Cell stripping method and cell stripping device

Abstract

Provided are a cell dissection method and a cell dissection device that make it possible to realize a vibration design suitable for target cells and to dissect attached cells at a high dissection rate. A cell peeling method for peeling cells adhering to a culture surface of a culture substrate from the culture surface comprises a peeling step for peeling the cells from the culture surface in a state in which a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and a vibrating body and in a state in which at least one of (a) and (b) is satisfied. The cells are exfoliated from the culture surface by vibrating the vibrating body: (a) the culture substrate is in contact with a first acoustic transmission medium and a second acoustic transmission medium; or (b) the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium. In the cell dissection method, a first acoustic transmission medium and a second acoustic transmission medium are different from each other, the first acoustic transmission medium is a solid at room temperature, and the second acoustic transmission medium is a liquid or a solid with fluidity at room temperature.

Description

Technical Field

[0001] This disclosure relates to a cell stripping method and a cell stripping device. Background Technology

[0002] In recent years, cell culture for regenerative medicine and biopharmaceutical manufacturing has been actively pursued. Cell culture is broadly classified into attachment culture and suspension culture, with attachment culture being a widely used method applicable to various cell types. However, during cell collection, enzymatic treatment is required to detach the cells, and in some treatment methods, cells may be damaged. Therefore, cell detachment through enzymatic treatment is also a cause of changes in cell quality. To address this, detachment conditions for collecting cells with high detachment rates without damaging the cells have been investigated.

[0003] Patent Document 1 discloses a patterning device that positions cells at specific locations by transmitting acoustic radiation pressure to a medium in the culture medium. However, using a liquid such as water as the acoustic transmission medium poses a risk of contamination when vibration is applied, as the liquid from the vibration transmission medium may enter the culture medium. Furthermore, when silicone rubber is used as the acoustic transmission medium, vibrations generated by the vibration source may not be successfully transmitted to the culture medium, or the vibrations may vary.

[0004] Citation List

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Publication No. 2018-42534 Summary of the Invention

[0007] Technical issues

[0008] This disclosure is made to address the above-mentioned problems, and this disclosure relates to providing a cell peeling method and a cell peeling apparatus that can achieve a vibration design suitable for target cells and can peel off attached cells with a high peeling rate.

[0009] Solution to the problem

[0010] According to this disclosure, a cell stripping method is provided for detaching cells attached to a culture surface from the culture surface, the cell stripping method comprising the following stripping steps: detaching the cells from the culture surface by vibrating the vibrator while a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture surface and a vibrator, and while satisfying at least one of (a) or (b):

[0011] (a) The culture medium is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or

[0012] (b) The vibrating body is in contact with the first sound transmission medium and the second sound transmission medium.

[0013] The first sound transmission medium and the second sound transmission medium are different from each other. The first sound transmission medium is a solid at room temperature, and the second sound transmission medium is a liquid or a fluid solid at room temperature.

[0014] According to this disclosure, the following cell stripping apparatus is also provided.

[0015] A cell peeling device for peeling cells from a culture surface by transmitting vibrations generated by a vibrator to cells disposed on a culture surface of a culture medium, the cell peeling device comprising:

[0016] Vibrating body;

[0017] The first sound transmission medium; and

[0018] Culture medium placement area, and

[0019] The cell stripping device also includes a supply unit for supplying a second acoustic transmission medium.

[0020] The supply unit is configured to supply the second acoustic transmission medium such that at least one of (a) or (b) is satisfied:

[0021] (a) The culture medium is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or

[0022] (b) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0023] Beneficial effects of the present invention

[0024] According to this disclosure, a cell peeling method can be provided that enables the implementation of a vibration design suitable for the target cells and peels off attached cells with a high peeling rate. Attached Figure Description

[0025] Figure 1A This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a first embodiment of the present disclosure.

[0026] Figure 1B This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a first embodiment of the present disclosure.

[0027] Figure 1CThis is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a first embodiment of the present disclosure.

[0028] Figure 2A This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a second embodiment of the present disclosure.

[0029] Figure 2B This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a second embodiment of the present disclosure.

[0030] Figure 2C This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a second embodiment of the present disclosure.

[0031] Figure 3A This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a third embodiment of the present disclosure.

[0032] Figure 3B This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a third embodiment of the present disclosure.

[0033] Figure 3C This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a third embodiment of the present disclosure.

[0034] Figure 4A This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a fourth embodiment of the present disclosure.

[0035] Figure 4B This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a fourth embodiment of the present disclosure.

[0036] Figure 4C This is a schematic cross-sectional view illustrating an example of the construction of a cell stripping device according to a fourth embodiment of the present disclosure.

[0037] Figure 5A This is a flowchart illustrating the steps of a cell stripping method according to the present disclosure.

[0038] Figure 5B This is a flowchart illustrating a specific example of the steps of a cell stripping method according to the present disclosure.

[0039] Figure 6A This is a view of an example of a device according to this disclosure.

[0040] Figure 6B This is a view of an example of a device according to this disclosure.

[0041] Figure 6CThis is a view of an example of an apparatus including an information processing device according to the present disclosure.

[0042] Figure 7A This is a view illustrating an example of the configuration steps according to the method of this disclosure.

[0043] Figure 7B This is a view illustrating an example of the configuration steps according to the method of this disclosure.

[0044] Figure 7C This is a view illustrating an example of the pressurization step according to the method of this disclosure.

[0045] Figure 7D This is a view illustrating an example of the stripping steps according to the method of this disclosure.

[0046] Figure 8 This is a schematic diagram illustrating an example of the culture medium in this disclosure.

[0047] Figure 9A This is a schematic diagram illustrating an example of a first acoustic transmission medium in this disclosure.

[0048] Figure 9B This is a schematic diagram illustrating an example of a first acoustic transmission medium in this disclosure.

[0049] Figure 9C This is a schematic diagram illustrating an example of a first acoustic transmission medium in this disclosure.

[0050] Figure 9D This is a schematic diagram illustrating an example of a first acoustic transmission medium in this disclosure.

[0051] Figure 9E This is a schematic diagram illustrating an example of a first acoustic transmission medium in this disclosure.

[0052] Figure 9F These are cross-sectional and top views illustrating an example of a first acoustic transmission medium in this disclosure.

[0053] Figure 10 This is a schematic diagram illustrating an example of a piezoelectric element in this disclosure.

[0054] Figure 11 This is a view showing the groove of the first acoustic transmission medium in this disclosure.

[0055] Figure 12 This is a table showing the results obtained by summarizing the respective configurations and evaluation results of the embodiments and comparative examples of this disclosure. Detailed Implementation

[0056] The following examples illustrate the cell stripping method according to the present disclosure, but the present disclosure is not limited to the following examples.

[0057] (Basic Structure)

[0058] The cell stripping method according to this disclosure is a cell stripping method for detaching cells attached to the culture surface of a culture medium from the culture surface.

[0059] The cell stripping method includes the following stripping steps: with a first acoustic transmission medium and a second acoustic transmission medium disposed between the culture medium and the vibrator, and under the condition that at least one of (a) or (b) is satisfied, the cells are stripped from the culture surface by vibrating the vibrator:

[0060] (a) The culture medium is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or

[0061] (b) The vibrating body is in contact with the first sound transmission medium and the second sound transmission medium.

[0062] Wherein, the first sound transmission medium and the second sound transmission medium are different from each other; the first sound transmission medium is solid at room temperature, and the second sound transmission medium is liquid or a fluid solid at room temperature. By achieving at least one of (a) or (b) according to the cell stripping method of this disclosure, a state as described above is obtained. Figure 5A An example of a cell stripping method according to this disclosure is shown. Additionally, in Figures 7A to 7D The diagram shows the concept diagrams for each step.

[0063] Figure 7A and Figure 7B This is a view of the steps involved in configuring the first and second acoustic transmission media. The first and second acoustic transmission media can be pre-configured, and this step is not a necessary step in the cell stripping method of this disclosure.

[0064] Figure 7A In this process, a first acoustic transmission medium is placed on a vibrating body, and a second acoustic transmission medium is further applied and placed on the first acoustic transmission medium. Figure 7B In this process, culture medium is placed on the configured acoustic transmission medium. Figure 7C This is a view of the pressurization step, where pressure is applied over the culture medium. The pressurization step is not necessary for the peeling method according to this disclosure. Figure 7D This is a view of the stripping process. In Figure 7DIn this process, when pressure is applied from above the culture medium, a vibrator is driven to apply ultrasonic vibration and peel off cells. The pressurization step facilitates contact between the first and second acoustic transmission media and the culture medium and the vibrator, and the first and second acoustic transmission media can transmit vibrations while in contact with the culture medium and the vibrator during the peeling step. Here, the phrase "the state in which the first and second acoustic transmission media are in contact with the culture medium and the vibrator" means that the first and second acoustic transmission media are in full contact with the culture medium and the vibrator so that the vibration of the vibrator is fully transmitted to the culture medium through the first and second acoustic transmission media, and also means that very small gaps or extremely small amounts of material can be inserted between the first or second acoustic transmission media and the culture medium or the vibrator, as long as the problem of this disclosure is solved. For example, even if only a few molecules of water are inserted between the first acoustic transmission media and the culture medium, it can be considered that the first acoustic transmission media and the culture medium are in contact with each other.

[0065] Culture media vary considerably between different manufacturers' models and production batches, and when both the culture media and the vibrator are to be bonded to the first acoustic transmission medium, it is difficult to eliminate alignment mismatches caused by subtle differences in shape. Therefore, it is difficult to perfectly bond both the culture media and the vibrator to the first acoustic transmission medium, which exhibits minute shape variations. Furthermore, different culture media are used for each culture, thus requiring bonding of the contact surfaces between the culture media and the first acoustic transmission medium for each use, complicating the process. In particular, when cells are placed on the vibrator and pressure is applied selectively to the center of the cell container after cell culture, cells may be damaged, making it difficult to achieve close contact between the culture media and the first acoustic transmission medium. Similarly, even when the culture media is brought into contact with the first acoustic transmission medium and then the first acoustic transmission medium is placed on the vibrator, it is difficult to achieve close contact between the vibrator and the first acoustic transmission medium. Therefore, the inventors of this application have solved the practical difficulty in improving the alignment between the culture media and the first acoustic transmission medium, and between the vibrator and the first acoustic transmission medium.

[0066] As a result of diligent research, the inventors of this application have discovered that it is important for the first and second sound transmission media to be in contact with at least one of the culture medium or the vibrator. In the cell stripping method according to this disclosure, a first and second sound transmission media are disposed between the vibrator and the culture medium in order to transmit the vibration of the vibrator to the culture medium. The first and second sound transmission media can complement each other to achieve close contact with the vibrator or the culture medium, thus it is inferred that this configuration can achieve effective vibration transmission.

[0067] The vibrating body, which acts as the vibration generator, includes a piezoelectric element and a vibrating plate, and has a vibration mechanism for generating flexural vibration, wherein the vibration is amplified by a combination of the stiffness of the piezoelectric element due to its own expansion and contraction and the stiffness of the vibrating plate with which it is joined. In this disclosure, large vibrations can be obtained at the resonant frequency by utilizing the resonance caused by the structural factors of the vibrating body.

[0068] (Sound transmission medium)

[0069] In the cell stripping method according to this disclosure, an acoustic transmission medium is used for alignment between a vibrating body that generates vibration and a culture medium to which the vibration is to be transmitted. For alignment, not only physical alignment such as acoustic impedance but also mechanical alignment such as adhesion is important. Preferably, a structure is provided that can solve the problems of this disclosure by achieving both mechanical and physical alignment between the first or second acoustic transmission medium and the culture medium or vibrating body due to contact between the first and second acoustic transmission medium and at least one of the culture medium or vibrating body. In this embodiment, the acoustic transmission medium may also be referred to as an acoustic transmission material.

[0070] The physical alignment between the vibrating body and the culture medium can be defined by a physical quantity called "acoustic impedance".

[0071] Specifically, the acoustic impedance Z of the acoustic transmission medium is defined as:

[0072] Z = (density of the sound transmission medium) × (speed of sound inside the sound transmission medium).

[0073] Furthermore, when a sound wave reaches the boundary between two substances, the transmittance of the sound wave is determined by the following formula:

[0074] Transmittance = (4×Z1×Z2) / (Z1+Z2)^2,

[0075] Z1 and Z2 are the acoustic impedances of the first and second materials that transmit sound waves.

[0076] For example, by substituting the impedances of water and air into the above equation, the transmittance between water and air, as well as the transmittance between silicone rubber and air, is almost zero:

[0077] (Acoustic impedance of water Z1) = 1.5 × 10^6 [kg / m^2 / s]

[0078] (Acoustic impedance of air Z2) = 4.08 × 10^2 [kg / m^2 / s]

[0079] (Acoustic impedance of silicone rubber Z3) = 1.0 × 10^6 [kg / m^2 / s].

[0080] In other words, the transmittance of sound waves is almost zero between water and air, and between silicone rubber and air, resulting in total internal reflection.

[0081] Polystyrene, a material commonly used as a culture medium, has an acoustic impedance of Z4 = 2.4 × 10^6 [kg / m^2 / s]. Therefore, regarding the physical alignment between the vibrator in the acoustic transmission medium and the culture medium in this disclosure, it is preferable to use materials with acoustic impedances close to the above values ​​for both the first and second acoustic transmission media.

[0082] Regarding the mechanical alignment between the vibrator and the culture medium, it is preferable that they are in close contact with each other via solids. In particular, in this disclosure, it is important to allow such mechanical alignment, which enables close contact over a wide area, in order to form the vibration waveform of the vibrator on the cell culture surface of the culture medium.

[0083] Suppose a commercially available culture dish with protrusions is used as the culture medium and placed directly on a vibrator. In this state, even when using a low molecular weight material (such as water or glycerol, which is easily deformed under low load) as the sound transmission medium, the protrusions of the culture medium will directly contact the vibrator, resulting in multiple vibration transmission paths. As a result, the generated vibration becomes complex and therefore difficult to control. In the cell stripping method according to this disclosure, active vibration control is performed. That is, by using the medium of a solid to keep the vibrator and the culture medium in close contact, the vibration transmission paths are kept constant, thereby facilitating vibration control.

