Cell detachment method and cell detachment device

Abstract

To provide a cell detachment method and a cell detachment device that enable vibration design tailored to target cells and can detach adherent cells with a high detachment rate.SOLUTION: Provided is a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, which includes a detachment step of detaching the cells from the culture surface by vibrating a vibrator in a state in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and the vibrator and in a state in which at least one of the following (a) and (b) is satisfied: (a) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; and (b) the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium, the first acoustic transmission medium and the second acoustic transmission medium being not the same, the first acoustic transmission medium being a solid at room temperature, and the second acoustic transmission medium being a liquid or a solid having fluidity at room temperature.SELECTED DRAWING: Figure 1A

Description

[Technical field]

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

[0002] In recent years, cell culture has become popular for culturing cells used in regenerative medicine and cells used in the production of biopharmaceuticals. Cell culture is broadly divided into adhesion culture and suspension culture, of which adhesion culture is a culture method suitable for various cell types and is widely used. However, when recovering cells, enzyme treatment is required to detach the cells, and depending on the treatment method, the cells may be damaged. Therefore, cell detachment by enzyme treatment is also a cause of variation in cell quality. Therefore, detachment conditions that can recover cells with a high detachment rate without damaging them are being investigated.

[0003] Patent Document 1 proposes a patterning device that arranges cells at specific positions through a medium that transmits acoustic radiation pressure to a culture substrate. However, since the device uses a liquid such as water as the acoustic transmission medium, depending on the amount of acoustic transmission medium used, the liquid of the vibration transmission medium may penetrate into the culture substrate when vibration is applied, which may cause contamination. In addition, when silicone rubber is used as the acoustic transmission medium, the vibration generated by the vibration source may not be transmitted well to the culture substrate, or the vibration may be changed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-42534 A Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure has been made to address the above-mentioned problems, and provides a cell detachment method and cell detachment device that enable vibration design tailored to the target cells and can detach adherent cells with a high detachment rate. [Means for solving the problem]

[0006] The present disclosure provides a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A state in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and the vibrator, and a detachment step of detaching the cells from the culture surface by vibrating the vibrator while satisfying at least one of the following conditions (a) and (b): (A) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; (A) the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium; having The present invention provides a cell detachment method, wherein the first acoustic transmission medium and the second acoustic transmission medium are not the same, the first acoustic transmission medium is a solid at room temperature, and the second acoustic transmission medium is a liquid or a solid having flowability at room temperature.

[0007] The present disclosure also provides the following cell detachment device. A cell detachment device that detaches cells from a culture surface of a culture substrate by transmitting vibrations generated by a vibrator to the cells, Vibration body, A first acoustic transmission medium; and A culture substrate installation section is provided in this order. Further, a supply means for supplying a second acoustic transmission medium is provided, A cell detachment device, wherein the supplying means supplies a second acoustic transmission medium so as to satisfy at least one of the following (a) and (b). (A) The culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium. (a) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium. Effect of the Invention

[0008] According to the present disclosure, it is possible to provide a cell detachment method that enables vibration design tailored to the target cells and can detach adherent cells with a high detachment rate. [Brief description of the drawings]

[0009] [Figure 1A] 1 is a schematic cross-sectional view showing an example of the configuration of a first embodiment of a cell peeling device according to the present disclosure. [Figure 1B] 1 is a schematic cross-sectional view showing an example of the configuration of a first embodiment of a cell peeling device according to the present disclosure. [Figure 1C] 1 is a schematic cross-sectional view showing an example of the configuration of a first embodiment of a cell peeling device according to the present disclosure. [Figure 2A] 11 is a schematic cross-sectional view showing an example of the configuration of a second embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 2B] 11 is a schematic cross-sectional view showing an example of the configuration of a second embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 2C] 11 is a schematic cross-sectional view showing an example of the configuration of a second embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 3A] 13 is a schematic cross-sectional view showing an example of the configuration of a third embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 3B] 13 is a schematic cross-sectional view showing an example of the configuration of a third embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 3C] 13 is a schematic cross-sectional view showing an example of the configuration of a third embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 4A] 13 is a schematic cross-sectional view showing an example of the configuration of a fourth embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 4B] 13 is a schematic cross-sectional view showing an example of the configuration of a fourth embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 4C] 13 is a schematic cross-sectional view showing an example of the configuration of a fourth embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 5A]1 illustrates steps of the cell detachment method of the present disclosure. [Figure 5B] Specific examples of steps of the cell detachment method of the present disclosure are shown below. [Figure 6A] 1 illustrates an example of an apparatus of the present disclosure. [Figure 6B] 1 illustrates an example of an apparatus of the present disclosure. [Figure 6C] 1 illustrates an example of an apparatus of the present disclosure that includes an information processing device. [Figure 7A] An example of the placement step of the method of the present disclosure will now be described. [Figure 7B] An example of the placement step of the method of the present disclosure will now be described. [Figure 7C] An example of the pressurizing step of the method of the present disclosure will now be described. [Figure 7D] An example of the peeling step of the method of the present disclosure will now be described. [Figure 8] FIG. 1 shows a schematic diagram of an example of a culture substrate according to the present disclosure. [Figure 9A] 1 is a schematic diagram illustrating an example of a first acoustic transmission medium of the present disclosure. [Figure 9B] 1 is a schematic diagram illustrating an example of a first acoustic transmission medium of the present disclosure. [Figure 9C] 1 is a schematic diagram illustrating an example of a first acoustic transmission medium of the present disclosure. [Figure 9D] 1 is a schematic diagram illustrating an example of a first acoustic transmission medium of the present disclosure. [Figure 9E] 1 is a schematic diagram illustrating an example of a first acoustic transmission medium of the present disclosure. [Figure 9F] 1A and 1B are a cross-sectional view and a top view illustrating an example of a first acoustic transmission medium according to the present disclosure. [Figure 10] FIG. 2 is a schematic diagram illustrating an example of a piezoelectric body according to the present disclosure. [Figure 11] 3A and 3B are diagrams illustrating grooves in the first acoustic transmission medium of the present disclosure. [Figure 12] 1 is a table showing the results of summarizing the configurations and evaluation results of examples and comparative examples of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The cell detachment method of the present disclosure will be described below with reference to examples, but the present disclosure is not limited to the following examples.

[0011] (Basic configuration) The cell detachment method of the present disclosure includes the steps of: A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A state in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and the vibrator, and a detachment step of detaching the cells from the culture surface by vibrating the vibrator while satisfying at least one of the following conditions (a) and (b): (A) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; (A) the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium; having The cell detachment method is such that the first acoustic transmission medium and the second acoustic transmission medium are not the same, the first acoustic transmission medium is a solid at room temperature, and the second acoustic transmission medium is a liquid or a solid having fluidity at room temperature. An example of the cell detachment method of the present disclosure is shown in FIG. 5A by achieving a state in which at least one of (a) and (b) is satisfied by the cell detachment method of the present disclosure. In addition, conceptual diagrams of each step are shown in FIG. 7A to FIG. 7D.

[0012] 7A and 7B show a step of arranging a first acoustic transmission medium and a second acoustic transmission medium. Note that the first acoustic transmission medium and the second acoustic transmission medium may be arranged in advance, and this step is not essential to the cell detachment method of the present disclosure. In FIG. 7A, a first acoustic transmission medium is placed on the vibrator, and a second acoustic transmission medium is applied and placed on the first acoustic transmission medium. In FIG. 7B, a culture substrate is placed on the placed acoustic transmission medium. FIG. 7C shows a pressurizing step, in which pressure is applied from above the culture substrate. Note that the pressurizing step is not essential to the detachment method of the present disclosure. FIG. 7D shows a detachment step. In FIG. 7D, the vibrator is driven while pressure is applied from above the culture substrate to apply ultrasonic vibrations to detach the cells. The pressurizing step makes it easier to bring the first acoustic transmission medium and the second acoustic transmission medium into contact with the culture substrate and the vibrator, and also during the detachment step, the first acoustic transmission medium and the second acoustic transmission medium can transmit vibrations while in contact with the culture substrate and the vibrator. Here, the first acoustic transmission medium or the second acoustic transmission medium is in contact with the culture substrate or the vibrator means that the first acoustic transmission medium or the second acoustic transmission medium is in sufficient contact to sufficiently transmit the vibration of the vibrator to the culture substrate through the first acoustic transmission medium or the second acoustic transmission medium, but an extremely small gap or an extremely small amount of substance may be present between the first acoustic transmission medium or the second acoustic transmission medium and the culture substrate or the vibrator to a degree that can solve the problems of the present invention. For example, even if a few molecules of water are present between the first acoustic transmission medium and the culture substrate, the first acoustic transmission medium and the culture substrate can be considered to be in contact.

[0013] Here, the culture substrate varies greatly depending on the model number of each manufacturer and on the lot, and if both the culture substrate and the vibrator are to be attached to the first sound transmission medium, it is difficult to eliminate the mismatch caused by the minute difference in shape. Therefore, it is difficult to fully attach both the culture substrate and the vibrator to the first sound transmission medium, which has a small variation in shape. In addition, since a different culture substrate is used for each culture, a work is required for each use to bond the contact surface between the culture substrate and the first sound transmission medium, which is troublesome. In particular, if the center of the cell container after being placed on the vibrator and then cultured is selectively pressed, the cells may be damaged, so it is difficult to make the culture substrate and the first sound transmission medium adhere closely to each other. Similarly, when the culture substrate is placed on the first sound transmission medium and then the first sound transmission medium is to be placed on the vibrator, it is difficult to make the vibrator and the first sound transmission medium adhere closely to each other. The inventors of the present application have thus come to realize the problem that it is difficult to improve the matching between the culture substrate and the first acoustic transmission medium, and between the vibrator and the first acoustic transmission medium.

[0014] As a result of intensive research, the inventors of the present application have found that it is important that the first acoustic transmission medium and the second acoustic transmission medium are in contact with at least one of the culture substrate and the vibrator. In the cell detachment method of the present disclosure, when transmitting the vibration of the vibrator to the culture substrate, the first acoustic transmission medium and the second acoustic transmission medium are provided between them. The first acoustic transmission medium and the second acoustic transmission medium complement each other and can be in close contact with the vibrator or the culture substrate, so it is presumed that this configuration enables efficient vibration transmission. The vibrating body, which is the vibration generating part, is equipped with a piezoelectric body and a vibration plate, and its vibration mechanism generates bending vibrations that are amplified by the combination of the hardness of the piezoelectric body, which expands and contracts, and the vibration plate to which it is adhered.In this disclosure, resonance due to structural factors of the vibrating body is utilized, and large vibrations can be obtained at the resonance frequency.

[0015] (acoustic transmission medium) In the cell detachment method of the present disclosure, an acoustic transmission medium is used to match the vibrating body that generates vibrations with the culture substrate to which the vibrations are transmitted. In addition to physical matching such as acoustic impedance, mechanical matching such as adhesion is also important for matching. The first acoustic transmission medium and the second acoustic transmission medium are in contact with at least one of the culture substrate and the vibrating body, so that mechanical matching and physical matching can be achieved between the first acoustic transmission medium and the second acoustic transmission medium and the culture substrate or the vibrating body, and the problem of the present invention can be solved. In this embodiment, the acoustic transmission medium can also be referred to as an acoustic transmission material.

[0016] The physical matching between the vibrator and the culture substrate can be determined by a physical quantity called "acoustic impedance Z." Specifically, the acoustic impedance Z of the acoustic transmission medium is Z = (density of acoustic transmission medium) x (speed of sound in the acoustic transmission medium) is defined as:

[0017] Also, when a sound wave reaches the boundary between two materials, the transmittance of the sound wave is, Transmittance=(4×Z1×Z2) / (Z1+Z2)^2 where Z1 and Z2 are the acoustic impedances of the first and second materials that transmit sound waves. For example, the permeability between water and air and between silicone rubber and air are respectively: Acoustic impedance of water Z1 = 1.5 x 10^6 [kg / m^2 / s] Acoustic impedance of air Z2=4.08×10^2[kg / m^2 / s] Acoustic impedance of silicone rubber Z3=1.0×10^6[kg / m^2 / s] If we substitute the impedance of water and air into the above equation for the transmittance, the transmittance becomes almost zero. That is, the transmittance of sound waves between water and air, and between silicone rubber and air, is nearly zero, resulting in total reflection. Polystyrene, which is often used as a material for culture substrates, has an acoustic impedance of Z4 = 2.4 x 10^6 [kg / m^2 / s]. Therefore, in terms of matching the physical properties of the vibrator and the culture substrate in the acoustic transmission medium of the present disclosure, it is preferable to use materials with acoustic impedances close to the above values ​​as the first acoustic transmission medium and the second acoustic transmission medium.

[0018] Regarding mechanical matching between the vibrator and the culture substrate, it is preferable that they are in close contact with each other via a solid. In particular, in the present disclosure, since the vibration waveform of the vibrator is formed on the cell culture surface of the culture substrate, mechanical matching that allows for close contact over a wide area is important.

[0019] Assume that a commercially available dish with protrusions is used as the culture substrate and placed directly on the vibrator. Even if a low molecular weight material such as water or glycerin that easily deforms under low load is used as the acoustic transmission medium, the protrusions of the culture substrate directly contact the vibrator, and multiple vibration transmission paths are generated. As a result, the generated vibration becomes complex and it becomes difficult to control the vibration. The cell detachment method of the present disclosure performs subjective vibration control. In other words, by bringing the vibrator and the culture substrate into close contact with a solid, the vibration transmission path is kept constant, making it easy to control the vibration.

