Cell detachment system and cell detachment method

Abstract

To provide a cell detachment system that efficiently detaches a cell sheet culture while reducing damage to the cell sheet culture.SOLUTION: A cell detachment system detaches a cell sheet culture adhering to a culture container from the culture container, the system comprising detachment start means that starts detachment of the cell sheet culture, information acquisition means that acquires information regarding a detachment progressing direction of the cell sheet culture after start of the detachment of the cell sheet culture, shaking means that shakes the culture container, and setting means that, on the basis of the information regarding the progressing direction, sets the progressing direction and a direction of the shaking to be substantially parallel to each other.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] In recent years, in the fields of regenerative medicine and cell therapy, attempts have been made to culture cells in the form of a sheet and transplant the sheet-like cell culture (cell sheet) into the affected area in order to repair damaged tissue. When producing cell sheets using adhesive cells, for example, the cells are cultured in the form of a sheet on a polystyrene dish, an example of a culture substrate, and then detached and collected in the sheet form from the culture substrate. Studies are being conducted to develop a manufacturing method that can efficiently and stably produce cell sheets while reducing damage such as wrinkles, tears, and holes in the cell sheet.

[0003] Patent document 1 discloses a cell detachment device that has a container holder for attaching a culture container to which cultured cells are attached, and a guide mechanism that guides the reciprocating movement of the container holder, and that collides the container holder against a non-collision member. [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] When detaching a cell sheet from a culture vessel holding a liquid and having a cell sheet attached thereto, if the direction of reciprocating movement of the culture vessel is the same as the direction of impact on the culture vessel, the cell sheet may be easily damaged.Furthermore, when conditions are set to detach the cell sheet more efficiently in a shorter time, the cell sheet may be easily damaged.

[0006] Therefore, an object of the present invention is to provide a cell detachment system that efficiently detaches a cell sheet while reducing damage to the cell sheet. Another object of the present invention is to provide a cell detachment method that efficiently detaches a cell sheet while reducing damage to the cell sheet. [Means for solving the problem]

[0007] The above object is achieved by the present invention, which provides a cell detachment system for detaching a cell sheet adhered to a culture vessel from the culture vessel, the cell detachment system comprising: a detachment initiation means for initiating detachment of the cell sheet; an information acquisition means for acquiring information about the direction of detachment of the cell sheet after detachment of the cell sheet has begun; a shaking means for shaking the culture vessel; and a setting means for setting the direction of detachment to be approximately parallel to the direction of shaking based on the information about the direction of detachment.

[0008] Furthermore, according to the present invention, there is provided a cell detachment method for detaching a cell sheet adhered to a culture vessel from the culture vessel using a cell detachment system, the cell detachment method comprising: a detachment initiation step for initiating detachment of the cell sheet; an information acquisition step for acquiring information regarding the direction of detachment of the cell sheet after detachment of the cell sheet has begun; a shaking step for shaking the culture vessel; and a setting step for setting the direction of detachment to be approximately parallel to the direction of shaking based on the information regarding the direction of detachment. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a cell detachment system that efficiently detaches a cell sheet while reducing damage to the cell sheet. Also, according to the present invention, it is possible to provide a cell detachment method that efficiently detaches a cell sheet while reducing damage to the cell sheet. [Brief explanation of the drawings]

[0010] [Figure 1]FIG. 1 is a schematic diagram showing a cell detachment system according to a first embodiment. [Figure 2] FIG. 3 is a cross-sectional view showing a tapping means in the first embodiment. [Figure 3] FIG. 2 is a schematic diagram showing a configuration for generating a local water flow in the first embodiment. [Figure 4] 3 is a schematic diagram showing a configuration for starting peeling by applying ultrasonic vibration in the first embodiment. FIG. [Figure 5] FIG. 2 is a schematic diagram showing the detached state of the cell sheet in the first embodiment. [Figure 6] FIG. 3 is a schematic diagram of a configuration for acquiring information about the direction of peeling progression by referring to a database in the first embodiment. [Figure 7] FIG. 2 is a schematic diagram showing a one-axis shaking mechanism in the first embodiment. [Figure 8] FIG. 2 is a schematic diagram showing a two-axis shaking mechanism in the first embodiment. [Figure 9] FIG. 2 is a schematic diagram showing a mechanism for changing the direction of striking in the first embodiment. [Figure 10] FIG. 2 is a schematic diagram showing a mechanism for rotating a culture vessel in the first embodiment. [Figure 11] 10 is a flowchart showing an outline of processing that does not include a direction change in the first embodiment. [Figure 12] 10 is a flowchart showing an outline of processing including a direction change in the first embodiment. [Figure 13] FIG. 2 is a cross-sectional view showing a mechanism for applying longitudinal vibration in the first embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in further detail below with reference to preferred embodiments.

[0012] The present inventors have investigated an efficient method for detaching a cell sheet, and have found that when a cell sheet begins to detach from a portion of its outer edge, detachment can be promoted while reducing damage to the cell sheet by directing a liquid flow in a specific direction within the culture vessel.

[0013] Specifically, it was found that shaking the culture vessel in a direction approximately parallel to the direction of detachment of the cell sheet after it has begun to detach can efficiently detach the cell sheet while reducing damage to the cell sheet. Here, "approximately parallel" means that at least one of the angles formed by the two is 45° or less, more preferably 30° or less, and even more preferably 15° or less.

