Sheet-shaped cell culture peeling method and sheet-shaped cell culture peeling system

Abstract

To provide a method for peeling a sheet-shaped cell culture, which can prevent damage to the sheet-shaped cell culture when peeling the sheet-shaped cell culture from a culture substrate and can peel the sheet-shaped cell culture more efficiently, and to provide a system for peeling the sheet-shaped cell culture.SOLUTION: A method for peeling a sheet-shaped cell culture includes the steps of: discharging a liquid L1 for peeling toward the vicinity of a peripheral edge 205 of a sheet-shaped cell culture 200 housed in a culture substrate 120, thereby forming a peeling start point 210 where at least a part of the peripheral edge of the sheet-shaped cell culture is curled up from the culture substrate; and applying the liquid for peeling from the peeling star point along the peripheral edge of the sheet-shaped cell culture, thereby peeling the sheet-shaped cell culture from the culture substrate.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for detaching a sheet-shaped cell culture and a system for detaching a sheet-shaped cell culture. [Background technology]

[0002] Sheet-shaped cell cultures (cell sheets) are widely known for use in fields such as regenerative medicine. Generally, sheet-shaped cell cultures are produced by storing and culturing cells collected from humans or non-human animals in a culture substrate (culture vessel) containing a culture medium, and then allowing them to adhere and aggregate into a sheet.

[0003] When using a sheet-shaped cell culture, medical professionals such as physicians use tools such as tweezers to remove the sheet-shaped cell culture from the culture substrate. The sheet-shaped cell culture is partially or entirely adhered to the culture substrate during the production process. Therefore, when removing the sheet-shaped cell culture from the culture substrate, it is necessary to peel the sheet-shaped cell culture from the culture substrate. However, because the sheet-shaped cell culture is constructed in the form of a thin membrane, it has low physical strength and is prone to breakage, such as tearing. Therefore, medical professionals must exercise extreme caution when removing the sheet-shaped cell culture from the culture substrate.

[0004] Patent Document 1 discloses a system for peeling off a sheet-shaped cell culture that can solve the above-mentioned problems. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2011-155869 Summary of the Invention [Problem to be solved by the invention]

[0006] The detachment system of Patent Document 1 includes a sensor unit that detects the detachment state of the sheet-shaped cell culture, and a nozzle unit that sprays a detachment liquid toward the sheet-shaped cell culture and / or culture substrate based on the results detected by the sensor unit. By using a liquid to detach the sheet-shaped cell culture, this system can smoothly detach the sheet-shaped cell culture from the culture substrate without damaging the sheet-shaped cell culture.

[0007] As a result of extensive research, the inventors of the present invention have invented a method for detaching sheet-shaped cell cultures using a detachment liquid, which allows for more efficient detachment of sheet-shaped cell cultures.

[0008] The present invention aims to provide a method for detaching a sheet-shaped cell culture, which can prevent damage to the sheet-shaped cell culture when detaching the sheet-shaped cell culture from the culture substrate and can detach the sheet-shaped cell culture more efficiently, and a system for detaching the sheet-shaped cell culture. [Means for solving the problem]

[0009] The present invention is achieved by any one of the following means (1) to (6).

[0010] (1) A method for detaching a sheet-shaped cell culture, comprising: a step of ejecting a detachment liquid toward the vicinity of the peripheral edge of a sheet-shaped cell culture contained in a culture substrate, thereby forming a detachment starting point where at least a part of the peripheral edge of the sheet-shaped cell culture is turned up from the culture substrate; and a step of applying the detachment liquid from the detachment starting point along the peripheral edge of the sheet-shaped cell culture, thereby detaching the sheet-shaped cell culture from the culture substrate.

[0011] (2) The method for detaching a sheet-shaped cell culture according to (1), wherein the detachment liquid is continuously applied along the entire periphery of the sheet-shaped cell culture from the detachment starting point.

[0012] (3) The method for detaching a sheet-shaped cell culture according to (1) or (2), further comprising a step of confirming the detachment state of the sheet-shaped cell culture by linearly oscillating the culture substrate in a direction parallel to the surface direction of the sheet-shaped cell culture.

