Cell detachment method and cell detachment device

Abstract

To provide a cell detachment method that combines ultrasonic vibration and beating to detach cells cultured on a culture substrate, shortening the detachment time and reducing the variation in detachment time.SOLUTION: A cell detachment method for detaching cells adhered to the culture surface of a culture substrate from the culture surface, includes an ultrasonic generation step in which ultrasonic generating means generates ultrasonic waves to vibrate the culture substrate, and a beating step in which the culture substrate is beaten, and the ultrasonic generation step and the beating step being performed with the culture substrate and the ultrasonic generating means positioned within a predetermined area.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] In the field of regenerative medicine, large quantities of cells are required, and there is a particular need for an efficient and stable supply of adherent cells, which account for a large proportion of biological tissues. In the cultivation of adherent cells, the target cells are obtained through the steps of culturing the cells on a culture substrate, such as a polystyrene dish, detaching the cells from the substrate, recovering the cells, and washing them. To further expand the cells, a portion of the acquired cells is transferred to a new substrate and cultured there, a so-called passaging procedure. During this series of steps, detachment methods have been investigated to recover cells at a high detachment rate without damaging them. Patent Document 1 discloses a cell detachment device that applies ultrasonic vibrations to a substrate holding cells and a medium or liquid, thereby detaching at least some of the cells from the substrate.

[0003] Patent Document 2 lists any suitable method including scraping, beating, and / or ultrasonic waves as a method for removing cells from a cell culture vessel, but does not provide details on combining beating and ultrasonic waves. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016-047368 [Patent Document 2] Japanese Patent Application Publication No. 2023-112147 Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to provide a cell detachment method that can shorten the detachment time and reduce the variation in the detachment time by combining ultrasonic vibration and beating on a culture substrate to detach cells that have been cultured on the culture substrate. [Means for solving the problem]

[0006] The cell detachment method of the present invention is a cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, and includes an ultrasonic wave generating step in which an ultrasonic wave generating means generates ultrasonic waves to vibrate the culture substrate, and a beating step in which the culture substrate is beaten, and the ultrasonic wave generating step and the beating step are carried out with the culture substrate and the ultrasonic wave generating means positioned within a predetermined area.

[0007] The cell detachment device of the present invention is a cell detachment device that detaches cells adhered to the culture surface of a culture substrate from the culture surface, and has an ultrasonic generating means that generates ultrasonic waves to impart vibrations in the ultrasonic band to the culture substrate, and a beating means that strikes the culture substrate, and the generation of ultrasonic waves and the beating are carried out with the culture substrate and the ultrasonic generating means positioned within a predetermined area. [Effects of the Invention]

[0008] According to the cell detachment method of the present invention, cells cultured on a culture substrate are detached by combining ultrasonic vibration and beating, which shortens the detachment time and reduces the variation in the detachment time. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a flowchart and a schematic diagram illustrating an example of a cell detachment method and a cell detachment device according to an embodiment of the present invention. [Figure 2] 1 is a schematic cross-sectional view of a configuration for performing a cell detachment method according to Example 1 of the present invention. [Figure 3] 1 is a projection view of an ultrasonic wave generating means 1 and a culture substrate 3 according to a first embodiment of the present invention, seen from above. [Figure 4]1 is a flowchart showing the steps of a cell detachment method according to Example 1 of the present invention. [Figure 5] 1 is a schematic cross-sectional view of a configuration for performing a cell detachment method according to Example 2 of the present invention. [Figure 6] 1 is a projection view of an ultrasonic wave generating means 1 and a culture substrate 3 according to a second embodiment of the present invention, seen from above. [Figure 7] 1 is a schematic cross-sectional view of a configuration for performing a cell detachment method according to Example 3 of the present invention. [Figure 8] 1 is a projection view of an ultrasonic wave generating means 1 and a culture substrate 3 according to a third embodiment of the present invention, seen from above. [Figure 9] 10 is a schematic cross-sectional view of a configuration for performing a cell detachment method according to Example 5 of the present invention. [Figure 10] 1 is a projection view of an ultrasonic generating means 1 and a culture substrate 3 according to a fifth embodiment of the present invention, seen from above. [Figure 11] 10 is a schematic cross-sectional view of a configuration for performing a cell detachment method according to Example 6 of the present invention. [Figure 12] 10 is a projection view of the ultrasonic generating means 1 and the culture substrate 3 of Example 6 of the present invention seen from above. [Figure 13] FIG. 10 is a schematic diagram showing an example of a configuration for performing a cell detachment method according to Example 7 of the present invention. [Figure 14] FIG. 10 is a schematic diagram showing an example of a configuration for performing a cell detachment method according to Example 8 of the present invention. [Figure 15] FIG. 10 is a schematic diagram showing an example of a configuration for performing a cell detachment method according to Example 9 of the present invention. [Figure 16] 10 is a flowchart showing the steps of a cell detachment method according to Example 9 of the present invention. [Figure 17] FIG. 1 is a schematic diagram showing an example of a configuration for performing a cell detachment method according to a comparative example. [Figure 18] Positional relationship between the ultrasonic generating means 1 and the culture substrate 3 in the beating process of the comparative example [Figure 19] 1 is a projection view of the ultrasonic generating means 1 and the culture substrate 3 in the beating step of the comparative example, seen from above. [Figure 20] 1 is a flowchart showing the steps of a cell detachment method according to a comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, a cell detachment method and a cell detachment device according to an embodiment of the present invention will be described, but the present invention is not limited thereto.

