Cell treatment device, cell treatment system and cell treatment method

Abstract

To provide a passaging culture technique for cells that enables more efficient production of higher-quality cells.SOLUTION: A cell treatment device 100 includes a culture controller 53 and a vibration controller 54. The culture controller 53 applies proteolytic enzymes to cultured cells to reduce adhesion. The vibration controller 54 adds a culture medium to a culture vessel containing cultured cells with reduced adhesion and applies physical stimulation, thereby detaching the cultured cells. The culture controller 53 cultures the cultured cells detached by the vibration controller 54 in a set culture condition, thereby performing cell passaging culture.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present invention relates to a cell processing device, a cell processing system, and a cell processing method. [Background technology]

[0002] 2. Description of the Related Art Conventionally, in the fields of medicine and biotechnology, techniques for culturing various types of cells according to purpose have been used. When culturing cells, subculture of the cells may be performed to obtain a desired number of cells. In cell subculture, in order to obtain a desired number of cells, it is necessary to maintain an appropriate cell density in the culture vessel, and in order to increase the number of cells by expanding the culture surface or the number of culture vessels, it has been necessary to recover and reseed the cells during subculture. For this reason, in cell subculture, it is generally considered that the detachment of cells from the culture surface using a protease (trypsin), centrifugation, suspension in a medium, and reseeding are essentially essential steps. Techniques for subculturing cells are described in, for example, Patent Document 1. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2004-097047 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, in cell subculture, the series of steps, which include detaching the cells from the culture surface using a proteolytic enzyme (trypsin), centrifuging, suspending them in medium, and reseeding, are labor-intensive processes that lead to reduced efficiency, hinder the automation of culture, and increase the risk of contamination. Although attempts have been made to omit or automate some of the above steps, a method for subculturing cells that can efficiently obtain high-quality cells has not yet been established.

[0005] An object of the present invention is to realize a cell subculture technique that can more efficiently obtain cells of higher quality. [Means for solving the problem]

[0006] In order to achieve the above object, a cell processing device according to one embodiment of the present invention comprises: A cell treatment device for culturing cells, comprising: A cell culture means for culturing the cultured cells to be treated, which are seeded in a culture vessel, in a set culture environment; an adhesive strength reducing means for reducing adhesive strength by applying a protease to the cultured cells; A physical stimulus applying means for adding a medium to the culture vessel containing the cultured cells with reduced adhesive strength, and applying a physical stimulus to the cultured cells to detach the cultured cells; Equipped with The cell culture means is characterized in that the cultured cells detached by the physical stimulus applying means are cultured in a set culture environment, thereby subculturing the cells. Effect of the Invention

[0007] According to the present invention, a cell subculture technique can be realized that can more efficiently obtain cells of higher quality. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a diagram showing the procedure of a cell treatment method according to one embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram showing the procedure of an adhesive strength reduction experiment. [Diagram 3] FIG. 1 is a schematic diagram showing the relationship between the time that a culture dish is left standing in an incubator and the cell detachment rate RT. [Figure 4] FIG. 1 is a schematic diagram showing the procedure of a cultured cell detachment experiment using physical stimulation. [Diagram 5] FIG. 1 is a schematic diagram showing the relationship between the vibration frequency and vibration time of forced vibration as a physical stimulus and the cell detachment rate RV. [Figure 6] FIG. 1 is a schematic diagram showing the procedure of a comparative experiment using the present invention and a conventional culture method. [Figure 7] FIG. 1 is a schematic diagram showing the viability of cells when cells are cultured by the present invention and a conventional culture method. [Figure 8] 1 is a schematic diagram showing a configuration example of a cell processing apparatus 100 to which the present invention is applied. [Figure 9] FIG. 2 is a schematic diagram showing the configuration of a variable-volume culture vessel 200. [Figure 10] 1 is a schematic diagram showing a usage form of the variable-volume culture vessel 200. FIG. [Figure 11] 2 is a schematic diagram showing the functional configuration of a control unit 130. FIG. [Figure 12] 3 is a flowchart showing the flow of a cell culture controlling process executed by the cell processing device 100. [Figure 13] FIG. 1 is a schematic diagram showing the system configuration of a cell processing system 300 configured by combining a plurality of devices. [Figure 14] 4 is a schematic diagram showing the functional configuration of a control device 410. FIG. [Figure 15] 13 is a flowchart showing the flow of a cell culture controlling process executed by the cell processing system 300. [Figure 16] 1A to 1C are schematic diagrams showing an example of steps of a cell culture method in which one medium region is provided in a variable-volume culture vessel 200, and multiple small regions are provided within the medium region. [Figure 17] FIG. 13 is a schematic diagram showing an example of steps of a cell culture method in which one medium region is provided in a variable-volume culture vessel 200, and multiple small regions are provided within the medium region, and different types of cells are cultured in the multiple small regions. [Figure 18] FIG. 13 is a schematic diagram showing an example of steps of a cell processing method in which the bottom surface of the small region is flat and a recess (semispherical depression) is formed in the bottom surface of the middle region to culture cultured cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. [Basic concept of the present invention] The present invention makes it possible to expand the cell culture volume while maintaining the cells on the culture surface by going through a step of reducing the adhesive force to the culture surface and a step of detaching the cells from the culture surface by vibration, without carrying out a series of steps such as detaching the cells from the culture surface using a proteolytic enzyme (e.g., trypsin), centrifugation, suspension in medium, and reseeding, when subculturing cells. This eliminates the need to completely detach the cells from the culture surface using the protease, thereby shortening the time for immersing the cells in the protease. Also, since there is no need to collect and reseede the cells, the culture volume can be expanded by expanding the culture vessel, etc. Furthermore, since the steps of collecting, centrifuging, suspending, and reseeding the cells are no longer necessary, the workload required for subculture of cells can be reduced. Therefore, a cell subculture technique can be realized that can more efficiently obtain higher quality cells. Specific embodiments of the present invention will now be described.

