Cell detachment method and cell detachment apparatus

Abstract

Provided are a cell detachment method and a cell detachment apparatus, with which it becomes possible to design right vibrations for a cell of interest by mechanically matching a culture substrate to a vibration body and it also becomes possible to detach adherent cells with a high detachment rate. The cell detachment method is for detaching cells adhered to a culture surface of a culture substrate from the culture surface, the cell detachment method including vibrating a vibration body in a state where an acoustical transmission medium is disposed between the culture substrate and the vibration body, thereby detaching the cells from the culture surface. The method is characterized in that the acoustical transmission medium includes a first acoustical transmission medium and a second acoustical transmission medium, wherein the first acoustical transmission medium is a solid material having elasticity at room temperature, and the second acoustical transmission medium is contained in the first acoustical transmission medium, and has a liquid form at room temperature or has a property that a tanδ value obtained when vibrations having a frequency of 0.1 Hz are applied to the second acoustical transmission medium at room temperature is larger than a tanδ value of the first acoustical transmission medium.

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] 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, particularly 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 listed 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 in cell detachment performance and reduction in variability are required. Patent Document 2 also proposes a medical ultrasound transmission tool composed of a sealed body. Although it is used for acoustic coupling between an ultrasound probe and a target site, there is no mention of using this ultrasound transmission means to transmit ultrasonic vibrations and detach cells.

[0005] JP 2018-42534 A Patent No. 6325363 A

[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.

[0007] 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: disposing an acoustic transmission medium between the culture substrate and a vibrating body and vibrating the vibrating body to detach the cells from the culture surface; wherein the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium; the first acoustic transmission medium is an elastic solid at room temperature; and the second acoustic transmission medium is encapsulated in the first acoustic transmission medium and is liquid at room temperature, or the tan δ of the second acoustic transmission medium when vibrations of a frequency of 0.1 Hz are applied to the second acoustic transmission medium at room temperature is greater than the tan δ of the first acoustic transmission medium.

[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, the cell detachment device comprising, in this order, a vibrator, an acoustic transmission medium, and a culture substrate mounting section, and configured so that vibrations of the vibrator are transmitted to the culture substrate via the acoustic transmission media, the acoustic transmission media including a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium being an elastic solid at room temperature, and the second acoustic transmission medium being encapsulated in the first acoustic transmission medium and being liquid at room temperature, or the tan δ of the second acoustic transmission medium when vibrations of a frequency of 0.1 Hz are applied to the second acoustic transmission medium at room temperature is larger than the tan δ of the first acoustic transmission medium.

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

[0010] 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 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. 14 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. 15 is a cross-sectional schematic FIG. 1 shows a schematic diagram of an example of a culture substrate according to the present disclosure; FIG. 2 shows a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure; FIG. 3 shows a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure; FIG. 4 shows a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure; FIG. 5 shows a cross-sectional view and a top view illustrating an example of an acoustic transmission medium according to the present disclosure; and FIG. 6 shows a schematic diagram illustrating an example of a piezoelectric body according to the present disclosure.

[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 is a cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, wherein an acoustic transmission medium is placed between the culture substrate and a vibrating body and the vibrating body is vibrated to detach the cells from the culture surface, wherein the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium, the first acoustic transmission medium is an elastic solid at room temperature, and the second acoustic transmission medium is encapsulated in the first acoustic transmission medium and is liquid at room temperature, or the 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.

[0013] Conceptual diagrams of the cell detachment method of the present disclosure are shown in FIGS. 7A to 7D. FIG. 7A shows the 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 the pressurizing step, in which pressure is applied from above the culture substrate 21. Note that the pressurizing step is not essential to the detachment method of the present disclosure. FIG. 7D shows the detachment step. In FIG. 7D, the vibrating body 101 is driven while applying pressure from above the culture substrate 21 to apply ultrasonic vibrations to detach the cells. The pressurizing step makes it easier for the first acoustic transmission medium to conform to the culture substrate and the vibrating body, and also allows the first acoustic transmission medium to transmit vibrations while conforming to the culture substrate and the vibrating body during the detachment process. Here, "matching" the first acoustic transmission medium with the culture substrate or the vibrator means that they are in sufficient contact so that the 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 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.

