Cell stripping system and cell stripping method

Abstract

The invention relates to a cell stripping system and a cell stripping method. A cell stripping system for stripping a cell sheet attached to a culture container from the culture container, the cell stripping system comprising: a stripping starting unit for starting stripping of the cell sheet; an information acquisition unit for acquiring information on a peeling progress direction of the cell sheet after the start of peeling of the cell sheet; a shaking unit for shaking the culture container; and a setting unit for setting the peeling progress direction and the rocking direction to be substantially parallel to each other in accordance with the information on the peeling progress direction.

Description

Technical Field

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

[0002] In the field of regenerative medicine and cell medicine, attempts have been made in recent years to culture cells in sheet form, and the sheet cells (cell sheets) of culture are transplanted to the affected part to repair damaged tissues, etc. When making a cell sheet by using adherent cells, for example, cells are cultured into sheets on a polystyrene culture dish as an example of a culture substrate, and then cells are peeled off and recovered from the culture substrate in the form of a sheet. The method for effectively and stably making a cell sheet while reducing wrinkles, tears, holes, etc. in the cell sheet has been studied.

[0003] Japanese Patent Laid-Open No. 2014-113133 discloses a cell detachment device including a vessel holder for mounting a culture vessel to which cultured cells are attached and a guide mechanism for guiding reciprocating motion of the vessel holder, wherein the vessel holder is caused to collide with a collision target member. Summary of the Invention

[0004] The present disclosure provides a cell peeling system that effectively peels off a cell sheet while reducing cell sheet tearing. The present disclosure also provides a cell peeling method that effectively peels off a cell sheet while reducing cell sheet tearing.

[0005] One aspect of the present disclosure provides a cell detachment system for detaching a cell sheet attached to a culture container from the culture container, the cell detachment system comprising: a detachment start unit for starting detachment of the cell sheet; an information acquisition unit for acquiring information about a detachment progress direction of the cell sheet after detachment of the cell sheet starts; a shaking unit for shaking the culture container; and a setting unit for setting the detachment progress direction and the shaking direction to be approximately parallel to each other based on the information about the detachment progress direction.

[0006] Another aspect of the present disclosure provides a cell detachment method for detaching a cell sheet attached to a culture container from the culture container by using a cell detachment system, the method comprising: a detachment start step for starting the detachment of the cell sheet; an information acquisition step for acquiring information about the detachment progress direction of the cell sheet after the detachment of the cell sheet is started; a shaking step for shaking the culture container; and a setting step for setting the detachment progress direction and the shaking direction to be approximately parallel to each other based on the information about the detachment progress direction.

[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 a cell detachment system according to a first embodiment.

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

[0010] Figure 3 FIG. 1 is a schematic diagram of a structure for generating a local water flow according to a first embodiment.

[0011] Figure 4 is a schematic diagram of a structure in which peeling is initiated by applying ultrasonic vibration according to the first embodiment.

[0012] Figure 5A and Figure 5B Each is a schematic diagram of a cell sheet peeling state according to the first embodiment.

[0013] Figure 6 is a schematic diagram of a structure for acquiring information on the direction of progress of peeling by referring to a database according to the first embodiment.

[0014] Figure 7A and Figure 7B is a schematic diagram of a uniaxial rocking mechanism according to the first embodiment.

[0015] Figure 8 is a schematic diagram of a biaxial pan mechanism according to the first embodiment.

[0016] Figure 9 is a schematic diagram of a mechanism for changing the striking direction according to the first embodiment.

[0017] Figure 10 is a schematic diagram of a mechanism for rotating a culture container according to the first embodiment.

[0018] Figure 11 is a flowchart schematically illustrating processing involving no direction change according to the first embodiment.

[0019] Figure 12 is a flowchart schematically illustrating processing including direction change according to the first embodiment.

[0020] Figure 13 is a sectional view of a mechanism for applying vertical vibration according to the first embodiment. DETAILED DESCRIPTION

[0021] When peeling a cell sheet from a culture vessel containing a liquid and to which a cell sheet is attached, the cell sheet may be easily torn if the culture vessel is reciprocated in the same direction as the impact applied to the culture vessel. This can also occur under conditions designed to efficiently peel the cell sheet in a short period of time.

[0022] The present disclosure will now be described in detail by way of examples.

[0023] The inventors of the present disclosure have studied a method for effectively peeling a cell sheet. As a result, they have discovered that, for a cell sheet that has begun to peel from a portion of its outer edge, flowing the liquid within the culture container in a specified direction can promote cell sheet peeling while reducing tearing of the cell sheet.

[0024] Specifically, it has been found that shaking the culture container in a direction substantially parallel to the direction in which the cell sheet has begun to detach can effectively detach the cell sheet while reducing tearing of the cell sheet. Here, "substantially parallel" means that at least one of the angles formed by the two directions is 45° or less, 30° or less, or 15° or less.

[0025] The reason why adjusting the angle between the peeling direction and the shaking direction allows for the peeling of the cell sheet while minimizing tearing is hypothesized to be as follows. The liquid flow generated by shaking the culture vessel enters the gap between the culture vessel and the partially peeled cell sheet, pushing the cell sheet upward in the direction of further peeling. This process, presumably because the water flow acts in the peeling direction relative to the partially peeled cell sheet, reduces excessive force concentration on the cell sheet and promotes peeling.

[0026] A cell detachment system according to one embodiment of the present disclosure will now be described.

