Vibration transmission member, method for manufacturing vibration transmission member, composite body, vibration imparting device, cell detachment method, and vibration imparting method

Abstract

Provided is a vibration transmission member for cell detachment which detaches cells adhered to a culture surface of a culture container from the culture surface by using ultrasonic vibration, and which has high detachment performance and maintains the high detachment performance even when cell culture containers differ from each other. The vibration transmission member for detaching cultured cells is characterized in that the vibration transmission member is an elastic body, and has a micro rubber hardness type C of 10 degrees to 30 degrees and a specific gravity of 0.95 to 1.30.

Description

Vibration transmission member, method for manufacturing a vibration transmission member, composite, vibration imparting device, cell detachment method, and vibration imparting method

[0001] The embodiments disclosed in the present specification and drawings relate to a vibration transmission member, a method for manufacturing a vibration transmission member, a composite, a vibration imparting device, a cell detachment method, and a vibration imparting method.

[0002] In recent years, cell culture has become popular for culturing cells used in regenerative medicine and cells used in the production of biopharmaceuticals. Cell culture is broadly divided into adherent culture and suspension culture, of which adherent culture is a culture method suitable for various cell types and is widely used. As a cell manipulation method for cultured cells, minimally invasive techniques have been investigated for detaching cells from the culture substrate, separating cell clumps into single cells, and analyzing and seeding them (Patent Document 1). However, cell manipulation methods can sometimes damage cells. Cell detachment by enzyme treatment can also cause variations in cell quality. Therefore, detachment conditions that allow for high detachment rates without damaging cells have been investigated.

[0003] As a method for applying ultrasound to cells on a culture substrate to detach them, for example, a method of applying ultrasound using an elastic material such as rubber as a matching layer (Patent Document 2) is known. Examples of matching layers include an acoustic coupler and an elastic material such as rubber, but no specific consideration has been given as to what type of transmission medium is preferable. Furthermore, an example of an acoustic coupler using silicone rubber for an ultrasound probe (Patent Document 3) is known.

[0004] Japanese Patent No. 4775218 Japanese Patent Application Laid-Open No. 2024-000178 International Publication No. 2016 / 088699

[0005] The inventors' investigation of vibration transmission media revealed that when a solid transmission medium is used as a vibration transmission member, the vibrations generated from the vibration source are attenuated, sometimes preventing sufficient ultrasound from being applied to cells on the culture substrate. In such cases, it is difficult to perform the desired cell manipulation (including separation). In particular, they found that repeated use or the use of different culture vessels changes the way vibrations are transmitted, resulting in variations in cell detachment performance. Here, a culture vessel is a vessel used for cell culture, and a culture substrate is the portion of the culture vessel where cells adhere. The culture surface located on the inner surface of the culture vessel functions as the culture substrate. For example, when the dish exemplified in this disclosure is used as a culture vessel, the culture substrate serves as the bottom surface of the dish.

[0006] The object of the present disclosure is to provide a vibration transmission member that applies ultrasonic vibrations that reduce the attenuation of ultrasonic vibrations and can reduce variation in cell detachment performance even when used repeatedly or for different culture vessels.

[0007] The present disclosure relates to a vibration transmission member for applying ultrasonic vibrations to a cell culture in a culture vessel, the vibration transmission member being an elastic body, having a micro-rubber hardness type C of 10 degrees or more and 30 degrees or less, a specific gravity of 0.95 or more and 1.30 or less, and a post-load distortion rate of 50% or less.

[0008] The present disclosure also relates to a composite body including the vibration transmission member and a functional layer. The present disclosure also relates to a vibration imparting device including an ultrasonic generator that generates vibrations in an ultrasonic band and the vibration transmission member.

[0009] The present disclosure also provides a vibration imparting method for detaching cells adhered to the culture surface of a culture vessel by imparting ultrasonic vibrations to the cells in the culture vessel via the vibration transmission member.

[0010] The present disclosure also provides a cell detachment method for detaching cells adhered to a culture surface by applying ultrasonic vibrations to the cells in a culture vessel via the vibration transmission member.

[0011] The present disclosure also provides a method for producing the vibration transmission member for applying ultrasonic vibrations to cells on a culture substrate, the method comprising the steps of: mixing at least (A) an organopolysiloxane having alkenyl groups at both ends and a molecular weight of 10,000 or more and 150,000 or less, (B) an organohydrogenpolysiloxane having hydrosilyl groups (SiH groups), (C) a solid additive whose content relative to the total volume of the vibration transmission member is 10 volume % or less, and (D) a silicone composition containing a crosslinking catalyst to obtain a mixture; and curing and molding the mixture.

[0012] According to the present disclosure, an object of the present disclosure is to provide a vibration transmission member that applies ultrasonic vibrations that reduce the attenuation of ultrasonic vibrations and can reduce variation in cell detachment performance even when used repeatedly or for different culture vessels.

[0013] Fig. 1 is a flowchart showing a vibration imparting method according to an embodiment of the present disclosure. Fig. 2 is a schematic configuration diagram of a vibration imparting device according to an embodiment of the present disclosure. Fig. 3 is a cross-sectional view schematically showing warping of the bottom surface of a culture vessel. Fig. 4 is a schematic cross-sectional view showing a mold for forming a vibration transmission member according to Example 1 of the present disclosure. Fig. 5 is a schematic cross-sectional view showing a mold for forming a vibration transmission member according to Example 1 of the present disclosure. Fig. 6 is a schematic cross-sectional view showing a mold for forming a composite according to Example 12 of the present disclosure. Fig. 7 is a schematic cross-sectional view showing a mold for forming a composite according to Example 12 of the present disclosure.

[0014] Hereinafter, the vibration imparting method, vibration imparting device, vibration transmission member, composite, and method for manufacturing a vibration transmission member will be described in detail with reference to the drawings, but the present disclosure is not limited to the following examples.

[0015] (1) Vibration Imparting Method A vibration imparting method according to this embodiment will be described. FIG. 1 is a flowchart showing the vibration imparting method according to this embodiment. In step S1, cells are cultured in a culture vessel. In step S2, a vibration transmission member is placed on an ultrasonic vibrator. In step S3, a culture vessel is placed on the vibration transmission member. In step S4, power is supplied to the ultrasonic vibrator. In step S5, vibration is imparted to the culture vessel via the vibration transmission member. In step S6, it is determined whether there is a culture vessel to be vibrated next. If there is another vessel to be vibrated, the process proceeds to step S7. In step S7, the previous culture vessel is removed, the next culture vessel is placed, and the process proceeds to step S4. If it is determined in step S6 that there is no culture vessel to be vibrated next, the process ends.

[0016] (2) Vibration Apparatus A vibration applicator according to this embodiment will be described. FIG. 2 is a schematic diagram of a vibration applicator 100 according to this embodiment. The vibration applicator 100 includes an ultrasonic generator 3 that generates ultrasonic vibrations, a controller 4 that supplies power to the ultrasonic generator, and a vibration transmission member 2 that transmits the generated vibrations to a culture vessel 1. Examples of vibrations generated by the ultrasonic generator include vibrations in a frequency band of 10 kHz or higher and 1 MHz or lower. The ultrasonic generator includes an ultrasonic generating means, which can be any means capable of applying ultrasonic vibrations to a culture substrate or cells, without particular limitations. One example is the use of an ultrasonic vibrator such as lead zirconate titanate (PZT) as the vibrator. Here, the vibrator can be directly contacted with the outer surface of a culture vessel filled with a culture medium and sealed to impart vibrations to the culture substrate. Alternatively, instead of directly contacting the ultrasonic generating means with the culture vessel, an ultrasonic transmitter can be interposed between the ultrasonic generating means and the target area, allowing ultrasonic waves to be incident on the cells to be ablated.