[0020] When the acoustic transmission medium of the present disclosure mechanically matches the vibrator and the culture substrate, the solid material is preferably a polymeric material having elasticity such as rubber or gel, and specific examples include silicone resin, urethane resin, diallyl dilate resin, unsaturated polyester, epoxy resin, phenol resin, urea resin, melamine resin, and natural rubber. These may be porous resin materials. Among these, silicone rubber is preferred because it has physical properties with little attenuation in the frequency band of the driving frequency (vibration frequency), which results in high transmission efficiency, and furthermore, heat generation of the acoustic transmission medium itself can be suppressed.

[0021] (First Acoustic Transmission Medium) The first acoustic transmission medium is a solid at room temperature. In this specification, room temperature means 20°C or higher and 30°C or lower, and preferably 25°C. Preferably, the solid has elasticity. Examples of the solid material having elasticity include polymeric materials such as rubber and gel.

[0022] Examples of polymeric rubber materials include rubber containing at least one resin selected from the group consisting of silicone resin, urethane resin, diallyl dylate resin, unsaturated polyester, epoxy resin, phenol resin, urea resin, and melamine resin, natural rubber, etc. More specifically, examples of the rubber material include silicone rubber, urethane rubber, butyl rubber, nitrile rubber, butadiene rubber, and latex rubber.

[0023] Examples of polymer gel materials include hydrogels and polymer gels. Examples of hydrogels include hydrogels obtained by combining hydrophilic polymer materials such as polyvinyl alcohol and polyacrylic acid with corresponding crosslinking agents. Examples of crosslinking agents include inorganic crosslinking agents such as borax and layered clay minerals (clay), and organic crosslinking agents such as dendritic polymers, which are multifunctional polymer materials. Examples of polymer gels include polymer gels obtained by combining polymer materials such as acrylic resins and urethane resins with corresponding multifunctional compounds, which are crosslinking agents.

[0024] It is preferable to use a first acoustic transmission medium that has a tan δ of less than 1.0 obtained by dynamic viscoelasticity measurement at room temperature when a vibration with a frequency of 1 Hz is applied to the first acoustic transmission medium.

[0025] The first acoustic transmission medium preferably contains at least one selected from the group consisting of silicone rubber, natural rubber, urethane gel, and gel materials, and more preferably contains silicone rubber. Silicone rubber has physical properties with little attenuation in the frequency band of the drive frequency (vibration frequency), so that the transmission efficiency is high, and further, heat generation of the acoustic transmission medium itself can be suppressed. Furthermore, for the purpose of facilitating removal, the surface of the first acoustic transmission medium facing the culture substrate or the vibrator may be subjected to a surface treatment such as a fluorine coating.

[0026] In order to directly observe the state of cell detachment, the acoustic transmission medium is preferably transparent to light in the visible light wavelength range.

[0027] Various additives may be added to the first acoustic transmission medium for the purpose of controlling physical properties such as viscosity, affinity with the second acoustic transmission medium, acoustic impedance with the second acoustic transmission medium, storage stability, antibacterial action, etc. Note that affinity with the second acoustic transmission medium refers to properties such as the contact angle and wettability between the first acoustic transmission medium and the second acoustic transmission medium, for example.

[0028] (Second Acoustic Transmission Medium) The second acoustic transmission medium is a material different from the first acoustic transmission medium, and is a liquid or a solid having fluidity at room temperature. Having fluidity means having the property of flowing or moving according to gravity when tilted. An example of a solid having fluidity is a solid whose tan δ obtained by dynamic viscoelasticity measurement at room temperature is 1 or more when a vibration with a frequency of 0.1 Hz is applied to the solid material. Being liquid at room temperature refers to being liquid at temperatures between 20°C and 30°C, and in particular, the intrinsic viscosity [η] at room temperature (25°C), or the complex viscosity η obtained by dynamic viscoelasticity measurement at room temperature (25°C) when vibration of 1 Hz frequency is applied. * Alternatively, the viscosity η measured by changing the shear rate using a Brookfield viscometer or rheometer can be made small.

[0029] When the second acoustic transmission medium is a fluid solid, it is preferable that the second acoustic transmission medium has a larger tanδ than the first acoustic transmission medium. That is, the second acoustic transmission medium is preferably a solid material or a liquid material having a larger tanδ than the first acoustic transmission medium. Specifically, when a vibration of 1 Hz is applied, and the tanδ of the first acoustic transmission medium and the second acoustic transmission medium obtained by dynamic viscoelasticity measurement at room temperature are defined as tanδ1 and tanδ2 respectively, it is preferable to satisfy tanδ1 < tanδ2. Alternatively, when the second acoustic transmission medium is a fluid solid, a solid having a tanδ of 1.0 or more obtained by dynamic viscoelasticity measurement at room temperature is preferable when a vibration of 0.1 Hz is applied.

[0030] Examples of solids satisfying these conditions include flexible gels. Similar to the gels exemplified as the first acoustic transmission medium, flexible gels are, for example, hydrogels combined with hydrophilic polymer materials such as polyvinyl alcohol and polyacrylic acid, and multi-branched polymer materials such as borax and layered clay minerals (clays), dendritic dendritic polymers, etc. as cross-linking components, and polymer gels combined with a polyfunctional compound as a cross-linking agent corresponding to a polymer material such as an acrylic resin and a urethane resin, and the like.

[0031] It is preferable to use the value of tanδ at the temperature and time scale when mechanically aligning the vibrating body and the culture substrate with the acoustic transmission medium. That is, for the measurement of tanδ, at room temperature, more preferably 25 °C, the dynamic viscoelasticity G' and G" when applying a vibration of 1 (Hz) or 0.1 (Hz) are measured with a rheometer or the like, and tanδ can be calculated by using the relational expression of tanδ = G" / G'. Here, G represents the shear modulus, G' represents the storage shear modulus, and G" represents the loss shear modulus. However, for the second acoustic transmission medium, it is only necessary to satisfy the above when tanδ is measured, and for the second acoustic transmission medium, there may be cases where tanδ cannot be measured. The second acoustic transmission medium may be a liquid at room temperature. In this case, tanδ may not be measurable.

[0032] If the second acoustic transmission medium is a liquid, it may have a high viscosity as long as it does not impair acoustic transmission. A low viscosity is preferable in terms of ease of handling, since excess liquid can be easily discharged from between the culture substrate or the vibrator and the acoustic transmission medium. The viscosity can be measured at room temperature, more preferably at 25°C, using a B-type viscometer or a rheometer. Furthermore, it is preferable to use the viscosity value at a shear rate of 1.0 (1 / s) when measuring with 25 mm parallel plates and a gap of 1.0 mm. Depending on the viscosity of the liquid, an appropriate torque value may not be obtained under the above conditions. In that case, it is possible to measure by changing the plate diameter, gap, etc. to obtain a similar shear rate.

[0033] Examples of the liquid include low-viscosity liquids such as water, physiological saline, ethanol, and diluted ethanol, mixtures of these, and high-viscosity liquids and gel materials such as glycerin, silicone oil, and polymer solutions, as well as mixtures of these. A polymer solution in which a gel is dispersed and swollen in a liquid such as water may be used. More specifically, an ultrasound gel in which viscoelasticity is adjusted by adding an additive such as a thickener such as PVA gel to a liquid such as glycerin is also preferred. Note that gel materials may be classified as solid or liquid. A gel as a solid has a high content of crosslinking components, and a gel as a liquid has a low content of crosslinking components. In this specification, a material in which tan δ can be measured by the measurement method performed by the inventors is considered to be solid, and a material in which it is not is considered to be liquid. This is thought to be related to the repulsion as an elastic body. In the following examples, PVA is a flexible gel as a solid, and ultrasound gel is a gel as a liquid.

[0034] As the second sound transmission medium, a hydrophilic liquid is particularly preferred. For example, an ethanol / water mixture in which water and ethanol are mixed in a volume ratio of 3:7 to 2:8 is preferred because it has a high bactericidal effect and prevents the growth of germs. The second sound transmission medium preferably contains at least one selected from the group consisting of water, physiological saline, ethanol, and diluted ethanol.

[0035] The second acoustic transmission medium may contain various additives for the purpose of controlling physical properties such as viscosity, affinity with the first acoustic transmission medium, acoustic impedance, surface tension, storage stability, and antibacterial action.

[0036] Examples of the additives include the following: Additives for adjusting the surface tension include anionic surfactants such as sodium dodecyl sulfate and sodium lauryl sulfate. Examples of cationic surfactants include ethyltrimethylammonium bromide, benzalkonium chloride, and dimethylaminopropylamide stearate. Examples of nonionic surfactants include Contaminon, Contaminon US, Pluronic F-68, Tween 20, and Tween 80. Examples of amphoteric surfactants include CHAPS and CHAPSO. Examples of the storage stabilizer include light stabilizers such as hindered amines, benzophenones, and benzotriazoles, and antioxidants such as amine-based, phenol-based, sulfur-based, and phosphorus-based antioxidants. Those that exhibit antibacterial activity include benzalkonium chloride, sodium azide, and antibiotics such as ampicillin, penicillin, and streptomycin.

[0037] (Combination of acoustic transmission media) The second acoustic transmission medium fills in the mismatched portion when the first acoustic transmission medium and the culture substrate, or the first acoustic transmission medium and the vibrator, are mechanically matched. Therefore, the first acoustic transmission medium and the second acoustic transmission medium complement each other, making it difficult for an air layer to be formed between the acoustic transmission medium and the culture substrate or the vibrator. A preferred example of a combination of the first acoustic transmission medium and the second acoustic transmission medium is one in which the first acoustic transmission medium includes silicone rubber and the second acoustic transmission medium includes at least one selected from the group consisting of water, physiological saline, ethanol, and diluted ethanol.

[0038] It is preferable to vibrate the vibrator in a state where the ratio of the area of ​​the part in contact with the first acoustic transmission medium on the surface of the culture substrate or the vibrator where the first acoustic transmission medium is in contact with the second acoustic transmission medium to the sum of the areas of the parts in contact with the first and second acoustic transmission media is 5% or more.

[0039] That is, it is preferable to satisfy at least one of the following: When the area of ​​the culture substrate in contact with the first acoustic transmission medium is S1 and the area of ​​the culture substrate in contact with the second acoustic transmission medium is S2, 0.05≦S1 / S1+S2 When the area of ​​the vibrating body in contact with the first acoustic transmission medium is S3 and the area of ​​the vibrating body in contact with the second acoustic transmission medium is S4, 0.05≦S3 / S3+S4.

[0040] By having the area of ​​the part that mechanically matches the first acoustic transmission medium (the area of ​​the part where the vibrator is in contact with the first acoustic transmission medium) be 5% or more, the vibrator or culture substrate can be brought into close contact with the solid first acoustic transmission medium, the vibration transmission path can be kept constant, vibration control becomes easier, and it is easy to control the vibration to a desired level. S1 / S1+S2 and S3 / S3+S4 are sometimes called "contact area ratio" or "contact area ratio of the first acoustic transmission medium". These are also sometimes expressed as a ratio or a percentage. That is, 0.05≦S1 / S1+S2 or 0.05≦S3 / S3+S4 may be expressed as a contact area ratio of 0.05 or more, or a contact area ratio of the first acoustic transmission medium of 5% or more, etc.

[0041] If the area of ​​the part that mechanically matches the first acoustic transmission medium (the area of ​​the part where the vibrator is in contact with the first acoustic transmission medium) is less than 5%, the area that brings the vibrator into solid contact with the culture substrate will be reduced, and it may become difficult to control the vibration to the desired level.

[0042] As a method for calculating the area of ​​the part where the first and second acoustic transmission media are in contact, for example, there is a method in which the second acoustic transmission medium is colored, and the first acoustic transmission medium is judged as an uncolored area and the second acoustic transmission medium is judged as a colored area, and the area is calculated. In addition, there is a method in which a contrast difference in the refractive index difference of the part where the first and second acoustic transmission media are in contact is created by devising a way of applying lighting during observation, and the area of ​​the area with different contrast is calculated. In addition, there is a method in which the vibrator and the culture substrate are brought into close contact with a solid and then the friction force in the lateral direction is measured, and the contact area of ​​the first acoustic transmission medium is estimated based on the increase in the friction force.

[0043] The portion where the first acoustic transmission medium and the culture substrate are mechanically matched is a region where the vibration of the vibrator can be efficiently transmitted to the culture substrate. In order to efficiently transmit the vibration of the vibrator to the culture substrate, it is preferable that the abdomen (region with a large amplitude value) in the vibration pattern of the vibrator overlaps with the region where the first acoustic transmission medium and the culture substrate are mechanically matched. If the abdomen in the vibration pattern of the vibrator does not overlap with the region where the first acoustic transmission medium and the culture substrate are mechanically matched, the vibration is difficult to transmit, which may result in a decrease in cell detachment efficiency or an increase in variation during detachment.

[0044] In order to mechanically match the desired region, for example, a method of designing the desired region on the culture substrate surface of the first acoustic transmission medium to have a convex shape compared to the surrounding area can be mentioned. Specifically, when a pattern having a strong vibration in the center is to be efficiently transmitted, the first acoustic transmission medium can be shaped so that the center part is convex, and when a pattern having a strong vibration in the peripheral part is to be efficiently transmitted, the first acoustic transmission medium can be shaped so that the peripheral part is convex. Examples of processing methods include cutting and processing flat silicone rubber, and preparing a mold that will have an arbitrary surface shape in advance and molding it.