[0027] First embodiment

[0028] Overview of Cell Detachment System

[0029] The cell peeling system according to the first embodiment peels off the cell sheet attached to the culture surface of the culture container from the culture surface through the following process. The cell peeling system of this embodiment performs: a peeling start process of starting peeling by a peeling start unit; and an information acquisition process of acquiring information about the direction of progress of cell sheet peeling by an information acquisition unit after starting peeling. The cell peeling system of this embodiment also performs: a shaking process of shaking the culture container by using a shaking unit; and a setting process of setting the peeling progress direction and the shaking direction to be roughly parallel to each other by a setting unit. Examples of the cell peeling start step include any one of the steps of tapping the culture container in which the cell sheet is cultured, causing a water flow to locally contact the cell sheet, and applying vibrations in an ultrasonic frequency band (ultrasonic vibrations) to the culture container in which the cell sheet is cultured.

[0030] cell sheets

[0031] The cell sheet in this embodiment refers to a membrane in which cells are connected to form a sheet. The cells constituting the cell sheet are not particularly limited as long as the cells can form a cell sheet. Examples of cells include adherent cells such as adherent somatic cells.

[0032] Examples of somatic cells include myoblasts (e.g., skeletal 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 such as cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS cells, synovial cells, chondrocytes, and epithelial cells (e.g., oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.).

[0033] 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.

[0034] In addition, somatic cells can be cells differentiated from iPS cells (iPS cell-derived cells). Examples of cells derived from iPS cells include cardiomyocytes, fibroblasts, myoblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gum cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc. derived from iPS cells.

[0035] Culture container

[0036] The culture container of this embodiment is not particularly limited, as long as it is a cell-attached culture container. For example, the culture container can be any one of a flask, a tissue culture flask, a culture dish, a petri dish, a tissue culture dish, a duplex culture dish, a microplate, a multiwell plate, a duplex culture plate, a culture bag, and a bottle.

[0037] The material of the culture container of the present embodiment can be any material that is chemically stable and can cultivate the required cells.The example of material comprises polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(methyl) acrylic acid, poly(methyl) acrylic acid derivatives, polyacrylonitrile, poly(methyl) acrylamide, poly(methyl) acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysiloxane, polymethylpentene, glass and metal.Wherein, from the viewpoint of stability, polystyrene can be used.

[0038] Alternatively, a temperature-responsive container having a culture surface whose hydrophilicity changes with temperature may be used as the culture container of this embodiment.

[0039] Cell detachment solution

[0040] The cell detachment solution herein refers to the solution maintained in the culture vessel during cell detachment using the cell detachment method disclosed herein. The cell detachment solution herein does not necessarily contain components that promote cell detachment. For example, a cell detachment solution that is substantially free of proteases may be used to protect the cell surface.

[0041] Here, "substantially containing no" means that the content is equal to or lower than 0.0005 mass%.

[0042] The pH value of the cell detachment solution in this embodiment can be in the neutral or acidic range.

[0043] The neutral range is suitable for cell culture, and cell viability can be maintained at a stable and high level. The pH value can be appropriately adjusted using hydrochloric acid, sodium hydroxide, etc. Various buffers can also be used to stabilize the pH value.

[0044] In the present embodiment, any buffer can be used without limitation, as long as the neutral range can be maintained. Examples thereof include Tris buffers such as Tris-HCl buffer, phosphate buffer, HEPES buffer, citrate phosphate buffer, glycylglycine sodium hydroxide buffer, Britton Robinson buffer, and GTA buffer. Among them, a phosphate buffer close to the in vivo environment can be used, for example, phosphate buffered saline (PBS) (PBS) obtained by adjusting the phosphate buffer to isotonicity with the intracellular fluid.

[0045] The viscosity of the cell detachment solution in this example can be 1.80 mPa·s or less. This is because the flow of the detachment solution generated by ultrasonic vibrations is not hindered, and the detachment efficiency can be maintained at a high level. The viscosity of the cell detachment solution can be appropriately adjusted by adding polymers, sugars, etc.

[0046] The cell detachment solution of this embodiment may contain protease, but the amount of protease relative to the total mass of the cell detachment solution may be 0.0005% by mass or less, or zero. This is because, while protease improves detachment efficiency by partially degrading cells, it may also degrade cell quality.

[0047] The protease in this embodiment is an enzyme that decomposes a part of cells to facilitate the detachment of cells from the substrate. Examples thereof include trypsin, coagulase, collagenase, natural protease, chymotrypsin, elastase, papain, pronase, and recombinant forms thereof.

[0048] The cell detachment solution in this embodiment can be a culture medium, but is not particularly limited. Alternatively, a solution containing a metal ion chelator (hereinafter referred to as a chelating agent) can be used. This is because using a cell detachment solution containing a chelating agent allows for more efficient cell detachment using ultrasonic vibrations.

[0049] The chelating agent in the present embodiment is not particularly limited. Examples of chelating agents include ethylenediaminetetraacetic acid (hereinafter also referred to as EDTA), ethylenediamine, ethylenediaminetetramethylenephosphoric acid, glycol ether diaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediamine disuccinic acid, etidronic acid, citric acid, gluconic acid and phosphonobutanetricarboxylic acid.

[0050] The chelating agent can form a chelate with a divalent cation, or with Ca2+ and Mg2+. The chelating agent can be ethylenediaminetetraacetic acid (EDTA). When EDTA is used as the chelating agent, the pH of the cell exfoliation solution can be between 7.0 and 8.0. This is because the pH is relatively high within the neutral range that maintains high cell viability, further enhancing the chelating ability of EDTA and improving exfoliation efficiency.

[0051] The chelating agent can be used alone or in combination. The chelating agent content can be 0.01 mM or more and 5.0 mM or less. Within this range, the chelating effect can be reliably obtained, and the activity reduction caused by the presence of excessive chelating agent can also be reduced.