[0010] [Cell treatment method] FIG. 1 is a diagram showing the procedure of a cell treatment method according to one embodiment of the present invention. As shown in FIG. 1, when the cell treatment method according to this embodiment is carried out, a culture vessel capable of expanding the culture area from a small area to a large area is prepared. Then, in step S1 of the cell processing method, primary cultured cells are seeded in a small area of ​​the prepared culture vessel (cultured cell seeding step). At this time, since the cultured cells are seeded in a small area of ​​the culture vessel, a limited number of primary cultured cells can be seeded at a constant cell density.

[0011] Next, in step S2, the culture vessel is placed in a culture environment, and cells are cultured under predetermined conditions (cell culture step). The predetermined conditions for culturing cells can be determined according to the cells to be cultured. When culturing cells, the success of the culture depends on achieving a certain cell density or higher in the early stages of culture (i.e., the cells are not excessively sparse). Therefore, by seeding the cultured cells in a concentrated manner in a small area, the probability of successful cell culture can be increased compared to when culture is started in a large area.

[0012] In step S3, a protease (trypsin in this example) is supplied to the cultured area of ​​the small region where culture has been completed to reduce the adhesive force of the cultured cells (adhesion reducing step). At this time, the time for which the cultured cells are immersed in the protease is set to be shorter than when the cultured cells are detached using the protease. That is, in the adhesion reducing step of this embodiment, the adherent cells are not detached using the protease, but the proteolytic ability of the protease is used to reduce the adhesive force of the adherent cells to the adhesive surface (while maintaining adhesion).

[0013] In step S4, after the set time has elapsed, the protease is removed from the small area of ​​the culture area (protease removal step). At this time, since the time for immersing the cultured cells in the protease is set to be shorter than when the cells are detached, most of the cultured cells are in a state of adhering to the adhesion surface with reduced adhesive strength. Also, compared to when the cells are detached by the protease, damage to the cells caused by the action of the protease can be suppressed.

[0014] In step S5, a medium necessary for subculture is supplied (added) to the small area after removal of the protease (medium supply step), whereby the cultured cells with reduced adhesive strength are immersed in the medium.

[0015] In step S6, a physical stimulus (here, forced vibration) is applied to the culture vessel, and the cultured cells that have adhered to the culture medium with reduced adhesive strength are detached from the culture medium by the action of the physical stimulus (cell detachment step). The physical stimulus applied at this time may be any stimulus that exerts a force on the cultured cells, and may be forced vibration, or may be, for example, an impulse (impact force) or a fluid stimulus (mechanical action due to fluid pressure, etc.) that can act to detach the adhered cells. By detaching the cells by the action of a physical stimulus, damage to the cells can be suppressed compared to when the cells are detached using the action of a proteolytic enzyme (chemical action).

