Cell detachment method and cell detachment device

Abstract

To provide a cell detachment method and a cell detachment device that enable vibration design tailored to target cells by mechanically matching a culture substrate and a vibrator, and can detach adherent cells with a high detachment rate.SOLUTION: Provided is a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, in which an acoustic transmission medium is placed between the culture substrate and a vibrator, and the vibrator is vibrated to detach the cells from the culture surface, the acoustic transmission medium including a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium being a solid having elasticity at room temperature, the second acoustic transmission medium being contained within the first acoustic transmission medium, and being a liquid at room temperature, or having a larger tan δ when a vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature than the tan δ of the first acoustic transmission medium.SELECTED DRAWING: Figure 1A

Description

[Technical field]

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

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

[0003] In cell detachment techniques that use ultrasound or acoustic radiation pressure, water is generally used as the acoustic transmission medium. In this case, there is a strong concern that water may breed bacteria, and so it tends to be avoided in medical settings.

[0004] Patent Document 1 proposes a patterning device that arranges cells at specific positions via a medium that transmits acoustic radiation pressure to a culture substrate. Silicone rubber is described as an example of an acoustic transmission medium, but when acoustic radiation pressure is transmitted to a culture substrate using this material, the cell detachment performance is insufficient compared to water, and further improvement of cell detachment performance and reduction of variation are required. Patent Document 2 also proposes a medical ultrasonic transmission tool composed of a sealed body. Although it is used for acoustic coupling between an ultrasonic probe and a target site, there is no description of using this ultrasonic transmission means to transmit ultrasonic vibrations and detach cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-42534 A [Patent Document 2] Patent No. 6325363 Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure has been made to address the above-mentioned problems, and provides a cell detachment method that enables vibration design tailored to the target cells and can detach adherent cells with a high detachment rate. [Means for solving the problem]

[0007] The present disclosure provides a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: a cell detachment method for detaching the cells from the culture surface by vibrating a vibrator while disposing an acoustic transmission medium between the culture substrate and the vibrator; However, the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium is a solid having elasticity at room temperature; The present invention provides a cell detachment method, wherein the second acoustic transmission medium is contained within the first acoustic transmission medium and is liquid at room temperature, or has a larger tan δ than the tan δ of the first acoustic transmission medium when vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature.

[0008] The present disclosure also provides a cell detachment device that detaches cells by transmitting vibrations generated by a vibrator to cells provided on a culture surface of a culture substrate, Vibration body, Acoustic transmission medium, and A culture substrate installation section is provided in this order. The vibration of the vibrator is transmitted to the culture substrate via the acoustic transmission medium, the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium; the first acoustic transmission medium is a solid having elasticity at room temperature; The second acoustic transmission medium is contained within the first acoustic transmission medium, and is liquid at room temperature, or has a larger tan δ than the tan δ of the first acoustic transmission medium when vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature. Effect of the Invention

[0009] According to the present disclosure, it is possible to provide a cell detachment method that enables vibration design tailored to the target cells and can detach adherent cells with a high detachment rate. [Brief description of the drawings]

[0010] [Figure 1A] 1 is a schematic cross-sectional view showing an example of the configuration of a first embodiment of a cell peeling device according to the present disclosure. [Figure 1B] 1 is a schematic cross-sectional view showing an example of the configuration of a first embodiment of a cell peeling device according to the present disclosure. [Figure 1C] 1 is a schematic cross-sectional view showing an example of the configuration of a first embodiment of a cell peeling device according to the present disclosure. [Figure 2A] 11 is a schematic cross-sectional view showing an example of the configuration of a second embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 2B] 11 is a schematic cross-sectional view showing an example of the configuration of a second embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 2C] 11 is a schematic cross-sectional view showing an example of the configuration of a second embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 3A] 13 is a schematic cross-sectional view showing an example of the configuration of a third embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 3B] 13 is a schematic cross-sectional view showing an example of the configuration of a third embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 3C] 13 is a schematic cross-sectional view showing an example of the configuration of a third embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 4A] 13 is a schematic cross-sectional view showing an example of the configuration of a fourth embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 4B] 13 is a schematic cross-sectional view showing an example of the configuration of a fourth embodiment of a cell peeling device according to the present disclosure. FIG. [Figure 4C] 13 is a schematic cross-sectional view showing an example of the configuration of a fourth embodiment of a cell peeling device according to the present disclosure. FIG. [Diagram 5] Specific examples of steps of the cell detachment method of the present disclosure are shown below. [Figure 6] 1 illustrates an example of an apparatus of the present disclosure. [Figure 7A] An example of the placement step of the method of the present disclosure will now be described. [Figure 7B] An example of the placement step of the method of the present disclosure will now be described. [Figure 7C] An example of the pressurizing step of the method of the present disclosure will now be described. [Figure 7D] An example of the peeling step of the method of the present disclosure will now be described. [Figure 8] FIG. 1 shows a schematic diagram of an example of a culture substrate according to the present disclosure. [Figure 9A] 1A and 1B are a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure. [Figure 9B] 1A and 1B are a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure. [Figure 9C] 1A and 1B are a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure. [Figure 9D] 1A and 1B are a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure. [Figure 10] FIG. 2 is a schematic diagram illustrating an example of a piezoelectric body according to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 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.

[0012] (Basic configuration) The cell detachment method of the present disclosure includes the steps of: A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: a cell detachment method for detaching the cells from the culture surface by vibrating a vibrator while disposing an acoustic transmission medium between the culture substrate and the vibrator; However, the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium is a solid having elasticity at room temperature; This is a cell detachment method, characterized in that the second acoustic transmission medium is contained within the first acoustic transmission medium and is liquid at room temperature, or has a larger tan δ than the tan δ of the first acoustic transmission medium when vibrations of a frequency of 0.1 Hz are applied to the second acoustic transmission medium at room temperature.

[0013] A conceptual diagram of the cell detachment method of the present disclosure is shown in Figures 7A to 7D. 7A shows a step of placing the acoustic transmission medium 13. Note that the acoustic transmission medium 13 may be placed in advance, and this step is not essential to the cell detachment method of the present disclosure. In FIG. 7A, the acoustic transmission medium 13 is placed on the vibrating body 101. In FIG. 7B, the culture substrate 21 is placed on the placed acoustic transmission medium 13. FIG. 7C shows a pressurizing step, in which pressure is applied from above the culture substrate 21. Note that the pressurizing step is not essential to the peeling method of the present disclosure. FIG. 7D shows a peeling step. In FIG. 7D, the vibrating body 101 is driven while applying pressure from above the culture substrate 21 to apply ultrasonic vibration to peel off the cells. The pressurizing step makes it easier for the first acoustic transmission medium to match the culture substrate or the vibrating body, and even in the peeling step, the first acoustic transmission medium can transmit vibration in a state where it is matched to the culture substrate or the vibrating body. Here, the first acoustic transmission medium is matched with the culture substrate or the vibrating body means that the first acoustic transmission medium is in sufficient contact with the culture substrate or the vibrating body so that the vibration of the vibrating body is sufficiently transmitted to the culture substrate through the first acoustic transmission medium or the second acoustic transmission medium, but an extremely small gap or an extremely small amount of material may be present between the first acoustic transmission medium and the culture substrate or the vibrating body to a degree that can solve the problem of the present invention. For example, even if a few molecules of water are present between the first acoustic transmission medium and the culture substrate, the first acoustic transmission medium and the culture substrate can be considered to be in contact with each other.