[0084] When using the acoustic transmission medium of this disclosure to mechanically align a vibrator with a culture medium, it is preferable to use an elastic material such as rubber or gel (which is a polymer material) as the solid material. Specific examples include silicone resins, polyurethane resins, diallyl phthalate resins, unsaturated polyesters, epoxy resins, phenolic resins, urea resins, melamine resins, and natural rubber. These materials can be porous resin materials. Silicone rubber is preferred because of its high transmission efficiency, due to its physical property of very low attenuation in the frequency band of the driving frequency (vibration frequency), and also because it can suppress the heating of the acoustic transmission medium itself.

[0085] (First sound transmission medium)

[0086] The first sound transmission medium is a solid at room temperature. The term "room temperature" as used herein refers to a temperature above 20°C and below 30°C, preferably 25°C.

[0087] The solid is preferably elastic. Examples of elastic solid materials may include rubber and gel, which are polymer materials.

[0088] Examples of rubber as a polymer material include: rubber containing at least one resin selected from silicone resin, polyurethane resin, diallyl phthalate resin, unsaturated polyester, epoxy resin, phenolic resin, urea resin, and melamine resin; and natural rubber. More specific examples include silicone rubber, polyurethane rubber, butyl rubber, nitrile rubber, butadiene rubber, and latex rubber.

[0089] Examples of gels as polymeric materials include hydrogels and polymer gels. Examples of hydrogels include those obtained by combining hydrophilic polymeric materials such as polyvinyl alcohol or polyacrylic acid with a crosslinking agent. Examples of crosslinking agents include inorganic reagents such as borax or layered clay minerals (clay), and organic reagents such as multifunctional polymeric materials, such as dendritic polymers. Examples of polymer gels include those obtained by combining polymeric materials such as acrylic resins or polyurethane resins with multifunctional compounds as the corresponding crosslinking agents.

[0090] Preferably, the following acoustic transmission medium is used as the first acoustic transmission medium: when a vibration with a frequency of 1 Hz is applied to the first acoustic transmission medium, the tanδ obtained by dynamic viscoelastic measurement at room temperature is less than 1.0.

[0091] The first sound transmission medium preferably comprises at least one selected from silicone rubber, natural rubber, polyurethane gel, and gel materials, and preferably includes silicone rubber. Silicone rubber has high transmission efficiency due to its low attenuation in the frequency range of the driving (vibration) frequency, and can also suppress the heating of the sound transmission medium itself.

[0092] In addition, to facilitate removal, a surface treatment, such as a fluorine coating, can be applied to the surface of the first sound transmission medium facing the culture medium or vibrator.

[0093] In addition, the acoustic transmission medium is preferably transparent to the visible light range so as to allow direct observation of the cell shedding state.

[0094] To control physical properties such as viscosity, affinity with the second sound transmission medium, acoustic impedance with the second sound transmission medium, storage stability, and antibacterial activity, various additives can be added to the first sound transmission medium. Affinity refers to properties such as the contact angle and wettability between the first and second sound transmission media.

[0095] (Second sound transmission medium)

[0096] The second sound transmission medium is a material different from the first sound transmission medium, and is either a liquid or a fluid solid at room temperature. The term "fluidity" refers to the ability to flow or move under gravity when tilted. Examples of fluid solids include solids with a tanδ of 1 or greater obtained by dynamic viscoelasticity measurements at room temperature when a vibration at a frequency of 0.1 Hz is applied to the solid material.

[0097] The phrase "liquid at room temperature" means liquid at 20°C to 30°C. The material preferably has one of the following characteristics: low intrinsic viscosity [η] at 25°C; or low complex viscosity η obtained by dynamic viscoelasticity measurement. ; or the low viscosity η measured by changing the shear rate using a type B viscometer or rheometer at room temperature of 25°C when a vibration at a frequency of 1 Hz is applied.

[0098] When the second sound transmission medium is a fluid solid, preferably, the tanδ of the second sound transmission medium is greater than the tanδ of the first sound transmission medium; that is, a solid or liquid material having a tanδ greater than that of the first sound transmission medium is preferably used as the second sound transmission medium. Specifically, it is preferable that tanδ1 < tanδ2, where tanδ1 and tanδ2 are the tanδ of the first sound transmission medium and the tanδ of the second sound transmission medium, respectively, obtained by dynamic viscoelasticity measurement at room temperature when a vibration at a frequency of 1 Hz is applied.

[0099] In another case, when the second sound transmission medium is a fluid solid, a solid with a tanδ of 1.0 or greater obtained by dynamic viscoelasticity measurement at room temperature when vibration at a frequency of 0.1 Hz is applied is preferred.

[0100] Examples of solids that meet these conditions include flexible gels. Flexible gels are the same as the gels exemplifying the first sound transmission medium, and examples include: hydrogels obtained by combining hydrophilic polymeric materials such as polyvinyl alcohol or polyacrylic acid with crosslinking components such as borax, layered clay minerals (clay), or hyperbranched polymeric materials such as dendritic polymers; and polymeric gels obtained by combining polymeric materials such as acrylic resins or polyurethane resins with multifunctional compounds as corresponding crosslinking agents.

[0101] Preferably, the tanδ value obtained on the temperature and time scales when the vibrating body and the culture medium are mechanically aligned using an acoustic transmission medium is used. That is, the tanδ value can be measured by measuring the dynamic viscoelasticity obtained when vibrations at a frequency of 1 Hz or 0.1 Hz are applied at room temperature (more preferably 25 °C) using a rheometer or similar instrument. and By using the relation tanδ= / The calculation is used to calculate tanδ, where G represents the shear modulus. Indicates the energy storage shear modulus. This represents the loss shear modulus. However, when the tanδ of the second sound transmission medium is measured, the above conditions must be met, and in some cases, the tanδ of the second sound transmission medium cannot be measured. The second sound transmission medium can be a liquid at room temperature. In this case, tanδ is not measurable.

[0102] When the second sound transmission medium is a liquid, the viscosity can be high enough not to impair sound transmission.

[0103] Low viscosity is preferred for ease of handling, as it facilitates the removal of excess liquid from between the culture medium or vibrator and the sound transmission medium.

[0104] Viscosity can be measured at room temperature, more preferably 25°C, using a type B viscometer or rheometer. Furthermore, when measuring using a 25 mm parallel plate with a 1.0 mm gap, a viscosity value obtained at a shear rate of 1.0 (1 / s) is preferred. Depending on the viscosity of the liquid, a suitable torque value may not be obtained under the above conditions; however, in this case, a similar shear rate can be achieved by changing the plate diameter and gap, etc.

[0105] Examples of liquids include low-viscosity liquids such as water, saline, ethanol, and diluted ethanol and mixtures thereof, as well as high-viscosity liquids such as glycerol, silicone oil, and polymer solutions, gel materials, and further mixtures thereof. Polymer solutions in which the gel is dispersed or swollen in a liquid (such as water) can also be used. More specifically, ultrasonic gels (e.g., PVA gels) in which additives such as thickeners are added to liquids such as glycerol to adjust viscoelasticity are also preferred. Gel materials can be classified as solid or liquid. Solid gels have a high proportion of crosslinking components, while liquid gels have a low proportion of crosslinking components. In this document, materials whose tanδ can be measured by the inventors' measurement methods are classified as solids, while materials whose tanδ cannot be measured are classified as liquids. This is believed to be related to the repulsive properties of the elastomer. In the following examples, PVA is a flexible gel as a solid, while the ultrasonic gel is a gel as a liquid.

[0106] The second sound transmission medium is particularly preferably a hydrophilic liquid. For example, an ethanol / water mixture obtained by mixing water and ethanol in a volume ratio of 3:7 to 2:8 is preferred due to its high sterilization activity and prevention of bacterial growth. The second sound transmission medium preferably includes at least one selected from water, physiological saline, ethanol, and diluted ethanol.

[0107] The second sound transmission medium may contain various additives to control physical properties such as viscosity, affinity and acoustic impedance to the first sound transmission medium, surface tension, storage stability and antibacterial activity.

[0108] Examples of the additives mentioned above may include the following.

[0109] Examples of additives used to regulate surface tension can include anionic surfactants such as sodium dodecyl sulfate and sodium lauryl sulfate.

[0110] Examples of cationic surfactants may include ethyltrimethylammonium bromide, benzalkonium chloride, and dimethylaminopropyl stearate.

[0111] Examples of nonionic surfactants may include Contaminon, Contaminon US, Pluronic (trademark) F-68, Tween 20, and Tween 80.

[0112] Examples of amphoteric surfactants may include CHAPS and CHAPSO.

[0113] Examples of storage stabilizers may include light stabilizers such as hindered amines, benzophenone, and benzotriazole, as well as antioxidants such as amine-based, phenol-based, sulfur-based, and phosphorus-based antioxidants.

[0114] Examples of additives that exhibit antibacterial activity include benzalkonium chloride, sodium azide, and antibiotics such as ampicillin, penicillin, and streptomycin.

[0115] (Combination of sound transmission media)

[0116] When the first acoustic transmission medium is mechanically aligned with the culture medium or with the vibrator, the second acoustic transmission medium fills the misalignment. For this reason, the first and second acoustic transmission media are complementary, making it unlikely that an air layer will exist between the acoustic transmission medium and the culture medium or vibrator. Preferred examples of the combination of the first and second acoustic transmission media may include instances where the first acoustic transmission medium comprises silicone rubber and the second acoustic transmission medium comprises at least one selected from water, saline solution, ethanol, and diluted ethanol.

[0117] Preferably, the vibrator is vibrated in the following condition: wherein on the surface of the culture medium or the vibrator that is in contact with the first sound transmission medium and the second sound transmission medium, the ratio of the area of ​​the portion in contact with the first sound transmission medium to the total area of ​​the portions in contact with the first sound transmission medium and the second sound transmission medium is 5% or more.

[0118] That is, preferably, at least one of the following conditions is met:

[0119] 0.05≤S1 / S1+S2,

[0120] Where S1 is the area of ​​the portion of the culture medium in contact with the first sound transmission medium, and S2 is the area of ​​the portion of the culture medium in contact with the second sound transmission medium; or

[0121] 0.05≤S3 / S3+S4

[0122] Wherein, S3 is the area of ​​the part of the vibrating body in contact with the first sound transmission medium, and S4 is the area of ​​the part of the vibrating body in contact with the second sound transmission medium.

[0123] When the area of ​​the portion of the first sound transmission medium that is mechanically aligned (the area of ​​the portion in contact with the vibrator and the first sound transmission medium) is 5% or more, the vibrator or culture medium can be in close contact with the first sound transmission medium, which is a solid, and the vibration transmission path remains constant. This further facilitates vibration control and makes it easier to achieve the desired vibration. S1 / S1+S2 and S3 / S3+S4 can be referred to as the "contact area ratio" or "contact area ratio of the first sound transmission medium". These contact area ratios can also be expressed as ratios or percentages. That is, 0.05≤S1 / S1+S2 or 0.05≤S3 / S3+S4 can be expressed as, for example, a contact area ratio of 0.05 or more, or a contact area ratio of the first sound transmission medium of 5% or more.

[0124] When the area of ​​the portion of the first sound transmission medium that is mechanically aligned (the area of ​​the portion of the vibrator in contact with the first sound transmission medium) is less than 5%, the area of ​​close contact between the vibrator and the culture medium through the solid medium is reduced, which may make it difficult to control in order to achieve the desired vibration.

[0125] As a method for calculating the area of ​​the portion where the first and second sound transmission media are in contact, there are methods for calculating the area by coloring the second sound transmission media to determine the first sound transmission media to be in the uncolored area and the second sound transmission media to be in the colored area. There are also methods that generate a contrast difference in refractive index at the portion where the first and second sound transmission media are in contact by adjusting, for example, the illumination during observation and calculating the area of ​​regions exhibiting different contrasts. For example, there are also methods that measure the lateral frictional force after bringing the vibrating body and the culture medium into close contact through the action of a solid medium, and thereby estimate the contact area of ​​the first sound transmission media based on the increase in frictional force.

[0126] The portion of the first acoustic transmission medium that is mechanically aligned with the culture substrate is the area where the vibration of the vibrator can be effectively transmitted to the culture substrate. For effective transmission of the vibration of the vibrator to the culture substrate, preferably, the antinodes (regions exhibiting large amplitude values) in the vibration pattern of the vibrator overlap with the area where the first acoustic transmission medium and the culture substrate are mechanically aligned. When the antinodes in the vibration pattern of the vibrator do not overlap with the area where the first acoustic transmission medium and the culture substrate are mechanically aligned, vibration transmission is hindered, and cell stripping efficiency may decrease or deviation during stripping may increase.

[0127] To mechanically align the desired area, for example, the desired area on the substrate surface of the first acoustic transmission medium is designed to be convex compared to the surrounding area. Specifically, for example, to effectively transmit patterns with strong vibrations at the center, the central portion of the first acoustic transmission medium is formed to be convex, and to effectively transmit patterns with strong vibrations at the periphery, the periphery of the first acoustic transmission medium is formed to be convex. Examples of processing methods include cutting and machining flat silicone rubber, and pre-preparing molds for any surface shape and performing molding.

[0128] The volume ratio of the second sound transmission medium to the first sound transmission medium is preferably 0.05 or more and 2.0 or less. When the volume ratio is less than 0.05, the volume of the second sound transmission medium is small, and when the first sound transmission medium is mechanically aligned with the culture medium, the amount of the first sound transmission medium used to fill the misaligned portion may be insufficient, which may hinder vibration transmission.

[0129] When the volume ratio is greater than 2.0, the volume of the second acoustic transmission medium increases, and the excess portion of the second acoustic transmission medium may overflow into the surrounding area during the aforementioned alignment process. If the overflowing second acoustic transmission medium comes into direct contact with both the vibrator and the culture medium, this can lead to an increase in the number of vibration transmission paths or adverse effects such as vibration suppression due to contact with the culture medium, making it difficult for the vibration of the vibrator to be transmitted properly.