[0045] The volume ratio of the second acoustic transmission medium to the first acoustic transmission medium is preferably 0.05 to 2.0. If the volume ratio is less than 0.05, the volume of the second acoustic transmission medium is small, and when the first acoustic transmission medium and the culture substrate are mechanically matched, the amount of the first acoustic transmission medium to fill the mismatched portion may be insufficient, which may make it difficult to transmit vibrations.

[0046] If the volume ratio is more than 2.0, the volume of the second acoustic transmission medium becomes large, and the excess second acoustic transmission medium may overflow into the surroundings during the above matching. The overflowing second acoustic transmission medium may come into direct contact with both the vibrator and the culture substrate. This may increase the number of vibration paths or suppress the vibration due to contact with the culture substrate, making it difficult to reflect the vibration of the vibrator.

[0047] In the disposing step, even if an excess of the second acoustic transmission medium is disposed, it is sufficient if the excess second acoustic transmission medium can be removed thereafter. The following methods can be given as a means for removing the excess second acoustic transmission medium.

[0048] By using diaphragm 11 having a convex shape as shown in FIG. 6B for the vibrating body, the excess second acoustic transmission medium does not remain around the acoustic transmission medium, but can be more easily discharged.

[0049] As shown in FIG. 11, the surface of the first acoustic transmission medium 131 facing the culture substrate or vibrator can be structured to have a continuous groove portion 30 extending from the center to the outer periphery, making it easier for excess second acoustic transmission medium to be discharged to the outside through the groove.

[0050] Alternatively, the contact angle between the first and second acoustic transmission media may be preferably 120° or less, more preferably 90° or less.

[0051] By reducing the contact angle between the first and second acoustic transmission media, excess second acoustic transmission medium is less likely to remain near the matching portion, making it less likely that a vibration transmission path will be created by the excess second acoustic transmission medium.

[0052] Furthermore, the contact angle between the second acoustic transmission medium and the surface of the culture substrate or the region where the vibrator is in contact with the second acoustic transmission medium can be made smaller than 40°.

[0053] Alternatively, in order to make it difficult for an excess of the second acoustic transmission medium to remain near the matching portion, a surface treatment can be applied to any of the surface of the culture substrate facing the first acoustic transmission medium, the surface of the vibrating body facing the first acoustic transmission medium, the surface of the first acoustic transmission medium facing the culture substrate, and the surface of the first acoustic transmission medium facing the vibrating body, and this process can be called a surface treatment process. By making it difficult for an excess of the second acoustic transmission medium to remain near the matching portion, it becomes difficult for a vibration transmission path to be generated by the excess of the second acoustic transmission medium.

[0054] Examples of the surface treatment include hydrophilization treatment, and more specifically, laser treatment, chemical treatment, plasma treatment, and the like.

[0055] The vibration path caused by the excess second acoustic transmission medium may occur when the vibrator and the culture substrate are directly matched by the second acoustic transmission medium without the first acoustic transmission medium being used. Therefore, the contact angle between the second acoustic transmission medium and the surface of the culture substrate or the vibrator in the area where the vibrator is in contact with the second acoustic transmission medium becomes small, so that the excess second acoustic transmission medium on the surface where the second acoustic transmission medium is in contact can be easily discharged.

[0056] The above-mentioned means for removing the excess second acoustic transmission medium may be combined in several ways, which may further increase the effect. As a means for adjusting the amount of the second acoustic transmission medium during matching, for example, the first acoustic transmission medium may be made of a porous resin material. By impregnating the second acoustic transmission medium into the porous resin material, it becomes possible to achieve matching by exuding the second acoustic transmission medium onto the matching surface when the acoustic transmission medium and the culture substrate are mechanically matched.

[0057] The degree of seepage of the second acoustic transmission medium during matching is controlled by the pore size of the pores contained in the porous resin material, the pore size distribution in the height direction, the proportion of the pores, and the amount of pressure applied in the pressure step.

[0058] In addition, the amount of volatilization of the second acoustic transmission medium when it is unused and has not been subjected to the peeling process can be controlled by the pore size and pore size distribution in the height direction of the voids contained in the porous resin material, the proportion of the voids, and the pore size and proportion of the voids on the surface of the resin material.

[0059] A known method is used to manufacture the porous resin material, and examples of such methods include a method in which a salt having a uniform particle size is added to a resin raw material, and a method in which minute droplets of a solvent or the like are dispersed and stabilized in the resin raw material.

[0060] In order to improve the compatibility of the contact surface between the culture substrate and the first acoustic transmission medium, or the contact surface between the vibrator and the first acoustic transmission medium, the entire surface or a part of either the contact surface between the culture substrate and the first acoustic transmission medium or the contact surface between the vibrator and the first acoustic transmission medium may be bonded using an adhesive or a pressure sensitive adhesive. When the above (A) is satisfied but (B) is not satisfied, the vibrator and the first culture substrate may be bonded with an adhesive or the like, and when the above (B) is satisfied but (A) is not satisfied, the vibrator and the first culture substrate may be bonded with an adhesive or the like. However, since the culture substrate is often used as a disposable material, a configuration in which the vibrator and the first acoustic transmission medium are bonded with an adhesive or the like is preferable to a configuration in which the culture substrate and the first acoustic transmission medium are bonded with an adhesive or the like.

[0061] Therefore, when either the culture substrate or the vibrator is bonded to the first acoustic transmission medium, it is preferable to bond the vibrator to the first acoustic transmission medium. As the adhesive or pressure-sensitive adhesive, a known adhesive or pressure-sensitive adhesive, double-sided tape, or the like is used.

[0062] (Culture substrate) The cell detachment method of the present disclosure is to detach cells attached to the culture surface of a culture substrate from the culture substrate. The adhesion of cells includes not only adhesion of adhesive cells, but also the presence of cells in contact with the culture substrate. That is, the adhesion of cells to a culture substrate includes, but is not limited to, adhesion of cells via an adhesion factor, and includes the cells remaining attached by weak interaction with the culture substrate, or the cells remaining simply attached by an external force such as gravity.

[0063] The culture substrate of the present disclosure is a substrate used for cell culture, and refers to any substrate including a surface to which cells adhere. The culture substrate includes, for example, a container for cell culture or a part thereof. Examples of cell containers include dishes (also called petri dishes), flasks, plates, tubes, etc. All of these are used as general-purpose disposable containers, for example, products of Corning, Thermo Fisher Scientific, AGC Techno Glass, etc. Among them, a dish is a typical example of the culture substrate of the present disclosure. A culture substrate with a removable lid is generally used. In this specification, the culture substrate may or may not include a part such as a lid. An example of the shape of a dish is as shown in the schematic diagram of FIG. 8. In particular, the bottom warp height 300 varies greatly depending on the model number of each manufacturer, the lot, and even within the lot. In order to mechanically match the culture substrate with the acoustic transmission medium, it is necessary to take into account the variation in the bottom warp height 300; however, by using the first acoustic transmission medium and the second acoustic transmission medium of the present disclosure in combination, mechanical matching becomes easier.

[0064] As shown in Figure 8, the bottom surface of a commercially available culture substrate has a protrusion on the outer periphery. The main roles of the protrusion are thought to be to prevent damage to the bottom surface of the substrate, to enable containers to be stacked on top of each other, and to prevent the temperature of the workbench from being directly transmitted to the bottom surface of the substrate when placed on the workbench. When mechanically matching the culture substrate and the acoustic transmission medium, the matching may be performed by avoiding this protrusion, or the matching may be performed by covering the protrusion.

[0065] The material of the culture substrate may be any material that is chemically stable and capable of culturing the desired cells, such as 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.

[0066] (Vibration body) The vibrating body of the present disclosure can be any one that generates vibration, and may be, for example, 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 range without being damaged as a vibrating body. The vibrating body can include an ultrasonic transducer.

[0067] 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. The thickness of the diaphragm is preferably such that when the piezoelectric body and the diaphragm are bonded together and the diaphragm flexes and vibrates, the midpoint of the thickness direction of the flexure, i.e., the neutral plane that is neither stretched nor compressed when the diaphragm flexes, is on the diaphragm side, in order to efficiently utilize the distortion of the piezoelectric body for flexure.

[0068] The shape of the diaphragm of the present disclosure can be a flat diaphragm 11 as shown in Fig. 6A or a convex diaphragm 11 as shown in Fig. 6B. Methods for producing a convex diaphragm include a method of bonding diaphragms of different diameters together, and a method of machining the diaphragm into a convex shape by cutting.

[0069] (Piezoelectric material) The piezoelectric body used in the vibrator of the present disclosure is preferably a ring-shaped piezoelectric body, but the shape of the piezoelectric body that deflects the vibrator may be a circular ring shape or a disk shape. In order to directly observe the cell detachment state, a circular ring shape is preferable because it ensures a field of view below the culture substrate.

[0070] Piezoelectric bodies exhibit piezoelectric properties when polarized, and as shown in Figure 10, the polarity of polarization can be changed according to the electrode pattern. The area where electrodes A1, A2, A3, B1, B2, and B3 are patterned is the driving phase that contributes to deformation, and the area between electrodes A1 and B1 is the sensor phase for detecting the degree of deformation. A polarized piezoelectric body can excite standing wave mode and traveling wave mode vibration modes by controlling the phase of the input AC voltage for each electrode pattern. Standing waves are generated by applying an AC voltage with a phase difference of 180 degrees to negatively polarized electrodes (A2, B2) relative to positively polarized electrodes (A1, A3, B1, B3). In contrast, as shown in Figure 10, when phase A (electrodes A1, A2, A3) and phase B (electrodes B1, B2, B3) 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 two-phase driven traveling wave. In Figure 10, GND refers to the ground, and is used to ground the electrodes placed on the back side.

[0071] 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.

[0072] The piezoelectric material used for the vibrator is PbZrTiO3 (PZT), but in consideration of environmental regulations, it is preferable to use a lead-free piezoelectric material that does not substantially contain Pb. Lead-free piezoelectric materials include BaTiO3 (BT), NaNbO3, BiNaTiO3, BiFeO3, etc., whose main components are BaTiO3 (BT), and combinations of these or metal elements may be added to the main components. 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, may also be used.

[0073] The thickness of the piezoelectric material in the vibrating body is determined by the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm. The thickness of the piezoelectric material in the vibrating body is determined by the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm. It is preferable that the midpoint of the thickness direction of the bending, that is, the neutral plane that is neither stretched nor compressed when bending, is located on the diaphragm side in order to efficiently utilize the distortion of the piezoelectric element in the bending. 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.

[0074] (Process) The cell detachment method of the present disclosure is characterized by having the following steps. Placement process: This is a step of disposing a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and the vibrator. Peeling process: This is a step of vibrating the vibrator while at least one of (a) and (b) is satisfied, thereby detaching the cells from the culture surface. (A) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; (i) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0075] Further, a further step includes a pressurizing step, which is a step of applying pressure so as to bring the culture substrate and the vibrating body closer to each other, and this step promotes contact between the culture substrate or the vibrating body and the first acoustic transmission medium and the second acoustic transmission medium.

[0076] One specific example of the cell detachment method of the present disclosure is shown in FIG. 5B. First, in a placement step, a first acoustic transmission medium is placed on a vibrating body (step S210). Then, the second acoustic transmission medium is placed on the first acoustic transmission medium (step S220). Next, in a pressurizing step, the first and second acoustic transmission media are brought into contact with the culture substrate (step S230), and then pressure is applied from the top surface of the culture substrate with a weight or the like (step S240). As the detachment step, ultrasonic application conditions are set (step S250). Next, ultrasonic application is started (step S260). Finally, the detached cells are collected (step S270). The application of pressure is not an essential step in the cell detachment method of the present disclosure. Each step will be described below.

[0077] (Placement process) In the arrangement step, the first acoustic transmission medium and the second acoustic transmission medium are arranged so as to be in contact with the vibrator or the culture substrate, respectively, depending on the state of vibration generated by the vibrator in the peeling step. Also, in the peeling step, either the first acoustic transmission medium or the second acoustic transmission medium can be arranged so as to be in contact with either the vibrator or the culture substrate, depending on the state of vibration excited in the vibrator in the peeling step. For example, when it is desired to make the center of the culture substrate imitate the vibration state of the vibrating body among the vibration states excited by the vibrating body, it is preferable to use a first acoustic transmission medium having a shape that allows the center of the culture substrate to come into contact with it.

[0078] The portions of the culture substrate where there is no contact between the first acoustic transmission medium can be brought into contact with the first and second acoustic transmission media by applying the second acoustic transmission medium in advance. The application method is preferably a method that can apply the second acoustic transmission medium in a volume that is just right for the area of ​​the contact portion and the gap that is generated, and with little error. For example, the second acoustic transmission medium may be applied by spraying it with a dispenser, nozzle, spray, etc., or by impregnating a sponge or the like with the second acoustic transmission medium and applying it.

[0079] (Pressure process) The pressurizing process in this embodiment may be a process of applying pressure so as to bring the culture substrate and the vibrating body closer together, and may involve displacing the culture substrate to bring it closer to the vibrating body, or displacing the vibrating body to bring it closer to the culture substrate, or displacing both the culture substrate and the vibrating body to bring them closer together. In the pressurizing step, pressure is applied from the culture substrate or the vibrator side so that the culture substrate and the acoustic transmission medium are mechanically aligned by contacting the culture substrate or the vibrator with the first acoustic transmission medium and the second acoustic transmission medium. Examples of pressure applying means for carrying out the pressure applying step include placing a weight on the lid of the culture substrate, applying a uniform load from above using a loading method such as a spring or leaf spring on the lid of the culture substrate container to fix the top of the culture substrate lid so that it does not move from above, and applying a load from below upward to the vibrating body.

