Cell separation device

Abstract

To provide a cell-peeling device constituted so as to stop a beating member at a position separated from a culture substrate, and facilitating the exchange of the culture substrate.SOLUTION: A cell separation device configured to separate cells that have adhered to a culture surface of a culture substrate from the culture surface by beating the culture substrate, the cell separation device comprising: a beating member configured to beat the culture substrate; and a beating control unit configured to move the beating member and control a position of the beating member, wherein the beating member is stopped at a position away from the culture substrate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to 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 are transferred to a new substrate and cultured, a process known as passaging. During this series of steps, detachment methods have been investigated to recover cells at a high detachment rate without damaging them.

[0003] Patent Document 1 discloses a method for removing cells from a culture vessel in which a vessel holder is caused to collide with a collision target member to impart vibration to the culture vessel. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-113133 Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors of the present application have found that the cell detachment device disclosed in Patent Document 1 has a problem in terms of ease of replacing the culture substrate. [Means for solving the problem]

[0006] The cell detachment device of the present invention is a cell detachment device that detaches cells adhered to a culture surface of a culture substrate from the culture surface by beating the culture substrate, and has a beating member that strikes the culture substrate, and a beating control unit that moves the beating member and controls the position of the beating member, and stops the beating member at a position separated from the culture substrate. [Effects of the Invention]

[0007] According to the cell detachment device of the present invention, the beating member is stopped at a position spaced apart from the culture substrate, which makes it easy to replace the culture substrate. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a schematic diagram of a cell detachment device according to an embodiment of the present invention. [Figure 2] 1 is a schematic cross-sectional view of a cell detachment device according to Example 1 of the present invention. [Figure 3] 4 is a flowchart showing control when the tapping member 2 according to the first embodiment of the present invention is stopped. [Figure 4] 10 is a flowchart showing control when the tapping member 2 according to the second embodiment of the present invention is stopped. [Figure 5] FIG. 10 is a schematic cross-sectional view showing the shape of a cam used in a cell separation device according to Example 3 of the present invention. [Figure 6] 10 is a flowchart showing control when the tapping member 2 is stopped according to the fifth embodiment of the present invention. [Figure 7] 10 is a flowchart showing control when the tapping member 2 is stopped according to the sixth embodiment of the present invention. [Figure 8] 10 is a flowchart showing control when the tapping member 2 according to the seventh embodiment of the present invention is stopped. [Figure 9] FIG. 10 is a schematic diagram of a cell detachment device according to Example 8 of the present invention. [Figure 10] 10 is a flowchart showing control when the tapping member 2 is stopped according to a comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, a cell detachment device according to an embodiment of the present invention will be described, but the present invention is not limited thereto.

[0010] (Cell detachment device) FIG. 1 is a schematic diagram illustrating a cell detachment device 100 according to an embodiment of the present invention. In this embodiment, the cell detachment device 100 vibrates the culture substrate 1 by bringing a beating member 2 into contact with the culture substrate 1 in order to detach cells adhered to the culture surface of the culture substrate 1 from the culture surface. The culture substrate 1 is placed on a mounting section, and the beating member 2 moves, bringing the culture substrate 1 and the beating member 2 into contact. The position and movement of the beating member 2 are controlled by a beating control section 3, and the timing at which the movement of the beating member 2 starts and stops, the drive speed, and other parameters can be freely set. When the position of the beating member 2 is stopped, it is stopped when the culture substrate 1 and the beating member 2 are separated from each other.

[0011] Here, the present inventors discovered that if the movement of the tapping member 2 is stopped while the culture substrate 1 and the tapping member 2 are in contact with each other, the culture substrate 1 and the tapping member 2 interfere with each other when replacing the culture substrate 1 with the next one from which cells are to be detached, resulting in reduced workability. Furthermore, they discovered that the vibration of the culture substrate 1 after beating may not be transmitted sufficiently. Furthermore, they discovered that by stopping the tapping member 2 while the culture substrate 1 and the tapping member 2 are separated, the culture substrate 1 and the tapping member 2 are less likely to get caught when replacing the culture substrate 1, causing the culture medium in the culture substrate 1 to spill. Furthermore, they discovered that when the culture substrate 1 and the tapping member 2 are not in contact with each other, the culture substrate 1 can vibrate with a greater degree of freedom, improving cell detachment performance.