[0016] In step S7, the members separating the small regions of the culture vessel are removed, and the culture volume is expanded to a large region (culture volume expansion process). At this time, the medium in which the cultured cells detached in the cell detachment process are suspended flows out from the small region into the large region, and the culture region expands to the large region. Since the number of cultured cells increased by culturing in the small region expands to the large region, a certain level of cell density is maintained even after the medium expands to the large region, creating a high probability of successful cell culture. Furthermore, compared to the case of extracting cultured cells by centrifugation, the workload required for subculture can be reduced and efficiency can be increased. After step S7, steps S2 and onwards are repeated as necessary to expand the culture region to a desired size.

[0017] [Verification experiment] When carrying out the above-mentioned cell treatment method, it is necessary to suitably achieve a reduction in adhesive force of the cultured cells by a protease (trypsin) (adhesion force reducing step) and detachment of the cultured cells by physical stimulation (cell detachment step). The results of verifying these steps are described below. Here, the experiment was carried out under the following conditions. Cell type: Chinese hamster ovary cells (CHO) Culture vessel: 60mm dish Number of seeded cells: 5×10 5 pieces Culture period: 48 hours

[0018] [Adhesion reduction experiment] FIG. 2 is a schematic diagram showing the procedure of the adhesive force reduction experiment. As shown in FIG. 2, in the adhesion reduction experiment, first, a culture dish on which cultured cells were cultured was prepared. Next, the medium in the culture dish was removed, and trypsin (0.05%, 1 mL) was added.

[0019] After 30 seconds had elapsed, the culture dish was placed in an incubator and the cultured cells were immersed in trypsin for a predetermined time, which was shorter than the time for detaching the cultured cells. Next, the culture dish was removed from the incubator, and after 30 seconds, the trypsin was collected. T The tissue contains exfoliated cells.

[0020] Next, in order to detach the cultured cells remaining on the culture dish, trypsin was added to the culture dish, and the dish was allowed to stand in an incubator for 5 minutes. Finally, trypsin was collected from the culture dish. At this time, the culture dish contained N R The cells remain. Here, the percentage of cultured cells that were detached by immersing the cultured cells in trypsin to reduce their adhesive strength was defined as the cell detachment rate R T is defined as shown in equation (1). Cell detachment rate R T =N T / (N T +N R ) (1)

[0021] Figure 3 shows the relationship between the time the culture dish was left in the incubator and the cell detachment rate R T FIG. In FIG. 3, the cell detachment rate R when the culture dish was left in the incubator (cultured cells were immersed in trypsin) for 0.5, 1.0, 1.5, and 2.0 minutes was T The time for which the cultured cells were immersed in trypsin outside the incubator was 1.0 minute for all the samples.

[0022] According to Figure 3, when the time the culture dish was left in the incubator was increased from 1.5 to 2.0 minutes, the cell detachment rate R T It can be seen that there is a rapid increase. Therefore, under the above experimental conditions, it is considered appropriate to immerse the cultured cells in trypsin for 1.5 minutes in order to reduce adhesion, which is slightly shorter than the 2.0 minutes at which the cultured cells are rapidly detached.

[0023] [Detachment of cultured cells by physical stimulation] FIG. 4 is a schematic diagram showing the procedure of a cultured cell detachment experiment using physical stimulation. As shown in FIG. 4, in the experiment of detaching cultured cells by physical stimulation, first, a culture dish on which cultured cells were cultured was prepared. Next, the medium in the culture dish was removed, and trypsin (0.05%, 1 mL) was added.

[0024] After 30 seconds had elapsed, the culture dish was placed in an incubator and the cultured cells were immersed in trypsin for a predetermined time, which was shorter than the time for detaching the cultured cells. Next, the culture dish was removed from the incubator, and after 30 seconds had passed, the trypsin was collected and an amount of medium required for subculture was supplied (added).

[0025] Next, a physical stimulus (here, forced vibration) was applied to the culture dish in order to detach the cultured cells remaining on the culture dish. As a result, the cultured cells, whose adhesive strength had decreased, were detached by the action of the physical stimulus. Next, the medium was collected from the culture dish. V The tissue contains exfoliated cells.