[0080] Regarding the pressure applying means, a weight is preferable in consideration of the insulation of vibration, but other loading methods can be selected as long as they do not impede vibration. For example, a constant force spring can be used as the pressure applying means. The weight placed on the lid of the culture substrate is preferably in the shape of a ring, for example, so that the cells in the container can be easily observed.

[0081] The load to be applied varies depending on the size of the culture substrate and the vibration pattern to be applied, but for example, in the case of a 35 mm dish, it is preferably between 10 g and 300 g, and in the case of a 60 mm dish, it is preferably between 100 g and 600 g. The load may be a weight or the weight of the culture vessel itself. If the weight is too heavy, it will not only suppress and inhibit the vibration itself, but also cause the culture substrate itself to bend, making the vibration transmission state uneven, which will be a factor in reducing the detachment rate.

[0082] (Peeling process) In the detachment step, the vibrator is vibrated while the culture substrate or the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium to detach the cells. Further, the detachment step can include a step of exciting the vibrator to vibration in the ultrasonic band.

[0083] In the arrangement step, the vibration of the vibrator is not started, and in the peeling step, the vibration of the vibrator is started. For example, in the example of the device shown in Fig. 6A and Fig. 6B, the vibrator is driven by supplying a voltage of a resonant frequency to generate a desired ultrasonic vibration mode. In this case, in the peeling step, the vibrator is driven by supplying a voltage of a resonant frequency to the vibrator to generate a desired ultrasonic vibration mode. Note that the power source that applies the above voltage is not shown in Fig. 6A and Fig. 6B.

[0084] The vibration pattern can be freely selected depending on the cell type and the detachment result. In addition, in the detachment step, these vibration patterns may be used alone or may be input by periodically changing them.

[0085] The vibration waveform is preferably one that can vibrate the cell culture surface of the culture substrate with a certain amplitude, for example, an amplitude of 1 μm. Basically, when the frequency is swept from low frequency to high frequency, for example, from 20 kHz to 150 kHz in the ultrasonic region, resonant vibrations can be detected, but the vibrations detected on the low frequency side have a large amplitude but a small acceleration. In contrast, the vibrations detected on the high frequency side have a small amplitude but a large acceleration. From this, the conditions for applying a force that weakens the adhesive force of the cultured cells to the culture substrate are largely dependent on the adhesive force of the cells and the size and shape of the cells. Furthermore, the flow generated in the liquid such as the detachment liquid in the cell culture substrate also differs depending on the amplitude and acceleration, so there are various combinations. In addition, simply large amplitude and large acceleration may not be preferable because they promote the occurrence of cavitation, which is a factor in reducing the survival rate of cells.

[0086] The vibration waveform can be a standing wave mode that is formed as a pattern with orders in the circumferential direction or concentric circles from the center of the disc-shaped vibration plate, or a traveling wave mode that forms a radial vibration waveform on the vibration plate by vibrating the piezoelectric body with a phase change in the radial direction. The vibration waveform can be formed by applying a voltage at the resonant frequency to the vibration body, and the resonant frequency and vibration waveform shape can be calculated by structural calculations using the shape of the vibration body and the material properties such as hardness and density.

[0087] In addition, when the vibrator is continuously driven, the resonant frequency shifts to the low frequency side due to heat generation from the piezoelectric body itself, so the amplitude becomes smaller if the frequency is fixed and the drive is continued. For this reason, sufficient amplitude can be obtained by using a drive method that suppresses temperature rise due to self-heating by providing a rest period after a certain period of oscillation, or by repeatedly sweeping from the high frequency side to the low frequency side in a certain frequency width, so that the resonant frequency is always reached even if heat is generated.

[0088] (Vibration evaluation) The vibration waveform of the diaphragm can be measured with a laser Doppler vibrometer (Graphtec AT7200). The measurement method will be explained using the cell detachment device shown in Figure 6A as an example. When the cell detachment device is driven at the desired resonance frequency and voltage and the surface of the diaphragm 11 is scanned two-dimensionally in the XY directions with a laser, the vibration velocity at each point is obtained and the amplitude in the Z direction can be calculated. As a result, the vibration waveform of the diaphragm 11 can be constructed three-dimensionally, and the positions of the antinodes with large vibration amplitude and the nodes that do not vibrate when driven can be dynamically grasped.

[0089] Vibration measurement of the cell culture surface of the culture substrate 21 can also be performed with a similar configuration. It is preferable to place the acoustic transmission medium 13 on the diaphragm 11, form a metal film, for example Ag, on the inner surface of the culture substrate 21 by vapor deposition so that the laser is reflected by the culture substrate 21, and place the acoustic transmission medium 13 without creating an air gap between the culture substrate 21 and the acoustic transmission medium 13. Note that in FIG. 6A, the first acoustic transmission medium and the second acoustic transmission medium are collectively shown as the acoustic transmission medium 13.

[0090] In the case of a standing wave mode in which the resonance to be driven by the cell peeling device, which is placed on the acoustic transmission medium 13, oscillates concentrically, the amplitude of the central part of the culture substrate 21 is swept near the desired resonance frequency, and then the entire cell culture surface of the culture substrate 21 is scanned two-dimensionally at the frequency at which the amplitude is largest, thereby making it possible to grasp the vibration waveform generated on the cell culture surface of the culture substrate 21 in three dimensions.

[0091] (cell) The cell detachment method of the present disclosure can be applied to any type of cell without limitation. Examples include 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, human cervical carcinoma-derived HeLa cells, and insect-derived cell lines; epithelial cells and endothelial cells that constitute various tissues and organs in the body; skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells that constitute the nervous system, glial cells, and fibroblasts; primary cultured cells such as hepatic parenchymal cells, non-parenchymal hepatic cells, and adipocytes that are involved in the metabolism of the body; induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, various stem cells such as mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germ stem cells; and progenitor cells of various tissues, as well as cells induced to differentiate from these.

[0092] The cell culture conditions can be appropriately selected according to the cells to be cultured. In general, a suitable medium is added to the culture substrate 21, and 1.0×10 1 cells / cm 2 ~5.0×10 4 cells / cm 2 The cells are seeded in such a manner that the cells are cultured in an environment at 37° C. and a CO2 concentration of 5%. At this time, it is preferable to culture the cells until the cell occupancy rate in the culture substrate 21 reaches about 70% to 80% of the surface area, that is, until the cells reach a so-called subconfluent state.

[0093] The culture medium (culture medium) for the cultured cells can be replaced with a cell detachment solution in the cell detachment step. Generally, the detachment solution contains a protease. Examples of the protease include trypsin, accutase, collagenase, natural protease, chymotrypsin, elastase, papain, pronase, or recombinant forms thereof. However, since protease dissolves cells, there is concern that it may have a negative effect on cells. In contrast, the cell detachment method of the present disclosure does not use protease, or even if it does, a very small amount of protease is used to achieve the same effect, thereby suppressing the negative effect on cells. Here, the cell detachment solution is a solution used when detaching cells, and may have the effect of reducing the adhesive strength of the cells. The pH of the cell detachment solution is preferably in the neutral range. This is because the neutral range is suitable for cell culture and can stably maintain a high cell viability. The pH can be appropriately adjusted with hydrochloric acid, sodium hydroxide, etc. In addition, various buffer solutions are preferably used to stably maintain the pH. The detachment solution may also be a culture medium. The viscosity of the cell detachment solution can be appropriately adjusted by adding polymers or sugars.

[0094] As the cell detachment solution, a solution containing a metal ion chelating agent (hereinafter, sometimes referred to as a chelating agent) is particularly preferable, because the adhesive force of cells can be effectively reduced by using a cell detachment solution containing a chelating agent. The chelating agent is not particularly limited, and examples thereof include ethylenediaminetetraacetic acid (hereinafter, sometimes referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycol ether diamine tetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethyl glutamic acid, ethylenediamine disuccinic acid, etidronic acid, citric acid, gluconic acid, and phosphonobutane triacetic acid. Among these, chelating agents that form chelates with divalent cations are preferred, chelating agents that form chelates with Ca2+ and Mg2+ are particularly preferred, and ethylenediaminetetraacetic acid is the most preferred. When ethylenediaminetetraacetic acid is used as the chelating agent, the pH of the cell detachment solution is preferably 7.0 or more and 8.0 or less. This is because the higher pH in the neutral region that can maintain a high cell viability can enhance the chelating ability of ethylenediaminetetraacetic acid, and the efficiency of reducing the adhesive force of cells can be increased. The chelating agents may be used alone or in combination of two or more kinds. The content of the chelating agent is preferably 0.01 mM or more and 5.0 mM or less. Within this range, the chelating effect can be reliably obtained, and a decrease in activity due to the presence of an excess chelating agent can be suppressed.

[0095] The cell detachment solution may contain a hydrophilic polymer containing a polyalkylene glycol structure. An example of a hydrophilic polymer containing a polyalkylene glycol structure is polyethylene glycol. The hydrophilic polymer preferably has a peak molecular weight Mp of 800 or more and 50,000 or less, more preferably 1,200 or more and 20,000 or less, as measured by gel permeation chromatography. This is because the polymer has little effect on cells and the thickening effect caused by the polymer can be suppressed.

[0096] (Peel test) A detachment test can be performed to evaluate the cell detachment method of the present disclosure. The detachment test can be performed, for example, as follows. The cells are cultured, and the substrate in which the culture medium (culture medium) has been replaced with the detachment liquid as necessary is subjected to a placement process, and the vibrator can be driven while being maintained at a constant temperature, for example, 25°C, in a warmer as necessary. The drive time is long enough for cell detachment. After driving, the detached cells are collected with a dropper, and cells that cannot be collected and are still attached to the culture substrate 21 are collected by a pipetting method. Note that the cells may be damaged by leaving them in a high-temperature environment after removing the culture medium (culture medium). Some cells are also damaged by exposure to changes in environmental temperature, so it is preferable to set the temperature when placing the cells in the detachment device or when detaching the cells according to the characteristics of the cells used. For the various measured values ​​described later, the physical properties of the acoustic transmission medium at the temperature when the cells in the culture vessel are placed in the detachment device are particularly important. Therefore, the first acoustic transmission medium is a solid at room temperature, and the second acoustic transmission medium is a liquid or a solid having fluidity at room temperature. Furthermore, it is preferable that the first acoustic transmission medium is a solid, and the second acoustic transmission medium is a liquid or a solid having fluidity, even at the temperature during detachment. In addition, it is preferable that the second acoustic transmission medium is a solid having a tan δ of 1.0 or more when vibration of a frequency of 0.1 Hz is applied at room temperature.

[0097] The detachment rate used below is the ratio of the number of cells detached by vibration to the total number of cells detached by vibration and the pipetting method, expressed as a percentage. The detachment rate was calculated for n=10, and the standard deviation of the detachment rate was graded as A for less than 5%, B for 5% to 7.5%, C for 7.5% to 10%, and D for 10% or more. The residual cells remaining on the bottom surface of the substrate after cell detachment can be observed using scattered light from a backlight.

[0098] (Cell detachment device) The cell detachment device of the present disclosure is A cell detachment device that detaches cells from a culture surface of a culture substrate by transmitting vibrations generated by a vibrator to the cells, Vibration body, A first acoustic transmission medium; and A culture substrate installation section is provided in this order. Further, a supply means for supplying a second acoustic transmission medium is provided, The supplying means supplies a second acoustic transmission medium so as to satisfy at least one of the following (a) and (b). (A) The culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium. (i) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0099] The cell detachment device configuration of the present disclosure is shown in FIG. 6A. As shown in FIG. 6A, the cell detachment device of the present disclosure includes a vibrating body 101, an acoustic transmission medium 13, and a supplying means 23 for supplying a second acoustic transmission medium. In this figure, the acoustic transmission medium 13 is collectively shown as the first acoustic transmission medium 131 and the second acoustic transmission medium supplied by the supplying means 23 as the acoustic transmission medium 13. The cell detachment device may further include an arm 24 for moving the supplying means 23 in the XYZ axis directions. The culture substrate installation section only needs to be able to recognize that a culture vessel is to be placed thereon. In the example of FIG. 6A, an opening formed by the vibrating body 101, the upper cover 15, and the cushioning material 16 is suitable for placing a general-purpose dish, and therefore this opening corresponds to the culture substrate installation section.

[0100] The cell peeling device in FIG. 6A shows an example in which a piezoelectric body 12 and a vibration plate 11 are provided as a vibration generating unit, i.e., a vibration body 101 provided with a piezoelectric body 12 and a vibration plate 11, generates bending vibrations in which the vibration is amplified by a combination of the hardness of the piezoelectric body 12, which expands and contracts, and the vibration plate 11 to which it is adhered. In this example, resonance due to the structural factor of the vibration body 101 is utilized, and large vibrations can be obtained at the resonance frequency. The vibration body can include a transducer such as an ultrasonic transducer. The above-mentioned expansion and contraction is caused by applying a voltage to the piezoelectric body 12 using a power source (not shown) (the same applies to FIG. 6B and FIG. 6C).

[0101] In FIG. 6A, a ring-shaped transducer is shown as a representative transducer 101, but a Langevin transducer or a rectangular transducer that can obtain a large amplitude may also be used.

[0102] In order to efficiently transmit the vibrations formed in the diaphragm by resonance to the culture substrate 21 on which the cells are cultured, an acoustic transmission medium 13 is interposed between the diaphragm 11 and the culture substrate 21. If the culture substrate 21 is placed directly on the diaphragm 11, the contact area will be a point contact, which will cause noise, heat generation, and other energy consumption other than vibration, resulting in a decrease in efficiency. Basically, it is preferable to place the acoustic transmission medium 13 with an area sufficient to cover the detachment area of ​​the cells attached to the culture substrate 21.