[0130] In the configuration step, even if the second acoustic transmission medium is over-configured, the excess portion of the second acoustic transmission medium can subsequently be removed. Examples of methods for removing the excess portion of the second acoustic transmission medium include the following methods.

[0131] By using, such Figure 6B The protruding shape of the vibrating plate 11 shown serves as the vibrating body, making it easier for excess portions of the second sound transmission medium to be discharged rather than remaining around the sound transmission medium.

[0132] like Figure 11As shown, the first acoustic transmission medium 131 may be configured to include continuous grooves 30 extending from the center of the surface to the outer periphery in its surface facing the culture medium or vibrator, thereby facilitating the discharge of excess portions of the second acoustic transmission medium to the outside through the grooves.

[0133] In another case, the contact angle between the first sound transmission medium and the second sound transmission medium may preferably be 120° or less, more preferably 90° or less.

[0134] When the contact angle between the first and second sound transmission media becomes smaller, the excess portion of the second sound transmission medium becomes less likely to remain near the alignment part, and therefore the vibration transmission path caused by the excess portion of the second sound transmission medium becomes less likely to occur.

[0135] In addition, the contact angle between the surface of the culture medium or vibrator and the second sound transmission medium in the area where the culture medium or vibrator contacts the second sound transmission medium can be set to less than 40°.

[0136] In another scenario, to reduce the likelihood of excess portions of the second acoustic transmission medium remaining near the alignment point, surface treatment can be applied to any one of the following: the surface of the culture medium facing the first acoustic transmission medium, the surface of the vibrator facing the first acoustic transmission medium, the surface of the first acoustic transmission medium facing the culture medium, or the surface of the first acoustic transmission medium facing the vibrator. This step can be referred to as the surface treatment step. Excess portions of the second acoustic transmission medium become less likely to remain near the alignment point, thus reducing the likelihood of vibration transmission paths caused by excess portions of the second acoustic transmission medium occurring.

[0137] Examples of surface treatments can include hydrophilic treatments, and more specific examples include laser treatments, chemical treatments, and plasma treatments.

[0138] When the vibrator and the culture medium are directly aligned via the second acoustic transmission medium without the mediating effect of the first acoustic transmission medium, a vibration path caused by the excess portion of the second acoustic transmission medium can also occur. Therefore, in the area where the culture medium or vibrator contacts the second acoustic transmission medium, the contact angle between the second acoustic transmission medium and the surface of the culture medium or vibrator decreases, making it easier for the excess portion of the second acoustic transmission medium on the surface in contact with the second acoustic transmission medium to be discharged.

[0139] The methods listed above for removing excess portions of the second acoustic transmission medium can also be a combination of multiple methods, and in this case, they can exhibit a further enhanced effect.

[0140] Furthermore, examples of methods for adjusting the amount of the second acoustic transmission medium during alignment include forming a first acoustic transmission medium of a porous resin material. By impregnating the second acoustic transmission medium into the porous resin material, the acoustic transmission medium and the culture medium can be mechanically aligned by allowing the second acoustic transmission medium to permeate to the alignment surfaces.

[0141] The extent of leakage of the second acoustic transmission medium during alignment is controlled by the following factors: the pore size and pore size distribution of the voids contained in the porous resin material in the height direction, the proportion of voids, and the amount of pressure applied during the pressurization step.

[0142] In addition, the amount of evaporation of the unused second acoustic transmission medium obtained when the stripping step is not performed can be controlled by the following: the pore size and pore size distribution of the voids contained in the porous resin material in the height direction, the proportion of voids, and the pore size and proportion of voids on the surface of the resin material.

[0143] As a method for manufacturing porous resin materials, known manufacturing methods are used, examples of which include methods for manufacturing porous resin materials by adding salts with uniform particle sizes to resin raw materials, and methods for manufacturing porous resin materials by dispersing and stabilizing micro-droplets such as solvents in resin raw materials.

[0144] Regarding the contact surfaces between the culture medium and the first acoustic transmission medium, or between the vibrator and the first acoustic transmission medium, to improve alignment, all or part of the surfaces of either contact surfaces can be joined using, for example, an adhesive or a pressure-sensitive adhesive. When condition (a) is satisfied but condition (b) is not, a structure in which the vibrator and the first culture medium are joined together using an adhesive or the like can be used; when condition (b) is satisfied but condition (a) is not, a structure in which the vibrator and the first culture medium are joined together using an adhesive or the like can be used. However, since the culture medium is typically for single use, a structure in which the vibrator and the first acoustic transmission medium are joined together using an adhesive or the like is preferred over a structure in which the culture medium and the first acoustic transmission medium are joined together using an adhesive or the like.

[0145] Therefore, in cases where either the culture medium or the vibrator is bonded to the first sound transmission medium, it is preferable to bond the vibrator to the first sound transmission medium. Known adhesives, pressure-sensitive adhesives, double-sided tapes, etc., are used as the adhesive or pressure-sensitive adhesive.

[0146] (Cultivation material)

[0147] In the cell stripping method according to this disclosure, cells attached to the culture surface of a culture medium are detached from the culture medium. The term "cell attachment" includes not only the attachment of adhesive cells, but also the simple contact of cells with a surface. That is, the phrase "cells attached to a culture medium" includes, but is not limited to, cells attaching to it via adhesion factors, and includes cells remaining on the surface due to weak interactions with the culture medium, or cells simply remaining due to external forces (such as gravity).

[0148] As used in this disclosure, the term "culture medium" refers to any substrate used for cell culture, including the surface on which cells attach. Culture medium includes, for example, containers or portions thereof used for cell culture. Examples of cell containers may include culture dishes (also known as Pietro plates), flasks, plates, and tubes. For all such purposes, products from companies such as Corning Incorporated, Thermo Fisher Scientific Inc., and AGC Techno Glass Co., Ltd. are used as general-purpose disposable containers. Among these, culture dishes are typically used as examples of culture mediums in this disclosure. Culture mediums with removable lids are commonly used. Culture mediums as used herein may or may not include components such as lids. Figure 8 The schematic diagram illustrates an example of the shape of a petri dish. In particular, the bottom warpage height 300 varies considerably between different manufacturers' models, between production batches, and even within the same production batch. Variations in the bottom warpage height 300 need to be considered to ensure mechanical alignment of the culture medium with the acoustic transmission medium; however, mechanical alignment is facilitated by combining the use of the first and second acoustic transmission media of this disclosure.

[0149] like Figure 8 As shown, the bottom surface of commercially available culture media has a protrusion on its outer periphery. The main function of this protrusion is believed to be, for example, to prevent damage to the bottom surface of the substrate, to allow containers to be stacked, and to prevent the temperature of the workbench from being directly transferred to the bottom surface of the substrate when placed on a workbench. When the culture media and the sound transmission medium are mechanically aligned, alignment can be performed by avoiding this protrusion or by covering the protrusion.

[0150] Materials used for culture media only need to be chemically stable and capable of culturing the desired cells, and examples 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, polysilicon, polymethylpentene, glass, and metals. Polystyrene is preferred.

[0151] (Vibrating body)

[0152] The vibrator in this disclosure can be used without limitation, as long as it generates vibration. Examples include a vibrator obtained by joining a piezoelectric element and a vibrating plate together. When the piezoelectric element is circular, glass, stainless steel (SUS), or quartz is preferably used for the vibrating plate. By using glass, stainless steel (SUS), or quartz for the vibrating plate, the vibrator can output large amplitude at a relatively high driving frequency (vibration frequency) in the ultrasonic region without damage. The vibrator may include an ultrasonic transducer.

[0153] In the case of a ring-shaped piezoelectric element, the outer diameter of the vibrating plate is preferably equal to the outer diameter of the piezoelectric element.

[0154] Regarding the thickness of the vibrating plate, it is preferable that when the piezoelectric body and the vibrating plate are joined together and flexural vibration occurs, the midpoint in the thickness direction of the flexure (i.e., the neutral plane that is neither in tension nor compression during flexure) is on the vibrating plate side, so as to effectively utilize the strain of the piezoelectric body for flexure.

[0155] Examples of the shapes of the vibrating plates in this disclosure include, for example: Figure 6A The flat vibrating plate 11 shown and as shown Figure 6B The convex vibrating plate 11 is shown. Examples of methods for manufacturing convex vibrating plates include methods for joining vibrating plates with different diameters and methods for performing machining to form a convex shape by cutting.

[0156] (Piezoelectric element)

[0157] In this disclosure, the piezoelectric element used for the vibrator is preferably a toroidal piezoelectric element, but the shape of the piezoelectric element that causes the vibrator to flex can be either circular or disc-shaped. For direct observation of the cell detachment state, a circular shape that ensures a clear field of view below the culture medium is preferred.

[0158] Piezoelectric materials exhibit piezoelectric properties through polarization treatment, but as Figure 10 As shown, the polarization polarity can be changed according to the electrode pattern. The patterned portions of electrodes A1, A2, A3, B1, B2, and B3 are the driving phases that facilitate deformation, and the region sandwiched between electrodes A1 and B1 is the sensor phase for detecting the degree of deformation. The polarized piezoelectric can excite standing wave and traveling wave vibration modes by controlling the phase of the input AC voltage to each electrode pattern. Standing waves are generated by applying an AC voltage with a 180-degree phase difference to the negative electrodes (A2 and B2) relative to the positive electrodes (A1, A3, B1, and B3). In contrast, when the electrodes are divided into... Figure 10When the single-point link line is connected to phase A (electrodes A1, A2, and A3) and phase B (electrodes B1, B2, and B3), a two-phase driven traveling wave is generated by applying an AC voltage with a 90-degree phase difference between phases A and B. Figure 10 In this context, GND stands for ground, which is used to ground the electrodes located on the back side.

[0159] Examples of forming electrode patterns using Ag through printing methods can be given, but the electrode material can be more than just Ag; it can also be noble metals such as Au, Pt, or Pd, or base metals such as Cu. The forming method can be printing, plating, or sputtering.

[0160] PbZrTiO3 (PZT) is used as a material composition for piezoelectric elements used in vibrators; however, considering environmental regulations, lead-free piezoelectric materials that essentially do not use Pb are preferred. Examples of main components of lead-free piezoelectric materials include BaTiO3 (BT), NaNbO3, BiNaTiO3, and BiFeO3, and combinations thereof, or metallic elements may be added to the main components. In particular, it has been shown that the vibrational performance of materials based on BaTiO3 (BT) is equivalent to that obtained using PbZrTiO3 (PZT). Furthermore, single-crystal materials or polymer-based piezoelectric materials can be used as materials other than ceramics.

[0161] For the thickness of the piezoelectric element in the vibrating body, it is preferable that when the joined and stacked piezoelectric element and the vibrating plate flexurally vibrate, the midpoint in the thickness direction of the flexural portion (i.e., the neutral surface where neither tension nor compression occurs during flexural) is on the vibrating plate side, so as to effectively utilize the strain of the piezoelectric element for flexural. Therefore, the combination of the thicknesses of the piezoelectric element and the vibrating plate is calculated based on the relationship between the hardness of the piezoelectric element and the hardness of the vibrating plate.

[0162] (step)

[0163] The cell stripping method according to this disclosure is characterized by including the following steps:

[0164] Configuration steps:

[0165] The steps of configuring a first acoustic transmission medium and a second acoustic transmission medium between the culture medium and the vibrator.

[0166] Peeling steps:

[0167] The step of peeling cells from the culture surface by vibrating a vibrator, provided that at least one of (a) or (b) is satisfied:

[0168] (a) The culture medium is in contact with the first and second sound transmission media, or

[0169] (b) The vibrating body is in contact with the first sound transmission medium and the second sound transmission medium.

[0170] In addition, a pressurization step can be provided as an additional step. The pressurization step is the step of applying pressure to bring the culture medium and the vibrator closer together, and this step promotes contact between the culture medium or vibrator and the first and second sound transmission media.

[0171] Figure 5B The diagram shows one specific example of a cell stripping method according to this disclosure.

[0172] First, as a configuration step, a first sound transmission medium is placed on the vibrator (step S210).

[0173] Subsequently, a second sound transmission medium is placed on the first sound transmission medium (step S220).

[0174] Next, as a pressurization step, the first and second acoustic transmission media are brought into contact with the culture medium (step S230). Thereafter, pressure is applied from above the culture medium using a weight or the like (step S240).

[0175] As part of the dissection step, the conditions for ultrasound application are set (step S250). Next, ultrasound application is started (step S260). Finally, the dissected cells are collected (step S270).

[0176] Applying pressure is not a necessary step in the cell stripping method according to this disclosure.

[0177] Each step is described below.

[0178] (Configuration Steps)

[0179] In the configuration step, the first and second acoustic transmission media are respectively configured to contact the vibrator or the culture medium, based on the state of vibration generated by the vibrator in the peeling step. Furthermore, in the peeling step, either the first or second acoustic transmission medium may be configured to contact either the vibrator or the culture medium, based on the state of vibration excited in the vibrator during the peeling step.

[0180] For example, in the state of vibration excited in a vibrating body, when it is desired that the center of the culture medium conforms to the vibration state of the vibrating body, it is preferable to use a first sound transmission medium having a shape that allows the center of the culture medium to contact it.

[0181] The second acoustic transmission medium is pre-applied to the portion of the culture medium in which the first acoustic transmission medium is not in contact with each other, thereby enabling contact with the first and second acoustic transmission media.

[0182] As an application method, a method that ensures that the volume relative to the contact area and the resulting gap is neither excessive nor insufficient is applied in a manner that ensures minimal deviation. Examples include methods that spray and apply the second sound transmission medium using a dispenser, nozzle, sprayer, etc., and methods that apply the second sound transmission medium by impregnating a sponge, etc., with the second sound transmission medium.

[0183] (Pressure application step)

[0184] The pressurization step in this embodiment can be any step that applies pressure to bring the culture medium and the vibrator closer to each other. The culture medium can be moved closer to the vibrator, or the vibrator can be moved closer to the culture medium, or both the culture medium and the vibrator can be moved closer to each other.

[0185] In the pressurization step, pressure is applied from the culture medium side or from the vibrator side to mechanically align the culture medium and the sound transmission medium by bringing the culture medium or vibrator into contact with the first sound transmission medium and the second sound transmission medium.

