Cell stripping device and cell stripping method

Abstract

A cell dissecting device according to an embodiment is provided with a culture container and a discharge unit. The culture vessel cultures cells. The ejection unit is provided at a position inside the culture container that is not immersed in the liquid, and ejects a plurality of liquid droplets onto the culture surface of the culture container, thereby peeling the cells from the culture surface.

Description

Technical Field

[0001] The embodiments disclosed in this specification and accompanying drawings relate to cell stripping apparatus and cell stripping methods. Background Technology

[0002] During cell culture, it is necessary to detach cells adhering to the bottom of the culture vessel. For example, technicians may manually and physically detach the cells using a cell scraper or pipette. However, manual operations carry risks such as microbial contamination and mechanical damage to the cells. Therefore, the detachment process must be performed carefully by skilled technicians while ensuring and maintaining a sterile environment.

[0003] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2011-155869 Summary of the Invention

[0004] The technical problem that the invention aims to solve One of the technical problems to be solved by the embodiments disclosed in this specification and accompanying drawings is the ease of cell dissection. However, the technical problems to be solved by the embodiments disclosed in this specification and accompanying drawings are not limited to the above-mentioned technical problems. It is also possible to identify other technical problems as corresponding to the effects of the structures shown in the embodiments described below.

[0005] Means for solving technical problems The cell stripping device of this embodiment includes a culture container and a spraying part. Cells are cultured in the culture container. The spraying part is located within the culture container at a position not immersed in liquid, and strips the cells from the culture surface by spraying multiple droplets onto the culture surface of the culture container.

[0006] Invention Effects According to the present invention, cells can be easily peeled off. Attached Figure Description

[0007] Figure 1 This is a schematic diagram illustrating a structural example of the cell stripping device according to the first embodiment.

[0008] Figure 2 This is a flowchart illustrating an example of the operation of the cell stripping device according to the first embodiment.

[0009] Figure 3 This is a schematic diagram illustrating an example of the operation of the cell stripping device according to the first embodiment.

[0010] Figure 4 This is a diagram showing the cell stripping results for each experimental condition in the first embodiment.

[0011] Figure 5 This is a schematic diagram illustrating a structural example of the cell stripping device according to the second embodiment.

[0012] Figure 6 This is a flowchart illustrating an example of the operation of the cell stripping device according to the second embodiment.

[0013] Figure 7 This is a schematic diagram illustrating an example of the operation of the cell stripping device according to the second embodiment. Detailed Implementation

[0014] Hereinafter, various embodiments will be described with reference to the accompanying drawings. In the following embodiments, parts marked with the same reference numerals are considered to perform the same operations, and repeated descriptions are omitted where appropriate.

[0015] (First Implementation) Figure 1 This is a schematic diagram showing a structural example of the cell peeling device 1 according to the first embodiment. The diagram is viewed horizontally and shows the cell peeling device 1. The cell peeling device 1 is a device for peeling cells. The cell peeling device 1 includes a culture container 2, a nozzle 3, a syringe 4, a pressing mechanism 5, a stop member 6, a support frame 7, a mounting member 8, a connecting member 9, and a control device 10.

[0016] Culture container 2 is a container for culturing cells C. Culture container 2 is formed of a material that prevents the liquid L adhering to its surface from permeating (impermeability). Examples of such materials include polyethylene, polypropylene, polycarbonate, polystyrene, and glass. Culture container 2 can be of any size and shape. For example, culture container 2 is a circular container with a diameter of 35 mm or more and less than 100 mm. Culture container 2 is an example of a culture section.

[0017] Cell C is a cell (adhesive cell) that is attached to the surface of culture vessel 2 via adhesive proteins. Examples of cell C include iPS (induced pluripotent stem) cells, epithelial cells, endothelial cells, synovial cells, cardiomyocytes, myoblasts, fibroblasts, and nerve cells. Cell C can be a single cell or a colony (cell cluster) composed of multiple cells.

[0018] Liquid L can be any type of liquid. Examples of liquid L include PBS (Phosphate Buffered Saline) and liquid culture media. Liquid L is preferably a liquid that does not chemically or physiologically damage cells C.

[0019] Nozzle 3 is a mechanical component that ejects liquid L. Nozzle 3 is formed of a material with high rigidity, such as metal. Nozzle 3 is physically connected to syringe 4 via connector 9. Preferably, the distance d1 between the tip of nozzle 3 and the bottom surface of culture container 2 is 10 mm or more and 50 mm or less. The tip of nozzle 3 is positioned on a straight line perpendicular to the bottom surface of culture container 2, passing through the center of the bottom surface. The bottom surface of culture container 2 functions as a culture surface for cell C adhesion. Nozzle 3 is positioned above culture container 2, i.e., at a position where it is not immersed in the liquid inside culture container 2. Nozzle 3 ejects liquid L as multiple droplets toward the bottom surface of culture container 2. Nozzle 3 is an example of an ejection part.