[0013] (4) The method for peeling off a sheet-shaped cell culture according to any one of (1) to (3), further comprising a step of adjusting the planar shape of the sheet-shaped cell culture into a predetermined shape by shaking the sheet-shaped cell culture.

[0014] (5) the sheet-shaped cell culture has a substantially circular planar shape in its natural state without the application of external force; The method for detaching a sheet-shaped cell culture according to any one of (1) to (4), wherein the detachment liquid is applied in a tangential direction to the peripheral edge of the sheet-shaped cell culture while the detachment origin is formed.

[0015] (6) a nozzle unit capable of ejecting a liquid for detaching the sheet-shaped cell culture from the culture substrate; a stage on which the culture substrate is placed; a control unit that controls the operation of the nozzle unit and the stage, The control unit ejects the detachment liquid from the nozzle unit toward the vicinity of the peripheral edge of the sheet-shaped cell culture contained in the culture substrate, thereby forming a detachment starting point where at least a part of the peripheral edge of the sheet-shaped cell culture is turned up from the culture substrate, and when the detachment starting point is formed, the nozzle unit and the stage are moved relative to each other to apply the detachment liquid from the detachment starting point along the peripheral edge of the sheet-shaped cell culture, thereby detaching the sheet-shaped cell culture from the culture substrate. [Effects of the Invention]

[0016] According to the sheet-shaped cell culture detachment method and sheet-shaped cell culture detachment system of the present invention, at least a portion of the peripheral edge of the sheet-shaped cell culture is turned up from the culture substrate to form a detachment starting point, and by applying a detachment liquid along the peripheral edge of the sheet-shaped cell culture from the detachment starting point, it is possible to prevent damage to the sheet-shaped cell culture when detaching it from the culture substrate and to detach the sheet-shaped cell culture more efficiently. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a block diagram showing a detachment system for a sheet-shaped cell culture according to an embodiment. [Figure 2] 1 is a flowchart showing each step of a sheet-shaped cell culture detachment method according to an embodiment. [Figure 3] FIG. 1 is a diagram illustrating a method for detaching a sheet-shaped cell culture according to an embodiment. [Figure 4] FIG. 1 is a diagram illustrating a method for detaching a sheet-shaped cell culture according to an embodiment. [Figure 5] FIG. 1 is a diagram illustrating a method for detaching a sheet-shaped cell culture according to an embodiment. [Figure 6] FIG. 10 is a plan view illustrating an example of the arrangement of a nozzle part when ejecting a liquid for peeling onto a sheet-shaped cell culture in the peeling method of a sheet-shaped cell culture according to an embodiment. [Figure 7] FIG. 10 is a perspective view illustrating an example of the arrangement of a nozzle part when ejecting a liquid for detachment onto a sheet-shaped cell culture in the detachment method of a sheet-shaped cell culture according to an embodiment. [Figure 8] FIG. 1 is a diagram illustrating a method for detaching a sheet-shaped cell culture according to an embodiment. [Figure 9] FIG. 1 is a diagram illustrating a method for detaching a sheet-shaped cell culture according to an embodiment. [Figure 10] FIG. 1 is a diagram illustrating a method for detaching a sheet-shaped cell culture according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the dimensional proportions in the drawings are exaggerated for the sake of explanation and may differ from the actual proportions.

[0019] FIG. 1 is a simplified block diagram showing the overall configuration of a detachment system 100 (hereinafter also simply referred to as the "detachment system") for a sheet-shaped cell culture 200 according to this embodiment. FIG. 2 is a flowchart showing each step of a detachment method for a sheet-shaped cell culture 200 according to this embodiment (hereinafter also simply referred to as the "detachment method"). FIGS. 3 to 10 are diagrams for explaining the detachment method. Note that FIGS. 3, 4, 6, and 8 to 10 are plan views of the sheet-shaped cell culture 200 and the culture substrate 120 as viewed from the direction of arrow 3A shown in FIG. 1.

[0020] The arrows X1, X2, Y1, and Y2 in each figure indicate directions parallel to the surface direction of the sheet-shaped cell culture 200, and the arrows Z1 and Z2 indicate the thickness direction (the same direction as the height direction of the culture substrate 120) perpendicular to the surface direction of the sheet-shaped cell culture 200.

