Cell detachment device and cell detachment method

Abstract

To allow for easy cell detachment.SOLUTION: A cell detachment device according to an embodiment of the present invention comprises a culture vessel and a discharge part. The culture vessel is used for culturing cells. The discharge part is provided at such a position as not to be immersed in liquid in the culture vessel, and discharges a plurality of droplets toward a culturing surface of the culture vessel to cause detachment of the cells from the culturing surface.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The embodiments disclosed in the present specification and drawings relate to a cell detachment device and a cell detachment method. [Background technology]

[0002] In the process of cell culture, it is necessary to detach cells that have adhered to the bottom of the culture vessel. For example, technicians manually and physically detach the cells using a cell scraper or pipette. However, manual work involves risks such as cell contamination by microorganisms and cell damage due to mechanical stimulation, so the detachment work must be performed by skilled technicians with the utmost care while ensuring and maintaining a sterile environment. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2011-155869 A Summary of the Invention [Problem to be solved by the invention]

[0004] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to easily detach cells. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described later can also be positioned as other problems. [Means for solving the problem]

[0005] A cell detachment device according to an embodiment includes a culture vessel and a discharge unit. The culture vessel cultures cells. The discharge unit is provided at a position in the culture vessel that is not immersed in liquid, and discharges a plurality of droplets toward a culture surface of the culture vessel to detach the cells from the culture surface. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is a schematic diagram showing a configuration example of a cell detachment device according to a first embodiment. [Diagram 2] 4 is a flowchart showing an example of the operation of the cell detachment device according to the first embodiment. [Diagram 3] FIG. 2 is a schematic diagram showing an example of operation of the cell detachment device according to the first embodiment. [Figure 4] FIG. 4 is a diagram showing cell detachment results under each experimental condition according to the first embodiment. [Diagram 5] FIG. 13 is a schematic diagram showing a configuration example of a cell detachment device according to a second embodiment. [Figure 6] 10 is a flowchart showing an example of the operation of the cell detachment device according to the second embodiment. [Figure 7] FIG. 11 is a schematic diagram showing an example of operation of the cell detachment device according to the second embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Hereinafter, each embodiment will be described with reference to the drawings. In the following embodiments, parts with the same reference numerals are assumed to perform the same operation, and duplicated descriptions will be omitted as appropriate.

[0008] (First embodiment) Fig. 1 is a schematic diagram showing a configuration example of a cell detachment device 1 according to a first embodiment. This diagram shows the cell detachment device 1 as viewed from a horizontal direction. The cell detachment device 1 is a device for detaching cells. The cell detachment device 1 includes a culture vessel 2, a nozzle 3, a syringe 4, a pressing mechanism 5, a stopper 6, a support frame 7, a mounting device 8, a connector 9, and a control device 10.

[0009] The culture vessel 2 is a vessel for culturing cells C. The culture vessel 2 is formed from a material that does not allow the liquid L attached to the surface to penetrate (is impermeable). Examples of such materials include polyethylene, polypropylene, polycarbonate, polystyrene, and glass. The culture vessel 2 may have any size and any shape. For example, the culture vessel 2 is a circular vessel with a diameter of 35 mm or more and 100 mm or less. The culture vessel 2 is an example of a culture section.

[0010] Cell C is a cell (adherent cell) that adheres to the surface of culture vessel 2 via an adhesive protein. Examples of cell C include iPS (induced pluripotent stem) cells, epithelial cells, endothelial cells, synovial cells, cardiomyocytes, myoblasts, fibroblasts, and neuroblasts. Cell C may be a single cell or a colony (cell mass) consisting of multiple cells.

[0011] The liquid L may be any type of liquid. Examples of the liquid L include PBS (Phosphate Buffered Saline) and liquid culture media. The liquid L is preferably a liquid that does not chemically or physiologically damage the cells C.

[0012] The nozzle 3 is a mechanical part that ejects the liquid L. The nozzle 3 is made of a material such as metal that has a relatively high rigidity. The nozzle 3 is physically connected to the syringe 4 by a connector 9. Preferably, the distance d1 between the tip of the nozzle 3 and the bottom surface of the culture vessel 2 is 10 mm or more and 50 mm or less. The tip of the nozzle 3 is disposed on a straight line that passes through the center of the bottom surface of the culture vessel 2 and is perpendicular to the bottom surface. The bottom surface of the culture vessel 2 functions as a culture surface to which the cells C adhere. The nozzle 3 is disposed above the culture vessel 2, that is, at a position that is not immersed in the liquid in the culture vessel 2. The nozzle 3 ejects the liquid L as a plurality of droplets toward the bottom surface of the culture vessel 2. The nozzle 3 is an example of an ejection unit.