[0020] For nozzle 3, the number, location, size, and shape of the orifices (ejection orifices) used for ejection are designed in such a way that the conditions involved in the ejection of liquid L (ejection conditions) are at desired values. Examples of ejection conditions include the average droplet size, average velocity, and average kinetic energy of multiple droplets of liquid L, the ejection volume of liquid L at one time, and the ejection time. By appropriately setting the pressing parameters of the pressing mechanism 5 (described later), nozzle 3 sprays liquid L in a mist onto the cells C cultured in culture container 2 under the desired ejection conditions. That is, nozzle 3 detaches cells C from culture container 2 by spraying liquid L onto cells C. The droplets of liquid L ejected from nozzle 3 fly toward the bottom surface of culture container 2 and collide with cells C, thereby transferring their kinetic energy to cells C, achieving the detachment of cells C.

[0021] Nozzle 3 "sprays liquid L in a mist" means that nozzle 3 sprays liquid L "as multiple tiny droplets". The average particle size of these multiple tiny droplets is greater than 50 μm and less than 600 μm (refer to...). Figure 4 That is, "mist" does not refer to the shape of the spatial range in which the droplets are ejected (e.g., conical, linear), but rather to the state related to the size of the droplets.

[0022] Nozzle 3 can also detach cells C from culture vessel 2 by spraying liquid L in a single pass. In this case, the volume of liquid L sprayed in a single pass, i.e., the total amount of droplets sprayed in a single pass, is preferably 3.0 mL or less. The spraying time of liquid L is preferably 0.2 seconds or less. The spray angle of liquid L from nozzle 3 can be designed or adjusted to ensure that the sprayed liquid L contacts the entire surface (preferably more than 95% of the area) of cell C. Typically, nozzle 3 sprays liquid L in a conical shape. Nozzle 3 can also spray liquid L in a straight line.

[0023] Nozzle 3 can also spray liquid L onto the air-contact portion of the surface of cells C cultured in culture container 2. That is, nozzle 3 sprays liquid L onto the air-contact portion of the surface of cells C, rather than the portion immersed in liquid L (immersion portion). Nozzle 3 transfers the kinetic energy of the sprayed liquid L directly to cells C without passing through the liquid L covering the surface of cells C, thus enabling effective detachment of cells C from culture container 2.

[0024] Syringe 4 is a device that stores liquid L and transfers kinetic energy to liquid L. Syringe 4 is formed, for example, of the same material as culture container 2. Syringe 4 is positioned horizontally (in...) Figure 1 (The middle is arranged in a left-right direction). The syringe 4 has a barrel 41, a flange 42, and a plunger 43.

[0025] The cylinder 41 is a stationary component for storing liquid L. The cylinder 41 is a hollow, cylindrical container. One end of the cylinder 41 is connected to the nozzle 3 via a connector 9. The other end of the cylinder 41 is connected to the plunger 43 via a flange 42. The lower part of the cylinder 41 is physically supported by a support frame 7. Liquid L can be pre-stored in the cylinder 41 by the user, or it can be automatically stored using a known structure. Liquid L can also be repeatedly stored in the cylinder 41. The cylinder 41 is an example of a storage unit.

[0026] Flange 42 is a stationary component that joins the cylinder 41 and the plunger 43. Flange 42 allows the plunger 43 to move along the long axis of the cylinder 41 (in... Figure 1 The cylinder 41 and plunger 43 move in a left-right direction. The flange 42 is an example of the joint.

[0027] The plunger 43 is an actuating component 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 also be integrally formed. The plunger 43 is an example of a transmission part.

[0028] Rod 431 is a rod that moves the liquid L stored in cylinder 41. Rod 431 is a cylinder with a diameter matching the inner diameter of cylinder 41. One end of rod 431 is installed inside cylinder 41. The other end of rod 431 is connected to pressure plate 432. By moving in the same direction as the moving direction of pressure plate 432, rod 431 pushes liquid L toward nozzle 3. Rod 431 directly transmits kinetic energy to liquid L.

[0029] The pressure plate 432 is a plate that receives pressure from the pressing mechanism 5. By moving the pressure plate 432 in the direction of the pressure, the rod 431 moves in the same direction.