[0186] Examples of pressurizing units used to perform the pressurization step include: a method of placing a weight on the lid of the culture medium, and a method of applying a load from below to above while fixing the upper part of the culture medium lid from above, so as not to move due to the uniform load applied from above by a loading method (such as a spring or leaf spring from above the lid of the culture medium container).

[0187] For the pressurization unit, heavy loading is preferred to isolate vibration, but other loading methods can be selected without suppressing vibration. For example, a constant force spring can be used as the pressurization unit.

[0188] Regarding the shape of the weight to be placed on the lid of the culture medium, it is preferable to use a weight with, for example, a ring shape, so that the cells in the container can be easily observed.

[0189] The load to be applied varies depending on the size of the culture medium and the vibration pattern to be applied. For example, in the case of a 35 mm culture dish, the load to be applied is preferably 10 g to 300 g, and in the case of a 60 mm culture dish, the load to be applied is preferably 100 g to 600 g. The load can be applied by using a heavy object, or it can be the weight of the culture vessel itself.

[0190] When the weight is too heavy, in addition to inhibiting and limiting the vibration itself, the culture medium itself may flex, resulting in an uneven vibration transmission state, which in turn leads to a decrease in the peeling rate.

[0191] (Stripping step)

[0192] In the peeling step, cells are peeled off by vibrating the vibrator while it is in contact with a first acoustic transmission medium and a second acoustic transmission medium. Alternatively, the peeling step may include exciting vibrations in the ultrasonic frequency band within the vibrator.

[0193] The vibration of the vibrator is not started in the configuration step, but is started in the stripping step. For example, in Figure 6A and Figure 6B In the example of the apparatus shown, the vibrator is driven to generate the desired ultrasonic vibration mode by supplying a voltage at the resonant frequency. In this case, during the stripping step, the vibrator is driven to generate the desired ultrasonic vibration mode by supplying a voltage to the vibrator at the resonant frequency. Figure 6A and Figure 6B The power source supplying the above voltage is not shown in the diagram.

[0194] The vibration pattern can be freely selected based on cell type and dissection results. Furthermore, such vibration patterns can be used alone during the dissection step, or they can be input by periodically changing them.

[0195] Preferably, the vibration waveform causes the cell culture surface of the culture medium to vibrate with a constant amplitude (e.g., 1 μm). Essentially, resonant vibrations can be detected when performing a frequency scan from low to high frequencies (e.g., from 20 kHz to 150 kHz) within the ultrasonic range, but the vibrations detected on the low-frequency side have large amplitudes but small accelerations. In contrast, the vibrations detected on the high-frequency side have small amplitudes but large accelerations. From the above, the conditions for applying force to weaken the adhesion of cultured cells to the culture medium depend largely on the cell adhesion as well as the size and shape of the cells. Furthermore, the flow rate generated in the liquid (e.g., stripping fluid) in the cell culture medium also varies depending on the amplitude and acceleration, thus various combinations exist. Moreover, simply using large amplitude and large acceleration may be undesirable because it promotes cavitation, which can reduce cell viability.

[0196] The vibration waveform can be configured as a standing wave mode formed concentrically from the center of the disc-shaped vibrating plate or in a certain order along its circumference, or as a traveling wave mode that forms a radial vibration waveform on the vibrating plate by vibrating a piezoelectric body with a phase that changes radially. For each mode, the vibration waveform can be formed by applying a voltage to the vibrating body at the resonant frequency, and the resonant frequency and the shape of the vibration waveform can be calculated using structural calculations based on the shape of the vibrating body and its material physical properties (such as hardness and density).

[0197] Furthermore, when the vibrating body is continuously driven, the resonant frequency shifts to the lower frequency side due to the heating of the piezoelectric body itself. Therefore, the amplitude decreases as the drive continues at a fixed frequency. To address this, sufficient amplitude can be obtained by using the following driving methods: a driving method that provides a pause time after vibration for a certain period of time to suppress the temperature rise caused by self-heating, or a driving method that repeatedly scans from the high-frequency side to the low-frequency side with a fixed frequency width so that the resonant frequency always passes even when heat is generated.

[0198] (Vibration Evaluation)

[0199] The vibration waveform of the vibrating plate can be measured using a laser Doppler vibrometer (obtained from Graphtec AT7200). Figure 6A The measurement method is illustrated using the cell peeling device shown as an example. When the cell peeling device is driven at the desired resonant frequency and voltage, and the surface of the vibrating plate 11 is scanned in two dimensions in the X and Y directions using a laser, the vibration velocity at each point can be obtained, and the amplitude in the Z direction can be calculated. Therefore, the vibration waveform of the vibrating plate 11 can be constructed in three dimensions, and the positions of antinodes exhibiting large vibration amplitudes and nodes exhibiting no vibration can be dynamically determined during driving.

[0200] A similar configuration can also be used to measure the vibration of the cell culture surface of the culture medium 21. Preferably, an acoustic transmission medium 13 is placed on the vibrating plate 11, a metal (e.g., Ag) film is formed on the inner surface of the culture medium 21 by vapor deposition so that the laser is reflected by the culture medium 21, and the culture medium 21 is positioned such that no air layer is introduced between the culture medium 21 and the acoustic transmission medium 13. Figure 6A In the image, the first sound transmission medium and the second sound transmission medium are collectively referred to as sound transmission medium 13.

[0201] In the case where the target resonance obtained by driving is a standing wave mode of resonance with concentric amplitude, the amplitude of the center part of the culture medium 21 placed on the acoustic transmission medium 13 is scanned near the target resonance frequency by the cell stripping device, and the entire cell culture surface of the culture medium 21 is scanned in two dimensions at the frequency when the amplitude is maximum, thereby enabling three-dimensional understanding of the vibration waveform generated on the cell culture surface of the culture medium 21.

[0202] (cell)

[0203] The cell stripping method disclosed herein can be applied without limitation to all types of cells. Examples include various cultured cell lines, such as CHO cells derived from Chinese hamster ovaries, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), normal diploid fibroblasts derived from human fetal lungs (TIG-3 cells), cells derived from human fetal kidneys (HEK293 cells), A549 cells derived from human alveolar basal epithelial adenocarcinoma, HeLa cells derived from human cervical cancer, and cell lines derived from insects, as well as epithelial and endothelial cells, skeletal muscle cells, and smooth muscle cells that form various tissues and organs in vivo. And contractile cardiomyocytes, neurons and glial cells that form the nervous system, fibroblasts, primary cultured cells such as hepatocytes, non-hepatocytes, and adipocytes involved in metabolism, various stem cells such as induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ cells (EG cells), embryonic carcinoma cells (EC cells), mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells and germline stem cells, and precursor cells of various tissues, as well as cells induced from their differentiation.

[0204] Cell culture conditions can be appropriately selected based on the cells to be cultured. Typically, an appropriate amount of culture medium is added to culture medium 21, and approximately 1.0 × 10⁻⁶ μL of culture medium is added. 1 cells / cm 2 To approximately 5.0 × 10 4 cells / cm 2 Cells are seeded in the culture medium and cultured at 37°C and 5% CO2. It is preferable to culture until the cell occupancy in the culture medium 21 reaches approximately 70% to approximately 80%, a state known as sub-confluence.

[0205] In the cell stripping step, cell stripping solution can be used to replace the culture medium (culture medium) of the cultured cells.

[0206] Typically, peeling solutions contain proteolytic enzymes. Examples of proteolytic enzymes include trypsin, Accutase, collagenase, native proteases, chymotrypsin, elastase, papain, streptomycin, and their recombinants. However, proteolytic enzymes can lyse cells, thus raising concerns about adverse effects on cells. In contrast, the cell peeling method according to this disclosure exhibits similar effects even without the use of proteolytic enzymes, or even when used, with only a very small amount of proteolytic enzymes, thus having the effect of suppressing adverse effects on cells.

[0207] Cell stripping fluid is a liquid used during cell stripping and can reduce cell adhesion.

[0208] The pH of the cell stripping solution is preferably within the neutral range. This is because a neutral range is suitable for cell culture and allows for a consistently high cell viability. The pH can be appropriately adjusted using hydrochloric acid, sodium hydroxide, etc. Furthermore, various buffer solutions can be appropriately used to stably maintain the pH. The stripping solution can also be a culture medium.

[0209] The viscosity of the cell stripping solution can be appropriately adjusted by, for example, adding polymers or sugars.

[0210] A solution containing a metal ion chelating agent (hereinafter referred to as "chelating agent") is particularly preferred as a cell stripping solution. This is because using a cell stripping solution containing a chelating agent can effectively reduce cell adhesion.

[0211] There are no particular limitations on the chelating agent, but examples include ethylenediaminetetraacetic acid (hereinafter referred to as "EDTA"), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycol ether diaminetetraacetic acid, hypozoxytriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediaminedisuccinic acid, hydroxyethyl phosphate, citric acid, gluconic acid, and phosphonobutanetriacetic acid. Among these, chelating agents that form chelates with divalent cations are preferred, and those that form chelates with Ca are particularly preferred. 2+ and Mg 2+ The preferred chelating agent for forming the chelate is ethylenediaminetetraacetic acid (EDTA). When EDTA is used as the chelating agent, the pH of the cell stripping solution is preferably above 7.0 and below 8.0. This is because the chelating ability of EDTA can be enhanced by a slightly higher pH within the neutral range, which allows for a higher cell viability and thus further improves the efficiency of reducing cell adhesion. The chelating agent can be used alone or in combination of two or more.

[0212] The chelating agent content is preferably 0.01 mM or more and 5.0 mM or less.

[0213] When the content falls within this range, chelation effect can be reliably obtained, and the reduction in activity due to the presence of excessive chelating agent can also be inhibited.

[0214] The cell stripping fluid may contain a hydrophilic polymer comprising a polyalkylene glycol structure. An example of a hydrophilic polymer comprising 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, as measured by gel permeation chromatography, more preferably a peak molecular weight Mp of 1200 or more and 20,000 or less. This is because the polymer has minimal impact on the cells and can suppress the thickening effect caused by the polymer.

[0215] (Peel test)

[0216] A peeling test can be performed to evaluate the cell peeling method according to this disclosure. For example, the peeling test can be performed as follows.

[0217] The preparation steps can be performed on the cell culture substrate, and a stripping solution can be used instead of the culture medium when needed. The vibrator can also be driven while the substrate is maintained at a constant temperature (e.g., 25°C) in an incubator, if necessary. The drive duration is sufficient for cell stripping. After drive, the stripped cells are collected with a pipette, and cells that could not be collected and remain attached to the culture medium 21 are removed by pipetting. Cells may be damaged due to exposure to high temperatures after removal of the culture medium. Furthermore, some cells may be damaged due to exposure to changes in ambient temperature; therefore, it is preferable to set the temperature according to the characteristics of the cells to be used when placing the cells in the stripping device or during cell stripping.

[0218] Regarding the various measurements described later, the physical properties of the acoustic transmission medium at the temperature at which the cells provided in the culture vessel are placed in the peeling device are particularly important. Therefore, the first acoustic transmission medium is solid at room temperature, and the second acoustic transmission medium is liquid or a fluid-flowing solid at room temperature. Furthermore, preferably, even at the temperature during peeling, the first acoustic transmission medium is solid, and the second acoustic transmission medium is liquid or a fluid-flowing solid. It is also preferred that, when a vibration with a frequency of 0.1 Hz is applied, the second acoustic transmission medium is a solid with a tanδ of 1.0 or higher at room temperature.

[0219] The detachment rate used below is expressed as the percentage of cells detached by vibration relative to the total number of cells detached by vibration and by aspiration. For deviations, the detachment rate is determined with n=10, and the standard deviation of the detachment rate is rated 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 more than 10%.

[0220] By using diffused light from the backlight, residual cells remaining on the substrate surface after cell peeling can be observed.

[0221] (Cell peeling device)

[0222] The cell peeling device according to this disclosure is a cell peeling device for peeling cells from the culture surface of a culture medium by transmitting vibrations generated by a vibrator to cells disposed on a culture surface, the cell peeling device comprising:

[0223] Vibrating body;

[0224] The first sound transmission medium;

[0225] Culture medium placement area, and

[0226] The cell stripping device also includes a supply unit for supplying a second acoustic transmission medium.

[0227] The supply unit supplies the second acoustic transmission medium such that at least one of (a) or (b) is satisfied:

[0228] (a) The culture medium is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or

[0229] (b) The vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium.

[0230] Figure 6A The structure of the cell stripping device according to this disclosure is shown.

[0231] like Figure 6A As shown, the cell stripping device according to this disclosure includes a vibrator 101, an acoustic transmission medium 13, and a supply unit 23 for supplying a second acoustic transmission medium. In this figure, the first acoustic transmission medium 131 and the second acoustic transmission medium supplied by the supply unit 23 are collectively shown as the acoustic transmission medium 13. The cell stripping device may further include arms 24 for moving the supply unit 23 in the X, Y, and Z axis directions. The culture medium placement section only needs to be recognized by the user as the location for placing culture containers. Figure 6A In the example, the opening defined by the vibrator 101, the cover 15 and the cushioning material 16 is suitable for placing a general-purpose culture dish, and thus the opening corresponds to the culture medium placement section.

[0232] exist Figure 6A In the cell stripping apparatus, an example is shown where the vibrating body 101, serving as the vibration generating unit, includes a piezoelectric element 12 and a vibrating plate 11. The vibrating body 101, including the piezoelectric element 12 and the vibrating plate 11, generates flexural vibrations, wherein the vibrations are amplified by a combination of the stiffness of the piezoelectric element 12, which expands and contracts itself, and the stiffness of the vibrating plate 11, which it is joined to. In this example, resonance caused by structural factors of the vibrating body 101 is used, thereby allowing a large vibration to be obtained at the resonant frequency. The vibrating body may include an oscillator, such as an ultrasonic oscillator. The aforementioned expansion and contraction (for...) is performed by applying a voltage to the piezoelectric element 12 using a power source (not shown). Figure 6B and Figure 6C The same applies).