[0052] The cell detachment solution of this embodiment may contain a hydrophilic polymer having a polyalkylene glycol structure. Polyalkylene glycol can improve cell survival in cell detachment methods using ultrasound. An example of a hydrophilic polymer having a polyalkylene glycol structure is polyethylene glycol. The hydrophilic polymer may have a peak molecular weight (Mp) of 800 to 50,000, or a peak molecular weight (Mp) of 1,200 to 20,000, as measured by gel permeation chromatography. This is because the polymer has a minimal effect on cells and can reduce the thickening effect of the culture medium caused by the polymer.

[0053] The type of culture medium may be any type, and examples thereof include Dulbecco's Modified Eagles's Medium (DMEM), Ham's Nutrition Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagles's Minimum Essential Medium (EMEM), α-Modified Eagle'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 Growth Medium.

[0054] Among them, a culture medium suitable for culturing each cell type can be used.

[0055] Serum or antibiotics can be added to the above culture medium. Examples of serum include fetal bovine serum (FBS), calf serum, bovine serum, horse serum, sheep serum, goat serum, porcine serum, chicken serum, rabbit serum and human serum, and FBS is generally used because of its easy availability. Alternatively, a serum-free culture medium containing no untreated or unpurified serum but containing purified blood-derived components or animal tissue-derived components (growth factors, etc.) can be used.

[0056] Examples of the antibiotics added to the culture medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, and amphotericin B.

[0057] Cell sheet culture conditions

[0058] The cell sheet culture conditions can be appropriately selected according to the cells to be cultured. Generally, appropriate culture medium is added to a culture dish and about 1.0×10 1 to 5.0×10 4 cells / cm 2 The cells are cultured at 37° C. and 5% CO 2 . Here, the culture can be carried out until the cell occupancy rate in the substrate is approximately 100%, in other words, until the substrate reaches a confluent state.

[0059] Cell sheet detachment start unit

[0060] Examples of the cell sheet detachment initiation means include tapping the culture container in which the cell sheet is cultured, locally contacting the cell sheet with a water stream, and applying ultrasonic frequency band vibration (ultrasonic vibration) to the culture container. These can be used alone or in combination.

[0061] Percussion Unit

[0062] By using a knocking unit as a peeling initiation unit and performing a knocking step of applying an impact force to a culture container in which a cell sheet is cultured, the peeling can be prompted to start.

[0063] The knocking step in this embodiment includes applying an impact force to the culture substrate to cause at least a portion of the cells attached to the culture surface of the culture substrate to detach from the culture surface. The knocking step may include, for example, performing at least one of the following steps: applying the impact force to the culture substrate by knocking the culture substrate itself; and applying the impact force to the culture substrate by moving the culture substrate and causing it to collide with a member constituting the cell detachment device.

[0064] The tapping process in this embodiment can be periodic or aperiodic, and the magnitude of the impact force applied to the culture substrate during the tapping process can be appropriately set. The tapping process in this embodiment can be performed continuously from the start to the completion of cell detachment, can be terminated before completion, or can be performed intermittently.

[0065] The striking unit in this embodiment may be any device capable of performing the aforementioned striking process. For example, the striking unit may include an object to collide with the culture substrate and a moving unit to move the object so that it collides with the culture substrate, or may include a moving unit to move the culture substrate so that it collides with a component of the cell detachment device. The object may have a mass sufficient to impart a suitable impact force to the culture substrate without tearing the culture substrate.

[0066] Examples of object shapes include rods, hammers, and balls. Examples of moving units include motors and solenoids that can generate a moving force to move an object or a culture substrate by energizing it. The object or culture substrate can be moved using only motors, solenoids, or the like, or they can be used in combination with a member that can store energy, such as a spring member.

[0067] Figure 2 The figure is a cross-sectional view of an example of a striking unit according to this embodiment. Cam 41 is connected to a motor and, when driven by the motor, rotates in the direction of the shaft's rotation. Hammer 42 contacts cam 41 at shaft 48 and is urged by spring 43 in the direction of striking culture container 8. After spring 43 is compressed by the rotation of cam 41, hammer 42 moves in the direction of striking culture container 8 according to the contour of cam 41. The frequency of the striking can be determined by the motor's rotational speed and detected by photocoupler 44.

[0068] Liquid discharge unit

[0069] A liquid discharge unit that discharges liquid can be used as a peeling initiation unit. The water flow generated by the liquid discharge is brought into contact with the cell sheet to promote the peeling of the cell sheet. The liquid discharge unit is, for example, a pipette. The unit for generating the water flow is not limited thereto, and structures such as a nozzle or a flow channel can be used. From the perspective of effectively peeling the cell sheet, the water flow generated by the liquid discharge can be brought into contact with the edge (outer edge) of the cell sheet.

[0070] Figure 3 Figure 1 is a schematic diagram of the liquid discharge unit of this embodiment. The electric pipette 12 is controlled by the controller 5 and can cause a localized flow of water to contact the edge of the culture container 8. The culture medium discharged from the electric pipette 12 generates a flow of water directed from the bottom end toward the center of the bottom of the culture container 8. This flow of water exerts a force that initiates the detachment of the cell sheet edge.

[0071] Start peeling by ultrasonic vibration

[0072] Another example of a detachment initiating unit is an ultrasonic wave generating unit that applies vibrations in an ultrasonic frequency band to the culture container to initiate detachment of a cell sheet attached to the culture container. Figure 4This is a schematic diagram of the structure for initiating exfoliation using ultrasonic vibrations in this embodiment. Ultrasonic wave generator 13 is controlled by controller 5 and applies ultrasonic waves to culture container 8. The ultrasonic vibrations exert a force that causes the cell sheet in culture container 8 to exfoliate from its outer edges.