[0030] The pressing mechanism 5 is a mechanism for pressing the pressure plate 432. The pressing mechanism 5 is formed of a material with high rigidity, such as metal. The pressing mechanism 5 is electrically connected to the control device 10 via wires. Under the control of the control device 10, the pressing mechanism 5 adjusts the parameters (pressing parameters) involved in pressing the pressure plate 432 to achieve the desired ejection conditions. Examples of pressing parameters include pressing force and pressing time. The pressing mechanism 5 presses the pressure plate 432 under the adjusted pressing parameters. The pressing mechanism 5 has a pressing plate 51, an arm 52, and a base 53. The pressing mechanism 5 is an example of a pressing part.

[0031] The pressing plate 51 is an actuating component that presses down on the pressure plate 432. One surface of the pressing plate 51 faces one surface of the pressure plate 432. The distance d2 between one surface of the pressing plate 51 and one surface of the pressure plate 432 is designed to be an arbitrary value. The other surface of the pressing plate 51 is engaged with the arm 52.

[0032] The arm 52 is an actuating component that moves the pressing plate 51. The arm 52 moves the pressing plate 51 in the same direction by extending and retracting along the long axis of the cylinder 41.

[0033] The base 53 is a stationary component that physically supports the arm 52. The base 53 houses a portion of the arm 52. The base 53 is fixed to the upper surface of the mounting member 8.

[0034] The stop member 6 is a device that stops the movement of the pressing plate 51. The stop member 6 is disposed between the pressing plate 51 and the flange 42. The stop member 6 has a absorbing member 61 and a supporting member 62. The stop member 6 is an example of a stopping part.

[0035] The absorber 61 is a stationary component that absorbs pressure from the pressure plate 51. The absorber 61 is formed of a cushioning material. Examples of such materials include polyurethane, rubber, and gel. The distance d3 between the absorber 61 and the surface of the pressure plate 51 is designed to be larger than the distance d2 (d3>d2). The distance obtained by subtracting the distance d2 from the distance d3 (d3-d2) corresponds to the distance that the plunger 43 can move.

[0036] Support member 62 is a stationary component that physically supports absorber 61. Support member 62 is formed of a material such as a metal with high rigidity. Support member 62 is L-shaped and has a vertical orientation (in...) Figure 1 The device consists of a first part extending vertically and a second part extending horizontally. The height of the first part is designed so that it does not obstruct the movement of the plunger 43 while allowing the pressure plate 51 stop member to move. An absorber 61 is disposed on the upper part of the first part. The second part is fixed to the upper surface of the mounting member 8.

[0037] The support frame 7 is a stationary component that physically supports the cylinder 41. The support frame 7 adjusts the height of the mounted cylinder 41. The support frame 7 is formed of a material such as a metal with high rigidity. The support frame 7 is fixed to the upper surface of the mounting member 8. The support frame 7 is an example of a support component.

[0038] The mounting member 8 is a device for mounting the pressing mechanism 5, the stopping device 6, and the support frame 7. The mounting member 8 is formed of a material with high rigidity, such as metal. The mounting member 8 has a base 81, a plate 82, and legs 83. The mounting member 8 is an example of a mounting part.

[0039] The base 81 is a stationary component that supports the base 53. The height of the base 81 is adjusted. The height of the base 81 is designed so that the pressing plate 51 can press against the pressure plate 432. The base 81 is fixed to the upper surface of the plate 82.

[0040] Plate 82 is a stationary component that holds the stop member 6 and the support frame 7. One end of plate 82 and the other end are connected to the leg 83.

[0041] Leg 83 is a stationary component that adjusts the height of plate 82 from the floor surface. Leg 83 rests on the floor surface. The user of cell peeling device 1 can insert their fingers into the gap formed between plate 82 and the floor surface to lift plate 82 off the floor surface.

[0042] Connector 9 is a device that connects nozzle 3 and cylinder 41 to each other. Connector 9 has a supply pipe 91 and a flow sensor 92. Connector 9 is an example of a connection part.

[0043] The supply pipe 91 is a pipe that supplies liquid L from the cylinder 41 to the nozzle 3. The supply pipe 91 is made of silicone rubber, polyethylene, metal, etc. A flow sensor 92 may also be installed on a part of the supply pipe 91.

[0044] Flow sensor 92 is a sensor that measures the flow rate of liquid L through supply pipe 91. Flow sensor 92 is electrically connected to control device 10 via wires. Flow sensor 92 measures the flow rate of liquid L under the control of control device 10 and sends the measured flow rate value to control device 10. Control device 10 can also determine the set value of the pressing parameter based on the relationship between the set pressing parameter and the flow rate measured by flow sensor 92. For example, control device 10 measures the flow rate for each spray action based on multiple pressing parameters, thereby calculating a conversion formula between pressing parameters and flow rate. Control device 10 can also calculate the pressing parameter corresponding to the flow rate that achieves the desired spray conditions based on this conversion formula, and set the calculated pressing parameter as the new set value.