[0233] exist Figure 6A The diagram shows a circular ring oscillator as a representative of the vibrating body 101, but a Langevin oscillator or a rectangular oscillator capable of producing large amplitudes can also be used.

[0234] To effectively transmit the vibrations generated on the vibrating plate through resonance to the culture medium 21 containing cells, an acoustic transmission medium 13 is inserted between the vibrating plate 11 and the culture medium 21. When the culture medium 21 is placed directly on the vibrating plate 11, the contact is in a point contact state, and energy is dissipated as non-vibrational energy, such as whistling or heating, resulting in reduced efficiency. Essentially, it is preferable to place the acoustic transmission medium 13 in an area sufficient to cover the region used for peeling cells attached to the culture medium 21.

[0235] To fix the vibrating body 101, the following methods are adopted: Figure 6A The structure consists of an upper cover 15, a lower cover 17, a cushioning material 16, and bolts 18 that clamp the end of the vibrator 101, but the pressure used to clamp the end is set to a pressure that allows tightening to a degree that does not suppress vibrations generated in the vibrating plate.

[0236] An example is shown where a weight 14 is provided above the culture medium and below the vibrator as a pressurizing unit. Pressure is applied and released by moving the weight 14 up and down. The pressurizing unit applies pressure to bring the culture medium and the vibrator closer together.

[0237] Figure 6B This is another example view. In Figure 6B In this process, the vibrating plate 11 has a protruding shape, and the excess portion of the second sound transmission medium is more easily discharged without remaining around the sound transmission medium.

[0238] The vibrator is driven by supplying voltage at the resonant frequency to generate the desired ultrasonic vibration mode.

[0239] To drive the vibrator, the cell dissection device can be driven by an AC power supply providing voltage at the resonant frequency. The desired amplitude is obtained by applying a voltage at a frequency that forms the desired vibration waveform on the cell culture surface of the culture medium. At this time, when the voltage of the S-phase (which is the sensor phase provided to the vibrator) is measured using an oscilloscope, the voltage generated by the strain of the vibrator itself can be directly monitored, allowing for simultaneous monitoring of the vibrator's vibration state and driving. Furthermore, when a thermocouple capable of measuring the temperature during the cell dissection step is installed in a position that does not affect the vibration, driving can be performed while monitoring the temperature. An infrared thermometer can also be used for temperature measurement.

[0240] The cell stripping apparatus according to this disclosure also includes an information processing device that can acquire information about the state of vibration to be excited in the vibrator and control the supply unit based on the acquired information.

[0241] Figure 6C This is a conceptual diagram showing a cell stripping device including an information processing unit.

[0242] exist Figure 6C In this process, the information processing device 205 acquires information about the state of the vibration to be excited in the vibrator 101. The information processing device 205 controls the configuration and quantity of the second acoustic transmission medium by controlling the supply unit 23 based on the acquired information, so as to peel off cells under optimal conditions.

[0243] Information processing device 205 includes a central processing unit (CPU) 206, random access memory (RAM) 207, read-only memory (ROM) 208, and hard disk drive (HDD) 209 to realize the functions of a computer performing calculations and storage. Information processing system 205 also includes a communication interface (I / F) 210, a display device 211, and an input device 212. The CPU 206, RAM 207, ROM 208, HDD 209, communication I / F 210, display device 211, and input device 212 are interconnected via bus 213. The display device 211 and input device 212 can be connected to bus 213 via a drive device (not shown) for driving these devices.

[0244] exist Figure 6C In the illustration, the various units forming the information processing system 205 are shown as an integrated device, but some of these functions may be constituted by external devices. For example, the display device 211 and the input device 212 may be external devices that are different from the parts that constitute the functions of a computer including the CPU 206, etc.

[0245] The CPU 206 executes predetermined operations according to programs stored in RAM 207, HDD 209, etc., and also has the function of controlling each unit of the information processing system 205. RAM 207 is formed of volatile storage medium and provides a temporary memory area required for the operation of the CPU 206. ROM 208 is formed of non-volatile storage medium and stores necessary information, such as programs for the operation of the information processing system 205. HDD 209 is formed of non-volatile storage medium and is a storage device for storing, for example, information about the vibration state to be excited in the vibrator 101.

[0246] The communication I / F 210 is a communication interface based on standards such as Wi-Fi (trademark) or 5G, and is a module used to perform communication to / from other devices. The display device 211 is a liquid crystal display, an organic light-emitting diode (OLED) display, etc., and is used to display moving images, still images, text, etc. The input device 212 is a button, touch panel, keyboard, pointing device, etc., and is used by the user to operate the information processing system 205. The display device 211 and the input device 212 can be integrated into a touch panel.

[0247] Figure 6C The hardware configuration shown is illustrated as an example, and additional devices may be added, and some devices may be omitted. Some devices may also be replaced by other devices with similar functionality. Furthermore, some of the functions may be provided via a network by other devices, and the functions constituting the cell stripping device according to this disclosure can be implemented in a distributed manner across multiple devices. For example, HDD 209 may be replaced by a solid-state drive (SSD) using semiconductor elements such as flash memory, or it may be replaced by cloud storage.

[0248] CPU 206 controls the supply unit 23 by loading a program stored in ROM 208, etc., into RAM 207 and executing the program. CPU 206 also controls the display unit 110 by controlling the display device 211. CPU 206 also controls the storage unit 111 by controlling HDD 209.

[0249] The cell stripping method according to this disclosure includes the following first to fourth embodiments. Each embodiment is described below.

[0250] <First Implementation Plan>

[0251] This disclosure provides the following cell stripping method as a first embodiment.

[0252] This embodiment is a cell stripping method for detaching cells from the culture surface attached to the culture medium, and includes:

[0253] The steps of configuring a first sound transmission medium and a second sound transmission medium between the culture medium and the vibrator; and

[0254] The cell stripping step involves vibrating the vibrator to peel off cells from the culture surface while the culture medium is in contact with the first and second acoustic transmission media.

[0255] When implementing the first embodiment of this disclosure, a structure is adopted in which the first acoustic transmission medium and the second acoustic transmission medium are in contact with the culture medium side.

[0256] The placement of the second sound transmission medium can be changed according to the shape of the bottom surface of the culture medium and the surface shape of the first sound transmission medium.

[0257] Figures 1A to 1C A structural view of the acoustic transmission medium used in the cell stripping method as a first embodiment of this disclosure is shown.

[0258] Figure 1AIt is a view of the configuration of a second acoustic transmission medium 132 in a combination of a culture medium 21 having a flat substrate bottom and a first acoustic transmission medium 131 having a raised central portion.

[0259] Figure 1B It is a view of the configuration of the second acoustic transmission medium 132 in a combination of a general-purpose culture medium 21 with a warped substrate bottom and a flat first acoustic transmission medium 131.

[0260] Figure 1C It is a view of the configuration of the second acoustic transmission medium 132 in a combination of a general-purpose culture medium 21 with a warped substrate bottom and a flat first acoustic transmission medium 131.

[0261] As mentioned above, the culture medium 21 varies considerably between models from different manufacturers and between production batches. In contrast, in this embodiment, the second acoustic transmission medium 132 is disposed on the side of the culture medium 21, and the first acoustic transmission medium 131 and the second acoustic transmission medium 132 complement each other to achieve close contact. Therefore, even when such variations exist in the culture medium, it is easy to apply appropriate vibrations accordingly.

[0262] In this implementation plan, such as Figures 1A to 1C As shown, the first acoustic transmission medium 131 and the second acoustic transmission medium 132 are preferably in contact with the culture medium 21 side, and the vibrator 101 is in contact with the first acoustic transmission medium 131.

[0263] Figure 5B This is a flowchart illustrating examples of more detailed steps in an embodiment of this disclosure.

[0264] First, as a configuration step, a first sound transmission medium is placed on the vibrator (step S210).

[0265] Subsequently, a second sound transmission medium is placed on top of the first sound transmission medium (step S220).

[0266] Next, as a pressurization step, the first and second acoustic transmission media are brought into contact with the culture medium (step S230).

[0267] Subsequently, pressure is applied from above the culture medium using weights or the like (step S240).

[0268] As a stripping step, ultrasonic application conditions are set (step S250).

[0269] Next, ultrasound application begins (step S260).

[0270] Finally, collect the detached cells (step S270).

[0271] <Second Implementation Plan>

[0272] This disclosure provides the following cell stripping method as a second embodiment.

[0273] This embodiment is a cell stripping method for detaching cells from the culture surface attached to the culture medium, and includes:

[0274] The steps of configuring a first sound transmission medium and a second sound transmission medium between the culture medium and the vibrator; and

[0275] The cell stripping step involves vibrating the vibrator to peel off cells from the culture surface while the culture medium is in contact with the first and second acoustic transmission media.

[0276] When implementing the second embodiment of this disclosure, a structure is adopted in which the first sound transmission medium and the second sound transmission medium are in contact with the vibrating body.

[0277] The placement of the second sound transmission medium can be changed according to the shape of the bottom surface of the culture medium, the shape of the upper surface of the vibrator, and the surface shape of the first sound transmission medium.

[0278] Figures 2A to 2C A structural view of the acoustic transmission medium used in the cell stripping method as a second embodiment of this disclosure is shown.

[0279] Figure 2A It is a view of the configuration of a second acoustic transmission medium 132 in a combination of a culture medium 21 having a flat substrate bottom surface, a vibrator having a flat upper surface, and a first acoustic transmission medium 131 having a downwardly convex central portion.

[0280] Figure 2B It is a view of the configuration of a second acoustic transmission medium 132 in a combination of a general-purpose culture medium 21 with a warped substrate bottom, a flat vibrator, and a first acoustic transmission medium 131 with an upper and lower convex center.

[0281] Figure 2C It is a view of the configuration of a second acoustic transmission medium 132 in a combination of a general-purpose culture medium 21 with a warped substrate bottom, a flat vibrator, and a first acoustic transmission medium 131 with a convex upper and concave lower center.

[0282] In this implementation plan, such as Figures 2A to 2C As shown, the first sound transmission medium 131 and the second sound transmission medium 132 are preferably in contact with the vibrator, and the culture medium 21 is in contact with the first sound transmission medium.

[0283] <Third Implementation Plan>

[0284] This disclosure provides the following cell stripping method as a third embodiment.

[0285] This embodiment is a cell stripping method for detaching cells from the culture surface attached to the culture medium, and includes:

[0286] The steps of configuring a first sound transmission medium and a second sound transmission medium between the culture medium and the vibrator; and

[0287] In the state where the culture medium is in contact with the first and second acoustic transmission media and the vibrator is in contact with the first and second acoustic transmission media, a peeling step is performed by vibrating the vibrator to peel off the cells from the culture surface.

[0288] When implementing the third embodiment of this disclosure, a structure is adopted in which the first sound transmission medium and the second sound transmission medium are in contact with both the culture medium and the vibrator.

[0289] The placement of the second sound transmission medium can be changed according to the shape of the bottom surface of the culture medium and the surface shape of the first sound transmission medium.

[0290] Figures 3A to 3C A structural view of the acoustic transmission medium used in the cell stripping method as a third embodiment of this disclosure is shown.

[0291] Figure 3A It is a view of the configuration of a second acoustic transmission medium 132 in a combination of a culture medium 21 having a flat substrate bottom surface and a first acoustic transmission medium 131 having an upper and lower convex central portion.

[0292] Figure 3B It is a view of the configuration of a second acoustic transmission medium 132 in a combination of a general-purpose culture medium 21 having a warped substrate bottom surface and a first acoustic transmission medium 131 having an upper and lower convex central portion.

[0293] Figure 3C It is a view of the configuration of a second acoustic transmission medium 132 in a combination of a general-purpose culture medium 21 having a warped substrate bottom surface and a first acoustic transmission medium 131 having a convex upper and concave lower central portion.

[0294] <Fourth Implementation Plan>

[0295] This disclosure provides the following cell stripping method as a fourth embodiment.

[0296] This embodiment is a cell stripping method for detaching cells from the culture surface attached to the culture medium, and includes:

[0297] The steps of configuring a first sound transmission medium and a second sound transmission medium between the culture medium and the vibrator; and

[0298] In the case where the culture medium is in contact with the first and second acoustic transmission media, the vibrator is in contact with the first and second acoustic transmission media, and the second acoustic transmission media are arranged in a vertically continuous manner, a cell stripping step is performed by vibrating the vibrator to peel off the cells from the culture surface.

[0299] When implementing the fourth embodiment of this disclosure, a structure is adopted in which the first sound transmission medium and the second sound transmission medium are in contact with both the culture medium and the vibrator.

[0300] The placement of the second sound transmission medium can be changed according to the shape of the bottom surface of the culture medium and the surface shape of the first sound transmission medium.

[0301] Figures 4A to 4C The diagram shows a structural view of the acoustic transmission medium used in the cell stripping method as a fourth embodiment of this disclosure.

[0302] Figure 4A It is a view of the configuration of a culture medium 21 having a flat substrate bottom surface and a second acoustic transmission medium 132 in a combination of a first acoustic transmission medium 131 divided into a central portion and an outer peripheral portion, both of which are flat.

[0303] Figure 4B It is a view of the configuration of a general-purpose culture medium 21 having a warped substrate bottom surface and a second acoustic transmission medium 132 in a combination of a first acoustic transmission medium 131 divided into a central portion and an outer peripheral portion, the central portion being flat and the outer peripheral portion tapering towards the outer edge.

[0304] Figure 4C This is a view showing the configuration of a general-purpose culture medium 21 with a warped substrate bottom and a second acoustic transmission medium 132 in a combination of a first acoustic transmission medium 131 divided into a central portion and an outer peripheral portion. The central portion is flat and has a warped substrate bottom. Figure 4B Different diameters, the outer periphery decreases towards the outer edge and has the same Figure 4B Different widths, and top views of the configured images.

[0305] When the antinodes in the vibration pattern of the vibrator appear near the center of the vibrator, effective peeling can be achieved, as in this embodiment, by providing a first acoustic transmission medium 131 in the center and ensuring close contact between the culture medium and the acoustic transmission medium.