[0014] In the cell detachment method of the present disclosure, an acoustic transmission medium is provided between the vibrator and the culture substrate when the vibrations of the vibrator are transmitted to the culture substrate. The acoustic transmission medium includes a first acoustic transmission medium that is elastic and solid at room temperature, and a second acoustic transmission medium that is either liquid at room temperature or has a larger tan δ when a vibration 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 (tan δ when a vibration of 0.1 Hz is applied to the first acoustic transmission medium at room temperature). The vibrator, which serves as a vibration generating unit, includes a piezoelectric body and a diaphragm, and its vibration mechanism generates bending vibrations that are amplified by the combination of the hardness of the piezoelectric body, which expands and contracts, and the diaphragm to which it is attached. In the present disclosure, this utilizes resonance due to structural factors of the vibrator, 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 vibrator 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 mechanical and physical matching is achieved between the acoustic transmission medium and the culture substrate or vibrator, 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 defined by a physical quantity called "acoustic impedance Z." 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.

[0018] 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 and second acoustic transmission media.

[0019] 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.

[0020] When using a commercially available dish-type container as the culture substrate, if the acoustic transmission medium is a low-molecular-weight material such as water or glycerin, which easily deforms under low load, the protrusions of the culture substrate directly contact the vibrator when the culture substrate is placed on the vibrator, resulting in multiple vibration transmission paths, making the generated vibrations complex and difficult to control. The cell detachment method disclosed herein controls the vibrations proactively. That is, by tightly contacting the vibrator and the culture substrate with a solid, the vibration transmission path is kept constant, making vibration control easier.

[0021] When the acoustic transmission medium of the present disclosure mechanically matches the vibrator and the culture substrate, the solid material is preferably a polymeric material having elasticity such as rubber or gel, and specific examples include silicone resin, urethane resin, diallyl dylate resin, unsaturated polyester, epoxy resin, phenolic 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 drive frequency (vibration frequency), resulting in high transmission efficiency, and furthermore, heat generation by the acoustic transmission medium itself can be suppressed.

[0022] (First Acoustic Transmission Medium) The first acoustic transmission medium is made of a solid material that has elasticity at room temperature. In this specification, room temperature is 20°C or higher and 30°C or lower, and preferably 25°C.

[0023] Examples of elastic solid materials include polymeric materials such as rubber and gel. Polymeric rubber materials include natural rubber and rubber containing at least one resin selected from the group consisting of silicone resin, urethane resin, diallyl dylate resin, unsaturated polyester, epoxy resin, phenolic resin, urea resin, and melamine resin. More specific examples 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. 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.

[0025] The first acoustic transmission medium can be one whose tan δ obtained by dynamic viscoelasticity measurement at room temperature when vibration of 1 Hz frequency is applied is less than 1.0. If 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 set to tan δ i , tanδ ii When this is the case, tan δ i <tan δ 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 result in low attenuation in the frequency band of the drive frequency, thereby providing high transmission efficiency and further suppressing heat generation by the acoustic transmission medium itself.

[0027] Furthermore, for the purpose of facilitating removal, the surface of the first acoustic transmission medium facing the culture substrate or the vibrator may be subjected to a surface treatment such as a fluorine coating. 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.

[0028] 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.

[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 deteriorates, and it may be torn or have holes, 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 vibrating body.

[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 a liquid at room temperature means being a liquid at 20°C or higher and 30°C or lower, 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 a frequency of 1 Hz is applied. * Alternatively, the viscosity η measured by changing the shear rate using a Brookfield viscometer or a rheometer can be made small.

[0032] Furthermore, when the second acoustic transmission medium is a solid at room temperature, the tan δ when vibrations of a frequency of 0.1 Hz are 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 the tan δ obtained by dynamic viscoelasticity measurement at room temperature when vibrations of a frequency of 0.1 Hz are applied is 1 or more. That is, when the second acoustic transmission medium is a solid, the tan δ of the first acoustic transmission medium and the second acoustic transmission medium obtained by dynamic viscoelasticity measurement at room temperature when vibrations of a frequency of 0.1 Hz are applied is tan δ i , tanδ ii When this is the case, tan δ i <tan δ ii and / or tan δ i It is preferred that ≧1.

[0033] A solid that can serve as the second acoustic transmission medium is a flexible gel. 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.

[0034] 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.

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

[0036] 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 solid, while materials for which it is not possible to measure tan δ are considered liquid. 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.