[0014] The reason why cell sheet damage can be reduced by adjusting the angle between the detachment direction and the shaking direction is presumed to be as follows: The liquid flow generated by shaking the culture vessel enters between the partially detached cell sheet and the culture vessel, pushing the cell sheet upward in a direction that further detaches the cell sheet. At this time, the water flow acts on the partially detached cell sheet in the detachment direction, which is presumed to promote detachment without excessively concentrating force on the cell sheet.

[0015] A cell detachment system according to an embodiment of the present invention will be described below.

[0016] [First embodiment] (Cell detachment system overview) The cell detachment system according to the first embodiment detaches a cell sheet adhered to the culture surface of a culture vessel from the culture surface through the following steps: The cell detachment system according to this embodiment performs a detachment initiation step in which detachment is initiated by a detachment initiation means, and an information acquisition step in which, after detachment has begun, information regarding the direction of detachment of the cell sheet is acquired by an information acquisition means. Furthermore, the cell detachment system according to this embodiment also performs a shaking step in which the culture vessel is shaken by a shaking means, and a setting step in which the setting means sets the direction of detachment and the shaking direction to be approximately parallel.

[0017] (cell sheet) In this embodiment, the cell sheet refers to a membrane in which cells are connected to each other to form a sheet. The cells constituting the cell sheet are not particularly limited as long as they can form a cell sheet. For example, adhesive cells such as adhesive somatic cells can be used.

[0018] Examples of somatic cells include myoblasts (e.g., skeletal myoblasts), muscle satellite cells, and mesenchymal stem cells (e.g., derived from bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue, endometrium, placenta, and umbilical cord blood). Other examples include tissue stem cells such as cardiomyocytes, fibroblasts, and cardiac stem cells, pluripotent stem cells such as embryonic stem cells and iPS cells, synovial cells, chondrocytes, and epithelial cells (e.g., oral mucosal epithelial cells, retinal pigment epithelial cells, and nasal mucosal epithelial cells). Still other examples include endothelial cells (e.g., vascular endothelial cells), hepatocytes (e.g., hepatic parenchymal cells), pancreatic cells (e.g., pancreatic islet cells), kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, and skin cells.

[0019] In addition, somatic cells may be cells differentiated from iPS cells (iPS cell-derived cells). Examples of iPS cell-derived cells include iPS cell-derived cardiomyocytes, fibroblasts, myoblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, and chondrocytes.

[0020] (Culture container) The culture vessel in this embodiment is not particularly limited as long as it is a cell-adherent culture vessel, and examples of the culture vessel include a flask, a tissue culture flask, a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a multi-well plate, a multi-plate, a Petri dish, a culture bag, and a bottle.

[0021] The material of the culture vessel in this embodiment may be any material that is chemically stable and capable of culturing the desired cells. Examples of such materials 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, and metal. Among these, polystyrene is preferred from the viewpoint of stability.

[0022] Furthermore, the culture vessel in this embodiment may be a temperature-responsive vessel in which the hydrophilicity of the culture surface changes depending on the temperature.

[0023] (Cell detachment solution) The cell detachment solution used herein refers to a solution retained in a culture vessel when cells are detached using the cell detachment method of the present invention. The cell detachment solution used herein does not necessarily need to contain components that promote the detachment of cell sheets. For example, it is more preferable to use a cell detachment solution that is substantially free of protease enzymes in order to protect the state of the cell surface. Here, "substantially free" refers to a content of 0.0005% by mass or less.

[0024] In this embodiment, the pH of the cell detachment solution is preferably in the neutral or acidic range. This is because the neutral range is suitable for cell culture and can maintain a stable high cell viability. The pH can be adjusted appropriately with hydrochloric acid, sodium hydroxide, or the like. In addition, various buffer solutions are preferably used to maintain a stable pH.

[0025] 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.

[0026] In this embodiment, the viscosity of the cell detachment solution is preferably 1.80 mPa·s or less. This is because the flow of the detachment solution generated by ultrasonic vibrations is not impeded, and high detachment efficiency can be maintained. The viscosity of the cell detachment solution can be adjusted as appropriate by adding polymers or sugars, for example.

[0027] The cell detachment solution in this embodiment may contain proteases, but the amount of proteases relative to the total mass of the cell detachment solution is preferably 0.0005% by mass or less, and more preferably does not contain proteases. This is because proteases partially degrade cells, thereby increasing detachment efficiency, but may also reduce the quality of the cells.

[0028] In this embodiment, the protease is, for example, an enzyme that decomposes a part of a cell to facilitate detachment of the cell from the substrate, and examples thereof include trypsin, accutase, collagenase, natural proteases, chymotrypsin, elastase, papain, pronase, and recombinant forms thereof.

[0029] In this embodiment, the cell detachment solution is preferably a culture medium, but is not limited thereto. A solution containing a metal ion chelating agent (hereinafter, sometimes referred to as a chelating agent) may also be used. This is because the use of a cell detachment solution containing a chelating agent allows cells to be effectively detached by ultrasonic vibration.

[0030] The chelating agent in this embodiment is not particularly limited. Examples of the chelating agent include ethylenediaminetetraacetic acid (hereinafter, sometimes referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycoletherdiaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediaminedisuccinic acid, etidronic acid, citric acid, gluconic acid, and phosphonobutanetriacetic acid.

[0031] Among these, chelating agents that form chelates with divalent cations are preferred, chelating agents that form chelates with Ca2+ and Mg2+ are particularly preferred, and ethylenediaminetetraacetic acid is the most preferred. When ethylenediaminetetraacetic acid is used as the chelating agent, the pH of the cell detachment solution is more preferably 7.0 or higher and 8.0 or lower. This is because a higher pH within the neutral range, which can maintain high cell viability, can enhance the chelating ability of ethylenediaminetetraacetic acid and thereby increase detachment efficiency.