[0045] The control device 10 is a device that controls the overall operation of the cell stripping device 1. The control device 10 has at least one processor. A processor can be a CPU (Central Processing Unit), GPU (Graphics Processing Unit), Application Specific Integrated Circuit (ASIC), or programmable logic device (e.g., Simple Programmable Logic Device (SPLD), Complex Programmable Logic Device (CPLD), Field Programmable Gate Array (FPGA)). When the processor is a CPU, the CPU implements each function by reading and executing the control program stored in memory. When the processor is an ASIC, each function is directly assembled into the ASIC circuit as logic circuitry. The processor can be configured as a single circuit or by combining multiple independent circuits. The control device 10 is an example of a control unit.

[0046] The control device 10 can also be a general-purpose computer or other information processing device. The control device 10 can also possess the components of a general-purpose computer (e.g., processor, memory, input devices, output devices, communication devices). The control device 10 can also receive various instructions from the user (e.g., discharge instructions, discharge instructions, end instructions) through the input devices. The control device 10 can also display the measured flow rate from the flow sensor 92 to the user in a prescribed manner through the output devices.

[0047] Figure 2 This is a flowchart illustrating an example of the operation of the cell stripping device 1 according to the first embodiment. In this example, the cell stripping device 1 sprays liquid L into the culture container 2 according to a spray instruction from the user.

[0048] (Step S11) First, the cell stripping device 1 determines whether a dispensing instruction has been received from the user. For example, the control device 10 determines whether the user has input a dispensing instruction using an input device (e.g., mouse, keyboard, communication terminal). If a dispensing instruction has been input (Step S11 - Yes), the process proceeds to step S12. If no dispensing instruction has been input (Step S11 - No), the process returns to step S11. That is, the control device 10 remains on standby until a dispensing instruction is received from the user. Additionally, the dispensing instruction may also include the dispensing conditions related to the dispensing of liquid L.

[0049] (Step S12) Finally, the cell dissection device 1 sprays liquid L into the culture container 2. Specifically, the control device 10 controls the pressing mechanism 5 to cause the pressing plate 51 to press the pressure plate 432, and under the desired spraying conditions, sprays liquid L from the nozzle 3 into the culture container 2 (see reference). Figure 3 ).

[0050] Figure 3 This is a schematic diagram illustrating an operation example of the cell stripping 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 plate 432 and presses the pressure plate 432 along direction D2, which is the same as direction D1. The pressure plate 432 pushes out the liquid L stored in the cylinder 41 along direction D2 by moving the rod 431 in direction D2. The movement of the pressing plate 51 is stopped by contacting the absorbent 61.

[0051] Liquid L, propelled from cylinder 41, passes through supply pipe 91 and reaches nozzle 3. Nozzle 3 sprays the supplied liquid L into culture container 2 under desired spraying conditions. Nozzle 3 detaches cells C from culture container 2 by spraying liquid L into culture container 2. Specifically, the cells C detached by the spraying of liquid L are separated into individual cells (single units). Flow sensor 92 measures the flow rate of liquid L through supply pipe 91 and sends the measured flow rate 920 to control device 10.

[0052] Figure 4 This is a graph showing the cell stripping results under each experimental condition of the first embodiment. Tables 100A and 100B show the cell stripping results obtained by spraying liquid L onto cell C through nozzle 3 under three spraying conditions (average particle size, average velocity, and average kinetic energy). The experimental procedures performed to obtain these stripping results are shown below.

[0053] Human iPS cells (specifically P0 cells) were used as cell type C. A 35mm diameter circular container (35mm culture dish) was used as culture vessel 2. First, iPS cells and liquid culture medium were added to the circular container, and after the iPS cells aggregated, all the liquid culture medium was aspirated. Next, 1 mL of PBS was added to the circular container, and all the PBS was aspirated to wash the aggregates. Then, 400 μL of EDTA (ethyllenediamine tetraacetic acid) was added to the circular container, and the aggregates were incubated at 37°C for 10 minutes, after which all the EDTA was aspirated. Finally, PBS was added to the circular container, and all the PBS was aspirated to wash the aggregates again.

[0054] Next, under three spraying conditions (average particle size, average velocity, and average kinetic energy), the experimenter sprayed 1 mL of PBS from nozzle 3 onto the colonies, thereby detaching the colonies from the circular container. The experimenter measured the "survival rate" of the detached colonies. Furthermore, the experimenter visually observed the detachment status of the colonies from the circular container using a microscope. Based on the observations, the experimenter judged the presence or absence of "full-area detachment" of the detached colonies and the quality of the "evaluation."