[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 at 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 surface tension include anionic surfactants such as sodium dodecyl sulfate and sodium lauryl sulfate; cationic surfactants such as ethyltrimethylammonium bromide, benzalkonium chloride, and dimethylaminopropylamide stearate; nonionic surfactants such as Contaminon (registered trademark), Contaminon (registered trademark) US, Pluronic (registered trademark) F-68, Tween (registered trademark) 20, and Tween (registered trademark) 80; amphoteric surfactants such as CHAPS and CHAPSO; storage stabilizers such as light stabilizers (hindered amines, benzophenones, and benzotriazoles), and antioxidants (amines, phenols, sulfurs, and phosphoruss). Antibacterial agents such as benzalkonium chloride, sodium azide, and antibiotics (ampicillin, penicillin, streptomycin, etc.) are also included.

[0040] (Encapsulation 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. "Encapsulation" refers to the presence of the 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 within 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. When the second acoustic transmission medium is circulated within the first acoustic transmission medium, it is preferable to encapsulate the second acoustic transmission medium within the first acoustic transmission medium in the placement step described below, and then use a valve or the like to prevent circulation during the pressurization and detachment steps.

[0041] As a method for enclosing the second acoustic transmission medium, for example, a bag-shaped first acoustic transmission medium is prepared, the second acoustic transmission medium is poured into the bag, and the pouring port is then sealed. Examples of sealing methods include a method of melting the first acoustic transmission medium with heat, a method of crimping the first acoustic transmission medium with pressure, a method of reacting the first acoustic transmission medium with an adhesive or a compound of the raw materials for the first acoustic transmission medium, and a method of physically clamping the first acoustic transmission medium.

[0042] Another method of encapsulation includes preparing upper and lower sheets of the first acoustic transmission medium, and then encapsulating the second acoustic transmission medium while closely contacting the upper and lower surfaces to seal them. The cell detachment method of the present disclosure can include, as the encapsulation step, an encapsulation step of encapsulating the second acoustic transmission medium in the first acoustic transmission medium.

[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 preferably has a shape that physically contacts the first acoustic transmission medium on the opposite surface when pressurized in the pressurizing step, and may be, for example, cylindrical, hemispherical, conical, or a structure combining a cylindrical or conical and hemispherical shape. 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 abdominal part (region with large amplitude value) of the ultrasonic vibration pattern to be applied, but it may also include a part with weak vibration or a part that does not vibrate. The number of protrusions also needs to be at least one, and for example, in order to correspond to a plurality of vibration patterns, a plurality of protrusions may be arranged at positions corresponding to the abdominal parts of each vibration pattern.

[0045] The first acoustic transmission medium may have at least one column portion that crosses and connects to the second acoustic transmission medium. The surface of the acoustic transmission medium of the present disclosure facing the vibrator may 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 that the second acoustic transmission medium is contained so that air is not contained.

[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 generating vibrations in the vibrating body.

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

[0049] To improve compatibility between the vibrator and the surface of the acoustic transmission medium of the present disclosure that faces 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 compatibility, the surface of the acoustic transmission medium of the present disclosure that faces the vibrating body may be entirely or partially adhered to the vibrating body using an adhesive or pressure-sensitive adhesive, etc. Known adhesives, pressure-sensitive adhesives, double-sided tape, etc. are used as the adhesive or pressure-sensitive adhesive.

[0051] (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 the 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" refers not only to the adhesion of adhesive cells, but also to the presence of cells in contact with the culture substrate. That is, this includes, but is not limited to, adhesion of cells to the culture substrate via an adhesion factor, and also includes cells remaining attached by weak interaction with the culture substrate, or cells remaining attached simply by an external force such as gravity.

[0052] 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 surface warpage height, but mechanical alignment 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 protrusions on the outer periphery of their bottom surfaces. The main roles 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.

[0054] The substrate material 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.

[0055] (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.

[0056] 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.