[0017] The ultrasonic wave generating means in this embodiment includes at least an ultrasonic oscillator and a vibration plate. The ultrasonic wave generating means is not limited as long as it generates ultrasonic vibrations. For example, the ultrasonic vibration generating means may be a piezoelectric body bonded to a vibration plate. When the piezoelectric body is circular, the vibration plate is preferably made of glass, SUS, or quartz. By using glass, SUS, or quartz as the vibration plate, it is possible to output a large amplitude at a relatively high driving frequency (vibration frequency) in the ultrasonic range without damaging the ultrasonic vibrator.

[0018] When the ultrasonic vibrator is a ring-shaped piezoelectric body, it is preferable that the outer diameter of the vibration plate, which is the ultrasonic vibrator, is equal to the outer diameter of the piezoelectric body. The thickness of the vibration plate is preferable when the piezoelectric body and the vibration plate are bonded together and the midpoint of the thickness direction of the bending when vibrating, that is, the neutral plane where there is neither tension nor compression during bending, is on the vibration plate side, because this allows the distortion of the piezoelectric body to be efficiently used for bending.

[0019] Furthermore, commercially available Langevin type transducers or rectangular type transducers can also be used as the ultrasonic transducer of this embodiment. Langevin type transducers include those in which a piezoelectric body is sandwiched between two metal blocks and fastened together with bolts or the like to form an integrated structure, and examples of these include those manufactured by Honda Electronics Co., Ltd. and Fuji Ceramics Co., Ltd.

[0020] (3) Vibration Transmission Member The vibration transmission member of this embodiment is an elastic body, characterized by a micro-rubber hardness type C of 10 degrees or more and 30 degrees or less, a specific gravity of 0.95 or more and 1.30 or less, and a post-load distortion rate of 50% or less. When the vibration transmission member of this embodiment satisfies all of these physical property values, attenuation of ultrasonic vibrations is reduced when ultrasonic vibrations are applied to a cell culture in a culture vessel, and the way in which the vibrations are transmitted is less likely to change during repeated use or between different culture vessels. As a result, ultrasonic vibrations can be applied with reduced variation in cell detachment performance.

[0021] (3-1) Elastic Body When changing the culture vessel and applying vibrations to cells, if the vibration transmission member is a deformable gel or water, it is necessary to reset the vibration transmission member each time. On the other hand, if the vibration transmission member is an elastic body, this effort can be eliminated. Whether the vibration transmission member is an elastic body can be confirmed by measuring the viscoelasticity tan δ. Specifically, when vibrations of a frequency of 1 Hz are applied at room temperature, it is preferable that tan δ obtained by dynamic viscoelasticity measurement is less than 1.0. Here, room temperature is preferably 25°C, and tan δ can be calculated by measuring the dynamic viscoelasticity G' and G" when vibrations of a frequency of 1 Hz are applied using a rheometer or the like, and calculating using the relational expression tan δ = G" / G'. Here, G represents the shear modulus, G' represents the storage shear modulus, and G" represents the loss shear modulus.

[0022] Tan δ in the present disclosure was measured by the following method: Using a modular compact rheometer (product name: MCR-302; manufactured by Anton Paar), 25 mmφ parallel plates were used to hold the sample so that the normal force was 0.1 N, and G′ and G″ were measured at 25°C while the frequency was changed from 0.1 to 5 Hz, and tan δ at 1 Hz was calculated.

[0023] (3-2) Micro Rubber Hardness Type C In this embodiment, micro rubber hardness type C refers to hardness measured using a micro rubber hardness tester (product name: Micro Rubber Hardness Tester MD-1 capa Type C; manufactured by Kobunshi Keiki Co., Ltd.). This hardness tester for measuring micro rubber hardness type C has a small probe structure, so it can easily measure the hardness of a thin test piece of about 1 to 2 mm. Furthermore, since the vibration transmission member of the present disclosure has a low hardness, the hardness is measured using a micro rubber hardness tester MD-1 capa Type C, which provides a value close to the Asker C hardness, which is a rubber hardness measurement method for low hardness rubber.

[0024] (3-3) Specific Gravity The specific gravity in this embodiment can be measured using a liquid immersion specific gravity measuring device (SGM-6, manufactured by Mettler Toledo K.K.). When the vibration transmission member is made of silicone rubber that does not contain solid additives, the specific gravity is about 0.95 to 0.98. Solid additives generally have a higher specific gravity than silicone rubber, and increasing the content of the solid additive increases the specific gravity of the vibration transmission member. As an example, alumina (density 3.9 g / cm3) is a common solid additive. ３ When the content of the acrylic resin is 10% by volume, the specific gravity is 1.3. From the viewpoint of the stability of the peeling rate, the specific gravity is preferably 0.95 or more and 1.10 or less.

[0025] (3-4) Post-load distortion rate In this embodiment, the post-load distortion rate refers to the recovery rate of the indented portion of the vibration transmission member after a certain period of indentation load is applied to the vibration transmission member with a stainless steel plate. In this embodiment, the post-load distortion rate can be measured as follows. (Method for measuring post-load distortion rate) The post-load distortion rate is measured by using a press machine to measure the rate of return of deformation when a stainless steel plate is indented to 50% of the thickness of the vibration transmission member. Specific steps are shown below. Step 1 (Preparation) The vibration transmission member is cut to a diameter of 10 mm, and the thickness at the center is measured. This thickness is defined as t1. Step 2 (Installation) The vibration transmission member is placed in the press machine, and a 3 mm wide and 10 mm deep indentation stainless steel plate is placed on top of it so that it overlaps the center line of the vibration transmission member. The thickness of the indentation stainless steel plate is defined as 50% of the thickness of the vibration transmission member, and this is defined as tS. To prevent excessive pressure, 10 mm diameter stainless steel plates, the same thickness as the vibration transmission member, were placed in the press as spacer stainless steel plates at four locations 90 degrees around the vibration transmission member. Step 3 (Pressing): Pressurize the vibration transmission member until the top surfaces of the press-in stainless steel plates and the vibration transmission member are flush with each other, and maintain this condition for 8 hours. Step 4 (Measurement of Deformation Rate): After releasing the pressure and leaving the vibration transmission member to stand for 1 hour, measure the thickness of the vibration transmission member at its center. This thickness is designated as t2. The post-load distortion rate is calculated using the following formula: Post-load distortion rate = (t1 - t2) / tS × 100 (%). To achieve a post-load distortion rate of 50% or less, it is preferable that the toluene elution rate be 40% or less, that the material be made of silicone rubber, and that the solid additive content be 10% or less by volume. This is described in detail below.