[0013] The nozzle 3 is designed with the number, position, size and shape of holes (discharge holes) for discharging the liquid L so that the conditions (discharge conditions) related to the discharge of the liquid L are set to desired values. Examples of the discharge conditions include the average particle size, average speed and average kinetic energy of multiple droplets of the liquid L, and the amount and duration of one discharge of the liquid L. The nozzle 3 discharges the liquid L in a mist form onto the cells C cultured in the culture vessel 2 under the desired discharge conditions by appropriately setting the pressing parameters of the pressing mechanism 5 described later. That is, the nozzle 3 sprays the liquid L onto the cells C, thereby detaching the cells C from the culture vessel 2. The droplets of the liquid L discharged from the nozzle 3 fly toward the bottom surface of the culture vessel 2 and collide with the cells C, thereby transmitting their kinetic energy to the cells C, thereby realizing the detachment of the cells C.

[0014] The nozzle 3 ejecting the liquid L in a "mist" means that the nozzle 3 ejects the liquid L "as a plurality of minute droplets." The average particle size of the plurality of minute droplets is 50 μm or more and 600 μm or less (see FIG. 4). In other words, "mist" refers to the state related to the size of the droplets, not the shape of the spatial range into which the droplets are ejected (e.g., cone-shaped, linear-shaped).

[0015] The nozzle 3 may detach the cells C from the culture vessel 2 by discharging the liquid L once. At this time, the amount of liquid L discharged once, i.e., the total amount of droplets discharged in one discharge, is preferably 3.0 mL or less. The time required for one discharge of the liquid L is preferably 0.2 seconds or less. The discharge angle of the liquid L from the nozzle 3 may be designed or adjusted so that the discharged liquid L contacts the entire surface of the cells C (preferably, 95% or more of the area). Typically, the nozzle 3 discharges the liquid L in a cone shape. The nozzle 3 may also discharge the liquid L in a straight line.

[0016] The nozzle 3 may eject the liquid L onto a portion of the surface of the cells C cultured in the culture vessel 2 that is in contact with the air. That is, the nozzle 3 ejects the liquid L onto the portion of the surface of the cells C that is in contact with the air, not onto the portion immersed in the liquid L (immersed portion). The nozzle 3 transmits the kinetic energy of the ejected liquid L directly to the cells C, not via the liquid L that covers the surface of the cells C, so that the cells C can be effectively detached from the culture vessel 2.

[0017] The syringe 4 is a device that stores the liquid L and transmits kinetic energy to the liquid L. The syringe 4 is formed, for example, from a material similar to that of the culture vessel 2. The syringe 4 is disposed in a horizontal direction (left-right direction in FIG. 1). The syringe 4 has a barrel 41, a flange 42, and a plunger 43.

[0018] The barrel 41 is a stationary component that stores the liquid L. The barrel 41 is a hollow, cylindrical container. One end of the barrel 41 is connected to the nozzle 3 by a connector 9. The other end of the barrel 41 is coupled to the plunger 43 by a flange 42. The lower part of the barrel 41 is physically supported by the support frame 7. The liquid L may be stored in the barrel 41 in advance by a user, or the liquid L may be automatically stored in the barrel 41 by a known configuration. The liquid L may be stored repeatedly in the barrel 41. The barrel 41 is an example of a storage section.

[0019] The flange 42 is a stationary component that connects the barrel 41 and the plunger 43. The flange 42 connects the barrel 41 and the plunger 43 so that the plunger 43 is movable along the longitudinal direction of the barrel 41 (the left-right direction in FIG. 1 ). The flange 42 is an example of a connecting portion.

[0020] The plunger 43 is an operating part that transmits kinetic energy to the liquid L. The plunger 43 has a rod 431 and a pressure receiving plate 432. The rod 431 and the pressure receiving plate 432 may be integrally formed. The plunger 43 is an example of a transmission part.

[0021] The rod 431 is a bar that moves the liquid L stored in the barrel 41. The rod 431 is a cylinder having a diameter that matches the inner diameter of the barrel 41. One end of the rod 431 is attached inside the barrel 41. The other end of the rod 431 is connected to the pressure-receiving plate 432. The rod 431 pushes out the liquid L toward the nozzle 3 by moving in the same direction as the movement direction of the pressure-receiving plate 432. The rod 431 directly transmits kinetic energy to the liquid L.