[0012] (slap) In this embodiment, beating involves beating the culture substrate 1 in order to detach at least a portion of the cells adhered to the culture surface of the culture substrate 1 from the culture surface. For example, beating can be performed by at least one of the following methods: beating the culture substrate 1 itself to impart an impact force to the culture substrate 1; or moving the culture substrate 1 and causing it to collide with components constituting the cell detachment device to betray the culture substrate 1. The beating may be performed by directly beating the culture substrate 1. If a holding container or the like is provided to hold the culture substrate 1, the impact force may be imparted to the culture substrate 1 by beating the holding container. The beating in this embodiment may be periodic or aperiodic, and the strength of the impact force imparted to the culture substrate by beating can be set appropriately. Furthermore, the beating 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.

[0013] (Striking member) The hitting member in this embodiment can be a member that can apply an appropriate impact force to the culture substrate and has a mass that does not damage the culture substrate. The shape of the hitting member can be a rod, hammer, or sphere.

[0014] (Tapping control unit) The beating control unit in this embodiment is not particularly limited as long as it can move the beating member and control the position of the beating member. For example, the beating control unit can control the movement of the beating member by controlling the moving means that moves the beating member to collide with the culture substrate. Alternatively, it can control the moving means that moves the culture substrate to collide with a component constituting the cell detachment device. Examples of the moving means include a motor, a solenoid, etc. that can generate power to move an object or a culture substrate when energized. The beating member or culture substrate may be moved using only a motor, a solenoid, etc., or a motor or a solenoid may be used in combination with a component that can store energy, such as a spring.

[0015] The tapping control unit in this embodiment may be configured to control the movement speed of the tapping member. The tapping control unit may also be configured to switch between a tapping drive mode, in which the tapping member taps the culture substrate at a first speed, and a tapping stop mode, in which the tapping member taps the culture substrate at a second speed slower than the first speed. By providing multiple modes, the movement speed of the tapping member can be controlled by switching from the tapping drive mode to the tapping stop mode. For example, the movement speed of the tapping member can be reduced by switching from the tapping drive mode to the tapping stop mode before stopping the movement of the tapping member. Furthermore, after switching to the tapping stop mode, the tapping control unit may control the movement of the tapping member for a predetermined time (Tk) before stopping the movement of the tapping member. Note that the control of the movement speed of the tapping member is not limited to mode switching, and the movement speed may be continuously changed.

[0016] The tapping control unit in this embodiment can move the tapping member so that the tapping member reciprocates at a constant amplitude. The reciprocating motion may be a reciprocating motion with a specific period, such as simple harmonic motion, or a reciprocating motion that includes multiple vibrations with different periods.

[0017] The hammering control unit in this embodiment may be configured to control the position of the hammering member so that the hammering member stops at a position where the distance between the hammering member and the culture substrate is at least half of the amplitude of the hammering member. Alternatively, the hammering member may be stopped at a position where the distance between the hammering member and the culture substrate is greatest during the constant amplitude reciprocating vibration. The details of the hammering member stopping position are described below.

[0018] (Stop control of tapping) In this embodiment, the control for stopping the beating is to stop the beating member 2 at a position separated from the culture substrate 1. If a holding container or the like is provided to hold the culture substrate 1, the beating member 2 is stopped in a state separated from the holding container. Whether the beating member 2 is in contact with the culture substrate 1 may be determined by directly observing the contact state using a commercially available camera or by detecting the position of the beating member 2, a part of the mechanism that imparts power to the beating member 2, the culture substrate 1, etc. When the beating member 2 is periodically moved using a power imparting mechanism such as a motor, the power imparting mechanism is controlled to stop the beating member 2 at a position where it does not come into contact with the culture substrate 1. The beating does not need to be stopped just once. If the beating member 2 and the culture substrate 1 were in contact when the beating member 2 was first stopped, the beating member 2 may be moved again in a direction away from the culture substrate 1 to separate them. Furthermore, when stopped, the beating member 2 may be driven with a force different from that used during beating to separate them. The force to be applied may be an electrically operated force such as that of a motor, or a means for releasing stored force such as a spring, or the force may be applied in a direction away from the culture substrate 1 by the weight of the striking member 2 itself.