[0032] The chelating agent may be used alone or in combination of two or more. The content of the chelating agent is preferably 0.01 mM or more and 5.0 mM or less. By keeping the content within this range, the chelating effect can be reliably obtained and a decrease in activity due to the presence of an excess chelating agent can be suppressed.

[0033] The cell detachment solution of this embodiment may contain a hydrophilic polymer containing a polyalkylene glycol structure. Polyalkylene glycol can increase the cell viability in cell detachment methods using ultrasound. An example of a hydrophilic polymer containing a polyalkylene glycol structure is polyethylene glycol. The hydrophilic polymer preferably has a peak molecular weight Mp measured by gel permeation chromatography of 800 to 50,000, more preferably 1,200 to 20,000. This is because the polymer has little effect on cells and can suppress the thickening effect of the culture medium caused by the polymer.

[0034] There are no particular limitations on the type of culture medium, and examples thereof include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemSpan H3000, StemSpanSFEM, Stemline II, Endothelial Cell Growth Medium 2 Kit, Mesenchymal Stem Cell Growth Medium 2, and MSCGM Bullet. Examples include Kit, mTeSR1, mTeSR2 medium, Repro FF, Repro FF2, NutriStem medium, MF-Medium mesenchymal stem cell growth medium, etc. Among these, it is preferable to use a medium suitable for culturing each cell type.

[0035] 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).

[0036] Examples of antibiotics added to the medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, amphotericin B, and the like.

[0037] (Cell sheet culture conditions) The culture conditions for the cell sheet can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to a dish, and 1.0 × 10 1 ~5.0×10 4 cells / cm 2 The cells are seeded at about 100 cells / well and cultured in an environment with a temperature of 37°C and a CO concentration of 5%. At this time, it is preferable to culture the cells until they reach a confluent state, where the cell occupies about 100% of the surface area of ​​the substrate.

[0038] (Means for initiating cell sheet detachment) The cell sheet can be detached by tapping the culture vessel in which the cell sheet is cultured, exposing the cell sheet to a localized water flow, or applying ultrasonic vibrations to the culture vessel. These methods can be used alone or in combination.

[0039] (beating means) The start of detachment can be promoted by carrying out a thumping step in which a striking force is applied to the culture vessel in which the cell sheet has been cultured using a thumping means as the detachment initiation means.

[0040] The beating step in this embodiment includes a step of applying an impact force to the culture substrate in order to detach at least a portion of the cells adhered to the culture surface of the culture substrate from the culture surface. The beating step may include, for example, at least one of a step of applying an impact force to the culture substrate by beating the culture substrate itself, and a step of applying an impact force to the culture substrate by moving the culture substrate and colliding it with a component constituting a cell detachment device.

[0041] 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 may 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.

[0042] The striking means in this embodiment is not particularly limited as long as it is capable of performing the striking step. For example, the striking means may include an object that strikes the culture substrate and a moving means that moves the object to strike the culture substrate, or a moving means that moves the culture substrate to strike a component of the cell detachment device. The object may have a mass that is sufficient to apply an appropriate striking force to the culture substrate without damaging the culture substrate.

[0043] The shape of the object may be rod-like, hammer-like, spherical, etc. Examples of the moving means include a motor, solenoid, etc., which can generate power to move the object or culture substrate when electrified. Here, the object or culture substrate may be moved using only the motor, solenoid, etc., or the motor or solenoid may be used in combination with a member that can store energy, such as a spring member.

[0044] 2 is a cross-sectional view showing an example of the tapping means in this embodiment. Cam 41 is connected to a motor and rotates in the direction of shaft rotation when driven by the motor. Hammer 42 is in contact with cam 41 at shaft 48 and is biased by spring 43 in the direction of tapping culture vessel 8. Hammer 42 compresses spring 43 as cam 41 rotates, and then moves in the direction of tapping culture vessel 8 according to the profile of cam 41. The tapping frequency can be determined by the number of rotations of the motor and can be detected by photocoupler 44.

[0045] (Liquid discharge means) A liquid ejection means for ejecting a liquid can be used as the detachment initiation means. By contacting the cell sheet with the water flow generated by the ejection of the liquid, the detachment of the cell sheet can be promoted. The liquid ejection means is, for example, a pipette. However, the means for generating the water flow is not limited to this, and configurations such as a nozzle or a flow channel can be used. In particular, it is preferable to contact the water flow generated by the ejection of the liquid with the edge (outer edge) of the cell sheet from the viewpoint of efficient detachment of the cell sheet.

[0046] 3 is a schematic diagram showing the configuration of the liquid discharge means in this embodiment. The electric pipette 12 is controlled by the control unit 5 and can bring a localized water flow into contact with the edge of the culture vessel 8. The medium discharged from the electric pipette 12 generates a water flow that flows from the edge of the bottom of the culture vessel 8 toward the center of the bottom. This water flow can apply a force to the edge of the cell sheet to initiate detachment.

[0047] (Peeling begins with ultrasonic vibration) Another example of the detachment initiation means is an ultrasonic wave generating means. The ultrasonic wave generating means applies vibrations in the ultrasonic band to the culture vessel, thereby initiating detachment of the cell sheet adhered to the culture vessel. Figure 4 is a schematic diagram showing the configuration for initiating detachment by ultrasonic vibration in this embodiment. The ultrasonic wave generating means 13 is controlled by the control unit 5 and can apply ultrasonic vibrations to the culture vessel 8. This ultrasonic vibration can provide a force that initiates detachment of the cell sheet in the culture vessel 8 from the outer edge.

