Cell detachment method and cell detachment system

Abstract

To provide a cell detachment method that efficiently detaches a cell sheet while reducing damage to the cell sheet.SOLUTION: A cell detachment method detaches a cell sheet adhering to a culture surface of a culture container from the culture container by using a cell detachment system, the method including a tapping step of tapping the culture container in a first direction that contains a component parallel to the culture surface, and a shaking step of shaking the culture container in a second direction that contains a component parallel to the culture surface, wherein the first direction and the second direction are different from each other as viewed in a direction perpendicular to the culture surface.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell detachment method and a cell detachment system. [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 sheets and transplant the sheet-like cell cultures (cell sheets) into the affected area in order to repair damaged tissues. When producing cell sheets using adhesive cells, for example, the cells are cultured in the form of a sheet on a polystyrene dish, which is an example of a culture substrate, and then detached and collected in the sheet form from the culture substrate. There is a need for a manufacturing method that can efficiently and stably produce cell sheets while reducing damage such as wrinkles, tears, and holes in the cell sheets.

[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 method 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 system 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 method for detaching a cell sheet adhered to a culture surface of a culture vessel from the culture vessel using a cell detachment system, the method comprising: a beating step of beating the culture vessel in a first direction that includes a component parallel to the culture surface; and a shaking step of shaking the culture vessel in a second direction that includes a component parallel to the culture surface, wherein the first direction and the second direction are different when viewed from a direction perpendicular to the culture surface.

[0008] Furthermore, according to the present invention, there is provided a cell detachment system, which is a cell detachment method for detaching a cell sheet adhered to the culture surface of a culture vessel from the culture vessel, and which comprises a beating means for beating the culture vessel in a first direction including a component parallel to the culture surface, and a shaking means for shaking the culture vessel in a second direction including a component parallel to the culture surface, and which is characterized in that the first direction and the second direction are different when viewed from a direction perpendicular to the culture surface. [Effects of the Invention]

[0009] 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. Also, 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. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing an example of 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 top view showing the relationship between the tapping direction and the shaking direction in the first embodiment. [Figure 4] FIG. 1 is a schematic diagram illustrating an example of a cell detachment system according to a first embodiment. [Figure 5] FIG. 1 is a schematic diagram illustrating an example of a cell detachment system according to a first embodiment. [Figure 6] FIG. 2 is a schematic diagram showing a one-axis shaking mechanism in the first embodiment. [Figure 7] 1 is a flowchart showing a cell detachment method according to a 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 a method for efficiently detaching a cell sheet while reducing damage to the cell sheet. As a result, they have found that a cell sheet can be efficiently detached by combining a beating step in which the culture vessel to which the cell sheet is attached is beaten with a shaking step in which the culture vessel is shaken. Furthermore, the present inventors have found that the cell sheet tends to start detaching from a position that forms a specific range of angles with respect to the direction of beating the culture vessel.

[0013] Based on the above findings, further investigations revealed that cell sheets can be detached more efficiently by shaking the culture vessel at an angle within a specific range relative to the direction of beating. Specifically, it was found that cell sheets can be detached more efficiently by making the direction of beating and the direction of shaking different when viewed perpendicular to the culture surface. The reason why the configuration of the present invention improves cell sheet detachment efficiency while reducing breakage is presumed to be as follows: The liquid flow generated by shaking the culture vessel penetrates 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 toward the partially detached portion of the cell sheet, allowing for efficient detachment while reducing damage to the cell sheet.

[0014] (Cell detachment method) The cell detachment method according to the first embodiment detaches a cell sheet adhered to the culture surface of a culture vessel from the culture surface by performing a beating step in which the culture vessel is beaten and a shaking step in which the culture vessel is shaken. The cell detachment method according to this embodiment is characterized in that, in the beating step, the culture vessel is beaten in a first direction that includes a component parallel to the culture surface, and in the shaking step, the culture vessel is shaken in a second direction that includes a component parallel to the culture surface. Because the first and second directions are different when viewed perpendicular to the culture surface, detachment can be performed efficiently while reducing damage to the cell sheet.

