Cell detachment method and cell detachment device

Abstract

The present invention provides a cell detachment method and a cell detachment device that enable vibration design tailored to a target cell and make it possible for adherent cells to be detached at a high detachment rate. Provided is a cell detachment method for detaching cells that adhere to a culture surface of a culture substrate from the culture surface, wherein the cell detachment method has a detachment step in which, in a state where a first acoustic transmission medium and a second acoustic transmission medium are arranged between the culture substrate and a vibrating body and where at least one of (a) or (b) below is satisfied, the vibrating body is caused to vibrate to detach 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; (b) the vibrating body 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 as each other, the first acoustic transmission medium being a solid at room temperature, and the second acoustic transmission medium being a liquid or a fluid solid at room temperature.

Description

Cell detachment method and cell detachment device

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

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

[0003] Patent Literature 1 proposes a patterning device that arranges cells at specific positions on a culture substrate via a medium that transmits acoustic radiation pressure. However, because the device uses a liquid such as water as the acoustic transmission medium, depending on the amount of acoustic transmission medium used, the liquid in the vibration transmission medium may penetrate into the culture substrate when vibration is applied, resulting in contamination. Furthermore, when silicone rubber is used as the acoustic transmission medium, vibrations generated by the vibration source may not be transmitted properly to the culture substrate, or the vibrations may be distorted.

[0004] JP 2018-42534 A

[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.

[0006] The present disclosure provides a cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, the method comprising: a detachment step in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and a vibrating body, and the vibrating body is vibrated to detach the cells from the culture surface while 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 vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium, wherein the first acoustic transmission medium and the second acoustic transmission medium are different, 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.

[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 provided on the culture surface, the cell detachment device comprising, in this order, a vibrator, a first acoustic transmission medium, and a culture substrate mounting section, and further comprising a supply means for supplying a second acoustic transmission medium, wherein the supply means supplies the 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. (B) The vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[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 is capable of detaching adherent cells at a high detachment rate.

[0009] FIG. 1 is a cross-sectional schematic diagram showing an example of the configuration of a first embodiment of a cell detachment device of the present disclosure. FIG. 1 is a cross-sectional schematic diagram showing an example of the configuration of a first embodiment of a cell detachment device of the present disclosure. FIG. 2 is a cross-sectional schematic diagram showing an example of the configuration of a second embodiment of a cell detachment device of the present disclosure. FIG. 3 is a cross-sectional schematic diagram showing an example of the configuration of a second embodiment of a cell detachment device of the present disclosure. FIG. 4 is a cross-sectional schematic diagram showing an example of the configuration of a third embodiment of a cell detachment device of the present disclosure. FIG. 5 is a cross-sectional schematic diagram showing an example of the configuration of a third embodiment of a cell detachment device of the present disclosure. FIG. 6 is a cross-sectional schematic diagram showing an example of the configuration of a third embodiment of a cell detachment device of the present disclosure. FIG. 7 is a cross-sectional schematic diagram showing an example of the configuration of a fourth embodiment of a cell detachment device of the present disclosure. FIG. 8 is a cross-sectional schematic diagram showing an example of the configuration of a fourth embodiment of a cell detachment device of the present disclosure. FIG. 9 is a cross-sectional schematic diagram showing an example of the configuration of a fourth embodiment of a cell detachment device of the present disclosure. FIG. 10 is a cross-sectional schematic diagram showing an example of the configuration of a fourth embodiment of a cell detachment device of the present disclosure. FIG. 11 is a cross-sectional schematic diagram showing an example of the configuration of a fourth embodiment of a cell detachment device of the present disclosure. FIG. 12 is a cross-sectional schematic diagram showing an example of the configuration of a fourth embodiment of a cell detachment device of the present disclosure. FIG. 13 shows steps of a cell detachment method of the present disclosure. FIG. 14 shows specific examples of steps of a cell detachment method of the present disclosure. FIG. 15 shows an example of an apparatus of the present disclosure. FIG. 16 shows an example of an apparatus of the present disclosure. FIG. 17 shows 1 illustrates an example of the arrangement step of the method of the present disclosure. 2 illustrates an example of the pressure step of the method of the present disclosure. 3 illustrates an example of the peeling step of the method of the present disclosure. 4 illustrates a schematic diagram of an example of a culture substrate of the present disclosure. 5 illustrates a schematic diagram of an example of a first acoustic transmission medium of the present disclosure. 6 illustrates a schematic diagram of an example of a first acoustic transmission medium of the present disclosure. 7 illustrates a schematic diagram of an example of a first acoustic transmission medium of the present disclosure. 8 illustrates a schematic diagram of an example of a first acoustic transmission medium of the present disclosure. 9 illustrates a cross-sectional view and a top view of an example of a first acoustic transmission medium of the present disclosure. 10 illustrates a schematic diagram of an example of a piezoelectric body of the present disclosure. 11 illustrates a diagram of a groove portion of the first acoustic transmission medium of the present disclosure. 12 illustrates a table summarizing the configurations and evaluation results of examples and comparative examples of the present disclosure.

[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 is a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: a detachment step in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and a vibrating body, and the vibrating body is vibrated to detach the cells from the culture surface while at least one of the following conditions (a) and (b) is met: (a) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; and (b) the vibrating body 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 are different, the first acoustic transmission medium is a solid at room temperature, and the second acoustic transmission medium is a liquid or a fluid solid at room temperature. An example of the cell detachment method of the present disclosure is shown in FIG. 5A by achieving a state where at least one of (a) and (b) is met using the cell detachment method of the present disclosure. Also, conceptual diagrams of each step are shown in FIGS. 7A to 7D.

[0012] 7A and 7B show steps for placing a first acoustic transmission medium and a second acoustic transmission medium. The first acoustic transmission medium and the second acoustic transmission medium may be placed in advance, and this step is not essential to the cell detachment method of the present disclosure. In FIG. 7A, the first acoustic transmission medium is placed on the vibrator, and then the 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 pressurization step in which pressure is applied from above the culture substrate. The pressurization 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, thereby detaching cells. The pressurization step facilitates contact of the first acoustic transmission medium and the second acoustic transmission medium with the culture substrate and the vibrator, and also allows the first acoustic transmission medium and the second acoustic transmission medium to transmit vibrations while in contact with the culture substrate and the vibrator during the detachment process. 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 so that vibrations of the vibrator are sufficiently transmitted to the culture substrate via the first acoustic transmission medium or the second acoustic transmission medium, but that 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 the problem of the present invention can be solved. 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, culture substrates vary greatly in model number and lot size depending on the manufacturer. Therefore, if one were to attempt to bond both the culture substrate and the vibrator to the first acoustic transmission medium, it would be difficult to resolve mismatches caused by minute differences in shape. Therefore, it is difficult to fully bond both the culture substrate and the vibrator to the first acoustic transmission medium, which has minimal variation in shape. Furthermore, because a different culture substrate is used for each culture, bonding the contact surfaces of the culture substrate and the first acoustic transmission medium requires a complicated process each time they are used. In particular, selectively applying pressure to the center of a cell container after it has been placed on the vibrator and then culturing cells could damage the cells, making it difficult to achieve close contact between the culture substrate and the first acoustic transmission medium. Similarly, even if one attempts to place the first acoustic transmission medium on the vibrator after placing the culture substrate on the first acoustic transmission medium, it is difficult to achieve close contact between the vibrator and the first acoustic transmission medium. The inventors of the present invention 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] After extensive research, the present inventors discovered 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 vibrating body. In the cell detachment method disclosed herein, when transmitting vibrations from the vibrating body to the culture substrate, a first acoustic transmission medium and a second acoustic transmission medium are provided between them. Because the first acoustic transmission medium and the second acoustic transmission medium complement each other and can be in close contact with the vibrating body or the culture substrate, this configuration is presumed to enable efficient vibration transmission. The vibrating body, which serves as a vibration generating unit, includes a piezoelectric body and a diaphragm. The vibration mechanism generates bending vibrations that are amplified by the combination of the stiffness of the piezoelectric body, which expands and contracts, and the hardness of the attached diaphragm. In the present disclosure, this utilizes resonance due to the structural factors of the vibrating body, allowing for large vibrations to be obtained at the resonant 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 matching physical properties such as acoustic impedance, mechanical matching such as adhesion is also important for matching. It is desirable 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 vibrating body, thereby achieving mechanical and physical matching between the first acoustic transmission medium and the second acoustic transmission medium and the culture substrate or the vibrating body, thereby achieving the object of the present invention. 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." Specifically, the acoustic impedance Z of an acoustic transmission medium is defined as Z = (density of the acoustic transmission medium) × (speed of sound in the acoustic transmission medium).

[0017] Furthermore, when a sound wave reaches the boundary between two materials, the transmittance of the sound wave is determined by the following equation: Transmittance = (4 x Z1 x Z2) / (Z1 + Z2)^2. Here, Z1 and Z2 are the acoustic impedances of the first and second materials transmitting the sound wave. For example, the transmittance 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 x 10^2 [kg / m^2 / s] Acoustic impedance of silicone rubber Z3 = 1.0 x 10^6 [kg / m^2 / s]. Substituting the impedances of water and air into the above transmittance equations results in a transmittance of almost zero. In other words, the transmittance of sound waves between water and air, and between silicone rubber and air, is almost zero, resulting in total reflection. The acoustic impedance of polystyrene, which is often used as a material for culture substrates, is Z4 = 2.4 × 10^6 [kg / m^2 / s]. Therefore, with regard to the physical property matching between 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 value as the first acoustic transmission medium and the second acoustic transmission medium.

[0018] Regarding mechanical matching between the vibrator and the culture substrate, a state of close contact via a solid is preferable. 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 close contact over a wide area is important.