[0306] Example

[0307] (Example 1)

[0308] Examples 1 to 28, 32 and 33 correspond to the first implementation scheme.

[0309] In Example 1, using Figure 1A The schematic diagram illustrates the construction of the acoustic transmission medium. A Nunc (trademark) Φ35-mm culture dish manufactured by Thermo Fisher Scientific Inc. was used as the culture medium. A glass plate with an outer diameter of 70 mm and a thickness of 4 mm was used as the vibrating plate of the cell dissection device. For the piezoelectric element, PZT was used as the material composition, and considering that the neutral plane was on the vibrating plate side, the outer diameter of the piezoelectric element was set to 70 mm (the same as the outer diameter of the vibrating plate), the inner diameter to be 57 mm, and the thickness to be 2 mm. The construction of the cell dissection device is as follows... Figure 6A As shown, a disk-shaped silicone rubber with a hardness of 30° and a central protrusion (e.g., 0.9 mm center height (1), 0.4 mm side height (2), and 32 mm diameter (3) is used. Figure 9A The silicone rubber (as shown in the schematic diagram) is placed on the vibrating plate as the first sound transmission medium, with its center aligned with the center of the vibrating plate. The silicone rubber is set as the first sound transmission medium. The tanδ of this silicone rubber, obtained by dynamic viscoelasticity measurement at room temperature when vibration at a frequency of 1 Hz is applied, is 0.13. Water, used as the second sound transmission medium, is dripped in 200 μL onto the center of the first sound transmission medium using a dispenser. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. is slowly placed on the vibrating plate, aligning the centers of both. Afterward, a 100g weight is placed on the lid of the culture medium to apply pressure. A schematic diagram illustrating the construction of the sound transmission medium after pressure application is shown. Figure 1B Regarding the contact areas of the first and second sound transmission media with the culture medium, water, used as the second sound transmission medium, was colored with food coloring. After applying pressure, photographs taken from above confirmed the absence of an air layer between the sound transmission media and the culture medium. The respective contact areas of the first and second sound transmission media were then calculated. The contact area ratio of the first sound transmission medium was 10%. Furthermore, the contact angle between the first and second sound transmission media was 110°.

[0310] For cell culture, at 10,000 cells / cm² 2Chinese hamster ovary (CHO) cells were seeded at a density suitable for incubation in Nunc (trademark) Φ35-mm culture dishes manufactured by Thermo Fisher Scientific Inc., and cultured at 37°C and 5% CO2. The culture medium used was Ham's F12 (manufactured by Thermo Fisher Scientific Inc.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10000 U / ml; manufactured by Thermo Fisher Scientific Inc.). After 48 hours of culture, cell morphology was observed using a phase-contrast microscope to examine cell attachment and proliferation. The cell occupancy of the culture dish was approximately 80%.

[0311] (Cell peeling)

[0312] Remove the culture medium from the culture dish, wash the cells with PBS(-), and then immerse them in PBS(-) as a stripping solution for 3 minutes.

[0313] Subsequently, the culture medium was placed in a cell stripping device at 25°C, and cell stripping was performed for 2 minutes at an ambient temperature of 25°C using a standing wave mode with swept-frequency vibration (frequency from 22 kHz to 27 kHz, sweep period of 1 second, voltage of 90 V). In this case, by relative to... Figure 10 The positive electrodes (A1, A3, B1, and B3) in the electrode array apply an alternating voltage with a 180-degree phase difference to the negative electrodes (A2 and B2) to generate standing waves. The vibration modes caused by this driving force are described later. Figure 12 This is referred to as "Vibration Mode A".

[0314] After collecting the detached cells, all cells that failed to be detached by ultrasound were removed from the culture dish after ultrasonic detachment using a cell scraper, and the cell count was measured using a hemocytometer. The total number of detached cells was defined as the sum of the number of cells detached by ultrasound and the number of cells detached by the cell scraper after ultrasonic detachment, and the detachment rate was defined as the ratio of the number of cells detached by ultrasound to the total number of detached cells, thus calculating the detachment rate value. For deviations, the detachment rate was determined with n=10, and the standard deviation of the detachment rate was evaluated: less than 5% was rated as A, more than 5% but less than 7.5% as B, more than 7.5% but less than 10% as C, and more than 10% as D. The evaluation result in Example 1 was a detachment rate of 97.4% and a deviation rating of A.

[0315] (Example 2)

[0316] Example 2 was carried out under the same conditions as Example 1, except that the first sound transmission medium was changed as described below and the amount of the second sound transmission medium was changed as described below. That is, a disk-shaped silicone rubber with a hardness of 30° (e.g., with a center height (1) = side height (2) of 0.9 mm and a diameter (3) of 32 mm) was used. Figure 9B (As shown in the schematic diagram) is used as the first sound transmission medium on the vibrating plate. Water is placed on the first sound transmission medium by uniformly spraying it at a rate of 70 μL using a spraying device, serving as the second sound transmission medium. The contact area ratio of the first sound transmission medium is 20%.

[0317] The evaluation results in Example 2 showed a stripping rate of 90.3% and a deviation rating of C.

[0318] (Example 3)

[0319] Example 3 was performed under the same conditions as Example 1, except that the cell stripping conditions were modified as described below. Cell stripping was performed for 2 minutes in standing wave mode under cell stripping conditions involving swept-frequency vibration (frequency from 67.5 kHz to 72.5 kHz, sweep period of 1 second, and voltage of 90 V). Although the frequency band differed from that of Example 1, a standing wave was generated under the same conditions to apply an AC voltage with a 180-degree phase difference to the negative electrodes (A2 and B2) relative to the positive electrodes (A1, A3, B1, and B3). The vibration mode caused by this drive is described later. Figure 12 This is referred to as "Vibration Mode B".

[0320] The evaluation results in Example 3 showed a stripping rate of 90.0% and a deviation rating of C.

[0321] (Example 4)

[0322] Example 4 was performed under the same conditions as Example 2, except that the cell stripping conditions were modified as described below. Cell stripping was performed for 2 minutes in standing wave mode under cell stripping conditions involving swept-frequency vibration (frequency from 67.5 kHz to 72.5 kHz, sweep period of 1 second, and voltage of 90 V). The evaluation result in Example 4 was a stripping rate of 96.8% and a deviation rating of A.

[0323] (Example 5)

[0324] Example 5 was carried out under the same conditions as Example 1, except that the first sound transmission medium was changed as described below. That is, the center height (1) was changed to 0.9 mm and the upper surface height (1) was changed to 0.9 mm. The side height (2) is 0.3 mm, the diameter (3) is 32 mm, and the inner diameter (3) is 0.3 mm. The silicone rubber with a hardness of 30° is used to modify the shape of the central raised disc by 5 mm. Figure 9C (As shown in the schematic diagram) is used as the first sound transmission medium on the vibrating plate. Water, which is used as the second sound transmission medium, is dripped onto the first sound transmission medium in a volume of 250 μL using a dispenser. The contact area ratio of the first sound transmission medium is 5%.

[0325] The evaluation results in Example 5 showed a stripping rate of 90.2% and a deviation rating of B.

[0326] (Example 6)

[0327] Example 6 was carried out under the same conditions as Example 1, except that the first sound transmission medium was changed as described below. That is, the center height (1) was changed to 0.9 mm, the side height (2) to 0.7 mm, and the side height (2) to 0.7 mm. It is 0.4 mm, the diameter (3) is 32 mm and the inner diameter (3) is 0.4 mm. Silicone rubber with a hardness of 30° and a 20 mm diameter disc shape (e.g.) Figure 9D (As shown in the schematic diagram) is used as the first sound transmission medium on the vibrating plate. Water, which will be used as the second sound transmission medium, is dripped onto the first sound transmission medium in a volume of 200 μL using a dispenser. The contact area ratio of the first sound transmission medium is 30%.

[0328] The evaluation results in Example 6 showed a stripping rate of 98.2% and a deviation rating of A.

[0329] (Example 7)

[0330] Example 7 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to PVA gel and the amount was changed.

[0331] PVA gel was prepared by the following method. Denka Poval (trademark) K-24E (manufactured by Denka Company Limited) was weighed to achieve a concentration of 3.7% by mass, and pure water was added to prepare a mixture with a total volume of 80 mL. The mixture was heated and stirred in a constant temperature chamber until the PVA was completely dissolved, yielding a PVA aqueous solution. Next, borax (sodium tetraborate decahydrate (premium grade), manufactured by Kishida Chemical Co., Ltd.) was weighed, and pure water was added to prepare an aqueous solution with a concentration of 1.0% by mass and a total volume of 16 mL. While stirring the PVA aqueous solution, the borax aqueous solution was gradually added dropwise, and the mixture was stirred until homogeneity was achieved, thus obtaining a PVA gel. When vibration at a frequency of 0.1 Hz was applied to the PVA gel, the tanδ obtained by dynamic viscoelasticity measurement at room temperature was 1.7, indicating that the PVA gel is a flowable solid. When vibration at a frequency of 1 Hz was applied, the tanδ obtained by dynamic viscoelasticity measurement at room temperature was 0.856.

[0332] 0.2 g of PVA gel was weighed and used as a second acoustic transmission medium, thinly spread into a 25 mm diameter disc, and placed on the first acoustic transmission medium. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. was slowly placed on the vibrating plate, aligning the centers of both. Afterward, a 100 g weight was placed on the lid of the culture medium to apply pressure.

[0333] The contact area ratio of the first sound transmission medium is 10%.

[0334] The evaluation results in Example 7 showed a stripping rate of 85.7% and a deviation rating of C.

[0335] (Example 8)

[0336] Example 8 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to an ultrasonic gel and the amount was changed.

[0337] As the second acoustic transmission medium, 0.2 g of ultrasonic gel (Pro Jelly for Ultrasonic Examination, Normal Type; manufactured by JEX Co., Ltd.) was weighed and set up by evenly spreading it within a 20 mm radius of the center of the first acoustic transmission medium. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells on it), manufactured by Thermo Fisher Scientific Inc., was slowly placed on the vibrating plate, aligning the centers of both. Afterward, a 100 g weight was placed on the lid of the culture medium to apply pressure.

[0338] The contact area ratio of the first sound transmission medium is 10%.

[0339] The evaluation results in Example 8 showed a stripping rate of 96.4% and a deviation rating of A.

[0340] (Example 9)

[0341] Example 9 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to glycerol and the amount was changed.

[0342] 0.27 g of glycerol (extra grade glycerol; manufactured by Kishida Chemical Co., Ltd.) was weighed as the second sound transmission medium and set up by spreading it evenly on the first sound transmission medium. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by ThermoFisher Scientific Inc. was slowly placed on the vibrating plate, ensuring the centers of both were aligned. Subsequently, a 100 g weight was placed on the lid of the culture medium to apply pressure.

[0343] The contact area ratio of the first sound transmission medium is 10%. Furthermore, the contact angle between the first and second sound transmission media is 100°.

[0344] The evaluation results in Example 9 showed a stripping rate of 94.3% and a deviation rating of A.

[0345] (Example 10)

[0346] Example 10 was carried out under the same conditions as Example 1, except that the first sound transmission medium was changed as described below. That is, as Figure 9AAs shown in the schematic diagram, a silicone rubber with a hardness of 30° and a centrally protruding disc shape, having a center height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm, is used as the first sound transmission medium on the vibrating plate. Twelve straight grooves, each 0.3 mm wide and 0.2 mm deep, are radially distributed from the center at 30° intervals on the surface of the substrate side of the silicone rubber. Water, used as the second sound transmission medium, is dripped onto the first sound transmission medium at a rate of 1400 μL using a dispenser. The contact area ratio of the first sound transmission medium is 10%.

[0347] The evaluation results in Example 10 showed a stripping rate of 92.1% and a deviation rating of A.

[0348] (Example 11)

[0349] Example 11 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to ethanol and the amount was changed.

[0350] Ethanol (premium ethanol; manufactured by Kishida Chemical Co., Ltd.) used as the second acoustic transmission medium was prepared by dripping 1400 μL onto the center of the first acoustic transmission medium using a dispenser. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by ThermoFisher Scientific Inc. was slowly placed on the vibrating plate, ensuring the centers of both were aligned. Subsequently, a 100 g weight was placed on the lid of the culture medium to apply pressure.

[0351] The contact area ratio of the first sound transmission medium is 10%. Furthermore, the contact angle between the first and second sound transmission media is 40°.

[0352] The evaluation results in Example 11 showed a stripping rate of 94.0% and a deviation rating of A.

[0353] (Example 12)

[0354] Example 12 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to a 70% aqueous ethanol solution and the amount was changed.

[0355] A 70% ethanol-water solution, obtained by mixing ethanol (premium ethanol; manufactured by Kishida Chemical Co., Ltd.) and pure water at a volume ratio of 70:30, was used as the second acoustic transmission medium. The obtained 70% ethanol-water solution was prepared by dripping 1400 μL dropwise onto the center of the first acoustic transmission medium using a dispenser. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. was slowly placed on the vibrating plate, ensuring the centers of both were aligned. Subsequently, a 100g weight was placed on the lid of the culture medium to apply pressure.

[0356] The contact area ratio of the first sound transmission medium is 10%. Furthermore, the contact angle between the first and second sound transmission media is 55°.

[0357] The evaluation results in Example 12 showed a stripping rate of 94.6% and a deviation rating of A.

[0358] (Example 13)

[0359] Example 13 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to an aqueous solution of surfactant 1 (water + surfactant 1) and the amount was changed.

[0360] An aqueous surfactant solution, serving as the second acoustic transmission medium, was prepared by mixing sodium lauryl sulfate (grade 1 sodium lauryl sulfate; manufactured by Kishida Chemical Co., Ltd.) in pure water to a concentration of 10 mmol / L. The obtained surfactant aqueous solution 1 (sodium lauryl sulfate aqueous solution) was added dropwise in 1400 μL amounts onto the center of the first acoustic transmission medium using a dispenser. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. was slowly placed on a vibrating plate, ensuring the centers of both were aligned. Subsequently, a 100g weight was placed on the lid of the culture medium to apply pressure.

[0361] The contact area ratio of the first sound transmission medium is 10%. Furthermore, the contact angle between the first and second sound transmission media is 50°.

