Cell stripping method and cell stripping system

Abstract

A cell stripping method for stripping a cell sheet adhered to a culture surface of a culture container from the culture container by using a cell stripping system includes: a knocking process of knocking the culture container in a first direction including a directional component parallel to the culture surface; and a vibration process of vibrating the culture vessel in a second direction containing a directional component parallel to the culture surface, in which the first direction and the second direction are different from each other when viewed in a direction perpendicular to the culture surface. The invention also relates to a cell stripping system.

Description

Technical Field

[0001] The present disclosure relates to a cell detachment method and a cell detachment system. Background Art

[0002] In the fields of regenerative medicine and cell medicine, attempts have been made in recent years to cultivate sheet-like cells and transplant the cultivated sheet-like cells (cell sheets) into affected areas to repair damaged tissue, for example. When producing cell sheets using adherent cells, for example, the cells are cultured as a sheet on a polystyrene culture dish (an example of a culture substrate), and then peeled from the substrate and recovered as a sheet. A method for efficiently and stably producing cell sheets while minimizing wrinkles, tears, holes, and the like in the cell sheets is desired.

[0003] Japanese Patent Laid-Open No. 2014-113133 discloses a cell detachment apparatus including a container holder for attaching a culture container to which cultured cells have adhered, and a guide mechanism that guides forward and backward movement of the container holder, wherein the container holder is allowed to collide with a collision target member. Summary of the Invention

[0004] The present disclosure provides a cell detachment method for effectively detaching a cell sheet while reducing tearing of the cell sheet. The present disclosure also provides a cell detachment system for effectively detaching a cell sheet while reducing tearing of the cell sheet.

[0005] One aspect of the present disclosure provides a cell detachment method, which detaches a cell sheet adhered to a culture surface of a culture container from a culture container by using a cell detachment system, the method including a tapping process of tapping the culture container in a first direction having a directional component parallel to the culture surface; and a vibration process of vibrating the culture container in a second direction having a directional component parallel to the culture surface, wherein the first direction and the second direction are different from each other when viewed in a direction perpendicular to the culture surface.

[0006] Another aspect of the present disclosure provides a cell detachment system that detaches a cell sheet adhered to a culture surface of the culture container from a culture container, the cell detachment system including: a knocking unit that knocks the culture container in a first direction having a directional component parallel to the culture surface; and a vibration unit that vibrates the culture container in a second direction having a directional component parallel to the culture surface, wherein the first direction and the second direction are different from each other when viewed in a direction perpendicular to the culture surface.

[0007] Further features of the present disclosure will become apparent from the following description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1is a schematic diagram of an example of a cell detachment system according to the first embodiment.

[0009] Figure 2 is a sectional view of a knocking unit according to the first embodiment.

[0010] Figure 3 is a top schematic diagram illustrating the relationship between the striking direction and the vibration direction according to the first embodiment.

[0011] Figure 4 is a schematic diagram of an example of a cell detachment system according to the first embodiment.

[0012] Figure 5 is a schematic diagram of an example of a cell detachment system according to the first embodiment.

[0013] Figure 6A and Figure 6B They are schematic diagrams of the uniaxial vibration mechanism according to the first embodiment.

[0014] Figure 7 is a flow chart of the cell detachment method according to the first embodiment. DETAILED DESCRIPTION

[0015] When a cell sheet is peeled from a culture vessel holding a liquid and to which the cell sheet adheres, or when the culture vessel is allowed to move back and forth in the same direction as the direction in which an impact is applied to the culture vessel, the cell sheet may be easily torn. Cell sheets may also be easily torn under conditions designed to effectively peel the cell sheet in a relatively short period of time.

[0016] The present disclosure will now be described in detail by way of examples.

[0017] The inventors of the present disclosure have studied a method for effectively peeling a cell sheet while reducing tearing of the cell sheet. As a result, the inventors found that the cell sheet can be effectively peeled by combining a tapping process of tapping a culture container to which the cell sheet is adhered and a vibration process of vibrating the culture container.

[0018] Furthermore, the inventors have discovered that the cell sheet tends to start detaching from a position forming an angle within a specific range relative to the direction in which the culture container is struck.

[0019] Further research has been conducted on this discovery, revealing that cell sheets can be more effectively exfoliated by vibrating the culture container at a specific range of angles relative to the tapping direction. Specifically, it has been discovered that cell sheets can be effectively exfoliated when the tapping direction and the vibration direction, when viewed perpendicular to the culture surface, differ. The reason why the aforementioned features of the present disclosure improve cell sheet exfoliation efficiency while reducing tearing is likely due to the following.

[0020] The liquid flow generated by vibrating the culture vessel enters the gap between the culture vessel and the partially peeled cell sheet, pushing the cell sheet further to peel. Here, when the water flows toward the partially peeled portion of the cell sheet, it can effectively peel the cell sheet while reducing tearing.