[0073] An example of vibration in the ultrasonic frequency band used in the peeling initiation unit is vibration with a frequency of 10 kHz or higher and 1 MHz or lower. The ultrasonic wave generating unit can be any unit capable of applying vibration in the ultrasonic frequency band to cells. One example is the use of an ultrasonic oscillator such as lead zirconate titanate (PZT) as the vibrator.

[0074] The ultrasonic vibrator can be any object capable of generating ultrasonic waves, an example of which is a piezoelectric element and a vibrating plate bonded together. When the piezoelectric element 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 a large amplitude at a relatively high drive frequency (vibration frequency) in the ultrasonic range without damaging the ultrasonic vibrator.

[0075] 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 bonded together and subjected to deflection vibration, the deflection midpoint in the thickness direction (i.e., the neutral plane where no stretching or contraction occurs during deflection) can be located on the vibration plate side because the strain of the piezoelectric body can be efficiently used for deflection.

[0076] Furthermore, a commercially available Langevin oscillator or rectangular oscillator can also be used as the ultrasonic vibrator of the present disclosure. An example of a Langevin oscillator is one in which a piezoelectric body is inserted between two metal blocks and clamped together with bolts or the like to form an integral structure.

[0077] The operation of the detachment starting unit may be performed periodically, or may be continued until the detachment of the cell sheet is completed.

[0078] Obtain information about the direction of peeling progress through the observation unit

[0079] An example of a unit that acquires information about the direction of detachment progression is an observation unit, such as a camera that observes the cell sheet and acquires the observation results as image information. Alternatively, a measurement unit, such as a distance measurement sensor, can be used that acquires the state of attachment of the cell sheet to the inner surface of the culture container as numerical information.

[0080] Figure 1 is a schematic diagram of the cell detachment system according to this embodiment, which includes an observation unit 11. The observation unit 11 is, for example, a commercially available camera or microscope, is controlled by the controller 5, and can observe the state of the cell sheet in the culture container 8.

[0081] Figure 5A and Figure 5B The peeling state of the cell sheet 9 is shown in FIG. 1 and FIG. 2 , respectively. The peeling boundary line AB represents the attachment-peeling boundary of the cell sheet 9. Figure 5A In the state shown, the peeling direction can be determined as, for example, a direction perpendicular to the peeling boundary line AB or a direction connecting the midpoint of the peeling boundary line AB and the center of gravity of the cell sheet 9. Figure 5B When the peeling boundary curve CD is formed into an arc, the peeling direction can be determined as, for example, the normal direction of the midpoint of the peeling boundary curve CD or the direction connecting the midpoint of the peeling boundary curve CD and the center of gravity of the cell sheet 9. In this embodiment, the normal direction of the midpoint of the peeling boundary curve CD is defined as the peeling direction.

[0082] Get information about the direction of the divestiture process through the database

[0083] In other cases, the information acquisition unit may refer to a database that stores information about the culture container and the direction of progress of the peeling process for each culture container in a correlated manner. The database that pre-correlates the direction of progress of the peeling process and information about the culture container may be stored in a storage area of ​​the peeling system or in a server connected via a network. The information about the culture container may include, for example, at least one of the size, shape, manufacturer, and pre-assigned serial number of the culture container.

[0084] Figure 6 This is a schematic diagram of the structure used in this embodiment to obtain information about the direction of peeling progress by referencing a database. This database-referencing technique enables obtaining information about the direction of peeling progress without observing a cell sheet, thereby reducing the number of structures and system size.

[0085] Information on the peeling progress direction may be acquired multiple times during peeling, or may be acquired even before peeling begins. To acquire a more accurate peeling direction, information acquisition may be performed multiple times according to the progress of peeling, and the information may be updated in response to the acquired peeling progress direction information.

[0086] Shake the culture container

[0087] In this embodiment, shaking can include a feature that generates flow in the liquid within the culture container by applying an acceleration component parallel to the culture surface of the culture container in which the cell sheet is cultured. For example, a feature can be used in which a motor generates rotational motion and a slide rail is used to perform uniaxial reciprocating motion. Alternatively, a feature can be used in which a linear motor is used to accelerate the culture container to apply an acceleration component parallel to the culture surface. As long as shaking can apply an acceleration component parallel to the culture surface of the culture container, the shaking axis itself does not necessarily need to be parallel to the culture surface.

[0088] In this embodiment, the amplitude of the shaking may be 0.1 mm to 300 mm, or 0.1 mm to 150 mm. For the reciprocating motion, the frequency may be 0.1 Hz to 20 Hz, or 0.1 Hz to 10 Hz.

[0089] Single-axis shaking mechanism

[0090] Figure 7A and Figure 7B The single-axis rocking mechanism according to this embodiment is shown. The guide rail 50 is fastened to Figure 1 The culture substrate 8 is reciprocally rocked on the base plate 7 shown. A base-side spring column 52, to which a pressure spring 51 is connected, is fixed to the base plate 7. The pressure spring 51 is connected to the base-side spring column 52. The other end of the pressure spring 51 is fixed to a rocked-side spring column 53 provided in the rocked portion. The pressure spring 51 can be selected as needed and has a detachable structure.

[0091] The rocking occurs when the rotation of the rocking motor 54 is transmitted to the rocking lever 56 via the gear 55. The rotating disk 57 is fixed to the rotating shaft of the rocking motor 54 and rotates as shown in the figure.

[0092] The direction of rotation can be reversed. A linear bushing bracket 58 is rotatably mounted at a position offset from the axis of rotation. A linear bushing 59 is assembled within the linear bushing bracket, and a rocking lever 56 is assembled within the linear bushing for linear movement. One end of the rocking lever 56 is fixed to a rotatable rocking fulcrum shaft 60. This converts the motion into reciprocating motion centered on the rocking fulcrum shaft 60. The frequency of this reciprocating motion is detected by a rotary photoelectric coupler 61.