[0048] An example of ultrasonic vibrations used in the ablation initiation means is vibrations with a frequency 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 cells. One example is the use of an ultrasonic oscillator such as lead zirconate titanate (PZT) as the vibrating body.

[0049] Any ultrasonic vibrator can be used as long as it generates ultrasonic vibrations, but examples include 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, the ultrasonic vibrator can output a large amplitude at a relatively high driving frequency (vibration frequency) in the ultrasonic range without being damaged.

[0050] In the case of a ring-shaped piezoelectric element, it is preferable that the outer diameter of the diaphragm is equal to that of the piezoelectric element. The thickness of the diaphragm is such that when the piezoelectric element and the diaphragm are bonded and the piezoelectric element vibrates, the midpoint of the thickness direction of the flexure, that is, the neutral plane where there is neither tension nor compression during the flexure, is located on the diaphragm side, in order to efficiently utilize the distortion of the piezoelectric element for flexure.

[0051] Furthermore, commercially available Langevin type transducers or rectangular type transducers can also be used as the ultrasonic transducer of the present invention. 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.

[0052] The operation of the detachment initiation means may be performed periodically, or may be performed continuously until the detachment of the cell sheet is completed.

[0053] (Obtaining information on the direction of separation progression using observation means) The means for acquiring information regarding the direction of detachment can be, for example, an observation means such as a camera that observes the cell sheet and acquires the observation results as image information. Alternatively, a measurement means such as a distance sensor that acquires numerical information regarding the adhesion state of the cell sheet to the inner surface of the culture vessel can be used.

[0054] 1 is a schematic diagram showing the cell detachment system of this embodiment, including the observation means 11. The observation means 11 is, for example, a commercially available camera or microscope, and is controlled by the control unit 5, allowing the state of the cell sheet in the culture vessel 8 to be observed.

[0055] FIG. 5 shows the state of detachment of a cell sheet, with the detachment boundary line AB indicating the boundary between adhesion and detachment of the cell sheet. For example, in the state shown in FIG. 5(a), the detachment direction can be determined as a direction perpendicular to the detachment boundary line AB or a direction connecting the midpoint of the detachment boundary line AB and the center of gravity of the cell sheet. Even when the detachment boundary curve CD forms an arc, as shown in FIG. 5(b), the detachment direction can be determined as a normal direction to the midpoint of the detachment boundary curve CD or a direction connecting the midpoint of the detachment boundary curve CD and the center of gravity of the cell sheet. In this embodiment, the normal direction to the midpoint of the detachment boundary curve CD is defined as the detachment direction.

[0056] (Acquisition of information on the direction of separation from the database) As another example, the information acquiring means can refer to a database linking information about the culture vessel with the direction of detachment. The database linking the direction of detachment with information about the culture vessel is preferably stored in a storage area of ​​the detachment system or on a server connected via a network. The information about the culture vessel is, for example, at least one of the size, shape, manufacturer, and pre-assigned number of the culture vessel.

[0057] 6 is a schematic diagram of a configuration for obtaining information about the direction of detachment by referencing a database according to this embodiment. The method of referring to a database allows for obtaining information about the direction of detachment without observing the cell sheet, thereby reducing the number and size of the components in the system.

[0058] The above-mentioned information regarding the direction of peeling may be acquired multiple times during peeling, or may be acquired before the start of peeling. In order to obtain a more accurate peeling direction, it is preferable to acquire information multiple times in accordance with the degree of peeling progress and update the peeling direction in accordance with the acquired information.

[0059] (Shaking the culture vessel) In this embodiment, the shaking can be configured to generate a flow in the liquid in the culture vessel by applying acceleration including a directional component parallel to the culture surface of the culture vessel in which the cell sheet is cultured. For example, a configuration can be used in which a motor is used to rotate the culture vessel, 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 vessel, thereby applying acceleration including a directional component parallel to the culture surface. As long as the shaking can apply acceleration including a directional component parallel to the culture surface of the culture vessel, the axis of shaking itself does not necessarily have to be parallel to the culture surface.

[0060] In this embodiment, the amplitude of the vibration is preferably 0.1 mm to 300 mm, more preferably 0.1 mm to 150 mm. In addition, in the case of reciprocating motion, the frequency is preferably 0.1 Hz to 20 Hz, more preferably 0.1 Hz to 10 Hz.

[0061] (One-axis shaking mechanism) Figure 7 shows a single-axis shaking mechanism in this embodiment. A guide rail 50 is fastened to the base plate 7 of Figure 1 so as to shake the culture substrate 8 back and forth. A base-side spring post 52 to which a pressure spring 51 is connected is fixed to the base plate 7, and the pressure spring 51 is connected to the base-side spring post 52. The opposite end of the pressure spring 51 is fixed to a shaking-side spring post 53 in the shaken part. The pressure spring 51 can be selected as needed and is configured to be removable.

[0062] The rotation of the vibration motor 54 is transmitted to the vibration rod 56 via gear 55, causing vibration. A rotating disk 57 is fixed to the rotating shaft of the vibration motor 54, and rotates as shown in the figure. The direction of rotation can also be reversed. A linear bush holder 58 is rotatably installed at a position offset from the rotating shaft, and a linear bush 59 is incorporated into it, with the vibration rod 56 incorporated so that it can move linearly. One end of the vibration rod 56 is fixed to a rotatable vibration fulcrum shaft 60. This converts the vibration into a reciprocating motion centered on the vibration fulcrum shaft 60. The frequency of the reciprocating motion is detected by a rotary photocoupler 61.

