Cell detachment device and cell detachment method

Abstract

A cell detachment device according to an embodiment of the present invention comprises a culture vessel and a discharge part. The culture vessel is for culturing cells. The discharge part is provided at a position so 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.

Description

Cell detachment device and cell detachment method

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

[0002] During cell culture, cells adhering to the bottom of a culture vessel must be detached. For example, technicians manually and physically detach cells using a cell scraper or pipette. However, manual procedures involve risks such as microbial contamination and mechanical damage to cells. Therefore, the detachment process must be performed meticulously by skilled technicians in a sterile environment.

[0003] JP 2011-155869 A

[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 below can also be positioned as other problems.

[0005] A cell detachment device according to an embodiment includes a culture vessel and a discharge unit. The culture vessel is used to culture cells. The discharge unit is located in a position within the culture vessel that is not immersed in liquid, and discharges multiple droplets toward a culture surface of the culture vessel to detach the cells from the culture surface.

[0006] According to the present invention, cells can be easily detached.

[0007] Schematic diagram showing a configuration example of a cell detachment device according to the first embodiment. Flowchart showing an operation example of the cell detachment device according to the first embodiment. Schematic diagram showing an operation example of the cell detachment device according to the first embodiment. Diagram showing cell detachment results for each experimental condition according to the first embodiment. Schematic diagram showing a configuration example of a cell detachment device according to the second embodiment. Flowchart showing an operation example of the cell detachment device according to the second embodiment. Schematic diagram showing an operation example of the cell detachment device according to the second embodiment.

[0008] Hereinafter, each embodiment will be described with reference to the drawings. In the following embodiments, parts with the same reference numerals will be considered to perform the same operation, and duplicated descriptions will be omitted as appropriate.

[0009] (First embodiment) Fig. 1 is a schematic diagram showing an example of the configuration of a cell detachment device 1 according to the first embodiment. This diagram shows the cell detachment device 1 as viewed from the 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.

[0010] The culture vessel 2 is a vessel for culturing cells C. The culture vessel 2 is made of a material that does not allow the liquid L adhering to its surface to penetrate (is impermeable). Examples of such materials include polyethylene, polypropylene, polycarbonate, polystyrene, and glass. The culture vessel 2 can 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.

[0011] Cells C are cells (adherent cells) that adhere to the surface of the culture vessel 2 via adhesive proteins. Examples of cells C include iPS (induced pluripotent stem) cells, epithelial cells, endothelial cells, synovial cells, cardiomyocytes, myoblasts, fibroblasts, and neuroblasts. Cells C may be a single cell or a colony (cell mass) consisting of multiple cells.

[0012] 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.

[0013] The nozzle 3 is a mechanical component 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 positioned 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 cells C adhere. The nozzle 3 is positioned above the culture vessel 2, i.e., at a position that is not immersed in the liquid in the culture vessel 2. The nozzle 3 ejects the liquid L as multiple droplets toward the bottom surface of the culture vessel 2. The nozzle 3 is an example of an ejection unit.

[0014] The nozzle 3 is designed with a number, position, size, and shape of holes (discharge holes) for discharging the liquid L so that 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 velocity, and average kinetic energy of multiple droplets of the liquid L, and the amount and duration of a single discharge of the liquid L. By appropriately setting the pressure parameters of the pressure mechanism 5 (described later), 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. 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 transferring their kinetic energy to the cells C, thereby detaching the cells C.

[0015] 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, "in a mist" refers to the state related to the size of the droplets, rather than the shape of the spatial range into which the droplets are ejected (e.g., conical or linear).

[0016] The nozzle 3 may detach the cells C from the culture vessel 2 by discharging the liquid L once. In this case, the amount of the liquid L discharged in one discharge, i.e., the total amount of droplets discharged in one discharge, is preferably 3.0 mL or less. The discharge time of the liquid L in one discharge 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.