[0014] In the cell detachment method of the present disclosure, when the vibration of the vibrator is transmitted to the culture substrate, an acoustic transmission medium is provided between them. The acoustic transmission medium includes a first acoustic transmission medium that is elastic and solid at room temperature, a second acoustic transmission medium that is liquid at room temperature, or a second acoustic transmission medium whose tan δ when a vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature is larger than the tan δ of the first acoustic transmission medium (tan δ when a vibration of a frequency of 0.1 Hz is applied to the first acoustic transmission medium at room temperature). The vibrator, which is a vibration generating unit, includes a piezoelectric body and a vibration plate, and the vibration mechanism generates bending vibration in which the vibration is amplified by a combination of the hardness of the piezoelectric body that expands and contracts itself and the vibration plate to which it is attached. In the present disclosure, the resonance due to the structural factor of the vibrator is utilized, and a large vibration can be obtained at the resonance frequency.

[0015] (acoustic transmission medium) In the cell detachment method of the present disclosure, an acoustic transmission medium is used to match the vibrating body that generates vibrations with the culture substrate to which the vibrations are transmitted. In addition to physical matching such as acoustic impedance, mechanical matching such as adhesion is also important for matching. It is preferable that mechanical matching and physical matching are achieved between the acoustic transmission medium and the culture substrate or the vibrating body, thereby achieving a configuration that can solve the problems 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 Z." Specifically, the acoustic impedance Z of the acoustic transmission medium is Z = (density of acoustic transmission medium) x (speed of sound in the acoustic transmission medium) is defined as:

[0017] Also, when a sound wave reaches the boundary between two materials, the transmittance of the sound wave is, Transmittance=(4×Z1×Z2) / (Z1+Z2)^2 where Z1 and Z2 are the acoustic impedances of the first and second materials that transmit sound waves. For example, the permeability between water and air and between silicone rubber and air are respectively: Acoustic impedance of water Z1 = 1.5 x 10^6 [kg / m^2 / s] Acoustic impedance of air Z2=4.08×10^2[kg / m^2 / s] Acoustic impedance of silicone rubber Z3=1.0×10^6[kg / m^2 / s] If we substitute the impedance of water and air into the above equation for the transmittance, the transmittance becomes almost zero. That is, the transmittance of sound waves between water and air, and between silicone rubber and air, is nearly zero, resulting in total reflection.

[0018] Polystyrene, which is often used as a material for culture substrates, has an acoustic impedance of Z4 = 2.4 x 10^6 [kg / m^2 / s]. Therefore, in terms of matching the physical properties of the vibrator and the culture substrate in the acoustic transmission medium of the present disclosure, it is preferable to use materials with acoustic impedances close to the above values ​​for the first and second acoustic transmission media.

[0019] Regarding mechanical matching between the vibrator and the culture substrate, it is preferable that they are in close contact with each other via a solid. In particular, in the present disclosure, since the vibration waveform of the vibrator is formed on the cell culture surface of the culture substrate, mechanical matching that allows for close contact over a wide area is important.

[0020] When a commercially available dish-type container is used as the culture substrate, and when the acoustic transmission medium is a low molecular weight material such as water or glycerin that easily deforms under low load, when the culture substrate is placed on the vibrator, the protrusions of the culture substrate directly contact the vibrator, resulting in multiple vibration transmission paths, which may make the generated vibration complex and difficult to control. The cell detachment method of the present disclosure performs subjective vibration control. In other words, by bringing the vibrator and the culture substrate into close contact with a solid, the vibration transmission path is kept constant, making it easier to control the vibration.

[0021] When the acoustic transmission medium of the present disclosure mechanically matches the vibrator and the culture substrate, the solid material is preferably a material having elasticity such as polymeric materials such as rubber or gel, and specific examples include silicone resin, urethane resin, diallyl dilate resin, unsaturated polyester, epoxy resin, phenol resin, urea resin, melamine resin, natural rubber, etc. Among these, silicone rubber is preferred because it has physical properties with little attenuation in the frequency band of the driving frequency (vibration frequency), resulting in high transmission efficiency, and furthermore, heat generation of the acoustic transmission medium itself can be suppressed.

[0022] (First Acoustic Transmission Medium) The first acoustic transmission medium is made of a solid material having elasticity at room temperature, which in this specification means a temperature between 20°C and 30°C, preferably 25°C.

[0023] Examples of solid materials having elasticity include polymeric materials such as rubber and gel. Examples of polymeric rubber materials include rubber containing at least one resin selected from the group consisting of silicone resin, urethane resin, diallyl dylate resin, unsaturated polyester, epoxy resin, phenol resin, urea resin, and melamine resin, natural rubber, etc. More specifically, examples of the rubber material include silicone rubber, urethane rubber, butyl rubber, nitrile rubber, butadiene rubber, and latex rubber.

[0024] Examples of polymer gel materials include hydrogels and polymer gels. Examples of hydrogels include hydrogels obtained by combining hydrophilic polymer materials such as polyvinyl alcohol and polyacrylic acid with corresponding crosslinking agents. Examples of crosslinking agents include inorganic crosslinking agents such as borax and layered clay minerals (clay), and organic crosslinking agents such as dendritic polymers, which are multifunctional polymer materials. Examples of polymer gels include polymer gels obtained by combining polymer materials such as acrylic resins and urethane resins with corresponding multifunctional compounds, which are crosslinking agents.

[0025] The first acoustic transmission medium can be one that has a tan δ of less than 1.0 obtained by dynamic viscoelasticity measurement at room temperature when vibration of 1 Hz frequency is applied. In addition, when the second acoustic transmission medium is solid at room temperature, the tan δ of the first acoustic transmission medium and the second acoustic transmission medium obtained by dynamic viscoelasticity measurement at room temperature when vibration of 0.1 Hz frequency is applied can be respectively tan δ i、 tan δ ii Then, tan δ i <tanδ ii and / or tan δ i It is preferred that ≧1.

[0026] The first acoustic transmission medium preferably contains at least one selected from the group consisting of silicone rubber, natural rubber, urethane gel, and gel materials, and more preferably contains silicone rubber. Silicone rubber has physical properties that cause little attenuation in the frequency band of the drive frequency, so that transmission efficiency is high, and further, heat generation of the acoustic transmission medium itself can be suppressed.

[0027] For the purpose of facilitating removal, the surface of the first acoustic transmission medium facing the culture substrate or the vibrator may be subjected to a surface treatment such as a fluorine coating. In order to directly observe the state of cell detachment, the acoustic transmission medium is preferably transparent to light in the visible light wavelength range.

[0028] Various additives may be added to the first acoustic transmission medium for the purpose of controlling physical properties such as viscosity, affinity with the second acoustic transmission medium, acoustic impedance with the second acoustic transmission medium, storage stability, antibacterial action, etc. Note that affinity with the second acoustic transmission medium refers to properties such as the contact angle and wettability between the first acoustic transmission medium and the second acoustic transmission medium, for example.

[0029] Regarding the thickness of the first acoustic transmission medium, if it is too thick, ultrasonic waves are likely to be reflected at the interface during ultrasonic vibration transmission, and vibration attenuation cannot be ignored. If it is too thin, the durability of the film containing the second acoustic transmission medium is poor, and it may be torn or have a hole, allowing the contained material to escape. Therefore, it is preferable that the total thickness of the first acoustic transmission medium is 5 mm or less when vibrating the vibrator.

[0030] (Second Acoustic Transmission Medium) The second acoustic transmission medium is liquid at room temperature, or has a larger tan δ than the first acoustic transmission medium when vibration at a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature.

[0031] Being liquid at room temperature means being liquid at temperatures between 20°C and 30°C, and in particular, the intrinsic viscosity [η] at room temperature (25°C) or the complex viscosity η obtained by dynamic viscoelasticity measurement at room temperature (25°C) when vibration of 1 Hz is applied. * Alternatively, the viscosity η measured by changing the shear rate using a Brookfield viscometer or rheometer can be made small.