[0063] A connecting rod 62 is fastened to the oscillation rod 56 in parallel with the oscillation rod 56 by a connecting rod fixture 63, and one end is fixed to the oscillation fulcrum shaft 60. An oscillation slide top 64 equipped with a base plate 7 abuts against the reciprocating connecting rod 62, causing it to perform a reciprocating oscillation motion. The oscillation slide top 64 is supported by an oscillation top slide shaft 66 arranged in parallel with an oscillation adjustment screw rod 65, and the oscillation stroke can be freely adjusted by rotating the oscillation adjustment screw rod 65. A stroke knob 67 is provided at the end of the oscillation adjustment screw rod 65, and can be adjusted by turning it manually.

[0064] (2-axis shaking mechanism) 8 is a schematic diagram showing the two-axis shaking mechanism of this embodiment. Linear rails 14 are arranged on two axes perpendicular to each other, allowing the shaking direction to be freely changed. The linear rails 14 are controlled by the control unit 5, and can be controlled so that the direction of peeling and the shaking direction are approximately parallel.

[0065] (Setting means for setting the peeling direction and shaking direction approximately parallel) In this embodiment, the setting means for setting the direction of detachment progress and the shaking direction to be approximately parallel can change the direction of the detachment start means to match the direction of detachment progress. Alternatively, the shaking direction can be changed to match the direction of detachment progress, or the orientation of the culture vessel can be changed to match the direction of detachment progress. Each of these may be performed individually, or two or more may be performed in combination.

[0066] (Change of striking direction) 9 is a schematic diagram showing a mechanism for changing the direction of beating in this embodiment. The beating means 4 is configured to be able to freely change the angle at which it strikes the culture vessel 8, thereby making it possible to freely change the direction of beating. By operating the beating means 4 in a state in which the beating direction of the beating means 4 has been changed based on the beating direction set by the setting means, it is possible to make the shaking direction and the direction of detachment approximately parallel.

[0067] (Change shaking direction) In the example shown in Figure 8, the shaking direction is controlled on two axes, and the shaking direction can be changed to any angle within the operating plane. By changing the shaking direction based on the shaking direction set by the setting means, the shaking direction and the peeling direction can be made approximately parallel to each other.

[0068] (Changing the orientation of the culture vessel) After the cell sheet begins to detach from the culture vessel 8, the shaking direction and the direction of detachment can be made approximately parallel by rotating the culture vessel 8. FIG. 10 is a schematic diagram showing a mechanism for rotating the culture vessel 8 in this embodiment. Drive roller 15 and roller 16 are in contact with the culture vessel 8, and the culture vessel 8 is rotated by drive roller 15 connected to a motor. Roller 16 is rotated in response to the rotation of the culture vessel 8. The rotation angle of drive roller 15 is controlled by control unit 5, and the shaking direction and the direction of detachment can be made approximately parallel.

[0069] (Cell detachment system operation) The operation of the cell detachment system according to this embodiment will be explained using an example in which a beating means is used as the detachment initiation means, but the configuration shown here is merely an example and is not intended to limit the scope of the present invention.Instead of the beating means, at least one of the following may be used: generation of a local water flow into the culture vessel by a liquid discharge means; and application of ultrasonic vibrations (ultrasonic vibrations) to the culture vessel.

[0070] FIG. 1 is a schematic diagram showing an example of the configuration of a cell detachment system according to this embodiment. The beating means 4 beats the culture vessel 8 as a means for initiating detachment. The observation means 11 observes the state of the cell sheet cultured in the culture vessel 8 as a means for acquiring information regarding the direction of detachment. The culture vessel 8 in which the cell sheet has been cultured is set on the shaking means 3, and the cell sheet can be detached by driving the beating means 4, observation means 11, and shaking means 3, which are controlled by the control unit 5. As an example of the operation, the following steps can be performed in order: the culture vessel 8 is beaten by the beating means 4, the detachment is detected by the observation means 11, and the culture vessel 8 is shaken by the shaking means 3 in a direction approximately parallel to the detachment direction.

[0071] However, in the present invention, it is important that the detachment direction and the shaking direction are approximately parallel, and the timing of these operations is not particularly limited. For example, after the cell detachment system is started, the shaking process, observation process, and beating process may be started simultaneously, or the shaking direction may be changed when the detachment direction is detected by the observation process.

[0072] The order in which beating and shaking are started and vertical vibration such as ultrasonic vibration is started does not matter. Performing the beating and shaking steps during or after the application of vertical vibration allows for the application of shear force while weakening the adhesive strength of the cells, resulting in more effective cell detachment. Furthermore, information about the cells adhered to the substrate may be measured and used to determine the transition between each step.

[0073] The cell detachment system of this embodiment may be configured to perform steps other than those described above, such as replacing the solution with a cell detachment solution, rinsing the cells with the cell detachment solution, diluting the detachment solution, and homogenizing the solution in the culture vessel.

[0074] The above steps may be repeated periodically or non-periodically. The timing may be unified or adjusted for each step. It is preferable that the ratio of the total time spent on the beating and shaking steps to the total time spent on the external stimulation step, such as ultrasound, is 0.01 or more and 100 or less.