[0017] 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 air. That is, the nozzle 3 ejects the liquid L onto a portion of the surface of the cells C that is in contact with air, rather than onto a portion immersed in the liquid L (immersed portion). The nozzle 3 transfers the kinetic energy of the ejected liquid L directly to the cells C without passing through the liquid L that covers the surface of the cells C, thereby enabling the cells C to be effectively detached from the culture vessel 2.

[0018] The syringe 4 is an instrument that stores the liquid L and transmits kinetic energy to the liquid L. The syringe 4 is formed, for example, from the same material as the culture vessel 2. The syringe 4 is disposed horizontally (left-right direction in FIG. 1 ). The syringe 4 has a barrel 41, a flange 42, and a plunger 43.

[0019] 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 stored automatically using a known configuration. The liquid L may be stored in the barrel 41 repeatedly. The barrel 41 is an example of a storage unit.

[0020] 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 axis direction of the barrel 41 (the left-right direction in FIG. 1 ). The flange 42 is an example of a connecting portion.

[0021] 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 plate 432. The rod 431 and the pressure plate 432 may be integrally molded. The plunger 43 is an example of a transmission part.

[0022] 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 to the inside of the barrel 41. The other end of the rod 431 is connected to the pressure plate 432. The rod 431 pushes the liquid L toward the nozzle 3 by moving in the same direction as the movement direction of the pressure plate 432. The rod 431 directly transfers kinetic energy to the liquid L.

[0023] 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.

[0024] 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 that has 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 achieve the desired discharge conditions. Examples of the pressing parameters include pressing force and pressing time. The pressing mechanism 5 presses the pressure receiving plate 432 at 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.

[0025] The pressure plate 51 is an operating part that presses the pressure plate 432. One surface of the pressure plate 51 faces one surface of the pressure plate 432. The distance d2 between one surface of the pressure plate 51 and one surface of the pressure plate 432 is designed to be any value. The other surface of the pressure plate 51 is connected to the arm portion 52.

[0026] The arm 52 is a moving part that moves the pressure plate 51. The arm 52 expands and contracts along the longitudinal direction of the barrel 41, thereby moving the pressure plate 51 in the same direction.

[0027] The base 53 is a stationary component that physically supports the arm 52. The base 53 houses a part of the arm 52. The base 53 is fixed to the upper surface of the mounting device 8.

[0028] 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 absorber 61 and a support member 62. The stopper 6 is an example of a stop portion.

[0029] The absorbent material 61 is a stationary component that absorbs pressure from the pressure plate 51. The absorbent material 61 is made of a material with cushioning properties. Examples of such materials include urethane, rubber, and gel. The distance d3 between the absorbent material 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.

[0030] The support member 62 is a stationary part that physically supports the absorber 61. The support member 62 is formed of a material such as metal that has a relatively high rigidity. The support member 62 is L-shaped and has a first portion that extends vertically (up and down in FIG. 1 ) and a second portion that extends horizontally. 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 pressure plate 51. The absorber 61 is disposed above the first portion. The second portion is fixed to the upper surface of the mounting device 8.

[0031] 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 device 8. The support frame 7 is an example of a support portion.

[0032] The mounting device 8 is a device on which the pressing mechanism 5, the stopping device 6, and the support frame 7 are mounted. The mounting device 8 is made of a material such as metal that has a relatively high rigidity. The mounting device 8 has a base 81, a plate 82, and legs 83. The mounting device 8 is an example of a mounting portion.

[0033] 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.

[0034] The plate 82 is a stationary part 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.

[0035] The legs 83 are stationary components that adjust the height of the plate 82 from the floor. The legs 83 are placed on the floor. A user of the cell detachment device 1 can insert their fingers into the gap formed between the plate 82 and the floor to lift the plate 82 from the floor.

[0036] The connector 9 is a tool 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.

[0037] 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.