[0019] Suppose 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, which easily deforms under low load, is used as the acoustic transmission medium, the protrusions on the culture substrate will directly contact the vibrator, creating multiple vibration transmission paths. As a result, the generated vibrations become complex and difficult to control. The cell detachment method disclosed herein performs active vibration control. In other words, by tightly contacting the vibrator and the culture substrate with a solid, the vibration transmission path is kept constant, making vibration control easier.

[0020] When the acoustic transmission medium of the present disclosure mechanically aligns the vibrator and the culture substrate, the solid material is preferably a polymeric material having elasticity, such as rubber or gel. Specific examples include silicone resin, urethane resin, diallyl dylate resin, unsaturated polyester, epoxy resin, phenolic resin, urea resin, melamine resin, and natural rubber. These may also be porous resin materials. Among these, silicone rubber is preferred because it has physical properties that result in low attenuation in the frequency band of the driving frequency (vibration frequency), resulting in high transmission efficiency, and furthermore, it can suppress heat generation in the acoustic transmission medium itself.

[0021] (First Acoustic Transmission Medium) The first acoustic transmission medium is solid at room temperature. In this specification, room temperature means 20°C or higher and 30°C or lower, preferably 25°C. Preferably, the solid has elasticity. Examples of elastic solid materials 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 specific examples 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. Hydrogels include hydrogels obtained by combining hydrophilic polymer materials such as polyvinyl alcohol and polyacrylic acid with corresponding crosslinkers. Examples of crosslinkers include inorganic crosslinkers such as borax and layered clay minerals (clay), and organic crosslinkers such as multifunctional polymer materials like dendritic polymers. Examples of polymer gels include polymer gels obtained by combining polymer materials such as acrylic resins and urethane resins with corresponding multifunctional compounds as crosslinkers.

[0024] The first acoustic transmission medium preferably has a tan δ of less than 1.0 as determined by dynamic viscoelasticity measurement at room temperature when vibrations at a frequency of 1 Hz are applied to the first acoustic transmission medium.

[0025] The first acoustic transmission medium preferably contains at least one material selected from the group consisting of silicone rubber, natural rubber, urethane gel, and gel materials, and particularly preferably contains silicone rubber. Silicone rubber has physical properties that result in low attenuation in the frequency band of the drive frequency (vibration frequency), thereby providing high transmission efficiency and suppressing heat generation by the acoustic transmission medium itself. Furthermore, to facilitate 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] Furthermore, in order to directly observe the state of cell detachment, the acoustic transmission medium is preferably transparent to light in the visible wavelength range.

[0027] Various additives may be added to the first acoustic transmission medium in order to control physical properties such as viscosity, affinity with the second acoustic transmission medium, acoustic impedance with the second acoustic transmission medium, storage stability, antibacterial activity, 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 that it has the property of flowing or moving according to gravity when tilted. An example of a solid having fluidity is a solid that has a tan δ of 1 or more obtained by dynamic viscoelasticity measurement at room temperature when a vibration of 0.1 Hz frequency is applied to the solid material. Being a liquid at room temperature means being a liquid at a temperature of 20°C or higher and 30°C or lower, and in particular, has an intrinsic viscosity [η] at room temperature of 25°C, or a complex viscosity η obtained by dynamic viscoelasticity measurement at room temperature of 25°C when a vibration of 1 Hz frequency is applied. * Alternatively, the viscosity η measured by changing the shear rate using a Brookfield viscometer or a rheometer can be made small.

[0029] When the second sound transmission medium is a fluid solid, it is preferable that the second sound transmission medium has a larger tan δ than the first sound transmission medium. That is, the second sound transmission medium is preferably a solid material or liquid material having a larger tan δ than the first sound transmission medium. Specifically, when a vibration of 1 Hz frequency is applied, and the tan δ of the first sound transmission medium and the tan δ of the second sound transmission medium obtained by dynamic viscoelasticity measurement at room temperature are tan δ1 and tan δ2, respectively, it is preferable that tan δ1 < tan δ2 be satisfied. Alternatively, when the second sound transmission medium is a fluid solid, it is preferable that the second sound transmission medium has a tan δ of 1.0 or more obtained by dynamic viscoelasticity measurement at room temperature when a vibration of 0.1 Hz frequency is applied.

[0030] A flexible gel is an example of a solid that satisfies these requirements. Similar to the gels exemplified as the first acoustic transmission medium, examples of the flexible gel include hydrogels that combine a hydrophilic polymer material such as polyvinyl alcohol or polyacrylic acid with a cross-linking component such as borax, layered clay minerals (clay), or a hyperbranched polymer material such as a dendritic polymer, and polymer gels that combine a polymer material such as an acrylic resin or urethane resin with a corresponding polyfunctional compound that serves as a cross-linking agent.

[0031] It is preferable to use the value of tan δ at the temperature and time scale when the vibrator and the culture substrate are mechanically aligned with each other using the acoustic transmission medium. That is, tan δ can be measured by measuring the dynamic viscoelasticity G' and G" using a rheometer or the like when vibrations of a frequency of 1 (Hz) or 0.1 (Hz) are applied at room temperature, preferably at 25°C, and calculating tan δ using the relational expression tan δ = 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, the above conditions only need to be met when tan δ is measured; there are cases where tan δ cannot be measured for the second acoustic transmission medium. The second acoustic transmission medium may be a liquid at room temperature. In this case, there are cases where tan δ cannot be measured.

[0032] When the second acoustic transmission medium is a liquid, the viscosity may be high as long as it does not impair acoustic transmission. A low viscosity is preferable from the standpoint of ease of handling, as it facilitates the discharge of excess liquid from between the culture substrate or vibrator and the acoustic transmission medium. The viscosity can be measured at room temperature, preferably 25°C, using a B-type viscometer or rheometer. Furthermore, it is preferable to use the viscosity value at a shear rate of 1.0 (1 / s) when measuring using 25 mm parallel plates with 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 such cases, measurements can be performed by changing the plate diameter, gap, or other values ​​to achieve a similar shear rate.

[0033] Examples of liquids include low-viscosity liquids such as water, saline, ethanol, and diluted ethanol, as well as mixtures of these, and high-viscosity liquids and gel materials such as glycerin, silicone oil, and polymer solutions, as well as mixtures of these. Polymer solutions in which gels are dispersed and swollen in liquids such as water are also suitable. More specifically, ultrasound gels in which viscoelasticity is adjusted by adding additives such as thickeners, such as PVA gel, to liquids such as glycerin are also preferred. Gel materials can be classified as either solid or liquid. Solid gels have a high cross-linking component content, while liquid gels have a low cross-linking component content. In this specification, materials for which tan δ can be measured using the measurement method used by the inventors are considered solids, while materials for which it is not possible to measure tan δ are considered liquids. This is thought to be related to the resilience of the elastic body. In the following examples, PVA is a flexible gel in the solid state, and ultrasound gel is a gel in the liquid state.

[0034] A hydrophilic liquid is particularly preferred as the second acoustic transmission medium. 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 various bacteria. The second acoustic transmission medium preferably contains at least one selected from the group consisting of water, 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: Anionic surfactants such as sodium dodecyl sulfate and sodium lauryl sulfate are additives for adjusting surface tension. Cationic surfactants such as ethyltrimethylammonium bromide, benzalkonium chloride, and dimethylaminopropylamide stearate are examples. Nonionic surfactants such as Contaminon, Contaminon US, Pluronic (registered trademark) F-68, Tween 20, and Tween 80 are examples. Amphoteric surfactants such as CHAPS and CHAPSO are examples. Storage stabilizers such as light stabilizers, including hindered amines, benzophenones, and benzotriazoles, and antioxidants such as amines, phenols, sulfurs, and phosphorus-based antioxidants are examples. Antibacterial agents such as benzalkonium chloride, sodium azide, and antibiotics such as ampicillin, penicillin, and streptomycin are examples.

[0037] (Combination of Acoustic Transmission Media) The second acoustic transmission medium fills in any mismatched areas when the first acoustic transmission medium and the culture substrate, or the first acoustic transmission medium and the vibrator, are mechanically aligned. Therefore, the first acoustic transmission medium and the second acoustic transmission medium complement each other, making it difficult for an air gap to form 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 portion of the culture substrate or the vibrator where the first acoustic transmission medium and the second acoustic transmission medium are in contact with the first acoustic transmission medium to the sum of the areas of the portions of the culture substrate or the vibrator where the first acoustic transmission medium and the second acoustic transmission medium are in contact with each other is 5% or more.

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

[0040] When the area of ​​the portion that mechanically matches the first acoustic transmission medium (the area of ​​the portion where the vibrator is in contact with the first acoustic transmission medium) is 5% or more, the vibrator or culture substrate can be closely attached to the solid first acoustic transmission medium, maintaining a constant vibration transmission path, making vibration control easier, and facilitating control to achieve the desired vibration. S1 / S1+S2 and S3 / S3+S4 are sometimes referred to as "contact area ratios" or "contact area ratios of the first acoustic transmission medium." These may also be expressed as ratios or percentages. For example, 0.05≦S1 / S1+S2 or 0.05≦S3 / S3+S4 may be interpreted 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.

[0041] If the area of ​​the part that mechanically aligns 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 solidly connects the vibrator to the culture substrate becomes smaller, and it may become difficult to control the vibration to achieve the desired level.