[0093] The connecting rod 62 is fastened to the rocking rod 56 in a manner parallel to the rocking rod 56 by a connecting rod fixing tool 63, and one end is fixed to the rocking fulcrum shaft 60. A rocking slider 64 equipped with a base plate 7 contacts the reciprocating connecting rod 62 to produce a reciprocating rocking motion. The rocking slider 64 is supported by a slider sliding shaft 66 parallel to the rocking adjustment screw 65, and the rocking stroke can be freely adjusted by rotating the rocking adjustment screw 65. A stroke lug 67 is mounted on the end of the rocking adjustment screw 65 and can be manually rotated for adjustment.

[0094] Dual-axis shaking mechanism

[0095] Figure 8 Figure 1 is a schematic diagram of a biaxial rocking mechanism according to this embodiment. Linear rails 14, orthogonal to each other, are arranged biaxially, enabling the rocking direction to be freely changed. The linear rails 14 are controlled by the controller 5, and the peeling progress direction and the rocking direction can be controlled to be approximately parallel to each other.

[0096] Setting unit that sets the peeling progress direction and the shaking direction to be approximately parallel to each other

[0097] In this embodiment, the setting unit, which sets the peeling progress direction and the shaking direction to be substantially parallel to each other, can change the direction of application of the peeling start unit according to the peeling progress direction. Furthermore, the shaking direction can be changed according to the peeling progress direction, and / or the orientation of the culture container can be changed according to the peeling progress direction. These two changes can be implemented separately, or two or more such changes can be implemented together.

[0098] Change tapping direction

[0099] Figure 9 This is a schematic diagram of the mechanism for changing the tapping direction in this embodiment. The tapping unit 4 is configured to freely change the angle at which it strikes the culture container 8, and this structure also allows for freely changing the tapping direction. When the tapping unit 4 is actuated while changing its tapping direction according to the tapping direction set by the setting unit, the rocking direction and the peeling progress direction can be made substantially parallel to each other.

[0100] Change the shaking direction

[0101] exist Figure 8 In the example shown, the rocking direction is biaxially controlled and can be changed to any angle within the operating plane. By changing the rocking direction according to the rocking direction set by the setting unit, the rocking direction and the peeling progress direction can be made substantially parallel to each other.

[0102] Changing the orientation of the culture vessel

[0103] By rotating the culture container 8 after the cell sheet starts to detach from the culture container 8 , the shaking direction and the detachment progress direction can be made substantially parallel to each other. Figure 10 This is a schematic diagram of the mechanism for rotating culture container 8 in this embodiment. Drive rollers 15 and 16 are in contact with culture container 8, and drive roller 15, connected to a motor, rotates culture container 8. Roller 16 rotates as culture container 8 rotates. The rotation angle of drive roller 15 is controlled by controller 5, and the rocking direction and the peeling direction are kept approximately parallel to each other.

[0104] Operation of the cell detachment system

[0105] The operation of the cell detachment system according to this embodiment will now be described using a knocking unit as an example of a detachment initiation unit; however, please note that the structures and features herein are merely examples and do not limit the scope of this disclosure. Instead of a knocking unit, at least one of generating a localized flow of water into the culture container using a liquid discharge unit and applying ultrasonic frequency band vibrations (ultrasonic vibrations) to the culture container may also be used.

[0106] Figure 1 The figure is a schematic diagram of an example of the structure of a cell detachment system according to this embodiment. The knocking unit 4 serves as a detachment initiation unit and knocks the culture container 8. The observation unit 11 serves as a unit for obtaining information on the direction of detachment progress and for observing the state of the cell sheet cultured within the culture container 8. The culture container 8, in which the cell sheet is cultured, is placed on the shaking unit 3. The cell sheet can be detached by driving the knocking unit 4, the observation unit 11, and the shaking unit 3, which are controlled by the controller 5. An example of this operation is to sequentially perform the following steps: knocking the culture container 8 with the knocking unit 4, detecting detachment with the observation unit 11, and shaking the culture container 8 in a direction generally parallel to the detachment direction with the shaking unit 3.

[0107] It is important that the exfoliation direction and the shaking direction are approximately parallel to each other, and the timing of the operations is not particularly limited. For example, after the cell exfoliation system is activated, the shaking step, the observation step, and the tapping step can be started simultaneously, or the shaking direction can be changed during the observation step to detect the exfoliation direction.

[0108] The order of starting tapping, shaking, and vertical vibrations such as ultrasonic vibrations can be any. Performing the tapping and shaking steps during or after the application of vertical vibrations can weaken the cell adhesion while simultaneously applying shear force, resulting in more efficient cell detachment. To determine whether to transfer to each step, information about cells attached to the substrate can be measured and used.

[0109] The cell detachment system according to this embodiment may also perform other steps in addition to the above steps. Examples of other steps include replacing the cell detachment solution, rinsing cells with the cell detachment solution, and diluting the cell detachment solution to homogenize the solution in the culture container.

[0110] The above steps may be repeated at a specific cycle or at an irregular cycle. The timing may be uniform or may be adjusted for each step. The ratio of the total time of performing the tapping step and the shaking step to the total time of performing the external stimulation step such as ultrasound may be greater than 0.01 and less than 100.

[0111] Processing procedures performed by the cell detachment system 1

[0112] Figure 11 1 is a flow chart schematically showing the process performed by the cell detachment system according to this embodiment. In step S11, cell detachment is started. In step S12, information on the detachment direction of the cell sheet in the culture container 8 is obtained.

