 61001 that detaches adherent cells cultured on a culture substrate 621, and a control unit 61002. The detachment unit 61001 includes a vibration applying unit 6102 that applies ultrasonic vibrations to the culture substrate 621, and a shear force applying unit 6103 that applies shear force to the culture surface of the culture substrate. The control unit 61002 controls the vibration applying unit 6102 so that the start of driving occurs before the start of driving the shear force applying unit 6103. In the example of FIG. 6, the vibration applying unit 6102 constituting the detachment unit 61001 includes a vibrating body 6101, a vibration transmission member 613, and a cover 615. The vibrating body 6101 includes a vibrating plate 611 and a piezoelectric body 612. The shear force applying unit 6103 includes a tapping unit 617 and a tapping member 616. The control unit 61002 is made up of a vibration control unit 619 that controls the vibration applying unit 6102, a shear force control unit 618 that controls the shear force applying unit, and a main control unit 620. The vibration control unit 619, shear force control unit 618, and main control unit 620 may be integrated or separated. Fig. 6 further shows an example in which the culture substrate 621 is covered with a culture substrate top cover 622, and a weight 614 is placed on top of it.

[0103] The cell detachment system 7001 illustrated in Figure 7 is a cell detachment system having a detachment unit 71001 that detaches adherent cells cultured on a culture substrate 721, which is a dish in this example, a control unit 71002, and a cell information acquisition unit 71003. The peeling unit 71001 has a vibration applying unit 7102 that applies ultrasonic vibrations to the culture substrate 721 held by the holding member 722, and a shear force applying unit 7103 that applies shear force to the culture surface of the culture substrate. In this example, the shear force applying unit 7103 is a biaxial vibration mechanism. The cell information acquisition unit 71003 has a mechanism for acquiring cell information about cells adhered to the culture substrate. The cell information acquired by the cell information acquisition unit 71003 may be processed by the control unit 71002, and the control unit 71002 may control the detachment unit 71001 based on the cell information. The acquired cell information is used to execute the information acquisition step (step S220) and can be used, for example, to determine whether to transition from the vibration application step to the shear force application step, or to determine whether to terminate the vibration application step or the shear force application step. In this example, the control unit 71002 also functions as a computer that performs calculations and storage.

[0104] The control unit 71002 includes a CPU (Central Processing Unit) 706, a RAM (Random Access Memory) 707, a ROM (Read Only Memory) 708, and an HDD (Hard Disk Drive) 709 to realize the functions of a computer that performs calculations and storage. The control unit 71002 also includes a communication I / F (Interface) 710, a display device 711, and an input device 712. The CPU 706, RAM 707, ROM 708, HDD 709, communication I / F 710, display device 711, and input device 712 are connected to one another via a bus 713. The display device 711 and input device 712 may be connected to the bus 713 via a drive device (not shown) for driving these devices.

[0105] 7, the components constituting the control unit 71002 are illustrated as an integrated device, but some of these functions may be configured by external devices. For example, the display device 711 and the input device 712 may be external devices separate from the components constituting the functions of the computer, including the CPU 706, etc.

[0106] The CPU 706 performs predetermined operations in accordance with programs stored in the RAM 707, HDD 709, etc., and also has the function of controlling each part of the control unit 71002. The RAM 707 is made up of a volatile storage medium and provides a temporary memory area necessary for the operation of the CPU 706. The ROM 708 is made up of a non-volatile storage medium and stores necessary information such as programs used in the operation of the information processing system 705. The HDD 709 is made up of a non-volatile storage medium and is a storage device that stores information regarding the number and positions of individual independent separation compartments, fluorescence intensity, etc.

[0107] The communication I / F 710 is a communication interface based on standards such as Wi-Fi (registered trademark) and 4G, and is a module for communicating with other devices. The display device 711 is a liquid crystal display, an OLED (Organic Light Emitting Diode) display, or the like, and is used to display moving images, still images, characters, and the like. The input device 712 is a button, a touch panel, a keyboard, a pointing device, or the like, and is used by a user to operate the information processing system 705. The display device 711 and the input device 712 may be integrally formed as a touch panel.

[0108] The hardware configuration shown in FIG. 7 is an example, and other devices may be added, or some devices may not be provided. Also, some devices may be replaced with other devices having similar functions. Furthermore, some functions may be provided by other devices via a network, or the functions constituting this embodiment may be distributed and realized among multiple devices. For example, the HDD 709 may be replaced with an SSD (Solid State Drive) using semiconductor elements such as flash memory, or may be replaced with cloud storage.

[0109] The CPU 706 loads a program stored in the ROM 708 or the like into the RAM 707 and executes it, thereby realizing control so that the start of driving of the vibration applying unit occurs before the start of driving of the shear force applying unit, and also realizing control of the vibration applying unit and the shear force applying unit. The CPU 706 also controls the display device 711 to realize the function of the display unit 711. The CPU 706 also controls the HDD 709 to realize the function of the storage unit. [Example]

[0110] The present disclosure will be explained in more detail below with reference to examples and comparative examples, but the present invention is not limited thereto in any way. The cells used in the examples of the present disclosure were obtained according to the following culture conditions. (Cultivation of CHO cells on a culture substrate) CHO-K1 (Chinese Hamster Ovary) cells were cultured on a Φ60 polystyrene dish (manufactured by Corning) at a density of 10,000 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). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupation rate on the culture substrate was approximately 80%.