[0055] The "survival rate" was specifically determined using the method described below. First, the experimenter randomly took 10 μL from 1 mL of the exfoliated cell suspension and mixed it with 10 μL of trypan blue to obtain a mixture. Next, the experimenter injected this mixture into an automated cell counter slide, inserted the slide into the automated cell counter (Bio-Rad, TC20 (registered trademark)), and used multifocal surface resolution to determine the cell number and survival rate.

[0056] The experimenter judged "complete peeling: present" as the condition where the colony was completely peeled off from the bottom of the circular container. If the "survival rate" of the peeled cells was above 80% and "complete peeling: present" was met, the experimenter judged it as "good".

[0057] Tables 100A and 100B highlight the records for which "Evaluation: Good". According to the highlighted records, the average particle size is 100 μm or more but less than 500 μm, the average velocity is 5 km / h or more but less than 50 km / h, and the average kinetic energy is 5.0 × 10⁻⁶. -13 J or higher and 6.3 × 10 -9 J and below. That is, when the above conditions are met under the three ejection conditions, it is possible to effectively detach iPS cell colonies from a circular container.

[0058] Furthermore, when PBS is sprayed onto the colonies while PBS remains in the circular container (i.e., the iPS cell colonies are immersed in PBS), the proportion of the total area of ​​the colony that is detached from the circular container (detachment area ratio) decreases. (i) With no PBS residue, the detachment area ratio is "98%". (ii) With 400 μL of PBS residue, the detachment area ratio is "45%". (iii) With 1 mL of PBS residue, the detachment area ratio is "5%". That is, it is preferable to fully drain the PBS from the circular container before detaching the colonies, thereby increasing the portion of the colony surface in contact with air.

[0059] That is, in the cell stripping apparatus 1 of the first embodiment, before the step (S12) of spraying liquid L into the culture container 2, the liquid volume in the culture container 2 can be reduced by discharging the liquid in the culture container 2 through a liquid port (not shown) driven by a liquid pump (not shown). In this case, the same structure as the liquid pump 25B and liquid port 211B of the second embodiment described later can be used as the liquid pump and liquid port, for example.

[0060] According to the first embodiment described above, the cell peeling device 1 peels off cells C by spraying liquid L from a nozzle 3 onto cells C cultured in a culture container 2. This allows the user to easily peel off cells C without complicated manual operations. Furthermore, the cell peeling device 1 has a nozzle 3 positioned where it does not immerse itself in the liquid L within the culture container 2. This allows the user to effectively peel off cells C using the impact of the droplets. Moreover, the cell peeling device 1 peels off cells C by spraying liquid L from the fixed nozzle 3. Therefore, the structure of the cell peeling device 1 can be simplified.

[0061] Furthermore, the cell stripping device 1 transfers the kinetic energy of the sprayed liquid L directly to the cells C without passing through the wall of the culture container 2. Therefore, the cell stripping device 1 can effectively strip cells C. In particular, the cell stripping device 1 can separate colonies of cell C into individual cells C (single-cell separation).

[0062] (Second Implementation) Figure 5 This is a schematic diagram showing a structural example of the cell peeling device 1 according to the second embodiment. The cell peeling device 1 of the second embodiment, except for the structures of the first embodiment (see reference...), Figure 1 In addition to the cell peeling container 200, gas pump 25A, and liquid pump 25B, it also includes a cell peeling container 200, a gas pump 25A, and a liquid pump 25B. The gas pump 25A and liquid pump 25B can also be electrically connected to the control device 10 via wires.

[0063] The cell peeling container 200 is a container used for peeling cells. The cell peeling container 200 is a container of a closed system. The cell peeling 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.

[0064] The upper cover 21 and the lower cover 22 are the lids that form the shell of the cell peeling container 200. The upper cover 21 and the lower cover 22 are formed of a material with high rigidity, such as a metal. The upper cover 21 and the lower cover 22 have a U-shaped cross-section, with a flat bottom surface and L-shaped sides. The upper cover 21 and the lower cover 22 are joined together with the sides of the upper cover 21 in contact with each other. Through this joining, the interior and exterior of the cell peeling container 200 are physically separated, maintaining an airtight seal. The upper cover 21 and the lower cover 22 can also be detachably joined together.

[0065] On the bottom surface of the upper cover 21, a gas port 211A, a liquid port 211B, and a nozzle 3 are disposed through the bottom surface. The nozzle 3 is disposed at the center of the bottom surface. The nozzle 3 may also be detachably disposed on the bottom surface. The bottom surface of the lower cover 22 is used to place the culture container 2. The bottom surface of the lower cover 22 may also be placed on a floor surface.