[0026] Furthermore, in order to detach the cultured cells remaining on the culture dish, trypsin was added to the culture dish, and the dish was left standing in an incubator for 5 minutes. Finally, trypsin was collected from the culture dish. At this time, the culture dish contained N R The cells remain. Here, the cell detachment rate R is the percentage of cultured cells that have detached due to a physical stimulus applied to cultured cells with reduced adhesiveness. V is expressed as equation (2). Cell detachment rate R V =N V / (N V +N R ) (2)

[0027] Figure 5 shows the relationship between the vibration frequency and vibration time of the forced vibration as a physical stimulus and the cell detachment rate R V FIG. In FIG. 5, the cell detachment rate R V are shown respectively.

[0028] According to Fig. 5, when the vibration frequency was 500 Hz, the cell detachment rate R V The cell detachment rate R V It can be seen that reaches a maximum value. Therefore, under the above experimental conditions, it is considered appropriate to apply forced vibration with a vibration frequency of 500 Hz as a physical stimulus to realize detachment of cultured cells, and a vibration time of 1.0 minute is sufficient. In addition, since it is believed that appropriate parameters will vary depending on the type of culture vessel or the type of cells being cultured, it is desirable to obtain suitable parameters for each application of the present invention by performing a process similar to the above verification.

[0029] [effect] Next, the effects of the cell treatment method according to this embodiment will be described. Below, the effect of using the cell treatment method of this embodiment will be verified from the viewpoint of cell viability. [Comparative experiment] FIG. 6 is a schematic diagram showing the procedure of a comparative experiment using the present invention and a conventional culture method. As shown in Figure 6, Chinese hamster ovary cells were used here, and the seeding cell number was 5 × 10 5 A comparative experiment was conducted using this as the initial value. First, the conventional method requires a small cultivation area (25 cm 2 The cultured cells were seeded into a culture flask (100 ml) and cultured for 30 hours.

[0030] Then, the cells were detached from the culture surface using a proteolytic enzyme (trypsin), centrifuged, suspended in medium, and reseeded to obtain a medium-sized culture surface (75 cm 2 The medium was transferred to a culture flask (2.2 x 10 cm) to expand the culture volume. After culturing for 30 hours, the cells were detached from the culture surface using a protease (e.g., trypsin), centrifuged, suspended in a medium, and reseeded to increase the culture area to a large area (225 cm2). 2 The medium was transferred to a culture flask (2.2 x 10 cm) to expand the culture volume. Thereafter, the cells were cultured for a predetermined period of time (for example, 30 hours), and the cultured cells were collected.

[0031] On the other hand, in the cell treatment method of the present invention, the culture area in the culture vessel is small (25 cm 2 Cultured cells were seeded in the area (small area) of the plate and cultured for 30 hours. Then, according to the above-mentioned cell treatment method, the adhesive strength of the cultured cells was reduced using a protease (trypsin), and the cultured cells were detached by applying a physical stimulus, after which the frame that had formed the small area was removed, and the culture area was increased to a medium area (75 cm 2 The culture volume was expanded to the 1000-well region (middle region). After culturing for 30 hours, the adhesion of the cultured cells was reduced using a protease (trypsin), and the cultured cells were detached by applying physical stimulation. The frame that formed the middle region was then removed, and the culture area was enlarged (225 cm). 2 The culture volume was expanded to the region of 1000 x 1000 mm (large region). Thereafter, the cells were cultured for a predetermined period of time (for example, 30 hours), and the cultured cells were collected.

[0032] [Viable cell rate] FIG. 7 is a schematic diagram showing the viability of cells cultured by the present invention and a conventional culture method. As shown in FIG. 7, when cells were cultured using the conventional method and the cell treatment method of the present invention, the cell viability was close to 100% in both cases. That is, it is evident that when cells are cultured using the cell treatment method of the present invention, a viable cell rate similar to that achieved by conventional methods can be achieved.

[0033] [First embodiment] Next, a first embodiment of the present invention will be described. In this embodiment, an example in which the present invention is realized as a cell processing device will be described.

[0034] [Cell processing equipment] FIG. 8 is a schematic diagram showing a configuration example of a cell processing apparatus 100 to which the present invention is applied. As shown in FIG. 8, the cell processing apparatus 100 includes an incubator 110, a vibration unit 120, and a control unit . The incubator 110 has a chamber that forms a sealed culture environment, and is capable of controlling the temperature and humidity in the chamber, and has a function of ventilating the chamber when necessary. The incubator 110 controls the environment in the chamber according to the set culture conditions, and executes various processes for culturing cells. Furthermore, the stage on which the culture vessel is placed in the incubator 110 is supported by a vibration unit 120, and the operation of the vibration unit 120 imparts vibration (vertical vibration or horizontal vibration) to the placed culture vessel. In this embodiment, when cell culture processing is performed by the cell processing device 100, a variable-volume culture vessel 200 in which the volume of the cell culture region is variable is used as the culture vessel.