[0026] (3-4-1) Crosslink Density and Toluene Swelling Degree The more crosslinking reaction sites in the elastomer, i.e., the higher the crosslink density, the lower the post-load strain rate, which is preferable. Crosslink density can be measured, for example, by measuring the toluene swelling degree using the toluene swelling method. After immersing the vibration transmission member in a sufficiently high toluene ratio (50 g toluene per 1 g of vibration transmission member) at 23°C for 72 hours, the volume V2 of the toluene-swollen vibration transmission member is measured, and the volume V1 of the removed vibration transmission member after air-drying for 72 hours to remove the toluene is measured. Note that V1 in the toluene swelling degree is the volume of the rubber polymer in the vibration transmission member. For V1 in the absence of solid additives, the volume value measured by immersion specific gravity after toluene drying is used as is. For V1 in the presence of solid additives, the volume value measured by immersion specific gravity measurement minus (volume value measured by immersion specific gravity measurement × solid additive content A) is used. The method for measuring the solid additive content A is described in the section (3-4-3) Silicone Rubber / Solid Additive Content. V2 is the weight M1 after drying the toluene, the weight M2 after wiping off the toluene from the surface after immersion in toluene, and the density of toluene, 0.87 g / cm ３ The following formula can be determined from V1: V2 = V1 + (M2 - M1) / 0.87 Using V2 and V1, the toluene swelling degree can be calculated by calculating (V2 / V1) x 100 (%). A lower value indicates a higher crosslink density, and a value of 3000% or less is preferable. If the toluene swelling degree exceeds 3000%, the crosslink density is extremely low, making it difficult to achieve a micro rubber hardness type C of 10 or more for the vibration transmission member. A more preferable range for the toluene swelling degree is 1200% or less. This increases the strength of the rubber itself against deformation, making it suitable for reducing the post-load strain rate. Note that the higher the molecular weight of (A) organopolysiloxane having alkenyl groups at both ends and a molecular weight of 10,000 or more and 150,000 or less, the higher the toluene swelling degree of the vibration transmission member tends to be. Furthermore, when the amount of (B) organohydrogenpolysiloxane having hydrosilyl groups (SiH groups) added is such that the proportion of hydrosilyl groups is small relative to the alkenyl groups in (A) organopolysiloxane, the degree of toluene swelling of the vibration transmitting member tends to be high.

[0027] (3-4-2) Toluene Elution Ratio The toluene elution ratio indicates the volume fraction of free oil contained in the vibration transmission member. When the free oil component is high, i.e., when the toluene elution ratio is high, the vibration transmission member contains a large amount of oil, making it difficult to restore its original shape when deformed. For example, examples of free oil include oils without crosslinkable functional groups and oils with crosslinkable functional groups but unreacted, which are added for purposes such as reducing the viscosity or hardness of the vibration transmission member to facilitate processing, and improving the surface smoothness of the vibration transmission member. The lower the volume change rate, the more preferable it is. However, particularly in the case of low-hardness elastomers such as those disclosed herein, the lower the crosslink density, making it easier for unreacted oil to remain. Therefore, to reduce the amount of free oil as much as possible, it is preferable to avoid the use of oils without crosslinkable functional groups. To achieve a post-load strain rate of 50% or less, the toluene elution ratio is preferably 40% or less.

[0028] The measurement method is as follows. First, the volume of the vibration transmission member is measured using an immersion specific gravity measuring device (SGM-6, manufactured by Mettler Toledo K.K.). This volume is designated as V0. Next, the vibration transmission member is immersed in toluene at a sufficiently high ratio of 50 g of toluene per 1 g of vibration transmission member at 23°C for 72 hours, thereby transferring the free oil contained in the vibration transmission member to the toluene. The vibration transmission member is then removed. The removed vibration transmission member is air-dried for 72 hours to remove the toluene, and the volume is measured using the method described above. This volume is designated as V1. The toluene elution rate is calculated using the obtained V0 and V1 using the formula {(V0 - V1) / V0} x 100 (%). Note that for V0 and V1 in the calculation of the toluene elution rate in the present disclosure, the volumes measured using the immersion specific gravity measuring device can be used as is, regardless of the presence or absence of solid additives. The denominator of the above equation is V0, i.e., the total volume of the vibration transmission member (including the volumes of rubber polymer, free oil, and solid additives), and the numerator of the above equation (V0-V1) essentially represents the free oil volume regardless of whether or not solid additives are present, and this equation shows the volume ratio of free oil to the total volume of the vibration transmission member.

[0029] (3-4-3) Content of Silicone Rubber and Solid Additives Silicone rubber is preferred as the material composition of the vibration transmission member from the viewpoint of its resistance to attenuation of ultrasonic vibrations and chemical stability. The composition of silicone rubber can be confirmed by performing attenuated total reflection (ATR) measurement using an infrared spectrometer (FT-IR) (for example, product name: Frontier FT IR, manufactured by PerkinElmer). The silicon-oxygen bond (Si—O), which is the main chain structure of silicone, undergoes stretching vibrations at a wave number of 1020 cm －１ In addition, the methyl group bonded to the silicon atom (Si-CH ３ ) is accompanied by a bending vibration due to its structure, and has a wave number of 1260 cm －１ Its presence can be confirmed by the strong infrared absorption in the vicinity.

[0030] Silicone rubber generally has low tear strength, so it can be reinforced by adding common solid additives such as silica and alumina. However, as the solid additive content increases, the post-load strain rate tends to increase. While the mechanism is unclear, it is thought that an increase in the solid additive content reduces the crosslink density of the vibration transmission member in terms of volume, or causes misalignment at the interface between the solid additive and the silicone rubber. Therefore, in order to achieve a post-load strain rate of 50% or less while maintaining low hardness, it is preferable that the solid additive content be 10% or less by volume relative to the total volume of the vibration transmission member.

[0031] The content of the solid additive contained in the vibration transmission member can be calculated by observing a cross section of the vibration transmission member and determining the content from the area ratio of the solid additive, or by the following formula: A = (Mf / ρf) / Vs. Here, A is the content of the solid additive, Mf is the weight of the solid additive, ρf is the density of the solid additive, and Vs is the volume of the vibration transmission member. Vs can be measured using an immersion specific gravity analyzer (SGM-6, manufactured by Mettler-Toledo). Mf can be confirmed using a thermogravimetric analyzer (TGA) (e.g., trade name: TGA851, manufactured by Mettler-Toledo). Specifically, a weight Mα is cut out from the weight Ms of the vibration transmission member with a razor blade and placed in an alumina pan used in the instrument. The alumina pan containing the sample is placed in the instrument and heated from room temperature to 800°C at a heating rate of 20°C per minute in a nitrogen atmosphere, and then held at 800°C for one hour. In a nitrogen atmosphere, as the temperature rises, the cured silicone rubber component is decomposed and removed by cracking without being oxidized, resulting in a decrease in the weight of the sample. The remaining sample weight, Mβ, is used to calculate Mf using the following formula: Mf = Ms × (Mβ / Mα). ρf can be used when the solid additive is known, e.g., 2.2 g / cm for silica. ３ , and 3.9 g / cm for alumina ３ In the case of an unknown solid additive, the density ρf can be calculated from the weight Mγ of samples collected by carrying out the heat treatment multiple times and the volume Vγ of the sample measured with a dry automatic density meter (trade name: Accupyc 1330-1, manufactured by Shimadzu Corporation).

[0032] (3-5) Method for Producing Vibration Transmission Member The vibration transmission member of the present disclosure comprises the steps of mixing the following silicone composition and curing and molding the silicone composition.

[0033] (3-5-1) Silicone Composition The silicone composition used to manufacture the vibration transmission member contains at least (A) an organopolysiloxane having alkenyl groups at both ends and a molecular weight of 10,000 or more and 150,000 or less, (B) an organohydrogenpolysiloxane having hydrosilyl groups (SiH groups), (C) a solid additive whose content is 10% by volume or less based on the total volume of the vibration transmission member, and (D) a crosslinking catalyst. An elastomer can be obtained by the curing reaction of (A) and (B) via (D). Details of (A), (B), (C), (D), and the optional (other) components are provided below.