[0111] (Culture of A549 cells on a culture substrate) Human alveolar basal epithelial adenocarcinoma cells, A549 cells, were cultured on a Φ60 polystyrene dish (manufactured by Corning) at 10,000 cells / cm. 2The 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 occupation rate on the culture substrate was approximately 80%.

[0112] (Culture of hMSC cells on a culture substrate) Human mesenchymal stem cells (hMSCs) were cultured at 4,000 cells / cm on a Φ60 polystyrene dish (Corning), an example of a culture substrate. 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 culture substrate was approximately 80%.

[0113] (Culture of mouse macrophage-like cells on a culture substrate) Mouse macrophage-like cells (RAW264.7) were cultured on a Φ60 polystyrene dish (Corning) at 20,000 cells / cm. 2The 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 occupation rate on the culture substrate was approximately 80%.

[0114] (Replacement with stripping solution) The medium in the culture substrate was removed, and phosphate buffered saline (PBS(-), manufactured by Thermo Fisher Scientific) was added to the culture substrate as a detachment solution.

[0115] (Adhesion strength evaluation method) An adhesion strength evaluation device 1700 was fabricated by combining a silicone tube 1701, a tube pump 1702, a reservoir tank 1703, a three-way valve 1704, a needle 1705, and a nozzle 1707 as shown in Figure 8. A culture substrate 1706 on which cells had been cultured was positioned so that the tip of the nozzle 1707 (1.04 mm inner diameter) was 17 mm away from the culture surface. PBS was sprayed perpendicularly from the nozzle 1707 at 0.65 mL / s for 3 seconds to apply shear force to the culture surface. After the spraying, the PBS was removed from the culture substrate, and the position of the boundary of the cells detached by the spraying (the size of the detached circle) was observed. The shear force generated at that position was calculated using Ansys Fluent and used as the adhesion strength.

[0116] (Cell adhesion area evaluation method) The culture substrate was placed under a phase-contrast microscope, and phase-contrast images were observed at a magnification of 40. The projected areas of the cells within the field of view of the phase-contrast images were calculated, and the average value was taken as the cell adhesion area.

[0117] (Method for evaluating cell brightness information) The culture substrate was set on a stage, and the average brightness value was calculated from an image of the culture surface of the dish observed under oblique incidence illumination, and this was used as brightness information of the cells.

[0118] (Each device) For the application of vibration and shear force, the vibration applying mechanisms of devices 1 to 8 or the shear force applying mechanisms of shaking, beating, rocking, vortex stirring, and jet flow were used, respectively.

[0119] The devices 1 to 9 used are shown in Figs. 9 to 17, respectively. 9, the device 1 is a device having a vibration imparting mechanism, and has a holding mechanism 11 for holding a culture substrate 10 and an ultrasonic band vibration imparting mechanism 12. Details of the ultrasonic band vibration imparting mechanism 12 are shown in Table 1.

[0120] As shown in FIG. 10, the device 2 is a device having a shear force applying mechanism, and has a holding mechanism 11 for holding the culture substrate 10 and a biaxial shaking mechanism 131.

[0121] 11, the apparatus 3 is an apparatus having a vibration imparting mechanism and a shear force imparting mechanism, and has a holding mechanism 11 for holding the culture substrate 10, an ultrasonic band vibration imparting mechanism 12, and a biaxial shaking mechanism 131. Details of the ultrasonic band vibration imparting mechanism 12 are shown in Table 1.

[0122] As shown in FIG. 12, the device 4 is a device having a shear force applying mechanism, and has a holding mechanism 11 for holding the culture substrate 10, and a rotating mechanism 132 and a beating mechanism 133 for applying shear force.

[0123] As shown in Fig. 13, the device 5 is an apparatus having a vibration imparting mechanism and a shear force imparting mechanism, and has a holding mechanism 11 for holding the culture substrate 10, an ultrasonic band vibration imparting mechanism 12, and a rotation mechanism 132 and a beating mechanism 133 for imparting shear force. Details of the ultrasonic band vibration imparting mechanism 12 are shown in Table 1.

[0124] As shown in FIGS. 14A and 14B, the device 6 is a device having a shear force applying mechanism, and includes a holding mechanism 11 for holding the culture substrate 10 and a swinging mechanism 134.

[0125] 15A and 15B, the apparatus 7 is an apparatus having a shear force applying mechanism, and includes a holding mechanism 11 for holding a culture substrate 10, a jet mechanism 135 for applying shear force, an XY moving stage 14, and a Z moving stage 15. Using the stage movement, jets were applied to the culture substrate at 10 mm intervals, as shown in FIG.

[0126] As shown in FIG. 16, the device 8 is a device having a shear force applying mechanism, and has a holding mechanism 11 for holding the culture substrate 10 and a flow mechanism 136 for applying shear force.