[0015] 7 shows a flowchart of the cell detachment method according to this embodiment. First, in a beating step (S11), the culture vessel to which the cell sheet is adhered is beaten by a beating means. Then, in a shaking step (S12), the culture vessel is shaken in a direction different from the direction of beating by the beating means. Note that the start times of the beating step and the shaking step are not limited to these, and there may be a time when both steps are performed simultaneously, or they may be performed alternately.

[0016] In this embodiment, the cell detachment method refers to a method of detaching a cell sheet adhered to a culture surface from a culture vessel using an external stimulus. Examples of external stimuli include beating, shaking, ultrasound, liquid ejection, and liquid agitation. Liquid ejection is a method of ejecting liquid from a needle or the like and applying it to the culture surface, thereby spreading the liquid along the culture surface and applying shear force. Liquid agitation is a method of applying shear force by creating a flow path along the culture surface and using a pump or syringe to send the liquid in one direction or by switching the direction. The cell detachment method may use only the external stimuli of beating or shaking, or may be combined with multiple other external stimuli.

[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 ~1.0×10 5 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] (Cell detachment system) 1 is a schematic diagram showing an example of a cell detachment system for carrying out the cell detachment method of this embodiment. A culture vessel 8 is pounded by a pounding means 3. The culture vessel 8 is also shaken by a shaking means 4. The cell detachment system of this embodiment may also have a function for applying other external stimuli, such as ultrasonic vibrations.

[0039] (pounding process) 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. Examples of the beating step include 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 components constituting a cell detachment device. The beating step in this embodiment may be periodic or aperiodic, and the strength of the impact force applied to the culture substrate by the beating step can be appropriately set. Furthermore, the beating step in this embodiment may be performed continuously from the start of cell detachment to the completion of detachment, or may be completed midway, or may be performed intermittently.

[0040] The time at which the beating is performed in the beating step can be set periodically or non-periodically. From the viewpoint of applying continuous impact to the entire container and facilitating the initiation of detachment, beating is preferably performed at periodic timing. The time and period of beating may be changed with the passage of time after the start of operation of the cell detachment system.

[0041] The beating frequency in the beating process in this embodiment refers to the period of time required for a cycle to return to a state at any given time point. The frequency is expressed as f (Hz), meaning the number of vibrations per second. The frequency can be expressed in units of rpm as the number of rotations per minute. This mainly refers to the number of times the same cycle is repeated per minute, assuming that one cycle in a rotating machine is one recursion. The frequency is not particularly limited, and in this embodiment, it is preferably 0.1 Hz to 20 Hz, and more preferably 0.1 Hz to 10 Hz. In this embodiment, the frequency range for the magnitude relationship between the beating process and the shaking process is not limited, but it is preferable that the frequency in the beating process / the frequency in the shaking process be 1 to 50 times, and more preferably 1 to 15 times. The frequency in the beating process may be changed over time.

[0042] The time for applying the continuous / intermittent impact in the beating step can be appropriately set depending on the cell characteristics of the cell sheet, the environmental temperature, the type of detachment solution, and the detachment conditions. The position of the beating in the beating step is not particularly limited, but it is preferable to apply the impact to the culture vessel or the holder, and it is more preferable to apply the impact to the side of the culture vessel.

[0043] The direction of beating (first direction) in the beating step is not particularly limited, but preferably includes a component horizontal to the culture surface. In particular, applying an impact to the culture vessel or holder can create inertial force and a flow of culture fluid. Applying the above force can detach the cell sheet. The impact direction may be changed over time after the cell detachment system begins operation.

[0044] The environmental temperature is not particularly limited, but from the viewpoint of maintaining cell viability, it is preferably 20° C. to 40° C. By keeping the temperature close to the temperature during culture, the effects of temperature changes on the cells can be reduced, and the viability can be maintained high.