[0034] (A) Organopolysiloxane having alkenyl groups at both ends and a molecular weight of 10,000 or more and 150,000 or less is represented by the following structural formula (1): In structural formula (1), R １ represents a monovalent unsubstituted or substituted hydrocarbon group bonded to a silicon atom, which does not contain an alkenyl group. １ Specific examples are given below.

[0035] Examples of unsubstituted hydrocarbon groups include alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, and hexyl, and aryl groups such as phenyl. Examples of substituted hydrocarbon groups include chloromethyl, 3-chloropropyl, 3,3,3-trifluoropropyl, 3-cyanopropyl, and 3-methoxypropyl. In particular, R １ It is preferable that 50% or more of R are methyl groups, and all of R １ is particularly preferably a methyl group.

[0036] Also, R ２ represents an alkenyl group bonded to a silicon atom. ２Specific examples of the group include vinyl, allyl, 3-butenyl, 4-pentenyl, and 5-hexenyl groups. Among these, vinyl groups are preferred because they are easy to synthesize and handle, and can easily undergo crosslinking reactions. The molecular weight of the organopolysiloxane of the present disclosure is 10,000 or more and 150,000 or less. The molecular weight varies depending on the number n in structural formula (1). When the molecular weight is less than 10,000, the crosslinking density is high even at low hardness, and the distance between crosslinking reaction points is short. Therefore, the vibration transmission member becomes brittle and may be destroyed when handled. On the other hand, when the molecular weight exceeds 150,000, the adhesive strength of the molded vibration transmission member is too high, and the member may be destroyed when released from the mold after molding or during handling, although the mechanism is unclear. The molecular weight can be measured using catalog values, or, if unknown, can be measured as a weight-average molecular weight converted to polystyrene using gel permeation chromatography (GPC).

[0037] (B) Organohydrogenpolysiloxane having a hydrosilyl group (SiH group) is represented by the following structural formula (2): R in structural formula (2) １ is as described above. m is preferably 3 or more on average. If it is less than 3, it becomes difficult to mold it into an elastomer. The organohydrogenpolysiloxane (B) functions as a curing agent (hereinafter also referred to as "crosslinking agent") that forms a crosslinked structure by reacting with the alkenyl groups of the organopolysiloxane (A) under the action of the crosslinking catalyst (D). Hydrogen nuclear magnetic resonance analysis ( １ The presence of hydrosilyl groups (SiH groups) can be confirmed by measurement using H-NMR (trade name: AL400 type FT-NMR; manufactured by JEOL Ltd.).

[0038] The molecular weight of (B) is not particularly limited. The viscosity of the organopolysiloxane at 25°C is preferably 10 mm ２ / s or more, 20,000mm ２ / s or less. This is because it does not volatilize during storage, preventing the desired crosslink density and physical properties of the molded product from being obtained, is easy to synthesize and handle, and can be easily and uniformly dispersed in the system. Furthermore, by appropriately changing the amount of (B) added, the hardness of the vibration transmission member can be adjusted. For example, by increasing the amount of (B) added, the hardness of the vibration transmission member can be increased.

[0039] (C) Solid Additives As long as the effects of the present disclosure are not impaired, the solid additive may contain a thermally conductive filler, a reinforcing filler, or the like. Silica or alumina is generally used. Furthermore, in order to increase strength, the surface of the solid additive may be subjected to a hydrophobic treatment that has high affinity with silicone rubber. The solid additive is contained in an amount of 10% by volume or less of the total volume of the vibration transmitting member to suppress attenuation of ultrasonic vibrations.

[0040] (D) Crosslinking catalyst Examples of the crosslinking catalyst include hydrosilylation catalysts. Known substances such as platinum compounds and rhodium compounds can be used as the hydrosilylation catalyst. The amount of the catalyst to be added can be appropriately determined and is not particularly limited.

[0041] (Other Components) A hardening retarder may be used as needed to prevent the mixture from hardening immediately after mixing. An example of a hardening retarder is tetravinyltetramethylcyclotetrasiloxane. Other ingredients such as pigments, dyes, antistatic agents, antioxidants, and flame retardants may also be added as needed.

[0042] (3-5-2) Step of Mixing the Silicone Composition The materials containing at least (A), (B), (C), and (D) can be mixed using known means such as a planetary universal mixer or a revolutionary mixer without stirring blades. If air bubbles are present in the vibration transmission member, vibration transmission may not be successful. Therefore, it is preferable to obtain a mixture by degassing the silicone composition by placing it in a vacuum environment during or after mixing.

[0043] (3-5-3) Step of curing and molding the silicone composition To mold the vibration transmission member, known methods can be used, such as a method in which the mixture is filled into a mold and then heated to cure and mold, or a method in which the silicone composition is coated on a workpiece and then heated to cure and mold.

[0044] (3-6) Shape and Layer Structure of Vibration Transmission Member Preferred embodiments of the shape and layer structure of the vibration transmission member are shown below. The layer structure refers to the structure of a composite provided with a functional layer for imparting a desired function to the vibration transmission member. Examples of functional layers include an adhesive layer that fixes the vibration transmission member to the ultrasonic generator, a high-hardness layer that reduces amplitude unevenness within the bottom surface of the culture vessel, a water-repellent layer or low-adhesion layer that prevents contamination by water and dust, and a release agent layer that prevents adhesion between the culture vessel and the vibration transmission member, and these can be provided as appropriate.

[0045] (3-6-1) Shape: The central portion preferably has a thickness of 0.5 mm or more and 4 mm or less, and a circular shape with a diameter of 5 mm or more and 200 mm or less, with the central portion being thicker than the peripheral portion. The bottom surface of a typical culture vessel is not perfectly flat and contains slight warping or undulation. It is desirable to ensure that there is no air between the vibration transmission member and the culture vessel, and the vibration transmission member is deformed to conform to the shape of the culture vessel bottom. To accommodate various culture vessels, a large amount of deformation is required, and it is important to ensure a certain degree of thickness. On the other hand, from the perspective of vibration transmission, excessive thickness hinders vibration transmission. A central thickness of 0.5 mm or more and 4 mm or less can optimally achieve both. Regarding the diameter, it is preferable to set the size so that it can widely cover the bottom surface of the culture vessel used. Furthermore, by making the central portion thicker than the peripheral portion, it is possible to reduce the amount of air trapped at the interface between the culture vessel and the vibration transmission member when the culture vessel is lowered onto it. The shape can be measured using a general vernier caliper and a multicolor laser coaxial displacement meter (CL-3000, manufactured by Keyence Corporation).

[0046] (3-6-2) Adhesion Force The vibrating member has a surface that contacts the ultrasonic generator and a surface that contacts the culture vessel. It is preferable that the adhesion force A1 on the surface that contacts the ultrasonic generator and the adhesion force A2 on the surface that contacts the culture vessel satisfy the relationship A1 > A2. Typically, when applying vibrations to cells in a culture vessel using a vibration imparting device, multiple samples with different cell types or culture conditions are prepared, and the culture vessels are transferred for continuous processing. When a culture vessel placed on a vibration transmission member is removed from the vibration transmission member, whether the vibration transmission member remains on the ultrasonic element or adheres to the culture vessel depends on the magnitude relationship between the adhesion forces A1 and A2. When performing continuous transfer operations, it is desirable for the vibration transmission member to remain on the ultrasonic element from the perspective of workability. By achieving the adhesion force A1 > A2 relationship, the vibration transmission member can remain on the device, improving workability.