[0127] (Method of applying ultrasonic vibration) The ultrasonic devices shown in Table 1 were used as the ultrasonic band vibration imparting mechanisms for each of Apparatus 1 (FIG. 9), Apparatus 3 (FIG. 11), and Apparatus 5 (FIG. 13), and the culture substrate was set on the holding mechanism installed on the ultrasonic device. Then, at an ambient temperature of 37°C, each ultrasonic device was driven under the vibration imparting conditions shown in Table 1 to impart ultrasonic band vibrations.

[0128] In this embodiment, when the ring device is used in standing wave mode, bending vibrations can be applied in a vibration mode in which the maximum amplitude acts on the center of the culture substrate in the used frequency band.When the ring device is used in traveling wave mode, bending vibrations can be applied in a vibration mode in which the maximum amplitude acts on a position 2 / 3 of the way from the center with respect to the outer diameter of the culture substrate in the used frequency band.Furthermore, by using the traveling wave mode, these bending vibrations can be applied in a vibration that rotates circumferentially around the center.When a Langevin device is used, vibrations with a nearly flat vibration distribution in which the center is the maximum amplitude can be applied to the entire area of ​​the culture substrate.

[0129] Table 1 below shows vibration conditions 1 to 8, which are the conditions used in the ultrasonic vibration applying mechanisms of devices 1, 3, and 5.

[0130] [Table 1]

[0131] (Method of applying shear force by shaking) Apparatus 2 (FIG. 10) and apparatus 3 (FIG. 11) had a biaxial shaking mechanism 131 as a shear force application mechanism. Apparatuses 2 and 3 were equipped with a holding mechanism for holding the culture substrate on the biaxial shaking mechanism, which could apply shear force by shaking using an automatic stage that moved independently in the x and y directions, or an ultrasonic vibration application mechanism with a holding mechanism already installed. The culture substrate was placed on the holding mechanism, and the biaxial shaking mechanism was operated at an ambient temperature of 37°C under the driving conditions shown in Table 2 to apply shear force.

[0132] Table 2 below shows movement conditions 1 to 4, which are the conditions used in the two-axis shaking mechanisms of devices 2 and 3. [Table 2]

[0133] (Method of applying shear force by beating) The device 4 (FIG. 12) or the device 5 (13) had a rotation mechanism 132 and a beating mechanism 133 as a shear force applying mechanism.

[0134] Apparatus 4 and Apparatus 5 combined a beating mechanism capable of applying shear force by beating, a culture substrate holding mechanism, a rotation mechanism capable of rotating the holding mechanism, and an ultrasonic vibration mechanism. Using this apparatus, the culture substrate was placed in the holding mechanism, and then shear force was applied to the outer periphery of the culture substrate at an ambient temperature of 37°C by varying the force, frequency, and force application position as shown in Table 3.

[0135] Table 3 below shows drive conditions 1 to 3, which are drive conditions for the rotation mechanisms of devices 4 and 5. [Table 3]

[0136] (Other methods of applying shear force) As other shear force applying mechanisms, device 6 had a rocking mechanism as shown in Figures 14A and 14B, device 7 had a jet mechanism 135 as shown in Figures 15A and 15B, and device 8 had a flow mechanism 136 as shown in Figure 16.

[0137] The shear force was applied by the following methods. For the rocking, device 6 was used, in which a culture substrate holding mechanism was installed on a rocking mechanism using a stage that moves on an arc with the center as a fulcrum, as shown in FIG. 14A.

[0138] An image of the stage movement during rocking is shown in Figure 14B. After using this device to set the culture substrate in the holding mechanism, shear force was applied at an ambient temperature of 37°C at the speed and with the operation shown in Operating Condition 1 in Table 4.

[0139] For eccentric rotation, a vortex mixer was used. The vortex mixer used was a culture substrate holding mechanism, in which the sample mounting section of a Vortex Genie 2 (SI-0286; Scientific Industries) had been modified to accommodate the culture substrate. The culture substrate was set and secured in the holding mechanism, and shear force was applied at the speed and operation shown in Operating Condition 2 in Table 4 at an ambient temperature of 37°C. Because the speed varied depending on factors such as the load weight on the mixer, a culture substrate containing the same amount of liquid was set on the holding mechanism in advance, and the mixer operating conditions were determined to achieve the set speed by observing it with a high-speed camera.

[0140] For the jet flow experiment, we used Apparatus 7, which was equipped with a culture substrate holding mechanism mounted on an XY-moving stage capable of moving in the X and Y directions, as shown in Figure 15A. This apparatus also included a Z-moving stage equipped with a jet flow mechanism using an electric pipettor. Using this apparatus, the culture substrate was placed in the holding mechanism. At an ambient temperature of 37°C, the pipette volume was set to 1 mL for each position on the culture substrate corresponding to the top view of the culture substrate shown in Figure 15B. Shear force was applied at the speed and operation specified in Operating Condition 3 in Table 4. For each position, suction and discharge were repeated twice over a 3-second period, followed by 1 second of movement to the next position, and the same operation was repeated. For example, there are a total of 21 positions corresponding to the above in the field of view shown in Figure 15B. If a jet flow were applied to all of these positions, the shear force application process would take 83 seconds.