[0038] 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 under control of the control device 10 and transmits the measured flow rate to the control device 10. The control device 10 may determine a set value of the pressure parameter based on the relationship between the set pressure parameter and the flow rate measured by the flow rate sensor 92. For example, the control device 10 calculates a conversion formula between the pressure parameter and the flow rate by measuring the flow rate for each of the ejection operations using a plurality of pressure parameters. The control device 10 may calculate a pressure parameter corresponding to a flow rate that achieves the desired ejection conditions based on the conversion formula and determine the calculated pressure parameter as a new set value.

[0039] The control device 10 controls the overall operation of the cell detachment device 1. The control device 10 has at least one processor. The processor refers to a circuit such as a central processing unit (CPU), a graphics processing unit (GPU), an application-specific integrated circuit (ASIC), or 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 memory. When the processor is an ASIC, each function is directly incorporated as a logic circuit within the ASIC circuit. The processor may be configured as a single circuit or may be configured by combining multiple independent circuits. The control device 10 is an example of a control unit.

[0040] 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 (e.g., a discharge instruction, a discharge instruction, an end instruction) from a user via an input device. The control device 10 may present the flow rate measurement value from the flow rate sensor 92 to the user in a predetermined format via an output device.

[0041] 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 vessel 2 in accordance with a discharge command from the user.

[0042] (Step S11) First, 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 (e.g., a mouse, keyboard, or communication terminal). If a discharge instruction has been input (step S11-YES), the process proceeds to step S12. If a discharge instruction has not been input (step S11-NO), the process returns to step S11. In other words, the control device 10 waits until a discharge instruction is received from the user. Note that the discharge instruction may include discharge conditions related to the discharge of the liquid L.

[0043] (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 ).

[0044] 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 direction D1, thereby moving the pressing plate 51 along direction D1. The pressing plate 51 contacts the pressure-receiving plate 432 and presses the pressure-receiving plate 432 along direction D2, which is the same as direction D1. The pressure-receiving plate 432 moves the rod 431 in direction D2, thereby pushing out the liquid L stored in the barrel 41 along direction D2. The movement of the pressing plate 51 stops when it comes into contact with the absorbent material 61.

[0045] The liquid L extruded 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 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 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.

[0046] 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 of three types of ejection conditions (average particle size, average velocity, and average kinetic energy). The experimental procedures performed to obtain these detachment results are described below.

[0047] Human-derived iPS cells (particularly, P0 cells) were used as cells C. A circular vessel (35 mm dish) with a diameter of 35 mm was used as 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 the entire liquid medium. Next, the experimenter added 1 mL of PBS to the circular vessel and aspirated the entire PBS to wash the colonies. 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 the entire EDTA. Next, the experimenter added PBS to the circular vessel and aspirated the entire PBS to wash the colonies again.

[0048] 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 determined whether the detached colonies were "completely detached" and whether the "evaluation" was good or bad.

[0049] Specifically, the "viability" was measured using the following method. First, the experimenter randomly selected 10 μL from 1 mL of the detached cell suspension and mixed it with 10 μL of trypan blue to obtain a mixture. Next, the experimenter poured the mixture into an automated cell counter slide, inserted the slide into an automated cell counter (Bio-Rad, TC20®), and measured the cell count and viability using multi-focal plane analysis.

[0050] The experimenter judged the colony as "completely detached from the bottom of the circular container" when it was completely detached from the bottom. The experimenter judged the colony as "good" when the "survival rate" of the cells after detachment was 80% or more and "completely detached" when it was "completely detached."

[0051] 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. That is, when the three types of discharge conditions satisfy the above-mentioned conditions, colonies of iPS cells can be detached particularly effectively from the circular container.

[0052] Furthermore, when PBS was sprayed onto the colonies while PBS remained in the circular container (i.e., the iPS cell colonies were immersed in PBS), the percentage of the area detached from the circular container relative to the total area of ​​the colonies (detached area ratio) decreased. (i) When no PBS remained, the detached area ratio was 98%. (ii) When 400 μL of PBS remained, the detached area ratio was 45%. (iii) When 1 mL of PBS remained, the detached area ratio was 5%. In other words, it is desirable to sufficiently drain the PBS from the circular container prior to detaching the colonies, thereby increasing the area of ​​the colony surface that comes into contact with air.