[0021] Cell detachment method

[0022] The cell detachment method according to the first embodiment removes a cell sheet adhered to the culture surface of the culture container from the culture surface by tapping the culture container and vibrating the culture container. Furthermore, in this embodiment, the tapping process involves tapping the culture container in a first direction having a directional component parallel to the culture surface, and the vibration process involves vibrating the culture container in a second direction having a directional component parallel to the culture surface. When the first and second directions differ when viewed perpendicular to the culture surface, the cell sheet can be effectively detached while minimizing tearing of the cell sheet.

[0023] Figure 7 The flowchart of the cell detachment method according to this embodiment is shown. First, in a tapping process (S11), a tapping unit taps the culture container with the cell sheet adhered thereto. Next, in a vibration process (S12), the culture container is vibrated in a direction different from the tapping by the tapping unit. The timing of the start of the tapping and vibration processes is not limited to this, and the two processes can be performed simultaneously or alternately within a certain time period.

[0024] In this example, the cell detachment method refers to a method for detaching a cell sheet adhered to a culture surface from a culture vessel by using an external stimulus. Examples of external stimuli include tapping, vibration, ultrasound, liquid discharge, and liquid agitation. Liquid discharge involves methods that discharge liquid from a needle, for example, so that the liquid impacts the culture surface, disperses along the culture surface, and applies shear force.

[0025] Agitation involves methods that create a flow channel along the culture surface and apply shear force by feeding the liquid in one direction or switching directions using a pump or syringe. Cell detachment methods can involve external stimuli alone, such as tapping and vibration, or a combination of other external stimuli.

[0026] cell sheets

[0027] The cell sheet in this embodiment refers to a membrane in which cells are connected to each other to form a sheet. There is no particular limitation on the cells that constitute the cell sheet, as long as the cells can form a cell sheet. Examples of cells include adhesive cells, such as adhesive somatic cells.

[0028] Examples of somatic cells include myoblasts (e.g., skeletal muscle myoblasts), muscle satellite cells, and mesenchymal stem cells (e.g., cells derived from bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue, endometrium, placenta, umbilical cord blood, etc.). Other examples include cardiomyocytes, fibroblasts, tissue stem cells (e.g., cardiac stem cells), embryonic stem cells, pluripotent stem cells (e.g., iPS cells), synoviocytes, chondrocytes, and epithelial cells (e.g., oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.).

[0029] Still other examples include endothelial cells (e.g., vascular endothelial cells, etc.), hepatocytes (e.g., hepatocytes, etc.), pancreatic cells (e.g., pancreatic islet cells, etc.), kidney cells, adrenal gland cells, periodontal ligament cells, gingival cells, periosteal cells, and skin cells.

[0030] In addition, the somatic cells may be cells differentiated from iPS cells (iPS cell-derived cells). Examples of iPS cell-derived cells include 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, chondrocytes, and the like derived from iPS cells.

[0031] Culture container

[0032] The culture container of this embodiment is not particularly limited as long as it is a cell-adhesive culture container. For example, the culture container is any one of a flask, a tissue culture flask, a culture dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a multi-well plate, a multi-plate, a culture bag, and a bottle.

[0033] The material used for the culture container in this embodiment can be any chemically stable material that can culture the desired cells. Examples 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, polysiloxane, polymethylpentene, glass, and metal. Polystyrene can be used from the perspective of stability.

[0034] Alternatively, a temperature-responsive container having a culture surface whose hydrophilicity changes with temperature can be used as the culture container of this embodiment.

[0035] Cell removal agent

[0036] The cell removal agent herein refers to a solution maintained in a culture vessel during cell removal using the cell removal method disclosed herein. The cell removal agent herein does not necessarily contain a component that promotes the removal of a cell sheet. For example, from the perspective of protecting the cell surface, a cell removal agent substantially free of proteolytic enzymes may be used.

[0037] Here, "substantially containing no" means that the content is equal to or lower than 0.0005% by mass.

[0038] The pH of the cell removing agent in this embodiment may be in the neutral or acidic range.

[0039] The neutral range is suitable for cell culture, and the cell survival rate can be stably maintained at a high level. The pH can be appropriately adjusted using hydrochloric acid, sodium hydroxide, etc. Various buffer solutions can also be used to stabilize the pH.

[0040] In this embodiment, any buffer solution can be used without limitation as long as it can maintain a neutral range. Examples include Tris buffer solutions (such as Tris-HCl buffer solutions), phosphate buffer solutions, HEPES buffer solutions, citric acid-phosphate buffer solutions, glycylglycine-sodium hydroxide buffer solutions, Britton-Robinson buffer solutions, and GTA buffer solutions. Phosphate buffer solutions that approximate the in vivo environment can be used, such as phosphate-buffered saline (PBS) obtained by adjusting a phosphate buffer solution to be isotonic with the intracellular fluid.

[0041] The viscosity of the cell removal agent in this embodiment can be 1.80 mPa·s or less. This is because the flow of the agent generated by ultrasonic vibration is not impeded, and the removal efficiency can be maintained high. The viscosity of the cell removal agent can be appropriately adjusted by adding polymers, sugars, etc.