[0022] The pressure-receiving plate 432 is a plate that receives pressure from the pressing mechanism 5. The pressure-receiving plate 432 moves in the direction in which it receives pressure, thereby moving the rod 431 in the same direction.

[0023] The pressing mechanism 5 is a mechanism that presses the pressure receiving plate 432. The pressing mechanism 5 is formed of a material such as metal having a relatively high rigidity. The pressing mechanism 5 is electrically connected to the control device 10 by an electric wire. The pressing mechanism 5 adjusts parameters (pressing parameters) related to the pressing of the pressure receiving plate 432 in accordance with the control of the control device 10 so as to realize the desired discharge conditions. Examples of the pressing parameters include the pressing force and pressing time. The pressing mechanism 5 presses the pressure receiving plate 432 with the adjusted pressing parameters. The pressing mechanism 5 has a pressing plate 51, an arm portion 52, and a base 53. The pressing mechanism 5 is an example of a pressing portion.

[0024] The pressure plate 51 is an operating part that presses the pressure receiving plate 432. One surface of the pressure plate 51 faces one surface of the pressure receiving plate 432. A distance d2 between one surface of the pressure plate 51 and one surface of the pressure receiving plate 432 is designed to be an arbitrary value. The other surface of the pressure plate 51 is connected to the arm portion 52.

[0025] The arm 52 is a moving part that moves the pressing plate 51. The arm 52 expands and contracts along the longitudinal direction of the barrel 41, thereby moving the pressing plate 51 in the same direction.

[0026] The base 53 is a stationary component that physically supports the arm portion 52. The base 53 houses a part of the arm portion 52. The base 53 is fixed to the upper surface of the mounting device 8.

[0027] The stopper 6 is a device that stops the movement of the pressure plate 51. The stopper 6 is disposed between the pressure plate 51 and the flange 42. The stopper 6 has an absorbent material 61 and a support material 62. The stopper 6 is an example of a stop portion.

[0028] The absorbent 61 is a stationary part that absorbs pressure from the pressure plate 51. The absorbent 61 is made of a material having cushioning properties. Examples of such materials include urethane, rubber, and gel. A distance d3 between the absorbent 61 and the surface of the pressure plate 51 is designed to be greater than the distance d2 (d3>d2). The distance d3 minus the distance d2 (d3-d2) corresponds to the distance that the plunger 43 can move.

[0029] The support material 62 is a stationary part that physically supports the absorbent material 61. The support material 62 is formed of a material such as metal having a relatively high rigidity. The support material 62 is L-shaped and has a first portion extending in the vertical direction (up and down direction in FIG. 1 ) and a second portion extending in the horizontal direction. The height of the first portion is designed so that the first portion does not interfere with the movement of the plunger 43 and stops the movement of the pressing plate 51. The absorbent material 61 is disposed on the upper part of the first portion. The second portion is fixed to the upper surface of the mounting device 8.

[0030] The support frame 7 is a stationary component that physically supports the barrel 41. The support frame 7 adjusts the height at which the barrel 41 is placed. The support frame 7 is made of a material such as metal that has relatively high rigidity. The support frame 7 is fixed to the upper surface of the mounting tool 8. The support frame 7 is an example of a support portion.

[0031] The mounting tool 8 is a device on which the pressing mechanism 5, the stopper 6, and the support frame 7 are mounted. The mounting tool 8 is made of a material such as metal having a relatively high rigidity. The mounting tool 8 has a base 81, a plate 82, and legs 83. The mounting tool 8 is an example of a mounting portion.

[0032] The pedestal 81 is a stationary component on which the base 53 is placed. The pedestal 81 adjusts the height at which the base 53 is placed. The height of the pedestal 81 is designed so that the pressing plate 51 can press the pressure receiving plate 432. The pedestal 81 is fixed to the upper surface of the plate 82.

[0033] The plate 82 is a stationary component on which the stopper 6 and the support frame 7 are placed. One end and the other end of the plate 82 are connected to the legs 83.

[0034] The legs 83 are stationary components that adjust the height of the plate 82 from the floor surface. The legs 83 are placed on the floor surface. A user of the cell detachment device 1 can insert a finger into a gap formed between the plate 82 and the floor surface to lift the plate 82 from the floor surface.