[0035] FIG. 9 is a schematic diagram showing the configuration of the variable-volume incubation vessel 200. As shown in FIG. FIG. 10 is a schematic diagram showing a mode of use of the variable-volume incubation vessel 200. As shown in FIG. As shown in FIGS. 9 and 10, the variable volume incubation vessel 200 includes a substrate 201, a sidewall member 202, an outer partition member 203, an inner partition member 204, and a top plate 205. The substrate 201 is a member (eg, a glass member) that constitutes the bottom plate of the variable volume culture vessel 200, and a substrate 201 having an area according to the maximum culture volume required for the variable volume culture vessel 200 is used.

[0036] The sidewall member 202 is a frame-shaped member (for example, a member made of PDMS (polydimethylsiloxane)) whose outer shape matches the plate surface of the substrate 201. By placing the sidewall member 202 on the substrate 201, a cell culture region (large region) with the substrate 201 as the bottom surface is formed within the frame of the sidewall member 202. Note that the sidewall member 202 is fixed to the substrate 201 during cell culture, and the substrate 201 and the sidewall member 202 are in close contact with each other to prevent liquid from leaking out.

[0037] The outer partition member 203 is a frame-shaped member (e.g., a member made of PDMS) whose outer shape matches the inner shape of the sidewall member 202 and has a square through-hole 203a in the center. The height of the outer partition member 203 is configured to match the height of the sidewall member 202. By placing the outer partition member 203 within the frame of the sidewall member 202, a cell culture region (middle region) with the substrate 201 as the bottom surface is formed within the frame of the outer partition member 203. Note that the outer partition member 203 is fixed to the substrate 201 during cell culture, and the substrate 201 and the outer partition member 203 are in close contact with each other to prevent liquid from leaking out.

[0038] The inner partition member 204 is a frame-shaped member (e.g., a member made of PDMS) whose outer shape matches the inner shape of the through-hole 203a of the outer partition member 203 and has a square through-hole 204a in the center. The height of the inner partition member 204 is configured to match the height of the side wall member 202. By placing the inner partition member 204 in the through-hole 203a of the outer partition member 203, a cell culture region (small region) with the substrate 201 as the bottom surface is formed within the frame of the inner partition member 204. Note that the inner partition member 204 is fixed to the substrate 201 during cell culture, and the substrate 201 and the inner partition member 204 are in close contact with each other to prevent liquid from leaking out.

[0039] The top plate 205 is a plate-like member (e.g., a member made of glass) whose outer shape matches the plate surface of the substrate 201, and is configured to cover as its top surface the sidewall member 202 that is installed with the substrate 201 as its bottom surface. A protrusion 205a is provided along one side of the sidewall member 202 on the lower surface of the top plate 205 (the surface that faces the substrate 201 when installed). The length of the protrusion 205a is formed to be equal to or less than the inner shape of the sidewall member 202, and when the top plate 205 is placed overlapping the sidewall member 202 so as to match in a top view, the outer periphery of the lower surface of the top plate 205 and the upper surface of the sidewall member 202 (the surface on the side not in contact with the substrate 201) are in close contact with each other, thereby blocking the inside of the frame of the sidewall member 202. On the other hand, when the top plate 205 is disposed at a position offset from the sidewall member 202 in a top view (for example, at a position where the protrusion 205a of the top plate 205 overlaps with the upper surface of the sidewall member 202 (such as the top of one side of the sidewall member 202)), a gap is formed between the lower surface of the top plate 205 and the upper surface of the sidewall member 202 (the surface on the side not in contact with the substrate 201), and the inside of the frame of the sidewall member 202 is ventilated with the outside. By utilizing such a configuration, the inside of the frame of the sidewall member 202 can be ventilated with the outside during cell culture, and the inside of the frame of the sidewall member 202 can be closed during forced vibration application.

[0040] Returning to Fig. 8, the vibration unit 120 generates vibrations (vertical or horizontal vibrations) by sound waves, and applies forced vibrations as physical stimuli to the culture stage on which the culture vessel is placed. Note that the vibration unit 120 can also be a device that generates vibrations by ultrasonic waves. Also, impulses (impact force) can be used as the vibrations applied to the culture stage.