[0057] (Piezoelectric Body) The piezoelectric body used in the vibrator of the present disclosure is preferably ring-shaped, but the shape of the piezoelectric body that deflects the vibrator may be annular or disc-shaped. To directly observe the cell detachment status, a ring-shaped body is preferable, as it ensures a clear view of the area below the culture substrate. Piezoelectric bodies exhibit piezoelectric properties when polarized, but the polarity of polarization may be changed depending on the electrode pattern, as shown in Figure 10. Polarized piezoelectric bodies 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 180-degree phase difference to a negatively polarized electrode relative to a positively polarized electrode. In contrast, when electrodes are separated into phase A and phase B on the left and right, as shown in Figure 10, applying an AC voltage with a 90-degree phase difference between phase A and phase B generates a two-phase driving traveling wave. In Figure 10, GND refers to ground and is used to ground the electrodes located on the back surface.

[0058] 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.

[0059] The piezoelectric material used for the vibrator is PbZrTiO３ 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.

[0060] 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.

[0061] (Steps) The cell detachment method of the present disclosure can include the following steps: Placement step: A step of placing an acoustic transmission medium on the vibrator, and if necessary, supplying a second acoustic transmission medium so that it is contained in the first acoustic transmission medium, and then placing a culture substrate; Pressurization step: A step of applying pressure from the culture substrate or vibrator side so that the culture substrate and vibrator come into contact with the acoustic transmission medium; Detachment step: A step of vibrating the vibrator while the culture substrate and vibrator are in contact with the acoustic transmission medium.

[0062] A specific example of a cell detachment method according to the present disclosure is shown in FIG. 5. First, in the placement step, a first acoustic transmission medium is placed on a vibrator (step S210). Then, a second acoustic transmission medium is supplied so that it is encapsulated in the first acoustic transmission medium (step S220). Then, a culture substrate is placed on the acoustic transmission medium (step S230). Next, in the pressure application step, 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).

[0063] Note that supplying the second acoustic transmission medium so that it is encapsulated in the first acoustic transmission medium, i.e., the encapsulation 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 encapsulated in advance in the first acoustic transmission medium 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) In the placement process, an acoustic transmission medium is placed on the vibrator, and if necessary, a second acoustic transmission medium is supplied so that it is encapsulated in the first acoustic transmission medium, and the culture substrate is then placed. If the acoustic transmission medium is configured to be able to supply and circulate 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 pressurization process and peeling process. 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, allowing for appropriate contact depending on the type and shape of the culture substrate, and circulating the second acoustic transmission medium also makes it possible to control the temperature of the acoustic transmission medium.

[0065] (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. This may involve displacing the culture substrate closer to the vibrator, displacing the vibrator closer to the culture substrate, or displacing both the culture substrate and the vibrator to bring them closer together. In this pressure step, the culture substrate and the vibrator are in contact with the acoustic transmission medium, and pressure is applied from either the culture substrate side or the vibrator side to mechanically align the culture substrate and the 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. Regarding the pressure step, weight loading is preferred in consideration of vibration insulation, 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 step.

[0066] The weight placed on the lid of the culture substrate is preferably, for example, a ring-shaped weight to facilitate observation of the cells in the container. The applied load varies depending on the size of the culture substrate and the vibration pattern 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 also cause the culture substrate itself to bend, resulting in uneven vibration transmission and a decrease in the detachment rate.

[0067] In the detachment step, the vibrator is vibrated while the culture substrate or the vibrator is in contact with the 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.

[0068] 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 Figure 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 process, the vibrator is driven by supplying a voltage of a resonant frequency to the vibrator to generate a desired ultrasonic vibration mode. Note that the power source that applies the voltage is not shown in Figure 6.

[0069] 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.

[0070] 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 generated in the detachment solution also varies depending on the amplitude and acceleration, so various combinations exist. Furthermore, simply using large amplitudes and large accelerations may be undesirable because they promote the occurrence of cavitation, which reduces cell viability.

[0071] 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.

[0072] 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.

[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 detachment device shown in Figure 6 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 where no vibration occurs during driving can be dynamically determined.

[0074] 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 Figure 6, the first acoustic transmission medium and the second acoustic transmission medium are collectively referred to as the acoustic transmission medium 13.

[0075] 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.

[0076] (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.

[0077] 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.

[0078] 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 lyses 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.

[0079] 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.

[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 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.

[0081] (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 vibrator can be driven while maintained at a constant temperature, e.g., 25°C, in an incubator. The drive time is sufficient for cell detachment. After activation, the detached cells are collected with a dropper, and any cells that were not collected and still adhered to the culture substrate 21 are collected by pipetting. The detachment rate used below is expressed as a percentage of the number of cells detached by vibration relative to the total number of cells detached by vibration and the number of cells detached by pipetting. Variation in the detachment rate was calculated using n = 10, and the standard deviation of the detachment rate was graded 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.