[0066] Gas port 211A and liquid port 211B are ports for exchanging fluid between the inside and outside of the cell peeling container 200. Gas port 211A and liquid port 211B are formed of materials such as metal, resin, and plastic. A gas pipe 212A is connected to one end of gas port 211A. A gas pump 25A is connected to the other end of gas port 211A via a pipe. A liquid pipe 212B is connected to one end of liquid port 211B. A liquid pump 25B is connected to the other end of liquid port 211B via a pipe.

[0067] Gas tube 212A and liquid tube 212B are tubes for exchanging fluid between the inside and outside of the cell exfoliation container 200. Gas tube 212A and liquid tube 212B are formed, for example, of the same material as the supply tube 91. The length of gas tube 212A is designed such that the tip of gas tube 212A does not contact the liquid L ejected from nozzle 3. The length of liquid tube 212B is designed such that the tip of liquid tube 212B almost contacts the bottom surface of culture container 2.

[0068] Gas pump 25A and liquid pump 25B are pumps that exchange fluid between the inside and outside of the cell peeling container 200. Gas pump 25A and liquid pump 25B can, under the control of control device 10, deliver fluid into and discharge fluid from the inside of the cell peeling container 200. Gas pump 25A delivers or discharges any type of gas. Liquid pump 25B delivers or discharges any type of liquid. Liquid pump 25B is an example of both a delivery unit and a discharge unit. Alternatively, a liquid pump different from liquid pump 25B can be added, with one of the two liquid pumps used for liquid delivery and the other for liquid discharge.

[0069] Figure 6 This is a flowchart illustrating an example of the operation of the cell peeling device 1 according to the second embodiment. The cell peeling device 1 of the second embodiment operates the same as that of the first embodiment (see [link]). Figure 2 In addition to the discharge instructions from the user, liquid L is discharged from culture container 2.

[0070] (Step S21) First, the cell stripping device 1 determines whether a dispensing instruction has been received from the user. Step S21 is the same as step S11. If a dispensing instruction has been input (step S21 - Yes), the process proceeds to step S22. If no dispensing instruction has been input (step S21 - No), the process proceeds to step S23.

[0071] (Step S22) Next, the cell peeling device 1 sprays liquid L into the culture container 2. Step S22 is the same as step S12.

[0072] (Step S23) Next, the cell stripping device 1 determines whether a discharge instruction has been received from the user. For example, the control device 10 determines whether 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 no discharge instruction has been input (Step S23 - No), the process proceeds to step S25.

[0073] (Step S24) Next, the cell stripping device 1 discharges liquid L from the culture container 2. Specifically, the control device 10 controls the liquid pump 25B to draw liquid L from the culture container 2. The liquid pump 25B discharges liquid L from the culture container 2 through the liquid port 211B and the liquid tube 212B (see...). Figure 7 ).

[0074] (Step S25) Finally, the cell peeling device 1 determines whether it has received an end instruction from the user. For example, the control device 10 determines whether 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 peeling device 1 ends a series of actions. If no end instruction has been input (Step S25 - No), the process returns to step S21. That is, the control device 10 repeats each process from S21 to S24 until an end instruction is received from the user.

[0075] Figure 7 This is a schematic diagram illustrating an operational example of the cell stripping device 1 according to the second embodiment. The diagram shows the cell stripping container 200 in various states along a time sequence.

[0076] like Figure 7 As shown in (A), liquid L remains in culture container 2. Cells C cultured in culture container 2 are immersed in liquid L. At this time, at least a portion of the surface of the cell C colonies can also be immersed in liquid L.

[0077] like Figure 7As shown in (B), liquid pump 25B discharges liquid L from culture vessel 2 through liquid port 211B and liquid pipe 212B. Liquid pump 25B discharges liquid L vertically upward along direction D3. With the discharge of liquid L, the amount of liquid L in culture vessel 2 decreases, and the portion of the surface of cell C in contact with air appears and expands.

[0078] like Figure 7 As shown in (C), nozzle 3 sprays liquid L onto cells C with almost no liquid L remaining in culture container 2. That is, nozzle 3 effectively detaches cells C from culture container 2 by spraying liquid L onto the portion of cells C in contact with air that expands due to the discharge of liquid L.

[0079] According to the second embodiment described above, the cell stripping device 1 discharges the liquid L remaining in the culture container 2 via the liquid pump 25B. The cell stripping device 1 sprays liquid L through the nozzle 3 when almost or completely free of liquid L remaining in the culture container 2. The cell stripping device 1 transfers the kinetic energy of the sprayed liquid L directly to the cell C without passing through the liquid L covering the surface of the cell C, thus enabling efficient stripping of the cell C from the culture container 2.