[0045] (beating means) The striking means in this embodiment is not particularly limited as long as it is capable of performing the striking step. The striking means may include, for example, an object that strikes the culture substrate and a moving means for moving the object to strike the culture substrate, or a moving means for moving the culture substrate to strike a component constituting the cell detachment device. The object may have a mass that is sufficient to impart an appropriate striking force to the culture substrate without damaging the culture substrate. Examples of the object's shape include rod-like, hammer-like, and spherical. Examples of the moving means include a motor, solenoid, or the like that can generate power to move the object or culture substrate when energized. Here, the object or culture substrate may be moved using only a motor, solenoid, or the like, or a motor or solenoid may be used in combination with a component capable of storing energy, such as a spring.

[0046] The beating means 3 starts detaching the cell sheet by beating the culture vessel 8. FIG. 2 is a cross-sectional view showing an example of the beating means in this embodiment. The cam 41 is connected to a motor and rotates in the direction of shaft rotation when driven by the motor. The hammer 42 is in contact with the cam 41 at a shaft 48 and is biased by a spring 43 in the direction of striking the culture vessel 8. The hammer 42 compresses the spring 43 in the contracting direction as the cam 41 rotates, and then moves in the direction of striking the culture vessel 8 according to the profile of the cam 41. The beating frequency can be determined by the number of rotations of the motor and can be detected by a photocoupler 44.

[0047] (Shaking process) In this embodiment, the shaking step refers to a step of moving the culture vessel or a part that holds the culture vessel. In the shaking step, the culture vessel is shaken to detach the cell sheet from the culture vessel.

[0048] The power source for the shaking step is not limited to a spring, but may include a magnet, an electromagnet, a motor, etc.

[0049] Figure 6 shows a single-axis shaking mechanism in this embodiment. A guide rail 50 is fastened to the base plate 2 of Figure 1 so as to shake the culture vessel 8 back and forth. A base-side spring post 52 to which a pressure spring 51 is connected is fixed to the base plate 2, and the pressure spring 51 is connected to this 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.

[0050] 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.

[0051] A connecting rod 62 is fastened to the oscillation rod 56 in parallel with the oscillation rod 56 by a connecting rod fastener 63, and one end is fixed to the oscillation fulcrum shaft 60. An oscillation slide top 64 equipped with a base plate 2 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.

[0052] Figure 1 is a schematic diagram showing an example of the shaking structure of this embodiment. Two multi-axis rails 5 are arranged perpendicular to each other, allowing the direction of the shaking process to be freely changed. The multi-axis rails 5 are controlled by a control unit 7, allowing the angle between the tapping direction and the shaking direction to be adjusted.

[0053] The shaking operation in the shaking step can be set to be periodic or aperiodic. From the viewpoint of applying a physical stimulus continuously to the entire culture vessel and facilitating the progress of detachment, it is preferable that the shaking in the shaking step be performed periodic. The time and period of shaking may be changed over time after the start of operation of the cell detachment system.

[0054] The shaking frequency in the shaking step in this embodiment refers to the period of time required for a cycle to return to a state at any given time point. The frequency is expressed as f (Hz), meaning the number of vibrations per second. The frequency can be expressed in units of rpm as the number of rotations per minute. This mainly refers to the number of times the same cycle is repeated per minute, assuming that one cycle in a rotating machine is one cycle. The frequency is not particularly limited, and in this embodiment, 0.1 Hz to 20 Hz is preferred, with 0.1 Hz to 10 Hz being more preferred. Furthermore, the shaking frequency is preferably lower than the frequency at which the culture vessel is beaten in the beating step. The shaking frequency by the shaking means may be changed over time after the start of operation of the cell detachment system.

[0055] The amount of displacement of the position of the culture vessel during the shaking step is preferably 0.1 mm to 300 mm, more preferably 0.1 mm to 150 mm, but is not particularly limited. The amount of displacement of the position of the culture vessel may change over time after the start of operation of the cell detachment system.

[0056] The shaking time in the shaking step can be appropriately set depending on the cell characteristics of the cell sheet, the environmental temperature, the type of detachment solution, and the detachment conditions.

[0057] The shaking direction (second direction) in the shaking step is not particularly limited, but in this embodiment, it preferably includes a component horizontal to the culture surface. This makes it possible to create a flow along the culture surface inside the culture vessel, allowing the cell sheet to be effectively detached. The shaking direction in the shaking step may be changed over time after the cell detachment system begins operation.