[0075] (First processing flow using the cell detachment system) FIG. 11 is a flowchart showing an outline of the processing performed by the cell detachment system according to this embodiment. Cell detachment begins in step S11. In step S12, information regarding the detachment direction of the cell sheet in the culture vessel 8 is acquired. The information regarding the detachment direction of the cell sheet is stored in a memory area within the control unit 5. In step S13, it is determined whether the cell sheet has started to detach from the culture vessel 8 based on the information regarding the detachment direction of the cell sheet.

[0076] The control unit 5 may be configured with a module executed by a CPU or MPU, or may be configured with a circuit that realizes a specific function, such as an ASIC. Note that the present invention may be realized by a cell detachment system to which multiple devices and computing units are connected, and a cell detachment device consisting of a single unit that realizes similar functions is also included in the present invention.

[0077] If detachment has not started, the process proceeds to step S12, and steps S12 and S13 are repeated until detachment has started. If detachment has started in step S13, the process proceeds to step S14. In step S14, the detachment direction is determined based on information regarding the detachment direction of the cell sheet. In step S15, the culture vessel 8 is shaken in a direction approximately parallel to the detachment direction obtained in step S14.

[0078] In step S16, it is determined whether the cell sheet has been completely detached from the culture vessel 8. When the cell sheet has lifted off the culture surface of the culture vessel 8, it is determined that detachment is complete. If detachment is not complete, the process proceeds to step S15, and steps S15 and S16 are repeated until detachment is complete. In step S16, when detachment is complete, the system operation is terminated.

[0079] (Second flow using cell detachment system) FIG. 12 is a flowchart showing an outline of the processing performed by the cell detachment system according to this embodiment. In step S11, cell detachment is initiated. In step S12, information regarding the detachment direction of the cell sheet is acquired. The acquired information regarding the detachment direction of the cell sheet is stored in a memory area within the control unit 5. The memory area can be configured using any memory or storage medium such as an optical disk. In step S13, it is determined whether the cell sheet has started to detach from the culture vessel 8 based on the information regarding the detachment direction of the cell sheet.

[0080] If detachment has not started, the process proceeds to step S12, and steps S12 and S13 are repeated until detachment starts. If detachment has started in step S13, the process proceeds to step S14. In step S14, the direction of detachment is determined based on information regarding the detachment direction of the cell sheet. In step S15, the culture vessel 8 is shaken in a direction approximately parallel to the detachment direction obtained in step S14.

[0081] In step S16, it is determined whether the cell sheet has been completely detached from the culture vessel 8. When the cell sheet has lifted off the culture surface of the culture vessel 8, it is determined that detachment is complete. If detachment is not complete, the process proceeds to step S12, where the state of the cell sheet is acquired. After the determination in step 13, the detachment direction is determined again in step 14. If the detachment direction has changed, in step S15, the cell sheet is shaken in a direction approximately parallel to the changed detachment direction. Steps S12 to S16 are repeated until detachment is complete. In step S16, when detachment is complete, the system operation is terminated.

[0082] (Additional longitudinal vibration by cell detachment system) 13 is a cross-sectional view showing the configuration of the cell detachment system according to this embodiment, which is combined with an additional mechanism for applying vertical vibration to the culture vessel 8. An ultrasonic element 17 is placed below the culture vessel 8, and applies vertical ultrasonic vibration to the culture vessel 8. The ultrasonic element 17 is controlled by the control unit 5, and the drive timing can be controlled as desired.

[0083] The cell sheet detachment method is appropriately selected from the above-mentioned process flow 1 or process flow 2. In addition, additional longitudinal vibration may be performed in addition to process flow 1 or process flow 2.

[0084] (Program for realizing cell processing method) The present invention can also be realized by executing the following process. That is, the present invention can be realized by supplying software (programs) that realize one or more functions of the various embodiments described above to a system or device via a network or a storage medium. The present invention can also be realized by a process in which a computer (or a CPU, MPU, or the like) of the system or device reads and executes the programs. The computer has one or more processors or circuits, and may include multiple separate computers or a network of multiple separate processors or circuits to read and execute computer-executable instructions.

[0085] In this case, the processor or circuitry may include a central processing unit (CPU), a microprocessing unit (MPU), a graphics processing unit (GPU), an application specific integrated circuit (ASIC), or a field programmable gateway (FPGA), a digital signal processor (DSP), a data flow processor (DFP), or a neural processing unit (NPU).

[0086] [Examples and Comparative Examples] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples in any way as long as the gist of the invention is not exceeded.

[0087] (Culture of A549 cells on a substrate) Human alveolar basal epithelial adenocarcinoma cells, A549 cells, were cultured in a Φ35 temperature-responsive dish (Upcell™, CellSeed) at 10,000 cells / cm. 2The cells were seeded at a density of 100 μg / ml 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 6 days, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 100%.

[0088] (C2C12 cell culture on a substrate) Mouse striated muscle cells, C2C12 cells, were cultured in a Φ35 temperature-responsive dish (Upcell™, CellSeed) at 65,000 cells / cm. 2 The cells were seeded at a density of 100 μg / ml and cultured at 37°C in a 5% CO2 environment. The culture medium used was DMEM / F12 medium (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 two 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 100%.

[0089] (Culture of MDCK cells on a substrate) MDCK cells, which are canine kidney tubular epithelial cells, were cultured in a Φ35 temperature-responsive dish (Upcell™, CellSeed) at 65,000 cells / cm. 2 The cells were seeded at a density of 100 μg / ml 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 8 days, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 100%.