[0042] The cell removing agent of this embodiment may contain a proteolytic enzyme, but the amount of the proteolytic enzyme relative to the total mass of the cell removing agent may be 0.0005% by mass or less, or zero. This is because while the removal efficiency may be improved as the proteolytic enzyme decomposes certain parts of the cells, the proteolytic enzyme may reduce the mass of the cells.

[0043] The proteolytic enzyme in this embodiment is an enzyme that decomposes certain parts of cells to facilitate the detachment of cells from the substrate. Examples thereof include trypsin, Accutase, collagenase, natural protease, chymotrypsin, elastase, papain, pronase, and recombinant forms thereof.

[0044] The cell removal agent in this embodiment can be a medium, but is not particularly limited. A solution containing a metal ion chelator (hereinafter also referred to as a chelating agent) can also be used. This is because using a cell removal agent containing a chelating agent can more effectively remove cells through ultrasonic vibration.

[0045] The chelating agent in this embodiment is not particularly limited. Examples of the chelating agent include ethylenediaminetetraacetic acid (hereinafter also referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycol ether diaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediamine disuccinic acid, hydroxyethylenediphosphonic acid, citric acid, gluconic acid, and phosphonic acid butane tricarboxylic acid.

[0046] The chelating agent may form a chelate with a divalent cation, or may form a chelate with Ca2+ and Mg2+, and the chelating agent may be ethylenediaminetetraacetic acid. When ethylenediaminetetraacetic acid is used as the chelating agent, the pH of the cell removal agent may be 7.0 or higher and 8.0 or lower. This is because this level is relatively high within the neutral range where cell viability can be maintained at a high level, thereby further enhancing the chelating ability of ethylenediaminetetraacetic acid and further increasing the removal efficiency.

[0047] The chelating agent can be used alone or in combination. The content of the chelating agent can be 0.01 mM or more and 5.0 mM or less. Within this range, the chelating effect can be safely obtained and the activity reduction caused by the presence of excessive chelating agent can also be reduced.

[0048] The cell removal agent of this embodiment may contain a hydrophilic polymer including a polyalkylene glycol structure. In a cell removal method using ultrasound, polyalkylene glycol can increase cell survival. An example of a hydrophilic polymer including a polyalkylene glycol structure is polyethylene glycol. As measured by gel permeation chromatography, the hydrophilic polymer may have a peak molecular weight Mp of 800 or more and 50,000 or less, or may have a peak molecular weight Mp of 1,200 or more and 20,000 or less. This is because the polymer has a lesser effect on cells and can reduce the medium thickening effect caused by the polymer.

[0049] The type of culture medium may be any, and examples thereof include Dulbecco's Modified Eagles's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagles's Minimum Essential Medium (EMEM), α Modified Eagles's Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM), RPMI-1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB 131 medium, William's E medium, IPL-41 medium, Fischer's medium, StemSpan H3000, StemSpan SFEM, Stemline II, Endothelial Cell Growth Medium 2 Kit, Mesenchymal Stem Cell Growth Medium 2, MSCGM Bullet Kit, mTeSR1, mTeSR2 medium, Repro FF, Repro FF2, NutriStem medium, and MF-Medium. Mesenchymal Stem Cell GrowthMedium.

[0050] Among them, a medium suitable for culturing each cell type can be used.

[0051] Serum or antibiotics may be added to the above media. Examples of serum include fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, porcine serum, chicken serum, rabbit serum, and human serum. FBS is commonly used due to its high availability. Alternatively, serum-free media can be used, which do not contain any unprocessed or unpurified serum but do contain purified blood-derived components or animal tissue-derived components (growth factors, etc.).

[0052] Examples of antibiotics added to the medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, and amphotericin B.

[0053] Cell sheet culture conditions

[0054] Cell sheet culture conditions suitable for culturing cells can be selected. Typically, appropriate medium is added to a culture dish and approximately 1.0 × 10 1to 1.0×10 5 cells / cm 2 The cells are cultured in an environment at a temperature of 37° C. and a CO 2 concentration of 5%. Here, the culture can be performed until the cell occupation area ratio in the substrate reaches approximately 100%, in other words, until a confluent state is reached.

[0055] Cell detachment system

[0056] Figure 1 This is a schematic diagram of an example of a cell detachment system for performing the cell detachment method according to this embodiment. Culture container 8 is struck by striking unit 3. Furthermore, culture container 8 is vibrated by vibration unit 4. The cell detachment system according to this embodiment may also be capable of applying external stimulation (such as ultrasonic vibration).