[0021] <Removal system 100> The peeling system 100 shown in FIG. 1 is configured as a system for carrying out a peeling method to be described later.

[0022] To give an overview with reference to Figures 1, 4, and 5, the peeling system 100 has a nozzle unit 140 capable of ejecting a peeling liquid L1 for peeling the sheet-shaped cell culture 200 from the culture substrate 120, a stage 110 on which the culture substrate 120 is placed, and a control unit 130 that controls the operation of the nozzle unit 140 and the stage 110.

[0023] The nozzle unit 140 is a liquid supply mechanism that discharges the exfoliation liquid L1. The nozzle unit 140 discharges the exfoliation liquid L1 at a predetermined flow rate, thereby generating a water flow used to exfoliate the sheet-shaped cell culture 200. The nozzle unit 140 is controlled to discharge the exfoliation liquid L1 at a rate of, for example, 0.01 ml / s to 1 ml / s.

[0024] The detachment liquid L1 is, for example, a culture medium (liquid medium), Hank's balanced salts, or PBS, but the type of detachment liquid L1 is not limited as long as it can detach the sheet-shaped cell culture 200 from the culture substrate 120 and does not cause damage to the sheet-shaped cell culture 200.

[0025] The stage 110 is controlled by the control unit 130 to perform a predetermined operation for peeling off the sheet-shaped cell culture 200. Specifically, the stage 110 is configured to be capable of rotating clockwise and counterclockwise around a predetermined rotation axis R, and swinging in a predetermined direction.

[0026] The rotation axis R of the stage 110 can be set, for example, at the center position in the planar direction of the stage 110. Furthermore, the center position in the planar direction of the stage 110 can be set at a position that overlaps with the center position in the planar direction of the culture substrate 120. In this embodiment, the center position of the culture substrate 120 is a position that overlaps with the center position O in the planar direction of the sheet-shaped cell culture 200 before detachment. Therefore, in this embodiment, the rotation axis R of the stage 110, the center position in the planar direction of the culture substrate 120, and the center position O in the planar direction of the sheet-shaped cell culture 200 before detachment are all positioned to overlap with each other.

[0027] The direction in which the stage 110 is oscillated is, for example, a direction parallel to the surface direction of the sheet-shaped cell culture 200. The control unit 130 can oscillate the stage 110 linearly along a direction parallel to the surface direction of the sheet-shaped cell culture 200. The above-mentioned "oscillate linearly" means oscillating in at least two directions (inward and outward in the radial direction from the center position O of the sheet-shaped cell culture 200) that are spaced apart from the center position O, for example, as shown by arrows a1-a1' and b1-b1' in the plan view of FIG. 9 .

[0028] The stage 110 can also be configured as part of a culture device (storage cabinet or storage equipment) used for culturing the sheet-shaped cell culture 200, for example. When the stage 110 is configured in this manner, when starting to detach the sheet-shaped cell culture 200, it becomes possible to immediately start the work of detaching the sheet-shaped cell culture 200 without transporting or moving the culture substrate 120 containing the sheet-shaped cell culture 200. In addition, a jig or mechanism for fixing the culture substrate 120 can be added to the stage 110 as appropriate.

[0029] The peeling system 100 can be equipped with an imaging unit 150 capable of acquiring image data of the sheet-shaped cell culture 200, and an image display unit 160 capable of displaying an image generated from the image data acquired by the imaging unit 150.

[0030] The imaging unit 150 may be configured with a known camera device capable of acquiring still image data and / or video data. The image display unit 160 may be configured with a known display device capable of displaying an image generated from the image data acquired by the imaging unit 150.

[0031] In addition, in the peeling system 100 of this embodiment, the imaging unit 150, image display unit 160, and control unit 130 can also be configured as a portable or non-portable information terminal device in which these functions are integrated.

[0032] The control unit 130 is configured to comprehensively control each unit of the peeling system 100. The control unit 130 includes a CPU and a memory unit. The memory unit includes a ROM for storing various programs and data, a RAM for temporarily storing programs and data as a working area, and a hard disk capable of storing various programs and data. The memory unit can store a series of programs required for controlling the operation of the peeling system 100. An algorithm for causing the control unit 130 to function as a determination unit 135 can be incorporated into the programs.