[0053] 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, the liquid in the culture vessel 2 may be discharged through a liquid port (not shown) by driving a liquid pump (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 the liquid pump 25B and liquid port 211B in the second embodiment described below.

[0054] According to the first embodiment described above, the cell detachment device 1 detaches cells C by ejecting liquid L from the nozzle 3 onto cells C cultured in the culture vessel 2. This allows the user to easily detach cells C without the need for complicated manual work. Furthermore, the cell detachment device 1 has the nozzle 3 in a position that is not immersed in the liquid L in the culture vessel 2. This allows the user to effectively detach cells C by utilizing the action of droplet collision. Furthermore, the cell detachment device 1 detaches cells C by ejecting liquid L from the fixed nozzle 3. This allows the configuration of the cell detachment device 1 to be simplified.

[0055] 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).

[0056] Second Embodiment Fig. 5 is a schematic diagram showing a configuration example of a cell detachment device 1 according to a 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.

[0057] 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 lid 21, a lower lid 22, a gas port 211A, a liquid port 211B, a gas tube 212A, and a liquid tube 212B.

[0058] 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 made 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, with 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. This joining physically isolates the interior of the cell detachment container 200 from the outside and keeps it airtight. The upper lid 21 and the lower lid 22 may be joined to each other in a detachable manner.

[0059] A gas port 211A, a liquid port 211B, and a nozzle 3 are disposed on the bottom surface of the upper lid 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 lid 22. The bottom surface of the lower lid 22 may be placed on the floor surface.

[0060] The gas port 211A and the liquid port 211B are ports for exchanging fluids between the inside and outside of the cell detachment container 200. The gas port 211A and the liquid port 211B are formed from materials such as metal, resin, and 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.

[0061] The gas tube 212A and the liquid tube 212B are tubes that exchange 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.

[0062] 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 cell detachment container 200 or 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 delivery unit and a discharge unit. Note that a liquid pump other than the liquid pump 25B may be added, and one of the two liquid pumps may be used to send liquid and the other to discharge liquid.

[0063] 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 operations as those of the first embodiment (see FIG. 2), the cell detachment device 1 according to the second embodiment also discharges the liquid L from the culture vessel 2 in accordance with a discharge command from the user.

[0064] (Step S21) First, the cell detachment device 1 determines whether or not a discharge instruction has been received from the 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.

[0065] (Step S22) Next, the cell detachment device 1 discharges the liquid L into the culture vessel 2. Step S22 is the same as step S12.

[0066] (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.

[0067] (Step S24) Next, the cell detachment device 1 discharges the liquid L from the culture vessel 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 vessel 2. The liquid pump 25B discharges the liquid L from the culture vessel 2 through the liquid port 211B and the liquid tube 212B (see FIG. 7 ).

[0068] (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 of the processes from step S21 to S24 until an end instruction is received from the user.

[0069] 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.

[0070] 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 cells C may be immersed in the liquid L.

[0071] 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. As liquid L is discharged, the amount of liquid L in culture vessel 2 decreases, and the portion of the surface of cells C that comes into contact with air appears and expands.

[0072] 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.

[0073] According to the second embodiment described above, the cell detachment device 1 uses the liquid pump 25B to discharge the liquid L remaining in the culture vessel 2. 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 transfers the kinetic energy of the ejected liquid L directly to the cells C without passing through the liquid L that covers the surfaces of the cells C, thereby enabling the cells C to be effectively detached from the culture vessel 2.

[0074] Furthermore, the cell detachment device 1 can automatically and sterilely detach 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 automatically and sterilely culture 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."

[0075] According to at least one of the embodiments described above, cells can be easily detached.

[0076] Although several 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 within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims.