[0035] The connector 9 is a device that connects the nozzle 3 and the barrel 41 to each other. The connector 9 has a supply tube 91 and a flow rate sensor 92. The connector 9 is an example of a connecting portion.

[0036] The supply tube 91 is a tube that supplies the liquid L delivered from the barrel 41 to the nozzle 3. The supply tube 91 is made of silicone rubber, polyethylene, metal, or the like. A flow rate sensor 92 may be disposed in a portion of the supply tube 91.

[0037] The flow rate sensor 92 is a sensor that measures the flow rate of the liquid L passing through the supply tube 91. The flow rate sensor 92 is electrically connected to the control device 10 by an electric wire. The flow rate sensor 92 measures the flow rate of the liquid L according to the control of the control device 10, and transmits the measured value of the flow rate to the control device 10. The control device 10 may determine the set value of the pressing parameter based on the relationship between the set pressing parameter and the flow rate measured by the flow rate sensor 92. For example, the control device 10 calculates a conversion formula between the pressing parameter and the flow rate by measuring the flow rate for each of the ejection operations based on a plurality of pressing parameters. The control device 10 may calculate a pressing parameter corresponding to a flow rate that realizes a desired ejection condition based on the conversion formula, and determine the calculated pressing parameter as a new set value.

[0038] The control device 10 is a device that controls the overall operation of the cell detachment device 1. The control device 10 has at least one processor. The processor means a circuit such as a central processing unit (CPU), a graphics processing unit (GPU), an application specific integrated circuit (ASIC), a programmable logic device (e.g., a simple programmable logic device (SPLD), a complex programmable logic device (CPLD), or a field programmable gate array (FPGA)). When the processor is a CPU, the CPU realizes each function by reading and executing a control program stored in a memory. When the processor is an ASIC, each function is directly incorporated as a logic circuit in the circuit of the ASIC. The processor may be configured as a single circuit, or may be configured by combining multiple independent circuits with each other. The control device 10 is an example of a control unit.

[0039] The control device 10 may be an information processing device such as a general-purpose computer. The control device 10 may have each component of a general-purpose computer (e.g., a processor, a memory, an input device, an output device, a communication device). The control device 10 may receive various instructions from a user (e.g., a discharge instruction, a discharge instruction, an end instruction) via an input device. The control device 10 may present the measured value of the flow rate from the flow sensor 92 to the user in a predetermined form via an output device.

[0040] 2 is a flowchart showing an example of the operation of the cell detachment device 1 according to the first embodiment. According to this example, the cell detachment device 1 discharges the liquid L into the culture container 2 in accordance with a discharge command from a user.

[0041] (Step S11) First, the cell detachment device 1 determines whether or not an ejection instruction has been received from the user. For example, the control device 10 determines whether or not the user has input an ejection instruction to the control device 10 using an input device (e.g., a mouse, a keyboard, a communication terminal). If an ejection instruction has been input (step S11-YES), the process proceeds to step S12. If an ejection instruction has not been input (step S11-NO), the process returns to step S11. That is, the control device 10 waits until an ejection instruction is received from the user. Note that the ejection instruction may include ejection conditions related to the ejection of the liquid L.

[0042] (Step S12) Finally, the cell detachment device 1 discharges the liquid L into the culture vessel 2. Specifically, the control device 10 controls the pressing mechanism 5 to cause the pressing plate 51 to press the pressure receiving plate 432, and discharges the liquid L from the nozzle 3 into the culture vessel 2 under the desired discharge conditions (see FIG. 3).

[0043] 3 is a schematic diagram showing an example of the operation of the cell detachment device 1 according to the first embodiment. As shown in this figure, the control device 10 extends the arm 52 of the pressing mechanism 5 along the direction D1, thereby moving the pressing plate 51 along the direction D1. The pressing plate 51 comes into contact with the pressure-receiving plate 432 and presses the pressure-receiving plate 432 along the same direction D2 as the direction D1. The pressure-receiving plate 432 moves the rod 431 in the direction D2, thereby pushing out the liquid L stored in the barrel 41 along the direction D2. The movement of the pressing plate 51 stops when it comes into contact with the absorbent 61.

[0044] The liquid L pushed out from the barrel 41 passes through the supply tube 91 and reaches the nozzle 3. The nozzle 3 discharges the supplied liquid L into the culture vessel 2 under the desired discharge conditions. The nozzle 3 detaches the cells C from the culture vessel 2 by spraying the liquid L onto the culture vessel 2. In particular, the cells C detached by the discharge of the liquid L are in a state of being separated into individual cells (single cells). The flow rate sensor 92 measures the flow rate of the liquid L passing through the supply tube 91, and transmits a measured value 920 of the flow rate to the control device 10.