[0113] The information on the cell sheet peeling direction is stored in the storage area of ​​the controller 5. In the process of step S13, based on the information on the cell sheet peeling direction, it is determined whether the cell sheet has started to peel from the culture container 8.

[0114] The controller 5 may be composed of a module implemented by a CPU or MPU, or may be composed of, for example, a circuit (such as an ASIC) that implements a specific function. It should be noted that the present disclosure can be implemented by a cell detachment system connected to multiple devices or computing devices, and the scope of the present disclosure includes a cell detachment device composed of a single device that implements the same function.

[0115] If peeling has not yet begun, the process proceeds to step S12, and steps S12 and S13 are repeated until peeling begins. If peeling has already begun in step S13, the process proceeds to step S14. In step S14, the peeling direction is determined based on the information regarding the peeling direction of the cell sheet. In step S15, the culture container 8 is shaken in a direction generally parallel to the peeling direction determined in step S14.

[0116] In step S16, it is determined whether the cell sheet has been completely detached from the culture container 8. Detaching is determined to be complete when the cell sheet floats from the culture surface of the culture container 8. If detachment is not complete, the process shifts to step S15, and steps S15 and S16 are repeated until detachment is complete. Completion of detachment in step S16 terminates system operation.

[0117] Processing procedures performed by the cell detachment system 2

[0118] Figure 12 is a flowchart schematically illustrating the steps performed by the cell detachment system according to this embodiment. In step S11, cell detachment begins. In step S12, information regarding the direction of cell sheet detachment is acquired. This acquired information regarding the direction of cell sheet detachment is stored in a storage area within controller 5. The storage area can be configured using any desired memory or storage medium, such as an optical disc. In step S13, based on the information regarding the direction of cell sheet detachment, it is determined whether the cell sheet has begun detaching from culture container 8.

[0119] If peeling has not yet begun, the process proceeds to step S12, and steps S12 and S13 are repeated until peeling begins. If peeling has already begun in step S13, the process proceeds to step S14. In step S14, the peeling direction is determined based on the information regarding the peeling direction of the cell sheet. In step S15, the culture container 8 is shaken in a direction generally parallel to the peeling direction determined in step S14.

[0120] In the process of step S16, it is determined whether the cell sheet has been completely peeled off from the culture container 8. The state in which the cell sheet floats from the culture surface of the culture container 8 is determined to be the completion of the peeling. If the peeling is not completed, the process is transferred to the process of step S12, and the state of the cell sheet is obtained. Through the judgment in the process of step S13, the peeling direction is determined again in the process of step S14. If the peeling direction deviates, the process of step S15 causes the culture container 8 to be shaken in a direction roughly parallel to the deviated peeling direction. The processes from step S12 to step S16 are repeated until the peeling is completed. In the process of step S16, the completion of the peeling ends the driving of the system.

[0121] Additional vertical vibration generated by the cell detachment system

[0122] Figure 13 This is a cross-sectional view of the structure of the cell detachment system of this embodiment, which adds a mechanism for applying vertical vibration to culture container 8. An ultrasonic element 17 is provided below culture container 8 to vertically apply ultrasonic vibration to culture container 8. The ultrasonic element 17 is controlled by the controller 5, and the driving timing can be freely controlled.

[0123] As the cell sheet detachment method, an appropriate one of the above-mentioned treatment flow 1 and treatment flow 2 is selected. Alternatively, in addition to treatment flow 1 or treatment flow 2, additional vertical vibration may be performed.

[0124] Procedures for implementing cell processing methods

[0125] In addition, the present disclosure is also implemented by performing the following processing. That is, the present disclosure can be implemented by providing a system or device with software (program) that implements one or more functions of the various embodiments described above via a network or storage medium. The present disclosure can also be implemented by a computer (or CPU, MPU, etc.) of the system or device reading out and executing the program. The computer includes one or more processors or circuits, and can include a network composed of multiple separate computers or multiple separate processors or circuits for reading and executing computer-executable instructions.

[0126] Here, the processor or circuit may include a central processing unit (CPU), a microprocessing unit (MPU), and a graphics processing unit (GPU). In addition, the processor or circuit may include an application-specific integrated circuit (ASIC) or a field-programmable gate array (FPGA). In addition, the processor or circuit may include a digital signal processor (DSP), a data flow processor (DFP), or a neural processing unit (NPU).

[0127] Examples and Comparative Examples

[0128] The present disclosure will now be described in more detail through examples and comparative examples; however, the present disclosure is not in any way limited by the following examples without departing from the gist of the present disclosure.

[0129] Culture of A549 cells on substrates

[0130] A549 cells (human lung epithelial adenocarcinoma cells) were plated at 10,000 cells / cm 2 The cells were sown at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark), manufactured by CellSeed Co., Ltd.) and cultured in an environment of 37°C and 5% CO2 concentration. The culture medium used was DMEM (manufactured by Thermo Fisher Scientific Co., Ltd.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co., Ltd.) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo-Fisher Scientific Co., Ltd.). The culture was carried out for 6 days, and the cell state was observed under a phase contrast microscope to confirm cell attachment and growth. The cell occupancy area ratio of the culture dish was approximately 100%.

[0131] Cultivation of C2C12 cells on substrates

[0132] C2C12 cells (mouse myoblasts) were cultured at 65,000 cells / cm 2The cells were sown at a density of 100 μg / ml in a Φ35 temperature-responsive culture dish (UpCell (registered trademark), manufactured by CellSeed Co., Ltd.) and cultured in an environment of 37°C and 5% CO2 concentration. The culture medium used was DMEM / F12 culture medium (manufactured by Thermo Fisher Scientific Co., Ltd.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co., Ltd.) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo-Fisher Scientific Co., Ltd.). The culture was carried out for 2 days, and the cell state was observed under a phase contrast microscope to confirm cell attachment and growth. The cell occupancy area ratio of the culture dish was approximately 100%.