[0011] (Cell detachment method) The cell detachment method of this embodiment includes an ultrasonic wave generating step (S101) in which ultrasonic waves generated by ultrasonic wave generating means 1 are transmitted to the culture substrate to vibrate the culture substrate in an ultrasonic band in order to detach cells adhered to the culture surface of the culture substrate 3 from the culture surface. Furthermore, a beating step (S102) is performed in which the culture substrate 3 is struck by a beating means 2 to generate an impact on the culture substrate (FIG. 1(a)). Note that the ultrasonic wave generating step and the beating step are performed with the culture substrate 3 and the ultrasonic wave generating means 1 positioned within a predetermined area (FIG. 1(c)).

[0012] Here, the inventors have found that if the ultrasonic generation step and the beating step are performed when the culture substrate and the ultrasonic generating means are outside of a predetermined range, the cell detachment time may be prolonged or may vary. While investigating a cell detachment method using ultrasonic vibrations and impacts caused by beating, the inventors have found that if ultrasonic generation and beating are performed when the culture substrate and the ultrasonic generating means are located within a predetermined range, the generated ultrasonic waves can be efficiently and stably transmitted to the culture substrate.

[0013] By positioning the culture substrate and the ultrasonic generating means within a specified area, the ultrasonic band vibrations generated by the ultrasonic generating means and the impact caused by beating can be applied to appropriate positions on the culture substrate, shortening the cell detachment time and reducing variation in the detachment rate.

[0014] (prescribed area) The predetermined area in this embodiment is not particularly limited as long as it is an area where vibrations in the ultrasonic band generated by the ultrasonic generating means 1 and impacts due to beating can be applied to appropriate positions on the culture substrate 3. An example of the predetermined area will be described below. In the following description, "viewed from above" means a view from the z direction in FIG. 1(b). "Viewed from above" can also be said to be a view from a direction perpendicular to the vibration plane of an ultrasonic vibrator (described below) included in the ultrasonic generating means 1. In this case, the projection drawings described below can be said to be drawings projected onto a plane parallel to the vibration plane.

[0015] In this embodiment, the predetermined region refers to, for example, a state in which, when the ultrasound generating means 1 and the culture substrate 3 are viewed from above, at least a portion of the projection of the ultrasound generating means 1 and the projection of the culture substrate 3 overlap (i.e., the two projections are in contact). Furthermore, overlapping is preferable to point or line contact between the projections of the ultrasound generating means and the culture substrate. Furthermore, by comparing the area Sd of the projection of the culture substrate 3 viewed from above with the area Su of the projection of the ultrasound generating means 1 viewed from above, the center of gravity of the smaller area may be contained within the larger area. However, when vibrating the culture substrate via a vibration transmitter that propagates ultrasonic vibrations generated by the ultrasound generating means 1, it is sufficient that the ultrasound generating means and the vibration transmitter, and the vibration transmitter and the culture substrate, are in contact. In other words, the projection of the ultrasound generating means and the projection of the culture substrate do not necessarily need to be in contact.

[0016] (Cell detachment device) The cell detachment device 100 according to this embodiment is a cell detachment device for detaching cells adhered to the culture surface of a culture substrate 3 from the culture surface, and includes an ultrasonic wave generating means 1 that generates ultrasonic waves to impart vibrations in the ultrasonic band to the culture substrate 3, and a beating means 2 that beates the culture substrate. Ultrasonic waves are generated and beating is performed while the culture substrate 3 and the ultrasonic wave generating means 1 are positioned within a predetermined area. For example, the control unit 5 adjusts the strength of the beating so that, as a result of beating the culture substrate 3 by the beating means 2, the culture substrate 3 and the ultrasonic wave generating means 1 are positioned within the predetermined area.

[0017] The cell detachment device 100 according to this embodiment may have a position restricting member, and the position of the culture substrate may be restricted by the position restricting member. The position restricting member may have a protrusion at the contact portion with the culture substrate, and may be configured to make point contact with the culture substrate.

[0018] An example of a cell detachment device for carrying out the cell detachment method according to this embodiment of the present invention is specifically shown. FIG. 1(b) is a schematic diagram showing an example of a configuration (cell detachment device) for carrying out the cell detachment method according to this embodiment of the present invention. Ultrasonic wave generating means 1 receives power supply from control means 5 to generate ultrasonic waves and impart vibrations in the ultrasonic band to the culture substrate 3. Hitting means 2 causes a hammer 23 to collide with the culture substrate 3.

[0019] The ultrasonic wave generating step and the beating step are performed with the culture substrate 3 and the ultrasonic wave generating means 1 positioned within a predetermined area. The positional relationship within the predetermined area may or may not change. The ultrasonic wave generating step and the beating step may be performed sequentially or simultaneously. One step may be performed intermittently, and the other step may be performed intermittently. The method of controlling both steps can be appropriately controlled by the practitioner depending on the type of cells to be detached.

[0020] (Ultrasonic wave generation process) Examples of ultrasonic vibrations used in the ultrasonic generation step include vibrations in the frequency band of 10 kHz to 1 MHz. Any ultrasonic generating means can be used without particular limitations, as long as it is capable of applying ultrasonic vibrations to the culture substrate and cells. One example is the use of an ultrasonic oscillator such as lead zirconate titanate (PZT) as the vibrating body. Here, the vibrating body can be directly applied to the outer surface of the culture substrate, which is filled with a medium and sealed, to apply vibrations to the culture substrate. Alternatively, instead of directly contacting the ultrasonic generating means with the culture substrate, an ultrasonic transmitter can be interposed between the ultrasonic generating means and the area to be treated, and ultrasonic waves can be incident on the cells to be ablated.

[0021] The ultrasonic wave generating means in this embodiment includes at least an ultrasonic vibrator 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.