[0141] For the flow, device 8 was used, and a flow mechanism 136 that can be installed on the culture substrate as shown in Figure 16 was used, and the device was connected to a pump that can set the flow rate to make the liquid flow. Using this device, the culture substrate was set in the holding mechanism, and shear force was applied at an ambient temperature of 37°C by performing the speed and operation shown in Operating Condition 4 in Table 4.

[0142] Table 4 below shows the operations under operating condition 1 (oscillation by device 6), operating condition 2 (eccentric rotation by vortex), operating condition 3 (jet by device 7), and operating condition 4 (flow by device 8).

[0143] [Table 4]

[0144] (Removal method) Peeling methods 1 to 33 were performed using the above-mentioned devices 1 to 8, with steps [1] and [2] set under the conditions shown in Table 5 below. Step [1] was started before step [2]. Details of each peeling method are described below.

[0145] [Table 5]

[0146] [Peeling Method 1, 8-12, 15-17, 20-27] The culture medium in the dish in which the cells were cultured was replaced with the detachment solution, and then the dish was set on the holding mechanism of an ultrasonic vibration imparting device, i.e., device 1, as shown in Table 5. For the detachment operation in step [1], ultrasonic vibration was applied to the dish under the conditions and for the duration shown in Table 5. Then, for the detachment operation in step [2], the dish was shaken when set on the holding mechanism of each shear force imparting device mechanism shown in Table 5, and it was confirmed that cell detachment was not complete in the detachment operation in step [1].

[0147] Next, for the peeling operation of step [2], a shear force was applied under each of the conditions shown in Table 5, starting 30 seconds after the application of ultrasonic vibrations was completed, using the mechanism of each shear force application device shown in Table 5.

[0148] [Peeling methods 2, 5-7, 13, 18, 19] The culture medium in the dish in which the cells were cultured was replaced with the detachment solution, and then the dish was set on the holding mechanism of either Apparatus 3 or Apparatus 5, which combines an ultrasonic vibration device and a shear force device, as shown in Table 5. As the detachment operation of step [1], ultrasonic vibration was applied to the dish under the conditions and for the duration shown in Table 5.

[0149] Next, before starting step [2], it was visually confirmed that cell detachment had not been completed in the detachment operation of step [1], and then, as the detachment operation of step [2], each shear force application device shown in Table 5 was operated under each condition to apply shear force, starting 30 seconds before the end of application of ultrasonic vibration.

[0150] [Removal method 3] The culture medium in the dish in which the cells were cultured was replaced with the detachment solution, and then the dish was placed on the holding mechanism of device 3, which combines an ultrasonic vibration device and a shear force device, as shown in Table 5. As the detachment operation of step [1], ultrasonic vibration was applied to the dish under the conditions and for the duration shown in Table 5. In this method, immediately after the application of vibration in step [1] was completed, the detachment operation of step [2] was performed under the conditions shown in Table 5 to apply shear force. After the detachment operation of step [2] was completed, the detachment operation of step [1] was performed, and these were repeated a total of six times.

[0151] [Removal method 4] The culture medium in the cell culture dish was replaced with the detachment solution, and then the dish was placed on the holding mechanism of device 3, which combines an ultrasonic vibration device and a shear force device, as shown in Table 5. For the detachment operation in step [1], ultrasonic vibration was applied to the dish under the conditions and for the duration shown in Table 5. This detachment method involves repeating the detachment operation in step [2] while continuing to apply vibration in step [1]. Twenty seconds after the start of the detachment operation in step [1], the detachment operation in step [2] was performed under the conditions shown in Table 5 to apply shear force.

[0152] [Removal method 14] The culture medium in the cell-cultured dish was replaced with the detachment solution. Then, as shown in Table 5, the dish was rocked for 30 seconds at room temperature (25°C) using the rocking mechanism of Apparatus 6 (Figures 14A and 14B) to thoroughly soak the detachment solution in the pre-operation step [1]. The dish was then placed on the holding mechanism of the ultrasonic vibration applicator (Apparatus 1) shown in Table 5, and ultrasonic vibrations were applied to the dish under the conditions and for the duration shown in Table 5 for the detachment operation in step [1]. Then, for the detachment operation in step [2], the dish was rocked when placed on the holding mechanism of the shear force applicator mechanism shown in Table 5, confirming that cell detachment was not complete in the detachment operation in step [1].

[0153] Next, for the peeling operation of step [2], a shear force was applied under each of the conditions shown in Table 5, starting 30 seconds after the application of ultrasonic vibrations was completed, using the mechanism of the shear force application device shown in Table 5.

[0154] [Removal method 28] The culture medium in the cell-cultured dish was replaced with the detachment solution, and then, as a pre-operation for step [1], the dish was left to stand in a 37°C incubator for 2 minutes to allow the detachment solution to fully soak in, as shown in Table 5. Next, the dish was placed on the holding mechanism of the ultrasonic vibration imparting device shown in Table 5, i.e., device 1, and ultrasonic vibration was applied to the dish under the conditions and for the application time shown in Table 5 as the detachment operation for step [1]. Thereafter, for the detachment operation for step [2], the dish was shaken when placed on the holding mechanism of the shear force imparting device shown in Table 5, i.e., device 2, to confirm that cell detachment was not complete in the detachment operation for step [1].