[0019] The separation distance is not particularly specified, but is preferably 1 mm or more. A separation distance of 3 mm or more is more preferable. When transferring the culture substrate 1, even if the culture substrate 1 shakes slightly, by separating it by a distance that prevents it from coming into contact with the tapping member 2, workability is improved. Furthermore, when transferring the culture substrate 1 using an automatic machine such as a robot arm, it is preferable to leave a space between the culture substrate 1 and the tapping member 2 so that the arm can be inserted.

[0020] (Vibration generating part) The cell detachment device of this embodiment may have a vibration generating unit that applies ultrasonic vibrations to the culture substrate. Examples of vibrations that can be applied in the ultrasonic band 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 can apply 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 contacted with the outer surface of a culture substrate filled and sealed with a medium 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 region to be treated, allowing ultrasonic waves to be incident on the cells to be detached.

[0021] The vibration generating unit in this embodiment includes at least an ultrasonic vibrator and a vibration plate. The vibration generating unit is not limited as long as it generates ultrasonic vibrations. For example, the vibration generating unit 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] (Position acquisition part) The cell detachment device in this embodiment may have a position acquisition unit that acquires information about the position of the beating member. The information about the position of the beating member may be the position of the beating member itself, or may be the relative position of a component of the cell detachment device (e.g., the culture substrate mounting unit, the ultrasonic generator) as a reference position.

[0025] The tapping control unit may be configured to control the stop position of the tapping member based on information about the position of the tapping member acquired by the position acquisition unit.

[0026] (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.

[0027] 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.

[0028] (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.

[0029] 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.

[0030] (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.

[0031] (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.).

[0032] Among these, it is preferable to use a medium suitable for culturing each cell.

[0033] (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).

[0034] (antibiotics) Examples of antibiotics added to the medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, amphotericin B, and the like.

[0035] (Cell culture conditions) The cell culture conditions can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to a dish, and the cells are cultured at a density of 1.0 × 10 to 5.0 × 10 cells / cm. 2 The cells are seeded in an amount of about 1000 cells per well and cultured in an environment at 37°C and a CO2 concentration of 5%. At this time, it is preferable to culture the cells until the cell occupancy rate in the substrate is about 70 to 80%, that is, until the substrate becomes subconfluent. [Example]

[0036] 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.

[0037] (CHO cell culture on a substrate) CHO cells (Chinese hamster ovary cells) were cultured in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) at a density of 15,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The medium used was Ham's F12 (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued for 48 hours, and the 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%.

[0038] (Culture of A549 cells on a substrate) A549 cells (human alveolar basal epithelial adenocarcinoma cells) were cultured in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) at a density of 15,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture 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). 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 occupation rate of the dish was approximately 80%.

[0039] (C2C12 cell culture on a substrate) C2C12 cells (mouse striated muscle cells) were cultured in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) at a density of 15,000 cells / cm. 2The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture medium used 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 occupation rate of the dish was approximately 80%.

[0040] (Culture of MDCK cells on a substrate) MDCK cells (canine renal tubular epithelial cells) were cultured in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) at a density of 20,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The 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 occupation rate of the dish was approximately 80%.

[0041] (Culture of HEK293 cells on a substrate) HEK293 cells (human embryonic kidney cells) were cultured in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) at a density of 20,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The 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 occupation rate of the dish was approximately 80%.

[0042] (Culture of BAEC cells on a substrate) BAEC cells (bovine aortic endothelial cells) were cultured in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) at a density of 20,000 cells / cm. 2 The cells were seeded at a density of 1000 and cultured at 37°C in a 5% CO2 environment. The culture 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). 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 occupation rate of the dish was approximately 80%.

[0043] (HMSC cell culture on a substrate) hMSC cells (mesenchymal stem cells) were cultured in a Φ35 temperature-responsive container (CellSeed: UpCell 3.5cm dish) at 4000 cells / cm 2 The cells were seeded at a density of 1000 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 the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell coverage of the dish was approximately 80%.