[0057] Tapping process

[0058] The knocking process in the present embodiment includes such a process, which applies an impact force to the culture substrate so that at least part of the cells on the culture surface that adhere to the culture substrate are peeled off from the culture surface. For example, the knocking process is such a process, which performs at least one of the process of applying an impact force to the culture substrate by knocking the culture substrate itself and the process of applying an impact force to the culture substrate by moving the culture substrate and causing the culture substrate to collide with the member constituting the cell stripping device. The knocking process in the present embodiment can be periodic or non-periodic, and the size of the impact force applied to the culture substrate in the knocking process can be appropriately set. The knocking process in the present embodiment can be performed continuously from the beginning of cell stripping to completion, can stop before cell stripping is completed, or can be performed intermittently.

[0059] The timing of the tapping during the tapping process can be set periodically or aperiodically. Since the impact is continuously applied to the entire container, tapping can be performed at a periodic timing to promote the start of detachment. After the operation of the cell detachment system is started, the timing and period of the tapping can be changed over time.

[0060] The tapping frequency in the tapping process of this embodiment refers to the cycle it takes for the state to return to the state at a specific point in time. Frequency is expressed as f (Hz) and is the number of vibrations per second. Frequency can be expressed in rpm (rotations per minute). This means that, primarily for rotating machines, one cycle is considered a return, and the number of times the same return is repeated within one minute is assumed to be the frequency. There are no specific restrictions on the frequency, and in this embodiment, the frequency can be 0.1 Hz to 20 Hz or 0.1 Hz to 10 Hz. There are no specific restrictions on the magnitude relationship between the frequency ranges of the tapping process and the vibration process in this embodiment, but the ratio of the frequency of the tapping process to the frequency of the vibration process can be 1 to 50 or 1 to 15. The frequency of the tapping process can change over time.

[0061] The time for continuously and / or intermittently applying the impact during the tapping process can be appropriately set according to the cell characteristics of the cell sheet, the ambient temperature, the type of the remover, and the peeling conditions. The tapping position during the tapping process is not particularly limited; however, the impact can be applied to the culture container or the holder, including the side surface of the culture container.

[0062] The direction of the impact (first direction) during the impact process is not particularly limited, but may include a directional component parallel to the culture surface. In particular, when impact is applied to the culture container or holder, inertial forces or the flow of the culture solution may be generated. This application of these forces can detach the cell sheet. After the cell detachment system begins operating, the direction of the impact may change over time.

[0063] The ambient temperature is not particularly limited and may be 20° C. to 40° C. from the viewpoint of maintaining cell viability. When the temperature is close to the temperature during culture, the influence of temperature changes on cells can be reduced, and a high viability can be maintained.

[0064] Percussion Unit

[0065] The knocking unit in this embodiment can be any feature that can perform the above-mentioned knocking process. The knocking unit includes, for example, an object to be collided with the culture substrate and a mobile unit that moves the object to collide with the culture substrate, or includes a mobile unit that moves the culture substrate to collide with a component constituting the cell stripping device. The object can have a mass that can apply an appropriate impact force to the culture substrate without tearing the culture substrate. Examples of the shape of the object include a rod shape, a hammer shape, and a spherical shape. Examples of the moving unit include a motor and a solenoid that can generate a moving force for moving the object or the culture substrate by energizing it. Here, the object or the culture substrate can be moved only by using a motor, a solenoid, etc., or the motor and the solenoid can be used in combination with a component that can store energy (such as a spring component).

[0066] The knocking unit 3 knocks the culture container 8 to start detachment of the cell sheet. Figure 2 4 is a cross-sectional view of an example of a knocking unit of this embodiment. Cam 41 is connected to a motor and rotates in the direction of the shaft's rotation when driven by the motor. Hammer 42 contacts cam 41 at shaft 48 and is urged by spring 43 in a direction to knock against culture container 8. After spring 43 is compressed by the rotation of cam 41, hammer 42 moves in a direction that causes the profile of cam 41 to knock against culture container 8. The frequency of the knocking can be determined by the rotation rate of the motor and can be detected by optical coupler 44.

[0067] Vibration process

[0068] In this embodiment, the vibration process refers to a process of moving the culture container or a portion holding the culture container. In the vibration process, the culture container is vibrated to detach the cell sheet from the culture container.

[0069] The driving force of the vibration process is not limited to a spring and may include a magnet, an electromagnet, a motor, or the like.

[0070] Figure 6A and Figure 6B The uniaxial vibration mechanism according to this embodiment is shown. The guide rail 50 is fixed to Figure 1 The base plate 2 shown in FIG. 1 is provided to vibrate the culture container 8 back and forth. A base-side spring column 52, to which a pressure spring 51 is connected, is fixed to the base plate 2, and the pressure spring 51 is connected to the base-side spring column 52. The opposite ends of the pressure spring 51 are fixed to a vibrated-side spring column 53 provided in the vibrated portion. The pressure spring 51 can be selected as needed and has a detachable structure.

[0071] Vibration occurs when the rotation of the vibration motor 54 is transmitted to the vibration rod 56 through the gear 55. The rotating disk 57 is fixed to the rotating shaft of the vibration motor 54, and rotates as shown in the figure.