[0155] Next, for the peeling operation of step [2], a shear force was applied under each of the conditions shown in Table 5, starting 30 seconds after the application of ultrasonic vibrations was completed, using the shear force application device shown in Table 5, i.e., device 2.

[0156] [Removal method 29] The culture medium in the dish in which the cells were cultured was replaced with the detachment solution, and then the dish was set on the holding mechanism of Apparatus 2, a shear force applying device, as shown in Table 5. For the detachment operation in step [1], shear force was applied to the dish under the conditions and for the duration shown in Table 5. Then, for the detachment operation in step [2], the dish was shaken when set on the holding mechanism of Apparatus 1, an ultrasonic vibration applying device shown in Table 5, and it was confirmed that some cells had detached during the detachment operation in step [1].

[0157] Next, for the peeling operation of step [2], ultrasonic band vibration was applied under each of the conditions shown in Table 5 using the ultrasonic band vibration application mechanism shown in Table 5, i.e., device 1, starting 30 seconds after the application of shear force was completed.

[0158] [Peeling Method 30, 31] The culture medium in the dish in which the cells were cultured was replaced with the detachment solution, and then the dish was placed on the holding mechanism of each shear force applying device, i.e., Device 2 or Device 4, as shown in Table 5. As the detachment operation in step [1], shear force was applied to the dish under the conditions and for the application time shown in Table 5. Step [2] was not performed.

[0159] [Peeling Method 32, 33] The culture medium in the dish in which the cells were cultured was replaced with the detachment solution, and then the dish was set on the holding mechanism of the ultrasonic vibration imparting device, i.e., Device 1, as shown in Table 5. As the detachment operation of step [1], ultrasonic vibration was imparted to the dish under the conditions and for the duration shown in Table 5. Step [2] was not performed.

[0160] [Cell information acquisition method 1] Cell adhesion was evaluated using the following method. Three additional dishes in which cells had been cultured under the same conditions were prepared for cell information acquisition, and the culture medium in the dishes was replaced with a detachment solution. One of the dishes was sprayed with PBS under the conditions described above using the adhesion evaluation device 1700 shown in Figure 8, as described in the adhesion evaluation method. After the PBS was removed from the culture substrate, the boundary position of the cells detached by the spraying was observed, and the shear force generated at that position was calculated using Ansys Fluent to determine adhesion A1. When sprayed using the adhesion evaluation device, cells on the culture surface of the dish detach in an approximately circular shape centered on the spraying position. The boundary position of the detached cells, which is a circular boundary, can also be referred to as the detachment circle. In the examples of the present disclosure, the shear force was calculated by averaging the vertical diameters of the obtained detachment circles. Next, the detachment operation of step [1] was performed on each of the remaining two dishes according to the detachment method described above. Just before starting step [2], the adhesion of one of the dishes was evaluated using the adhesion evaluation device in the same manner as described above. The adhesive strength at this time was designated as A2.

[0161] For the remaining dish, the peeling operation of step [2] was carried out, and then the adhesive strength was evaluated in the same manner as above using the adhesive strength evaluation device. The adhesive strength at this time was designated as A3.

[0162] In this embodiment, the adhesive force can be evaluated within the range of 1 to 80 Pa under the above-mentioned conditions. When PBS is injected during the measurement of the adhesive force, if the boundary of the peeling circle generated by the injection is not detected, or when the adhesive force evaluated by the small diameter of the peeling circle is 80 Pa or more, the adhesive force is regarded as 80 Pa or more. When the diameter of the peeling circle becomes large and the adhesive force is calculated as less than 1 Pa, it is uniformly regarded as less than 1 Pa. Regarding the magnitude relationship of the adhesive force when it is less than 1 Pa, it was determined that the larger the diameter of the peeling circle generated by the injection, the smaller the adhesive force. It was confirmed whether the relationship A1 > A2 > A3 was satisfied from the obtained adhesive forces A1, A2, and A3.

[0163] [Cell information acquisition method 2] The evaluation of the cell adhesion area was performed by the following method. After replacing the culture medium in the dish in which the cells were cultured with a stripping solution, it was set on a phase-contrast microscope (Axio Obserer 7; manufactured by Carl Zeiss) as described in the above-mentioned cell adhesion area evaluation method, and phase-contrast observation was performed. The average value was calculated from the sum of the projected areas of the cells in the field of view at a magnification of 40 times, and this was taken as the cell adhesion area B1. Next, the peeling operation of step [1] was performed according to the above-mentioned peeling method, and phase-contrast observation was performed with a phase-contrast microscope immediately before starting step [2], and the projected area of the cells was calculated as before. This value was taken as the cell adhesion area B2. Next, after performing the peeling operation of step [2], phase-contrast observation was performed with a phase-contrast microscope, and the projected area of the cells was calculated as before. Regarding the cells whose contours were blurred at this time, they were regarded as not adhered and floating, and the adhesion area was regarded as 0 and averaged. This value was taken as the cell adhesion area B3. It was confirmed whether the relationships B2 < 0.5 × B1 and 0 ≤ B3 < B2 were satisfied from the obtained cell adhesion areas B1, B2, and B3.