[0362] The evaluation results in Example 13 showed a stripping rate of 93.9% and a deviation rating of A.

[0363] (Example 14)

[0364] Example 14 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to an aqueous solution of surfactant 2 (water + surfactant 2) and the amount was changed.

[0365] An aqueous solution of surfactant, used as the second acoustic transmission medium, was prepared by mixing Pluronic F-68 (Pluronic F-68 Non-ionic Surfactant (100X); manufactured by Thermo Fisher Scientific Inc.) in pure water to prepare a solution with a solids content of 0.1% by mass. The obtained surfactant aqueous solution 2 was then added dropwise in 1400 μL amounts onto the center of the first acoustic transmission medium using a dispenser. Next, a Nunc Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. was slowly placed on a vibrating plate, aligning the centers of both. Subsequently, a 100g weight was placed on the lid of the culture medium to apply pressure.

[0366] The contact area ratio of the first sound transmission medium is 10%. Furthermore, the contact angle between the first and second sound transmission media is 75°.

[0367] The evaluation results in Example 14 showed a stripping rate of 93.2% and a deviation rating of A.

[0368] (Example 15)

[0369] Example 15 was carried out under the same conditions as Example 1, except that the first sound transmission medium was changed as described below. That is, the center height (1) of Example 1 was changed to 0.9 mm, the side height (2) to 0.4 mm, and the diameter (3) to 32 mm. Figure 9A A silicone rubber with a hardness of 30°, shaped like a central convex disc (as shown in the schematic diagram), is used as the first sound transmission medium on the vibrating plate. This silicone rubber undergoes a hydrophilic treatment on its convex surface. A water droplet, which serves as the second sound transmission medium, is added to the hydrophilicated silicone rubber surface, and the contact angle is measured to be 25°.

[0370] Water, serving as the second sound transmission medium, is added dropwise in a volume of 1400 μL to the center of the first sound transmission medium using a dispenser. The contact area ratio of the first sound transmission medium is 10%.

[0371] The evaluation result of Example 15 was a stripping rate of 95.5% and a deviation rating of A.

[0372] (Example 16)

[0373] Example 16 was carried out under the same conditions as Example 1, except that by... Figure 6A The vibrating plate 11 in the device construction diagram is changed to glass with a hydrophilic treatment on the upper surface for assembly.

[0374] Water was used as a second sound transmission medium and dropped onto a glass surface that had undergone hydrophilic treatment. The contact angle was measured to be 20°.

[0375] As the second sound transmission medium, water was placed on the center of the first sound transmission medium by dripping it in a volume of 1400 μL using a dispenser. The contact area ratio of the first sound transmission medium was 10%.

[0376] The evaluation results in Example 16 showed a stripping rate of 95.1% and a deviation rating of A.

[0377] (Example 17)

[0378] Example 17 was carried out under the same conditions as Example 1, except that... Figure 6A The culture medium 21 in the device construction diagram is changed to a culture medium that has undergone hydrophilic treatment on the outer surface including the side surface and the bottom surface.

[0379] Water was used as a second sound transmission medium and dropped onto the bottom surface of the culture medium that had undergone hydrophilic treatment. The contact angle was measured to be 25°.

[0380] Water was placed on the center of the first sound transmission medium in a 1400 μL droplet using a dispenser as the second sound transmission medium. The contact area ratio of the first sound transmission medium was 10%.

[0381] The evaluation results in Example 17 showed a stripping rate of 94.8% and a deviation rating of A.

[0382] (Example 18)

[0383] Example 18 was carried out under the same conditions as Example 1, except that the vibrating plate 11 was changed to Figure 6B The convex glass in the device construction diagram is used for assembly.

[0384] The convex glass used is obtained by joining a glass with an outer diameter of 32 mm and a thickness of 2 mm with a glass with an outer diameter of 70 mm and a thickness of 4 mm.

[0385] Water was placed on the center of the first sound transmission medium in a 1400 μL droplet using a dispenser as the second sound transmission medium. The contact area ratio of the first sound transmission medium was 10%.

[0386] The evaluation results in Example 18 showed a stripping rate of 95.8% and a deviation rating of A.

[0387] (Example 19)

[0388] Example 19 was carried out under the same conditions as Example 18, except that the second sound transmission medium was changed to a 70% aqueous ethanol solution.

[0389] In other words, as the second sound transmission medium, a 70% ethanol aqueous solution was placed on the center of the first sound transmission medium by dripping 1400 μL of the solution onto it using a dispenser. The contact area ratio of the first sound transmission medium was 10%.

[0390] The evaluation results in Example 19 showed a stripping rate of 97.4% and a deviation rating of A.

[0391] (Example 20)

[0392] Example 20 was carried out under the same conditions as Example 1, except that the second sound transmission medium was changed to a 70% aqueous ethanol solution, and the amount used was also changed.

[0393] That is, the same 70% ethanol aqueous solution used in Example 12 was used as the second sound transmission medium. The 70% ethanol aqueous solution was placed by dripping 1400 μL of liquid onto the center of the first sound transmission medium using a dispenser. The contact area ratio of the first sound transmission medium was 10%.

[0394] The evaluation results in Example 20 showed a stripping rate of 97.1% and a deviation rating of A.

[0395] (Example 21)

[0396] Example 21 was carried out under the same conditions as Example 1, except that the amount of water used as the second sound transmission medium was changed.

[0397] In other words, water is placed on the center of the first sound transmission medium in a 1200 μL droplet using a dispenser, serving as the second sound transmission medium. The contact area ratio of the first sound transmission medium is 10%.

[0398] The evaluation results in Example 21 showed a stripping rate of 91.1% and a deviation rating of B.

[0399] (Example 22)

[0400] Example 22 was carried out under the same conditions as Example 2, except that the amount of water used as the second sound transmission medium was changed.

[0401] In other words, water is placed on the first sound transmission medium by uniformly spraying it at a rate of 40 μL using a spray device, serving as the second sound transmission medium. The contact area of ​​the first sound transmission medium is 20%.

[0402] The evaluation results in Example 22 showed a stripping rate of 90.2% and a deviation rating of C.

[0403] (Example 23)

[0404] Example 23 was carried out under the same conditions as Example 1, except that the amount of water used as the second sound transmission medium was changed.

[0405] In other words, water is placed on the center of the first sound transmission medium in a volume of 1400 μL using a dispenser, serving as the second sound transmission medium. The contact area ratio of the first sound transmission medium is 10%.

[0406] The evaluation results in Example 23 showed a stripping rate of 90.4% and a deviation rating of B.

[0407] (Example 24)

[0408] Example 24 was carried out under the same conditions as Example 2, except that the amount of water used as the second sound transmission medium was changed.

[0409] In other words, water was placed on the first sound transmission medium by uniformly spraying it at a rate of 35 μL using a spray device as the second sound transmission medium. The contact area of ​​the first sound transmission medium was 20%. It was also confirmed that the second sound transmission medium did not spread over the entire surface, and that 10% of the total area was an air layer.

[0410] The evaluation results in Example 24 showed a stripping rate of 84.2% and a deviation rating of C.

[0411] (Example 25)

[0412] Example 25 was carried out under the same conditions as Example 5, except that the amount of water used as the second sound transmission medium was changed.

[0413] In other words, water was placed on the first sound transmission medium by uniformly spraying it at a rate of 25 μL using a spray device as the second sound transmission medium. The contact area of ​​the first sound transmission medium was 10%. It was also confirmed that the second sound transmission medium did not spread over the entire surface, and that 20% of the total area was an air layer.

[0414] The evaluation results in Example 25 showed a stripping rate of 83.5% and a deviation rating of C.

[0415] (Example 26)

[0416] Example 26 was carried out under the same conditions as Example 1, except that the center height (1) used in Example 1 was 0.9 mm, the side height (2) was 0.4 mm, and the diameter (3) was 32 mm. Figure 9A A polyurethane rubber in the shape of a central raised disk (as shown in the schematic diagram) is used as the first sound transmission medium on the vibrating plate. The tanδ of this polyurethane rubber, obtained by viscoelastic measurement at room temperature when subjected to vibration at a frequency of 1 Hz, is 0.23.

[0417] Water was placed on the center of the first sound transmission medium in a 200 μL droplet using a dispenser as the second sound transmission medium. The contact area ratio of the first sound transmission medium was 10%. Furthermore, the contact angle between the first and second sound transmission media was 80°.

[0418] The evaluation results in Example 26 showed a stripping rate of 90.1% and a deviation rating of B.

[0419] (Example 27)

[0420] Example 27 was carried out under the same conditions as Example 1, except that the center height (1) used in Example 1 was 0.9 mm, the side height (2) was 0.4 mm, and the diameter (3) was 32 mm. Figure 9A A silicone rubber with a hardness of 10° and a central convex disc shape (as shown in the schematic diagram) is used as the first sound transmission medium on the vibrating plate. The tanδ of this silicone rubber, obtained by viscoelastic measurement at room temperature when vibration at a frequency of 1 Hz is applied to it, is 0.14.

[0421] Water was placed on the center of the first sound transmission medium in a 200 μL droplet using a dispenser as the second sound transmission medium. The contact area ratio of the first sound transmission medium was 10%. Furthermore, the contact angle between the first and second sound transmission media was 110°.

[0422] The evaluation results in Example 27 showed a stripping rate of 93.3% and a deviation rating of B.

[0423] (Example 28)

[0424] Example 28 was carried out under the same conditions as Example 1, except that the center height (1) used in Example 1 was 0.9 mm, the side height (2) was 0.4 mm, and the diameter (3) was 32 mm. Figure 9A A silicone rubber with a hardness of 50°, shaped like a central convex disk (as shown in the schematic diagram), is used as the first sound transmission medium on the vibrating plate. The tanδ of this silicone rubber, obtained by viscoelastic measurement at room temperature when vibration is applied at a frequency of 1 Hz, is 0.05.

[0425] Water was placed on the center of the first sound transmission medium in a 200 μL droplet using a dispenser as the second sound transmission medium. The contact area ratio of the first sound transmission medium was 10%. Furthermore, the contact angle between the first and second sound transmission media was 110°.

[0426] The evaluation results in Example 28 showed a stripping rate of 96.4% and a deviation rating of A.

[0427] (Example 29)

[0428] Example 29 corresponds to the second implementation scheme.

[0429] Example 29 was carried out under the same conditions as Example 1, except that the steps during the placement period were changed and the following method was used. Figure 2A The diagram shows the structure of the acoustic transmission medium.

[0430] Nunc (trademark) Φ35-mm culture dishes, manufactured by Thermo Fisher Scientific Inc. and used as culture medium for culturing cells, are brought into contact from their bottom or back surface with the convex surface of a first sound transmission medium similar to that in Example 1, ensuring center alignment and that the entire surface is in close contact with each other to prevent the introduction of an air layer. Next, using a dispenser, 200 μL of water, used as a second sound transmission medium, is dripped onto the center of the vibrating plate, and the Φ35-mm culture dish, with its bottom surface in close contact with the first sound transmission medium, is slowly placed on the vibrating plate, ensuring center alignment. Subsequently, a 100g weight is placed on the lid of the culture medium to apply pressure. A schematic diagram illustrating the construction of the sound transmission medium after pressure application is shown below. Figure 2C Regarding the contact areas of the first and second sound transmission media with the culture medium, water, used as the second sound transmission medium, was colored with food coloring. Photographs taken from below the vibrating plate after pressure was applied confirmed the absence of an air layer between the sound transmission media and the culture medium. The respective contact areas of the first and second sound transmission media were then calculated. The contact area ratio of the first sound transmission medium was 10%.

[0431] Cell stripping conditions and cell stripping examination were performed using the same method as in Example 1.

[0432] The evaluation results in Example 29 showed a stripping rate of 97.2% and a deviation rating of A.

[0433] (Example 30)

[0434] Example 30 corresponds to the third implementation scheme.

[0435] In Example 30, a Φ35-mm petri dish manufactured by Corning Incorporated was used as the culture medium. Silicone rubber with a hardness of 10° and a center height (1) of 1.0mm, a side height (2) of 0.3mm, a diameter (3) of 32mm, a top surface height (4) of 0.4mm, and a bottom surface height (5) of 0.3mm was prepared as the first sound transmission medium. Figure 9E The schematic diagram is shown. Assemble the cell dissection device, and use a dispenser to drop 100 μL of water, used as the second sound transmission medium, onto the center of the vibrating plate. Next, slowly place the silicone rubber, used as the first sound transmission medium, onto the vibrating plate, aligning the lower surface of the silicone rubber with the center of the vibrating plate. Furthermore, use a dispenser to drop 150 μL of water, used as the second sound transmission medium, onto the center of the surface of the first sound transmission medium. Slowly place a Φ35-mm culture dish, manufactured by Corning Incorporated, as the culture medium for culturing cells, onto the vibrating plate, aligning the centers of both. Then, place a 100g weight on the lid of the culture medium to apply pressure. The schematic diagram illustrating the construction of the sound transmission medium after pressure application is shown below. Figure 3B Regarding the contact area between the first and second sound transmission media and the culture medium, water, used as the second sound transmission medium, was colored with food coloring. Photographs taken from above and below after pressure was applied confirmed that there was no air layer between the sound transmission media and the culture medium. Then, the contact areas of the upper and lower surfaces of the first and second sound transmission media were calculated. The contact area ratio of the first sound transmission medium was 10% on the upper surface and 50% on the lower surface.

[0436] Cell stripping conditions and cell stripping examination were performed using the same method as in Example 1.

[0437] The evaluation results in Example 30 showed a stripping rate of 94.4% and a deviation rating of B.

[0438] (Example 31)

[0439] Example 31 corresponds to the fourth implementation scheme.

[0440] In Example 31, a ΦIWAKI 35-mm culture dish manufactured by AGC Techno Glass Co., Ltd. was used as the culture medium. Figure 9F As shown in the schematic diagram, a circle with a thickness (6) of 0.6 mm and a diameter (7) of 3.0 mm is used. 0.6 mm, width (8 It is 4.0mm and the diameter is (7 Silicone rubber with a hardness of 10° was prepared as the first sound transmission medium for a 32mm ring. Figure 4C The cell stripping device is constructed in the same way as described above, and circular and annular silicone rubbers, which serve as the first sound transmission medium, are placed on the center of the vibrating plate.