[0033] The operational control executed by the control unit 130 includes a predetermined detachment operation (detachment method). In carrying out this detachment operation, as shown in FIGS. 4 to 6, the control unit 130 discharges a detachment liquid L1 from the nozzle unit 140 toward the vicinity of the peripheral edge 205 of the sheet-shaped cell culture 200 contained in the culture substrate 120, thereby forming a detachment starting point 210 in which at least a portion of the peripheral edge 205 of the sheet-shaped cell culture 200 is turned up from the culture substrate 120, and then, with the detachment starting point 210 formed, moves the nozzle unit 140 and the stage 110 relative to each other to apply the detachment liquid L1 from the detachment starting point 210 along the peripheral edge 205 of the sheet-shaped cell culture 200, thereby detaching the sheet-shaped cell culture 200 from the culture substrate 120. Note that the specific procedure of the detachment operation will be described later.

[0034] <Sheet-shaped cell culture 200> The sheet-shaped cell culture 200 can contain any cells capable of forming the sheet-shaped cell culture 200. There are no particular limitations on the specific types of such cells, but examples include myoblasts (e.g., skeletal myoblasts), cardiomyocytes, fibroblasts, synovial cells, epithelial cells, and endothelial cells. Furthermore, the term "sheet-shaped cell culture" as used herein refers to cells that are connected to one another and configured in a sheet shape (thin film).

[0035] In this embodiment, the sheet-shaped cell culture 200 is configured to have a substantially circular planar shape in its natural state (not detached from the culture substrate 120) when no external force is applied. The sheet-shaped cell culture 200 covers the entire bottom 125 of the culture substrate 120 and is adhered to the bottom 125 of the culture substrate 120. The peripheral edge 205 of the sheet-shaped cell culture 200 is in contact with the inner surface of the side wall 123 of the culture substrate 120.

[0036] The sheet-shaped cell culture 200 has a surface 201, a back surface 203 located opposite the surface 201 and arranged to face the bottom 125 of the culture substrate 120, and a peripheral portion 205 having a circular outer shape.

[0037] The planar shape of the sheet-shaped cell culture 200 is not limited to a circle, and may be, for example, a rectangle, a triangle, an oval, or any other geometric shape. There are also no particular limitations on the specific thickness of the sheet-shaped cell culture 200.

[0038] <Culture substrate 120> The culture substrate 120 is not particularly limited as long as it is configured to allow cells to form a cell culture. The culture substrate 120 may include, for example, an object having a container structure, a solid or semi-solid surface portion disposed in a container, or the like.

[0039] In this embodiment, as shown in FIGS. 1 and 3, the culture substrate 120 is made up of a container having a structure and material that is impermeable to liquids such as the culture solution L2.

[0040] As shown in Figures 1 and 3, the culture substrate 120 has a storage space 121 for storing the sheet-shaped cell culture 200 and the culture solution L2, a side wall 123 surrounding the storage space 121, and a bottom 125 on which the sheet-shaped cell culture 200 is placed.

[0041] The culture substrate 120 has a substantially circular shape in the plan view shown in Fig. 3. The bottom 125 of the culture substrate 120 has a size (inner diameter) that is the same as or larger than that of the sheet-shaped cell culture 200.

[0042] The sheet-shaped cell culture 200 is prepared (provided) together with the culture substrate 120 after cell culture is completed. The back surface 203 of the sheet-shaped cell culture 200 is partially or entirely adhered to the bottom surface 125 of the culture substrate 120 during the manufacturing process. Therefore, the sheet-shaped cell culture 200, which is prepared while housed in the culture substrate 120, remains adhered to the culture substrate 120.

[0043] In addition, with regard to the specific form of the sheet-shaped cell culture 200 (manufacturing method, physical properties, functions, addition of support, etc.), and the specific form of the culture substrate 120 (material, functions, structure and shape, type of culture solution L2 used, addition of coating, etc.), it is possible to refer to publicly known content (e.g., Patent Publication No. 2011-155869, Patent Publication No. 2022-106986, Patent Publication No. 2021-106609, etc.) as appropriate.