[0045] 4 is a diagram showing cell detachment results for each experimental condition according to the first embodiment. Tables 100A and 100B show the detachment results of cells C obtained by ejecting liquid L onto cells C from nozzle 3 under each value related to three types of ejection conditions (average particle size, average velocity, average kinetic energy). The experimental operations performed to obtain these detachment results are described below.

[0046] Human-derived iPS cells (particularly, P0 cells) were used as the cells C. A circular vessel (35 mm dish) with a diameter of 35 mm was used as the culture vessel 2. First, the experimenter added iPS cells and liquid medium to the circular vessel, allowed the iPS cells to form colonies, and then aspirated all of the liquid medium. Next, the experimenter washed the colonies by adding 1 mL of PBS to the circular vessel and aspirating all of the PBS. Next, the experimenter added 400 μL of EDTA (Ethylenediaminetetraacetic acid) to the circular vessel, incubated the colonies at 37° C. for 10 minutes, and then aspirated all of the EDTA. Next, the experimenter added PBS to the circular vessel and washed the colonies again by aspirating all of the PBS.

[0047] Next, the experimenter detached the colonies from the circular container by spraying 1 mL of PBS from nozzle 3 onto the colonies under each of the three discharge conditions (average particle size, average velocity, and average kinetic energy). The experimenter measured the "survival rate" of the detached colonies. Furthermore, the experimenter visually observed the detachment state of the colonies from the circular container using a microscope. Based on the observation results, the experimenter judged whether the detached colonies were "completely detached" and whether the "evaluation" was good or bad.

[0048] Specifically, the "survival rate" was measured by the following method. First, the experimenter randomly took 10 μL from 1 mL of the cell suspension after detachment, and mixed it with 10 μL of trypan blue to obtain a mixed solution. Next, the experimenter poured the mixed solution into an automatic cell counter slide, inserted the slide into an automatic cell counter (Bio-Rad, TC20 (registered trademark)), and measured the cell number and survival rate using multi-focal plane analysis.

[0049] If the colony was completely detached from the bottom of the circular container, the experimenter judged it as "complete detachment: yes." If the "survival rate" of the cells after detachment was 80% or more and "complete detachment: yes," the experimenter judged it as "good."

[0050] Tables 100A and 100B highlight records related to "Evaluation: Good." According to the highlighted records, the average particle size is 100 μm or more and 500 μm or less, the average speed is 5 km / h or more and 50 km / h or less, and the average kinetic energy is 5.0×10 -13 J or above 6.3×10 -9 J or less. In other words, when the three types of discharge conditions satisfy the above-mentioned respective conditions, a colony of iPS cells can be detached particularly effectively from the circular container.

[0051] When PBS remained in the circular container (i.e., the iPS cell colonies were immersed in PBS) and PBS was sprayed onto the colonies, the percentage of the area that had detached from the circular container to the total area of ​​the colonies (detached area rate) decreased. (i) When no PBS remained, the detached area rate was 98%. (ii) When 400 μL of PBS remained, the detached area rate was 45%. (iii) When 1 mL of PBS remained, the detached area rate was 5%. In other words, it is desirable to fully drain the PBS from the circular container prior to detaching the colonies, thereby expanding the area of ​​the colony surface that comes into contact with the air.

[0052] That is, in the cell detachment device 1 according to the first embodiment, prior to the step (S12) of discharging the liquid L into the culture vessel 2, a liquid pump (not shown) may be driven to discharge the liquid in the culture vessel 2 through a liquid port (not shown) to reduce the amount of liquid in the culture vessel 2. In this case, the liquid pump and liquid port may be, for example, the same as liquid pump 25B and liquid port 211B in the second embodiment described below.

[0053] According to the first embodiment described above, the cell detachment device 1 detaches the cells C by ejecting the liquid L from the nozzle 3 onto the cells C cultured in the culture vessel 2. This allows the user to easily detach the cells C without the need for complicated manual work. Furthermore, the cell detachment device 1 has the nozzle 3 at a position in the culture vessel 2 where it is not immersed in the liquid L. This allows the user to effectively detach the cells C by using the action of droplet collision. Furthermore, the cell detachment device 1 ejects the liquid L from the fixed nozzle 3 to detach the cells C. This allows the configuration of the cell detachment device 1 to be simplified.