[0077] With respect to the above embodiment, the following supplementary notes are disclosed as one aspect and optional features of the invention.

[0078] (Supplementary Note 1) A cell detachment device comprising: a culture vessel for culturing cells; and a discharge unit disposed in a position not immersed in liquid within the culture vessel, which discharges a plurality of droplets toward a culture surface of the culture vessel, thereby detaching the cells from the culture surface.

[0079] (Supplementary Note 2) The ejection unit may eject the plurality of droplets onto a portion of the surface of the cell that is in contact with air.

[0080] (Note 3) The average particle size of the plurality of droplets may be 100 μm or more and 500 μm or less.

[0081] (Supplementary Note 4) The average speed of the plurality of droplets may be 5 km / h or more and 50 km / h or less.

[0082] (Supplementary Note 5) The average kinetic energy of the plurality of droplets is 5.0 × 10 －１３ J or above 6.3×10 －９ It may be J or less.

[0083] (Note 6) The total amount of the droplets ejected in one ejection may be 3.0 mL or less.

[0084] (Supplementary Note 7) The ejection time of the plurality of droplets in one ejection may be 0.2 seconds or less.

[0085] (Supplementary Note 8) The distance between the culture surface and the discharge portion may be 10 mm or more and 50 mm or less.

[0086] (Supplementary Note 9) The cell detachment device may further include a storage unit connected to the discharge unit and configured to store a liquid. The cell detachment device may further include a transmission unit configured to transmit kinetic energy to the liquid stored in the storage unit. The liquid stored in the storage unit may be discharged by the discharge unit as the plurality of droplets with the kinetic energy transmitted by the transmission unit.

[0087] (Supplementary Note 10) The reservoir may be a hollow cylindrical barrel having one end connected to the discharge portion, and the transfer portion may be a plunger attached inside the barrel and moving the liquid toward the one end.

[0088] (Supplementary Note 11) The cell detachment device may further include a discharge unit that discharges liquid from the culture vessel. The discharge unit may discharge the liquid from the culture vessel to reduce the amount of liquid in the culture vessel, thereby expanding a contact area of ​​the surface of the cells with air, and the discharge unit may then discharge the plurality of liquid droplets onto the expanded contact area.

[0089] (Supplementary Note 12) A cell detachment method, comprising 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.

[0090] (Supplementary Note 13) In the cell detachment method, the plurality of droplets may be ejected onto a portion of the surface of the cell that is in contact with air.

[0091] (Supplementary Note 14) The plurality of droplets may have an average particle size of 100 μm or more and 500 μm or less.

[0092] (Supplementary Note 15) The average speed of the plurality of droplets may be 5 km / h or more and 50 km / h or less.

[0093] (Supplementary Note 16) The average kinetic energy of the plurality of droplets is 5.0 × 10 －１３ J or above 6.3×10 －９ It may be J or less.

[0094] (Supplementary Note 17) The total amount of the droplets ejected in one ejection may be 3.0 mL or less.

[0095] (Supplementary Note 18) The ejection time of the plurality of droplets in one ejection may be 0.2 seconds or less.

[0096] (Supplementary Note 19) The distance between the culture surface and the position may be 10 mm or more and 50 mm or less.

[0097] (Supplementary Note 20) The cell detachment method may include discharging liquid from the culture vessel to reduce the amount of liquid in the culture vessel, thereby expanding the contact area of ​​the cell surface with air, and then ejecting the multiple droplets onto the expanded contact area.