[0164] [Cell information acquisition method 3] The brightness information of the cells was evaluated by the following method. After replacing the culture medium in the dish on which the cells were cultured with the detachment solution, optical measurement was performed as in the above-described method for evaluating the brightness information of the cells. That is, the dish was set on the stage, oblique incident illumination was applied to the upper surface of the dish, and the camera was set on the lower surface of the dish to obtain an image of the culture surface of the dish. The average brightness value of the culture surface was calculated from the image thus obtained and used as the brightness information C1 of the cells. Next, the detachment operation of step [1] was performed according to the above-described detachment method. Immediately before starting step [2], the dish was set on the stage in the same manner as in the previous stage, and the average brightness value was calculated from the image of the dish culture surface observed by oblique incident illumination. This value was used as the brightness information C2 of the cells. From the obtained brightness information C1 and C2 of the cells, it was confirmed whether the relationship C1 < C2 was satisfied.

[0165] [Example 1] Using detachment method 1, CHO cells on the dish were detached. After the detached cells were collected, the cell count was measured using a hemocytometer, and the survival rate was calculated using the method for determining life and death by trypan blue staining. In addition, for the dish after ultrasonic detachment, all the cells that could not be detached by ultrasonic treatment were detached using a cell scraper, and the cell count was measured using a hemocytometer. The total number of detached cells was obtained by adding the number of cells detached by ultrasonic treatment and the number of cells detached by the cell scraper thereafter, and the ratio of the number of cells detached by ultrasonic treatment to the total number of detached cells was defined as the detachment rate and calculated. As a result, the survival rate was 96.0% and the detachment rate was 98.0%. In addition, both the survival rate and the detachment rate were judged to be good at 90% or more. The cell type used, the detachment method, the calculated survival rate, and the detachment rate are shown in Table 6.

[0166] In addition, in Example 1, among the above-described cell information, the adhesion force, the adhesion area, and the brightness information were obtained. The adhesion force was obtained according to the method shown in the above-described cell information acquisition method 1. The adhesion force A1 was evaluated using the above-described adhesion force evaluation apparatus after replacing the culture solution in the dish with the stripping solution. Since no peeling circle where the cells were peeled off by injection was observed, A1 > 80 Pa. The adhesion force A2 was evaluated immediately before starting step [2]. Since the diameter of the peeling circle by injection was large, A2 < 1 Pa. The adhesion force A3 was evaluated after the completion of step [2]. Since the diameter of the peeling circle was large as described above, A3 < 1 Pa. Since the diameter of the peeling circle at the time of evaluating A2 and A3 was such that A3 > A2, it was determined that the relationship of the adhesion force was A2 > A3. From the above, it was confirmed that the relationship of the adhesion force in Example 1 satisfies the relationship of A1 > A2 > A3.

[0167] Regarding the adhesion area of the cells, the average area in the visual field was obtained according to the method shown in the above-described cell information acquisition method 2. The adhesion area B1 was evaluated after replacing the culture solution in the dish with the stripping solution, and B1 = 528 μm 2 It was. The adhesion area B2 was evaluated immediately before starting step [2], and it was 192 μm 2 It was. Similarly, the adhesion area B3 was evaluated after the completion of step [2]. Considering that most of the cells were peeled off, the average was 2 μm 2 It became. It was confirmed that these values satisfy the relationships of B2 < 0.5 × B1 and 0 ≦ B3 < B2.

[0168] Regarding the luminance information of the cells, it was obtained according to the method shown in the above-described cell information acquisition method 3. The luminance information C1 was evaluated after replacing the culture solution in the dish with the stripping solution, and C1 = 78.5. The luminance information C2 was evaluated immediately before starting step [2], and it was 108.1. It was confirmed that these values satisfy the relationship of C1 < C2.

[0169] [Examples 2 to 34, Comparative Examples 1 to 6] In Examples 2 to 34 and Comparative Examples 1 to 6, the combination of the cell type and the peeling method was changed, and the evaluation after peeling was performed in the same manner as in Example 1. The results are shown in Table 6.

[0170] Table 7 also shows the results of cell information obtained for each representative example of the present disclosure. From these results, a threshold value for cell information used to determine whether to proceed to the shear force application process can be determined. For example, the threshold value for brightness information, which can be obtained using a simple optical system, can be determined by referring to the smallest value in the brightness information column C2 in Table 7. In this example, if the cell type is CHO cells, the brightness information threshold can be set to 105. By monitoring the brightness information values ​​over time using the set threshold, it becomes possible to determine the timing to proceed to the shear force application process. Since the brightness information threshold may vary depending on the cell type and culture conditions, the threshold value can be set in advance using experimental data. The amount of change in brightness information per unit time can also be set as the threshold. Ultrasound intensity can also be determined based on the change in brightness information over time.