[0032] Furthermore, when the second acoustic transmission medium is a solid at room temperature, tan δ when vibration of 0.1 Hz frequency is applied at room temperature is larger than that of the first acoustic transmission medium. Preferably, the second acoustic transmission medium is a solid in which tan δ obtained by dynamic viscoelasticity measurement at room temperature when vibration of 0.1 Hz frequency is applied is 1 or more. That is, when the second acoustic transmission medium is a solid, tan δ of the first acoustic transmission medium and the second acoustic transmission medium obtained by dynamic viscoelasticity measurement at room temperature when vibration of 0.1 Hz frequency is applied is 1 or more. i、 tan δ ii Then, tan δ i <tanδ ii and / or tan δ i It is preferred that ≧1.

[0033] A solid that can be the second acoustic transmission medium is a flexible gel. As with the gel exemplified as the first acoustic transmission medium, the flexible gel can be, for example, a hydrogel that combines a hydrophilic polymer material such as polyvinyl alcohol or polyacrylic acid with a cross-linking component such as borax, layered clay mineral (clay), or a multi-branched polymer material such as a dendritic polymer, or a polymer gel that combines a polymer material such as an acrylic resin or urethane resin with a multifunctional compound that is a corresponding cross-linking agent.

[0034] For tan δ, it is preferable to use a value at a temperature and time scale when the vibrator and the culture substrate are mechanically matched with the acoustic transmission medium. That is, tan δ can be measured by measuring the dynamic viscoelasticity G' and G" when vibration of a frequency of 1 (Hz) or 0.1 (Hz) is applied at room temperature, more preferably at 25°C, using a rheometer or the like, 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 satisfied when tan δ is measured, and 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.

[0035] If the second acoustic transmission medium is a liquid, it may have a high viscosity as long as it does not impair acoustic transmission. The viscosity can be measured at room temperature, more preferably at 25°C, using a B-type viscometer or a rheometer. Furthermore, it is preferable to use the viscosity value at a shear rate of 1.0 (1 / s) when measuring with 25 mm parallel plates and a gap of 1.0 mm. Depending on the viscosity of the liquid, an appropriate torque value may not be obtained under the above conditions. In that case, it is possible to measure by changing the plate diameter, gap, etc. to obtain a similar shear rate.

[0036] Examples of the liquid include low-viscosity liquids such as water, physiological saline, ethanol, and diluted ethanol, mixtures of these, and high-viscosity liquids and gel materials such as glycerin, silicone oil, and polymer solutions, as well as mixtures of these. A polymer solution in which a gel is dispersed and swollen in a liquid such as water may be used. More specifically, an ultrasound gel in which viscoelasticity is adjusted by adding an additive such as a thickener such as PVA gel to a liquid such as glycerin is also preferred. Note that gel materials may be classified as solid or liquid. A gel as a solid has a high content of crosslinking components, and a gel as a liquid has a low content of crosslinking components. In this specification, a material in which tan δ can be measured by the measurement method performed by the inventors is considered to be solid, and a material in which it is not is considered to be liquid. This is thought to be related to the repulsion as an elastic body. In the following examples, PVA is a flexible gel as a solid, and ultrasound gel is a gel as a liquid.

[0037] In order to prevent leakage of the contents when the first acoustic transmission medium containing the second acoustic transmission medium is damaged, it is preferable that the second acoustic transmission medium be 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.

[0038] 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.

[0039] Examples of additives include the following: Additives for adjusting the surface tension include anionic surfactants such as sodium dodecyl sulfate and sodium lauryl sulfate. Examples of cationic surfactants include ethyltrimethylammonium bromide, benzalkonium chloride, and dimethylaminopropylamide stearate. Examples of nonionic surfactants include Contaminon, Contaminon US, Pluronic F-68, Tween 20, and Tween 80. Examples of amphoteric surfactants include CHAPS and CHAPSO. Examples of the storage stabilizer include light stabilizers such as hindered amines, benzophenones, and benzotriazoles, and antioxidants such as amine-based, phenol-based, sulfur-based, and phosphorus-based antioxidants. Those that exhibit antibacterial activity include benzalkonium chloride, sodium azide, and antibiotics such as ampicillin, penicillin, and streptomycin.

[0040] (Inclusion of acoustic transmission medium) The acoustic transmission medium used in the cell detachment method of the present disclosure has a configuration in which a second acoustic transmission medium is enclosed within a first acoustic transmission medium. The term "encapsulation" refers to the presence of a second acoustic transmission medium inside the first acoustic transmission medium between the vibrator and the culture substrate. For example, the second acoustic transmission medium may be sealed inside a bag-shaped first acoustic transmission medium, or the bag-shaped first acoustic transmission medium may be provided with a circulation tube through which the second acoustic transmission medium can be supplied and circulated so as not to impair vibration transmission. In the case where the second acoustic transmission medium can be circulated inside the first acoustic transmission medium, it is preferable to encapsulate the second acoustic transmission medium in the first acoustic transmission medium in the arrangement step described below, and then use a valve or the like to prevent circulation in the pressurization and peeling steps.

[0041] As a method of containing the first acoustic transmission medium, for example, a bag-shaped first acoustic transmission medium is prepared, the second acoustic transmission medium is injected into the bag, and the injection port is then sealed. Examples of the sealing method include a method of sealing by melting with heat, a method of crimping with pressure, a method of sealing by reaction with an adhesive or a raw material compound of the first acoustic transmission medium, and a method of sealing by physical clamping.

[0042] As another method of encapsulation, a method of preparing sheets of the first acoustic transmission medium on the upper and lower surfaces in advance, and then sealing the upper and lower surfaces by closely contacting them while encapsulating the second acoustic transmission medium can be mentioned. The cell detachment method of the present disclosure can include an encapsulation step of encapsulating the second acoustic transmission medium in the first acoustic transmission medium as the encapsulation step.

[0043] The first acoustic transmission medium may have at least one protrusion that is convex in a direction facing the second acoustic transmission medium. The protrusion is preferably shaped so that it is in physical contact with the first acoustic transmission medium on the opposite side when pressurized in the pressurizing step, and may be, for example, cylindrical, hemispherical, conical, or a structure combining a cylindrical or conical shape with a hemisphere. The protrusion is preferably made of the same material as the first acoustic transmission medium.

[0044] The position of the protrusion is preferably a position corresponding to the abdomen (area with large amplitude value) of the ultrasonic vibration pattern to be applied, but it may include a part with weak vibration or a part that does not vibrate. The number of protrusions is also at least one, and for example, in order to correspond to multiple vibration patterns, multiple protrusions may be arranged at positions corresponding to the abdomen of each vibration pattern.

[0045] The first acoustically transmitting medium may have at least one post extending across said second acoustically transmitting medium. In the acoustic transmission medium of the present disclosure, the surface facing the vibrator can have a higher Young's modulus than the surface facing the culture substrate. A high Young's modulus is synonymous with a high elastic modulus, and the Young's modulus can be increased, for example, by increasing the thickness of the first acoustic transmission medium or by increasing the amount of branched components during the manufacture of the first acoustic transmission medium.

[0046] It is preferable that the difference in acoustic impedance between the first acoustic transmission medium and the second acoustic transmission medium is small. Also, it is preferable that the acoustic transmission medium does not contain air. Therefore, when the second acoustic transmission medium is contained in the first acoustic transmission medium, it is preferable to contain the second acoustic transmission medium in such a way that no air is contained therein.

[0047] The thickness of the acoustic transmission medium of the present disclosure is preferably 10 mm or less from the viewpoint of reducing vibration attenuation and preventing ultrasonic waves from being reflected at the interface, and is preferably 5 mm or less when vibration is generated in the vibrating body.

[0048] Moreover, the aspect ratio (thickness / diagonal size) of the acoustic transmission medium is preferably 0.3 or less, which increases stability when installed.

[0049] In order to improve the compatibility of the acoustic transmission medium of the present disclosure with the surface of the vibrator facing the culture substrate, a liquid such as water may be applied before use. The liquid is preferably a liquid with low surface tension.