[0072] The direction of rotation can be reversed. A linear bushing holder 58 is rotatably mounted at a position offset from the axis of rotation, with a linear bushing 59 incorporated therein. The vibration rod 56 is also incorporated therein so as to be linearly movable. One end of the vibration rod 56 is fixed to a rotatable vibration fulcrum shaft 60. This translates into forward and backward movement centered on the vibration fulcrum shaft 60. The frequency of this forward and backward movement is detected by a rotary optical coupler 61.

[0073] The connecting rod 62 is fixed to the vibration rod 56 parallel to the vibration rod 56 using a connecting rod fixing tool 63, and one end is fixed to the vibration fulcrum shaft 60. A vibration sliding part 64 equipped with the base plate 2 contacts the connecting rod 62, which moves back and forth, to generate a back and forth vibration movement. The vibration sliding part 64 is supported by a part sliding shaft 66 parallel to the vibration adjustment screw 65, and the vibration stroke can be freely adjusted by rotating the vibration adjustment screw 65. A stroke knob 67 is installed at one end of the vibration adjustment screw 65 and can be manually rotated to perform adjustments.

[0074] Figure 1 Schematic diagram of an example of a vibration structure according to this embodiment. Multi-axis guide rails 5 are arranged biaxially, orthogonal to each other, so that the direction of the vibration process can be freely changed. The multi-axis guide rails 5 are controlled by a controller 7, and the angle between the tapping direction and the vibration direction can be adjusted.

[0075] The vibration operation during the vibration process can be set to be periodic or aperiodic. Since physical impact is continuously applied to the entire culture container, the vibration during the vibration process can be performed periodically from the perspective of promoting the detachment process. After the operation of the cell detachment system is started, the timing and period of the vibration can be changed over time.

[0076] The vibration frequency during the vibration process in this embodiment refers to the cycle it takes for the state to return to the state at a specific point in time. Frequency is expressed as f (Hz) and is the number of vibrations per second. Frequency can be expressed in rpm (rotations per minute). This means that, primarily for rotating machines, one cycle is considered a return, and the number of times the same return is repeated within one minute is assumed to be the frequency. The frequency is not particularly limited and, in this embodiment, can be 0.1 Hz to 20 Hz or 0.1 Hz to 10 Hz. The vibration frequency can be lower than the frequency of the culture container being struck during the tapping process. After the cell detachment system begins operating, the vibration frequency of the vibration unit can be changed over time.

[0077] The displacement of the position of the culture container during vibration may be 0.1 mm to 300 mm or 0.1 mm to 150 mm, but is not particularly limited. After the operation of the cell detachment system is started, the displacement of the position of the culture container may change over time.

[0078] The time for performing vibration during the vibration process can be appropriately set according to the cell characteristics of the cell sheet, the ambient temperature, the type of the removing agent, and the peeling conditions.

[0079] The vibration direction (second direction) during the vibration process is not particularly limited, but in this embodiment, it can include a directional component parallel to the culture surface. This generates a flow along the culture surface within the culture container, effectively detaching the cell sheet. After the cell detachment system begins operating, the vibration direction during the vibration process can change over time.

[0080] The speed of the vibration is not particularly limited, but when the speed at the side surface of the culture dish is measured, the speed can be 0.1 m / s to 1.0 m / s. After the operation of the cell detachment system is started, the speed during the vibration process can be changed over time.

[0081] The ambient temperature is not particularly limited, but from the viewpoint of maintaining cell viability, it may be 20° C. to 40° C. or may be 37° C. When cells are treated at a temperature close to the culture temperature, the effect of temperature changes on cells can be reduced, and a high viability can be maintained.

[0082] The angle between the striking process and the vibration process

[0083] Figure 3 This represents the relationship between the tapping direction during tapping and the vibration direction during vibration. The angle θ formed between the tapping and vibration directions is the angle θ formed between the tapping and vibration directions when viewed perpendicular to the culture surface. The directions of tapping and vibration may change over time.

[0084] The inventors of the present disclosure have discovered that cell sheets tend to begin peeling from positions that are within a specific range of angles relative to the tapping direction. Here, the specific range of angles is an angle of 45° or greater and 135° or less. Furthermore, the inventors of the present disclosure have discovered that generating a water flow opposite the position where the cell sheet has begun peeling is effective in effectively peeling the cell sheet while reducing cell tearing.

[0085] In other words, the striking direction and the vibration direction may be different from each other and may form an angle θ of 30° or more and 150° or less, or may form an angle θ of 45° or more and 135° or less.

[0086] Vibrations in the ultrasonic frequency band

[0087] In addition to the vibration process and the tapping process, the cell detachment method according to this embodiment may further include an ultrasonic wave generating process of applying vibration in an ultrasonic frequency band to the culture container.