[0082] (Cell detachment device) The cell detachment device disclosed herein detaches cells by transmitting vibrations generated by a vibrator to cells arranged on the culture surface of a culture substrate, the cell detachment device comprising a vibrator, an acoustic transmission medium, and a culture substrate mounting section, in that order, configured so 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 encapsulated within the first acoustic transmission medium and being liquid at room temperature, or the second acoustic transmission medium having a larger tan δ than 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. The configuration of the cell detachment device disclosed herein is shown in Figure 6.

[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 placement section only needs to be recognizable as a location where a user can place a culture vessel. In the example of FIG. 6 , the opening formed by the vibrating body 101, upper cover 15, and cushioning material 16 is suitable for placing a general-purpose dish, and thus corresponds to the culture substrate placement section. The cell detachment device of FIG. 6 shows an example in which the vibrating body 101, which serves as a vibration generating section, includes a piezoelectric body 12 and a vibration plate 11. The vibrating body 101, which includes the piezoelectric body 12 and the vibration plate 11, generates bending vibrations that are amplified by the combination of the piezoelectric body 12, which expands and contracts, and the stiffness of the adhered vibration plate 11. This example utilizes resonance due to the structural factors of the vibrating body 101, and can generate large vibrations at the resonant 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). In FIG. 6, a ring-shaped vibrator is shown as a typical vibrating body 101, but a Langevin vibrator or a rectangular vibrator that can obtain a large amplitude may also be used.

[0084] To efficiently transmit the vibrations generated by the vibration plate through resonance to the culture substrate 21 on which cells are cultured, an acoustic transmission medium 13 is interposed between the vibration plate 11 and the culture substrate 21. Placing the culture substrate 21 directly on the vibration plate 11 results in point contact, resulting in energy consumption other than vibration, such as noise and heat generation, which reduces efficiency. The acoustic transmission medium 13 is preferably installed over an area sufficient to cover the detachment area of ​​cells attached to the culture substrate 21. To secure the vibration body 101, the upper cover 15, lower cover 17, buffer material 16, and bolts 18 shown in Figure 6 clamp the ends of the vibration body 101, but the clamping pressure at the ends is tightened to a degree that does not impede the vibration generated by the vibration plate. An example is shown in which a weight 14, which serves as a pressure means for the pressure process, is provided above the culture substrate and below the vibration body. The weight 14 moves up and down to apply and remove pressure.

[0085] By supplying a voltage at the resonant frequency, the vibrator is driven to generate the desired ultrasonic vibration mode. To drive the vibrator, an AC power source that supplies a voltage at the resonant frequency is used to apply a voltage at a vibration mode frequency that can form the desired vibration waveform on the cell culture surface of the culture substrate, and at a voltage that can obtain the desired amplitude, thereby driving the cell detachment device. At this time, by measuring the voltage of the S-phase, which is the sensor phase provided in 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, 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 9D.

[0087] (First Example) The configuration diagrams of a first example of an acoustic transmission medium are shown in Figures 1A to 1C and 9A. In the first example of the acoustic transmission medium, a second acoustic transmission medium is contained within a first acoustic transmission medium. Figure 1A shows a configuration in which the bottom surface is in contact with a flat culture substrate and a vibrating body. Figure 1B shows a configuration in which the bottom surface is in contact with a curved culture substrate and a vibrating body. Figure 1C further shows how 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 vibrating plate. Figure 9A illustrates a top view and a cross-sectional view of the first example of an acoustic transmission medium.

[0088] (Second Example) The configuration diagrams of a second example of an acoustic transmission medium are shown in Figures 2A to 2C and Figure 9B. In this second example of an acoustic transmission medium, a second acoustic transmission medium is enclosed within a first acoustic transmission medium, and the first acoustic transmission medium has a protrusion that is convex in the direction facing the second acoustic transmission medium. Figure 2A shows a configuration in which an acoustic transmission medium having a protrusion on its upper surface facing the first acoustic transmission medium is in contact with a culture substrate and a vibrator with a flat bottom. Figure 2B shows a configuration in which an acoustic transmission medium having a protrusion on its upper surface facing the first acoustic transmission medium is in contact with a culture substrate and a vibrator with a curved bottom. Figure 2C further shows the acoustic transmission medium of the present disclosure conforming to the shape of the bottom surface of the culture substrate by applying pressure from the culture substrate or diaphragm. As shown in the figure, it is preferable that the protrusion be configured to contact the opposing first acoustic transmission medium in a pressurized state. Figure 9B illustrates a top view and a cross-sectional view of the second example of an acoustic transmission medium. In the figure, 207 indicates the projection diameter, and 208 indicates the projection height.