[0044] (Evaluation of effectiveness) The effectiveness was evaluated based on three criteria: the ease of transferring the culture substrate 1, the variability in the peeling time, and the variability in the stopping position of the beating member 2. The ease of transferring the culture substrate 1 was evaluated based on the time from stopping the beating member 2 to completing the transfer of the culture substrate 1. Specifically, the beating member 2 was stopped, the previously placed culture substrate 1 was removed, and a new culture substrate 1 was placed. If the beating member 2 came into contact with the culture substrate 1 and the medium in the culture substrate 1 was spilled, a cleaning process was performed and the time until the new culture substrate 1 was placed was evaluated. The peeling operation was performed five times for each of CHO cells, A549 cells, C2C12 cells, MDCK cells, HEK293 cells, BAEC cells, and hMSC cells. The ease of transferring the culture substrate 1 was evaluated based on the longest time required (in seconds). In this embodiment, the evaluation was based on the following criteria. A: Very good (changeover in less than 5 seconds) B: Good (change in 5 to 10 seconds) C: Effective (replacement in 10 to 15 seconds) D: Poor (change in 15 seconds or more) It was determined that the present invention was effective if the transfer time was less than 15 seconds.

[0045] Variation in detachment time was evaluated based on the variation in the time required for detachment. CHO cells, A549 cells, C2C12 cells, MDCK cells, HEK293 cells, BAEC cells, and hMSC cells were each detached five times. The standard deviation of the detachment time for each cell was divided by the average value of the five times to obtain the individual cell variation value, and evaluation was based on the maximum individual cell variation value. In this embodiment, the evaluation of variation in detachment time was determined based on 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) If the standard deviation / average value is less than 0.5, it is determined that the present invention is effective.

[0046] The variation in the stopping position of the beating member 2 was evaluated by measuring the distance between the culture substrate 1 and the beating member 2 when the beating member 2 was stopped, and then evaluating the variation. First, the distance between the culture substrate 1 and the beating member 2 was measured when five detachments were performed for each of CHO cells, A549 cells, C2C12 cells, MDCK cells, HEK293 cells, BAEC cells, and hMSC cells, and the value was divided by the maximum movement distance of the beating member 2 to calculate the value. Next, the variation in the stopping position was evaluated by calculating the standard deviation of the distance between the culture substrate 1 and the beating member 2 / the maximum movement distance of the beating member 2 obtained from five detachments for each of seven types of cells, for a total of 35 stop controls. In this embodiment, the evaluation of the variation in the stopping position was determined based on the following criteria. A: Very good (standard deviation less than 0.1) B: Good (standard deviation 0.1 or more and less than 0.3) C: Effective (standard deviation 0.3 or more and less than 0.5) D: Poor (standard deviation is 0.5 or more) If the standard deviation / average value is less than 0.5, it is determined that the present invention is effective.

[0047] Example 1 FIG. 2 shows a schematic cross-sectional view of a cell detachment device according to Example 1 of the present invention. A culture substrate 1 placed on an ultrasonic vibrator 4 that generates vibrations in the ultrasonic band is struck by a beating member 2. During detachment, vibration is applied by the ultrasonic vibrator 4 shown in FIG. 1. A beating motor 21 in FIG. 2 is connected to a beating control unit 3 and is supplied with power when beating. Upon receiving power, the beating motor 21 begins to rotate in the direction indicated by the rotation direction of the beating motor in FIG. 2. A cam 22 connected to the beating motor 21 is in contact with the beating member 2, and the beating member 2 can be moved in response to the rotation of the beating motor 21. The beating member 2 is biased by a beating spring 23 in a direction that brings it into contact with the cam 22. The contraction length of the beating spring 23 is regulated by a contraction length adjustment member 26, and the position of the contraction length adjustment member 26 can be adjusted using a contraction length adjustment screw 27. The tapping member 2, pressurized by the tapping spring 23, moves along the slider 24 and taps the culture substrate 1. A photointerrupter 28 connected to the tapping control unit 3 can detect the position information of the cam 22, that is, the position information of the tapping member 2. By applying a voltage of 24 V to the motor 21, the cam 22 is rotated at 150 rpm, and tapping is performed at a frequency of 5 Hz.