[0047] (Method for measuring adhesive force) Adhesion forces A1 and A2 are measured using a tacking tester manufactured by RHESCA. Specifically, a vibration transmission member cut to a diameter of 30 mm is placed with the measurement surface facing the probe, and measurement is performed under the following conditions. The vibration transmission member is fixed using double-sided tape to prevent it from floating when the probe is lifted. Probe: SUS Probe diameter: 10 mm Pressing speed: 1 mm / s Pressing load: 200 gf Pressing hold time: 10 s Lifting speed: 1 mm / s After measurement, the integral value of the load when the probe is lifted is calculated using analysis software TAC1000, and this is taken as the adhesive force.

[0048] One method for achieving a relationship between the adhesive force A1 on the surface contacting the ultrasonic generator and the adhesive force A2 on the surface contacting the culture vessel, A1 > A2, is to form an adhesive layer on the surface with adhesive force A1. Known adhesives, such as acrylic adhesives and silicone adhesives, can be used. Silicone adhesives that cure using a hydrosilylation catalyst are preferred. This is because they provide high interlayer adhesion through self-adhesion, which forms chemical bonds with unreacted crosslinkable functional groups in the main silicone rubber layer of the vibration transmission member. Known methods for forming the adhesive layer include coating followed by curing and curing using a mold. The thickness of the adhesive layer can be appropriately set as long as it does not impair the effects of the present disclosure. Another method for achieving a relationship between A1 and A2 is to reduce the adhesive force by subjecting the surface to be used as the A2 surface to UV treatment, hydrogen peroxide plasma treatment, or the like.

[0049] (3-6-3) Composites In another embodiment, a composite having a laminated structure in which a functional layer is further provided on the vibration transmission member is preferred. Among these, a composite having the aforementioned adhesive layer or high-hardness layer provided on the surface that contacts the culture vessel is preferred. The high-hardness layer is a layer that is harder and thinner than the vibration transmission member, and can be provided on the vibration transmission member. Materials for the high-hardness layer may be any material that does not significantly impair the effects of the vibration transmission member, such as silicone rubber, butadiene rubber, urethane rubber, natural rubber, and fluororubber. Among these, materials that are resistant to environments such as chemicals and sterilizing UV and have acoustic impedance similar to that of the vibration transmission member are preferred. When the vibration transmission member is made of silicone rubber, high-hardness silicone rubber is preferred. Methods for increasing the hardness of silicone rubber include methods disclosed in the present disclosure, such as increasing the amount of curing agent or adding a solid additive. The provision of a high-hardness layer reduces unevenness in the amplitude within the bottom surface of the culture vessel, enabling cells that could not be detached due to poor amplitude to be detached, thereby improving the average detachment rate. Although the mechanism by which the provision of a high-hardness layer makes the amplitude uniform is unclear, it is believed that the high-hardness layer suppresses the inherent vibration of the bottom surface of the culture vessel. By providing a high-hardness layer, the value H1 of the micro-rubber hardness type C on the surface in contact with the culture vessel and the value H2 of the micro-rubber hardness type C on the opposing surface become H1 > H2. Furthermore, to significantly demonstrate the effect of the amplitude uniformity, it is preferable that H1 / H2 ≥ 1.4.

[0050] Furthermore, from the viewpoint of improving adhesion between the culture vessel and the composite, when the composite has a high-hardness layer, H1 is preferably ≦37. Furthermore, the thickness of the high-hardness layer is preferably 1 / 25 to 1 / 4 of the thickness of the composite. The thickness of the high-hardness layer is measured by cutting the composite with a razor from the surface in contact with the culture vessel to the opposing surface, passing through the center of the composite, and measuring the cross section using an optical microscope (product name: DSX1000, manufactured by Olympus Corporation). The thickness was measured at nine equally spaced positions, including both ends and the center, in the direction connecting the ends of the composite perpendicular to the cutting direction. The arithmetic mean value of these measurements was used as the thickness of the high-hardness layer. In the case of a vibration transmission member, a micro-rubber hardness type C of 30 or less is required. However, in the case of a composite, even if the micro-rubber hardness type C, i.e., H1, on the surface in contact with the culture vessel is 37, the high-hardness layer is sufficiently thin, and adhesion is ensured by the underlying vibration transmission member. In the measurement of H1, the influence of the hardness of the vicinity of the measurement surface, i.e., the high-hardness layer, is easily reflected, but the adhesion to the culture vessel is largely influenced by the deformation of the entire composite, especially the vibration transmission member, which has a low hardness and a relatively large thickness ratio. The high-hardness layer may be bonded to the vibration transmission member and integrated therewith.

[0051] The specific gravity, post-load strain rate, toluene elution rate, and solid additive content of the vibration transmission member in the composite are measured using values ​​obtained after removing the high-hardness layer. The removal method is not limited, but an example is removal using a razor. The toluene elution rate can be measured using the removed high-hardness layer. When the toluene elution rate of the vibration transmission member is X1 (%) and the toluene elution rate of the high-hardness layer is X2 (%), satisfying the following formula makes it less likely to be affected by volume changes due to the diffusion and movement of free oil between layers. Therefore, even during long-term use, this is suitable for suppressing warpage of the composite and maintaining its shape. Formula |X1-X2|≦10

[0052] (3-6-4) Manufacturing Method of Composite Examples of manufacturing methods of the composite include a method of manufacturing the composite by molding a high-hardness layer and a vibration transmission member separately and stacking them, and a manufacturing method of molding a high-hardness layer and a vibration transmission member simultaneously. As long as a laminated structure can be formed, there are no particular limitations. However, a composite can be manufactured by a method in which composition A, which will become the high-hardness layer, is applied to a desired thickness on the surface of a mold that molds and transfers the surface that contacts the culture vessel, and then composition B, which will become the low-hardness layer, is applied on top of composition A, and composition A and composition B are cured simultaneously (FIGS. 5A and 5B).

[0053] (4) Cell Culture (4-1) Cells The cells in this embodiment are not particularly limited as long as they can be cultured in vitro on a culture vessel. Examples include various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), human alveolar basal epithelial adenocarcinoma-derived A549 cells, mouse macrophage-like cells (RAW264.7), and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, and contractile cells. Examples of cells include skeletal muscle cells, smooth muscle cells, cardiac muscle cells, neuronal cells that constitute the nervous system, glial cells, and fibroblasts; hepatic parenchymal cells involved in the metabolism of living organisms; non-hepatic parenchymal cells and adipocytes; and cells with differentiation potential, such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, as well as progenitor cells of various tissues, and cells induced to differentiate therefrom. In this embodiment, the cells may be individual cells (so-called single cells) or sheet-like cell cultures (cell sheets). The cell detachment method according to this embodiment is particularly suitable for cells with strong intercellular bonds, cells with high adhesive strength to a substrate (e.g., the culture surface of a culture vessel), and cells with high trypsin sensitivity. Given the need for large-scale cell culture, this method is particularly suitable for, for example, CHO cells used for protein production and mesenchymal stem cells that can be used in cell therapy.

[0054] (4-2) Culture Vessel In this embodiment, the culture vessel refers to a culture vessel used for cell culture. The culture vessel is not particularly limited as long as it is a cell-adherent culture vessel, and examples thereof include a flask, a flask for tissue culture, a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a multi-well plate, a multi-plate, a Petri dish, a culture bag, a bottle, and the like.