[0133] MDCK cell culture on substrate

[0134] MDCK cells (Madin-Darby canine kidney epithelial cells) were cultured at a density of 65,000 cells / cm 2 The cells were sown at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark), manufactured by CellSeed Co., Ltd.) and cultured in an environment of 37°C and 5% CO2 concentration. The culture medium used was Eagle's MEM medium (manufactured by FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co., Ltd.) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Co., Ltd.). The culture was carried out for 8 days, and the cell state was observed under a phase contrast microscope to confirm cell attachment and growth. The cell occupancy area ratio of the culture dish was approximately 100%.

[0135] HEK293 cell culture on substrates

[0136] HEK293 cells (human embryonic kidney cells) were cultured at 65,000 cells / cm 2The cells were sown at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark), manufactured by CellSeed Co., Ltd.) and cultured in an environment of 37°C and 5% CO2 concentration. The culture medium used was Eagle's MEM medium (manufactured by FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co., Ltd.) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Co., Ltd.). The culture was carried out for 9 days, and the cell state was observed under a phase contrast microscope to confirm cell attachment and growth. The cell occupancy area ratio of the culture dish was approximately 100%.

[0137] BAEC cell culture on substrate

[0138] BAEC cells (bovine aortic endothelial cells) were cultured at a density of 20,000 cells / cm 2 Cells were seeded at a density of 100 μg / ml in Φ35 polystyrene culture dishes (manufactured by Corning) and cultured at 37°C in an environment with a 5% CO2 concentration. The culture medium used was DMEM (manufactured by Thermo Fisher Scientific Co., Ltd.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co., Ltd.) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo-Fisher Scientific Co., Ltd.). Culture was performed for 7 days, and the cell state was observed using a phase contrast microscope to confirm cell attachment and growth. The cell occupancy ratio of the culture dish was approximately 100%.

[0139] hMSC cell culture on substrate

[0140] hMSC cells (human mesenchymal stem cells) were cultured at 30,000 cells / cm 2 The cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark), manufactured by CellSeed Co., Ltd.) and cultured in an environment of 37°C and 5% CO2 concentration. The culture medium used was mesenchymal stem cell growth medium 2 (manufactured by PromoCell Co., Ltd.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co., Ltd.) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Co., Ltd.). The culture was carried out for 8 days, and the cell state was observed under a phase contrast microscope to confirm cell attachment and growth. The cell occupancy area ratio of the culture dish was approximately 100%.

[0141] Culture of HUVEC cells on substrates

[0142] HUVEC cells (human umbilical vein endothelial cells) were cultured at 65,000 cells / cm 2 The cells were seeded at a density of 100 μm in a Φ35 temperature-responsive culture dish (UpCell (registered trademark), manufactured by CellSeed Co., Ltd.) and cultured at 37°C and 5% CO2. The culture medium used was an endothelial cell growth medium set 2 (manufactured by PromoCell Co., Ltd.) supplemented with 10% fetal bovine serum (manufactured by Sigma-Aldrich Co., Ltd.) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific Co., Ltd.). The culture was carried out for 7 days, and the cell state was observed under a phase contrast microscope to confirm cell attachment and growth. The cell occupancy rate of the culture dish was approximately 100%.

[0143] Stripping start unit 1: tapping the culture vessel

[0144] use Figure 2 The knocking unit shown serves as the peeling initiation unit 1 and knocks the culture container 8 at a frequency of 5 Hz.

[0145] Stripping Start Unit 2: Local Water Flow

[0146] By using Figure 3 The liquid discharge unit shown serves as the stripping start unit 2 and discharges 1 mL of the culture medium from the bottom end portion to the bottom center portion of the culture container 8 within 1 second.

[0147] Peeling start unit 3: Ultrasonic vibration

[0148] By using Figure 4 The ultrasonic wave generating unit shown serves as the peeling starting unit 3 , and applies an AC voltage having a voltage of 50 V and a frequency of 30 kHz to the Langevin oscillator via the controller 5 to apply ultrasonic vibration to the culture container.

[0149] Shaking unit 1: Single axis shaking

[0150] use Figure 7A and Figure 7B The uniaxial rocking mechanism shown serves as the rocking unit 1 , and the motor rotates at 120 rpm and performs reciprocating motion at a frequency of 2 Hz.

[0151] Shaking unit 2: dual-axis shaking

[0152] By using Figure 8 The biaxial shaking mechanism shown serves as the shaking unit 2 to shake the culture container.

[0153] Information acquisition unit 1 for acquiring information on the direction of detachment progress: observation of cell sheet

[0154] Will Figure 1 The observation unit 11 shown in FIG. 1 is used as the information acquisition unit 1 to acquire the cell detachment progress direction.

[0155] Information acquisition unit 2 for acquiring information on the direction of peeling progress: referring to a database

[0156] As the information acquisition unit 2, a database stored in the cell detachment system is referred to to acquire the cell detachment progress direction.

[0157] Change Unit 1: Change the tapping direction

[0158] As a change unit 1, use Figure 9 The mechanism shown is used to change the knocking direction so that the shaking direction and the peeling progress direction are roughly parallel to each other.

[0159] Change Unit 2: Change the shaking direction

[0160] As a change unit 2, use Figure 9 The mechanism shown in is used to change the rocking direction so that the rocking direction and the peeling progress direction are approximately parallel to each other.

[0161] Change Unit 3: Rotating the Culture Vessel

[0162] As a change unit 3, use Figure 10 The mechanism shown is used to change the orientation (rotation) of the culture container so that the rocking direction and the peeling progress direction are approximately parallel to each other.