[0089] (Third Example) The configuration diagrams of a third example of an acoustic transmission medium are shown in Figures 3A to 3C and Figure 9C. In this third example of an acoustic transmission medium, a second acoustic transmission medium is contained within a first acoustic transmission medium, and the first acoustic transmission medium has a columnar portion extending across the second acoustic transmission medium. Figure 3A shows a configuration in which the first acoustic transmission medium has a columnar portion extending across the second acoustic transmission medium, and the columnar portion is in contact with a culture substrate and a vibrating body having a flat bottom surface. Figure 3B shows a configuration in which the first acoustic transmission medium has a columnar portion extending across the second acoustic transmission medium, and the columnar portion is in contact with a culture substrate and a vibrating body having a curved bottom surface. Figure 3C further shows 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. Figure 9C shows a top view and a cross-sectional view of the acoustic transmission medium of the third example. 209 in the figure indicates the column diameter.

[0090] (Fourth Example) The configuration diagrams of a fourth example of the acoustic transmission medium are shown in Figures 4A to 4C and Figure 9D. This fourth example of the acoustic transmission medium has a second acoustic transmission medium supply pipe 133 for encapsulating a second acoustic transmission medium within a first acoustic transmission medium. Figure 4A shows a configuration in which the acoustic transmission medium having the second acoustic transmission medium supply pipe 133 is in contact with a culture substrate and a vibrator having a flat bottom. Figure 4B shows a configuration in which the acoustic transmission medium having the second acoustic transmission medium supply pipe 133 is in contact with a culture substrate and a vibrator having a curved bottom. Figure 4C further shows the acoustic transmission medium of the present disclosure conforming to the shape of the bottom 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. Figure 9D illustrates a top view and a cross-sectional view of the fourth example of the acoustic transmission medium.

[0091] Example 1 The following example demonstrates the use of a first example of the acoustic transmission medium shown in the schematic diagram of FIG. 1A. 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 was PZT, with an outer diameter of 70 mm, the same as the diaphragm, an inner diameter of 57 mm, and a thickness of 2 mm, considering that the neutral plane was located on the diaphragm side. The cell detachment device was assembled as shown in FIG. 6, and the acoustic transmission medium was placed on the diaphragm with the following shape: 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 mm, diameter (203) = 30 mm, and water sealed inside, as shown in the schematic diagram of FIG. 9A.

[0092] The acoustic transmission medium was fabricated by preparing a mold corresponding to the upper and lower surface shapes of the acoustic transmission medium, pouring a silicone rubber raw material (product name: KE-1950-10; manufactured by Shin-Etsu Chemical Co., Ltd.) into the mold, and then heating and curing the mold to obtain the upper and lower silicone rubber surfaces, which served as the first acoustic transmission medium. The rubber on both sides was then bonded together with adhesive, and water was injected into the mold through the injection port as the second acoustic transmission medium, which was then sealed with adhesive. The excess injection port was then removed to obtain the acoustic transmission medium. The resulting acoustic transmission medium was placed so that its center was aligned with the diaphragm. Next, a Thermo Fisher Scientific Nunc® Φ35 mm dish, a culture substrate on which cells had been cultured, was slowly placed on the acoustic transmission medium, with its center aligned with the center of the diaphragm. A schematic diagram of the configuration at this stage is shown in Figure 1B. A 100 g weight was then placed on the lid of the culture substrate to apply pressure. A schematic diagram of the configuration of the acoustic transmission medium after pressure application is shown in Figure 1C.

[0093] The cells were cultured as follows: CHO (Chinese Hamster Ovary) cells were cultured in a Thermo Fisher Scientific Nunc (registered trademark) 35 mm diameter dish at a density of 10,000 cells / cm. ２ The cells were seeded at a density of 1000× ... and incubated at 37° ２ The 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%.