[0048] The ultrasonic vibrator 4 is connected to a control means (not shown), and power is supplied from the control means when vibrations in the ultrasonic band are applied. The ultrasonic vibrator 4 and the bottom surface of the culture substrate 1 are in contact, and when power is supplied, the ultrasonic vibrator 4 vibrates the culture substrate 1 at an ultrasonic frequency. A bolt-tightened Langevin vibrator (Honda Electronics Co., Ltd.: HEC-30502) was used as the ultrasonic vibrator 4. An AC voltage of ±20 V at 50 kHz was applied to the ultrasonic vibrator 4 to vibrate the culture substrate 1.

[0049] FIG. 3 is a flowchart showing the control of stopping the tapping member 2 according to Example 1 of the present invention. Detachment was performed on CHO cells, A549 cells, C2C12 cells, MDCK cells, HEK293 cells, BAEC cells, and hMSC cells cultured on the culture substrate 1. At the start of detachment, the tapping control unit 3 applies a normal driving voltage of 24 V to the motor 21 (step S1). The voltages shown in FIG. 3 are the voltages used for detaching CHO cells, and different voltages are used for detaching each type of cell. 30 V was used for A549 cells, 20 V for C2C12 cells, 30 V for MDCK cells, 12 V for HEK293 cells, 12 V for BAEC cells, and 24 V for hMSC cells. After detachment is complete, the tapping control unit receives a stop signal (step S2). Upon receiving the stop signal, the tapping control unit applies a stop driving voltage of 8 V to the motor 21 (step S3). As a result, the motor, which had been rotating at 150 rpm, rotates at 50 rpm. The speed at which the tapping member 2 moves also slows accordingly. The voltage is set to a stop drive voltage, and position information for the tapping member 2 is obtained in the decelerated state from a position detection signal from the photointerrupter 28 (step S4). A detection signal is obtained when the cam 22 interrupts the light from the photointerrupter 28, and at that moment the tapping member 2 is at its farthest point from the culture substrate 1. By setting the voltage to the motor 21 to zero immediately after obtaining the position detection signal in step S4, the tapping member 2 can be stopped with the culture substrate 1 and the tapping member 2 spaced apart (step S5).

[0050] Example 2 FIG. 4 is a flowchart showing the control of stopping the beating member 2 according to Example 2 of the present invention. The cell detachment device was configured similarly to Example 1, and detachment and stop control of the beating member 2 were performed. At the start of detachment, the beating control unit 3 applies 24 V to the motor 21 as a normal drive voltage (step S1). The voltages in FIG. 4 indicate the voltages used to detach CHO cells, as in Example 1. The voltages for each cell type were set similarly to Example 1. After detachment is completed, the beating control unit receives a stop signal (step S2). Upon receiving the stop signal, the beating control unit applies 8 V to the motor 21 as a stop drive voltage (step S3). As a result, the motor, which had been rotating at 150 rpm until then, rotates at 50 rpm. The speed of the beating member 2 also slows accordingly. The stop drive voltage is applied, and position information for the beating member 2 is obtained from the position detection signal from the photointerrupter 28 while it is decelerating (step S4). The beating member is driven at the stop drive voltage until a specified time Tk of 0.5 seconds has elapsed (step S5). The voltage applied to the tapping motor 21 is turned to zero, and the tapping member 2 is stopped (step S6).

[0051] If the specified time Tk is not set, the photointerrupter 28 interrupts light during the stop control process from step S4 onward, causing the photointerrupter to issue a detection signal. The tapping control unit 3 receives the detection signal, determines that the applied voltage to the tapping motor should be zero, and then stops the power supply to the motor. The motor then rotates by inertia and stops. The delay time between the cam 22 interrupting the light from the photointerrupter 28 and the motor actually stopping may vary with each stop control. Accordingly, the distance between the tapping member 2 and the culture substrate 1 may also vary slightly. The purpose of stopping the tapping member 2 after driving for the specified time Tk is to eliminate the delay between the cam 22 interrupting the light from the photointerrupter 28 and the power supply to the motor being stopped. In other words, by controlling the uncontrollable delay time using the specified time Tk, the stopping position of the tapping member 2 can be more accurately controlled.