[0022] When the ultrasonic vibrator is a ring-shaped piezoelectric body, it is preferable that the outer diameter of the vibration plate is equal to that 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 utilized for bending.

[0023] Furthermore, commercially available Langevin type transducers or rectangular type transducers can also be used as the ultrasonic transducer of this embodiment. Langevin type transducers have a piezoelectric body sandwiched between two metal blocks and fastened together with bolts or the like to form an integrated structure, and are available from Honda Electronics and Fuji Ceramics, for example.

[0024] (pounding process) The beating step in this embodiment includes beating the culture substrate to detach at least a portion of the cells adhered to the culture surface of the culture substrate from the culture surface. Examples of the beating step include at least one of the following: applying an impact force to the culture substrate by beating the culture substrate itself; and moving the culture substrate and causing it to collide with components constituting a cell detachment device, thereby beating the culture substrate. The beating step in this embodiment may be periodic or aperiodic, and the strength of the impact force applied to the culture substrate by the beating step can be appropriately set. Furthermore, the beating step in this embodiment may be performed continuously from the start of cell detachment to the completion of detachment, or may be completed midway, or may be performed intermittently.

[0025] (beating means) The striking means in this embodiment is not particularly limited as long as it is capable of performing the striking step. The striking means may include, for example, an object that strikes the culture substrate and a moving means for moving the object to strike the culture substrate, or a moving means for moving the culture substrate to strike a component constituting the cell detachment device. The object may have a mass that is sufficient to impart an appropriate striking force to the culture substrate without damaging the culture substrate. Examples of the object's shape include rod-like, hammer-like, and spherical. Examples of the moving means include a motor, solenoid, or the like that can generate power to move the object or culture substrate when energized. Here, the object or culture substrate may be moved using only a motor, solenoid, or the like, or a motor or solenoid may be used in combination with a component capable of storing energy, such as a spring.

[0026] The beating used in the beating step in this embodiment includes a step of applying an impact force to the culture substrate in order to detach cells adhered to the culture surface of the culture substrate from the culture surface. In particular, applying an impact force to the culture substrate can promote detachment. For example, an impact force can be applied to the culture substrate by colliding an object of appropriate mass with the culture substrate. Here, the object to be collided may be rod-shaped, hammer-shaped, or spherical, but the means for applying the impact force are not limited to these. Examples of power sources for driving the object to be collided include methods that generate force by applying electricity, such as a motor or solenoid. These power sources may be used directly or via a member that can store energy, such as a spring.

[0027] The beating step may be configured to apply an impact force to the culture substrate by moving the culture substrate and colliding it with an object. The beating step may be performed periodically. Alternatively, the beating step may be performed continuously until cell detachment is complete. In this case, the period and strength of the beating may be changed as desired. The ultrasonic wave generating step and the beating step may be performed simultaneously at least at a certain time.

[0028] (Other processes) In this embodiment, other steps may be performed in addition to the ultrasonic generation step and the beating step. For example, at least one of a shaking step for shaking the culture substrate, a shaking step for oscillating the culture substrate, and a pipetting step for generating a localized water flow within the culture substrate may be performed.

[0029] In this embodiment, the shaking step can be configured to generate a flow in the medium within the culture substrate by applying acceleration to the culture substrate, the acceleration including a component parallel to the culture surface of the culture substrate on which the cells are cultured. For example, a configuration can be used in which a motor is used to rotate the culture substrate, followed by a uniaxial reciprocating motion using a slide rail or the like. Alternatively, a linear motor or the like can be used to accelerate the culture substrate, thereby applying an acceleration including a component parallel to the culture surface. As long as the shaking can apply an acceleration including a component parallel to the culture surface of the culture substrate, the axis of shaking itself does not necessarily have to be parallel to the culture surface.

[0030] In the pipetting process, a localized water flow is generated to promote detachment of cells from the culture substrate. For example, a method of discharging a liquid using a pipette-like instrument can be used, but the means for generating the water flow is not limited to this.

[0031] These steps may be carried out independently, or may be carried out simultaneously with the ultrasonic wave generating step and the beating step described above, or may not be carried out simultaneously.

[0032] (cell) The cells in this embodiment are not particularly limited as long as they can be cultured in vitro on a culture vessel. For example, 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 can be used. In addition, for example, epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile cells, and the like can be used. Examples of cells that can differentiate include skeletal muscle cells, smooth muscle cells, cardiac muscle cells, neuron cells that make up the nervous system, glial cells, and fibroblasts, hepatic parenchymal cells that are involved in the metabolism of the living body, non-parenchymal liver cells, and adipocytes, as well as various stem cells such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, as well as progenitor cells of various tissues, and cells induced to differentiate from these.

[0033] The cells in this embodiment may be individual cells (so-called single cells) or sheet-like cell cultures (cell sheets). The cell detachment method according to this embodiment is suitable for cells with strong intercellular bonds, cells with high adhesive strength to a substrate, and cells with high trypsin sensitivity. Given the need for mass 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.

[0034] (Culture substrate) The substrate in this embodiment refers to a culture substrate used for cell culture. The culture substrate is not particularly limited as long as it is a cell-adherent culture substrate, and examples thereof include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, multi-well plates, multi-plates, Petri dishes, culture bags, bottles, etc.

[0035] The material of the culture substrate 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.

[0036] (buffer) 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 solutions, phosphate buffer solutions, HEPES buffer solutions, citrate-phosphate buffer solutions, glycylglycine-sodium hydroxide buffer solutions, Britton-Robinson buffer solutions, and GTA buffer solutions. Among these, phosphate buffer solutions that are close to the in vivo environment are preferred, and phosphate-buffered saline (PBS), which has been adjusted to be isotonic with intracellular fluid, is more preferably used.