[0171] [Table 6]

[0172] [Table 7]

[0173] Embodiments of the present disclosure include the following methods and compositions. (Method 1) A method for detaching adherent cells cultured on a culture substrate, comprising: a vibration applying step of applying ultrasonic vibration to the culture substrate; A shear force application step of applying shear force to the culture surface of the culture substrate. Includes the vibration applying step starts before the shear force applying step starts, A method for detaching cells, wherein the shear force is applied by a means other than ultrasonic vibration. (Method 2) The shear force may be applied to the culture substrate by beating, shaking, rocking, and eccentric rotation; The cell detachment method described in Method 1, characterized in that the cell is applied by at least one method selected from the group consisting of discharging a liquid onto the culture substrate. (Method 3) The cell detachment method described in Method 1 or 2, wherein the ultrasonic vibration applied in the vibration application step is produced by a vibration source having a maximum amplitude of 10 μm or less. (Method 4) The adhesive force between the cells and the culture substrate at the start time of the vibration application step is A1 The adhesive force between the cells and the culture substrate at the start time of the shear force application step is A2 A cell detachment method described in any one of methods 1 to 3, characterized in that when the adhesive force between the cells and the culture substrate at the time when both the vibration application process and the shear force application process are completed is A3, the relationship between A1, A2 and A3 satisfies Equation 1. A1>A2>A3 (formula 1) (Method 5) The adhesion area between the cells and the culture substrate at the start time of the vibration application step is defined as B1 The adhesion area between the cells and the culture substrate at the start time of the shear force application step is defined as B2 5. The cell detachment method according to any one of methods 1 to 4, characterized in that when the adhesion area at the time when both the vibration application step and the shear force application step are completed is defined as B3, the relationship between B1, B2, and B3 satisfies Equation 2 and Equation 3. B2<0.5×B1 (formula 2) 0≦B3 <b2(式3)(Method 6) The brightness information obtained by optical measurement of the cell at the start time of the vibration application step is C1 The brightness information obtained by optical measurement of the cell at the start time of the shear force application step is C2 6. The cell detachment method according to any one of Methods 1 to 5, wherein the relationship between C1 and C2 satisfies Formula 4 when C1 <c2(式4)(Method 7) 7. The cell detachment method according to any one of Methods 1 to 6, wherein the magnitude of the shear force applied in the shear force application step changes over time. (Method 8) 8. The cell detachment method according to any one of Methods 1 to 7, wherein the magnitude of the shear force applied in the shear force application step is changed periodically. (Method 9) 9. The cell detachment method according to any one of methods 1 to 8, wherein the direction of the applied shear force changes over time in the shear force application step. (Method 10) 10. The cell detachment method according to any one of Methods 1 to 9, wherein the shear force application step includes a step of applying a plurality of shear forces in different directions. (Method 11) 11. The cell detachment method according to any one of methods 1 to 10, wherein in the shear force application step, the direction of the shear force to be applied is determined based on cell information. (Method 12) A cell information acquisition step of acquiring information on cells in a state adhered to the culture substrate; and a shear force application step start time determination step of determining a start time of the shear force application step based at least on the information of the cells; A cell detachment method described in any one of methods 1 to 11, characterized in that the cell information acquisition process and the shear force application process start time determination process are both performed before the start of the vibration application process. (Method 13) The cell detachment method described in Method 12, characterized in that the cell information includes at least one selected from the group consisting of the size, shape, adhesion area, and brightness value of the cell. (Method 14) In the shear force applying step, 14. The cell detachment method described in Method 12 or 13, characterized in that the conditions for applying the shear force are determined according to information about the cells. (Method 15) 15. The cell detachment method according to any one of Methods 1 to 14, wherein the vibration application step is carried out for a period of time ranging from 1 second to 1 hour. (Method 16) 16. The cell detachment method according to any one of Methods 1 to 15, wherein the shear force application step is carried out for a period of time of 1 second to 1 hour. (Method 17) 17. A cell detachment method according to any one of methods 1 to 16, characterized in that when the time during which the vibration application process is performed is a and the time during which the shear force application process is performed is b, the relationship between a and b satisfies Equation 5. 0.01≦a / b≦100 (formula 5) (Method 18) 18. A cell detachment method according to any one of methods 1 to 17, characterized in that the difference between the time when the vibration application process starts and the time when the shear force application process starts is 1 second or more and 1 hour or less. (Method 19) The cell detachment method described in Method 1, characterized in that the difference between the time when the vibration application step starts and the time when the shear force application step ends is 10 seconds or more and 30 minutes or less. (Configuration 1) A cell detachment system having a detachment unit that detaches adherent cells cultured on a culture substrate, and a control unit, The peeling unit has a vibration applying unit that applies vibrations in an ultrasonic band to the culture substrate, and a shear force applying unit that applies shear force to the culture surface of the culture substrate, The cell detachment system is characterized in that the control unit controls the start of driving of the vibration applying unit to occur before the start of driving of the shear force applying unit. (Configuration 2) The cell detachment system described in Configuration 1 is characterized in that the shear force applying unit applies shear force to the culture surface by at least one means selected from beating, shaking, rocking, and eccentric rotation applied to the culture substrate. (Configuration 3) The cell detachment system described in configuration 1 or 2, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit so as to change the magnitude of the shear force over time. (Configuration 4) The cell detachment system described in any one of configurations 1 to 3, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit so as to periodically change the magnitude of the shear force. (Configuration 5) A cell detachment system described in any one of configurations 1 to 4, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit so as to change the direction of the shear force over time. (Configuration 6) The cell detachment system described in any one of configurations 1 to 5, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit to change the direction of the shear force applied based on at least one selected from the group consisting of the size, shape, adhesion area, and brightness value of the cell.