[0044] Next, a peeling method according to this embodiment will be described.

[0045] Explaining the outline with reference to Figures 4, 5 and 6, the detachment method according to this embodiment includes the steps of ejecting a detachment liquid L1 toward the vicinity of the peripheral edge 205 of a sheet-shaped cell culture 200 contained in a culture substrate 120, thereby forming a detachment starting point 210 in which at least a portion of the peripheral edge 205 of the sheet-shaped cell culture 200 is turned up from the culture substrate 120, and applying the detachment liquid L1 from the detachment starting point 210 along the peripheral edge 205 of the sheet-shaped cell culture 200, thereby detaching the sheet-shaped cell culture 200 from the culture substrate 120.

[0046] Specific work content of each step will be described with reference to the flowchart shown in Fig. 2 and Figs. 3 to 10. In the following description, an example will be described in which the delamination system 100 shown in Fig. 1 automatically performs each step of the delamination method. However, in the delamination method according to this embodiment, some or all of the steps can also be performed by an operator.

[0047] 2, before starting the detachment method, a culture substrate 120 to which cells are adhered is prepared (step S10). The culture substrate 120 is placed on a stage 110 (see FIG. 1).

[0048] Next, a water flow is used to form a detachment starting point 210 on the sheet-shaped cell culture 200 (step S11). At the stage when the culture substrate 120 with cells adhered thereto is prepared as shown in FIG. 3, a part or the entire back surface 203 of the sheet-shaped cell culture 200 is adhered to the bottom 125 of the culture substrate 120. In this state, as shown in FIG. 4, a detachment liquid L1 is discharged toward the peripheral edge 205 of the sheet-shaped cell culture 200. As a result, the water flow formed by the detachment liquid L1 forms a detachment starting point 210 as shown in FIG.

[0049] The above-mentioned "detachment starting point 210" refers to the part on the rear surface 203 of the sheet-shaped cell culture 200 that has been detached from the bottom surface 125 of the culture substrate 120 and turned up from the bottom surface 125 side.

[0050] The range of "near the peripheral edge 205 of the sheet-shaped cell culture 200" where the detachment liquid L1 is discharged when forming the detachment origins 210 is not particularly limited as long as the detachment origins 210 can be formed. For example, the detachment origins 210 can be formed by directing a water flow toward a position near the peripheral edge 205, a gap (space) between the peripheral edge 205 and the sidewall 123, a position near the sidewall 123, or the like. More specifically, the step of forming the detachment origins 210 can include at least one of discharging the detachment liquid L1 directly onto the portion where the peripheral edge (edge) 205 of the sheet-shaped cell culture 200 and the culture substrate 120 are adhered, discharging L1 outside the peripheral edge (edge) 205 to form the detachment origins 210 by water pressure, or discharging the detachment liquid L1 onto the portion where the peripheral edge (edge) 205 and the inner side of the sidewall 123 of the culture substrate 120 are in contact.

[0051] Next, a water flow is applied in the tangential direction of the sheet-shaped cell culture 200 (step S12). With the detachment starting points 210 formed, the water flow is applied along the peripheral edge 205 of the sheet-shaped cell culture 200, thereby allowing the peripheral edge 205 to be smoothly detached along the direction in which detachment progresses.

[0052] In step S12, with the detachment starting point 210 formed as shown in Figure 5, a water flow is applied in the tangential direction of the sheet-shaped cell culture 200 (the tangential direction in the plan view shown in Figure 3). By applying the water flow in this manner, the peripheral portion 205 of the sheet-shaped cell culture 200 is continuously detached from the culture substrate 120 along the peripheral portion 205 of the sheet-shaped cell culture 200 from the detachment starting point 210. Note that the dashed line A shown in Figure 5 is an imaginary line indicating the tangent line of the sheet-shaped cell culture 200.

[0053] Specifically, in step S12, a water flow is applied toward the end 210a in the peeling direction of the peeling starting point 210 formed on the peripheral edge 205. This allows the sheet-shaped cell culture 200 to be smoothly peeled from the culture substrate 120 along the peeling direction (the direction along the peripheral edge 205 in this embodiment) from the peeling starting point 210.