[0037] (Culture medium) The type of culture medium is not particularly limited, 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), alpha Modified Eagle's Minimum Essential Medium (αMEM), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemSpan H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma-Aldrich), Endothelial Cell Growth Examples include Medium 2 Kit (Promocell), Mesenchymal Stem Cell Growth Medium 2 (Promocell), MSCGM Bullet Kit (Lonza), mTeSR1 or 2 medium (Stem Cell Technology), Repro FF or Repro FF2 (ReproCell), NutriStem medium (Biological Industries), and MF-Medium mesenchymal stem cell growth medium (Toyobo Co., Ltd.).

[0038] Among these, it is preferable to use a medium suitable for culturing each cell.

[0039] (serum) The above-mentioned medium may contain serum or antibiotics. 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 contain neither raw nor unpurified serum, but may contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0040] (antibiotics) Examples of antibiotics added to the medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, amphotericin B, and the like.

[0041] (Cell culture conditions) Cell culture conditions can be selected appropriately depending on the cells to be cultured. Generally, an appropriate medium is added to a dish, and cells are seeded therein at approximately 1.0 x 10 to 5.0 x 10 cells / cm. The cells are then cultured at 37°C and in a CO2 concentration of 5%. Preferably, the cells are cultured until the cell occupies approximately 70% to 80% of the surface area of ​​the substrate, a so-called subconfluent state. [Example]

[0042] Examples to which the present invention is applied will be described below, but unless otherwise specified, the scope of the present invention is not intended to be limited to these examples.

[0043] (CHO cell culture on a substrate) CHO (Chinese Hamster Ovary Cell) cells were seeded at a density of 15,000 cells / cm2 in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) and cultured at 37°C in a 5% CO2 environment. The culture 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 continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage of the dish was approximately 80%.

[0044] (Culture of A549 cells on a substrate) A549 cells (human alveolar basal epithelial adenocarcinoma cells) were seeded at a density of 15,000 cells / cm2 in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) and cultured at 37°C in a 5% CO2 environment. The culture medium 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 continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage of the dish was approximately 80%.

[0045] (C2C12 cell culture on a substrate) C2C12 cells (mouse striated muscle cells) were seeded at a density of 15,000 cells / cm2 in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) and cultured at 37°C in a 5% CO2 environment. The culture medium was DMEM / F12 (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage of the dish was approximately 80%.

[0046] (Culture of MDCK cells on a substrate) MDCK cells (canine renal tubular epithelial cells) were seeded at a density of 20,000 cells / cm2 in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) and cultured at 37°C in a 5% CO2 environment. The culture medium used was Eagle's MEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage of the dish was approximately 80%.

[0047] (Culture of HEK293 cells on a substrate) HEK293 cells (human embryonic kidney cells) were seeded at a density of 20,000 cells / cm2 in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) and cultured at 37°C in a 5% CO2 environment. The culture medium used was Eagle's MEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage of the dish was approximately 80%.

[0048] (Culture of BAEC cells on a substrate) BAEC (bovine aortic endothelial cells) cells were seeded at a density of 20,000 cells / cm2 in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) and cultured at 37°C in a 5% CO2 environment. The culture medium 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 continued for 48 hours, and the cells were observed under a phase-contrast microscope to confirm the formation of a cell sheet. The cell coverage of the dish was approximately 80%.

[0049] (HMSC cell culture on a substrate) hMSC cells (mesenchymal stem cells) were seeded at a density of 4000 cells / cm2 in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) and cultured at 37°C in a 5% CO2 environment. The culture medium used was Mesenchymal Stem Cell Growth Medium 2 (Promocell) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). The culture medium was changed every 48 hours for 7 days, and cell adhesion and proliferation were confirmed by observing the cells under a phase-contrast microscope. The cell coverage of the dish was approximately 80%.

[0050] (Evaluation of effectiveness) The effectiveness was evaluated based on two criteria: detachment time and variability in detachment time. The detachment time was evaluated based on the time required to complete detachment of cells cultured on the culture substrate. However, since the time required to complete detachment varies depending on the target cell type, the degree of effectiveness was evaluated by comparison with the comparative example described below. In this embodiment, the evaluation of detachment time was determined based on the following criteria. A: Very good (peeling time 21% or more shorter than the comparative example) B: Good (peeling time 11 to 20% shorter than the comparative example) C: Effective (peeling time 1 to 10% shorter than the comparative example) D: Inferior (peel time is the same as the comparative example)

[0051] If the peeling time was 1% or more shorter than that of the comparative example, it was determined that the present invention was effective.

[0052] The variation in detachment time was evaluated by dividing the standard deviation of the detachment time when the cell culture was detached five times by the average value of the five times. In this embodiment, the evaluation of the variation in detachment time was judged according to the following criteria. A: Very good (standard deviation / average value less than 0.1) B: Good (standard deviation / average value is 0.1 or more and less than 0.3) C: Effective (standard deviation / average value is 0.3 or more and less than 0.5) D: Poor (standard deviation / average value is 0.5 or more)

[0053] If the standard deviation / average value is less than 0.5, it is determined that the present invention is effective.