[0050] In order to improve the compatibility of the surface of the acoustic transmission medium of the present disclosure that faces the vibrating body, the surface may be entirely or partially bonded to the vibrating body using an adhesive or a pressure-sensitive adhesive, etc. As the adhesive or pressure-sensitive adhesive, a known adhesive or pressure-sensitive adhesive, double-sided tape, etc. may be used.

[0051] (Culture substrate) The cell detachment method of the present disclosure is to detach cells attached to the culture surface of a culture substrate from the culture substrate. The adhesion of cells includes not only adhesion of adhesive cells, but also the presence of cells in contact. That is, the adhesion of cells to a culture substrate includes not only adhesion of adhesive cells, but also the presence of cells in contact. That is, the adhesion of cells to a culture substrate includes, but is not limited to adhesion, the adhesion of cells to a culture substrate via an adhesion factor, and includes the retention of cells by weak interaction with the culture substrate, or the retention of cells simply by external forces such as gravity.

[0052] The culture substrate of the present disclosure is a substrate used for cell culture, and refers to any substrate including a surface to which cells adhere. The culture substrate includes, for example, a container for cell culture or a part thereof. Examples of cell containers include dishes (also called petri dishes), flasks, plates, tubes, etc. All of these are used as general-purpose disposable containers, for example, products of Corning, Thermo Fisher Scientific, AGC Techno Glass, etc. Among them, a dish is a typical example of the culture substrate of the present disclosure. A culture substrate with a removable lid is generally used. In this specification, the culture substrate may or may not include a part such as a lid. An example of the shape of a dish is as shown in the schematic diagram of FIG. 8. In particular, the bottom warp height 300 varies greatly depending on the model number of each manufacturer, the lot, and even within the lot. In order to mechanically match the culture substrate with the acoustic transmission medium, it is necessary to take into account the variation in bottom warp height, but mechanical matching is made easier by using the first acoustic transmission medium and the second acoustic transmission medium of the present disclosure in combination.

[0053] As shown in the schematic diagram of Figure 8, commercially available culture substrates have a protrusion on the outer periphery of their bottom surface. The main roles of the protrusion are thought to be to prevent damage to the bottom surface of the substrate, to enable containers to be stacked on top of each other, and to prevent the temperature of the workbench from being directly transmitted to the bottom surface of the substrate when placed on the workbench. When mechanically matching the culture substrate and the acoustic transmission medium, the matching may be performed by avoiding this protrusion, or the matching may be performed by covering the protrusion.

[0054] The material of the substrate may be any material that is chemically stable and capable of culturing the desired cells, such as polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(meth)acrylic acid, poly(meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, glass, metal, etc. Among these, polystyrene is preferred.

[0055] (Vibration body) The vibrating body of the present disclosure can be any one that generates vibration, and may be, for example, a piezoelectric body bonded to a vibration plate. When the piezoelectric body is circular, the vibration plate is preferably made of glass, SUS, or quartz. By using glass, SUS, or quartz as the vibration plate, a large amplitude can be output at a relatively high driving frequency (vibration frequency) in the ultrasonic range without being damaged as a vibrating body. The vibrating body can include an ultrasonic transducer.

[0056] In the case of a ring-shaped piezoelectric body, it is preferable that the outer diameter of the diaphragm is equal to the outer diameter of the piezoelectric body. The thickness of the diaphragm is preferably such that when the piezoelectric body and the diaphragm are bonded together and the diaphragm flexes and vibrates, the midpoint of the thickness direction of the flexure, i.e., the neutral plane that is neither stretched nor compressed when the diaphragm flexes, is on the diaphragm side, in order to efficiently utilize the distortion of the piezoelectric body for flexure.

[0057] (Piezoelectric material) The piezoelectric body used in the vibrator of the present disclosure is preferably a ring-shaped piezoelectric body, but the shape of the piezoelectric body that deflects the vibrator may be a circular ring shape or a disk shape. In order to directly observe the cell detachment state, a circular ring shape is preferable because it ensures a field of view below the culture substrate. Piezoelectric materials can exhibit piezoelectric properties by being polarized, and as shown in Figure 10, the polarity of polarization can be changed according to the electrode pattern. 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. Standing waves are generated by applying an AC voltage with a phase difference of 180 degrees to a negatively polarized electrode relative to a positively polarized electrode. In contrast, when the electrodes are separated into phases A and B on the left and right as shown in Figure 10, applying an AC voltage with a phase difference of 90 degrees between phases A and B generates a two-phase driven traveling wave. In Figure 10, GND refers to ground and is used to ground the electrodes placed on the back side.

[0058] The electrode pattern may be formed by printing Ag, but the electrode material may be other than Ag, such as noble metals as Au, Pt, Pd, etc., or base metals such as Cu, etc. The electrode may be formed by printing, plating, or sputtering.

[0059] The piezoelectric material used for the vibrator is PbZrTiO3 (PZT), but in consideration of environmental regulations, it is preferable to use a lead-free piezoelectric material that does not substantially contain Pb. Lead-free piezoelectric materials include BaTiO3 (BT), NaNbO3, BiNaTiO3, BiFeO3, etc., whose main components are BaTiO3 (BT), and combinations of these or metal elements may be added to the main components. In particular, BaTiO3 (BT)-based materials have been confirmed to have vibration performance equivalent to that of PbZrTiO3 (PZT). Materials other than ceramics, such as single crystal materials and polymer-based piezoelectric materials, may also be used.

[0060] The thickness of the piezoelectric material in the vibrating body is determined by the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm. The thickness of the piezoelectric material in the vibrating body is determined by the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm. It is preferable that the midpoint of the thickness direction of the bending, that is, the neutral plane that is neither stretched nor compressed when bending, is located on the diaphragm side in order to efficiently utilize the distortion of the piezoelectric element in the bending. Therefore, the combination of the thickness of the piezoelectric material and the diaphragm was calculated based on the relationship between the hardness of the piezoelectric material and the hardness of the diaphragm.

[0061] (Process) The cell detachment method of the present disclosure may include the following steps. Placement process: A step of placing an acoustic transmission medium on the vibrator, supplying a second acoustic transmission medium so that the second acoustic transmission medium is contained in the first acoustic transmission medium as necessary, and further placing a culture substrate. Pressurization process: A step of applying pressure from the culture substrate or the vibrator side so that the culture substrate and the vibrator are in contact with the acoustic transmission medium. Peeling process: A step of vibrating the vibrator while the culture substrate and the vibrator are in contact with an acoustic transmission medium.

[0062] A specific example of the cell detachment method of the present disclosure is shown in FIG. First, in a placement step, a first acoustic transmission medium is placed on a vibrating body (step S210). Thereafter, the second acoustic transmission medium is supplied so as to be enclosed within the first acoustic transmission medium (step S220). Further, after that, a culture substrate is placed on the acoustic transmission medium (step S230). Next, as a pressurizing step, pressure is applied from the upper surface of the culture substrate with a weight or the like (step S240). For the peeling process, ultrasonic application conditions are set (step S250). Next, ultrasonic application is started (step S260). Finally, the detached cells are collected (step S270).

[0063] It should be noted that supplying the second acoustic transmission medium so that it is contained within the first acoustic transmission medium, i.e., the containment step, is not an essential step for the cell detachment method of the present disclosure, and the supply may be performed separately, or an acoustic transmission medium in which the second acoustic transmission medium is contained in the first acoustic transmission medium in advance may be used. Furthermore, applying pressure, i.e., the pressurization step, is also not an essential step for the cell detachment method of the present disclosure.