[0055] The material of the culture vessel in this embodiment may be any material that is chemically stable and capable of culturing the desired cells, and examples thereof include polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(meth)acrylic acid, poly(meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, glass, metal, etc. Among these, polystyrene is preferred.

[0056] Figure 3 is a cross-sectional view showing the warpage of the bottom surface of a culture vessel. Generally, the bottom surface of a culture vessel is warped so that the center is convex upward. The amount of warpage was measured using a multicolor laser coaxial displacement meter (CL-3000, manufactured by Keyence Corporation).

[0057] (4-3) Buffer Solution In this embodiment, the buffer solution can be used without limitation as long as it can maintain a neutral pH range. Examples include Tris buffer solutions such as Tris-HCl buffer solution, phosphate buffer solution, HEPES buffer solution, citrate-phosphate buffer solution, glycylglycine-sodium hydroxide buffer solution, Britton-Robinson buffer solution, and GTA buffer solution. Among these, phosphate buffer solutions that are close to the in vivo environment are preferred, and phosphate-buffered saline (PBS) adjusted to be isotonic with intracellular fluid is more preferably used as the phosphate buffer solution.

[0058] (4-4) Culture Medium There is no particular limitation on the type of culture medium, and examples thereof include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A Medium, Eagle's Minimum Essential Medium (EMEM), αMEM (alpha Modified Eagle's Minimum Essential Medium; αMEM), and MEM (Minimum Essential Medium). Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemSpan H3000 (Stem Cell Technology), StemSpan SFEM (Stem Cell Technology), Stemline II (Sigma-Aldrich), Endothelial Cell Growth Medium 2 Kit (Promocell), Mesenchymal Stem Cell Growth Medium 2 (Promocell), MSCGM Bullet Examples of suitable medium include Repro FF Medium (manufactured by ReproCell), Repro FF Medium (manufactured by ReproCell), NutriStem Medium (manufactured by Biological Industries), and MF-Medium Mesenchymal Stem Cell Growth Medium (manufactured by Toyobo Co., Ltd.). Among these, it is preferable to use a medium suitable for culturing the respective cells.

[0059] (4-5) Serum Serum or antibiotics may be added to the above-mentioned medium. Examples of serum include fetal bovine serum (FBS), baby calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum, with FBS being commonly used due to its ease of availability. Alternatively, the medium may be serum-free, not containing raw or unpurified serum, but containing purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0060] (4-6) Antibiotics Examples of antibiotics added to the medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, amphotericin B, and the like.

[0061] (4-7) Cell Culture Conditions Cell culture conditions can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to a dish, and 1.0 × 10 １ ~5.0 x 10 ４ cells / cm ２ The cells were seeded at 37°C and CO ２ The cells are cultured in an environment with a concentration of 5%. At this time, it is preferable to culture the cells until the cell occupancy rate in the culture vessel is about 70 to 80% (so-called subconfluent state).

[0062] Examples to which the present disclosure is applied will be described below, but unless otherwise specified, the scope of the present disclosure is not intended to be limited to only these examples.

[0063] (Culturing of CHO cells) CHO cells (Chinese hamster ovary cells) were cultured in a culture vessel at a density of 15,000 cells / cm. ２ The cells were seeded at a density of 1000× ... and incubated at 37°C in CO ２The cells were cultured in an environment with a 5% cell concentration. The medium used was Ham's F12 (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The culture was carried out for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupation area of ​​the dish was approximately 80%.

[0064] (Culturing of A549 cells) A549 cells (human alveolar basal epithelial adenocarcinoma cells) were cultured in a Φ35 polystyrene dish (manufactured by Corning) at a density of 10,000 cells / cm. ２ The cells were seeded at a density of 1000× ... and incubated at 37°C in CO ２ The cells were cultured in an environment with a 5% cell concentration. The medium used was DMEM (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The culture was carried out for 48 hours, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate of the dish was approximately 80%.

[0065] (Culture vessel) Three types of culture vessels were used: i) Corning 35 mm cell culture surface-treated dish (430165, Corning Incorporated), ii) Nunc Easy Dish 35 mm (150460, Thermo Fisher Scientific), and iii) IWAKI tissue culture dish 35 mm (3000-035, AGC Technoglass). Ten of each culture vessel were prepared, and the amount of warping of the bottom surface was measured and the average value was calculated. i) Corning 35 mm cell culture surface-treated dish was 120 μm, ii) Nunc Easy Dish 35 mm was 20 μm, and iii) IWAKI tissue culture dish 35 mm was 60 μm.

[0066] (Application of Ultrasonic Vibration) Ultrasonic vibration was applied to the cell culture of CHO cells obtained under the above culture conditions in the following manner. The culture medium in the culture vessel in which the cells were cultured was replaced with PBS(-), a detachment solution, and the cells were incubated at 37°C for 2 minutes. Next, the vibration transmission member 2 obtained in each Example and Comparative Example was placed on the ultrasonic generator 3 of the vibration imparting device 100 illustrated in FIG. 2, and the culture vessel was placed on the vibration transmission member, and the ultrasonic generator was driven. In this Example, a Langevin-type ultrasonic vibrator was used for the ultrasonic generator, and the cells were detached by driving the vibrator for 2 minutes at a resonant frequency of 34.5 kHz and an applied voltage of approximately 20 V, while adjusting the voltage and driving frequency so that the amplitude of the bottom surface of the culture vessel placed on the vibrator was 5.0 μm during driving. Ultrasonic waves were applied to the cell culture of A549 cells obtained under the above culture conditions in the same manner as for CHO cells, except that the incubation time was changed to 5 minutes and the ultrasonic driving time was changed to 3 minutes.

[0067] (Evaluation of Effect) The effect was evaluated based on three items: the average detachment rate of cells adhered to the culture substrate, the variation in detachment rate between different containers, and the decrease in detachment rate after repeated use.

[0068] The average detachment rate of detached cells can be calculated by measuring how many cells cultured in a culture vessel (Corning 35 mm cell culture surface-treated dish) were detached by vibration. After vibration, the detached cells were recovered together with the solution, and the number of cells was counted, which was designated as N_det. The cells remaining on the culture substrate were detached using trypsin, recovered, and the number of cells was counted, which was designated as N_rest. The detachment rate for one detachment trial was calculated as N_det / (N_det + N_rest) x 100 (%), and in this evaluation, the average value of three detachment trials was designated as the average detachment rate. The evaluation of the average detachment rate was judged according to the following criteria. An average detachment rate of 90% or more, or B or higher, was deemed to be effective according to the present disclosure. A: detachment rate of 95% or more B: detachment rate of 90% or more but less than 95% C: detachment rate of 85% or more but less than 90% D: detachment rate less than 85%

[0069] Variations in detachment rates between different containers were evaluated by detaching cells cultured in three containers each: i) Corning 35mm cell culture surface-treated dish (430165, Corning Incorporated), ii) Nunc Easy Dish 35mm (150460, Thermo Fisher Scientific), and iii) IWAKI Tissue Culture Dish 35mm (3000-035, AGC Technoglass). Individual detachment rates were calculated for each container, and the standard deviation of the detachment rates measured nine times was used for evaluation. Variations in detachment rates between different containers were evaluated according to the following criteria. A standard deviation of less than 5, or B or higher, was deemed to be effective according to the present disclosure. A: Standard deviation less than 3 B: Standard deviation 3 or more but less than 5 C: Standard deviation 5 or more but less than 7 D: Standard deviation 7 or more