[0054] More specifically, as shown in Fig. 6, the nozzle part 140 is arranged along a tangent line A that forms a perpendicular angle with a perpendicular line H1 extending circumferentially from the center of the culture substrate 120 (the same position as the center O of the sheet-shaped cell culture 200). Also, as shown in Fig. 7, the angle θ of the nozzle part 140 is preferably 0° to 60°, and more preferably 0° to 45°, from the horizontal plane of the sheet-shaped cell culture 200.

[0055] When performing step S12, the control unit 130 controls the operation of the nozzle unit 140 and / or the stage 110, as shown in Fig. 8. In this embodiment, with the position of the nozzle unit 140 fixed, the stage 110 is rotated as shown by arrow r1 to apply a water flow along the peripheral edge 205. At this time, the culture substrate 120 or the stage 110 is rotated in the direction opposite to the end 210a side of the peeling direction, that is, the rotation is performed in the opposite direction to the direction of peeling.

[0056] In step S12, the detachment liquid L1 can be continuously applied along the entire circumference of the peripheral portion 205 of the sheet-shaped cell culture 200, starting from the detachment starting point 210. At this time, the nozzle unit 140 or the culture substrate 120 is rotated once in the tangential direction, and the water flow is applied to the entire circumference of the peripheral portion 205 of the sheet-shaped cell culture 200. By performing such an operation, the sheet-shaped cell culture 200 can be more reliably detached from the culture substrate 120.

[0057] When the sheet-shaped cell culture 200 does not have a substantially circular planar shape (for example, when it has a rectangular, triangular, or other planar shape), after forming the detachment starting points 210, the detachment liquid L1 can be discharged along each linear side located at the peripheral edge. By performing this operation, the sheet-shaped cell culture 200 can be smoothly detached along the peripheral edge 205. When the sheet-shaped cell culture 200 has an elliptical planar shape, it is preferable to apply the water flow in the tangential direction of the ellipse, as in the case of a substantially circular planar shape.

[0058] When an operator performs the above-described step S11 and / or step S12, a known discharge tool such as a pipette can be used instead of the nozzle portion 140. When the operator performs step S12, the operator can move the discharge tool along the peripheral portion 205 and / or rotate the culture substrate 120, thereby smoothly peeling the peripheral portion 205 from the culture substrate 120. When the operator manually performs step S11 and / or step S12, the operator can easily perform the work by rotating the culture substrate 120 rather than moving the discharge tool. Therefore, it is preferable to perform the work of rotating the culture substrate 120 while fixing the position of the discharge tool.

[0059] Next, the culture substrate 120 is rocked (Step S13). In this step, the culture substrate 120 is rocked linearly in a direction parallel to the surface direction of the sheet-shaped cell culture 200, for example, as shown by arrows a1-a1' and b1-b1' in FIG. 9. Furthermore, while the culture substrate 120 is rocked, the detachment state of the sheet-shaped cell culture 200 is checked (Step S14). If, when the culture substrate 120 is rocked, the sheet-shaped cell culture 200 gradually detaches or the entire culture substrate 120 moves in a direction parallel to the surface direction, it is determined that there is a tendency for detachment (Step S14: YES). If it is determined that there is a tendency for detachment, proceed to Step S15.

[0060] On the other hand, if it is determined that there is no tendency for detachment to occur (step S14: NO), the process returns to step S12, and the water flow is applied again to the peripheral portion 205. At this time, the water flow may be applied to the space generated between the detached peripheral portion 205 of the sheet-shaped cell culture 200 and the culture substrate 120, or to the bottom 125 of the culture substrate 120. By applying the water flow again, the entire area of ​​the peripheral portion 205 of the sheet-shaped cell culture 200 can be more reliably detached from the culture substrate 120. Steps S12, S13, and S14 are then repeated until a tendency for detachment is confirmed.

[0061] The peeling system 100 can make the determination in step S14 based on the image data (still image data and / or video data) captured by the imaging unit 150. When an operator makes the above determination, the operator can make the determination based on the image displayed on the image display unit 160 while manually performing step S13, or by directly visually checking the state of the sheet-shaped cell culture 200.