[0054] Example 1 Figure 2 shows a schematic cross-sectional view of the configuration of a cell detachment device for carrying out the cell detachment method according to Example 1 of the present invention. The ultrasonic wave generating means 1 is connected to the control means 5, and power is supplied from the control means 5 when the ultrasonic wave generation step is carried out. The ultrasonic wave generating means 1 and the culture substrate 3 are in contact with each other at the bottom surface of the culture substrate 3, and the ultrasonic wave generating means 1 supplied with power ultrasonically vibrates the culture substrate 3. A bolt-tightened Langevin (HEC-30502, manufactured by Honda Electronics Co., Ltd.) was used as the ultrasonic wave generating means 1. An AC voltage of ±20 V at 50 kHz was applied to the ultrasonic wave generating means 1 to vibrate the culture substrate 3.

[0055] The motor 21 of the tapping means 2 is connected to the control means 5, and is supplied with power when the tapping process is performed. When power is supplied, the motor 21 begins to rotate in the direction indicated by the rotation direction of the shaft in FIG. 2. A cam 22 connected to the motor 21 is in contact with a hammer 23, and the hammer 23 can be moved as the motor 21 rotates. The hammer 23 is pressurized by a tapping spring 24 in a direction that brings it into contact with the cam 22. The contraction length of the tapping spring 24 is regulated by a contraction length adjustment member 25, and the position of the contraction length adjustment member 25 can be adjusted by a contraction length adjustment screw 26. The hammer 23, pressurized by the tapping spring 24, taps the culture substrate 3 along a slider 27. By applying a voltage of 24 V to the motor 21, the cam rotated at 150 rpm, and tapping was performed at a frequency of 5 Hz.

[0056] FIG. 3 shows a projection view of the ultrasound generating means 1 and culture substrate 3 of Example 1, viewed from above. Comparing the areas of the projection views of the ultrasound generating means 1 and the culture substrate 3, the area Su of the ultrasound generating means 1 is smaller than the area Sd of the culture substrate 3. Furthermore, in the arrangement of Example 1, the center of gravity of the projection view of the ultrasound generating means 1 is contained within the projection view of the culture substrate 3. In this way, by ensuring that the center of gravity on the smaller area side is contained within the projection view on the larger area side, the ultrasound generated by the ultrasound generating means 1 can vibrate the entire culture substrate 3. Furthermore, by performing the ultrasound generation step and the beating step with this arrangement, cells can be detached stably in a shorter detachment time.

[0057] CHO cells cultured on a culture substrate 3 were subjected to detachment. FIG. 4 is a flowchart showing the steps of the cell detachment method according to Example 1. After detachment begins, in step S1, the culture substrate 3 is set in a predetermined position. In step S2, an ultrasonic wave generation step and a beating step are performed. In step S3, it is visually confirmed whether cell detachment is complete. If detachment is complete, the detachment operation is terminated. If detachment is not complete, the process returns to step S2, and the ultrasonic wave generation step, the beating step, and confirmation of detachment completion are repeatedly performed until detachment is complete.

[0058] Example 2 Figure 5 shows a schematic cross-sectional view of the configuration for performing the cell detachment method according to Example 2 of the present invention. The ultrasonic wave generating means 1 is connected to the control means 5, and power is supplied from the control means 5 when performing the ultrasonic wave generation step. The ultrasonic wave generating means 1 and the culture substrate 3 are in contact with each other at the side of the culture substrate 3, and the ultrasonic wave generating means 1 supplied with power ultrasonically vibrates the culture substrate 3. A bolt-tightened Langevin (HEC-1340P4BF, manufactured by Honda Electronics Co., Ltd.) was used as the ultrasonic wave generating means 1. An AC voltage of ±40 V at 40 kHz was applied to the ultrasonic wave generating means 1 to vibrate the culture substrate 3.

[0059] 6 shows a projection view of the ultrasonic wave generating means 1 and culture substrate 3 of Example 2 of the present invention seen from above. The projection view of the ultrasonic wave generating means 1 and culture substrate 3 shows that they are in contact with each other just at the outer periphery of the culture substrate 3. The ultrasonic waves emitted from the ultrasonic wave generating means 1 travel along the side of the culture substrate 3 and ultrasonically vibrate the bottom surface on which the cells are cultured. By performing the ultrasonic wave generating step and the beating step in an arrangement in which the ultrasonic wave generating means 1 and culture substrate 3 are in contact with each other, cells can be detached stably in a shorter time.

[0060] The configuration of the beating means 2, the driving conditions of the beating means 2, the procedure of the detachment method, and the cell type to be cultured on the culture substrate 3 were the same as in Example 1, and the detachment was carried out.

[0061] Example 3 FIG. 7 shows a schematic cross-sectional view of the configuration for performing the cell detachment method according to Example 3 of the present invention. The ultrasonic wave generating means 1 is connected to the control means 5, and power is supplied from the control means 5 when performing the ultrasonic wave generation step. The ultrasonic wave generating means 1 and the culture substrate 3 are in contact with each other while being offset from the bottom surface of the culture substrate 3, and the ultrasonic wave generating means 1 supplied with power ultrasonically vibrates the culture substrate 3. A bolt-tightened Langevin (HEC-30502, manufactured by Honda Electronics Co., Ltd.) was used as the ultrasonic wave generating means 1. An AC voltage of ±20 V at 50 kHz was applied to the ultrasonic wave generating means 1 to vibrate the culture substrate 3.

[0062] 8 shows a projection view of the ultrasonic wave generating means 1 and culture substrate 3 of Example 3 of the present invention, viewed from above. The projection views of the ultrasonic wave generating means 1 and culture substrate 3 are arranged so that they partially overlap. By bringing them into contact in an offset state like this, ultrasonic vibrations can be generated in a focused manner at a specific location on the bottom surface of the culture substrate 3. By performing the ultrasonic wave generating step and the beating step in an arrangement where the projection views of the ultrasonic wave generating means 1 and culture substrate 3 partially overlap, cells can be detached stably in a shorter time.