[0064] (Placement process) The placement step involves placing an acoustic transmission medium on the vibrator, and if necessary, supplying a second acoustic transmission medium so that it is contained within the first acoustic transmission medium, and then placing the culture substrate. In the case where the acoustic transmission medium has a structure capable of supplying and circulating the second acoustic transmission medium, the second acoustic transmission medium is supplied and circulated in this process, and the valve is closed to stop the supply and circulation during the subsequent pressurizing and peeling processes. By supplying and circulating the second acoustic transmission medium, the amount of the second acoustic transmission medium in the acoustic transmission medium can be controlled, and appropriate contact can be achieved according to the type and shape of the culture substrate, and the temperature of the acoustic transmission medium can also be controlled by circulating the second acoustic transmission medium.

[0065] (Pressure process) The pressurizing process in this embodiment may be a process of applying pressure so as to bring the culture substrate and the vibrating body closer together, and may involve displacing the culture substrate to bring it closer to the vibrating body, or displacing the vibrating body to bring it closer to the culture substrate, or displacing both the culture substrate and the vibrating body to bring them closer together. In the pressurizing step, pressure is applied from the culture substrate side or the vibrator side so as to mechanically match the culture substrate and the acoustic transmission medium with the culture substrate and the vibrator in contact with the acoustic transmission medium. Examples of pressure applying means for carrying out the pressure applying step include placing a weight on the lid of the culture substrate, applying a uniform load from above using a loading method such as a spring or leaf spring on the lid of the culture substrate container to fix the top of the culture substrate lid so that it does not move from above, and applying a load from below upward to the vibrating body. Regarding the pressure applying means, a weight is preferable in consideration of the insulation of vibration, but other loading methods can be selected as long as they do not impede vibration. For example, a constant force spring can be used as the pressure applying means.

[0066] The weight placed on the lid of the culture substrate is preferably in the shape of a ring, for example, so that the cells in the container can be easily observed. The load to be applied varies depending on the size of the culture substrate and the vibration pattern to be applied, but for example, in the case of a 35 mm dish, it is preferably between 10 g and 300 g, and in the case of a 60 mm dish, it is preferably between 100 g and 600 g. The load may be a weight or the weight of the culture vessel itself. If the weight is too heavy, it will not only suppress and inhibit the vibration itself, but also cause the culture substrate itself to bend, making the vibration transmission state uneven, which will be a factor in reducing the detachment rate.

[0067] (Peeling process) In the detachment step, the vibrator is vibrated in a state where the culture substrate or the vibrator is in contact with the acoustic transmission medium to detach the cells. Further, the detachment step can include a step of exciting the vibrator to vibration in the ultrasonic band.

[0068] In the arrangement step, the vibration of the vibrator is not started, and in the peeling step, the vibration of the vibrator is started. For example, in the example of the device shown in FIG. 6, the vibrator is driven by supplying a voltage of a resonant frequency to generate a desired ultrasonic vibration mode. In this case, in the peeling step, the vibrator is driven by supplying a voltage of a resonant frequency to generate a desired ultrasonic vibration mode. Note that a power source that applies the above voltage is not shown in FIG. 6.

[0069] The vibration pattern can be freely selected depending on the cell type and the detachment result. In addition, in the detachment step, these vibration patterns may be used alone or may be input by periodically changing them.

[0070] The vibration waveform is preferably one that can vibrate the cell culture surface of the culture substrate with a certain amplitude, for example, an amplitude of 1 μm. Basically, when the frequency is swept from low frequency to high frequency, for example, from 20 kHz to 150 kHz in the ultrasonic region, resonant vibrations can be detected, but the vibrations detected on the low frequency side have a large amplitude but a small acceleration. In contrast, the vibrations detected on the high frequency side have a small amplitude but a large acceleration. From this, the conditions for applying a force that weakens the adhesive force of the cultured cells to the culture substrate depend largely on the adhesive force of the cells and the size and shape of the cells. Furthermore, the flow generated in the detachment solution also differs depending on the amplitude and acceleration, so there are various combinations. In addition, simply large amplitude and large acceleration may not be preferable because they promote the occurrence of cavitation, which is a factor in reducing the survival rate of cells.

[0071] The vibration waveform can be a standing wave mode that is formed as a pattern with orders in the circumferential direction or concentric circles from the center of the disc-shaped vibration plate, or a traveling wave mode that forms a radial vibration waveform on the vibration plate by vibrating the piezoelectric body with a phase change in the radial direction. The vibration waveform can be formed by applying a voltage at the resonant frequency to the vibration body, and the resonant frequency and vibration waveform shape can be calculated by structural calculations using the shape of the vibration body and the material properties such as hardness and density.

[0072] In addition, when the vibrator is continuously driven, the resonant frequency shifts to the low frequency side due to heat generation from the piezoelectric body itself, so the amplitude becomes smaller if the frequency is fixed and the drive is continued. For this reason, sufficient amplitude can be obtained by using a drive method that suppresses temperature rise due to self-heating by providing a rest period after a certain period of oscillation, or by repeatedly sweeping from the high frequency side to the low frequency side in a certain frequency width, so that the resonant frequency is always reached even if heat is generated.

[0073] (Vibration evaluation) The vibration waveform of the diaphragm can be measured with a laser Doppler vibrometer (Graphtec AT7200). The measurement method will be explained using the cell peeling device shown in Figure 6 as an example. The cell peeling device is driven at the desired resonance frequency and voltage, and the surface of the diaphragm 11 is scanned two-dimensionally in the XY directions with a laser, obtaining the vibration velocity at each point, and the amplitude in the Z direction can be calculated. As a result, the vibration waveform of the diaphragm 11 can be constructed three-dimensionally, and the positions of the antinodes with large amplitude of vibration during driving and the nodes that do not vibrate can be dynamically grasped.

[0074] Vibration measurement of the cell culture surface of the culture substrate 21 can also be performed with a similar configuration. It is preferable to place the acoustic transmission medium 13 on the diaphragm 11, form a metal film, for example Ag, on the inner surface of the culture substrate 21 by vapor deposition so that the laser is reflected by the culture substrate 21, and place the acoustic transmission medium 13 without creating an air gap between the culture substrate 21 and the acoustic transmission medium 13. Note that in FIG. 6, the first acoustic transmission medium and the second acoustic transmission medium are collectively shown as the acoustic transmission medium 13.

[0075] In the case of a standing wave mode in which the resonance to be driven by the cell peeling device, which is placed on an acoustic transmission medium 13, oscillates concentrically, the amplitude of the central part of the culture substrate 21 is swept near the desired resonance frequency, and the entire cell culture surface of the culture substrate 21 is scanned two-dimensionally at the frequency at which the amplitude is largest, thereby making it possible to grasp the vibration waveform generated on the cell culture surface of the culture substrate 21 in three dimensions.

[0076] (cell) The cell detachment method of the present disclosure can be applied to any type of cell without limitation. Examples include various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), human alveolar basal epithelial adenocarcinoma-derived A549 cells, human cervical carcinoma-derived HeLa cells, and insect-derived cell lines; epithelial cells and endothelial cells that constitute various tissues and organs in the body; skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells that constitute the nervous system, glial cells, and fibroblasts; primary cultured cells such as hepatic parenchymal cells, non-parenchymal hepatic cells, and adipocytes that are involved in the metabolism of the body; induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, various stem cells such as mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germ stem cells; and progenitor cells of various tissues, as well as cells induced to differentiate from these.

[0077] The cell culture conditions can be appropriately selected according to the cells to be cultured. In general, a suitable medium is added to the culture substrate 21, and 1.0×10 1 cells / cm 2 ~5.0×10 4 cells / cm 2 The cells are seeded in such a manner that the cells are cultured in an environment at 37° C. and a CO2 concentration of 5%. At this time, it is preferable to culture the cells until the cell occupancy rate in the culture substrate 21 reaches about 70% to 80% of the surface area, that is, until the cells reach a so-called subconfluent state.