[0058] The shaking speed is not particularly limited, but is preferably 0.1 m / s to 1.0 m / s when measuring the speed of the side of the dish. The shaking speed may be changed over time after the start of operation of the cell detachment system.

[0059] The environmental temperature is not particularly limited, but from the viewpoint of maintaining cell viability, it is preferably 20 to 40° C., and more preferably 37° C. By manipulating the cells at a temperature close to the temperature during culture, the effects of temperature changes on the cells can be reduced, and the viability can be maintained at a high level.

[0060] (Angle between beating process and shaking process) Figure 3 shows the relationship between the direction of beating in the beating step and the direction of shaking in the shaking step. The angle between the direction of beating and the direction of shaking refers to the angle θ between the direction of beating and the direction of shaking when viewed from a direction perpendicular to the culture surface. The direction of beating and shaking may be changed over time.

[0061] The present inventors have found that cell sheets tend to begin to detach from positions within a specific angle range relative to the direction of the beating. Here, the specific angle range is between 45° and 135°. Furthermore, the present inventors have found that generating a water flow opposite the position where the cell sheet begins to detach is effective for efficient detachment while reducing cell damage.

[0062] That is, it is preferable that the tapping direction and the shaking direction are different, and the angle θ between the tapping direction and the shaking direction is more preferably 30° or more and 150° or less, and even more preferably 45° or more and 135° or less.

[0063] (Ultrasonic vibration) The cell detachment method of this embodiment may include, in addition to the shaking step and the beating step, an ultrasonic wave generating step of applying vibrations in the ultrasonic band to the culture vessel.

[0064] FIG. 4 is a cross-sectional view showing an example of the configuration of the cell detachment system according to this embodiment, which additionally combines a mechanism for applying vertical vibrations to the culture vessel 8. It is preferable to use an ultrasonic wave generating means as the mechanism for applying vertical vibrations. An ultrasonic element 17, which is an ultrasonic wave generating means, is placed below the culture vessel 8, and applies vertical ultrasonic vibrations to the culture vessel 8. The ultrasonic element 17 is controlled by the control unit 11, and the drive timing can be controlled as desired.

[0065] An example of ultrasonic vibration is a vibration with a frequency of 10 kHz to 1 MHz. The vibration generating means can be any means capable of applying ultrasonic vibration to cells, and is not particularly limited. One example is the use of an ultrasonic oscillator such as lead zirconate titanate (PZT) as the vibrating body.

[0066] 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.

[0067] 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.

[0068] 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 include those in which a piezoelectric body is sandwiched between two metal blocks and fastened together with bolts or the like to form an integrated structure.

[0069] (Time of external stimulus application) There are no particular limitations on the timing of initiating external stimuli other than the beating and shaking steps. The order in which beating and shaking and external stimuli such as ultrasound are initiated does not matter. When applying ultrasound stimulation, performing the beating and shaking steps during or after application of ultrasound or other stimuli allows for the application of shear force while weakening the adhesive strength of the cells, resulting in more effective cell detachment. Furthermore, information about cells adhered to the substrate may be measured and the measurement information may be used to determine the transition of the external stimuli.

[0070] Other steps may also be included, 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 detachment solution.

[0071] The steps may be repeated periodically or non-periodically, and the timing may be unified or adjusted for each step.

[0072] It is preferable that the ratio of the total time during which the step of applying external stimulation such as ultrasound is carried out to the total time during which the step of beating and shaking is carried out is 0.01 or more and 100 or less.

[0073] (Process for changing the direction of shaking and beating) The cell detachment method according to this embodiment may include, in addition to the shaking step and beating step, a step of changing the direction of beating or shaking.

[0074] For example, since detachment is initiated by applying a detachment initiation means, the direction of detachment can be changed by changing the direction in which the detachment initiation means is applied to the culture vessel. Figure 5 is a schematic diagram showing an example of a mechanism for changing the direction of beating in this embodiment. The beating means 3 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. The change in the beating direction of the beating means 3 is controlled by the control unit 7, and by changing the beating direction, the shaking direction and the direction of detachment can be made approximately parallel. The control unit can adjust the direction of beating and shaking over time.