[0063] The configuration of the beating means 2, the driving conditions of the beating means 2, the procedure of the detachment method, and the cell type to be cultured on the culture substrate 3 were the same as in Example 1, and the detachment was carried out.

[0064] Example 4 FIG. 9 shows a schematic cross-sectional view of a configuration for performing a cell detachment method according to Example 4 of the present invention. The ultrasound generating means 1 is connected to a control means 5, and power is supplied from the control means 5 when performing the ultrasound generation step. The ultrasound generating means 1 and the culture substrate 3 are in contact with each other at the side of the culture substrate 3 via an ultrasound transmitter 4. The ultrasound generating means 1, to which power is supplied, ultrasonically vibrates the culture substrate 3 via the ultrasound transmitter 4. A bolt-tightened Langevin (HEC-1340P4BF, manufactured by Honda Electronics Co., Ltd.) was used as the ultrasound generating means 1. Silicone rubber with a thickness of 1 mm was used as the ultrasound transmitter 4. An AC voltage of ±40 V at 40 kHz was applied to the ultrasound generating means 1 to vibrate the culture substrate 3.

[0065] FIG. 10 shows a projection view of the ultrasonic wave generating means 1 and culture substrate 3 of Example 4, seen from above. The projection view of the ultrasonic wave generating means 1 and culture substrate 3 shows that they are not in direct contact. However, they are in contact via an ultrasonic wave transmitting material 4. The ultrasonic waves emitted from the ultrasonic wave generating means 1 vibrate the side of the culture substrate 3 via the ultrasonic wave transmitting material 4, causing ultrasonic vibrations in the bottom surface connected to the side. In this way, even when the ultrasonic wave generating means 1 and culture substrate 3 are not in direct contact, ultrasonic vibrations can be transmitted via the intervening material. Furthermore, by performing the ultrasonic wave generating step and the beating step in this state, cells can be detached stably in a shorter time.

[0066] The configuration of the beating means 2, the driving conditions of the beating means 2, the procedure of the detachment method, and the cell type to be cultured on the culture substrate 3 were the same as in Example 1, and the detachment was carried out.

[0067] Example 5 11 shows a schematic cross-sectional view of the configuration for carrying out the cell detachment method according to Example 5 of the present invention. The ultrasonic wave generating means 1 is connected to the control means 5, and power is supplied from the control means 5 when the ultrasonic wave generation step is carried out. The ultrasonic wave generating means 1 and the culture substrate 3 are in contact with each other at the bottom surface of the culture substrate 3, and the ultrasonic wave generating means 1 supplied with power ultrasonically vibrates the culture substrate 3. A bolt-tightened Langevin (HEC-45402, manufactured by Honda Electronics Co., Ltd.) was used as the ultrasonic wave generating means 1. An AC voltage of ±20 V at 40 kHz was applied to the ultrasonic wave generating means 1 to vibrate the culture substrate 3.

[0068] FIG. 12 shows a top view of the ultrasound generating means 1 and culture substrate 3 of Example 5 of the present invention. Comparing the areas of the ultrasound generating means 1 and culture substrate 3, the area Sd of the culture substrate 3 is smaller than the area Su of the ultrasound generating means. Furthermore, in the arrangement of Example 5, the center of gravity of the projection of the culture substrate 3 is contained within the projection of the culture substrate 3. In this way, by ensuring that the center of gravity of the smaller area side is contained within the projection of the larger area side, the ultrasound generated by the ultrasound generating means 1 can vibrate the entire culture substrate 3. Furthermore, by performing the ultrasound generation step and the beating step with this arrangement, cells can be detached stably in a shorter detachment time.

[0069] The configuration of the beating means 2, the driving conditions of the beating means 2, the procedure of the detachment method, and the cell type to be cultured on the culture substrate 3 were the same as in Example 1, and the detachment was carried out.

[0070] Example 6 An example of a configuration (cell detachment device) for performing a cell detachment method according to Example 6 of the present invention will be described with reference to FIG. 13. FIG. 13(a) is a cross-sectional view of the cell detachment device, and FIG. 13(b) is a partial top view (viewed from the z-direction) of the cell detachment device shown in FIG. 13(a). The position control member 6 is made of aluminum and is fixed to the housing 7. The position control member 6 is positioned with a slight gap from the culture substrate 3 to prevent the culture substrate 3 from moving during the beating process, thereby preventing the desired positional relationship between the culture substrate 3 and the ultrasonic generating means 1 from deviating from the desired state. This allows the desired positional relationship between the culture substrate 3 and the ultrasonic generating means 1 to be maintained even when the beating process is repeatedly performed over a long period of time, enabling stable cell detachment in a short detachment time (reducing variability in cell detachment time).

[0071] The positional relationship between the ultrasonic generating means 1 and the culture substrate 3, the driving conditions of the ultrasonic generating means 1, the configuration of the beating means 2, the driving conditions of the beating means 2, the procedure of the detachment method, and the cell type to be cultured on the culture substrate 3 were the same as in Example 3, and the detachment was performed.

[0072] Example 7 1 shows a schematic diagram illustrating an example of a configuration for performing a cell detachment method according to Example 7 of the present invention. The position restriction member 6 is made of aluminum and is fixed to a housing 7. The position restriction member 6 has a protrusion 61 on the curved surface facing the culture substrate 3. When the culture substrate 3 moves toward the position restriction member 6 during the beating step, the culture substrate 3 and the position restriction member 6 come into point contact. If the culture substrate 3, which is ultrasonically vibrating during the ultrasonic generation step, comes into surface contact with the position restriction member 6, the ultrasonic vibration is inhibited. However, if the contact between the two is point contact, the ultrasonic vibration is not significantly inhibited, and the effects of the beating step and the ultrasonic generation step can be maximized.