[0163] Processing Flow 1: No Direction Correction

[0164] In process 1, follow Figure 11 The cell sheets were processed according to the protocol shown.

[0165] Process 2: Direction Correction

[0166] In process 2, follow Figure 12 The cell sheets were processed according to the protocol shown.

[0167] Additional vertical vibration

[0168] By using Figure 13 The structure shown applies ultrasonic vibration as an additional vertical vibration to the culture container.

[0169] Peelability evaluation

[0170] The peeling performance evaluation evaluated three criteria: peeling time, peeling time deviation, and cell sheet quality. The peeling time was measured from the time the culture vessel 8 was placed in the cell peeling system and the system was activated until peeling was complete and the activation was terminated. Because the peeling time required for each cell type varies, the peeling time was evaluated by comparing the peeling time of the example to the peeling time of the comparative example for the same cell type. The peeling time was evaluated according to the following criteria.

[0171] A: Excellent (peeling completion time is 21% or more shorter than the comparative example)

[0172] B: Good (peeling completion time is 11% to 20% shorter than that of the comparative example)

[0173] C: Effective (peeling completion time is 1% to 10% shorter than that of the comparative example)

[0174] D: Poor (peeling time is the same as that of the comparative example)

[0175] The detachment time variation was evaluated by dividing the standard deviation of the detachment time observed when the cell sheet was detached five times by the average value of the five times. The detachment time variation was evaluated according to the following criteria.

[0176] A: Excellent (standard deviation / mean less than 0.1)

[0177] B: Good (standard deviation / average value is 0.1 or more and less than 0.3)

[0178] C: Valid (standard deviation / average value is 0.3 or more and less than 0.5)

[0179] D: Poor (standard deviation / average value is 0.5 or more)

[0180] The quality of the cell sheet was evaluated by observing the exfoliated cell sheet using a phase contrast microscope to check for holes and tears. A cell sheet was considered to have holes if a hole with a diameter of 500 μm or greater was observed. A cell sheet was considered to have tears if a tear with a size of 1 mm or greater was observed.

[0181] The quality of the cell sheets was evaluated according to the following criteria.

[0182] A: Excellent (no holes or tears)

[0183] B: Good (no tearing)

[0184] C: Effective (no hole)

[0185] D: Poor (holes and tears)

[0186] Example 1

[0187] C2C12 cells cultured in a culture vessel 8 were detached using the detachment initiation unit 1, the shaking unit 1, the information acquisition unit 1, and the change unit 1. The detachment time was evaluated, and it took 12 minutes to complete the detachment. Furthermore, the detachment time variation was evaluated, and the standard deviation was 1.33. The quality of the C2C12 cell sheet was evaluated, and after detachment, there were no holes, tears, or stretches, but some wrinkles were present.

[0188] Examples 2 to 14 and Comparative Examples 1 to 7

[0189] The peeling time, peeling time deviation, and cell sheet quality were evaluated based on the combination of the peeling start unit, shaking unit, information acquisition unit, change unit, processing flow, additional vertical vibration, and cell type shown in Table 1. The evaluation results are shown in Table 2.

[0190] Table 1: Processing conditions in Examples 1 to 14 and Comparative Examples 1 to 7

[0191]

[0192] Table 2: Evaluation results of Examples 1 to 14 and Comparative Examples 1 to 7

[0193]

[0194] 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. In addition, 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.

[0195] While the present disclosure has been described with reference to exemplary embodiments, it is to be understood that the present 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 modifications and equivalent structures and functions.

Claims

1. A cell detachment system for detaching a cell sheet attached to a culture container from the culture container, the cell detachment system comprising: a peeling start unit, used for starting peeling of the cell sheet; an information acquisition unit, configured to acquire information on a direction in which the cell sheet is peeled off after the cell sheet peeling begins; a shaking unit, for shaking the culture container; and A setting unit is configured to set the peeling progress direction and the rocking direction to be substantially parallel to each other based on the information on the peeling progress direction.

2. The cell detachment system according to claim 1, wherein: The peeling initiation unit includes a knocking unit that knocks the culture container.

3. The cell detachment system according to claim 2, wherein: The setting unit sets the tapping direction based on the information on the peeling progress direction; and The knocking unit knocks the culture container according to the set knocking direction.

4. The cell detachment system according to claim 1, wherein: The peeling start unit includes a liquid discharge unit that discharges liquid to allow a water flow to contact the cell sheet.

5. The cell detachment system according to claim 1, wherein: The setting unit sets the shaking direction based on the information on the peeling progress direction; and The shaking unit shakes the culture container according to a set shaking direction.

6. The cell detachment system according to claim 1, wherein: The setting unit sets the shaking direction based on the information on the peeling progress direction; and The cell detachment system further includes a changing unit that changes the orientation of the culture container according to a set shaking direction.

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

8. The cell detachment system according to claim 1, wherein: The information acquisition unit refers to a database containing information on the culture container and information on the direction in which the peeling progresses.

9. The cell detachment system according to claim 1, wherein: The peeling initiation unit applies vibrations in an ultrasonic frequency band to the culture container. 10 . The cell detachment system according to claim 1 , further comprising an ultrasonic wave generating unit configured to apply vibrations in an ultrasonic wave frequency band to the culture container.

11. A cell detachment method, comprising detaching a cell sheet attached to a culture container from the culture container using a cell detachment system, the method comprising: The peeling start process is used to start the peeling of the cell sheet; an information acquisition step for acquiring information on the progress direction of cell sheet detachment after the detachment of the cell sheet is started; A shaking process for shaking the culture container; and The setting step is for setting the peeling progress direction and the rocking direction to be substantially parallel to each other based on the information on the peeling progress direction.

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