[0042] Examples of methods for calculating the area of ​​the contact area between the first and second acoustic transmission media include a method of coloring the second acoustic transmission medium, determining that the first acoustic transmission medium is an uncolored region and the second acoustic transmission medium is a colored region, and then calculating the area. Another method involves creating a contrast in the refractive index difference between the contact area between the first and second acoustic transmission media by devising a lighting method during observation, and then calculating the area of ​​the region with different contrast. Another method involves bringing the vibrator and the culture substrate into solid contact with each other, measuring the lateral friction force, and estimating the contact area of ​​the first acoustic transmission medium based on the increase in 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 antinode (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 antinode 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 less likely to be transmitted, which may result in a decrease in cell detachment efficiency or increased variation during detachment.

[0044] To mechanically align the desired region, for example, a method can be used in which the desired region on the culture substrate surface of the first acoustic transmission medium is designed to have a convex shape compared to the surrounding area. Specifically, to efficiently transmit a pattern with strong vibrations in the center, the first acoustic transmission medium can be shaped so that the central portion is convex, while to efficiently transmit a pattern with strong vibrations in the peripheral portion, the first acoustic transmission medium can be shaped so that the peripheral portion is convex. Processing methods include cutting and processing flat silicone rubber, or preparing a mold that can produce the desired surface shape and then 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 reduced, and when the first acoustic transmission medium and the culture substrate are mechanically aligned, the amount of the first acoustic transmission medium to fill the misaligned portion may be insufficient, resulting in poor vibration transmission.

[0046] If the volume ratio is greater than 2.0, the volume of the second acoustic transmission medium becomes large, and excess second acoustic transmission medium may overflow into the surrounding area during the above-mentioned 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 vibration due to contact with the culture substrate, making it difficult for the vibration of the vibrator to be reflected.

[0047] In the disposing step, even if an excess amount of the second acoustic transmission medium is disposed, it is sufficient if the excess amount of the second acoustic transmission medium can be removed thereafter. The following methods can be used to remove the excess amount of the second acoustic transmission medium.

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

[0049] As shown in Figure 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 outside through the groove.

[0050] Alternatively, the contact angle between the first and second acoustic transmission media can 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, to prevent excess second acoustic transmission medium from remaining near the matching portion, a surface treatment can be performed on 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 preventing excess second acoustic transmission medium from remaining near the matching portion, it becomes less likely that a vibration transmission path will be created by the excess second acoustic transmission medium.

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

[0055] The vibration path caused by the excess second acoustic transmission medium may also occur when the vibrator and the culture substrate are directly matched by the second acoustic transmission medium without going through the first acoustic transmission medium. Therefore, the contact angle between the second acoustic transmission medium and the surface of the culture substrate or the vibrator in contact with the second acoustic transmission medium becomes small, making it easier to discharge the excess second acoustic transmission medium from the surface in contact with the second acoustic transmission medium.

[0056] The above-mentioned means for removing excess second acoustic transmission medium can be combined, which may further enhance the effect. Furthermore, as a means for adjusting the amount of second acoustic transmission medium during matching, for example, the first acoustic transmission medium can be made of a porous resin material. By impregnating the porous resin material with the second acoustic transmission medium, it becomes possible to bleed the second acoustic transmission medium into the matching surface when mechanically matching the acoustic transmission medium and the culture substrate.

[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] Furthermore, the amount of evaporation of the second acoustic transmission medium when it is unused and has not undergone the peeling process can be controlled by the pore size of the voids contained in the porous resin material, the pore size distribution in the height direction, the proportion of the voids, and the pore size and proportion of the voids on the surface of the resin material.

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

[0060] In order to improve compatibility, the entire or a portion 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, may be bonded using an adhesive or pressure-sensitive adhesive. When the above (a) is satisfied but (b) is not, the vibrator and the first culture substrate may be bonded using an adhesive or the like. When the above (b) is satisfied but (a) is not, the vibrator and the first culture substrate may be bonded using an adhesive or the like. However, because culture substrates are often disposable, a configuration in which the vibrator and the first acoustic transmission medium are bonded using an adhesive or the like is preferable to a configuration in which the culture substrate and the first acoustic transmission medium are bonded using 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, pressure-sensitive adhesive, double-sided tape, etc. can be used.

[0062] (Culture substrate) The cell detachment method of the present disclosure involves detaching cells attached to the culture surface of a culture substrate from the culture substrate. "Cell adhesion" refers not only to adhesion of adhesive cells, but also to the presence of cells in contact with the culture substrate. That is, "cell adhesion to a culture substrate" includes, but is not limited to, adhesion of cells via adhesion factors, and also includes cells remaining attached by weak interactions with the culture substrate, or cells remaining attached simply by external forces such as gravity.

[0063] The culture substrate of the present disclosure refers to a substrate used for cell culture and refers to any substrate that includes a surface to which cells adhere. The culture substrate includes, for example, a container or a portion thereof for cell culture. Examples of cell containers include dishes (also called petri dishes), flasks, plates, and tubes. All of these are used as general-purpose disposable containers, such as those manufactured by Corning, Thermo Fisher Scientific, and AGC Technoglass. Among these, a dish is a typical example of the culture substrate of the present disclosure. Culture substrates with removable lids are commonly used. The culture substrate herein may or may not include a lid-like component. An example of a dish shape is shown in the schematic diagram of Figure 8. In particular, the bottom warpage height 300 varies significantly between different manufacturer model numbers, between different lots, and even within a single lot. In order to mechanically align the culture substrate with the acoustic transmission medium, it is necessary to take into account variations in the bottom warp height 300, but mechanical alignment becomes easier by using the first acoustic transmission medium and the second acoustic transmission medium of the present disclosure in combination.

[0064] As shown in Figure 8, commercially available culture substrates have protrusions on the outer periphery of their bottom surfaces. The main functions of the protrusions are thought to be to prevent damage to the bottom surface of the substrate, to allow containers to be stacked on top of each other, and to prevent the temperature of the workbench from being directly transmitted to the bottom of the substrate when placed on the workbench. When mechanically aligning the culture substrate and the acoustic transmission medium, the alignment may be performed by avoiding the protrusions, or by covering the protrusions.

[0065] The material of the culture substrate may be any material that is chemically stable and capable of culturing the desired cells, and examples thereof include polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(meth)acrylic acid, poly(meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, glass, metal, etc. Among these, polystyrene is preferred.

[0066] (Vibration Body) The vibration body of the present disclosure can be any vibration-generating body, and examples thereof include a piezoelectric body and a vibration plate bonded together. 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 vibration body. The vibration body can include an ultrasonic vibrator.

[0067] In the case of a ring-shaped piezoelectric element, it is preferable that the outer diameter of the diaphragm is equal to that of the piezoelectric element. The thickness of the diaphragm is such that when the piezoelectric element and the diaphragm are bonded and the piezoelectric element vibrates, the midpoint of the thickness direction of the flexure, that is, the neutral plane where there is neither tension nor compression during the flexure, is located on the diaphragm side, in order to efficiently utilize the distortion of the piezoelectric element 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 processing the diaphragm into a convex shape by cutting.

[0069] (Piezoelectric Body) 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 annular or disc-shaped. In order to directly observe the cell detachment status, an annular shape is preferred, as it ensures a field of view below the culture substrate.

[0070] Piezoelectric materials exhibit piezoelectric properties when polarized, and as shown in Figure 10, the polarity of polarization can be changed depending on 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 sandwiched between electrodes A1 and B1 is the sensor phase that detects the degree of deformation. A polarized piezoelectric material can excite standing wave and traveling wave vibration modes by controlling the phase of the input AC voltage for each electrode pattern. A standing wave is generated by applying an AC voltage with a 180-degree phase difference to the negatively polarized electrodes (A2 and B2) relative to the positively polarized electrodes (A1, A3, B1, and 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 traveling wave of two-phase drive. In Figure 10, GND refers to the ground, and is used to ground the electrodes arranged on the back surface.

[0071] The electrode pattern may be formed by printing Ag, but the electrode material may be other than Ag, such as a noble metal as Au, Pt, or Pd, or a base metal as Cu. The electrode may be formed by printing, plating, or sputtering.

[0072] The piezoelectric material used for the vibrator is PbZrTiO ３ However, in consideration of environmental regulations, a lead-free piezoelectric material that does not substantially contain Pb is preferable. The lead-free piezoelectric material is preferably BaTiO ３ (BT), NaNbO ３ , BiNaTiO ３ , BiFeO ３ These may be combined or may contain metal elements added to the main components. ３ (BT)-based material, PbZrTiO ３ It was confirmed that the vibration performance was equivalent to that of PZT. Furthermore, 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 such that when the bonded and stacked piezoelectric material and the diaphragm flex and vibrate, the midpoint of the thickness direction of the flexure, that is, the neutral plane that is neither tensile nor compressed when flexing, is located on the diaphragm side, in order to efficiently utilize the distortion of the piezoelectric element for flexure.Therefore, the combination of the thickness of the piezoelectric material and the diaphragm was calculated based on the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm.

[0074] (Steps) The cell detachment method of the present disclosure is characterized by having the following steps: Arranging step: A step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and the vibrating body. Detachment step: A step of vibrating the vibrating body to detach the cells from the culture surface while at least one of (a) and (b) is satisfied. (a) The culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium, (b) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0075] Furthermore, a further step includes a pressurizing step, in which pressure is applied to bring the culture substrate and the vibrating body closer together, 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] FIG. 5B shows a specific example of a cell detachment method according to the present disclosure. First, in the placement step, a first acoustic transmission medium is placed on a vibrator (step S210). Then, a second acoustic transmission medium is placed on the first acoustic transmission medium (step S220). Next, in the pressure application step, the first and second acoustic transmission media are brought into contact with the culture substrate (step S230). Then, pressure is applied from the top surface of the culture substrate using a weight or the like (step S240). In the detachment step, ultrasound application conditions are set (step S250). Next, ultrasound application is initiated (step S260). Finally, the detached cells are collected (step S270). Applying pressure is not an essential step for the cell detachment method according to the present disclosure. Each step is described below.