Claims

1. A method for detaching adherent cells cultured on a culture substrate, comprising: a vibration applying step of applying ultrasonic vibration to the culture substrate; A shear force application step of applying shear force to the culture surface of the culture substrate. Includes the vibration applying step starts before the shear force applying step starts, A method for detaching cells, wherein the shear force is applied by a means other than ultrasonic vibration.

2. The cell detachment method according to claim 1, wherein the shear force is applied by at least one selected from the group consisting of beating, shaking, rocking, and eccentric rotation applied to the culture substrate, and discharging a liquid onto the culture substrate.

3. 2. The cell detachment method according to claim 1, wherein the ultrasonic vibration applied in the vibration application step is produced by a vibration source having a maximum amplitude of 10 μm or less.

4. The adhesive strength between the cells and the culture substrate at the start time of the vibration applying step is A1 The adhesive strength between the cells and the culture substrate at the start time of the shear force application step is A2 The cell detachment method described in claim 1, characterized in that when the adhesive force between the cells and the culture substrate at the time when both the vibration application process and the shear force application process are completed is A3, the relationship between A1, A2 and A3 satisfies Equation 1. A1>A2>A3 (Formula 1)

5. The adhesion area between the cells and the culture substrate at the start time of the vibration applying step is defined as B1 The adhesion area between the cells and the culture substrate at the start time of the shear force application step is defined as B2 The cell detachment method according to claim 1, characterized in that when the adhesion area at the time when both the vibration application process and the shear force application process are completed is defined as B3, the relationship between B1, B2, and B3 satisfies Equation 2 and Equation 3. B2<0.5×B1 (Formula 2) 0≦B3<B2 (Formula 3)

6. The brightness information obtained by optical measurement of the cell at the start time of the vibration application step is C1 The brightness information obtained by optical measurement of the cells at the start time of the shear force application step is C2 2. The cell detachment method according to claim 1, wherein the relationship between C1 and C2 satisfies Equation 4. C1<C2 (Formula 4)

7. 2. The cell detachment method according to claim 1, wherein the magnitude of the shear force applied in the shear force application step changes over time.

8. 2. The cell detachment method according to claim 1, wherein the magnitude of the shear force applied in the shear force application step is changed periodically.

9. 2. The cell detachment method according to claim 1, wherein the direction of the applied shear force changes over time in the shear force application step.

10. The cell detachment method according to claim 1 , wherein the shear force application step includes a step of applying a plurality of shear forces in different directions.

11. 2. The cell detachment method according to claim 1, wherein in the shear force application step, the direction of the shear force to be applied is determined based on cell information.

12. A cell information acquisition step of acquiring information on cells in a state adhered to the culture substrate; and a shear force application step start time determination step of determining a start time of the shear force application step based at least on the information of the cells; The cell detachment method according to claim 1 , wherein the cell information acquisition step and the shear force application step start time determination step are both executed before the vibration application step is started.

13. The cell detachment method according to claim 1 , wherein the cell information includes at least one selected from the group consisting of the size, shape, adhesion area, and brightness value of the cell.

14. In the shear force applying step, The cell detachment method according to claim 12, wherein the conditions for applying the shear force are determined according to information about the cells.

15. The cell detachment method according to claim 1, wherein the vibration application step is performed for a period of time ranging from 1 second to 1 hour.

16. The cell detachment method according to claim 1, wherein the time for which the shear force application step is carried out is from 1 second to 1 hour.

17. The cell detachment method according to claim 1, characterized in that, when the time during which the vibration applying step is performed is a and the time during which the shear force applying step is b, the relationship between a and b satisfies Equation 5. 0.01≦a / b≦100 (Formula 5)

18. 2. The cell detachment method according to claim 1, wherein the difference between the time when the vibration application step is started and the time when the shear force application step is started is 1 second or more and 1 hour or less.

19. 2. The cell detachment method according to claim 1, wherein the difference between the time when the vibration application step starts and the time when the shear force application step ends is 10 seconds or more and 30 minutes or less.

20. A cell detachment system having a detachment unit that detaches adherent cells cultured on a culture substrate, and a control unit, The peeling unit has a vibration applying unit that applies vibrations in an ultrasonic band to the culture substrate, and a shear force applying unit that applies shear force to the culture surface of the culture substrate, The cell detachment system is characterized in that the control unit controls the start of driving of the vibration applying unit to occur before the start of driving of the shear force applying unit.

21. The cell detachment system of claim 20, wherein the shear force applying unit applies shear force to the culture surface by at least one means selected from beating, shaking, rocking, and eccentric rotation applied to the culture substrate.

22. The cell detachment system described in claim 20, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit so as to change the magnitude of the shear force over time.

23. The cell detachment system described in claim 20, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit so as to periodically change the magnitude of the shear force.

24. The cell detachment system described in claim 20, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit so as to change the direction of the shear force over time.

25. The cell detachment system described in claim 20, characterized in that the control unit has a shear force control unit that controls the shear force application unit, and the shear force control unit controls the shear force application unit to change the direction of the shear force applied based on at least one selected from the group consisting of the size, shape, adhesion area, and brightness value of the cell.

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