[0041] The control unit 130 is configured with an information processing device such as a PC (Personal Computer) or a PLC (Programmable Logic Controller), and controls the entire cell processing device 100. For example, the control unit 130 controls the culture environment (temperature, humidity, ventilation state, etc.) in the incubator 110, and controls the vibration generated by the vibration unit 120 (vibration strength, time, etc.). In this embodiment, the control unit 130 realizes the following functional configuration by executing a control program using a processor.

[0042] FIG. 11 is a schematic diagram showing the functional configuration of the control unit 130. As shown in FIG. As shown in FIG. 11, the control unit 130 includes, as functional components, a parameter setting section 51, a sensor information acquiring section 52, a culture control section 53, and a vibration control section 54. The parameter setting unit 51 sets parameters for controlling the culture environment in the incubator 110 (such as set values ​​for temperature, humidity, or ventilation) and parameters for vibration control in the vibration unit 120 (such as set values ​​for vibration strength and time) in accordance with the operation of the operator. In addition to setting various parameters in accordance with the operation of the operator, the parameter setting unit 51 can also set parameters by reading a setting file in which set values ​​for various parameters are stored in advance.

[0043] The sensor information acquiring unit 52 acquires data representing detection results from various sensors (temperature sensor, humidity sensor, imaging sensor, etc.) that measure the inside of the chamber of the incubator 110. The type of sensor to be installed in the cell processing device 100 can be appropriately selected according to the type of data to be managed in the cell processing device 100. The detection results (captured images) of the imaging sensors can be used for various purposes, for example, to determine whether a culture vessel is installed in the incubator 110, to check the state of the cultured cells in the culture vessel (progress of cell culture, etc.), and to check the state of the culture vessel (whether it is sealed, ventilated, etc.).

[0044] The culture control unit 53 controls the culture environment in the incubator 110 by operating a heater, a humidifier, or the like according to the parameters for culture environment control set by the parameter setting unit 51. The culture control unit 53 also refers to data representing the detection results of various sensors acquired by the sensor information acquisition unit 52, and manages each step of the cell processing method executed in the cell processing device 100 (each step of cell culture in the incubator 110). For example, the culture control unit 53 detects that a culture container is placed in the incubator 110, determines that one of the steps of the cell processing method has been completed, and notifies the operator of these detection results or determination results (by sound, light, image, etc.). The vibration control unit 54 controls the vibration in the vibration applying unit 120 by vibrating the transducer of the vibration applying unit 120 in accordance with the vibration control parameters set by the parameter setting unit 51 .

[0045] [Operation of the cell processing device 100] Next, the operation of the cell processing apparatus 100 will be described. FIG. 12 is a flow chart showing the flow of the cell culture controlling process executed by the cell processing device 100. The cell culture control process is started in response to an instruction to execute the cell culture control process being input via the input unit of the control unit 130. Prior to the start of the cell culture control process, a culture vessel in which primary cultured cells are seeded in a small area is prepared (cultured cell seeding process).

[0046] When the cell culture control process is started, in step S11, the parameter setting unit 51 sets parameters for culture environment control in the incubator 110 (such as set values ​​for temperature, humidity, or ventilation) and parameters for vibration control in the vibration unit 120 (such as set values ​​for vibration strength and time). In step S12, the culture control unit 53 detects that a culture container (in its initial state with cultured cells seeded therein) is placed in the incubator 110 based on data representing the detection results of an imaging sensor that captures images of the inside of the chamber of the incubator 110 acquired by the sensor information acquisition unit 52 (data of the captured image inside the incubator 110).

[0047] In step S13, the culture environment in the incubator 110 is controlled by operating a heater, a humidifier, or the like according to the parameters for culture environment control set by the parameter setting unit 51. This completes cell culture in one culture area of ​​the variable-volume culture vessel 200 (cell culture step). In step S14, the culture control unit 53 notifies that the cell culture process has ended. In step S14, in response to the notification of the end of the cell culture process, the culture vessel is removed from the incubator 110. Then, a protease (trypsin) is supplied to the culture area where the cell culture process has ended, and after a set time has elapsed (adhesive strength reducing step), the protease (trypsin) is removed (degradative enzyme removing step). Then, a culture medium required for subculture is supplied (added) to the culture area of ​​the culture vessel (culture medium supplying step), and the culture vessel is subjected to the next step. At this time, the cultured cells in the culture vessel are in a state of reduced adhesive strength.