[0052] Example 3 Example 3 of the present invention uses a cell detachment device that is basically the same as Example 1, but the shape of the cam 22 is different. The control of the beating member 2 when stopped was the same as in Example 1, and cells were detached. Figure 5 shows the shape of the cam used in the cell detachment device of Example 3 of the present invention. When the tip point P of the beating member and point A on the cam 22 are in contact, the separation distance between the culture substrate 1 and the beating member 2 is half the maximum separation distance. When the tip point P of the beating member 2 is in contact with the cam 22 between points C and B, the separation distance between the culture substrate 1 and the beating member 2 is the maximum separation distance. By using a cam 22 with this shape, the separation distance between the beating member 2 and the culture substrate 1 is more than half the maximum separation distance as long as the tip point P of the beating member 2 and the cam 22 are in contact between points A and B.

[0053] Example 4 In Example 4 of the present invention, cell detachment was carried out using a cell detachment device similar to that in Example 3. In Figure 5, when the tip point P of the beating member 2 is in contact with the cam 22 between points A and C, the distance between the beating member 2 and the culture substrate 1 gradually increases from point A to point C. In this example, the beating member 2 is stopped in a state in which the tip point P is in contact with the cam 22 between points A and C. This allows the contracted length of the beating spring 23 when stopped to be longer than when it is fully compressed, thereby improving the durability of the beating spring 23.

[0054] Example 5 In Example 5 of the present invention, cell detachment was performed using a cell detachment device similar to that in Example 1, but the control of stopping the beating member 2 was different. Figure 6 is a flowchart showing the control of stopping the beating member 2 in this example. At the start of detachment, the beating control unit 3 applies 20 V to the motor 21 as a normal driving voltage (Step S1). The voltages in Figure 6 are the voltages used to detach CHO cells, and different voltages were used to detach each type of cell. 22 V was used for A549 cells, 18 V for C2C12 cells, 22 V for MDCK cells, 12 V for HEK293 cells, 12 V for BAEC cells, and 20 V for hMSC cells. Afterward, when detachment is complete, the beating control unit receives a stop signal (Step S2). Upon receiving the stop signal, the beating control unit applies 24 V to the motor 21 as a stop driving voltage (Step S3). As a result, the motor, which had been rotating at 125 rpm until then, rotates at 150 rpm. The speed of the beating member 2 also increases accordingly. The voltage for stopping the tapping member 2 is applied, and position information for the tapping member 2 is obtained from the position detection signal from the photointerrupter 28 (step S4). The cam 22 interrupts the light from the photointerrupter 28, generating a detection signal. At that moment, the tapping member 2 is at its farthest point from the culture substrate 1. The tapping member 2 moves periodically, and signals are periodically sent from the photointerrupter 28. In step S5, the timing of the next signal is predicted based on the interval between the position detection signals. Because the timing of the signal indicates when the culture substrate 1 and the tapping member 2 separate, the delay between receiving the signal and the actual stop is taken into account, and the stop position of the tapping member 2 due to the delay is estimated. In the case of CHO cell detachment in this example, the voltage to the motor was stopped 100 msec earlier than the timing of the next signal, allowing the tapping member 2 to stop when the tapping member 2 and the culture substrate 1 separated (step S6). For other cells as well, by controlling the hitting at an appropriate timing, the hitting member 2 can be stopped in a state where the hitting member 2 and the culture substrate 1 are separated from each other.

[0055] Example 6 In Example 6 of the present invention, cell detachment was performed using a cell detachment device similar to that of Example 1, but the control of stopping the beating member 2 was different. Figure 7 is a flowchart showing the control of stopping the beating member 2 in this example. At the start of detachment, the beating control unit 3 applies 24 V to the motor 21 as a normal driving voltage (step S1). The voltage in Figure 7 indicates the voltage used to detach CHO cells, as in Example 1. The voltage for each cell type was set similarly to Example 1. Afterwards, when detachment is complete, the beating control unit receives a stop signal (step S2). Position information for the beating member 2 is obtained from a position detection signal from the photointerrupter 28 (step S3). A detection signal is obtained when the cam 22 interrupts the light from the photointerrupter 28, and at that moment the beating member 2 is furthest from the culture substrate 1. The beating member 2 moves periodically, and periodic signals are sent from the photointerrupter 28. In step S4, the timing of the next signal is predicted based on the interval between the position detection signals. Because the timing of the signal corresponds to the timing when the culture substrate 1 and the tapping member 2 separate, the stop position of the tapping member 2 due to the delay is estimated, taking into account the delay between the reception of the signal and the actual stop. In the case of CHO cell detachment in this example, by stopping the voltage to the motor 100 msec earlier than the timing of the next signal, it was possible to stop the tapping member 2 when the tapping member 2 and the culture substrate 1 were separated (step S5). For other cells, by controlling at the appropriate timing, it is possible to stop the tapping member 2 when the tapping member 2 and the culture substrate 1 are separated.