[0077] (Arrangement process) In the arrangement process, 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 process. Furthermore, in the peeling process, 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, based on the state of vibration excited in the vibrator in the peeling process. For example, if it is desired to make the center of the culture substrate imitate the vibration state of the vibrator among the vibration states excited in the vibrator, 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] In areas where the culture substrate and the first acoustic transmission medium do not come into contact, a second acoustic transmission medium can be applied in advance to allow the first and second acoustic transmission media to come into contact. A preferred application method is one that applies a volume that is just right for the area of ​​the contact area and the resulting gap, and that minimizes error. Examples of such methods include spraying the second acoustic transmission medium using a dispenser, nozzle, or spray, or impregnating a sponge or similar with the second acoustic transmission medium and applying it.

[0079] (Pressure Step) The pressure step in this embodiment may be any step of applying pressure to bring the culture substrate and the vibrator closer together. The culture substrate may be displaced to bring it closer to the vibrator, or the vibrator may be displaced to bring it closer to the culture substrate. Alternatively, both the culture substrate and the vibrator may be displaced to bring them closer together. In the pressure step, the culture substrate or the vibrator is pressed from the culture substrate or the vibrator side so that the culture substrate and the acoustic transmission medium are mechanically aligned by contacting the first acoustic transmission medium and the second acoustic transmission medium. Examples of pressure methods for performing the pressure step include placing a weight on the lid of the culture substrate, applying a uniform load from above using a spring or leaf spring on the lid of the culture substrate container to fix the top of the lid of the culture substrate so that it does not move from above, and then applying a load from below to the vibrator.

[0080] Regarding the pressure applying means, a weight is preferable in consideration of the vibration insulation, but other loading methods can be selected as long as they do not impede the 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 ring-shaped, for example, to make it easier to observe the cells in the container.

[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, for a 35 mm dish, it is preferably between 10 g and 300 g, and for 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 will also cause the culture substrate itself to bend, resulting in uneven vibration transmission and a decrease in the detachment rate.

[0082] 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. The detachment step can further include a step of exciting the vibrator to vibrations in the ultrasonic band.

[0083] In the placement process, the vibration of the vibrator is not initiated, but is initiated in the peeling process. For example, in the example of the device shown in Figures 6A and 6B, the vibrator is driven by supplying a voltage at a resonant frequency to generate a desired ultrasonic vibration mode. In this case, in the peeling process, the vibrator is driven by supplying a voltage at a resonant frequency to generate a desired ultrasonic vibration mode. Note that the power source that applies the voltage is not shown in Figures 6A and 6B.

[0084] The vibration pattern can be freely selected depending on the cell type and the detachment result. In the detachment step, these vibration patterns may be used alone or may be input while being periodically changed.

[0085] Preferably, the vibration waveform can vibrate the cell culture surface of the culture substrate at a constant amplitude, for example, 1 μm. Basically, when frequencies are swept from low to high frequencies, for example, from 20 kHz to 150 kHz in the ultrasonic range, resonant vibrations can be detected. However, vibrations detected on the low-frequency side have large amplitudes but small accelerations. In contrast, vibrations detected on the high-frequency side have small amplitudes but large accelerations. Therefore, the conditions for applying a force that weakens the adhesive strength of cultured cells to the culture substrate depend largely on the adhesive strength of the cells and the size and shape of the cells. Furthermore, the flow occurring in liquids such as detachment solutions in the cell culture substrate also varies depending on the amplitude and acceleration, so various combinations exist. Furthermore, simply using large amplitudes and large accelerations can be undesirable because they promote the occurrence of cavitation, which can reduce cell viability.

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

[0087] Furthermore, when the vibrator is continuously driven, the resonant frequency shifts to the lower frequency side due to heat generation from the piezoelectric body itself, so if the frequency is kept fixed and the vibrator is continuously driven, the amplitude will decrease. For this reason, sufficient amplitude can be obtained by using a driving method that oscillates for a certain period of time, then provides a rest period to suppress temperature rise due to self-heating, 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 using a laser Doppler vibrometer (Graphtec AT7200). The measurement method will be explained using the cell detachment device shown in FIG. 6A as an example. By driving the cell detachment device at the desired resonant frequency and voltage and scanning the surface of the diaphragm 11 two-dimensionally in the X and Y directions with a laser, the vibration velocity at each point can be 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 antinodes with large vibration amplitudes and nodes without vibration during driving can be dynamically determined.

[0089] Vibration measurement of the cell culture surface of the culture substrate 21 can also be performed using a similar configuration. It is preferable to place the acoustic transmission medium 13 on the diaphragm 11, and form a metal film, such as Ag, on the inner surface of the culture substrate 21 by vapor deposition so that the laser is reflected from the culture substrate 21, and to place the acoustic transmission medium 13 so that no air gap is formed between the culture substrate 21 and the acoustic transmission medium 13. In Fig. 6A, the first acoustic transmission medium and the second acoustic transmission medium are collectively referred to as the acoustic transmission medium 13.

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

[0091] (Cells) 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 cancer-derived HeLa cells, and insect-derived cell lines; epithelial cells and endothelial cells that constitute various tissues and organs in the body; contractile skeletal muscle cells; Examples include smooth muscle cells, cardiac muscle cells, neuron cells that make up the nervous system, glial cells, fibroblasts, hepatic parenchymal cells involved in the metabolism of living organisms, primary cultured cells such as non-parenchymal liver cells and adipocytes, various stem cells such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells and germline stem cells, or progenitor cells of various tissues, as well as cells induced to differentiate from these.

[0092] The cell culture conditions can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to the culture substrate 21, and 1.0 × 10 １ cells / cm ２ ~5.0 x 10 ４ cells / cm ２ The cells were seeded at 37°C and CO ２ The cells are cultured in an environment with a 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, ie, until the cells reach a subconfluent state.

[0093] The culture medium (culture medium) for cultured cells can be replaced with a cell detachment solution during the cell detachment process. Typically, the detachment solution contains a protease. Examples of protease include trypsin, accutase, collagenase, natural proteases, chymotrypsin, elastase, papain, pronase, or recombinant forms thereof. However, protease lysis can lyse cells, raising concerns about its adverse effects on cells. In contrast, the cell detachment method disclosed herein does not use protease, or even if used, achieves the same effect with minimal amounts, thereby suppressing adverse effects on cells. The cell detachment solution is a solution used for cell detachment and may have the effect of reducing cell adhesion. 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 maintain a stable, high cell viability. The pH can be adjusted appropriately with hydrochloric acid, sodium hydroxide, etc. Various buffer solutions are also suitable for maintaining a stable pH. The detachment solution may also be culture medium. The viscosity of the cell detachment solution can be adjusted appropriately by adding polymers or sugars.

[0094] A solution containing a metal ion chelating agent (hereinafter sometimes referred to as a chelating agent) is particularly preferred as a cell detachment solution. This is because using a cell detachment solution containing a chelating agent can effectively reduce the adhesive strength of cells. The chelating agent is not particularly limited, but examples include ethylenediaminetetraacetic acid (hereinafter sometimes referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycoletherdiaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediaminedisuccinic acid, etidronic acid, citric acid, gluconic acid, and phosphonobutanetriacetic acid. Among these, chelating agents that form chelates with divalent cations are preferred, with chelating agents that form chelates with Ca and Mg being particularly preferred, and ethylenediaminetetraacetic acid being most preferred. When ethylenediaminetetraacetic acid is used as the chelating agent, the pH of the cell detachment solution is preferably 7.0 or higher and 8.0 or lower. This is because a higher pH within the neutral range, which can maintain high cell viability, can enhance the chelating ability of ethylenediaminetetraacetic acid, thereby enabling a higher efficiency in reducing cell adhesion. The chelating agent may be used alone or in combination with two or more other agents. The content of the chelating agent is preferably 0.01 mM or more and 5.0 mM or less. This range ensures a reliable chelating effect and also prevents a decrease in activity due to the presence of an excess chelating agent.

[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 measured by gel permeation chromatography of 800 to 50,000, more preferably 1,200 to 20,000. This is because the polymer has little effect on cells and the thickening effect of the polymer can be suppressed.

[0096] (Detachment 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. Cells are cultured on a substrate, and if necessary, the culture medium (culture medium) is replaced with a detachment solution. The placement process can be performed on the substrate, and if necessary, the vibrator can be driven while the substrate is maintained at a constant temperature, e.g., 25°C, in an incubator. The vibrator is driven for a time sufficient for cell detachment. After activation, the detached cells are collected with a dropper, and any cells that remain attached to the culture substrate 21 are collected by pipetting. Note that cells may be damaged if left in a high-temperature environment after removing the culture medium (culture medium). Furthermore, because some cells are damaged by exposure to changes in environmental temperature, it is preferable to set the temperature when the cells are placed in the detachment device or when the cells are detached according to the characteristics of the cells being used. For the various measurement values ​​described below, the physical properties of the acoustic transmission medium at the temperature when the cells placed 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 fluid solid 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 peeling. Also, 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 number of cells detached by pipetting, expressed as a percentage. Variation was determined for n = 10, and the standard deviation of the detachment rate was evaluated as A for less than 5%, B for 5% to less than 7.5%, C for 7.5% to less than 10%, and D for 10% or more. 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 provided on the culture surface, and has, in this order, a vibrator, a first acoustic transmission medium, and a culture substrate mounting section, and further has a supply means for supplying a second acoustic transmission medium, wherein the supply means supplies the 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. (B) The vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0099] The configuration of the cell detachment device 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 collectively refers to the first acoustic transmission medium 131 and the second acoustic transmission medium supplied by the supplying means 23 as acoustic transmission medium 13. The cell detachment device may further include an arm 24 for moving the supplying means 23 in the X, Y, and Z axes. The culture substrate placement section only needs to be recognizable as a location where a user can place a culture vessel. In the example of FIG. 6A , the opening formed by the vibrating body 101, upper cover 15, and cushioning material 16 is suitable for placing a commonly used dish, and therefore this opening corresponds to the culture substrate placement section.