[0054] Furthermore, the cell detachment device 1 transmits the kinetic energy of the discharged liquid L directly to the cells C without passing through the wall surface of the culture vessel 2. Therefore, the cell detachment device 1 can effectively detach the cells C. In particular, the cell detachment device 1 can separate a colony of cells C into individual cells C (single cells).

[0055] Second embodiment 5 is a schematic diagram showing a configuration example of a cell detachment device 1 according to the second embodiment. In addition to the components according to the first embodiment (see FIG. 1), the cell detachment device 1 according to the second embodiment includes a cell detachment container 200, a gas pump 25A, and a liquid pump 25B. The gas pump 25A and the liquid pump 25B may be electrically connected to the control device 10 by electric wires.

[0056] The cell detachment container 200 is a container for detaching cells. The cell detachment container 200 is a closed container. The cell detachment container 200 has an upper cover 21, a lower cover 22, a gas port 211A, a liquid port 211B, a gas tube 212A, and a liquid tube 212B.

[0057] The upper lid 21 and the lower lid 22 are lids that form the housing of the cell detachment container 200. The upper lid 21 and the lower lid 22 are formed of a material such as metal that has a relatively high rigidity. The upper lid 21 and the lower lid 22 have a U-shaped cross section, and have a flat bottom surface and an L-shaped side surface. The upper lid 21 and the lower lid 22 are joined to each other with the side surface of the upper lid 21 and the side surface of the lower lid 22 in contact with each other. With this joining, the inside of the cell detachment container 200 is physically isolated from the outside and kept airtight. The upper lid 21 and the lower lid 22 may be joined to each other in a detachable manner.

[0058] A gas port 211A, a liquid port 211B, and a nozzle 3 are disposed on the bottom surface of the upper cover 21, penetrating the bottom surface. The nozzle 3 is disposed at the center of the bottom surface. The nozzle 3 may be disposed on the bottom surface in a detachable manner. The culture vessel 2 is placed on the bottom surface of the lower cover 22. The bottom surface of the lower cover 22 may be placed on the floor surface.

[0059] The gas port 211A and the liquid port 211B are ports for exchanging fluid between the inside and outside of the cell peeling container 200. The gas port 211A and the liquid port 211B are formed from a material such as metal, resin, or plastic. A gas tube 212A is connected to one end of the gas port 211A. A gas pump 25A is connected to the other end of the gas port 211A via a tube. A liquid tube 212B is connected to one end of the liquid port 211B. A liquid pump 25B is connected to the other end of the liquid port 211B via a tube.

[0060] The gas tube 212A and the liquid tube 212B are tubes for exchanging fluid between the inside and outside of the cell detachment container 200. The gas tube 212A and the liquid tube 212B are formed, for example, from the same material as the supply tube 91. The length of the gas tube 212A is designed so that the tip of the gas tube 212A does not come into contact with the liquid L discharged from the nozzle 3. The length of the liquid tube 212B is designed so that the tip of the liquid tube 212B almost comes into contact with the bottom surface of the culture container 2.

[0061] The gas pump 25A and the liquid pump 25B are pumps that exchange fluid between the inside and outside of the cell detachment container 200. The gas pump 25A and the liquid pump 25B may send fluid into the inside of the cell detachment container 200 or may discharge fluid from the inside of the cell detachment container 200 under the control of the control device 10. The gas pump 25A sends or discharges any type of gas. The liquid pump 25B sends or discharges any type of liquid. The liquid pump 25B is an example of a sending section and a discharging section. It is also possible to add a liquid pump other than the liquid pump 25B, and use one of the two liquid pumps to send the liquid and the other to discharge the liquid.

[0062] 6 is a flowchart showing an example of the operation of the cell detachment device 1 according to the second embodiment. In addition to the same operation as in the first embodiment (see FIG. 2), the cell detachment device 1 according to the second embodiment discharges the liquid L from the culture container 2 according to a discharge command from a user.

[0063] (Step S21) First, the cell detachment device 1 determines whether or not a discharge instruction has been received from a user. Step S21 is the same as step S11. If a discharge instruction has been input (step S21-YES), the process proceeds to step S22. If a discharge instruction has not been input (step S21-NO), the process proceeds to step S23.

[0064] (Step S22) Next, the cell detachment device 1 discharges the liquid L into the culture container 2. Step S22 is similar to step S12.