[0062] In step S13, as described above, the culture substrate 120 is oscillated linearly in a direction parallel to the surface direction of the sheet-shaped cell culture 200. For example, the presence or absence of a tendency to detachment can be determined by checking the state of the sheet-shaped cell culture 200 when the culture substrate 120 is rotated and oscillated. However, oscillating the culture substrate 120 while rotating it can easily damage the sheet-shaped cell culture 200. Therefore, when oscillating the culture substrate 120, it is preferable to oscillate the culture substrate 120 linearly while stopping the operation of rotating the culture substrate 120 (for example, the operation of rotating the culture substrate 120 to apply a water flow to the peripheral portion 205 of the sheet-shaped cell culture 200).

[0063] Steps S13 and S14 are preferably performed after applying the detachment liquid L1 along the entire circumference of the peripheral edge 205 of the sheet-shaped cell culture 200 from the detachment starting point 210. By performing steps S13 and S14 after such an operation, it becomes possible to more accurately determine whether the sheet-shaped cell culture 200 has been detached from the culture substrate 120.

[0064] Next, the culture substrate 120 is rocked to detach the sheet-shaped cell culture 200 (step S15). In this step, the culture substrate 120 is rocked linearly in a direction parallel to the surface direction of the sheet-shaped cell culture 200, for example, as shown by arrows a1-a1' and b1-b1' in Figure 9. After the tendency for detachment is confirmed in step S14, the culture substrate 120 is further rocked to more reliably detach the portions of the sheet-shaped cell culture 200 adhered to the culture substrate 120 other than the peripheral edge 205.

[0065] If a tendency to peeling is confirmed in step S14, step S15 may be performed continuously without stopping the oscillation performed in step S13 (or may be performed as substantially the same operation as step S13).

[0066] Next, the culture substrate 120 is rocked to adjust the shape of the sheet-shaped cell culture 200 (step S16). In this step, the culture substrate 120 can be rocked linearly along a direction parallel to the surface direction of the sheet-shaped cell culture 200, as shown by arrows a1-a1' and b1-b1' in Fig. 10. Alternatively, as shown by arrows r1 and r2 in Fig. 10, methods of rocking the sheet-shaped cell culture 200 by rotation may be combined as appropriate.

[0067] After the sheet-shaped cell culture 200 is detached from the culture substrate 120 in step S15, the culture substrate 120 can be further rocked to return the planar shape of the sheet-shaped cell culture 200 to a predetermined shape (its natural shape). For example, in a method of detaching the sheet-shaped cell culture 200 by applying a water flow to the peripheral edge 205, the water flow is concentrated near the peripheral edge 205, and therefore, after detachment is complete, the peripheral edge 205 may be curled, warped, wrinkled, or the like. Therefore, by performing step S16 after detachment is complete, the sheet-shaped cell culture 200 can be shaped into a predetermined shape (a substantially circular planar shape in this embodiment) by removing the curled, warped, wrinkled, or the like. By performing step S16 to shape the sheet-shaped cell culture 200, damage to the sheet-shaped cell culture 200 when removing it from the culture substrate 120 and the laborious removal process can be prevented.

[0068] By carrying out each of the above steps, the sheet-shaped cell culture 200 can be properly and reliably detached from the culture substrate 120. After detaching the sheet-shaped cell culture 200 from the culture substrate 120, the operator can remove the sheet-shaped cell culture 200 from the culture substrate 120 using a tool such as tweezers or a spatula, as necessary.

[0069] As described above, the detachment method according to this embodiment includes the steps of ejecting a detachment liquid L1 toward the vicinity of the peripheral edge 205 of the sheet-shaped cell culture 200 contained in the culture substrate 120, thereby forming a detachment starting point 210 where at least a portion of the peripheral edge 205 of the sheet-shaped cell culture 200 is turned up from the culture substrate 120, and of applying the detachment liquid L1 from the detachment starting point 210 along the peripheral edge 205 of the sheet-shaped cell culture 200, thereby detaching the sheet-shaped cell culture 200 from the culture substrate 120.