[0094] (Cell Detachment) The medium in the dish was removed, and the cells were washed with PBS(-) and then immersed in 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 using a 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. After collecting the detached cells, cells that could not be detached by ultrasound were detached from the dish after ultrasonic detachment using a cell scraper. The number of detached cells was measured using a hemocytometer. The total number of detached cells was calculated by adding the number of cells detached by ultrasound and the number of cells subsequently detached with a cell scraper. The detachment rate was defined as the ratio of the number of ultrasonically detached cells to the total number of detached cells and was calculated. Variation in the detachment rate was determined using 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 second acoustic transmission medium was changed as follows. That is, in this example, ultrasound 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 a peeling rate of 96.1% and a variation rating of A.

[0096] Example 3 Example 3 was performed under the same conditions as Example 1, except that the type of second acoustic transmission medium was changed as follows. That is, PVA gel was used as the second acoustic transmission medium. The PVA gel was produced by the following method. 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 make a total of 80 mL. The solution was stirred while applying heat in a thermostatic chamber 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 make a total of 16 mL to make a 1.0% by mass solution. While stirring the PVA aqueous solution, the borax aqueous solution was gradually added dropwise and stirred until uniform, yielding a PVA gel. The evaluation results showed a peeling rate of 93.0% and a variation rating of A.

[0097] Example 4 Example 4 was performed 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 Figure 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 that the peeling rate was 94.6%, and the variation was rated A.

[0098] Comparative Example 1 In Comparative Example 1, an investigation 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 rating of D.

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

[0100]

[0101] 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.

[0102] This application claims priority based on Japanese Patent Application No. 2023-118584, filed on July 20, 2023, the entire contents of which are incorporated herein by reference.

[0103] 11: Vibration plate 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 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 comprising: disposing an acoustic transmission medium between the culture substrate and a vibrating body; and vibrating the vibrating body to detach the cells from the culture surface; wherein the acoustic transmission medium includes a first acoustic transmission medium and a second acoustic transmission medium; the first acoustic transmission medium is an elastic solid at room temperature; and the second acoustic transmission medium is contained within the first acoustic transmission medium and is 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 greater than the tan δ of the first acoustic transmission medium.

2. A cell detachment method as described in claim 1, characterized in that the first acoustic transmission medium has at least one protrusion that is convex in a direction facing the second acoustic transmission medium.

3. The cell detachment method according to claim 1, wherein the first acoustic transmission medium has at least one pillar portion extending across and connecting to the second acoustic transmission medium.

4. A cell detachment method described in any one of claims 1 to 3, characterized in that the first acoustic transmission medium includes at least one selected from the group consisting of silicone rubber, natural rubber, urethane gel, and gel material.

5. A cell detachment method described in any one of claims 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 vibrations of a frequency of 0.1 Hz at room temperature.

6. A cell detachment method according to any one of claims 1 to 5, characterized in that the second acoustic transmission medium is liquid at room temperature.

7. A cell detachment method described in any one of claims 1 to 6, characterized in that the second acoustic transmission medium includes at least one selected from the group consisting of liquid, polymer solution, and gel material.

8. A cell detachment method according to any one of claims 1 to 7, characterized in that the first acoustic transmission medium comprises silicone rubber and the second acoustic transmission medium comprises water.

9. A cell detachment method described in any one of claims 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.

10. A cell detachment method according to any one of claims 1 to 9, characterized in that it includes an encapsulation step of encapsulating the second acoustic transmission medium in the first acoustic transmission medium.

11. A cell detachment method according to any one of claims 1 to 10, characterized in that the vibrator generates vibrations in the ultrasonic band.

12. A cell detachment method according to any one of claims 1 to 11, further comprising a pressurizing step for applying pressure so as to bring the culture substrate and the vibrator closer together.

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, the cell detachment device having, in that order, a vibrator, an acoustic transmission medium, and a culture substrate mounting section, configured so that vibrations of the vibrator are transmitted to the culture substrate via the acoustic transmission media, 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 when a vibration of a frequency of 0.1 Hz is applied to the second acoustic transmission medium at room temperature is greater than the tan δ of the first acoustic transmission medium.

14. The cell detachment device according to claim 13, wherein the vibrating body 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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