[0088] Figure 4This is a schematic diagram illustrating an example of a structure in which a mechanism for applying vertical vibrations to the culture vessel 8 is added to and combined with the cell detachment system of this embodiment. An ultrasonic wave generating unit can be used as the mechanism for applying vertical vibrations. An ultrasonic wave element 17 serving as the ultrasonic wave generating unit is positioned below the culture vessel 8 and vertically applies ultrasonic vibrations to the culture vessel 8. The ultrasonic wave element 17 is controlled by a controller 11, and its driving timing can be freely controlled.

[0089] An example of vibration in the ultrasonic frequency band is vibration with a frequency of 10 kHz or higher and 1 MHz or lower. The vibration generating unit can be any device capable of applying vibration in the ultrasonic frequency band to cells. One example is using an ultrasonic oscillator made of lead zirconate titanate (PZT) as the vibrating element.

[0090] The ultrasonic vibrator can be any body capable of generating ultrasonic waves, and an example is a piezoelectric body and a vibrating plate combined together. When the piezoelectric body is circular, the vibrating plate can be made of glass, SUS, or quartz. When the vibrating plate is made of glass, SUS, or quartz, it can output large amplitudes at relatively high drive frequencies (vibration frequencies) in the ultrasonic range without damaging the ultrasonic vibrator.

[0091] In the case of an annular piezoelectric body, the outer diameter of the vibration plate can be equal to the outer diameter of the piezoelectric body. The thickness of the vibration plate can be such that when the vibration plate and the piezoelectric body are combined and subjected to flexural vibration, the midpoint of the flexure in the thickness direction (i.e., the neutral plane that does not experience stretching or contraction during flexure) can be located on the vibration plate side, because the strain in the piezoelectric body can be efficiently used for flexure.

[0092] In addition, a commercially available Langevin oscillator or rectangular oscillator can also be used as the ultrasonic vibration body of the present disclosure. An example of a Langevin oscillator is an oscillator in which a piezoelectric body is placed between two metal blocks and clamped together with bolts or the like to form an integral structure.

[0093] Time of applying external stimulus

[0094] There is no particular limitation on the timing of starting external stimulation other than the tapping process and the vibration process.

[0095] The order in which tapping, vibration, and external stimulation such as ultrasound are initiated can be arbitrary. When ultrasound stimulation is applied, the adhesion of cells can be weakened while shear force is applied. Therefore, when the tapping and vibration processes are performed during or after the application of stimulation such as ultrasound, cells can be more effectively detached. To determine the transition to external stimulation, information about cells adhered to the substrate can be measured and used.

[0096] In addition, other processes may be included. Examples of other processes include a process of replacing the cell removing agent, a process of washing cells with the cell removing agent, and a process of diluting and homogenizing the removing agent.

[0097] The aforementioned process can be repeated at a specific cycle or at irregular cycles. The timing can be uniform or can be adjusted for each process.

[0098] The ratio of the total time of performing the tapping process and the vibration process to the total time of the process of the external stimulation (such as ultrasonic waves) may be 0.01 or more and 100 or less.

[0099] The process of changing the vibration direction and the knocking direction

[0100] The cell detachment method according to the present embodiment may include a process of changing the tapping direction and the vibration direction in addition to the vibration process and the tapping process.

[0101] For example, since the peeling is started by applying the peeling starting unit, the peeling progress direction can be changed by changing the direction in which the peeling starting unit is applied to the culture container. Figure 5 is a schematic diagram of an example of a mechanism for changing the knocking direction in the present embodiment. The knocking unit 3 is constructed so that the angle of knocking the culture container 8 can be freely changed, and due to this structure the knocking direction can be freely changed. The change of the knocking direction of the knocking unit 3 is controlled by the controller 7, and the vibration direction and the peeling direction can be made substantially parallel to each other by changing the knocking direction. Over time, the knocking and vibration directions can be adjusted by the controller. The direction changing unit 9 can be a unit for changing the angle formed by the knocking direction of the knocking unit 3 and the vibration direction of the vibration unit 5, or can be a unit for changing the knocking direction relative to the culture container 8. The direction changing unit 9 can be, for example, a rotatable mounting table.

[0102] In another example, Figure 1 In the cell detachment system shown in , the vibration direction is biaxially controlled, so the vibration direction can be changed to any angle within the operation plane. By changing the vibration direction, the vibration direction and the detachment direction can be made substantially parallel to each other.

[0103] Example

[0104] The present disclosure will now be described in more detail through examples and comparative examples; however, the present disclosure is not limited by the following examples in any way without departing from the gist of the present disclosure.

[0105] Cultivation of C2C12 cells on substrates

[0106] C2C12 cells (mouse myoblasts) were plated at 65,000 cells / cm 2Cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark) manufactured by CellSeed Inc.) and cultured at 37°C in an environment with a CO2 concentration of 5%. The medium used was DMEM / F12 medium (manufactured by Thermo Fisher Scientific Inc.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Inc.). Culture was performed for 2 days, and the state of the cells was observed using a phase contrast microscope to confirm cell adhesion and growth. The cell occupancy ratio of the culture dish was approximately 100%.