[0090] (Culture of HEK293 cells on a substrate) HEK293 cells, human embryonic kidney cells, were cultured in a Φ35 temperature-responsive dish (Upcell™, CellSeed) at 65,000 cells / cm. 2 The cells were seeded at a density of 100 μg / ml 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 9 days, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell occupancy rate on the dish was approximately 100%.

[0091] (Culture of BAEC cells on a substrate) BAEC cells, bovine aortic endothelial cells, were cultured in a Φ35 polystyrene dish (Corning) at 20,000 cells / cm. 2 The cells were seeded at a density of 100 μg / ml 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 7 days, and the cells were observed under a phase-contrast microscope to confirm cell adhesion and proliferation. The cell area coverage of the dish was approximately 100%.

[0092] (HMSC cell culture on a substrate) Human mesenchymal stem cells (hMSCs) were cultured in a Φ35 temperature-responsive dish (Upcell™, CellSeed) at 30,000 cells / cm. 2The cells were seeded at a density of 100 μg / ml 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). Culture was continued for 8 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 100%.

[0093] (Culture of HUVEC cells on a substrate) Human umbilical vein endothelial cells (HUVEC) were cultured in a Φ35 temperature-responsive dish (Upcell™, CellSeed) at 65,000 cells / cm. 2 The cells were seeded at a density of 100 μg / ml and cultured at 37°C in a 5% CO2 environment. The culture medium used was Endothelial Cell Growth Medium 2 Kit (Promocell) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, Thermo Fisher Scientific). Culture was continued 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 100%.

[0094] (Peeling initiation means 1: hitting the culture vessel) As the detachment initiation means 1, the culture vessel 8 was struck at a frequency of 5 Hz using the striking means shown in FIG.

[0095] (Separation initiation method 2: localized water flow) Using the liquid discharge means shown in FIG. 3 as the peeling initiation means 2, 1 ml of medium was discharged in one second from the bottom edge of the culture vessel 8 toward the bottom center.

[0096] (Peeling initiation means 3: ultrasonic vibration) As the detachment initiation means 3, an ultrasonic generating means shown in Figure 4 was used, and an AC voltage of 50 V and a frequency of 30 kHz was applied to the Langevin transducer by the control unit 5, thereby applying ultrasonic vibrations to the culture vessel.

[0097] (Shaking method: 1:1 axial shaking) As the shaking means 1, a single-shaft shaking mechanism as shown in FIG. 7 was used, and the motor was rotated at 120 rpm to perform a reciprocating motion at a frequency of 2 Hz.

[0098] (Shaking method 2: 2-axis shaking) The culture vessel was shaken using a two-axis shaking mechanism as shown in FIG. 8 as the shaking means 2.

[0099] (Method 1 for obtaining information on the direction of detachment: Observation of cell sheet) As the information acquisition means 1, the observation means 11 shown in FIG. 1 was used to acquire the direction of cell detachment.

[0100] (Method 2 for obtaining information on the direction of separation: referencing the database) The information acquisition means 2 acquired the direction of cell detachment by referring to a database stored in the cell detachment system.

[0101] (Change method 1: Change the direction of the strike) As a modification means 1, the mechanism shown in FIG. 9 was used to change the direction of the beating, so that the shaking direction and the direction of peeling were made approximately parallel to each other.

[0102] (Change method 2: Change the shaking direction) As a second changer, the mechanism shown in FIG. 9 was used to change the shaking direction, so that the shaking direction and the direction of peeling were made approximately parallel to each other.

[0103] (Modification 3: Rotation of culture vessel) As changing means 3, the mechanism shown in FIG. 10 was used to change (rotate) the orientation of the culture vessel, so that the shaking direction and the direction of detachment were made approximately parallel.

[0104] (Processing flow 1: No direction correction) In treatment flow 1, the cell sheet was treated according to the flow shown in FIG.

[0105] (Processing flow 2: direction correction) In treatment flow 2, the cell sheet was treated according to the flow shown in FIG.

[0106] (additional longitudinal vibration) As an additional longitudinal vibration, ultrasonic vibration was applied to the culture vessel using the configuration shown in FIG.

[0107] (Removability evaluation) Detachability was evaluated based on three items: detachment time, variability in detachment time, and cell sheet quality. The detachment time can be determined by setting the culture vessel 8 in the cell detachment system and measuring the time from when drive is initiated until detachment is complete and drive is completed. Since the time required for detachment differs for each cell type, the detachment time was evaluated by calculating the ratio of the detachment time for each example to the time required for each comparative example. The detachment time was evaluated 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 equivalent to that of the comparative example)

[0108] The variation in detachment time was evaluated by dividing the standard deviation of the detachment time when the cell sheet was detached five times by the average value of the five times. 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)

[0109] The quality of the cell sheet was evaluated by observing the detached cell sheet under a phase-contrast microscope to check for the presence of holes or tears within the sheet. Holes were considered to be present if they were 500 μm or larger in diameter. Tears were considered to be present if they were 1 mm or larger.

[0110] The quality of the cell sheets was judged according to the following criteria. A: Very good (no holes or tears) B: Good (no tears) C: Effective (no holes) D: Poor (holes, tears)

[0111] Example 1 C2C12 cells cultured on a culture vessel 8 were detached using detachment initiation means 1, shaking means 1, information acquisition means 1, and change means 1. Evaluation of the detachment time showed that it took 12 minutes to complete detachment. Evaluation of the variability in detachment time showed that the standard deviation was 1.33. Evaluation of the quality of the sheet-like C2C12 cells showed that there were no holes or tears after detachment, no stretched areas, and some wrinkled areas.