[0065] (Step S23) Next, the cell detachment device 1 determines whether or not a discharge instruction has been received from the user. For example, the control device 10 determines whether or not the user has input a discharge instruction to the control device 10 using an input device. If a discharge instruction has been input (step S23-YES), the process proceeds to step S24. If a discharge instruction has not been input (step S23-NO), the process proceeds to step S25.

[0066] (Step S24) Next, the cell detachment device 1 discharges the liquid L from the culture container 2. Specifically, the control device 10 controls the liquid pump 25B to cause the liquid pump 25B to suck the liquid L in the culture container 2. The liquid pump 25B discharges the liquid L from the culture container 2 through the liquid port 211B and the liquid tube 212B (see FIG. 7).

[0067] (Step S25) Finally, the cell detachment device 1 determines whether or not an end instruction has been received from the user. For example, the control device 10 determines whether or not the user has input an end instruction to the control device 10 using an input device. If an end instruction has been input (step S25-YES), the cell detachment device 1 ends the series of operations. If an end instruction has not been input (step S25-NO), the process returns to step S21. That is, the control device 10 repeats each process from steps S21 to S24 until an end instruction is received from the user.

[0068] 7 is a schematic diagram showing an example of the operation of the cell separation device 1 according to the second embodiment. This diagram shows the cell separation container 200 in each state in chronological order.

[0069] 7(A), liquid L remains in the culture vessel 2. The cells C cultured in the culture vessel 2 are immersed in the liquid L. At this time, at least a part of the surface of the colony of the cells C may be immersed in the liquid L.

[0070] 7(B), liquid pump 25B discharges liquid L from culture vessel 2 through liquid port 211B and liquid tube 212B. Liquid pump 25B discharges liquid L vertically upward along direction D3. By discharging liquid L, the amount of liquid L in culture vessel 2 decreases, and the portion of the surface of cell C that is in contact with air appears and expands.

[0071] 7(C), the nozzle 3 sprays the liquid L onto the cells C in a state where almost no liquid L remains in the culture vessel 2. That is, the nozzle 3 sprays the liquid L onto the portion of the cells C that is in contact with the air and that has expanded due to the discharge of the liquid L, thereby effectively detaching the cells C from the culture vessel 2.

[0072] According to the second embodiment described above, the cell detachment device 1 discharges the liquid L remaining in the culture vessel 2 by the liquid pump 25B. The cell detachment device 1 ejects the liquid L from the nozzle 3 when almost no or no liquid L remains in the culture vessel 2. The cell detachment device 1 transmits the kinetic energy of the ejected liquid L directly to the cells C without passing through the liquid L covering the surfaces of the cells C, and therefore can effectively detach the cells C from the culture vessel 2.

[0073] Furthermore, the cell detachment device 1 can aseptically and automatically detach the cells C using the airtight cell detachment container 200. The cell detachment device 1 exchanges gas between the inside and outside of the cell detachment container 200 using a gas pump 25A and exchanges liquid using a liquid pump 25B. Therefore, the cell detachment device 1 can aseptically and automatically culture the cells C inside the cell detachment container 200. From this perspective, the cell detachment device 1 can also be referred to as a "cell culture device" or a "cell production device".

[0074] According to at least one of the embodiments described above, cells can be easily detached.

[0075] Although some embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, modifications, and combinations of embodiments can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents as described in the claims, as well as in the scope and spirit of the invention.

[0076] Regarding the above embodiment, the following supplementary notes are disclosed as one aspect and optional features of the invention.

[0077] (Appendix 1) A culture vessel for culturing cells; a discharge unit that is provided at a position not immersed in the liquid in the culture vessel and discharges a plurality of droplets toward a culture surface of the culture vessel to detach the cells from the culture surface; A cell detachment device comprising:

[0078] (Appendix 2) The ejection section may eject the plurality of droplets onto a portion of the surface of the cell that is in contact with air.

[0079] (Appendix 3) The plurality of droplets may have an average particle size of 100 μm or more and 500 μm or less.

[0080] (Appendix 4) The average velocity of the plurality of droplets may be between 5km / h and 50km / h.

[0081] (Appendix 5) The average kinetic energy of the droplets is 5.0×10 -13 J or above 6.3×10 -9 J or less is fine.

[0082] (Appendix 6) The total amount of the droplets ejected in one ejection may be 3.0 mL or less.

[0083] (Appendix 7) The ejection time of the plurality of droplets in one ejection may be 0.2 seconds or less.