[0100] The cell detachment device in Figure 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 vibrating body 101. The vibrating body 101, which includes a piezoelectric body 12 and a vibration plate 11, generates bending vibrations that are amplified by the combination of the piezoelectric body 12, which expands and contracts itself, and the hardness of the adhered vibration plate 11. In this example, resonance due to the structural factors of the vibrating body 101 is utilized, and large vibrations can be obtained at the resonance frequency. The vibrating body can include a vibrator such as an ultrasonic vibrator. The above-mentioned expansion and contraction is caused by applying a voltage to the piezoelectric body 12 using a power supply (not shown) (the same applies to Figures 6B and 6C).

[0101] In FIG. 6A, a ring-shaped vibrator is shown as a representative vibrator 101, but a Langevin vibrator or a rectangular vibrator 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 that the acoustic transmission medium 13 be placed over 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, buffer material 16, and bolts 18 shown in FIG. 6A, and the pressure applied to the ends is tightened to a degree that does not inhibit the vibrations generated in the diaphragm.

[0104] An example is shown in which a vibrator is attached above the culture substrate and a weight 14, which acts as a pressure means, is attached below the vibrator. The weight 14 moves up and down to apply and remove pressure. The pressure means applies pressure so as to bring the culture substrate and the vibrator closer together.

[0105] 6B shows another example. In FIG. 6B, the diaphragm 11 has a convex shape, which makes it easier for excess second acoustic transmission medium to be discharged without remaining around the acoustic transmission medium. By supplying a voltage at the resonant frequency, the vibrator is driven to generate the desired ultrasonic vibration mode.

[0106] The vibrator is driven using an AC power source that supplies a voltage at a resonant frequency. The cell detachment device can be driven by applying a voltage at a frequency in a vibration mode that generates a desired vibration waveform on the cell culture surface of the culture substrate and at a desired amplitude. 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, allowing the vibrator to be driven while checking its vibration state. Furthermore, by installing a thermocouple that can measure the temperature during the cell detachment process in a location that does not affect the vibration, the device can be driven while being monitored. An infrared thermometer can also be used to measure the temperature.

[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] 6C shows a conceptual diagram of a cell detachment device including an information processing device. In FIG. 6C, the information processing device 205 acquires information regarding 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, thereby controlling the placement 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 function as a computer that performs calculations and storage. The information processing system 205 also includes a communication interface (I / F) 210, a display device 211, and an input device 212. The CPU 206, RAM 207, ROM 208, HDD 209, communication I / F 210, display device 211, and input device 212 are interconnected via a bus 213. The display device 211 and 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 the information processing system 205 are illustrated as an integrated device, but some of these functions may be configured by external devices. For example, the display device 211 and the input device 212 may be external devices separate from the components constituting the functions of the computer, including the CPU 206, etc.

[0111] The CPU 206 performs predetermined operations in accordance with programs stored in the RAM 207, HDD 209, etc., and also has the function of controlling each unit of the information processing system 205. The RAM 207 is made up of a volatile storage medium and provides a temporary memory area necessary for the operation of the CPU 206. The ROM 208 is made up of a non-volatile storage medium and stores necessary information such as programs used in the operation of the information processing system 205. The HDD 209 is made up 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 body 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 the 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 Figure 6C is an example, and other devices may be added, or some devices may not be provided. Also, some devices may be replaced with other devices having similar functions. Furthermore, some functions may be provided by other devices via a network, or the functions constituting the cell detachment 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 controls the supply unit 23 by loading a program stored in the ROM 208 or the like into the RAM 207 and executing the program. The CPU 206 also controls the display device 211 to realize the function of the display unit 110. The CPU 206 also controls the HDD 209 to realize the function of the storage unit 111.

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

[0116] <First Embodiment> The present disclosure provides, as a first embodiment, the following cell detachment method. This embodiment is a cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, comprising: an arrangement step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and a vibrating body; and a detachment step of vibrating the vibrating body while the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium, thereby detaching the cells from the culture surface.

[0117] When implementing the first embodiment of the present disclosure, the first and second acoustic transmission media are configured to be in contact with the culture substrate side. The position where the second acoustic transmission medium is disposed may change 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 structural diagrams of acoustic transmission media used in the cell detachment method according to the first embodiment of the present disclosure. FIG. 1A shows an image of the arrangement of a second acoustic transmission medium 132 when a culture substrate 21 having a flat bottom surface is combined with a first acoustic transmission medium 131 having a convex center. FIG. 1B shows an image of the arrangement of the 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 the 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. In contrast, 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 are in close contact. Therefore, even if there is variation in the culture substrate, it is easy to apply appropriate vibrations in response to the variation. In this embodiment, as shown in FIGS. 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 of the steps of the embodiment of the present disclosure is shown in Figure 5B. First, in the placement step, a first acoustic transmission medium is placed on a vibrator (step S210). Then, a second acoustic transmission medium is placed on the first acoustic transmission medium (step S220). Next, in the pressure application step, the first and second acoustic transmission media are brought into contact with the culture substrate (step S230). Then, pressure is applied from the top surface of the culture substrate using a weight or the like (step S240). In the detachment step, ultrasound application conditions are set (step S250). Next, ultrasound application is started (step S260). Finally, the detached cells are collected (step S270).

[0121] <Second Embodiment> The present disclosure provides, as a second embodiment, the following cell detachment method. This embodiment is a cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, including: an arrangement step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and a vibrating body; and a detachment step of vibrating the vibrating body while the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium, thereby detaching the cells from the culture surface.

[0122] When implementing the second embodiment of the present disclosure, the first and second acoustic transmission media are configured to be in contact with the vibrator side. The position where the second acoustic transmission medium is disposed may vary depending on the shape of the bottom surface of the culture substrate, the shape of the top surface of the vibrator, and the surface shape of the first acoustic transmission medium.

[0123] 2A to 2C show configuration diagrams of acoustic transmission media used in a cell detachment method according to a second embodiment of the present disclosure. Fig. 2A shows an image of the arrangement of a second acoustic transmission medium 132 when a culture substrate 21 having a flat bottom surface is combined with a vibrator having a flat top surface and a first acoustic transmission medium 131 having a downwardly convex center. Fig. 2B 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 vibrator and a first acoustic transmission medium 131 having a vertically convex center. Fig. 2C 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 vibrator and a first acoustic transmission medium 131 having an upwardly convex and downwardly concave center. 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 vibrator, and the culture substrate 21 is in contact with the first acoustic transmission medium.

[0124] <Third Embodiment> The present disclosure provides, as a third embodiment, the following cell detachment method. This embodiment is a cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, including: an arrangement step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and a vibrating body; and a detachment step of vibrating the vibrating body in a state in which the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium and the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium, thereby detaching the cells from the culture surface.

[0125] When implementing the third embodiment of the present disclosure, the first and second acoustic transmission media are configured to be in contact with both the culture substrate and the vibrator. The position where 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 acoustic transmission media used in a cell detachment method according to a third embodiment of the present disclosure. Fig. 3A shows an image of the arrangement of a second acoustic transmission medium 132 when a culture substrate 21 having a flat bottom surface is combined with a first acoustic transmission medium 131 having a vertically convex central portion. Fig. 3B 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 first acoustic transmission medium 131 having a vertically convex central portion. Fig. 3C 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 first acoustic transmission medium 131 having a vertically convex central portion.

[0127] <Fourth Embodiment> The present disclosure provides, as a fourth embodiment, the following cell detachment method. This embodiment is a cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, including: an arrangement step of arranging a first acoustic transmission medium and a second acoustic transmission medium between the culture substrate and a vibrating body; and a detachment step of vibrating the vibrating body to detach the cells from the culture surface, with the culture substrate in contact with the first acoustic transmission medium and the second acoustic transmission medium, and the vibrating body in contact with the first acoustic transmission medium and the second acoustic transmission medium, and the second acoustic transmission medium being continuous above and below.

[0128] When implementing the fourth embodiment of the present disclosure, the first and second acoustic transmission media are configured to be in contact with both the culture substrate and the vibrator. The position where 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 acoustic transmission media used in a cell detachment method according to a fourth embodiment of the present disclosure. Fig. 4A shows an image of the arrangement of a second acoustic transmission medium 132 when a culture substrate 21 having a flat bottom surface is combined with a first acoustic transmission medium 131 that is divided into a central portion and an outer periphery, both of which are flat. Fig. 4B 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 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 tapering toward the outer edge. Fig. 4C shows an image of the arrangement of a second acoustic transmission medium 132 and a top view thereof when a general-purpose culture substrate 21 having a curved bottom surface is combined with a first acoustic transmission medium 131 that is divided into a central portion and an outer periphery, with the central portion being flat and having a different diameter than in Fig. 4B, and the outer periphery tapering toward the outer edge and having a different width than in Fig. 4B. When the abdomen in the vibration pattern of the vibrator appears near the center of the vibrator, efficient peeling is possible 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.