[0103] In order to fix the vibrating body 101, the ends of the vibrating body 101 are sandwiched between the upper cover 15, lower cover 17, cushioning material 16, and bolts 18 in FIG. 6A, and the pressure applied to the ends is such that the vibration generated in the diaphragm is not inhibited.

[0104] An example is shown in which a vibrator is attached to the top of the culture substrate and a weight 14 is attached to the bottom of the vibrator as a pressurizing means. Pressurization and depressurization are possible by moving the weight 14 up and down. The pressurizing means applies pressure to bring the culture substrate and the vibrator closer to each other.

[0105] Fig. 6B shows another example, in which the diaphragm 11 has a convex shape, so that the excess second acoustic transmission medium does not remain around the acoustic transmission medium but is more easily discharged. By supplying a voltage at a resonant frequency, the vibrator is driven to generate a desired ultrasonic vibration mode.

[0106] The vibrator can be driven by applying a voltage at a frequency of a vibration mode that can form a desired vibration waveform on the cell culture surface of the culture substrate and at a desired amplitude using an AC power source that supplies a voltage at a resonant frequency, thereby driving the cell detachment device. At this time, by measuring the voltage of the S-phase, which is the sensor phase of the vibrator, with an oscilloscope, the voltage generated by the distortion of the vibrator itself can be directly monitored, and the vibrator can be driven while checking its vibration state. In addition, by installing a thermocouple that can measure the temperature during the cell detachment process at a location that does not affect the vibration, it is possible to drive the device while monitoring it. An infrared thermometer may be used for temperature measurement.

[0107] The cell detachment device of the present disclosure further includes an information processing device, which acquires information relating to the state of vibration excited in the vibrating body, and can control the supply unit based on that information.

[0108] FIG. 6C shows a conceptual diagram of a cell detachment device including an information processing device. 6C, the information processing device 205 acquires information on the state of vibration excited in the vibrating body 101. Based on the acquired information, the information processing device 205 controls the supply unit 23 to control the arrangement and amount of the second acoustic transmission medium so that the cells are detached under optimal conditions.

[0109] The information processing device 205 includes a central processing unit (CPU) 206, a random access memory (RAM) 207, a read only memory (ROM) 208, and a hard disk drive (HDD) 209 to realize functions as a computer that performs calculations and storage. The information processing system 205 also includes a communication I / F (interface) 210, a display device 211, and an input device 212. The CPU 206, the RAM 207, the ROM 208, the HDD 209, the communication I / F 210, the display device 211, and the input device 212 are connected to each other via a bus 213. The display device 211 and the input device 212 may be connected to the bus 213 via a drive device (not shown) for driving these devices.

[0110] 6C, the components constituting information processing system 205 are illustrated as an integrated device, but some of these functions may be configured by external devices. For example, display device 211 and input device 212 may be external devices separate from the components constituting the functions of the computer including CPU 206, etc.

[0111] The CPU 206 performs a predetermined operation according to programs stored in the RAM 207, HDD 209, etc., and also has a function of controlling each part of the information processing system 205. The RAM 207 is composed of a volatile storage medium, and provides a temporary memory area necessary for the operation of the CPU 206. The ROM 208 is composed of a non-volatile storage medium, and stores necessary information such as programs used in the operation of the information processing system 205. The HDD 209 is composed of a non-volatile storage medium, and is a storage device that stores information regarding the state of vibration to be excited in the moving object 101, etc.

[0112] The communication I / F 210 is a communication interface based on standards such as Wi-Fi (registered trademark) and 5G, and is a module for communicating with other devices. The display device 211 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 212 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 205. The display device 211 and the input device 212 may be integrally formed as a touch panel.

[0113] Note that the hardware configuration shown in FIG. 6C is an example, and other devices may be added, or some devices may not be provided. Some devices may be replaced with other devices having similar functions. Furthermore, some functions may be provided by other devices via a network, and the functions constituting the cell peeling device of the present disclosure may be distributed and realized among multiple devices. For example, the HDD 209 may be replaced with an SSD (Solid State Drive) using semiconductor elements such as flash memory, or may be replaced with cloud storage.

[0114] The CPU 206 loads a program stored in the ROM 208 or the like into the RAM 207 and executes it to realize control of the supply unit 23. The CPU 206 also realizes the function of the display unit 110 by controlling the display device 211. The CPU 206 also realizes the function of the storage unit 111 by controlling the HDD 209.

[0115] The cell detachment method of the present disclosure includes the following first to fourth embodiments. Each embodiment will be described below.

[0116] <First embodiment> As a first embodiment, the present disclosure provides the following cell detachment method. The present embodiment is a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and the vibrator; and The method includes a detachment step of detaching the cells from the culture surface by vibrating the vibrator while the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0117] When implementing the first embodiment of the present disclosure, a configuration is adopted in which the first and second acoustic transmission media are in contact with the culture substrate side. The position at which the second acoustic transmission medium is disposed may vary depending on the shape of the bottom surface of the culture substrate and the surface shape of the first acoustic transmission medium.

[0118] 1A to 1C show configuration diagrams of an acoustic transmission medium used in the cell detachment method according to the first embodiment of the present disclosure. FIG. 1A shows an image of the arrangement of second acoustic transmission medium 132 when culture substrate 21 having a flat bottom surface is combined with first acoustic transmission medium 131 having a convex central portion. FIG. 1B shows an image of the arrangement of a second acoustic transmission medium 132 when a general-purpose culture substrate 21 having a curved bottom surface is combined with a flat first acoustic transmission medium 131. FIG. 1C shows an image of the arrangement of second acoustic transmission medium 132 when a general-purpose culture substrate 21 having a curved bottom surface is combined with a flat first acoustic transmission medium 131.

[0119] As described above, the culture substrate 21 varies greatly depending on the model number of each manufacturer and on the lot. On the other hand, in this embodiment, the second acoustic transmission medium 132 is disposed on the culture substrate 21 side, and the first acoustic transmission medium 131 and the second acoustic transmission medium 132 complement each other and come into close contact with each other. Therefore, even if there is such variation in the culture substrate, it is easy to apply appropriate vibration according to the variation. In this embodiment, as shown in Figures 1A to 1C, it is preferable that the first acoustic transmission medium 131 and the second acoustic transmission medium 132 are in contact with the culture substrate 21 side, and the vibrating body 101 is in contact with the first acoustic transmission medium 131.

[0120] A more detailed example process of an embodiment of the present disclosure is shown in FIG. 5B. First, in a placement step, a first acoustic transmission medium is placed on a vibrating body (step S210). Then, the second acoustic transmission medium is placed on the first acoustic transmission medium (step S220). Next, as a pressurizing step, The first and second acoustic transmission media are brought into contact with the culture substrate (step S230). Thereafter, pressure is applied from the top surface of the culture substrate with a weight or the like (step S240). As a peeling process, The ultrasonic wave application conditions are set (step S250). Next, application of ultrasonic waves is started (step S260). Finally, the detached cells are collected (step S270).

[0121] Second Embodiment As a second embodiment, the present disclosure provides the following cell detachment method. The present embodiment is a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and the vibrator; and The method includes a detachment step of detaching the cells from the culture surface by vibrating the vibrator while the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0122] When implementing the second embodiment of the present disclosure, a configuration is adopted in which the first and second acoustic transmission media are in contact with the vibrating body side. The position at which the second acoustic transmission medium is disposed may change depending on the shape of the bottom surface of the culture substrate, the shape of the top surface of the vibrating body, and the surface shape of the first acoustic transmission medium.

[0123] 2A to 2C show configuration diagrams of an acoustic transmission medium used in the cell detachment method according to the second embodiment of the present disclosure. FIG. 2A shows an image of the arrangement of second acoustic transmission medium 132 when a culture substrate 21 with a flat bottom surface is combined with a vibrator with a flat top surface and first acoustic transmission medium 131 with a downwardly convex center. FIG. 2B shows an image of the arrangement of second acoustic transmission medium 132 when a general-purpose culture substrate 21 with a warped bottom surface is combined with a flat vibrator and first acoustic transmission medium 131 with a vertically convex center. FIG. 2C shows an image of the arrangement of second acoustic transmission medium 132 when a general-purpose culture substrate 21 with a warped bottom surface is combined with a flat vibrating body and a first acoustic transmission medium 131 with a central portion that is convex upward and concave downward. In this embodiment, as shown in Figures 2A to 2C, it is preferable that the first acoustic transmission medium 131 and the second acoustic transmission medium 132 are in contact with the vibrating body, and the culture substrate 21 is in contact with the first acoustic transmission medium.

[0124] <Third embodiment> As a third embodiment, the present disclosure provides the following cell detachment method. The present embodiment is a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and the vibrator; and The method includes a detachment step of detaching the cells from the culture surface by vibrating the vibrator while the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium and while the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0125] When implementing the third embodiment of the present disclosure, a configuration is adopted in which the first and second acoustic transmission media are in contact with both the culture substrate and the vibrator. The position at which the second acoustic transmission medium is disposed may vary depending on the shape of the bottom surface of the culture substrate and the surface shape of the first acoustic transmission medium.

[0126] 3A to 3C show configuration diagrams of an acoustic transmission medium used in a cell detachment method according to a third embodiment of the present disclosure. FIG. 3A shows an image of the arrangement of second acoustic transmission medium 132 when culture substrate 21 having a flat bottom surface is combined with first acoustic transmission medium 131 having a central portion that is convex upward and downward. FIG. 3B shows an image of the arrangement of second acoustic transmission medium 132 when a general-purpose culture substrate 21 with a warped bottom surface is combined with first acoustic transmission medium 131 with a central portion that is convex upward and downward. FIG. 3C shows an image of the arrangement of second acoustic transmission medium 132 when a general-purpose culture substrate 21 with a warped bottom surface is combined with first acoustic transmission medium 131 with a central portion that is upwardly convex and downwardly concave.

[0127] <Fourth embodiment> As a fourth embodiment, the present disclosure provides the following cell detachment method. The present embodiment is a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and the vibrator; and The method includes a detachment step of detaching the cells from the culture surface by vibrating the vibrator while the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium, the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium, and the second acoustic transmission medium is continuous above and below.

[0128] When the fourth embodiment of the present disclosure is implemented, a configuration is adopted in which the first and second acoustic transmission media are in contact with both the culture substrate and the vibrator. The position at which the second acoustic transmission medium is disposed may vary depending on the shape of the bottom surface of the culture substrate and the surface shape of the first acoustic transmission medium.

[0129] 4A to 4C show configuration diagrams of an acoustic transmission medium used in the cell detachment method according to the fourth embodiment of the present disclosure. FIG. 4A shows an image of the arrangement of second acoustic transmission medium 132 when combined with culture substrate 21, which has a flat bottom surface, and first acoustic transmission medium 131, which is divided into a central portion and an outer periphery, both of which are flat. Figure 4B shows an image of the arrangement of second acoustic transmission medium 132 when combined with a general-purpose culture substrate 21 with a warped bottom surface of the substrate and a first acoustic transmission medium 131 that is divided into a central portion and an outer periphery, with the central portion being flat and the outer periphery becoming lower toward the outer edge. Figure 4C shows an image of the arrangement of second acoustic transmission medium 132 when combined with a general-purpose culture substrate 21 with a warped bottom surface of the substrate and a first acoustic transmission medium 131 that is divided into a central portion and an outer periphery, the central portion being flat and having a different diameter than that in Figure 4B, and the outer periphery becoming lower toward the outer edge and having a different width than that in Figure 4B, and a top view of the arrangement. When the abdomen in the vibration pattern of the vibrating body appears near the center of the vibrating body, efficient peeling can be achieved by providing a first acoustic transmission medium 131 at the center as in this embodiment and ensuring close contact between the culture substrate and the acoustic transmission medium. EXAMPLES

[0130] Example 1 Examples 1 to 28, 32, and 33 correspond to the first embodiment. In Example 1, the configuration of the acoustic transmission medium shown in the schematic diagram of FIG. 1A was used. A Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish was used as the culture substrate. The vibration plate of the cell detachment device was made of glass with an outer diameter of 70 mm and a thickness of 4 mm. The material composition of the piezoelectric body was PZT, the outer diameter was the same as the vibration plate, 70 mm, the inner diameter was 57 mm, and the thickness was 2 mm considering that the neutral plane was on the vibration plate side. The cell detachment device was configured as shown in FIG. 6A, and a silicone rubber with a hardness of 30° and a convex disc shape with a center height (1) = 0.9 mm, a side height (2) = 0.4 mm, and a diameter (3) = 32 mm was installed on the vibration plate as the first acoustic transmission medium, as shown in the schematic diagram of FIG. 9A, so that the center was aligned with the vibration plate. This silicone rubber was used as the first acoustic transmission medium. When vibration of a frequency of 1 Hz was applied to this silicone rubber, the tan δ obtained by dynamic viscoelasticity measurement at room temperature was 0.13. 200 μL of water, which is the second sound transmission medium, was dropped onto the center of the first sound transmission medium using a dispenser. Next, the center of a Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish, which is a culture substrate on which cells were cultured, was slowly placed so that it was aligned with the center of the diaphragm. Then, a 100 g weight was placed on the lid of the culture substrate and pressure was applied. A schematic diagram showing the configuration of the sound transmission medium after pressure application is shown in FIG. 1B. The contact areas of the first and second sound transmission media and the culture substrate were calculated by coloring the water, which is the second sound transmission medium, with food coloring, and confirming that there was no air layer between the sound transmission medium and the culture substrate from a photograph taken from above after pressure application. The contact area rate of the first sound transmission medium was 10%. In addition, the contact angle between the first sound transmission medium and the second sound transmission medium was 110°.