[0084] (Appendix 8) The distance between the culture surface and the discharge portion may be 10 mm or more and 50 mm or less.

[0085] (Appendix 9) The cell detachment device may further include a storage section connected to the discharge section and configured to store liquid. The cell detachment device may further include a transmission unit that transmits kinetic energy to the liquid stored in the storage unit. The liquid stored in the storage section may be ejected as the plurality of droplets by the ejection section in a state in which the kinetic energy is transmitted by the transmission section.

[0086] (Appendix 10) The reservoir may be a hollow, cylindrical barrel having one end connected to the discharge portion. The transmission part may be a plunger that is mounted inside the barrel and moves the liquid toward the one end.

[0087] (Appendix 11) The cell detachment device may further include a discharge unit that discharges liquid from the culture vessel. The discharge unit may discharge liquid from the culture vessel to reduce the amount of liquid in the culture vessel, thereby expanding the contact area of ​​the surface of the cell with the air, and then the discharge unit may discharge the multiple droplets onto the expanded contact area.

[0088] (Appendix 12) Discharging a plurality of droplets onto a culture surface of a culture vessel from a position where the droplets are not immersed in the liquid in the culture vessel, thereby detaching the cells from the culture surface. Cell exfoliation methods. [Explanation of symbols]

[0089] 1. Cell detachment device 2 Culture container 3 Nozzles 4 Syringes 5 Pressing mechanism 6 Stops 7 Support Frame 8 Mounting device 9 Connectors 10 Control device 21 Top lid 22 Lower lid 25A Gas Pump 25B Liquid Pump 41 barrels 42 Flange 43 Plunger 51 Pressure plate 52 Arm 53 Foundation 61 Absorbent material 62 Support material 81 Pedestal 82 Plate 83 Legs 91 Supply Tube 92 Flow Sensor 100A, 100B Table 200 Cell Dissection Container 211A Gas Port 211B Liquid Port 212A Gas Tube 212B Liquid Tube 431 Rod 432 Pressure Plate 920 Measurements C cell d1,d2,d3 distance D1,D2,D3 direction L liquid

Claims

1. A culture vessel for culturing cells; a discharge unit that is provided at a position not immersed in the liquid in the culture vessel and discharges a plurality of droplets toward a culture surface of the culture vessel to detach the cells from the culture surface; A cell detachment device comprising:

2. The cell detachment device according to claim 1 , wherein the ejection unit ejects the plurality of droplets onto a portion of the surface of the cell that is in contact with air.

3. The cell detachment device according to claim 1 , wherein the average particle size of the plurality of droplets is 100 μm or more and 500 μm or less.

4. The cell detachment apparatus according to claim 1 , wherein an average speed of the plurality of droplets is 5 km / h or more and 50 km / h or less.

5. The average kinetic energy of the droplets is 5.0×10 －１３ J or above 6.3×10 －９ The cell detachment device according to claim 1 , wherein the length is J or less.

6. The cell detachment device according to claim 1 , wherein a total amount of the droplets ejected in one ejection is 3.0 mL or less.

7. The cell detachment device according to claim 1 , wherein the ejection time of the plurality of droplets in one ejection is 0.2 seconds or less.

8. The cell detachment device according to claim 1 , wherein the distance between the culture surface and the discharge portion is 10 mm or more and 50 mm or less.

9. A storage portion connected to the discharge portion and configured to store liquid; A transmission unit that transmits kinetic energy to the liquid stored in the storage unit; Further comprising: The liquid stored in the storage unit is discharged as the plurality of droplets by the discharge unit in a state in which the kinetic energy is transmitted by the transmission unit. The cell detachment device according to claim 1 .

10. the reservoir is a hollow, cylindrical barrel having one end connected to the discharge portion; The transmission part is a plunger that is mounted inside the barrel and moves the liquid toward the one end. The cell detachment device according to claim 9 .

11. A discharge part for discharging liquid from the culture vessel; Further comprising: The discharge unit discharges liquid from the culture vessel to reduce the amount of liquid in the culture vessel, thereby expanding a contact area between the surface of the cell and air, and then the discharge unit discharges the plurality of droplets onto the expanded contact area. The cell detachment device according to claim 1 .

12. a plurality of droplets are discharged from a position not immersed in the liquid in the culture vessel toward a culture surface of the culture vessel, thereby detaching the cells from the culture surface; Cell exfoliation methods.

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