[0441] In addition, 400 μL of water, used as the second sound transmission medium, was dripped onto the center of the surface of the first sound transmission medium using a dispenser. A ΦIWAKI Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by AGC Techno Glass Co., Ltd. was slowly placed on the vibrating plate, aligning the centers of both. Afterward, a 100g weight was placed on the lid of the culture medium to apply pressure. A schematic diagram illustrating the construction of the sound transmission medium after pressure application is shown below. Figure 4C Regarding the contact area between the first sound transmission medium and the culture medium, food coloring was used to color the water used as the second sound transmission medium. A photograph taken from above after pressure was applied confirmed that there was no air layer between the sound transmission medium and the culture medium. The contact area of ​​the upper surface of the first sound transmission medium was then calculated. The contact area of ​​the first sound transmission medium accounted for 30% of the total.

[0442] Cell stripping conditions and cell stripping examination were performed using the same method as in Example 1.

[0443] The evaluation results in Example 31 showed a stripping rate of 92.3% and a deviation rating of C.

[0444] (Example 32)

[0445] Example 32 was performed under the same conditions as Example 1, except that the cell stripping conditions were modified as described below. Cell stripping was performed for 2 minutes in the traveling wave mode described above under cell stripping conditions involving swept-frequency vibration (frequency from 67.5 kHz to 72.5 kHz, sweep period of 1 second, and voltage of 90 V). Figure 10 An alternating voltage with a 90-degree phase difference between phases A and B generates a two-phase driven traveling wave. The vibration modes caused by this drive are described later. Figure 12 This is referred to as "vibration mode C".

[0446] The evaluation results in Example 32 showed a stripping rate of 99.4% and a deviation rating of A.

[0447] (Example 33)

[0448] Example 33 was carried out under the same conditions as Example 1, except that the pressurization step of placing a pressurizing weight in Example 1 was omitted, and the amount of water used as the second sound transmission medium was reduced.

[0449] That is, after placing the same first acoustic transmission medium as in Example 1 with its center aligned with the center of the vibrating plate, water, used as the second acoustic transmission medium, is dripped in a volume of 160 μL onto the center of the first acoustic transmission medium using a dispenser. A Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. is slowly placed on the vibrating plate, ensuring that the centers of both are aligned. The contact area ratio of the first acoustic transmission medium is 3%.

[0450] The evaluation results in Example 33 showed a stripping rate of 84.1% and a deviation rating of C.

[0451] (Comparative Example)

[0452] (Comparative Example 1)

[0453] Comparative Example 1 was conducted under the same conditions as Example 5, except that a second sound transmission medium was not used.

[0454] The same silicone rubber with a modified central raised disc shape and a hardness of 30° as in Example 5 (e.g.) Figure 9C The first acoustic transmission medium (as shown in the diagram) is placed so that its center is aligned with the center of the vibrating plate. Next, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells on it), manufactured by Thermo Fisher Scientific Inc., is slowly placed on the vibrating plate, ensuring that the centers of both are aligned. Afterward, a 100g weight is placed on the lid of the culture medium to apply pressure. An air layer around the contact area of ​​the first acoustic transmission medium is confirmed by a photograph taken from above after the pressure has been applied.

[0455] The contact area between the first acoustic transmission medium and the culture medium was calculated in the same manner as in Example 1, and the contact area was 5%.

[0456] The evaluation result in Comparative Example 1 was a stripping rate of 81.2% and a deviation rating of D.

[0457] (Comparative Example 2)

[0458] Comparative Example 2 was performed under the same conditions as Comparative Example 1, except that cell peeling was performed for 2 minutes under cell peeling conditions involving a sweeping vibration in standing wave mode (frequency from 67.5 kHz to 72.5 kHz, sweep period of 1 second, and voltage of 90 V).

[0459] The air layer around the contact portion of the first acoustic transmission medium was confirmed by photographs of the culture medium taken from above after pressure was applied, in the same manner as in Comparative Example 1.

[0460] The contact area between the first acoustic transmission medium and the culture medium was calculated in the same manner as in Example 1, and the contact area was 5%.

[0461] The evaluation result in Comparative Example 2 was a stripping rate of 70.8% and a deviation rating of D.

[0462] (Comparative Example 3)

[0463] Comparative Example 3 was carried out under the same conditions as in Example 1, but neither the culture medium nor the vibrator came into contact with the first sound transmission medium.

[0464] That is, in the same manner as in Example 1, a silicone rubber with a hardness of 30° in the shape of a centrally protruding disc with a center height (1) of 0.9 mm, a side height (2) of 0.4 mm, and a diameter (3) of 32 mm is used. Figure 9A The first acoustic transmission medium (as shown in the diagram) is placed on the center of the vibrating plate. Next, water, used as the second acoustic transmission medium, is added dropwise in equal portions of 50 μL each, for a total of 300 μL. Then, a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. is slowly placed on the vibrating plate, aligning the centers of both. A photograph taken from above the culture medium confirms that there is no contact area between the first and second acoustic transmission media, and that there is no air layer between the culture medium and the second acoustic transmission medium.

[0465] The contact area between the first sound transmission medium and the culture medium is 0%.

[0466] The evaluation result in Comparative Example 3 was a stripping rate of 58.4% and a deviation rating of D.

[0467] (Comparative Example 4)

[0468] Comparative Example 4 was conducted under the same conditions as in Example 1, except that the first sound transmission medium was not used and the amount of water used as the second sound transmission medium was changed.

[0469] As a secondary sound transmission medium, 4.0 mL of water was placed on the vibrator using a dispenser. After the water was evenly spread on the vibrator, the center of a Nunc (trademark) Φ35-mm culture dish (used as the culture medium for culturing cells) manufactured by Thermo Fisher Scientific Inc. was slowly placed on the vibrator, ensuring that the centers of both were aligned so that no air layer was introduced onto the bottom surface of the culture medium. Subsequently, a 100g weight was placed on the lid of the culture medium to apply pressure. A photograph taken from above after applying pressure confirmed that the entire bottom surface of the culture medium on the vibrator side was filled with the secondary sound transmission medium, and that there was no air layer.

[0470] The contact area between the first sound transmission medium and the culture medium is 0%.

[0471] The evaluation result in Comparative Example 4 is a stripping rate of 82.0% and a deviation rating of D.

[0472] (in conclusion)

[0473] exist Figure 12 The results obtained by summarizing the respective construction and evaluation results of the above embodiments and comparative examples are shown in the figure. Figure 12 In this context, tanδ represents the value obtained at room temperature when a vibration with a frequency of 0.1 Hz is applied.

[0474] This disclosure is not limited to the above-described embodiments, and various changes and modifications are possible without departing from the spirit and scope of the invention. Therefore, the claims are appended herein to disclose the scope of the invention.

[0475] This application claims priority based on Japanese Patent Application No. 2023-118581, filed on July 20, 2023, the entire contents of which are incorporated herein by reference.

[0476] List of reference numerals

[0477] 11: Vibrating plate

[0478] 12: Piezoelectric element

[0479] 13: Sound transmission medium

[0480] 131: First Sound Transmission Medium

[0481] 132: Second sound transmission medium

[0482] 14: Heavy objects

[0483] 15: Top Cover

[0484] 16: Cushioning material

[0485] 17: Bottom Cover

[0486] 18: Bolts

[0487] 21: Culture Material

[0488] 22: Cover of culture medium

[0489] 23: Supply Unit

[0490] 24: Arm

[0491] 30: Ditch

[0492] 101: Vibrating body

[0493] 205: Information processing device

[0494] 206: CPU

[0495] 207: RAM

[0496] 208: ROM

[0497] 209: HDD

[0498] 210: Communication I / F

[0499] 211: Display device

[0500] 212: Input device

Claims

1. A cell detachment method of detaching cells attached to a culture surface of a culture substrate from the culture surface, the cell detachment method comprising a detachment step of detaching the cells from the culture surface by vibrating a vibrating body in a state where a first acoustic transmission medium and a second acoustic transmission medium are disposed between the culture substrate and the vibrating body, and in a state where at least one of the following (a) or (b) is satisfied: (a) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or (b) the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium, wherein the first acoustic transmission medium and the second acoustic transmission medium being different from each other, the first acoustic transmission medium being a solid at room temperature, and the second acoustic transmission medium being a liquid or a solid having fluidity at room temperature.

2. The cell lift-off method according to claim 1, wherein, the detachment step includes detaching the cells from the culture surface by vibrating the vibrating body in the state where the (a) is satisfied.

3. The cell lift-off method according to claim 1 or 2, wherein, the detachment step includes detaching the cells from the culture surface by vibrating the vibrating body in the state where the (a) is satisfied, and in a state where the vibrating body is in contact with the first acoustic transmission medium.

4. The cell lift-off method according to claim 2 or 3, wherein, In the detachment step, the following expression is satisfied: 0.05 ≤ S1 / S1+S2, where S1 is an area of a portion of the culture substrate in contact with the first acoustic transmission medium, and S2 is an area of a portion of the culture substrate in contact with the second acoustic transmission medium.

5. The cell lift-off method according to claim 1, wherein, the detachment step includes detaching the cells from the culture surface by vibrating the vibrating body in the state where the (b) is satisfied.

6. The cell lift-off method according to claim 5, wherein, In the detachment step, the following expression is satisfied: 0.05 ≤ S3 / S3+S4, where S3 is an area of a portion of the vibrating body in contact with the first acoustic transmission medium, and S4 is an area of a portion of the vibrating body in contact with the second acoustic transmission medium.

7. The cell detachment method according to any one of claims 1 to 6, satisfying the following expression: tan δ1 < tan δ2, where tan δ1 and tan δ2 are tan δ of the first acoustic transmission medium and tan δ of the second acoustic transmission medium, respectively, obtained by dynamic viscoelasticity measurement at room temperature and at a frequency of 1 Hz.

8. The cell lift-off method according to any one of claims 1 to 7, wherein, the second acoustic transmission medium is a solid having tan δ of 1.0 or more obtained when a vibration at a frequency of 0.1 Hz is applied at room temperature.

9. The cell lift-off method according to any one of claims 1 to 8, wherein, the second acoustic transmission medium is a liquid at room temperature.

10. The cell detachment method according to any one of claims 1 to 9, wherein the second acoustic transmission medium is a liquid, and wherein a contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 120° or less.

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

12. The cell detachment method according to any one of claims 1 to 11, wherein the second acoustic transmission medium is a liquid, and At least one of a contact angle at an interface between the culture substrate and the second sound transmission medium or a contact angle at an interface between the vibration body and the second sound transmission medium is less than 40°.

13. The cell lift-off method according to any one of claims 1 to 12, wherein, The first sound transmission medium includes, in at least one of a surface facing the culture substrate or a surface facing the vibration body, a continuous groove portion extending from a central portion of the at least one of the surfaces to an outer peripheral portion.

14. The cell lift-off method according to any one of claims 1 to 13, wherein, The peeling step includes setting a volume ratio of the second sound transmission medium to the first sound transmission medium to be 0.05 or more and 2.0 or less.

15. The cell delamination method according to any one of claims 1 to 14, wherein, The first sound transmission medium includes silicone rubber.

16. The cell delamination method according to any one of claims 1 to 15, wherein, The first sound transmission medium includes a porous resin material.

17. The cell delamination method according to any one of claims 1 to 16, wherein, The second sound transmission medium includes at least one selected from the group consisting of water, physiological saline, ethanol, and diluted ethanol.

18. The cell delamination method according to any one of claims 1 to 17, wherein, The peeling step includes a step of exciting a vibration in an ultrasonic wave band in the vibration body.

19. The cell peeling method according to any one of claims 1 to 18, further comprising a configuration step of disposing the first sound transmission medium and the second sound transmission medium between the culture substrate and the vibration body before the peeling step.

20. The cell lift-off method according to claim 19, wherein, The configuration step includes configuring either of the first sound transmission medium or the second sound transmission medium so as to be in contact with either of the vibration body or the culture substrate, based on a state of a vibration to be excited in the vibration body in the peeling step.

21. The cell delaminating method according to claim 19 or 20, wherein, The configuration step includes supplying the second sound transmission medium by a supply unit including at least one selected from the group consisting of a dispenser, a nozzle, a sprayer, and a sponge, and configuring the second sound transmission medium so as to be in contact with the first sound transmission medium.

22. The cell peeling method according to any one of claims 1 to 21, further comprising a pressurization step of applying pressure to bring the culture substrate and the vibration body close to each other.

23. The cell peeling method according to any one of claims 1 to 22, further comprising a surface treatment step of applying a surface treatment to at least one surface selected from the group consisting of: a surface of the culture substrate facing the first sound transmission medium; a surface of the vibration body facing the first sound transmission medium; a surface of the first sound transmission medium facing the culture substrate; and a surface of the first sound transmission medium facing the vibration body.

24. A cell peeling apparatus for peeling cells provided on a culture surface of a culture substrate from the culture surface by transmitting a vibration generated by a vibration body to the cells, the cell peeling apparatus comprising, in this order: a vibration body; a first sound transmission medium; and a culture substrate placement portion, and the cell peeling apparatus further comprising a supply unit for supplying a second sound transmission medium, wherein the supply unit is configured to supply the second sound transmission medium so that at least one of the following (a) or (b) is satisfied: (a) the culture substrate is in contact with the first sound transmission medium and the second sound transmission medium; or (b) the vibration body is in contact with the first sound transmission medium and the second sound transmission medium.

25. The cell stripping apparatus according to claim 24, further comprising an information processing apparatus, wherein the information processing apparatus is configured to: obtain information on a state of vibration to be excited in the vibration body; and control the supply unit based on the information.

26. The cell delaminating device of claim 24 or 25, wherein, the vibration body includes an ultrasonic vibrator.

27. The cell stripping apparatus according to any one of claims 24 to 26, comprising a pressurizing unit for applying pressure to bring the culture substrate and the vibration body close to each other.

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