[0073] The positional relationship between the ultrasonic generating means 1 and the culture substrate 3, the driving conditions of the ultrasonic generating means 1, the configuration of the beating means 2, the driving conditions of the beating means 2, the procedure of the detachment method, and the cell type to be cultured on the culture substrate 3 were the same as in Example 3, and the detachment was performed.

[0074] Example 8 1 shows a schematic diagram illustrating an example of a configuration for performing a cell detachment method according to Example 8 of the present invention. The housing 7 is supported by linear rails 8 and can be shaken in a shaking step. In the shaking step, shaking was performed under conditions of an amplitude of 20 mm and a frequency of 2 Hz.

[0075] 16 is a flowchart showing the steps of the cell detachment method according to Example 8. After detachment begins, in step S1, the culture substrate 3 is set in a predetermined position. In step S2, an ultrasonic wave generation step, a beating step, and a shaking step are performed. In step S3, it is visually confirmed whether cell detachment is complete. If detachment is complete, the detachment operation is terminated. If detachment is not complete, the process returns to step S2, and the ultrasonic wave generation step, the beating step, and confirmation of detachment completion are repeatedly performed until detachment is complete.

[0076] The positional relationship between the ultrasonic generating means 1 and the culture substrate 3, the driving conditions of the ultrasonic generating means 1, the configuration of the beating means 2, the driving conditions of the beating means 2, and the cell type to be cultured on the culture substrate 3 were the same as in Example 3, and the detachment was performed.

[0077] (Examples 9 to 14) In Examples 9 to 14 of the present invention, the same detachment method as in Example 1 was used. The only difference was that the cell types to be cultured were as shown in Table 1.

[0078] (Comparative Examples 1 to 7) Fig. 17 is a schematic diagram showing an example of a configuration for performing a cell detachment method according to a comparative example. When performing the beating step, the culture substrate 3 is placed within reach of the hammer 23 of the beating means 2. When performing the ultrasonic wave generating step, the culture substrate 3 is placed directly above the ultrasonic wave generating means 1. Fig. 18 shows the positional relationship between the ultrasonic wave generating means 1 and the culture substrate 3 when performing the beating step. Fig. 19 shows a projection view of the ultrasonic wave generating means 1 and the culture substrate 3 viewed from above when performing the beating step. The two projection views are separated, and in this state, the ultrasonic vibrations generated by the ultrasonic wave generating means 1 cannot be sufficiently transmitted to the culture substrate 3.

[0079] 20 is a flowchart showing the steps of a cell detachment method according to a comparative example. After detachment begins, in step S1, the culture substrate 3 is set on the ultrasonic wave generating means 1. In step S2, the ultrasonic wave generating step is carried out. In step S3, the culture substrate 3 is set next to the beating means 2. In step S4, the beating step is carried out. In step S5, it is visually confirmed whether cell detachment is complete. If detachment is complete, the detachment operation is terminated. If detachment is not complete, the process returns to step S1, and the ultrasonic wave generating step, beating step, and confirmation of detachment completion are repeated while replacing the culture substrate 3 until detachment is complete.

[0080] The driving conditions of the ultrasonic wave generating means 1, the configuration of the beating means 2, and the driving conditions of the beating means 2 were the same as in Example 1, and peeling was carried out.

[0081] [Table 1]

[0082] Table 1 shows the results of evaluation of the peeling time and the variation in peeling time for Examples 1 to 14 of the present invention and Comparative Examples 1 to 7.

[0083] The peeling time in Example 1 was 25% shorter than that in Comparative Example 1, which was an extremely good result. The standard deviation / average value for the variation in peeling time was also very good at 0.09. This is thought to be because the center of gravity of the projection of the ultrasonic generating means 1 is contained within the projection of the culture substrate 3, and the entire culture substrate 3 can be firmly vibrated by the ultrasonic waves generated by the ultrasonic generating means 1. Furthermore, by performing the beating process in this state, the peeling time was short and the variation in the peeling time was small, which is thought to be an extremely good result.

[0084] In Example 2, the ultrasonic wave generating means 1 and the culture substrate 3 come into contact at one point on the outer periphery of the culture substrate 3. When transmitting ultrasonic vibrations, the vibrations are transmitted from a portion of the surface on which the cells in the culture substrate 3 are cultured, so the detachment time is longer than in Example 1. For the same reason, the detachment time also varies more than in Example 1. However, because the ultrasonic wave generating means 1 and the culture substrate 3 come into contact, albeit at one point, the detachment time and the detachment time variation were both more effective than in Comparative Example 1.

[0085] In Example 3, the contact area between the ultrasonic generating means 1 and the culture substrate 3 is increased compared to Example 2, and as a result, the peeling time is shortened and the variation in peeling time is reduced.

[0086] In Example 4, the ultrasonic generating means 1 is not in contact with the culture substrate 3. However, since the ultrasonic waves generated by the ultrasonic generating means 1 are transmitted to the culture substrate 3 via a transmitter, the effects of the peeling time and the variation in the peeling time are the same as those in Example 2.

[0087] In Example 5, the center of gravity of the projection of the culture substrate 3 is contained within the projection of the ultrasonic generating means 1. Therefore, the entire culture substrate 3 can be firmly vibrated by the ultrasonic waves generated by the ultrasonic generating means 1, and by carrying out the beating step in this state, excellent results were obtained, with a short peeling time and little variation in the peeling time.