[0107] Culture of A549 cells on substrates

[0108] A549 cells (human lung epithelial adenocarcinoma cells) were plated at 65,000 cells / cm 2 Cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark) manufactured by CellSeed Inc.) and cultured at 37°C in an environment with a CO2 concentration of 5%. The medium used was DMEM (manufactured by Thermo Fisher Scientific Inc.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Inc.). Culture was performed for 7 days, and the cells were observed using a phase contrast microscope to confirm cell adhesion and growth. The cell occupancy rate of the culture dish was approximately 100%.

[0109] BAEC cell culture on substrate

[0110] BAEC cells (bovine aortic endothelial cells) were cultured at 20,000 cells / cm 2Cells were seeded at a density of 100 μm in Φ35 polystyrene culture dishes (manufactured by Corning Incorporated) and cultured at 37°C in an environment with a 5% CO2 concentration. The medium used was DMEM (manufactured by Thermo Fisher Scientific Inc.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Inc.). Culture was performed for 7 days, and the cells were observed using a phase contrast microscope to confirm cell adhesion and growth. The cell occupancy rate of the culture dish was approximately 100%.

[0111] HEK293 cell culture on substrates

[0112] HEK293 cells (human embryonic kidney cells) were plated at 65,000 cells / cm 2 Cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark) manufactured by CellSeed Inc.) and cultured at 37°C in an environment with a CO2 concentration of 5%. The medium used was Eagle's MEM (manufactured by FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Inc.). Culture was performed for 9 days, and the cells were observed using a phase contrast microscope to confirm cell adhesion and growth. The cell occupancy rate of the culture dish was approximately 100%.

[0113] Culture of HUVEC on substrate

[0114] HUVEC cells (human umbilical vein endothelial cells) were cultured at 65,000 cells / cm 2Cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark) manufactured by CellSeed Inc.) and cultured in an environment of 37°C and 5% CO2 concentration. The medium used was Endothelial Cell Growth Medium 2 Kit (manufactured by PromoCell GmbH), to which 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Inc.) were added. The culture was carried out for 7 days, and the state of the cells was observed using a phase contrast microscope to confirm cell adhesion and growth. The cell occupancy ratio of the culture dish was approximately 100%.

[0115] MDCK cell culture on substrate

[0116] MDCK cells (Madin-Darby canine kidney cells) were cultured at 65,000 cells / cm 2 Cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark) manufactured by CellSeed Inc.) and cultured at 37°C in an environment with a CO2 concentration of 5%. The medium used was Eagle's MEM (manufactured by FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Inc.). Culture was performed for 8 days, and the state of the cells was observed using a phase contrast microscope to confirm cell adhesion and growth. The cell occupancy rate of the culture dish was approximately 100%.

[0117] hMSC cell culture on substrate

[0118] hMSC cells (human mesenchymal stem cells) were cultured at 30,000 cells / cm 2Cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark) manufactured by CellSeed Inc.) and cultured at 37°C and a CO2 concentration of 5%. The medium used was Mesenchymal Stem Cell Growth Medium 2 (manufactured by PromoCell GmbH) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co. LLC) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Inc.). Culture was performed for 7 days, and the state of the cells was observed using a phase contrast microscope to confirm cell adhesion and growth. The cell occupancy ratio of the culture dish was approximately 100%.

[0119] Drive method

[0120] Use at an ambient temperature of 37°C Figure 5 The cell detachment system shown in drives the knocking unit 3, the vibration unit 5, and the ultrasonic wave generating unit to detach a cell sheet adhered to a culture container.

[0121] Additional vertical vibration: ultrasonic vibration

[0122] Place the culture dish from above into the Figure 5 The device of the Langevin element is shown in FIG. A Langevin oscillator with a resonance frequency of 36 kHz was used as the ultrasonic element, and ultrasonic waves were continuously applied from the bottom at a frequency of 36 kHz and an input voltage of 20 V.

[0123] Evaluation of peelability

[0124] The peelability was evaluated from two items: the quality evaluation of the cell sheet in the peeled state and the peeling time. In each example, the peeling state of the cell sheet refers to the state including the tearing of the cell sheet after peeling from the culture surface. The quality of the cell sheet in each example was evaluated by observing the cell sheet in the peeled state. In addition, the peeling time in each example refers to the time it takes for the cell sheet to peel off from the culture surface. In the example, the state in which the cell sheet has been peeled means that the entire surface of the cell sheet has floated from the culture surface of the culture container. Since the time required for peeling is different for each cell type, the peeling time is evaluated by determining the ratio of the peeling time of the example for the same cell type to the peeling time of the control example. The evaluation is performed by observation using the naked eye, a camera, a phase contrast microscope, a fluorescence microscope using a dye liquid, etc. The quality and peeling time of the cell sheet are evaluated based on the following criteria, and those rated C or above are determined to have the effect of the present disclosure.

[0125] Quality assessment of cell sheets

[0126] A: No holes or tears were generated.