[0056] Example 7 In Example 7 of the present invention, cell detachment was performed using a cell detachment device similar to that of Example 1, but the control of stopping the beating member 2 was different. Figure 8 is a flowchart showing the control of stopping the beating member 2 in this example. At the start of detachment, the beating control unit 3 applies 24 V to the motor 21 as a normal driving voltage (step S1). The voltages in Figure 8 indicate the voltages used to detach CHO cells, as in Example 1. The voltages for each cell type were set similarly to Example 1. After detachment is completed, the beating control unit receives a stop signal (step S2). Upon receiving the stop signal, the beating control unit applies 8 V to the motor 21 as a stop driving voltage (step S3). As a result, the motor, which had been rotating at 150 rpm until then, rotates at 50 rpm. The speed of the beating member 2 also slows accordingly. After the stop driving voltage is set, the voltage to the motor 21 is set to zero, stopping the beating member 2 with the culture substrate 1 and the beating member 2 separated (step S5).

[0057] Example 8 9 is a schematic diagram of a cell detachment device according to Example 8 of the present invention. This example differs from Example 1 in that no vibration in the ultrasonic band is applied. Control when the beating member 2 is stopped is performed in the same manner as in Example 1.

[0058] (Comparative Example) FIG. 11 is a flowchart showing control of stopping the tapping member 2 according to the comparative example. At the start of detachment, the tapping control unit 3 applies 24 V to the motor 21 as a normal driving voltage (step S1). The voltages in FIG. 11 are those used when detaching CHO cells, as in Example 1. The voltages for each cell type were set similarly to Example 1. Thereafter, when detachment is complete, the tapping control unit receives a stop signal (step S2). Position information for the tapping member 2 is obtained from a position detection signal from the photointerrupter 28 (step S3). A detection signal is obtained when the cam 22 interrupts the light from the photointerrupter 28. At that moment, the tapping member 2 is at its farthest point from the culture substrate 1. Immediately after receiving the position detection signal in step S4, the voltage to the motor 21 is set to zero (step S4). There is a delay between the signal output from the photointerrupter 28 and the actual stopping of the motor 21, resulting in variations in the stop position depending on factors such as the communication timing and the driving speed of the tapping member 2. When the device was started and stopped five times for each cell, it stopped at least once with the beating member 2 and culture substrate 1 in contact. When the device stopped in a contacting state, it took more than 15 seconds to transfer the cells, resulting in poor transfer workability. Furthermore, when the beating member 2 and culture substrate 1 stopped in a contacting state, the vibrations caused by beating and ultrasonic vibrations were inhibited, which worsened the detachment time and resulted in poor variability in the detachment time. Furthermore, because the driving speed of the beating member 2 differed for each cell, there was also poor variability in the stopping position of the beating member 2.

[0059] [Table 1]

[0060] Table 1 shows the results of evaluation of the ease of replacing the culture substrate (ease of replacing the culture substrate), variation in detachment time, and variation in the stopping position of the beating member for Examples 1 to 8 of the present invention and the comparative example. The results show that the cell detachment device of this example allows for easy replacement of the culture substrate (high ease of replacement).