[0088] In Example 6, the variation in peeling time is smaller than in Example 3. This is due to the effect of the position control member 6. The positional relationship between the ultrasonic generating means 1 and the culture substrate 3 may shift slightly during the beating process, but the position control member 6 can reduce this shift. As a result, the variation in peeling time can be reduced. The deterioration in peeling time is due to the surface contact between the culture substrate 3 and the position control member 6, which slightly inhibits the ultrasonic vibration.

[0089] The peeling time in Example 7 is shorter than that in Example 6. When the culture substrate 3, which is ultrasonically vibrated by the ultrasonic generation process, comes into surface contact with the position control member 6, the ultrasonic vibration is inhibited. However, if the contact between the two is point contact, the ultrasonic vibration is not inhibited, which contributes to shortening the peeling time.

[0090] The peeling time in Example 8 is shorter than that in Example 6. This is due to the effect of the shaking process. Shaking the culture substrate 3 generates a flow in the liquid in the culture substrate 3, which promotes peeling.

[0091] In Comparative Examples 1 to 7, the position of the culture substrate 3 is changed between the ultrasonic generation step and the beating step. The time loss caused by the repositioning and the variation in peeling time due to the variation in position resulted in no effect of the present invention.

[0092] Embodiments of the present disclosure include the following methods and compositions.

[0093] [Method 1] A cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, the method comprising: an ultrasonic wave generating step in which an ultrasonic wave generating means generates ultrasonic waves to vibrate the culture substrate; and a beating step in which the culture substrate is beated, wherein the ultrasonic wave generating step and the beating step are carried out with the culture substrate and the ultrasonic wave generating means positioned within a predetermined area.

[0094] [Method 2] The cell detachment method described in Method 1, wherein the state in which the culture substrate is located in the specified area is a state in which a projection image of the culture substrate viewed from above and a projection image of the ultrasound generating means viewed from above at least partially overlap.

[0095] [Method 3] The state of being located within the predetermined area is the area Sd of a projection view of the culture substrate viewed from above, A cell detachment method according to Method 1, wherein the area Su of the projection of the ultrasonic generating means viewed from above is such that the center of gravity of the side with the smaller area is contained within the projection of the side with the larger area.

[0096] [Method 4] The cell detachment method described in Method 1, wherein the ultrasonic wave generating step and the beating step are carried out simultaneously at least at some point in time.

[0097] [Method 5] The cell detachment method according to Method 1, wherein the position of the culture substrate is regulated by a position regulating member.

[0098] [Method 6] The cell detachment method described in Method 1, wherein the position control member has a protrusion at a contact portion with the culture substrate and is configured to make point contact with the culture substrate.

[0099] [Configuration 1] A cell detachment device that detaches cells adhered to a culture surface of a culture substrate from the culture surface, the cell detachment device having an ultrasonic generating means that generates ultrasonic waves to impart vibrations in the ultrasonic band to the culture substrate, and a beating means that strikes the culture substrate, wherein the ultrasonic generation and beating are carried out while the culture substrate and the ultrasonic generating means are positioned within a predetermined area.

[0100] [Configuration 2] The cell detachment device according to configuration 1, wherein the cell detachment device has a position control member, and the position of the culture substrate is controlled by the position control member.

[0101] [Configuration 3] 3. The cell detachment device according to claim 2, wherein the position restriction member has a protrusion at a contact portion with the culture substrate and is configured to come into point contact with the culture substrate. [Explanation of symbols]

[0102] 1. Ultrasonic wave generating means 2 Hitting means 3 Culture substrate 5. Control measures 100 Cell detachment device

Claims

1. A cell detachment method for detaching cells adhered to a culture surface of a culture substrate from the culture surface, the method comprising: an ultrasonic wave generating step in which an ultrasonic wave generating means generates ultrasonic waves to vibrate the culture substrate; and a beating step in which the culture substrate is beated; The cell detachment method, wherein the ultrasonic wave generating step and the beating step are carried out with the culture substrate and the ultrasonic wave generating means positioned within a predetermined area.

2. The cell detachment method described in claim 1, wherein the state in which the culture substrate is located in the predetermined area is a state in which a projection image of the culture substrate viewed from above and a projection image of the ultrasonic generating means viewed from above at least partially overlap.

3. The state of being located within the predetermined area is the area Sd of a projection view of the culture substrate viewed from above, 2. The cell detachment method according to claim 1, wherein the area Su of the projection of the ultrasonic generating means viewed from above is such that the center of gravity of the side with the smaller area is included within the projection of the side with the larger area.

4. The cell detachment method according to claim 1 , wherein the ultrasonic wave generating step and the beating step are carried out simultaneously at least at some point in time.

5. The cell detachment method according to claim 1 , wherein the position of the culture substrate is restricted by a position restricting member.

6. The cell detachment method according to claim 1 , wherein the position restriction member has a protrusion at a contact portion with the culture substrate and is configured to make point contact with the culture substrate.

7. A cell detachment device that detaches cells adhered to a culture surface of a culture substrate from the culture surface, the cell detachment device having an ultrasonic generating means that generates ultrasonic waves to impart vibrations in the ultrasonic band to the culture substrate, and a beating means that strikes the culture substrate, wherein the ultrasonic generation and beating are carried out while the culture substrate and the ultrasonic generating means are positioned within a predetermined area.

8. The cell detachment device according to claim 7 , further comprising a position control member, and the position of the culture substrate is controlled by the position control member.

9. The cell detachment device according to claim 8 , wherein the position restriction member has a protrusion at a contact portion with the culture substrate and is configured to make point contact with the culture substrate.

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