[0080] Furthermore, the cell peeling device 1 can sterilely and automatically peel off cells C using a cell peeling container 200 that is kept airtight. The cell peeling device 1 exchanges gases between the inside and outside of the cell peeling container 200 via a gas pump 25A and liquids via a liquid pump 25B. Therefore, the cell peeling device 1 can sterilely and automatically culture cells C inside the cell peeling container 200. From this perspective, the cell peeling device 1 is also referred to as a "cell culture device" or a "cell production device".

[0081] According to at least one of the embodiments described above, cells can be easily peeled off.

[0082] Several embodiments of the present invention have been described, but these embodiments are given by way of example and are not intended to limit the scope of the invention. These embodiments can be implemented in various other ways, and various omissions, substitutions, modifications, and combinations of embodiments are possible without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the scope of the invention as set forth in the claims and its equivalents.

[0083] Regarding the above-described embodiments, as an aspect of the invention and as an optional feature, the following notes are disclosed.

[0084] (Note 1) A cell peeling device, comprising: Culture containers for culturing cells; and The ejector is located in a position within the culture container that is not immersed in the liquid. It ejects multiple droplets toward the culture surface of the culture container, thereby detaching the cells from the culture surface.

[0085] (Note 2) Alternatively, the ejector may eject the plurality of droplets onto the air-contact portion of the cell's surface.

[0086] (Note 3) Alternatively, the average particle size of the plurality of droplets may be greater than 100 μm and less than 500 μm.

[0087] (Note 4) Alternatively, the average velocity of the multiple droplets may be above 5 km / h and below 50 km.

[0088] (Note 5) Alternatively, the average kinetic energy of the plurality of droplets may be 5.0 × 10⁻⁶. -13 J or higher and 6.3 × 10 -9 Below J.

[0089] (Note 6) Alternatively, the total amount of the multiple droplets ejected in a single spray is less than 3.0 mL.

[0090] (Note 7) Alternatively, the ejection time of the multiple droplets ejected at one time may be less than 0.2 seconds.

[0091] (Postscript 8) Alternatively, the distance between the culture surface and the ejection part can be 10 mm or more and 50 mm or less.

[0092] (Note 9) Alternatively, the cell stripping device may also include a storage section connected to the ejection section for storing liquid.

[0093] Alternatively, the cell stripping device may also include a transfer unit that transfers kinetic energy to the liquid stored in the storage unit.

[0094] Alternatively, the liquid stored in the storage section may be ejected from the ejection section as a plurality of droplets after the kinetic energy has been transferred through the transmission section.

[0095] (Postscript 10) Alternatively, the storage section may be a hollow, cylindrical body having one end connected to the ejection section.

[0096] Alternatively, the transfer unit may be a plunger installed inside the cylinder that moves the liquid toward one end.

[0097] (Postscript 11) Alternatively, the cell stripping device may also include a discharge section for discharging liquid from the culture container.

[0098] Alternatively, the discharge section may discharge liquid from the culture container, thereby reducing the liquid volume in the culture container. This expands the contact area between the cells and the air, and the ejection section then ejects the plurality of droplets onto the expanded contact area.

[0099] (Postscript 12) A cell stripping method, The cells are detached from the culture surface by spraying multiple droplets from a position inside the culture container that is not immersed in the liquid toward the culture surface of the culture container.

[0100] (Postscript 13) Alternatively, the cell stripping method may spray the plurality of droplets onto the air-contact portion of the cell surface.

[0101] (Postscript 14) Alternatively, the average particle size of the plurality of droplets may be greater than 100 μm and less than 500 μm.

[0102] (Postscript 15) Alternatively, the average velocity of the plurality of droplets may be greater than 5 km / h and less than 50 km / h.

[0103] (Postscript 16) Alternatively, the average kinetic energy of the plurality of droplets may be 5.0 × 10⁻⁶. -13 J or higher and 6.3 × 10 -9 Below J.

[0104] (Postscript 17) Alternatively, the total amount of the multiple droplets ejected in a single spray is less than 3.0 mL.

[0105] (Postscript 18) Alternatively, the ejection time of the multiple droplets ejected at one time may be less than 0.2 seconds.

[0106] (Postscript 19) Alternatively, the distance between the culture surface and the location may be more than 10 mm and less than 50 mm.

[0107] (Postscript 20) Alternatively, in the cell stripping method, the volume of liquid in the culture container is reduced by draining liquid from the culture container, thereby expanding the contact portion of the cell surface with air, and then spraying the plurality of droplets onto the expanded contact portion.