[0131] For cell culture, CHO (Chinese Hamster Ovary) cells were cultured in Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dishes at 10,000 cells / cm. 2The cells were seeded at a density of 1000 and cultured at 37℃ in an environment with a CO2 concentration of 5%. The 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 state of the cells was observed under a phase contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate of the dish was about 80%.

[0132] (Cell detachment) The medium in the dish was removed, and the cells were washed with PBS(-) and then immersed in PBS(-) as a detachment solution for 3 minutes. The culture substrate was then placed in a cell detachment device at 25°C, and cells were detached for 2 minutes in standing wave mode using frequency sweep vibration (frequency 22-27kHz, frequency sweep period 1s, voltage 90V) at an ambient temperature of 25°C. A standing wave was generated by applying an AC voltage with a phase difference of 180 degrees to the negatively polarized electrodes (A2, B2) compared to the positively polarized electrodes (A1, A3, B1, B3) in Fig. 10. The vibration mode driven in this way is referred to as vibration mode A in Fig. 12 described below. After collecting the detached cells, a cell scraper was used to detach all the cells that could not be detached by ultrasound from the dish after detachment by ultrasound, and the number of cells was measured using a hemocytometer. The number of cells detached by ultrasound and the number of cells detached by a cell scraper after detachment by ultrasound were combined to determine the total number of detached cells, and the ratio of the number of ultrasonically detached cells to the total number of detached cells was defined as the detachment rate, and the value of the detachment rate was calculated. Regarding the variation, the detachment rate was calculated with n=10, and the standard deviation of the detachment rate was evaluated as A for less than 5%, B for 5% or more and less than 7.5%, C for 7.5% or more and less than 10%, and D for 10% or more. The evaluation result of Example 1 was a detachment rate of 97.4%, and the variation was rated A.

[0133] Example 2 In Example 2, the first acoustic transmission medium was changed as follows, and the amount of the second acoustic transmission medium was changed as follows, but the same conditions as Example 1 were used. That is, a silicone rubber with a disk shape and hardness of 30°, with a central height (1) = side height (2) = 0.9 mm and a diameter (3) = 32 mm, as shown in the schematic diagram of FIG. 9B, was used as the first acoustic transmission medium on the diaphragm. 70 μL of water was sprayed uniformly on the first acoustic transmission medium with a spray device and placed on the first acoustic transmission medium. The contact area ratio of the first acoustic transmission medium was 20%. The evaluation results for Example 2 were a peel rate of 90.3% and a C rating for variation.

[0134] Example 3 In Example 3, the cell detachment conditions were changed as follows, but the conditions were the same as those in Example 1. The cell detachment conditions were frequency sweep vibration (frequency 67.5-72.5 kHz, frequency sweep period 1 s, voltage 90 V) in standing wave mode for 2 minutes to detach the cells. Although the frequency band was different from that in Example 1, the conditions were the same, that is, an AC voltage with a phase difference of 180 degrees was applied to the negatively polarized electrodes (A2, B2) relative to the positively polarized electrodes (A1, A3, B1, B3), to generate a standing wave. The vibration mode by such driving is referred to as vibration mode B in FIG. 12 described later. The evaluation results for Example 3 were a peel rate of 90.0% and a C rating for variation.

[0135] Example 4 In Example 4, the cell detachment conditions were changed as follows, but the conditions were the same as those in Example 2. The cell detachment conditions were frequency sweep vibration in standing wave mode for 2 minutes (frequency 67.5-72.5 kHz, frequency sweep period 1 s, voltage 90 V), and the cells were detached. The evaluation results for Example 4 were a detachment rate of 96.8% and a variation rating of A.

[0136] Example 5 In Example 5, the first acoustic transmission medium was changed as follows, but the conditions were the same as those of Example 1. That is, on the diaphragm, silicone rubber with a hardness of 30° and a deformed convex disk shape with a center height (1) of 0.9 mm, a top surface height (1') of 0.3 mm, a side surface height (2) of 0.3 mm, a diameter (3) of 32 mm, and an inner diameter (3') of 5 mm was used as the first acoustic transmission medium. 250 μL of water, which is the second acoustic transmission medium, was dropped onto the first acoustic transmission medium using a dispenser. The contact area ratio of the first acoustic transmission medium was 5%. The evaluation results for Example 5 were a peel rate of 90.2% and a B rating for variation.

[0137] Example 6 In Example 6, the first acoustic transmission medium was changed as follows, but the conditions were the same as those of Example 1. That is, on the diaphragm, a silicone rubber with a hardness of 30° and a deformed disk shape with a center height (1) of 0.9 mm, a side height (2) of 0.7 mm, a side height (2') of 0.4 mm, a diameter (3) of 32 mm, and an inner diameter (3') of 20 mm was used as the first acoustic transmission medium. 200 μL of water, which is the second acoustic transmission medium, was dropped on the first acoustic transmission medium with a dispenser. The contact area ratio of the first acoustic transmission medium was 30%. The evaluation results for Example 6 were a peel rate of 98.2% and an A rating for variation.

[0138] Example 7 In Example 7, the second acoustic transmission medium was changed to PVA gel, and the conditions were the same as in Example 1, except that the amount used was changed. PVA gel was prepared by the following method. Denka Poval (trademark) K-24E (manufactured by Denka Co., Ltd.) was weighed out to 3.7% by mass, and pure water was added to prepare a total of 80 mL. The PVA was completely dissolved by stirring while heating in a thermostatic bath, and a PVA aqueous solution was obtained. Next, borax (special grade sodium tetraborate (decahydrate); manufactured by Kishida Chemical Co., Ltd.) was weighed out, and pure water was added to prepare a total of 16 mL to 1.0% by mass. The borax aqueous solution was gradually added dropwise while stirring the PVA aqueous solution, and the mixture was stirred until it became uniform, thereby obtaining a PVA gel. When vibration of a frequency of 0.1 Hz was applied to this PVA gel, the tan δ obtained by dynamic viscoelasticity measurement at room temperature was 1.7, and it can be considered as a solid with fluidity. When vibration of a frequency of 1 Hz was applied, the tan δ obtained by dynamic viscoelasticity measurement at room temperature was 0.856. 0.2 g of PVA gel, the second acoustic transmission medium, was weighed out, thinly spread into a circular sheet with a diameter of 25 mm, and placed on the first acoustic transmission medium. Next, the center of a Nunc (registered trademark) Φ35 mm dish made by Thermo Fisher Scientific, which was a culture substrate on which cells were cultured, was aligned with the center of the diaphragm and slowly placed. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 7 were a peel rate of 85.7% and a C rating for variation.

[0139] Example 8 In Example 8, the second acoustic transmission medium was changed to ultrasonic gel, and the amount used was changed, but the same conditions as in Example 1 were used. As the second acoustic transmission medium, 0.2 g of ultrasound gel (normal type ultrasound gel; manufactured by Gex) was weighed and evenly spread over a range of 20 mm from the center on the first acoustic transmission medium. Next, the center of a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which is a culture substrate on which cells were cultured, was aligned with the center of the diaphragm and slowly placed. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 8 were a peel rate of 96.4% and an A rating for variation.

[0140] Example 9 In Example 9, the second acoustic transmission medium was changed to glycerin, and the conditions were the same as in Example 1, except that the amount used was changed. 0.27 g of glycerin (special grade glycerin; Kishida Chemical Co., Ltd.) was weighed as the second acoustic transmission medium and evenly spread on the first acoustic transmission medium. Next, the center of a Nunc (registered trademark) Φ35 mm dish (a culture substrate on which cells were cultured; manufactured by Thermo Fisher Scientific Co., Ltd.) was slowly placed on the center of the diaphragm. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The contact area ratio of the first acoustic transmission medium was 10%, and the contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 100°. The evaluation results for Example 9 were a peel rate of 94.3% and a variation of A.

[0141] Example 10 In Example 10, the first sound transmission medium was changed as follows, but the same conditions as in Example 1 were used. That is, on the diaphragm, a silicone rubber having a convex disc shape with a hardness of 30° and a center height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm was used as the first sound transmission medium. The silicone rubber had 12 linear grooves with a width of 0.3 mm and a depth of 0.2 mm radially from the center at intervals of 30°, as shown in the schematic diagram of FIG. 9A. Water, which is the second sound transmission medium, was dropped on the first sound transmission medium at 1400 μL using a dispenser. The contact area ratio of the first sound transmission medium was 10%. The evaluation results for Example 10 were a peel rate of 92.1% and an A rating for variation.

[0142] Example 11 In Example 11, the second acoustic transmission medium was changed to ethanol, and the experiment was carried out under the same conditions as in Example 1, except that the amount of ethanol used was changed. 1400 μL of ethanol (special grade ethanol; Kishida Chemical Co., Ltd.), which is the second acoustic transmission medium, was dropped onto the center of the first acoustic transmission medium using a dispenser. Next, the center of a Nunc (registered trademark) Φ35 mm dish (a culture substrate on which cells were cultured; manufactured by Thermo Fisher Scientific Co., Ltd.) was slowly placed on the center of the diaphragm. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The contact area ratio of the first acoustic transmission medium was 10%, and the contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 40°. The evaluation results for Example 11 were a peel rate of 94.0% and a variation of A.

[0143] Example 12 In Example 12, the second acoustic transmission medium was changed to a 70% aqueous ethanol solution, and the experiment was carried out under the same conditions as in Example 1, except that the amount used was changed. The 70% ethanol aqueous solution, which is the second acoustic transmission medium, was obtained by mixing ethanol (special grade ethanol; manufactured by Kishida Chemical) and pure water in a volume ratio of 70:30. 1400 μL of the obtained 70% ethanol aqueous solution was dropped onto the center of the first acoustic transmission medium using a dispenser. Next, the center of a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which is a culture substrate on which cells were cultured, was aligned with the center of the diaphragm and slowly placed. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The contact area ratio of the first acoustic transmission medium was 10%, and the contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 55°. The evaluation results for Example 12 were a peel rate of 94.6% and a variation of A.

[0144] Example 13 In Example 13, the second acoustic transmission medium was changed to surfactant aqueous solution 1 (water + surfactant 1), and the conditions were the same as in Example 1, except that the amount used was changed. The surfactant aqueous solution, which is the second acoustic transmission medium, was obtained by mixing sodium lauryl sulfate (primary sodium lauryl sulfate; manufactured by Kishida Chemical) in pure water to a concentration of 10 mmol / L. 1400 μL of the obtained surfactant aqueous solution 2 (sodium lauryl sulfate aqueous solution) was dropped onto the center of the first acoustic transmission medium using a dispenser. Next, the center of a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which is a culture substrate on which cells were cultured, was aligned with the center of the diaphragm and slowly placed. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The contact area ratio of the first acoustic transmission medium was 10%, and the contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 50°. The evaluation results for Example 13 were a peel rate of 93.9% and a variation of A.

[0145] Example 14 In Example 14, the second acoustic transmission medium was changed to the surfactant aqueous solution 2 (water + surfactant 2), and the conditions were the same as in Example 1, except that the amount used was changed. The second acoustic transmission medium, a surfactant solution, was prepared by dissolving Pluronic F-68 in pure water. TM F-68 Non-ionic Surfactant (100X; manufactured by Thermo Fisher Scientific) was mixed to a solid content of 0.1% by mass. 1400 μL of the obtained surfactant aqueous solution 2 was dropped onto the center of the first acoustic transmission medium using a dispenser. Next, the center of a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which is a culture substrate on which cells were cultured, was aligned with the center of the diaphragm and slowly placed. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The contact area ratio of the first acoustic transmission medium was 10%, and the contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 75°. The evaluation results for Example 14 were a peel rate of 93.2% and a variation of A.

[0146] Example 15 In Example 15, the first acoustic transmission medium was changed as follows, and the same conditions as in Example 1 were used. That is, the first acoustic transmission medium was a silicone rubber (schematic diagram FIG. 9A) having a convex surface with a hardness of 30° and a central convex disc shape with a center height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm, which was used in Example 1, was placed on the diaphragm and hydrophilized. Water, which was the second acoustic transmission medium, was dropped onto the hydrophilized silicone rubber surface, and the contact angle was measured, which was 25°. As the second acoustic transmission medium, 1400 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 15 were a peel rate of 95.5% and a variation of A.

[0147] (Example 16) In Example 16, the investigation was carried out under the same conditions as in Example 1, except that diaphragm 11 in the device configuration diagram of FIG. 6A was changed to glass with an upper surface subjected to hydrophilic treatment. When water, the second acoustic transmission medium, was dropped onto the hydrophilized glass surface, the contact angle was measured and found to be 20°. As the second acoustic transmission medium, 1400 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 16 were a peel rate of 95.1% and a variation of A.

[0148] (Example 17) In Example 17, the study was carried out under the same conditions as in Example 1, except that the culture substrate 21 in the device configuration diagram in FIG. 6A was changed to a culture substrate whose outer surface, including the side and bottom surfaces, had been subjected to a hydrophilization treatment. When water, which is the second acoustic transmission medium, was dropped onto the bottom surface of the hydrophilized culture substrate, the contact angle was measured and found to be 25°. As the second acoustic transmission medium, 1400 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser. The contact area rate of the first acoustic transmission medium was 10%. The evaluation results for Example 17 were a peel rate of 94.8% and a variation of A.