[0075] In another example, in the cell detachment system shown in Figure 1, the shaking direction is controlled along two axes, and the shaking direction can be changed to any angle within the operating plane. By changing the shaking direction, the shaking direction and the detachment direction can be made approximately parallel.

[0076] [Example] 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.

[0077] (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%.

[0078] (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 65,000 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 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%.

[0079] (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%.

[0080] (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%.

[0081] (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%.

[0082] (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%.

[0083] (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 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%.

[0084] (Drive method) Using the cell detachment system shown in FIG. 5, the beating means 3, the shaking means 5, and the ultrasonic generating means were each driven at an environmental temperature of 37° C. to detach the cell sheet adhered to the culture vessel.

[0085] (Additional longitudinal vibration: ultrasonic vibration) The dish was placed from above in the device incorporating the Langevin element shown in Figure 5. A Langevin transducer with a resonant frequency of 36 kHz was used as the ultrasonic element, and ultrasonic waves were continuously irradiated from the bottom under conditions of a frequency of 36 kHz and an input voltage of 20 V.

[0086] (Removability evaluation) The detachment evaluation was performed based on two items: the quality of the cell sheet in the detached state and the detachment time. In each example, the detached state of the cell sheet refers to the state of the cell sheet after detachment from the culture surface, including any tearing. The quality of the cell sheet in each example was determined by observing the detached cell sheet. Furthermore, the detachment time in this example refers to the time required for the cell sheet to detach from the culture surface. In this example, the detached state of the cell sheet refers to the state in which the entire cell sheet has lifted off the culture surface of the culture vessel. Because the time required for detachment differs for each cell type, the detachment time was determined by calculating the ratio of the detachment time in each example to the time required in each comparative example. These evaluations were performed visually, using a camera, a phase-contrast microscope, or even a fluorescent microscope using a staining solution, depending on the situation. The cell sheet quality and detachment time were evaluated according to the following criteria. A rating of C or higher was deemed to be effective according to the present invention.

[0087] (Quality evaluation of cell sheets) A: No holes or tears have occurred. B: At least one of small holes and tears less than 500 μm occurred C: At least one of holes and tears of 500 μm or more and less than 1 mm has occurred D: At least one of a hole or tear of 1 mm or more has occurred

[0088] (peeling time evaluation) AA: Reduced by more than 30% A: Reduced by more than 20% but less than 30% B: Reduced by 10% or more but less than 20% C: Reduced by 1% or more but less than 10% D: It took the same peeling time as the comparative example

[0089] [Example 1] A detachment study was performed on a C2C12 cell sheet on a culture vessel using the following method. The C2C12 cell sheet was cultured under the culture conditions described above. After confirming that the cells had formed into a sheet, the culture medium in the culture vessel was replaced with fresh medium 4 hours before the detachment study, and the culture was continued in a 37°C incubator. The culture vessel was then removed from the 37°C incubator, placed in the detachment system shown in Figure 5, and driven under the driving conditions described above using the various conditions listed in Table 1. In this example, the driving was performed using the beating means 3 and shaking means 5, but not the ultrasonic generation means 8.

[0090] The angle θ between the beating direction and the shaking direction was changed by controlling the rotation stage that changes the beating direction according to the above-mentioned changing means 1. In this example, the angle θ was set to 90°.

[0091] The device was driven under these conditions, and after the cell sheet was detached, the detachment property was evaluated. The quality of the cell sheet was evaluated as A. Furthermore, the detachment time was also evaluated as A when compared with the results of Comparative Example 1, which was under the conditions of the same cell type. Since each evaluation was C or higher, it was determined that the present invention was effective.

[0092] [Examples 2 to 17] Except for changing the combination of cell type and driving conditions of each mechanism as shown in Table 1, cell sheet detachment was investigated in the same manner as in Example 1. The ultrasonic generation means used for applying additional vibration in Example 11 used the ultrasonic generation conditions described above.

[0093] The detachment properties of each example were evaluated in comparison with the conditions of Comparative Examples 1 to 7, which were the same cell type conditions. The results are shown in Table 2. Each example achieved a result of C or higher, and it was determined that the present invention was effective.