[0130] Example 1 Examples 1 to 28, 32, and 33 correspond to the first embodiment. In Example 1, the acoustic transmission medium configuration 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 diaphragm of the cell detachment device was made of glass with an outer diameter of 70 mm and a thickness of 4 mm. The piezoelectric material composition was PZT, the outer diameter was the same as the diaphragm (70 mm), the inner diameter was 57 mm, and the thickness was 2 mm, considering that the neutral plane was located on the diaphragm side. The cell detachment device was configured as shown in FIG. 6A. A convex disc-shaped silicone rubber 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 placed on the diaphragm so that its center was aligned with the diaphragm, as shown in the schematic diagram of FIG. 9A. This silicone rubber was used as the first acoustic transmission medium. When this silicone rubber was subjected to vibration at a frequency of 1 Hz, the tan δ obtained by dynamic viscoelasticity measurement at room temperature was 0.13. 200 μL of water, the second acoustic transmission medium, was dropped onto the center of the first acoustic transmission medium using a dispenser. Next, a Thermo Fisher Scientific Nunc® Φ35 mm dish, a culture substrate on which cells had been cultured, was slowly placed so that its center was aligned with the center of the vibration plate. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. Figure 1B shows a schematic diagram of the acoustic transmission medium configuration after pressure application. The contact areas of the first and second acoustic transmission media and the culture substrate were calculated by coloring the water, the second acoustic transmission medium, with food coloring and taking a photograph from above after pressure application to confirm that there was no air gap between the acoustic transmission medium and the culture substrate. 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°.

[0131] For cell culture, CHO (Chinese Hamster Ovary) cells were cultured in a Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish at a density of 10,000 cells / cm. ２ The cells were seeded at a density of 1000× ... and incubated at 37° ２The cells were cultured in an environment with a 5% cell concentration. 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). The culture was carried out for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell area occupied by the dish was approximately 80%.

[0132] (Cell Detachment) The medium in the dish was removed, and the cells were washed with PBS(-). Then, the cells were 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 using frequency sweep vibration (frequency 22-27 kHz, frequency sweep period 1 s, voltage 90 V) in standing wave mode for 2 minutes at an ambient temperature of 25°C. A standing wave was generated by applying an AC voltage with a 180-degree phase difference to the negatively polarized electrodes (A2, B2) relative to the positively polarized electrodes (A1, A3, B1, B3) shown in FIG. 10 . This vibration mode is referred to as vibration mode A in FIG. 12 (described below). After recovering the detached cells, a cell scraper was used to detach any cells that could not be detached by ultrasound from the dish, and the cell count was measured using a hemocytometer. The total number of cells detached by ultrasound and the number of cells detached with a cell scraper after ultrasound detachment were combined to form 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 detachment rate value was calculated. Variation was determined for n = 10, and the standard deviation of the detachment rate was evaluated as A for less than 5%, B for 5% to less than 7.5%, C for 7.5% to less than 10%, and D for 10% or more. The evaluation results for Example 1 showed a detachment rate of 97.4%, and the variance was rated A.

[0133] Example 2 Example 2 was performed under the same conditions as Example 1, except that the first acoustic transmission medium was changed as follows, and the amount of the second acoustic transmission medium used was changed as follows. Specifically, a disk-shaped silicone rubber with a hardness of 30°, having a center 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 onto the first acoustic transmission medium using 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 peeling rate of 90.3% and a variation rating of C.

[0134] Example 3 In Example 3, the cell detachment conditions were the same as in Example 1, except for the following changes. Cell detachment was performed under standing wave mode with frequency sweep vibration for 2 minutes (frequency 67.5-72.5 kHz, frequency sweep period 1 s, voltage 90 V). Although the frequency band was different from that in Example 1, standing waves were generated under the same conditions: AC voltage with a 180-degree phase difference was applied to negatively polarized electrodes (A2, B2) relative to positively polarized electrodes (A1, A3, B1, B3). The vibration mode achieved by such driving is referred to as vibration mode B in FIG. 12 described below. The evaluation results for Example 3 showed a detachment rate of 90.0% and a variation rating of C.

[0135] Example 4 In Example 4, the cell detachment conditions were the same as those in Example 2, except that they were changed as follows. Cell detachment was performed under standing wave mode with frequency sweep vibration for 2 minutes (frequency 67.5-72.5 kHz, frequency sweep period 1 s, voltage 90 V). 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 test was performed under the same conditions as Example 1, except that the first acoustic transmission medium was changed as follows. Specifically, a silicone rubber with a hardness of 30° and a deformed convex disc shape was used on the diaphragm as the first acoustic transmission medium, as shown in the schematic diagram of FIG. 9C , 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. 250 μL of water, which was 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 showed a peeling rate of 90.2% and a variation rating of B.

[0137] Example 6 Example 6 was performed under the same conditions as Example 1, except that the first acoustic transmission medium was changed as follows. Specifically, a silicone rubber with a deformed disk shape and a hardness of 30°, as shown in the schematic diagram of FIG. 9D , was used as the first acoustic transmission medium on the diaphragm. The dimensions were a central 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. 200 μL of water, which was 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 30%. The evaluation results for Example 6 showed a peeling rate of 98.2% and a variation rating of A.

[0138] Example 7 In Example 7, the second acoustic transmission medium was changed to PVA gel, and the experiment was carried out under the same conditions as Example 1, except that the amount used was changed. The PVA gel was prepared as follows. Denka Poval (registered 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 solution was heated in a thermostatic bath while stirring to completely dissolve the PVA, yielding a PVA aqueous solution. Next, borax (special grade sodium tetraborate (decahydrate); manufactured by Kishida Chemical Co., Ltd.) was weighed out, and pure water was added to prepare a 1.0% by mass solution to prepare a total of 16 mL. While stirring the PVA aqueous solution, the borax aqueous solution was gradually added dropwise, and the mixture was stirred until homogeneous, yielding a PVA gel. When vibrations of 0.1 Hz were applied to this PVA gel, the tan δ obtained by dynamic viscoelasticity measurement at room temperature was 1.7, indicating that the PVA can be considered a fluid solid. When vibrations of 1 Hz were 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, a Nunc (registered trademark) Φ35 mm dish (manufactured by Thermo Fisher Scientific) used as a culture substrate on which cells were cultured was slowly placed with its center aligned with the center of the vibration plate. A 100 g weight was then 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 showed a peeling rate of 85.7% and a variation rating of C.

[0139] Example 8: In Example 8, the second acoustic transmission medium was changed to ultrasound gel, and the experiment was conducted under the same conditions as Example 1, except that the amount used was changed. 0.2 g of ultrasound gel (Ultrasound Examination Pro Jelly Normal Type; manufactured by Gex) was weighed as the second acoustic transmission medium and uniformly spread over a 20 mm area from the center on the first acoustic transmission medium. Next, a Nunc® 35 mm Φ dish (manufactured by Thermo Fisher Scientific) serving as a culture substrate on which cells had been cultured was slowly placed with its center aligned with the center of the diaphragm. A 100 g weight was then 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 showed a detachment rate of 96.4% and a variation rating of A.

[0140] Example 9 In Example 9, the second acoustic transmission medium was changed to glycerin, and the experiment was conducted under the same conditions as Example 1, except that the amount used was changed. 0.27 g of glycerin (special grade glycerin; manufactured by Kishida Chemical Co., Ltd.) was weighed as the second acoustic transmission medium and evenly spread and placed on the first acoustic transmission medium. Next, a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which served as a culture substrate on which cells had been cultured, was slowly placed with its center aligned with the center of the diaphragm. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. 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 100°. The evaluation results for Example 9 showed a peeling rate of 94.3% and a variation of A.

[0141] Example 10: In Example 10, the experiment was conducted under the same conditions as Example 1, except that the first acoustic transmission medium was changed as follows. Specifically, the first acoustic transmission medium was a silicone rubber diaphragm with a convex disc shape (30° hardness) and a central height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm, as shown in the schematic diagram of FIG. 9A. The surface facing the culture substrate had 12 linear grooves, each 0.3 mm wide and 0.2 mm deep, spaced at 30° intervals and radiating from the center. 1400 μL of water, 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 10%. The evaluation results for Example 10 showed a peeling rate of 92.1% and a variation rating of A.

[0142] Example 11 In Example 11, the second acoustic transmission medium was changed to ethanol, and the experiment was conducted under the same conditions as Example 1, except that the amount used was changed. 1400 μL of ethanol (special grade ethanol; manufactured by Kishida Chemical Co., Ltd.), the second acoustic transmission medium, was dropped onto the center of the first acoustic transmission medium using a dispenser. Next, a Nunc (registered trademark) Φ35 mm dish (manufactured by Thermo Fisher Scientific Inc.) serving as a culture substrate on which cells had been cultured was slowly placed so that its center was aligned with the center of the diaphragm. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. 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 40°. The evaluation results for Example 11 showed a peeling 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% ethanol aqueous solution, and the experiment was conducted under the same conditions as Example 1, except that the amount used was changed. The 70% ethanol aqueous solution, which was the second acoustic transmission medium, was obtained by mixing ethanol (special grade ethanol; manufactured by Kishida Chemical) and pure water at 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, a Nunc® Φ35 mm dish manufactured by Thermo Fisher Scientific, which served as a culture substrate on which cells had been cultured, was slowly placed so that its center was aligned with the center of the diaphragm. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. 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 55°. The evaluation results for Example 12 showed a peeling 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 experiment was conducted under the same conditions as Example 1, except for the amount used. The surfactant aqueous solution, which was 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 resulting surfactant aqueous solution 2 (sodium lauryl sulfate aqueous solution) was dropped onto the center of the first acoustic transmission medium using a dispenser. Next, a Nunc (registered trademark) Φ35 mm dish (manufactured by Thermo Fisher Scientific) used as a culture substrate on which cells were cultured was slowly placed so that its center was aligned with the center of the diaphragm. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. 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 50°. The evaluation results for Example 13 showed a peeling rate of 93.9% and a variation of A.