[0078] The culture medium (culture medium) for the cultured cells can be replaced with a cell detachment solution in the cell detachment step. Generally, the detachment solution contains a protease. Examples of the protease include trypsin, accutase, collagenase, natural protease, chymotrypsin, elastase, papain, pronase, or recombinant forms thereof. However, since protease dissolves cells, there is concern that it may have a negative effect on cells. In contrast, the cell detachment method of the present disclosure does not use protease, or even if it does, a very small amount of protease is used to achieve the same effect, thereby suppressing the negative effect on cells. The cell detachment solution is a solution used when detaching cells, and may have the effect of reducing the adhesive strength of the cells. The pH of the cell detachment solution is preferably in the neutral range. This is because the neutral range is suitable for cell culture and can stably maintain a high cell viability. The pH can be appropriately adjusted with hydrochloric acid, sodium hydroxide, etc. In addition, various buffer solutions are preferably used to stably maintain the pH. The detachment solution may also be a culture medium. The viscosity of the cell detachment solution can be appropriately adjusted by adding polymers or sugars.

[0079] As the cell detachment solution, a solution containing a metal ion chelating agent (hereinafter, sometimes referred to as a chelating agent) is particularly preferable, because the adhesive force of cells can be effectively reduced by using a cell detachment solution containing a chelating agent. The chelating agent is not particularly limited, and examples thereof include ethylenediaminetetraacetic acid (hereinafter, sometimes referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycol ether diamine tetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethyl glutamic acid, ethylenediamine disuccinic acid, etidronic acid, citric acid, gluconic acid, and phosphonobutane triacetic acid. Among these, chelating agents that form chelates with divalent cations are preferred, chelating agents that form chelates with Ca2+ and Mg2+ are particularly preferred, and ethylenediaminetetraacetic acid is the most preferred. When ethylenediaminetetraacetic acid is used as the chelating agent, the pH of the cell detachment solution is preferably 7.0 or more and 8.0 or less. This is because the higher pH in the neutral region that can maintain a high cell viability can enhance the chelating ability of ethylenediaminetetraacetic acid, and the efficiency of reducing the adhesive force of cells can be increased. The chelating agents may be used alone or in combination of two or more kinds. The content of the chelating agent is preferably 0.01 mM or more and 5.0 mM or less. Within this range, the chelating effect can be reliably obtained, and a decrease in activity due to the presence of an excess chelating agent can be suppressed.

[0080] The cell detachment solution may contain a hydrophilic polymer containing a polyalkylene glycol structure. An example of a hydrophilic polymer containing a polyalkylene glycol structure is polyethylene glycol. The hydrophilic polymer preferably has a peak molecular weight Mp of 800 or more and 50,000 or less, more preferably 1,200 or more and 20,000 or less, as measured by gel permeation chromatography. This is because the polymer has little effect on cells and the thickening effect caused by the polymer can be suppressed.

[0081] (Peel test) A detachment test can be performed to evaluate the cell detachment method of the present disclosure. The detachment test can be performed, for example, as follows. The cells are cultured, and the substrate in which the culture medium (culture medium) is replaced with the detachment liquid as necessary is subjected to the placement process, and the vibrator can be driven while being maintained at a constant temperature, for example, 25°C, in an incubator as necessary. The drive time is long enough for cell detachment. After driving, the detached cells are collected with a dropper, and the cells that could not be collected and are still attached to the culture substrate 21 are collected by pipetting. The detachment rate used below is the ratio of the number of cells detached by vibration to the total number of cells detached by vibration and the pipetting method, expressed as a percentage. The detachment rate was calculated for n=10, and the standard deviation of the detachment rate was graded as A for less than 5%, B for 5% to 7.5%, C for 7.5% to 10%, and D for 10% or more. The residual cells remaining on the bottom surface of the substrate after cell detachment can be observed using scattered light from a backlight.

[0082] (Cell detachment device) The cell detachment device of the present disclosure is a cell detachment device that detaches cells by transmitting vibrations generated by a vibrator to cells provided on a culture surface of a culture substrate, the cell detachment device having a vibrator, an acoustic transmission medium, and a culture substrate mounting section, in that order, and configured such that vibrations of the vibrator are transmitted to the culture substrate via the acoustic transmission medium, the acoustic transmission medium including a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium being an elastic solid at room temperature, the second acoustic transmission medium being contained within the first acoustic transmission medium and being liquid at room temperature, or the tan δ of the second acoustic transmission medium when a vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature is larger than that of the first acoustic transmission medium. The configuration of the device of the present disclosure is shown in FIG.

[0083] As shown in Fig. 6, the cell detachment device of the present disclosure includes a vibrating body 101 and an acoustic transmission medium 13. The culture substrate installation section only needs to be able to recognize that a user will place a culture vessel thereon, and in the example of Fig. 6, an opening formed by the vibrating body 101, upper cover 15, and cushioning material 16 is suitable for placing a general-purpose dish, and so this opening corresponds to the culture substrate installation section. The cell peeling device in FIG. 6 shows an example in which a piezoelectric body 12 and a vibration plate 11 are provided as a vibration generating unit, i.e., a vibration body 101 provided with a piezoelectric body 12 and a vibration plate 11, generates bending vibrations in which the vibration is amplified by a combination of the hardness of the piezoelectric body 12, which expands and contracts, and the vibration plate 11 to which it is bonded. In this example, resonance due to the structural factor of the vibration body 101 is utilized, and large vibrations can be obtained at the resonance frequency. The vibration body can include a transducer such as an ultrasonic transducer. The above-mentioned expansion and contraction is caused by applying a voltage to the piezoelectric body 12 using a power source (not shown). In FIG. 6, a ring-shaped transducer is shown as a representative transducer 101, but a Langevin transducer or a rectangular transducer that can obtain a large amplitude may also be used.

[0084] In order to efficiently transmit the vibrations formed in the diaphragm by resonance to the culture substrate 21 on which the cells are cultured, an acoustic transmission medium 13 is interposed between the diaphragm 11 and the culture substrate 21. If the culture substrate 21 is placed directly on the diaphragm 11, the contact area will be a point contact, which will cause noise, heat generation, and other energy consumption other than vibration, resulting in a decrease in efficiency. Basically, it is preferable to place the acoustic transmission medium 13 with an area sufficient to cover the detachment area of ​​the cells attached to the culture substrate 21. In order to fix the vibrating body 101, the ends of the vibrating body 101 are sandwiched between the upper cover 15, lower cover 17, cushioning material 16, and bolts 18 shown in FIG. 6, and the pressure applied to the ends is such that the vibration generated in the diaphragm is not inhibited. An example is shown in which a vibrator is attached to the top of the culture substrate and a weight 14 is attached to the bottom of the vibrator as a pressurizing means for the pressurizing step. Pressurization and depressurization are possible by the weight 14 moving up and down.

[0085] By supplying a voltage at a resonant frequency, the vibrator is driven to generate a desired ultrasonic vibration mode. The vibrator can be driven by applying a voltage at a frequency of a vibration mode that can form a desired vibration waveform on the cell culture surface of the culture substrate and at a desired amplitude using an AC power source that supplies a voltage at a resonant frequency, thereby driving the cell detachment device. At this time, by measuring the voltage of the S-phase, which is the sensor phase of the vibrator, with an oscilloscope, the voltage generated by the distortion of the vibrator itself can be directly monitored, and the vibrator can be driven while checking its vibration state. In addition, by installing a thermocouple that can measure the temperature during the cell detachment process at a location that does not affect the vibration, it is possible to drive the device while monitoring it. An infrared thermometer can also be used to measure the temperature.

[0086] Examples of acoustic transmission media that can be used in the cell detachment method of the present disclosure are shown in FIGS. 1A to 1C, 2A to 2C, 3A to 3C, 4A to 4C, and 9A to D.