[0070] Furthermore, the detachment system 100 according to this embodiment includes a nozzle unit 140 capable of ejecting a detachment liquid L1 for detaching the sheet-shaped cell culture 200 from the culture substrate 120, a stage 110 on which the culture substrate 120 is placed, and a control unit 130 for controlling the operation of the nozzle unit 140 and the stage 110. The control unit 130 is configured to eject the detachment liquid L1 from the nozzle unit 140 toward the vicinity of a peripheral portion 205 of the sheet-shaped cell culture 200 contained in the culture substrate 120, thereby forming a detachment starting point 210 in which at least a portion of the peripheral portion 205 of the sheet-shaped cell culture 200 is turned up from the culture substrate 120, and to detach the sheet-shaped cell culture 200 from the culture substrate 120 by operating the nozzle unit 140 and the stage 110 relative to each other when the detachment starting point 210 is formed, by applying the detachment liquid L1 from the detachment starting point 210 along the peripheral portion 205 of the sheet-shaped cell culture 200.

[0071] According to the above-described peeling method and peeling system 100, at least a portion of the peripheral edge 205 of the sheet-shaped cell culture 200 is peeled up from the culture substrate 120 to form a peeling starting point 210, and by applying the peeling liquid L1 from the peeling starting point 210 along the peripheral edge 205 of the sheet-shaped cell culture 200, it is possible to prevent the sheet-shaped cell culture 200 from being damaged when peeling the sheet-shaped cell culture 200 from the culture substrate 120 and to peel the sheet-shaped cell culture 200 more efficiently.

[0072] Although the sheet-shaped cell culture peeling method and sheet-shaped cell culture peeling system according to the present invention have been described through the embodiments, the present invention is not limited to the configurations described in the embodiments, and can be modified as appropriate based on the claims. [Explanation of symbols]

[0073] 100 Peeling System 110 Stages 120 Culture substrate 121 Containment Space 130 control section 135 Judgment section 140 Nozzle section 150 Imaging unit 160 Image display unit 200 Sheet-shaped cell cultures 205 Periphery of sheet-shaped cell culture 210 Peeling origin L1 Stripping liquid L2 culture solution R rotation axis O Center position of the sheet-shaped cell culture in the plane direction

Claims

1. a step of discharging a liquid for detachment toward the vicinity of the peripheral edge of the sheet-shaped cell culture accommodated in a culture substrate, whereby at least a part of the peripheral edge of the sheet-shaped cell culture is peeled up from the culture substrate to form a detachment starting point; and applying the detachment liquid along the peripheral edge of the sheet-shaped cell culture from the detachment starting point to detach the sheet-shaped cell culture from the culture substrate.

2. The method for detaching a sheet-shaped cell culture according to claim 1 , wherein the detachment liquid is continuously applied along the entire periphery of the sheet-shaped cell culture from the detachment starting point.

3. The method for detaching a sheet-shaped cell culture according to claim 1, further comprising a step of confirming the detachment state of the sheet-shaped cell culture by linearly oscillating the culture substrate in a direction parallel to the surface direction of the sheet-shaped cell culture.

4. The method for detaching a sheet-shaped cell culture according to claim 1 , further comprising a step of adjusting the planar shape of the sheet-shaped cell culture into a predetermined shape by shaking the sheet-shaped cell culture.

5. the sheet-shaped cell culture has a substantially circular planar shape in its natural state without the application of external force; The method for detaching a sheet-shaped cell culture according to any one of claims 1 to 4, wherein the detachment liquid is applied in a tangential direction to the peripheral edge of the sheet-shaped cell culture while the detachment origin is formed.

6. a nozzle unit capable of ejecting a liquid for detaching the sheet-shaped cell culture from the culture substrate; a stage on which the culture substrate is placed; a control unit that controls the operation of the nozzle unit and the stage, The control unit ejects the detachment liquid from the nozzle unit toward the vicinity of the peripheral edge of the sheet-shaped cell culture contained in the culture substrate, thereby forming a detachment starting point where at least a part of the peripheral edge of the sheet-shaped cell culture is turned up from the culture substrate, and when the detachment starting point is formed, the nozzle unit and the stage are moved relative to each other to apply the detachment liquid from the detachment starting point along the peripheral edge of the sheet-shaped cell culture, thereby detaching the sheet-shaped cell culture from the culture substrate.

Images