[0094] [Comparative Examples 1 to 7] Cell sheet detachment was investigated in the same manner as in Example 1, except that the cell type of the cell sheet and the combination of operating conditions for each mechanism were changed as shown in Table 1. The quality evaluation results for all cell sheets were rated D. The detachment times were 9 minutes for C2C12, 44 minutes for A549, 33 minutes for HEK293, 30 minutes for HUVEC, 90 minutes for MDCK, 43 minutes for hMSC, and 102 minutes for BAEC, resulting in a rating of D.

[0095] [Table 1]

[0096] [Table 2]

[0097] The disclosure of this embodiment includes the following methods and configurations.

[0098] (Method 1) A cell detachment method for detaching a cell sheet adhered to a culture surface of a culture vessel from the culture vessel using a cell detachment system, comprising: A beating step of beating the culture vessel in a first direction including a component parallel to the culture surface; a shaking step of shaking the culture vessel in a second direction including a component parallel to the culture surface; A cell detachment method, characterized in that the first direction and the second direction are different when viewed from a direction perpendicular to the culture surface.

[0099] (Method 2) The cell detachment method described in Method 1, wherein the angle between the first direction and the second direction when viewed from a direction perpendicular to the culture surface is 30° or more and 150° or less.

[0100] (Method 3) The cell detachment method described in Method 1 or 2, wherein the angle between the first direction and the second direction when viewed from a direction perpendicular to the culture surface is 45° or more and 135° or less.

[0101] (Method 4) 4. The cell detachment method according to any one of Methods 1 to 3, wherein the beating step includes a step of periodically beating the culture vessel.

[0102] (Method 5) The cell detachment method according to Method 4, wherein the frequency at which the culture vessel is beaten in the beating step is higher than the frequency at which the culture vessel is shaken in the shaking step.

[0103] (Method 6) 6. The cell detachment method according to any one of Methods 1 to 5, further comprising a vibration step of applying ultrasonic vibrations to the culture vessel.

[0104] (Configuration 7) A cell detachment method for detaching a cell sheet adhered to a culture surface of a culture vessel from the culture vessel, comprising: A striking means for striking the culture vessel in a first direction including a component parallel to the culture surface; a shaking means for shaking the culture vessel in a second direction including a component parallel to the culture surface; A cell detachment system, wherein the first direction and the second direction are different when viewed from a direction perpendicular to the culture surface. [Explanation of symbols]

[0105] 3 Hitting means 4 Shaking means 7 Control Unit 8 Culture vessel

Claims

1. A cell detachment method for detaching a cell sheet adhered to a culture surface of a culture vessel from the culture vessel using a cell detachment system, comprising: A beating step of beating the culture vessel in a first direction including a component parallel to the culture surface; a shaking step of shaking the culture vessel in a second direction including a component parallel to the culture surface; A cell detachment method, characterized in that the first direction and the second direction are different when viewed from a direction perpendicular to the culture surface.

2. 2. The cell detachment method according to claim 1, wherein the angle between the first direction and the second direction when viewed from a direction perpendicular to the culture surface is 30° or more and 150° or less.

3. 2. The cell detachment method according to claim 1, wherein the angle between the first direction and the second direction when viewed from a direction perpendicular to the culture surface is 45° or more and 135° or less.

4. The cell detachment method according to claim 1 , wherein the beating step includes a step of periodically beating the culture vessel.

5. 5. The cell detachment method according to claim 4, wherein the frequency at which the culture vessel is beaten in the beating step is higher than the frequency at which the culture vessel is shaken in the shaking step.

6. 2. The cell detachment method according to claim 1, further comprising a vibration step of applying ultrasonic vibrations to the culture vessel.

7. A cell detachment method for detaching a cell sheet adhered to a culture surface of a culture vessel from the culture vessel, comprising: A beating means for beating the culture vessel in a first direction including a component parallel to the culture surface; a shaking means for shaking the culture vessel in a second direction including a component parallel to the culture surface; A cell detachment system, characterized in that the first direction and the second direction are different when viewed from a direction perpendicular to the culture surface.

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