[0145] Example 14 In Example 14, the second acoustic transmission medium was changed to surfactant aqueous solution 2 (water + surfactant 2), and the experiment was carried out under the same conditions as Example 1, except that the amount used was changed. The surfactant aqueous solution, which was the second acoustic transmission medium, was obtained by mixing Pluronic (registered trademark) F-68 (Pluronic (registered trademark) F-68 Non-ionic Surfactant (100X); manufactured by Thermo Fisher Scientific) with pure water to a solids 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, a Nunc (registered trademark) Φ35 mm dish manufactured by Thermo Fisher Scientific, which was a culture substrate on which cells had been cultured, was slowly placed so that its center 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. 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 75.degree.. The evaluation results for Example 14 showed that the peeling rate was 93.2% and the variation was A.

[0146] Example 15: In Example 15, the study was conducted under the same conditions as Example 1, except that the first acoustic transmission medium was changed as follows. Specifically, the first acoustic transmission medium was a silicone rubber (schematic diagram: Figure 9A ) used in Example 1, which had a convex disc shape with a hardness of 30° and a central convexity, a center height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm, and was hydrophilically treated on the convex surface. Water, the second acoustic transmission medium, was dropped onto the hydrophilically treated silicone rubber surface, and the contact angle was measured, resulting in a value of 25°. 1400 μL of water, the second acoustic transmission medium, 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 showed a peeling rate of 95.5% and a variation of A.

[0147] Example 16 In Example 16, the diaphragm 11 in the device configuration diagram of FIG. 6A was replaced with glass whose upper surface had been hydrophilized, and the device was assembled under the same conditions as Example 1. Water, the second acoustic transmission medium, was dropped onto the hydrophilized glass surface, and the contact angle was measured, which was 20°. 1400 μL of water as the second acoustic transmission medium 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 peeling rate of 95.1% and a variation of A.

[0148] Example 17 In Example 17, the culture substrate 21 in the device configuration diagram of FIG. 6A was replaced with a culture substrate whose outer surface, including the sides and bottom, had been hydrophilized. Water, serving as the second acoustic transmission medium, was dropped onto the hydrophilized bottom surface of the culture substrate, and the contact angle was measured, yielding a value of 25°. 1,400 μL of water, serving as the second acoustic transmission medium, was dropped onto the center of the first acoustic transmission medium using a dispenser and placed thereon. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 17 showed a peeling rate of 94.8% and a variation of A.

[0149] Example 18 In Example 18, the diaphragm 11 was replaced with a convex-shaped glass plate, as shown in the device configuration diagram of FIG. 6B. The convex-shaped glass plate used was a glass plate with an outer diameter of 70 mm and a thickness of 4 mm, to which a glass plate with an outer diameter of 32 mm and a thickness of 2 mm was bonded. 1400 μL of water was dropped onto the center of the first acoustic transmission medium using a dispenser, and the second acoustic transmission medium was placed. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 18 showed a peeling rate of 95.8% and a variation of A.

[0150] Example 19: Example 19 was studied 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. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 19 showed a peeling 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 amount used was also changed, but the study was conducted under the same conditions as in Example 1. That is, the same 70% ethanol used as the second acoustic transmission medium as in Example 12 was used. A liquid volume of 1400 μL 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 20 were a peeling rate of 97.1% and a variation of A.

[0152] Example 21 In Example 21, the study was conducted 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 as the second acoustic transmission medium was dropped onto the center of the first acoustic transmission medium using a dispenser and placed thereon. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 21 showed a peeling rate of 91.1% and a variation of B.

[0153] Example 22 In Example 22, the study was conducted 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 used as the second acoustic transmission medium, and the second acoustic transmission medium was sprayed uniformly onto the first 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 peeling rate of 90.2% and a variation of C.

[0154] Example 23 In Example 23, the study was conducted 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 as the second acoustic transmission medium was dropped onto the center of the first acoustic transmission medium using a dispenser and placed thereon. The contact area ratio of the first acoustic transmission medium was 10%. The evaluation results for Example 23 were a peeling rate of 90.4% and a variation of B.

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

[0156] Example 25: In Example 25, the study was conducted 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 used as the second acoustic transmission medium, and the water was sprayed evenly onto the first acoustic transmission medium using a spray device. The contact area ratio 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, with 20% of the total area being an air layer. The evaluation results for Example 25 were a peeling 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 disc-shaped urethane rubber (schematic diagram, Figure 9A ) similar to that used in Example 1, with a central height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm. A vibration of 1 Hz was applied to this silicone rubber, and viscoelasticity measurements were performed at room temperature. Tan δ was 0.23. As the second acoustic transmission medium, 200 μL of water was dispensed 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 contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 80°. The evaluation results for Example 26 showed a peeling rate of 90.1% and a variation of B.

[0158] Example 27: In Example 27, the same conditions as in Example 1 were used. The silicone rubber used in Example 1 was used as the first acoustic transmission medium on the diaphragm. The silicone rubber had 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° (schematic diagram: Figure 9A). A vibration of 1 Hz was applied to this silicone rubber, and viscoelasticity measurements were performed at room temperature. Tan δ was found to be 0.14. As the second acoustic transmission medium, 200 μL of water was dispensed 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 contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 110°. The evaluation results for Example 27 showed a peeling rate of 93.3% and a variation of B.

[0159] Example 28: In Example 28, the same conditions as in Example 1 were used, except that the silicone rubber used in Example 1, with a convex disc shape (center height (1) = 0.9 mm, side height (2) = 0.4 mm, and diameter (3) = 32 mm and a hardness of 50° (schematic diagram: Figure 9A), was used as the first acoustic transmission medium on the diaphragm. A vibration frequency of 1 Hz was applied to this silicone rubber, and viscoelasticity measurements were performed at room temperature. Tan δ was 0.05. As the second acoustic transmission medium, 200 μL of water was dispensed 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 contact angle between the first acoustic transmission medium and the second acoustic transmission medium was 110°. The evaluation results for Example 28 showed a peeling rate of 96.4% and a variation of A.

[0160] Example 29 corresponds to the second embodiment. Example 29 was similar to Example 1, except for the modified installation process, which used the acoustic transmission medium configuration shown in the schematic diagram of FIG. 2A. The convex surface of the first acoustic transmission medium, similar to that of Example 1, was brought into contact with the bottom surface of a Thermo Fisher Scientific Nunc® Φ35 mm dish, a culture substrate on which cells were cultured, from the backside, with the center aligned, and the entire surface was tightly attached to prevent air gaps from forming. Next, 200 μL of water, the second acoustic transmission medium, was dropped onto the center of the diaphragm using a dispenser, and the Φ35 mm dish with the first acoustic transmission medium tightly attached to its bottom surface was slowly placed so that its center 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. A schematic diagram showing the configuration of the acoustic transmission medium after pressure application is shown in FIG. 2C. The contact areas between the first and second acoustic transmission media and the culture substrate were calculated by coloring the second acoustic transmission medium, water, with food coloring, and then confirming that there was no air gap between the acoustic transmission medium and the culture substrate from a photograph taken from below the diaphragm after pressure application. The contact area rate of the first acoustic transmission medium was 10%. The cell detachment conditions and detached cell testing were performed using the same methods as in Example 1. The evaluation results for Example 29 showed a detachment rate of 97.2% and a variation of A.

[0161] Example 30 corresponds to the third embodiment. In Example 30, a Corning Φ35 mm dish was used as the culture substrate. As the first acoustic transmission medium, a silicone rubber with a hardness of 10° was prepared, as shown in the schematic diagram of FIG. 9E, with a central height (1) of 1.0 mm, a side height (2) of 0.3 mm, a diameter (3) of 32 mm, a top surface height (4) of 0.4 mm, and a bottom surface height (5) of 0.3 mm. The cell detachment device was assembled, and 100 μL of water, the second acoustic transmission medium, was dispensed onto the center of the diaphragm using a dispenser. Next, the bottom surface of the silicone rubber, the first acoustic transmission medium, was slowly placed so that its center was aligned with the center of the diaphragm. Furthermore, 150 μL of water, the second acoustic transmission medium, was dispensed onto the center of the surface of the first acoustic transmission medium using a dispenser. The center of the Corning Φ35 mm dish, the culture substrate on which the cells were cultured, was slowly placed so that its center was aligned with the center of the diaphragm. A 100g weight was then placed on the lid of the culture substrate and pressure was applied. Figure 3B shows a schematic diagram of the configuration of the acoustic transmission medium after pressure application. The contact areas of the first and second acoustic transmission media and the culture substrate were calculated by coloring water, the second acoustic transmission medium, with food coloring. After pressure application, photographs were taken from above and below to confirm that there was no air gap 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 detached cell testing were performed using the same methods as in Example 1. The evaluation results for Example 30 showed a detachment rate of 94.4% and a variation of B.