[0108] Explanation of reference numerals in the attached figures 1…cell peeling device, 2…culture container, 3…nozzle, 4…syringe, 5…pressing mechanism, 6…stop component, 7…support frame, 8…carrier, 9…connector, 10…control device, 21…top cover, 22…bottom cover, 25A…gas pump, 25B…liquid pump, 41…cylinder, 42…flange, 43…plunger, 51…pressing plate, 52…arm, 53…base, 61…absorbent, 62…support component, 81… …base, 82…plate, 83…leg, 91…supply pipe, 92…flow sensor, 100A, 100B…meter, 200…cell peeling container, 211A…gas port, 211B…liquid port, 212A…gas pipe, 212B…liquid pipe, 431…rod, 432…pressure plate, 920…measurement value, C…cell, d1, d2, d3…distance, D1, D2, D3…direction, L…liquid.

Claims

1. A cell peeling device, comprising: Culture containers for culturing cells; and The ejector is located in a position within the culture container that is not immersed in the liquid. It ejects multiple droplets toward the culture surface of the culture container, thereby detaching the cells from the culture surface.

2. The cell peeling device according to claim 1, wherein, The ejector sprays the plurality of droplets onto the air-contact portion of the cell's surface.

3. The cell peeling device according to claim 1 or claim 2, wherein, The average particle size of the plurality of droplets is greater than 100 μm and less than 500 μm.

4. The cell peeling device according to any one of claims 1 to 3, wherein, The average velocity of the multiple droplets is above 5 km / h and below 50 km / h.

5. The cell peeling device according to any one of claims 1 to 4, wherein, The average kinetic energy of the multiple droplets is 5.0 × 10⁻⁶. -13 J or higher and 6.3 × 10 -9 Below J.

6. The cell peeling device according to any one of claims 1 to 5, wherein, The total volume of the multiple droplets ejected in a single spray is less than 3.0 mL.

7. The cell peeling device according to any one of claims 1 to 6, wherein, The ejection time of the multiple droplets ejected in one go is less than 0.2 seconds.

8. The cell peeling device according to any one of claims 1 to 7, wherein, The distance between the culture surface and the ejection part is more than 10 mm and less than 50 mm.

9. The cell peeling device according to any one of claims 1 to 8, wherein, It also has: A storage unit, connected to the ejection unit, stores the liquid; and The transfer unit transfers kinetic energy to the liquid stored in the storage unit. The liquid stored in the storage section, having had its kinetic energy transferred by the transmission section, is ejected from the ejection section as a plurality of droplets.

10. The cell peeling device according to claim 9, wherein, The storage section is a hollow, cylindrical body having one end connected to the ejection section, and the delivery section is a plunger installed inside the cylinder that moves the liquid toward the one end.

11. The cell peeling device according to any one of claims 1 to 10, wherein, It also includes a discharge section that discharges liquid from the culture container. The discharge section discharges liquid from the culture container, reducing the liquid volume in the culture container. As a result, the ejection section sprays the plurality of droplets onto the enlarged contact portion of the cell surface, thereby expanding the contact portion.

12. A cell stripping method, comprising the following steps: The cells are detached from the culture surface by spraying multiple droplets from a position inside the culture container that is not immersed in the liquid toward the culture surface of the culture container.

13. The cell stripping method according to claim 12, wherein, The plurality of droplets are sprayed onto the air-contact portion of the cell's surface.

14. The cell stripping method according to claim 12 or claim 13, wherein, The average particle size of the plurality of droplets is greater than 100 μm and less than 500 μm.

15. The cell stripping method according to any one of claims 12 to 14, wherein, The average velocity of the multiple droplets is above 5 km / h and below 50 km / h.

16. The cell stripping method according to any one of claims 12 to 15, wherein, The average kinetic energy of the multiple droplets is 5.0 × 10⁻⁶. -13 J or higher and 6.3 × 10 -9 Below J.

17. The cell stripping method according to any one of claims 12 to 16, wherein, The total volume of the multiple droplets ejected in a single spray is less than 3.0 mL.

18. The cell stripping method according to any one of claims 12 to 17, wherein, The ejection time of the multiple droplets ejected in one go is less than 0.2 seconds.

19. The cell stripping method according to any one of claims 12 to 18, wherein, The distance between the culture surface and the location is more than 10 mm and less than 50 mm.

20. The cell stripping method according to any one of claims 12 to 19, wherein, By discharging liquid from the culture vessel, the volume of liquid in the culture vessel is reduced, thereby expanding the contact portion with air on the surface of the cells, and the plurality of droplets are sprayed out onto the expanded contact portion.

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