[0149] (Example 18) In Example 18, the investigation was carried out under the same conditions as in Example 1, except that the diaphragm 11 was changed to glass having a convex shape as shown in the device configuration diagram of FIG. 6B. The convex-shaped glass was made by bonding a piece of glass with an outer diameter of 32 mm and a thickness of 2 mm to a piece of glass with an outer diameter of 70 mm and a thickness of 4 mm. As the second acoustic transmission medium, 1400 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser. The contact area rate of the first acoustic transmission medium was 10%. The evaluation results for Example 18 were a peel rate of 95.8% and a variation of A.

[0150] (Example 19) Example 19 was examined under the same conditions as Example 18, except that the second acoustic transmission medium was changed to 70% ethanol. That is, 1400 μL of 70% ethanol was dropped onto the center of the first acoustic transmission medium using a dispenser as the second acoustic transmission medium. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 19 were a peel rate of 97.4% and a variation of A.

[0151] (Example 20) In Example 20, the second acoustic transmission medium was changed to 70% ethanol, and the study was carried out under the same conditions as in Example 1, except that the amount used was also changed. That is, the 70% ethanol used as the second acoustic transmission medium was the same as that used in Example 12. The liquid volume was 1400 μL, and it was dropped onto the center of the first acoustic transmission medium with a dispenser. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 20 were a peel rate of 97.1% and a variation of A.

[0152] Example 21 In Example 21, the study was carried out under the same conditions as in Example 1, except that the amount of water used as the second acoustic transmission medium was changed. That is, 1200 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser as the second acoustic transmission medium. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 21 were a peel rate of 91.1% and a variation of B.

[0153] Example 22 In Example 22, the study was carried out under the same conditions as in Example 2, except that the amount of water used as the second acoustic transmission medium was changed. That is, 40 μL of water was sprayed uniformly onto the first acoustic transmission medium as the second acoustic transmission medium using a spray device. The contact area ratio of the first acoustic transmission medium was 20%. The evaluation results for Example 22 were a peel rate of 90.2% and a variation of C.

[0154] (Example 23) In Example 23, the study was carried out under the same conditions as in Example 1, except that the amount of water used as the second acoustic transmission medium was changed. That is, 1400 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser as the second acoustic transmission medium. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 23 were a peel rate of 90.4% and a variation of B.

[0155] Example 24 In Example 24, the study was carried out under the same conditions as in Example 2, except that the amount of water used as the second acoustic transmission medium was changed. That is, 35 μL of water was sprayed evenly onto the first acoustic transmission medium as the second acoustic transmission medium using a spray device. The contact area rate of the first acoustic transmission medium was 20%. It was also confirmed that the second acoustic transmission medium did not spread over the entire surface, and 10% of the total area was an air layer. The evaluation results for Example 24 were a peel rate of 84.2% and a variation of C.

[0156] (Example 25) In Example 25, the study was carried out under the same conditions as in Example 5, except that the amount of water used as the second acoustic transmission medium was changed. That is, 25 μL of water was sprayed evenly onto the first acoustic transmission medium as the second acoustic transmission medium using a spray device. The contact area rate of the first acoustic transmission medium was 10%. It was also confirmed that the second acoustic transmission medium did not spread throughout the entire surface, and 20% of the total area was an air layer. The evaluation results for Example 25 were a peel rate of 83.5% and a variation of C.

[0157] (Example 26) In Example 26, the same conditions as in Example 1 were used, except that the first acoustic transmission medium was a convex circular urethane rubber (schematic diagram, FIG. 9A) with a center height (1) of 0.9 mm, side height (2) of 0.4 mm, and diameter (3) of 32 mm, which was used in Example 1. When a vibration of 1 Hz frequency was applied to the silicone rubber of this urethane rubber and a viscoelasticity measurement was performed at room temperature, the tan δ was 0.23. 200 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser as the second acoustic transmission medium. The contact area ratio of the first acoustic transmission medium was 10%. The contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 80°. The evaluation results for Example 26 were a peel rate of 90.1% and a variation of B.

[0158] Example 27 In Example 27, the study was carried out under the same conditions as in Example 1, and the silicone rubber (schematic diagram, FIG. 9A) used in Example 1 was used as the first acoustic transmission medium on the diaphragm, the silicone rubber having a convex disc shape with a center height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm and a hardness of 10°. When a vibration with a frequency of 1 Hz was applied to this silicone rubber and a viscoelasticity measurement was carried out at room temperature, the tan δ was 0.14. 200 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser as the second acoustic transmission medium. The contact area ratio of the first acoustic transmission medium was 10%. The contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 110°. The evaluation results for Example 27 were a peel rate of 93.3% and a variation of B.

[0159] (Example 28) In Example 28, the study was carried out under the same conditions as in Example 1, except that the silicone rubber (schematic diagram, FIG. 9A) used in Example 1, which has a convex disc shape with a center height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm and a hardness of 50°, was used as the first acoustic transmission medium on the diaphragm. When a vibration with a frequency of 1 Hz was applied to this silicone rubber and a viscoelasticity measurement was carried out at room temperature, the tan δ was 0.05. 200 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser as the second acoustic transmission medium. The contact area ratio of the first acoustic transmission medium was 10%. The contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 110°. The evaluation results for Example 28 were a peel rate of 96.4% and a variation of A.

[0160] (Example 29) Example 29 corresponds to the second embodiment. Example 29 was similar to Example 1, but the installation process was changed and the acoustic transmission medium configuration shown in the schematic diagram of FIG. 2A was used. The convex surface of the first acoustic transmission medium as in Example 1 was brought into contact with the bottom surface of a Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish, which is a culture substrate on which cells were cultured, from the back side, with the center aligned, and the entire surface was adhered to prevent air layers from entering. Next, 200 μL of water, which is the second acoustic transmission medium, was dropped onto the center of the diaphragm using a dispenser, and the center of the Φ35 mm dish with the first acoustic transmission medium adhered to the bottom surface was slowly placed so that it was aligned with the center of the diaphragm. Then, a weight of 100 g was placed on the lid of the culture substrate to apply pressure. A schematic diagram showing the configuration of the acoustic transmission medium after pressure application is shown in FIG. 2C. The contact areas of the first and second acoustic transmission media and the culture substrate were calculated by coloring the water, which is the second acoustic transmission medium, with food coloring, and confirming that there was no air layer between the acoustic transmission medium and the culture substrate from a photograph taken from the bottom of the diaphragm after pressure application. The contact area ratio of the first acoustic transmission medium was 10%. The cell detachment conditions and the detached cell test were performed in the same manner as in Example 1. The evaluation results for Example 29 were a peel rate of 97.2% and a variation of A.

[0161] (Example 30) Example 30 corresponds to the third embodiment. In Example 30, a Φ35 mm dish manufactured by Corning was used as the culture substrate. As the first acoustic transmission medium, silicone rubber with a hardness of 10° was prepared, with a central height (1) of 1.0 mm, a side height (2) of 0.3 mm, a diameter (3) of 32 mm, an upper surface height (4) of 0.4 mm, and a lower surface height (5) of 0.3 mm, as shown in the schematic diagram of FIG. 9E. The cell detachment device was assembled as shown in FIG. 1, and 100 μL of water, which is the second acoustic transmission medium, was dropped onto the center of the diaphragm using a dispenser. Next, the lower surface of the silicone rubber, which is the first acoustic transmission medium, was slowly placed so that it was aligned with the center of the diaphragm. Furthermore, 150 μL of water, which is the second acoustic transmission medium, was dropped onto the center of the surface of the first acoustic transmission medium using a dispenser. The center of the Φ35 mm dish manufactured by Corning, which is the culture substrate on which the cells were cultured, was slowly placed so that it was aligned with the center of the diaphragm. Then, a weight of 100 g was placed on the lid of the culture substrate to apply pressure. A schematic diagram showing the configuration of the acoustic transmission medium after pressure application is shown in Figure 3B. The contact areas of the first and second acoustic transmission medium and the culture substrate were calculated by coloring the second acoustic transmission medium, water, with food coloring, and confirming from photographs taken from above and below after pressure application that there was no air space between the acoustic transmission medium and the culture substrate. The contact area ratio of the first acoustic transmission medium was 10% on the top surface and 50% on the bottom surface. The cell detachment conditions and the detached cell test were performed in the same manner as in Example 1. The evaluation results for Example 30 were a peel rate of 94.4% and a variation of B.

[0162] (Example 31) Example 31 corresponds to the fourth embodiment. In Example 31, a ΦIWAKI 35 mm dish manufactured by AGC Techno Glass was used as the culture substrate. As the first acoustic transmission medium, a circular shape with a thickness (6) of 0.6 mm and a diameter (7) of 3.0 mm and a ring shape with a thickness (6') of 0.6 mm, a width (8') of 4.0 mm and a diameter (7') of 32 mm and a hardness of 10° silicone rubber were prepared as shown in the schematic diagram of FIG. 9F. A cell detachment device was constructed as shown in FIG. 4C, and the circular and ring-shaped silicone rubbers as the first acoustic transmission medium were placed in the center of the diaphragm. Furthermore, 400 μL of water, which is the second acoustic transmission medium, was dropped onto the center of the surface of the first acoustic transmission medium using a dispenser. The center of the ΦIWAKI Φ35mm dish manufactured by AGC Techno Glass, which is the culture substrate on which the cells were cultured, was aligned slowly with the center of the diaphragm. Then, a 100 g weight was placed on the lid of the culture substrate and pressure was applied. A schematic diagram showing the configuration of the acoustic transmission medium after pressure application is shown in Figure 4C. The contact area between the first acoustic transmission medium and the culture substrate was calculated by coloring the water, which is the second acoustic transmission medium, with food coloring, and confirming that there was no air layer between the acoustic transmission medium and the culture substrate from a photograph taken from above after pressure application. The contact area ratio of the first acoustic transmission medium was 30% in total. The cell detachment conditions and the detached cell test were performed in the same manner as in Example 1. The evaluation results for Example 31 were a peel rate of 92.3% and a variation of C.

[0163] Example 32 In Example 32, the cell detachment conditions were changed as follows, but the conditions were the same as in Example 1. The cell detachment conditions were frequency sweep vibration for 2 minutes in the traveling wave mode (frequency 67.5-72.5 kHz, frequency sweep period 1 s, voltage 90 V), and cells were detached. An AC voltage with a phase difference of 90 degrees between phases A and B in FIG. 10 was applied to generate a two-phase driven traveling wave. The vibration mode by such driving is referred to as vibration mode C in FIG. 12 described later. The evaluation results of Example 32 were a detachment rate of 99.4% and a variation rating of A.

[0164] (Example 33) In Example 33, the study was conducted under the same conditions as Example 1, except that the pressurizing process of installing the weight used for pressurization in Example 1 was eliminated, and the amount of water as the second acoustic transmission medium was reduced. That is, after the first acoustic transmission medium similar to that in Example 1 was placed so that its center was aligned with the diaphragm, 160 μL of water, which was the second acoustic transmission medium, was dropped onto the center of the first acoustic transmission medium using a dispenser. The center of a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which was a culture substrate on which cells were cultured, was slowly placed so that the center of the dish was aligned with the center of the diaphragm. The contact area rate of the first acoustic transmission medium was 3%. The evaluation results for Example 33 were a peel rate of 84.1% and a variation of C.

[0165] (Comparative Example) Comparative Example 1 In Comparative Example 1, the study was carried out under the same conditions as in Example 5, except that the second acoustic transmission medium was not used. As the first acoustic transmission medium, a silicone rubber with a hardness of 30° and the same deformed convex disc shape as in Example 5, as shown in the schematic diagram of FIG. 9C, was placed so that its center was aligned with the diaphragm. Next, the center of a Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish, which is a culture substrate on which cells were cultured, was slowly placed so that it was aligned with the center of the diaphragm. Then, a 100 g weight was placed on the lid of the culture substrate to apply pressure. From a photograph taken from above after applying pressure, an air layer was confirmed around the contact part of the first acoustic transmission medium. The contact area between the first acoustic transmission medium and the culture substrate was calculated in the same manner as in Example 1 and was 5%. The evaluation results for Comparative Example 1 were a peel rate of 81.2% and a variation of D.

[0166] Comparative Example 2 In Comparative Example 2, the cell detachment conditions were the same as those of Comparative Example 1, except that cell detachment was performed using frequency sweep vibration in standing wave mode for 2 minutes (frequency 67.5-72.5 kHz, frequency sweep period 1 s, voltage 90 V). From a photograph taken from above after pressurizing the culture substrate, an air layer was confirmed around the contact portion of the first acoustic transmission medium, as in Comparative Example 1. The contact area between the first acoustic transmission medium and the culture substrate was calculated in the same manner as in Example 1 and was 5%. The evaluation results for Comparative Example 2 were a peel rate of 70.8% and a variation of D.

[0167] Comparative Example 3 In Comparative Example 3, the study was carried out under the same conditions as in Example 1, but neither the culture substrate nor the vibrator was in contact with the first acoustic transmission medium. That is, in the center of the diaphragm, as in Example 1, a silicone rubber with a hardness of 30° was placed as a first acoustic transmission medium, as shown in the schematic diagram of FIG. 9A, with a central height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm. Next, as a second acoustic transmission medium, a total of 300 μL of water was dropped evenly in 50 μL increments using a dispenser. Next, the center of a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which is a culture substrate on which cells have been cultured, was slowly placed so that it was aligned with the center of the diaphragm, and a photograph taken from above the culture substrate confirmed that there was no contact portion of the first acoustic transmission medium and that there was no air layer between the culture substrate and the second acoustic transmission medium. The contact area between the first acoustic transmission medium and the culture substrate was 0%. The evaluation results for Comparative Example 3 were a peel rate of 58.4% and a variation of D.