[0070] The reduction in detachment rate after repeated use was evaluated by the difference between the average detachment rate of the first 10 attempts and the average detachment rate of the last 10 attempts when 50 attempts were made to detach cells cultured on a culture vessel (Corning 35 mm cell culture surface-treated dish). The reduction in detachment rate after repeated use was evaluated according to the following criteria. A difference in detachment rate of less than 10, or B or higher, was determined to be effective according to the present disclosure. A: Difference in detachment rate less than 5 B: Difference in detachment rate 5 or more but less than 10 C: Difference in detachment rate 10 or more but less than 15 D: Difference in detachment rate 15 or more The effectiveness of the composite having a laminated structure including a vibration transmission member was evaluated in three items: the improvement rate of the average detachment rate due to lamination, the variation in detachment rate between different vessels, and the reduction in detachment rate after repeated use. Of these, the improvement rate of the average detachment rate due to lamination was evaluated as follows, and the other two items were evaluated in the same way as when no lamination was performed. The improvement rate of the average peeling rate due to lamination is calculated by calculating the average peeling rate DRs when a vibration transmission member that has not been laminated is used and the average peeling rate DRc when a laminated composite is used using the method for calculating the average peeling rate described above, and then calculating it by (DRc-DRs) / Ds x 100(%). The improvement rate of the average peeling rate due to lamination was evaluated based on the following criteria. In this evaluation, an improvement rate of the average peeling rate due to lamination of 15% or more, or a rating of B or higher, was determined to be effective in accordance with the present disclosure. A: Improvement rate of 25% or more B: Improvement rate of 15% or more but less than 25% C: Improvement rate of less than 15%

[0071] Example 1 10 g of organopolysiloxane (manufactured by Gelest, trade name: DMS-V31) having vinyl groups at both ends and a molecular weight of 28,000, 0.1 g of organohydrogenpolysiloxane (manufactured by Gelest, trade name: HMS-301) having a hydrosilyl group (SiH group), 0.001 g of platinum compound solution (manufactured by Gelest, trade name: SIP6829.2) as a crosslinking catalyst, and 0.02 g of cure retarder (manufactured by Gelest, trade name: SIT7900.0) were weighed out and mixed to form a silicone composition. Mixing was carried out in a vacuum reduced pressure mode using a mixing device (trade name: ARV-310, manufactured by Thinky Corporation) at 2000 rpm for 2 minutes while degassing. The mixed silicone composition was filled into the mold in the state shown in Figure 4A, and the upper mold was closed to form the state shown in Figure 4B, after which the molds were fastened together with screws (not shown). In this state, the silicone composition was cured by heating in a hot air oven at 130°C for 30 minutes, and then the mold was removed from the oven and the upper mold was opened to obtain the vibration transmission member of Example 1. The mold used had a center L1 of 2.1 mm, a peripheral L2 of 2.0 mm, and a diameter of 32 mm.

[0072] (Examples 2 to 8) For micro rubber hardness type C, the amount of crosslinking agent was appropriately adjusted, and for the content of solid additive, the amount of solid additive added was appropriately adjusted, so as to obtain the results shown in Table 1. Otherwise, vibration transmission members for Examples 2 to 8 were obtained in the same manner as Example 1. Note that, for the solid additive, "Resonac Corporation, product name: CB-P10" was used in Examples 3, 4, and 8, and "AEROSIL RX200, product name: Nippon Aerosil Co., Ltd." was used in Examples 5 and 6.

[0073] Example 9 A vibration transmission member of Example 9 was obtained in the same manner as in Example 7, except that 2.2 g of an oil having no crosslinkable functional groups (manufactured by Shin-Etsu Chemical Co., Ltd., product name: KF-96A-100CS) was added to 10 g of organopolysiloxane.

[0074] (Examples 10 and 11) As organopolysiloxane having vinyl groups attached to both ends, a molecular weight of 17,200 (manufactured by Gelest, product name: DMS-V25) was used in Example 10, and a molecular weight of 100,000 (manufactured by Gelest, product name: DMS-V51) was used in Example 11, and the amount of crosslinking agent was appropriately adjusted so as to achieve a micro rubber hardness of type C as shown in Table 1. Otherwise, the vibration transmission members of Examples 10 and 11 were obtained in the same manner as in Example 1.

[0075] Comparative Example 1 A vibration transmission member of Comparative Example 1 was obtained in the same manner as in Example 7, except that the silicone composition was stirred with a spatula to incorporate air bubbles into the silicone composition, and the silicone composition was filled into the mold in the state shown in Figure 4A without being degassed after mixing, and molded.

[0076] (Comparative Examples 2 to 4) For micro rubber hardness type C, the amount of crosslinking agent was appropriately adjusted, and the content of solid additive was appropriately adjusted by adjusting the amount of solid additive added, so as to obtain the results shown in Table 1. Otherwise, vibration transmission members for Comparative Examples 2 to 4 were obtained in the same manner as in Example 1. The solid additive used was "Product Name: AEROSIL RX200, manufactured by Nippon Aerosil Co., Ltd."

[0077] Comparative Example 5 A vibration transmission member of Comparative Example 5 was obtained in the same manner as in Example 7, except that 4.6 g of an oil having no crosslinkable functional groups (manufactured by Shin-Etsu Chemical Co., Ltd., product name: KF-96A-100CS) was added to 10 g of organopolysiloxane.

[0078] Comparative Examples 6 and 7: An organopolysiloxane having vinyl groups attached to both ends, with a molecular weight of 6,000 (manufactured by Gelest, product name: DMS-V21) was used in Comparative Example 6, and a molecular weight of 165,000 (manufactured by Gelest, product name: DMS-V52) was used in Comparative Example 7, and the amount of crosslinking agent was appropriately adjusted to achieve a micro rubber hardness of type C as shown in Table 1. Other than that, the vibration transmission members of Comparative Examples 6 and 7 were obtained in the same manner as in Example 1.

[0079] The tan δ of the vibration transmission members obtained in Examples 1 to 11 and Comparative Examples 1 to 7 was calculated using the above-described measurement method. The members were confirmed to be elastic and were marked with "○" in Table 1. The micro rubber hardness type C, specific gravity, post-load strain rate, toluene elution rate, and solid additive content were also determined using the above-described measurement methods. Furthermore, the obtained vibration transmission members were used to perform cell detachment using CHO cells, and their effects were evaluated. The respective physical properties and evaluation results are shown in Table 1. For Comparative Examples 3, 6, and 7, measurements of tan δ, micro rubber hardness type C, and specific gravity were possible, but the members broke during handling for effect evaluation, so the evaluation was discontinued. The toluene swelling degrees of Examples 7 and 10 were determined using the above-described measurement method. As a result, the toluene swelling degree of Example 7 was 792%, and the toluene swelling degree of Example 10 was 1150%.