[0127] B: At least one of a hole smaller than 500 μm and a tear occurs.

[0128] C: At least one of a hole or a tear of 500 μm or more but less than 1 mm is generated.

[0129] D: At least one of a hole of 1 mm or more and a tear occurs.

[0130] Evaluation of peeling time

[0131] AA: Time is shortened by more than 30%.

[0132] A: The time is shortened by more than 20% and less than 30%.

[0133] B: The time is shortened by more than 10% and less than 20%.

[0134] C: The time is shortened by more than 1% and less than 10%.

[0135] D: The peeling time was as long as that in the comparative example.

[0136] Example 1

[0137] The detachment of C2C12 cell sheets from the culture vessel was studied as follows. C2C12 cell sheets were cultured under the above-described culture conditions. After confirming the formation of cell sheets, 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 and placed in a Figure 5 , and the system was driven under the aforementioned driving conditions using the conditions shown in Table 1. In this example, the knocking unit 3 and the vibration unit 5 were used, but the ultrasonic wave generating unit 8 was not used for driving.

[0138] The angle θ between the striking direction and the vibration direction is changed by controlling the rotation table that changes the striking direction according to the above-mentioned changing unit 1. In this example, the angle θ is set to 90°.

[0139] Drive was performed under these conditions, and the peelability was evaluated after the cell sheet was peeled. The quality of the cell sheet was evaluated as A. Compared with the results of Comparative Example 1 under the same cell type conditions, the peeling time was rated as A. Since all evaluation items were rated C or higher, it was determined that the effects of the present disclosure were demonstrated.

[0140] Examples 2 to 17

[0141] The detachment of the cell sheet was studied as in Example 1, except that the combination of cell types and driving conditions of the mechanism was changed as shown in Table 1. The aforementioned ultrasonic wave generation conditions were used as the ultrasonic wave generation unit for applying additional vibration in Example 11.

[0142] The peelability of each example was evaluated by comparing with the conditions of Comparative Examples 1 to 7 using the same cell type conditions. The results are shown in Table 2. In all examples, the results were rated C or higher, and it was determined that the effects of the present disclosure were exhibited.

[0143] Comparative Examples 1 to 7

[0144] The detachment of the cell sheet was studied as in Example 1, except that the combination of the cell type of the cell sheet and the driving conditions of the mechanism was changed as shown in Table 1. In all comparative examples, the quality of the cell sheet was evaluated as D. In addition, the detachment time of C2C12 was 9 minutes, the detachment time of A549 was 44 minutes, the detachment time of HEK293 was 33 minutes, the detachment time of HUVEC was 30 minutes, the detachment time of MDCK was 90 minutes, the detachment time of hMSC was 43 minutes, and the detachment time of BAEC was 102 minutes, and the detachment time was evaluated as D.

[0145] Table 1: Processing conditions of Examples 1 to 17 and Comparative Examples 1 to 7

[0146] Table 2: Evaluation results of Examples 1 to 17 and Comparative Examples 1 to 7

[0147] According to the present disclosure, a cell detachment method can be provided that can effectively detach a cell sheet while reducing tearing of the cell sheet. In addition, according to the present disclosure, a cell detachment system can be provided that can effectively detach a cell sheet while reducing tearing of the cell sheet.

[0148] While the present disclosure has been described with reference to exemplary embodiments, it is to be understood that the disclosure is not limited to the disclosed exemplary embodiments. The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A cell detachment method for detaching a cell sheet adhered to a culture surface of a culture container from a culture container by using a cell detachment system, the cell detachment method comprising: a knocking process of knocking the culture container in a first direction having a directional component parallel to the culture surface; and a vibration process of vibrating the culture container in a second direction having a directional component parallel to the culture surface, Wherein, when viewed in a direction perpendicular to the culture surface, the first direction and the second direction are different from each other.

2. The cell detachment method according to claim 1, wherein When viewed in a direction perpendicular to the culture surface, the first direction and the second direction form an angle of 30° or more and 150° or less.

3. The cell detachment method according to claim 1, wherein When viewed in a direction perpendicular to the culture surface, the first direction and the second direction form an angle of 45° or more and 135° or less.

4. The cell detachment method according to claim 1, wherein The knocking process includes a process of periodically knocking the culture container.

5. The cell detachment method according to claim 4, wherein In the knocking process, the culture container is knocked at a frequency higher than a frequency at which the culture container is vibrated in the vibration process. The cell detachment method according to claim 1 , further comprising applying a vibration in an ultrasonic frequency band to the culture container.

7. A cell detachment system for detaching a cell sheet adhered to a culture surface of a culture container from a culture container, the cell detachment system comprising: a knocking unit configured to knock the culture container in a first direction including a directional component parallel to the culture surface; and a vibration unit that vibrates the culture container in a second direction having a direction component parallel to the culture surface, Wherein, when viewed in a direction perpendicular to the culture surface, the first direction and the second direction are different from each other.

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