[0112] (Examples 2 to 14, Comparative Examples 1 to 7) The detachment time, variability in detachment time, and sheet quality were evaluated for the combinations of detachment initiation means, shaking means, information acquisition means, change means, processing flow, additional vertical vibration, and cell type shown in Table 1. The evaluation results are shown in Table 2.

[0113] [Table 1]

[0114] [Table 2]

[0115] The disclosure of this embodiment includes the following configurations and methods.

[0116] (Configuration 1) A cell detachment system for detaching a cell sheet adhered to a culture vessel from the culture vessel, comprising: a detachment initiation means for initiating detachment of the cell sheet; an information acquiring means for acquiring information regarding the direction of detachment of the cell sheet after the start of detachment of the cell sheet; A shaking means for shaking the culture vessel; and A cell detachment system comprising a setting means for setting the direction of movement and the direction of shaking to be approximately parallel based on information regarding the direction of movement.

[0117] (Configuration 2) 2. The cell detachment system according to claim 1, wherein the detachment initiation means includes a beating means for beating the culture vessel.

[0118] (Configuration 3) the setting means sets the direction of the beating based on information about the direction of travel; The cell detachment system according to configuration 2, wherein the beating means strikes the culture vessel based on the set direction of beating.

[0119] (Configuration 4) 4. The cell detachment system according to any one of configurations 1 to 3, wherein the detachment initiation means includes a liquid ejection means for ejecting a liquid to bring a water flow into contact with the cell sheet.

[0120] (Configuration 5) the setting means sets the direction of shaking based on the information about the traveling direction, 5. The cell detachment system according to any one of configurations 1 to 4, wherein the shaking means shakes the culture vessel based on the set shaking direction.

[0121] (Configuration 6) the setting means sets the direction of shaking based on the information about the traveling direction, 6. The cell detachment system according to any one of configurations 1 to 5, further comprising a change means for changing the orientation of the culture vessel based on the set shaking direction.

[0122] (Configuration 7) 7. The cell detachment system according to any one of configurations 1 to 6, wherein the information acquisition means acquires the observation results of the cell sheet.

[0123] (Configuration 8) 7. The cell detachment system according to any one of configurations 1 to 6, wherein the information acquisition means refers to a database configured to include information about the culture vessel and information about the traveling direction.

[0124] (Configuration 9) 9. The cell detachment system according to any one of configurations 1 to 8, wherein the detachment initiation means applies vibrations in the ultrasonic band to the culture vessel.

[0125] (Configuration 10) 9. The cell detachment system according to any one of configurations 1 to 8, further comprising an ultrasonic wave generating means for applying vibrations in an ultrasonic band to the culture vessel.

[0126] (Method 1) A cell detachment method for detaching a cell sheet adhered to a culture vessel from the culture vessel using a cell detachment system, comprising: a detachment initiation step of initiating detachment of the cell sheet; an information acquiring step of acquiring information regarding the direction of detachment of the cell sheet after the start of detachment of the cell sheet; A shaking step of shaking the culture vessel; and A cell detachment method comprising a setting step of setting the direction of movement and the direction of shaking to be approximately parallel based on information regarding the direction of movement. [Explanation of symbols]

[0127] 3 Shaking means 4 Hitting means 5. Control section 8 Culture vessel 11 Observation methods

Claims

1. A cell detachment system for detaching a cell sheet adhered to a culture vessel from the culture vessel, comprising: a detachment initiation means for initiating detachment of the cell sheet; an information acquiring means for acquiring information regarding the direction of detachment of the cell sheet after the start of detachment of the cell sheet; A shaking means for shaking the culture vessel; and A cell detachment system comprising a setting means for setting the direction of movement and the direction of shaking to be approximately parallel based on information regarding the direction of movement.

2. 2. The cell detachment system according to claim 1, wherein the detachment initiation means includes a beating means for beating the culture vessel.

3. the setting means sets the direction of the beating based on information about the direction of travel; The cell detachment system according to claim 2 , wherein the beating means strikes the culture vessel based on the set beating direction.

4. 2. The cell detachment system according to claim 1, wherein the detachment initiation means includes a liquid ejection means for ejecting a liquid to bring a water flow into contact with the cell sheet.

5. the setting means sets the direction of shaking based on the information about the traveling direction, 2. The cell detachment system according to claim 1, wherein the shaking means shakes the culture vessel based on the set shaking direction.

6. the setting means sets the direction of shaking based on the information about the traveling direction, The cell detachment system according to claim 1, further comprising a change unit for changing the orientation of the culture vessel based on the set shaking direction.

7. The cell detachment system according to claim 1 , wherein the information acquisition means acquires observation results of the cell sheet.

8. 2. The cell detachment system according to claim 1, wherein the information acquisition means refers to a database configured to contain information about the culture vessel and information about the traveling direction.

9. 2. The cell detachment system according to claim 1, wherein the detachment initiation means applies vibrations in the ultrasonic band to the culture vessel.

10. 2. The cell detachment system according to claim 1, further comprising an ultrasonic wave generating means for applying vibrations in an ultrasonic band to the culture vessel.

11. A cell detachment method for detaching a cell sheet adhered to a culture vessel from the culture vessel using a cell detachment system, comprising: a detachment initiation step of initiating detachment of the cell sheet; an information acquiring step of acquiring information regarding the direction of detachment of the cell sheet after the start of detachment of the cell sheet; A shaking step of shaking the culture vessel; and A cell detachment method comprising a setting step of setting the direction of movement and the direction of shaking to be approximately parallel based on information regarding the direction of movement.

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