[0080]

[0081] (Examples 12, 13, 14) 10 g of organopolysiloxane (Gelest, trade name: DMS-V31) with vinyl groups at both ends and a molecular weight of 28,000, 0.6 g of organohydrogenpolysiloxane (Gelest, trade name: HMS-301) with hydrosilyl groups (SiH groups), 0.001 g of platinum compound solution (Gelest, trade name: SIP6829.2) as a crosslinking catalyst, 0.02 g of cure retarder (Gelest, trade name: SIT7900.0), 1.9 g of solid additive "trade name: AEROSIL RX200, Nippon Aerosil Co., Ltd.", and 2.7 g of oil (Shin-Etsu Chemical Co., Ltd., trade name: KF-96-1000CS) without crosslinkable functional groups were weighed and mixed to form a silicone composition. The mixing was carried out in vacuum decompression mode using a mixing device (product name: ARV-310, manufactured by Thinky Corporation) at 2000 rpm for 2 minutes while degassing, to obtain Composition A. Composition A was applied to the mold in the state shown in FIG. 5A, and then uncured Composition B, the same as that used for the vibration transmission member of Example 7, was applied. The upper mold was closed to obtain the state shown in FIG. 5B, and the molds were then secured together with screws (not shown). In this state, the mold was heated in a hot air oven at 130°C for 30 minutes to cure Composition A and Composition B. The mold was then removed from the oven and the upper mold was opened to obtain the composites of Examples 12, 13, and 14. The mold used had a center L1 of 2.1 mm, a peripheral L2 of 2.0 mm, and a diameter of 32 mm. The thickness of the applied Composition A was varied in Examples 12, 13, and 14 to achieve the hardness H1 shown in Table 2.

[0082] (Example 15) 10 g of organopolysiloxane (manufactured by Gelest, trade name: DMS-V31) having vinyl groups at both ends and a molecular weight of 28,000, 0.6 g of organohydrogenpolysiloxane (manufactured by Gelest, trade name: HMS-301) having a hydrosilyl group (SiH group), 0.001 g of platinum compound solution (manufactured by Gelest, trade name: SIP6829.2) as a crosslinking catalyst, 0.02 g of cure retarder (manufactured by Gelest, trade name: SIT7900.0), and 1.5 g of solid additive "trade name: AEROSIL RX200, manufactured by Nippon Aerosil Co., Ltd." were weighed and mixed to form a silicone composition. The composite of Example 15 was obtained in the same manner as in Example 12, except that mixing was carried out while degassing using a mixing device (product name: ARV-310, manufactured by Thinky Corporation) in a vacuum reduced pressure mode at 2000 rpm for 2 minutes to obtain composition A.

[0083] Comparative Example 8 A composite of Comparative Example 8 was obtained in the same manner as in Example 13, except that the same composition as in Comparative Example 2 was used as composition B.

[0084] For the vibration transmission members obtained in Example 7 and Comparative Example 2, and the composites obtained in Examples 12-14 and Comparative Example 8, cell detachment was performed using A549 cells, which have stronger adhesion to the culture substrate than CHO cells, and their effectiveness was evaluated. The evaluation results for each are shown in Table 2. Furthermore, the tan δ, micro rubber hardness type C, specific gravity, post-load strain rate, toluene elution rate, and solid additive content of the vibration transmission members in the laminated composites were measured after removing the laminated high-hardness layer with a razor or similar tool immediately after production, confirming that they were equivalent to those of the unlaminated vibration transmission members alone. In Examples 12 and 15, the composites of the vibration transmission members and high-hardness layers obtained were stored at 23°C for two weeks, and then the variation in detachment rate between different containers was evaluated. While Example 12 showed no change from two weeks prior, Example 15 showed a tendency for a decrease in the average detachment rate in a Nunc Easy Dish 35 mm, making the detachment rate more likely to vary between different containers. The reason for this is believed to be that in the composite of Example 15, the toluene elution rate, i.e., the amount of free oil, was significantly different between the high-hardness layer and the vibration transmission member, causing the free oil to move between the layers and resulting in slight deformation of the shape. Examples 12, 13, 14, and Comparative Example 8 were manufactured again in the same manner, and only the high-hardness layer was recovered with a razor. The toluene elution rate was measured, and all were 17%. Example 15 was also manufactured again in the same manner, and the toluene elution rate of the high-hardness layer was measured, and was 3%. In Table 2, if the evaluation object was an elastomer, composite, or hardness H1 > hardness H2, it was marked with "○", and if not, it was marked with "×".

[0085]

[0086] The present disclosure is not limited to the above-described embodiments, and various modifications and variations are possible without departing from the spirit and scope of the present disclosure. Therefore, the following claims are appended to apprise the public of the scope of the present disclosure.

[0087] This application claims priority based on Japanese Patent Application No. 2024-101950 filed on June 25, 2024 and Japanese Patent Application No. 2025-006915 filed on January 17, 2025, the entire contents of which are incorporated herein by reference.

[0088] REFERENCE SIGNS LIST 1 culture vessel 2 vibration transmission member 3 ultrasonic generator 4 control unit 100 vibration imparting device

Claims

1. A vibration transmission member for applying ultrasonic vibrations to a cell culture in a culture vessel, characterized in that the vibration transmission member is an elastic body, has a micro-rubber hardness type C of 10 degrees or more and 30 degrees or less, a specific gravity of 0.95 or more and 1.30 or less, and a post-load distortion rate of 50% or less.

2. The vibration transmission member according to claim 1, characterized in that the toluene elution rate of said vibration transmission member is 40% or less.

3. A vibration transmission member according to claim 1 or 2, characterized in that the vibration transmission member is made of silicone rubber and contains 10% by volume or less of solid additives.

4. A vibration transmission member according to any one of claims 1 to 3, characterized in that the vibration transmission member has a specific gravity of 0.95 or more and 1.10 or less.

5. The vibration transmission member according to any one of claims 1 to 4, characterized in that it has a surface that contacts the ultrasonic generator and a surface that contacts the culture vessel, and the adhesive force A1 on the surface that contacts the ultrasonic generator and the adhesive force A2 on the surface that contacts the culture vessel satisfy the relationship A1 > A2.

6. A vibration transmission member according to any one of claims 1 to 5, characterized in that the degree of swelling with toluene of the vibration transmission member is 3000% or less, as calculated by (V2 / V1) x 100(%), where V2 is the volume of the vibration transmission member swollen with toluene and V1 is the volume of the vibration transmission member after drying with toluene, as measured by the toluene swelling method.

7. A composite comprising the vibration transmission member according to any one of claims 1 to 6 and a functional layer.

8. The composite described in claim 7, characterized in that it has a high hardness layer as the functional layer, and when the micro-rubber hardness type C of the layer of the composite having a surface in contact with the culture vessel is H1 and the micro-rubber hardness type C of the layer having the opposing surface is H2, H1 > H2 is satisfied.

9. The composite according to claim 8, wherein the toluene elution rate of the vibration transmission member is X1 (%) and the toluene elution rate of the high-hardness layer is X2 (%), and the composite satisfies |X1-X2|≦10.

10. A vibration imparting device comprising an ultrasonic generator for generating vibrations in the ultrasonic band and a vibration transmitting member according to any one of claims 1 to 6.

11. A vibration imparting method comprising imparting ultrasonic vibrations to cells in the culture vessel via a vibration transmission member according to any one of claims 1 to 6, thereby detaching cells adhered to the culture surface of the culture vessel.

12. A cell detachment method comprising applying ultrasonic vibrations to cells in the culture vessel via a vibration transmission member according to any one of claims 1 to 6, thereby detaching cells adhered to the culture surface of the culture vessel.

13. A method for producing a vibration transmission member for applying ultrasonic vibrations to cells on a culture substrate, the method comprising the steps of: mixing a silicone composition containing at least: (A) an organopolysiloxane having alkenyl groups at both ends and a molecular weight of 10,000 or more and 150,000 or less; (B) an organohydrogenpolysiloxane having hydrosilyl groups (SiH groups); (C) a solid additive whose content relative to the total volume of the vibration transmission member is 10 volume % or less; and (D) a crosslinking catalyst to obtain a mixture; and curing and molding the mixture.

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