[0061] Embodiments of the present disclosure include the following methods and compositions. (Configuration 1) A cell detachment device for detaching cells adhered to a culture surface of a culture substrate from the culture surface by beating the culture substrate, the cell detachment device comprising: a beating member for beating the culture substrate; and a beating control unit for moving the beating member and controlling the position of the beating member. A cell detachment device that stops the beating member at a position separated from the culture substrate. (Configuration 2) 2. The cell detachment device according to claim 1, wherein the beating control unit controls the moving speed of the beating member. (Configuration 3) 3. The cell detachment device according to configuration 1 or 2, further comprising a vibration generating unit that applies vibrations in the ultrasonic band to the culture substrate. (Configuration 4) a position acquisition unit that acquires information about the position of the tapping member; The tapping control unit is 4. The cell detachment device according to any one of configurations 1 to 3, wherein the stopping position of the hitting member is controlled based on information about the position of the hitting member. (Configuration 5) The tapping control unit is a beating drive mode in which the beating member is caused to bend against the culture substrate at a first speed; 4. The cell detachment device according to any one of configurations 1 to 3, wherein the moving speed of the beating member is controlled by switching between a beating stop mode in which the beating member is caused to strike the culture substrate at a second speed that is lower than the first speed and a beating stop mode in which the beating member is caused to strike the culture substrate at a second speed that is lower than the first speed. (Configuration 6) The tapping control unit is The cell detachment device according to configuration 5, wherein before stopping the movement of the beating member, the mode is switched from the beating drive mode to the beating stop mode to reduce the movement speed of the beating member. (Configuration 7) The tapping control unit is The cell detachment device according to configuration 6, characterized in that after switching to the beating stop mode, the beating member is moved for a predetermined time (Tk) and then the movement of the beating member is stopped. (Configuration 8) The tapping control unit is 8. The cell detachment device according to any one of configurations 1 to 7, wherein the beating member is moved so as to reciprocate at a constant amplitude. (Configuration 9) The tapping control unit is 9. The cell detachment device according to configuration 8, wherein the position of the beating member is controlled so as to stop the beating member at a position where the distance between the beating member and the culture substrate is equal to or greater than half of the amplitude. (Configuration 10) The tapping control unit is 9. The cell detachment device according to configuration 8, wherein the beating member is stopped at a position where the distance between the beating member and the culture substrate is the greatest. [Explanation of symbols]

[0062] 1 Culture substrate 2. Striking member 3. Tap control section 4. Vibration generating unit 100 Cell detachment device

Claims

1. A cell detachment device that detaches cells adhered to a culture surface of a culture substrate from the culture surface by beating the culture substrate, the cell detachment device comprising: a beating member that strikes the culture substrate; and a beating control unit that moves the beating member and controls the position of the beating member; A cell detachment device that stops the beating member at a position separated from the culture substrate.

2. The cell detachment device according to claim 1 , wherein the beating control unit controls the moving speed of the beating member.

3. The cell detachment device according to claim 1, further comprising a vibration generating unit that applies vibrations in an ultrasonic band to the culture substrate.

4. a position acquisition unit that acquires information about the position of the tapping member; The tapping control unit is The cell detachment device according to claim 1 , wherein the stopping position of the beating member is controlled based on information about the position of the beating member.

5. The tapping control unit is a beating drive mode in which the beating member is caused to bend against the culture substrate at a first speed; 2. The cell detachment device according to claim 1, wherein the moving speed of the beating member is controlled by switching between a beating stop mode in which the beating member is caused to strike the culture substrate at a second speed lower than the first speed and a beating stop mode in which the beating member is caused to strike the culture substrate at a second speed lower than the first speed.

6. The tapping control unit is The cell detachment device according to claim 5, wherein the moving speed of the beating member is reduced by switching from the beating drive mode to the beating stop mode before the movement of the beating member is stopped.

7. The tapping control unit is 7. The cell detachment device according to claim 6, wherein after switching to the beating stop mode, the beating member is moved for a predetermined time (Tk) and then the movement of the beating member is stopped.

8. The tapping control unit is The cell detachment device according to claim 1, wherein the beating member is moved so as to reciprocate at a constant amplitude.

9. The tapping control unit is The cell detachment device according to claim 8, wherein the position of the beating member is controlled so that the beating member is stopped at a position where the distance between the beating member and the culture substrate is equal to or greater than half of the amplitude.

10. The tapping control unit is The cell detachment device according to claim 8, wherein the beating member is stopped at a position where the distance between the beating member and the culture substrate is greatest.

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