[0087] (First Example) The configuration diagram of a first example of an acoustic transmission medium is shown in FIG. 1A to FIG. 1C and FIG. 9A. In the first example of the acoustic transmission medium, a second acoustic transmission medium is contained within a first acoustic transmission medium. FIG. 1A shows a configuration in which the bottom surface is in contact with a flat culture substrate and a vibrating body. FIG. 1B shows a configuration in which the bottom surface is in contact with a warped culture substrate and a vibrator. FIG. 1C further shows that the acoustic transmission medium conforms to the shape of the bottom surface of the culture substrate by applying pressure from the culture substrate or the diaphragm. FIG. 9A illustrates a top view and a cross-sectional view of a first example acoustic transmission medium.

[0088] (Second example) A configuration diagram of a second example of an acoustic transmission medium is shown in FIGS. 2A to 2C and 9B. In a second example of the acoustic transmission medium, a second acoustic transmission medium is contained within a first acoustic transmission medium, and the first acoustic transmission medium has a protrusion that is convex in a direction facing the second acoustic transmission medium. FIG. 2A shows a configuration in which a first acoustic transmission medium having a protrusion on the upper surface side thereof is in contact with a culture substrate having a flat bottom surface and a vibrator. FIG. 2B shows a configuration in which a first acoustic transmission medium having a protrusion on the upper surface side thereof is in contact with a culture substrate having a curved bottom surface and a vibrator. 2C further shows that the acoustic transmission medium of the present disclosure conforms to the shape of the bottom surface of the culture substrate by applying pressure from the culture substrate or the diaphragm. As shown in the figure, it is preferable that the protrusion is structured to contact the facing first acoustic transmission medium in a pressurized state. 9B shows a top view and a cross-sectional view of the acoustic transmission medium of the second example. In the figure, 207 indicates the projection diameter, and 208 indicates the projection height.

[0089] (Third example) The configuration diagram of a third example of the acoustic transmission medium is shown in FIG. 3A to FIG. 3C and FIG. 9C. A third example of the acoustic transmission medium is a second acoustic transmission medium that is contained within a first acoustic transmission medium, and the first acoustic transmission medium has a post portion that crosses and connects to the second acoustic transmission medium. FIG. 3A shows a configuration in which a first acoustic transmission medium having a column portion connected across a second acoustic transmission medium is in contact with a culture substrate having a flat bottom surface and a vibrator. FIG. 3B shows a configuration in which a first acoustic transmission medium having a column portion connected across a second acoustic transmission medium is in contact with a culture substrate having a curved bottom surface and a vibrating body. FIG. 3C further illustrates how the acoustic transmission medium of the present disclosure conforms to the shape of the bottom surface of the culture substrate by applying pressure from the culture substrate or the diaphragm. 9C illustrates a top view and a cross-sectional view of the third example of the acoustic transmission medium. In the figure, 209 indicates the diameter of the column.

[0090] (Fourth example) A configuration diagram of a fourth example of the acoustic transmission medium is shown in FIGS. 4A to 4C and 9D. The fourth example of the acoustic transmission medium has a second acoustic transmission medium supply pipe 133 for containing the second acoustic transmission medium in the first acoustic transmission medium. FIG. 4A shows a configuration in which an acoustic transmission medium having a second acoustic transmission medium supply pipe 133 is in contact with a culture substrate having a flat bottom surface and a vibrator. FIG. 4B shows a configuration in which an acoustic transmission medium having a second acoustic transmission medium supply pipe 133 is in contact with a culture substrate having a curved bottom surface and a vibrator. 4C further shows that the acoustic transmission medium of the present disclosure conforms to the shape of the bottom surface of the culture substrate by applying pressure from the culture substrate or the diaphragm. In this state, it is preferable that the valve of the second acoustic transmission medium supply pipe 133 is closed. FIG. 9D illustrates a top view and a cross-sectional view of a fourth example acoustic transmission medium. EXAMPLES

[0091] Example 1 In the following examples, an example using the configuration of the first example of the acoustic transmission medium shown in the schematic diagram of FIG. 1A is shown. A Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish was used as the culture substrate. The vibration plate of the cell detachment device was made of glass with an outer diameter of 70 mm and a thickness of 4 mm. The material composition of the piezoelectric body was PZT, the outer diameter was 70 mm, the inner diameter was 57 mm, and the thickness was 2 mm considering that the neutral surface was on the vibration plate side. The cell detachment device was assembled as shown in FIG. 6, and the acoustic transmission medium was used on the vibration plate with the shape shown in the schematic diagram of FIG. 9A, height (205) = 2.0 mm, upper surface height (204) = 1.0 mm, lower surface height (206) = 1.0 mm, upper surface thickness (201) = 0.2 mm, lower surface thickness (202) = 0.2, diameter (203) = 30 mm, and water was sealed inside.

[0092] The method for producing the sound transmission medium was to prepare a mold that corresponded to the upper and lower surface shapes of the sound transmission medium, prepare and pour silicone rubber raw material (product name: KE-1950-10; manufactured by Shin-Etsu Chemical) into it, heat it to harden, and obtain the silicone rubber for the upper and lower surfaces, which became the first sound transmission medium. Next, both sides of the rubber were bonded together with adhesive, and water was injected into it through an injection port as the second sound transmission medium, and the injection port was sealed with adhesive. The excess injection port was cut off to obtain the sound transmission medium. The obtained acoustic transmission medium was placed so that its center was aligned with the diaphragm, and then the center of a Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish, a culture substrate on which cells were cultured, was aligned with the center of the diaphragm and slowly placed on the acoustic transmission medium. The schematic diagram showing the configuration at this time is shown in FIG. 1B. After that, a 100 g weight was placed on the lid of the culture substrate to apply pressure. The schematic diagram showing the configuration of the acoustic transmission medium after pressure was applied is shown in FIG. 1C.

[0093] The cells were cultured as follows: CHO (Chinese Hamster Ovary) cells were cultured in a Thermo Fisher Scientific Nunc (registered trademark) Φ35 mm dish at 10,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37℃ in an environment with a CO2 concentration of 5%. The medium used was Ham's F12 (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the state of the cells was observed under a phase contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate of the dish was about 80%.

[0094] (Cell detachment) The medium in the dish was removed, and the cells were washed with PBS(-) and then immersed in a PBS(-) detachment solution for 3 minutes. The culture substrate was then placed in a cell detachment device at an ambient temperature of 25°C, and cells were detached for 2 minutes using frequency sweep vibration (frequency 22-27 kHz, frequency sweep period 1 s, voltage 90 V) in standing wave mode at an ambient temperature of 25°C. After collecting the detached cells, a cell scraper was used to detach cells that could not be detached by ultrasound from the dish after ultrasonic detachment. The number of detached cells was measured using a hemocytometer. The total number of detached cells was calculated by adding up the number of cells detached by ultrasound and the number of cells detached with the cell scraper, and the ratio of the number of ultrasonically detached cells to the total number of detached cells was defined as the detachment rate, which was calculated. Variation was calculated 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 showed that the peeling rate was 95.4% and the variation was rated A.

[0095] Example 2 Example 2 was carried out under the same conditions as Example 1, except that the type of the second acoustic transmission medium was changed as follows: That is, in this example, ultrasonic gel (Pro Jelly for Ultrasound Examination, Normal Type; manufactured by Gex Co., Ltd.) was used as the second acoustic transmission medium. The evaluation results showed that the peeling rate was 96.1% and the variation was rated A.