[0168] Comparative Example 4 In Comparative Example 4, the study was carried out under the same conditions as in Example 1, except that the first acoustic transmission medium was not used and the amount of water used as the second acoustic transmission medium was changed. As the second acoustic transmission medium, 4.0 mL of water was placed on the vibrator using a dispenser. After the water was uniformly spread on the vibrator, the center of the Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which is a culture substrate on which cells were cultured, was aligned with the center of the vibration plate and slowly placed so that no air layer was created on the bottom of the culture substrate. Then, a 100 g weight was placed on the lid of the culture substrate to apply pressure. From a photograph taken from above after pressure application, it was confirmed that the entire bottom surface of the culture substrate on the vibrator side was filled with the second acoustic transmission medium and there was no air layer. The contact area between the first acoustic transmission medium and the culture substrate was 0%. The evaluation results for Comparative Example 4 were a peel rate of 82.0% and a variation of D.

[0169] (summary) The configurations and evaluation results of the above-mentioned examples and comparative examples are summarized in Fig. 12. In Fig. 12, tan δ is the value at room temperature when vibration of frequency 0.1 Hz is applied.

[0170] Embodiments of the present disclosure include the following methods and compositions. [Method 1] A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A state in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and the vibrator, and a detachment step of detaching the cells from the culture surface by vibrating the vibrator while satisfying at least one of the following conditions (a) and (b): (A) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; (A) the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium; having A cell detachment method, wherein the first acoustic transmission medium and the second acoustic transmission medium are not the same, the first acoustic transmission medium is a solid at room temperature, and the second acoustic transmission medium is a liquid or a solid having flowability at room temperature. [Method 2] The cell detachment method according to method 1, characterized in that in the detachment step, the vibrator is vibrated while (a) is filled, thereby detaching the cells from the culture surface. [Method 3] In the peeling step, the condition (A) is satisfied, and With the vibrator in contact with the first acoustic transmission medium, The cell detachment method according to Method 1 or Method 2, characterized in that the vibrator is vibrated to detach the cells from the culture surface. [Method 4] In the peeling step, when the area of the portion of the culture substrate in contact with the first acoustic transmission medium is S1 and the area of the portion of the culture substrate in contact with the second acoustic transmission medium is S2, the cell peeling method according to Method 3, characterized in that 0.05 ≦ S1 / (S1 + S2) is satisfied. [Method 5] In the peeling step, in a state where the above (i) is satisfied, the cell peeling method according to Method 1, characterized in that the vibrator is vibrated to peel the cells from the culture surface. [Method 6] In the peeling step, when the area of the portion of the vibrator in contact with the first acoustic transmission medium is S3 and the area of the portion of the vibrator in contact with the second acoustic transmission medium is S4, the cell peeling method according to Method 5, characterized in that 0.05 ≦ S3 / (S3 + S4) is satisfied. [Method 7] Let the tanδ of the first acoustic transmission medium and the second acoustic transmission medium obtained by dynamic viscoelasticity measurement at a frequency of 1 Hz and room temperature be tanδ 1、 tanδ2, respectively. The cell peeling method according to any one of Methods 1 to 6, characterized in that tanδ1 < tanδ2 is satisfied. [Method 8] The second acoustic transmission medium is a solid having a tanδ of 1.0 or more when a vibration at a frequency of 0.1 Hz is applied at room temperature. The cell peeling method according to any one of Methods 1 to 7. [Method 9] The second acoustic transmission medium is a liquid at room temperature. The cell peeling method according to any one of Methods 1 to 8. [Method 10] The second acoustic transmission medium is a liquid, The contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 120° or less. The cell peeling method according to any one of Methods 1 to 9. [Method 11] The second acoustic transmission medium is a liquid, 11. The cell detachment method according to any one of methods 1 to 10, wherein the contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 90° or less. [Method 12] the second acoustic transmission medium is a liquid; A contact angle at a contact surface between the culture substrate and the second acoustic transmission medium, and A contact angle at a contact surface between the vibrator and the second acoustic transmission medium. 12. The cell detachment method according to any one of methods 1 to 11, wherein at least one of the angles is smaller than 40°. [Method 13] 13. The cell detachment method according to any one of Methods 1 to 12, wherein the first acoustic transmission medium has a continuous groove extending from the center to the outer periphery of at least one of the surfaces facing the culture substrate and the surface facing the vibrating body. [Method 14] 14. A cell detachment method according to any one of methods 1 to 13, characterized in that in the detachment step, the volume ratio of the second acoustic transmission medium to the first acoustic transmission medium is 0.05 or more and 2.0 or less. [Method 15] 15. The cell detachment method according to any one of methods 1 to 14, wherein the first acoustic transmission medium comprises silicone rubber. [Method 16] 16. The cell detachment method according to any one of Methods 1 to 15, wherein the first acoustic transmission medium comprises a porous resin material. [Method 17] 17. The cell detachment method according to any one of methods 1 to 16, wherein the second acoustic transmission medium comprises at least one selected from the group consisting of water, saline, ethanol, and diluted ethanol. [Method 18] 18. The cell detachment method according to any one of Methods 1 to 17, wherein the detachment step includes a step of exciting the vibrator to vibrations in the ultrasonic band. [Method 19] 19. The cell detachment method according to claim 1, further comprising a step of arranging the first acoustic transmission medium and the second acoustic transmission medium between the culture substrate and the vibrator prior to the detachment step. [Method 20] The cell detachment method described in Method 19, characterized in that in the placing step, either the first acoustic transmission medium or the second acoustic transmission medium is placed in contact with either the vibrating body or the culture substrate based on the state of vibration excited in the vibrating body in the detachment step. [Method 21] The cell detachment method described in Method 19 or 20, characterized in that in the placing step, the second acoustic transmission medium is supplied by a supply means including at least one selected from the group consisting of a dispenser, a nozzle, a spray, and a sponge, and placed in contact with the first acoustic transmission medium. [Method 22] 22. The cell detachment method according to any one of Methods 1 to 21, comprising a pressurizing step of applying pressure so as to bring the culture substrate and the vibrator closer together. [Method 23] A surface of the culture substrate facing the first acoustic transmission medium A surface of the vibrating body facing the first acoustic transmission medium A surface of the first acoustic transmission medium facing the culture substrate; and A surface of the first acoustic transmission medium facing the vibrator 23. The cell exfoliation method according to any one of Methods 1 to 22, comprising a surface treatment step of subjecting at least any surface selected from the group consisting of: [Configuration 1] A cell detachment device that detaches cells from a culture surface of a culture substrate by transmitting vibrations generated by a vibrator to the cells, Vibration body, A first acoustic transmission medium; and A culture substrate installation section is provided in this order. Further, a supply means for supplying a second acoustic transmission medium is provided, A cell detachment device, wherein the supplying means supplies a second acoustic transmission medium so as to satisfy at least one of the following (a) and (b). (A) The culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium. (i) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium. [Configuration 2] Further, the information processing device is provided. The information processing device acquires information regarding a state of vibration excited in the vibrating body, The cell detachment device according to configuration 1, characterized in that the supply unit is controlled based on the information. [Configuration 3] 3. The cell detachment device according to claim 1 or 2, wherein the vibrator includes an ultrasonic vibrator. [Configuration 4] The cell detachment device according to any one of configurations 1 to 3, further comprising a pressure applying means for applying pressure so as to bring the culture substrate and the vibrator closer to each other. [Explanation of symbols]

[0171] 11: Vibration plate 12: Piezoelectric material 13: Acoustic transmission medium 131: First acoustic transmission medium 132: Secondary Acoustic Transmission Medium 14: Weight 15: Top cover 16: Cushioning material 17: Lower cover 18: Bolt 21:Culture substrate 22:Culture substrate top lid 23: Supply section 24: Arm 30: Groove 101: Vibration body 205: Information processing device 206: CPU 207:RAM 208:ROM 209: HDD 210: Communication I / F 211:Display device 212: Input device

Claims

1. A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: A state in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and the vibrator, and a detachment step of detaching the cells from the culture surface by vibrating the vibrator while satisfying at least one of the following conditions (a) and (b): (A) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; (A) the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium; having A cell detachment method, wherein the first acoustic transmission medium and the second acoustic transmission medium are not the same, the first acoustic transmission medium is a solid at room temperature, and the second acoustic transmission medium is a liquid or a solid having flowability at room temperature.

2. 2. The cell detachment method according to claim 1, wherein in the detachment step, the vibrator is vibrated while (a) is filled, to detach the cells from the culture surface.

3. In the peeling step, the condition (A) is satisfied, and With the vibrator in contact with the first acoustic transmission medium, The cell detachment method according to claim 1 , wherein the cells are detached from the culture surface by vibrating the vibrator.

4. 3. The cell detachment method according to claim 2, characterized in that, in the detachment step, when an area of ​​the portion of the culture substrate in contact with the first acoustic transmission medium is S1 and an area of ​​the portion of the culture substrate in contact with the second acoustic transmission medium is S2, 0.05≦S1 / S1+S2 is satisfied.

5. 2. The cell detachment method according to claim 1, wherein in the detachment step, the vibrator is vibrated in a state where (i) is filled, to detach the cells from the culture surface.

6. 6. The cell detachment method according to claim 5, wherein, in the detachment step, when an area of ​​a portion of the vibrating body in contact with the first acoustic transmission medium is S3 and an area of ​​a portion of the vibrating body in contact with the second acoustic transmission medium is S4, 0.05≦S3 / S3+S4 is satisfied.

7. The tan δ of the first acoustic transmission medium and the second acoustic transmission medium obtained by dynamic viscoelasticity measurement at a frequency of 1 Hz and room temperature is tan δ １、 tan δ ２ Then, tan δ １ <tan δ ２ The cell detachment method according to any one of claims 1 to 6, characterized in that the above-mentioned condition is satisfied.

8. The cell detachment method according to any one of claims 1 to 6, characterized in that the second acoustic transmission medium is a solid having a tan δ of 1.0 or more when subjected to vibration at a frequency of 0.1 Hz at room temperature.

9. 7. The cell detachment method according to claim 1, wherein the second acoustic transmission medium is liquid at room temperature.

10. the second acoustic transmission medium is a liquid; 7. The cell detachment method according to claim 1, wherein a contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 120° or less.

11. the second acoustic transmission medium is a liquid; 10. The cell detachment method according to claim 9, wherein a contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 90° or less.

12. the second acoustic transmission medium is a liquid; A contact angle at a contact surface between the culture substrate and the second acoustic transmission medium, and A contact angle at a contact surface between the vibrator and the second acoustic transmission medium.

7. The cell detachment method according to claim 1, wherein at least one of the angles is smaller than 40°.

13. The cell detachment method according to any one of claims 1 to 6, characterized in that the first acoustic transmission medium has a continuous groove portion extending from the center to the outer periphery of at least one of the surfaces facing the culture substrate and the surface facing the vibrating body.

14. 7. The cell detachment method according to claim 1, wherein in the detachment step, a volume ratio of the second acoustic transmission medium to the first acoustic transmission medium is 0.05 or more and 2.0 or less.

15. 7. The cell detachment method according to claim 1, wherein the first acoustic transmission medium includes silicone rubber.

16. 7. The cell detachment method according to claim 1, wherein the first acoustic transmission medium includes a porous resin material.

17. 7. The cell detachment method according to claim 1, wherein the second acoustic transmission medium contains at least one selected from the group consisting of water, physiological saline, ethanol, and diluted ethanol.

18. 7. The cell detachment method according to claim 1, wherein the detachment step includes a step of exciting the vibrator with vibrations in an ultrasonic band.

19. The cell detachment method according to any one of claims 1 to 6, further comprising a step of arranging the first acoustic transmission medium and the second acoustic transmission medium between the culture substrate and the vibrator prior to the detachment step.

20. The cell detachment method according to claim 19, characterized in that in the placing step, either the first acoustic transmission medium or the second acoustic transmission medium is placed in contact with either the vibrating body or the culture substrate based on the state of vibration excited in the vibrating body in the detachment step.

21. The cell detachment method according to claim 19, characterized in that in the placing step, the second acoustic transmission medium is supplied by a supplying means including at least one selected from the group consisting of a dispenser, a nozzle, a spray, and a sponge, and placed in contact with the first acoustic transmission medium.

22. The cell detachment method according to claim 1 , further comprising a pressurizing step of applying pressure so as to bring the culture substrate and the vibrator closer to each other.

23. A surface of the culture substrate facing the first acoustic transmission medium A surface of the vibrating body facing the first acoustic transmission medium A surface of the first acoustic transmission medium facing the culture substrate; and A surface of the first acoustic transmission medium facing the vibrator 7. The cell detachment method according to claim 1, further comprising a surface treatment step of subjecting at least any surface selected from the group consisting of:

24. A cell detachment device that detaches cells from a culture surface of a culture substrate by transmitting vibrations generated by a vibrator to the cells, Vibration body, A first acoustic transmission medium; and A culture substrate installation section is provided in this order. Further, a supply means for supplying a second acoustic transmission medium is provided, A cell detachment device, wherein the supplying means supplies a second acoustic transmission medium so as to satisfy at least one of the following (a) and (b). (A) The culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium. (i) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

25. Further, the information processing device is provided. The information processing device acquires information regarding a state of vibration excited in the vibrating body, The cell detachment apparatus according to claim 24, characterized in that the supply unit is controlled based on the information.

26. The cell detachment device according to claim 24 or 25, wherein the vibrator includes an ultrasonic vibrator.

27. The cell detachment device according to claim 24 or 25, further comprising a pressure applying means for applying pressure so as to bring the culture substrate and the vibrator closer to each other.

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