[0162] Example 31 corresponds to the fourth embodiment. In Example 31, a ΦIWAKI Φ35 mm dish manufactured by AGC Techno Glass Co., Ltd. was used as the culture substrate. As the first acoustic transmission medium, a circular silicone rubber piece with a thickness (6) of 0.6 mm and a diameter (7) of 3.0 mm and a ring-shaped silicone rubber piece with a hardness of 10° and a thickness (6') of 0.6 mm, a width (8') of 4.0 mm, and a diameter (7') of 32 mm, as shown in the schematic diagram of FIG. 9F, was prepared. A cell detachment device was configured as shown in FIG. 4C, and the circular and ring-shaped silicone rubber pieces serving as the first acoustic transmission medium were placed at the center of the diaphragm. Furthermore, 400 μL of water serving as the second acoustic transmission medium was dropped onto the center of the surface of the first acoustic transmission medium using a dispenser. The ΦIWAKI Φ35 mm dish manufactured by AGC Techno Glass Co., Ltd., which served as the culture substrate on which the cells were cultured, was slowly placed so that its center was aligned with the center of the diaphragm. A 100g weight was then placed on the lid of the culture substrate and pressure was applied. Figure 4C shows a schematic diagram of the configuration of the acoustic transmission medium after pressure application. The contact area between the first acoustic transmission medium and the culture substrate was calculated by coloring the water used as the second acoustic transmission medium with food coloring, and then confirming the absence of an air gap between the acoustic transmission medium and the culture substrate using a photograph taken from above after pressure application. The total contact area rate of the first acoustic transmission medium was 30%. The cell detachment conditions and detached cell testing were performed using the same methods as in Example 1. The evaluation results for Example 31 showed a detachment rate of 92.3% and a variation of C.

[0163] Example 32 In Example 32, the cell detachment conditions were the same as those in Example 1, except that they were changed as follows. Cell detachment was performed under the conditions of frequency sweep vibration (frequency 67.5-72.5 kHz, frequency sweep period 1 s, voltage 90 V) in the traveling wave mode described above for 2 minutes. An AC voltage with a 90-degree phase difference between phases A and B in FIG. 10 was applied to generate a two-phase driven traveling wave. The vibration mode achieved by such drive is referred to as vibration mode C in FIG. 12 described below. The evaluation results for Example 32 showed a detachment rate of 99.4% and a variation rating of A.

[0164] Example 33 In Example 33, the same conditions as in Example 1 were used for the study, except that the pressurization step of installing the weight used in Example 1 for pressurization was omitted and the amount of water, the second acoustic transmission medium, was reduced. Specifically, a first acoustic transmission medium similar to that in Example 1 was placed so that its center was aligned with the diaphragm, and then 160 μL of water, the second acoustic transmission medium, was dropped onto the center of the first acoustic transmission medium using a dispenser. A Nunc® 35 mm Φ dish (manufactured by Thermo Fisher Scientific) serving as a culture substrate on which cells were cultured was slowly placed so that its center was aligned with the center of the diaphragm. The contact area ratio of the first acoustic transmission medium was 3%. The evaluation results for Example 33 showed a peeling rate of 84.1% and a variation of C.

[0165] (Comparative Examples) (Comparative Example 1) In Comparative Example 1, the study was conducted 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 30° hardness and a deformed convex disc shape, the same 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, a Nunc (registered trademark) Φ35 mm dish (manufactured by Thermo Fisher Scientific) serving as a culture substrate on which cells were cultured was slowly placed so that its center was aligned with the center of the diaphragm. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. A photograph taken from above after pressure application confirmed an air gap around the contact area 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 found to be 5%. The evaluation results for Comparative Example 1 showed a peeling 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 the 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). As in Comparative Example 1, a photograph taken from above after pressurizing the culture substrate confirmed an air layer around the contact area 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 found to be 5%. The evaluation results for Comparative Example 2 showed a detachment rate of 70.8% and a variation of D.

[0167] Comparative Example 3: In Comparative Example 3, the study was conducted under the same conditions as in Example 1, except that neither the culture substrate nor the vibrator was in contact with the first acoustic transmission medium. Specifically, as in Example 1, a convex, circular-shaped silicone rubber with a hardness of 30°, measuring 0.9 mm in center height (1), 0.4 mm in side height (2), and 32 mm in diameter (3), as shown in the schematic diagram of FIG. 9A, was placed at the center of the diaphragm as the first acoustic transmission medium. Next, as the second acoustic transmission medium, a total of 300 μL of water was dispensed evenly in 50 μL increments using a dispenser. Next, a Thermo Fisher Scientific Nunc® 35 mm Φ dish, which served as the culture substrate on which the cells had been cultured, was slowly placed with its center aligned with the center of the diaphragm. A photograph taken from above the culture substrate confirmed that there was no contact with the first acoustic transmission medium and that there was no air gap 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 peeling rate of 58.4% and a variation of D.

[0168] Comparative Example 4: In Comparative Example 4, the same conditions as in Example 1 were used, except that the first acoustic transmission medium was not used and the amount of water used as the second acoustic transmission medium was changed. 4.0 mL of water was placed on the vibrator using a dispenser as the second acoustic transmission medium. After the water was uniformly spread on the vibrator, the center of a Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish, which served as a culture substrate on which cells had been cultured, was aligned with the center of the vibration plate and slowly placed on the substrate to prevent air pockets from forming on the bottom of the culture substrate. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. A photograph taken from above after pressure application confirmed that the entire bottom surface of the culture substrate on the vibrator side was filled with the second acoustic transmission medium and no air pockets were present. The contact area between the first acoustic transmission medium and the culture substrate was 0%. The evaluation results for Comparative Example 4 showed a peeling rate of 82.0% and a variation of D.

[0169] (Summary) The configurations and evaluation results of the above-described Examples and Comparative Examples are summarized in Fig. 12. In Fig. 12, tan δ indicates the value at room temperature when vibration of a frequency of 0.1 Hz is applied.

[0170] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0171] This application claims priority based on Japanese Patent Application No. 2023-118581, filed on July 20, 2023, the entire contents of which are incorporated herein by reference.

[0172] 11: Diaphragm 12: Piezoelectric body 13: Acoustic transmission medium 131: First acoustic transmission medium 132: Second acoustic transmission medium 14: Weight 15: Upper cover 16: Cushioning material 17: Lower cover 18: Bolt 21: Culture substrate 22: Culture substrate upper cover 23: Supply unit 24: Arm 30: Groove portion 101: Vibrating 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 detachment step of vibrating the vibrator to detach the cells from the culture surface in a state in which a first acoustic transmission medium and a second acoustic transmission medium are placed 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, 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 fluidity at room temperature.

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

3. The cell detachment method according to claim 1 or 2, characterized in that in the detachment step, while (a) is satisfied and the vibrator is in contact with the first acoustic transmission medium, the vibrator is vibrated to detach the cells from the culture surface.

4. A cell detachment method as described in claim 2 or 3, characterized in that in the detachment 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 relationship 0.05≦S1 / S1+S2 is satisfied.

5. A cell detachment method according to claim 1, characterized in that in the detachment step, the vibrator is vibrated while (i) is filled, thereby detaching the cells from the culture surface.

6. A cell detachment method as described in claim 5, characterized in that in the detachment process, when the area of ​​the portion of the vibrating body in contact with the first acoustic transmission medium is S3 and the area of ​​the portion of the vibrating body in contact with the second acoustic transmission medium is S4, the relationship 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. A cell detachment method described in any one of claims 1 to 7, characterized in that the second acoustic transmission medium is a solid having a tan δ of 1.0 or more when subjected to vibrations of a frequency of 0.1 Hz at room temperature.

9. A cell detachment method according to any one of claims 1 to 8, wherein the second acoustic transmission medium is liquid at room temperature.

10. A cell detachment method described in any one of claims 1 to 9, characterized in that the second acoustic transmission medium is a liquid, and the contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 120° or less.

11. The cell detachment method described in claim 10, characterized in that the second acoustic transmission medium is a liquid, and the contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 90° or less.

12. A cell detachment method described in any one of claims 1 to 11, characterized in that the second acoustic transmission medium is liquid, and at least one of the contact angle at the contact surface between the culture substrate and the second acoustic transmission medium and the contact angle at the contact surface between the vibrator and the second acoustic transmission medium is smaller than 40°.

13. A cell detachment method described in any one of claims 1 to 12, 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. A cell detachment method described in any one of claims 1 to 13, characterized in that in the detachment process, 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.

15. A cell detachment method according to any one of claims 1 to 14, characterized in that the first acoustic transmission medium includes silicone rubber.

16. A cell detachment method according to any one of claims 1 to 15, characterized in that the first acoustic transmission medium includes a porous resin material.

17. A cell detachment method described in any one of claims 1 to 16, characterized in that the second acoustic transmission medium contains at least one selected from the group consisting of water, saline, ethanol, and diluted ethanol.

18. A cell detachment method according to any one of claims 1 to 17, characterized in that the detachment step includes a step of exciting vibrations in the ultrasonic band in the vibrator.

19. A cell detachment method described in any one of claims 1 to 18, characterized in that it includes a positioning step of positioning the first acoustic transmission medium and the second acoustic transmission medium between the culture substrate and the vibrating body prior to the detachment process.

20. The cell detachment method described in claim 19, characterized in that in the placement 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. A cell detachment method as described in claim 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.

22. A cell detachment method according to any one of claims 1 to 21, further comprising a pressurizing step for applying pressure so as to bring the culture substrate and the vibrator closer together.

23. A cell detachment method according to any one of claims 1 to 22, characterized in that it includes a surface treatment step of applying a surface treatment to at least one surface selected from the group consisting of: a surface of the culture substrate facing the first acoustic transmission medium, a surface of the vibrator 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.

24. A cell detachment device for detaching cells from a culture surface of a culture substrate by transmitting vibrations generated by a vibrator to the cells provided on the culture surface, the cell detachment device having, in this order, a vibrator, a first acoustic transmission medium, and a culture substrate mounting section, and further having a supply means for supplying a second acoustic transmission medium, the supply means supplying the 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. (b) the vibrator is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

25. The cell peeling device according to claim 24, further comprising an information processing device which acquires information relating to the state of vibration excited in the vibrating body and controls the supply unit based on the information.

26. The cell detachment device according to claim 24 or 25, characterized in that the vibrating body includes an ultrasonic vibrator.

27. The cell detachment device according to any one of claims 24 to 26, 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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