[0096] Example 3 Example 3 was carried out under the same conditions as Example 1, except that the type of the second acoustic transmission medium was changed as follows: That is, PVA gel was used as the second acoustic transmission medium. The PVA gel was prepared by the following method. Denka Poval (trademark) K-24E (manufactured by Denka Co., Ltd.) was weighed out to 3.7% by mass, and pure water was added to prepare a total of 80 mL. The PVA was completely dissolved by stirring while applying heat in a thermostatic bath, and an aqueous PVA solution was obtained. Next, borax (special grade sodium tetraborate (decahydrate); manufactured by Kishida Chemical Co., Ltd.) was weighed out, and pure water was added to prepare a total of 1.0% by mass, and a total of 16 mL was obtained. While stirring the aqueous PVA solution, the aqueous borax solution was gradually added dropwise, and the mixture was stirred until it became uniform, thereby obtaining a PVA gel. The evaluation results showed that the peeling rate was 93.0% and the variation was rated A.

[0097] Example 4 Example 4 was carried out under the same conditions as Example 1, except that the shape of the first acoustic transmission medium was changed as follows: That is, the acoustic transmission medium used had the following shape as shown in the schematic diagram of Fig. 9A: height (205) = 2.0 mm, upper surface height (204) = 1.0 mm, lower surface height (206) = 1.0 mm, upper surface thickness (201) = 0.2 mm, lower surface thickness (202) = 0.4 mm, and diameter (203) = 30 mm. The evaluation results showed a peeling rate of 94.6% and an A rating for variation.

[0098] Comparative Example 1 In Comparative Example 1, the study was carried out under the same conditions as in Example 1, except that silicone rubber was used as the acoustic transmission medium. Silicone rubber with a thickness of 2.0 mm and a hardness of 30° was used as the acoustic transmission medium. The evaluation results showed a peeling rate of 80.1% and a variation of D.

[0099] (summary) Table 1 shows the results of summarizing the configurations of the above-mentioned Examples and Comparative Examples.

[0100] [Table 1]

[0101] Embodiments of the present disclosure include the following methods and compositions. [Method 1] A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: a cell detachment method for detaching the cells from the culture surface by vibrating a vibrator while disposing an acoustic transmission medium between the culture substrate and the vibrator; However, the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium is a solid having elasticity at room temperature; A cell detachment method, characterized in that the second acoustic transmission medium is contained within the first acoustic transmission medium and is liquid at room temperature, or has a larger tan δ than the first acoustic transmission medium when vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature. [Method 2] The cell detachment method described in Method 1, wherein the first acoustic transmission medium has at least one protrusion that is convex in a direction facing the second acoustic transmission medium. [Method 3] The cell detachment method described in Method 1, wherein the first acoustic transmission medium has at least one pillar portion extending across and connecting to the second acoustic transmission medium. [Method 4] 4. The cell detachment method according to any one of Methods 1 to 3, wherein the first acoustic transmission medium comprises at least one material selected from the group consisting of silicone rubber, natural rubber, urethane gel, and gel materials. [Method 5] A cell detachment method described in any one of Methods 1 to 4, characterized in that the second acoustic transmission medium is a solid having a tan δ of 1.0 or more when subjected to vibration at a frequency of 0.1 Hz at room temperature. [Method 6] A cell detachment method described in any one of methods 1 to 5, characterized in that the second acoustic transmission medium is liquid at room temperature. [Method 7] The cell detachment method described in any one of Methods 1 to 6, wherein the second acoustic transmission medium includes at least one selected from the group consisting of a liquid, a polymer solution, and a gel material. [Method 8] 8. The cell detachment method according to any one of methods 1 to 7, wherein the first acoustic transmission medium comprises silicone rubber and the second acoustic transmission medium comprises water. [Method 9] A cell detachment method described in any one of Methods 1 to 8, characterized in that the surface of the acoustic transmission medium facing the vibrator has a higher Young's modulus than the surface of the acoustic transmission medium facing the culture substrate. [Method 10] 10. The cell detachment method according to any one of methods 1 to 9, further comprising an encapsulation step of encapsulating the second acoustic transmission medium in the first acoustic transmission medium. [Method 11] 11. The cell detachment method according to any one of Methods 1 to 10, characterized in that the vibrator generates vibrations in the ultrasonic band. [Method 12] 12. The cell detachment method according to any one of Methods 1 to 11, further comprising a pressurizing step of applying pressure so as to bring the culture substrate and the vibrator closer to each other. [Configuration 1] A cell detachment device that detaches cells by transmitting vibrations generated by a vibrator to cells provided on a culture surface of a culture substrate, Vibration body, Acoustic transmission medium, and A culture substrate installation section is provided in this order. The vibration of the vibrator is transmitted to the culture substrate via the acoustic transmission medium, the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium; the first acoustic transmission medium is a solid having elasticity at room temperature; A cell detachment device characterized in that the second acoustic transmission medium is contained within the first acoustic transmission medium, is liquid at room temperature, or has a larger tan δ than the tan δ of the first acoustic transmission medium when a vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature. [Configuration 2] The cell detachment device according to configuration 1, wherein the vibrator includes an ultrasonic vibrator. [Configuration 3] The cell detachment device according to configuration 1 or 2, further comprising a pressure applying means for applying pressure so as to bring the culture substrate and the vibrator closer to each other. [Explanation of symbols]

[0102] 11: Vibration plate 12: Piezoelectric material 13: Acoustic transmission medium 131: First Acoustic Transmission Medium 132: Second Acoustic Transmission Medium 14: Weight 15: Top cover 16: Cushioning material 17: Lower cover 18: Bolt 21:Culture substrate 22:Culture substrate top lid 101: Vibration body

Claims

1. A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, comprising: a cell detachment method for detaching the cells from the culture surface by vibrating a vibrator while disposing an acoustic transmission medium between the culture substrate and the vibrator; However, the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium is a solid having elasticity at room temperature; A cell detachment method, characterized in that the second acoustic transmission medium is contained within the first acoustic transmission medium and is liquid at room temperature, or has a larger tan δ than the tan δ of the first acoustic transmission medium when a vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature.

2. 2. The cell detachment method according to claim 1, wherein the first acoustic transmission medium has at least one protrusion that is convex in a direction facing the second acoustic transmission medium.

3. 2. The method of claim 1, wherein the first acoustic transmission medium has at least one pillar portion extending across the second acoustic transmission medium.

4. 4. The cell detachment method according to claim 1, wherein the first acoustic transmission medium includes at least one selected from the group consisting of silicone rubber, natural rubber, urethane gel, and a gel material.

5. 4. The cell detachment method according to claim 1, wherein 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.

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

7. 4. The cell detachment method according to claim 1, wherein the second acoustic transmission medium includes at least one selected from the group consisting of a liquid, a polymer solution, and a gel material.

8. 4. The cell detachment method according to claim 1, wherein the first acoustic transmission medium includes silicone rubber, and the second acoustic transmission medium includes water.

9. The cell detachment method according to any one of claims 1 to 3, characterized in that the surface of the acoustic transmission medium facing the vibrator has a higher Young's modulus than the surface of the acoustic transmission medium facing the culture substrate.

10. 4. The cell detachment method according to claim 1, further comprising an encapsulation step of encapsulating the second acoustic transmission medium in the first acoustic transmission medium.

11. The cell detachment method according to any one of claims 1 to 3, wherein the vibrator generates vibrations in an ultrasonic band.

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

13. A cell detachment device that detaches cells by transmitting vibrations generated by a vibrator to cells provided on a culture surface of a culture substrate, Vibration body, Acoustic transmission medium, and A culture substrate installation section is provided in this order. The vibration of the vibrator is transmitted to the culture substrate via the acoustic transmission medium, the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium; the first acoustic transmission medium is a solid having elasticity at room temperature; A cell detachment device characterized in that the second acoustic transmission medium is contained within the first acoustic transmission medium, is liquid at room temperature, or has a larger tan δ than the tan δ of the first acoustic transmission medium when a vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature.

14. The cell detachment device according to claim 13 , wherein the vibrator includes an ultrasonic vibrator.

15. The cell detachment device according to claim 13 or 14, 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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