[0098] DESCRIPTION OF SYMBOLS 1...Cell detachment device, 2...Culture vessel, 3...Nozzle, 4...Syringe, 5...Pressing mechanism, 6...Stopping device, 7...Support frame, 8...Placing device, 9...Connecting device, 10...Control device, 21...Upper cover, 22...Lower cover, 25A...Gas pump, 25B...Liquid pump, 41...Barrel, 42...Flange, 43...Plunger, 51...Pressing plate, 52...Arm, 53...Base, 61...Absorbing material, 62...Supporting material, 81...Pedestal , 82...plate, 83...leg, 91...supply tube, 92...flow rate sensor, 100A, 100B...table, 200...cell detachment container, 211A...gas port, 211B...liquid port, 212A...gas tube, 212B...liquid tube, 431...rod, 432...pressure receiving plate, 920...measurement value, C...cell, d1, d2, d3...distance, D1, D2, D3...direction, L...liquid

Claims

1. A cell detachment device comprising: a culture vessel for culturing cells; and a discharge unit that is provided in a position within the culture vessel that is not submerged in liquid, and that discharges a plurality of liquid droplets toward a culture surface of the culture vessel, thereby detaching the cells from the culture surface.

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

3. A cell peeling device as described in claim 1 or claim 2, wherein the average particle size of the multiple droplets is 100 μm or more and 500 μm or less.

4. A cell detachment device described in any one of claims 1 to 3, wherein the average speed of the multiple droplets is 5 km / h or more and 50 km / h or less.

5. The average kinetic energy of the droplets is 5.0 x 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. A cell detachment device as described in any one of claims 1 to 5, wherein the total amount of the multiple droplets ejected in one ejection is 3.0 mL or less.

7. A cell detachment device as described in any one of claims 1 to 6, wherein the ejection time of the multiple droplets in one ejection is 0.2 seconds or less.

8. A cell detachment device according to any one of claims 1 to 7, wherein the distance between the culture surface and the discharge portion is 10 mm or more and 50 mm or less.

9. A cell detachment device as described in any one of claims 1 to 8, further comprising: a storage section connected to the discharge section for storing liquid; and a transmission section for transmitting kinetic energy to the liquid stored in the storage section, wherein the liquid stored in the storage section is discharged by the discharge section as the multiple droplets with the kinetic energy transmitted by the transmission section.

10. The cell detachment device described in claim 9, wherein the storage portion is a hollow, cylindrical barrel having one end connected to the discharge portion, and the transmission portion is a plunger mounted inside the barrel and moving the liquid toward the one end.

11. A cell detachment device as described in any one of claims 1 to 10, further comprising: a discharge section that discharges liquid from the culture vessel, wherein the discharge section discharges liquid from the culture vessel to reduce the amount of liquid in the culture vessel, thereby expanding a contact area of ​​the surface of the cell with the air, and the discharge section then discharges the multiple droplets onto the expanded contact area.

12. A cell detachment method, comprising discharging a plurality of liquid droplets onto a culture surface of a culture vessel from a position that is not immersed in the liquid in the culture vessel, thereby detaching the cells from the culture surface.

13. The cell detachment method according to claim 12, wherein the plurality of droplets are ejected onto a portion of the surface of the cell that is in contact with air.

14. A cell detachment method as described in claim 12 or claim 13, wherein the average particle size of the multiple droplets is 100 μm or more and 500 μm or less.

15. A cell detachment method described in any one of claims 12 to 14, wherein the average speed of the multiple droplets is 5 km / h or more and 50 km / h or less.

16. The average kinetic energy of the droplets is 5.0 x 10 －１３ J or above 6.3×10 －９ The cell detachment method according to any one of claims 12 to 15, wherein the cell detachment length is J or less.

17. A cell detachment method described in any one of claims 12 to 16, wherein the total amount of the multiple droplets ejected in one ejection is 3.0 mL or less.

18. A cell detachment method according to any one of claims 12 to 17, wherein the ejection time of the multiple droplets in one ejection is 0.2 seconds or less.

19. A cell detachment method according to any one of claims 12 to 18, wherein the distance between the culture surface and the position is 10 mm or more and 50 mm or less.

20. A cell detachment method described in any one of claims 12 to 19, comprising discharging liquid from the culture vessel to reduce the amount of liquid in the culture vessel, thereby expanding the contact area of ​​the cell surface with air, and then ejecting the multiple droplets onto the expanded contact area.

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