[0048] In step S15, the culture control unit 53 detects that the culture container (after the adhesive force has decreased) is placed in the incubator 110 based on data representing the detection results of the imaging sensor that captures images of the inside of the chamber of the incubator 110 acquired by the sensor information acquisition unit 52 (data of the captured image inside the incubator 110). In step S16, the vibration control unit 54 generates vibrations (vertical or horizontal vibrations) using sound waves, and applies forced vibrations as physical stimuli to the culture stage on which the culture vessel is placed (cell detachment step). As a result, the cultured cells, whose adhesive strength has been reduced, are detached by the action of the forced vibrations.

[0049] In step S17, the culture control unit 53 notifies the completion of the cell detachment process and accepts an operation to expand the culture area in the culture vessel. In step S17, in response to the notification of the completion of the cell detachment process, the culture vessel is removed from the incubator 110. Then, the member that separates the current culture area of ​​the culture vessel is removed, and the culture area is expanded to a larger area (i.e., the culture capacity is expanded) (culture capacity expansion process). At this time, the medium in which the cultured cells are suspended has flowed out into the expanded culture area. This culture vessel is then provided for the next process.

[0050] In step S18, the culture control unit 53 controls the culture environment in the incubator 110 by operating a heater, a humidifier, or the like, according to the parameters for culture environment control set by the parameter setting unit 51. This completes the subculture of cells in the expanded culture area in the variable-capacity culture vessel 200 (cell culture process in the expanded culture area).

[0051] In step S19, the culture control unit 53 notifies the completion of the cell culture process (subculture) in the enlarged culture area. In step S19, in response to the notification of the completion of the cell culture process in the enlarged culture area, the culture vessel is removed from the incubator 110. If further subculture is to be performed, a protease (trypsin) is supplied to the culture area where the cell culture process in the enlarged culture area has been completed, and after a set time has elapsed (adhesive strength reducing step), the protease (trypsin) is removed (degradative enzyme removing step). Then, a medium required for subculture is supplied (added) to the culture area of ​​the culture vessel (medium supplying step), and the culture vessel is subjected to the next step. At this time, the cultured cells in the culture vessel are in a state of reduced adhesive strength.

[0052] In step S20, the culture control unit 53 determines whether or not an instruction to end the cell culture controlling process has been issued. If the end of the cell culture controlling process has not been instructed, the result of the determination in step S20 is NO, and the process proceeds to step S15. On the other hand, when an instruction to end the cell culture controlling process is given, the result of the determination in step S20 is YES, and the cell culture controlling process ends. When the cell culture control process is completed, the culture vessel is appropriately given a physical stimulus, for example, to recover the cultured cells.

[0053] As described above, when subculturing cells, the cell processing device 100 in this embodiment cultures primary cultured cells, then performs a process to reduce the adhesive strength of the cultured cells in the culture vessel using a protease (trypsin, etc.), adds a medium to the cultured cells with reduced adhesive strength, and then detaches the cultured cells by applying a physical stimulus (forced vibration, etc.).Then, the cell processing device 100 expands the culture volume in the same culture vessel with the expanded culture area and performs subculturing. Therefore, compared to the case where cells are detached using a protease, damage to cells caused by the action of the protease can be suppressed, and compared to the case where cultured cells are removed by centrifugation or the like, the workload required for subculture can be reduced and the efficiency can be increased. Therefore, the cell processing apparatus 100 of the present embodiment can realize a cell subculture technique that can more efficiently obtain higher quality cells.

[0054] [Second embodiment] Next, a second embodiment of the present invention will be described. In the first embodiment, the present invention has been described as being realized as a single cell processing device 100, but the present invention can also be realized as a system combining multiple devices. For example, by realizing the present invention as a system combining multiple devices, each step of the cell processing method can be automated and mass production of cultured cells can be facilitated.

[0055] [System configuration of cell processing system 300] FIG. 13 is a schematic diagram showing the system configuration of a cell processing system 300 configured by combining a plurality of devices. 13, the cell processing system 300 includes a transport device 310, a transfer device 320, a cultured cell supplying device 330, a culture medium supplying device 340, a culture medium removing device 350, a protease supplying device 360, a protease removing device 370, a vibration applying unit 380, a culture volume managing device 390, an incubator 400, and a control device 410. When the cell processing system 300 performs cell culture processing, a variable-volume culture vessel 200 in which the volume of the cell culture region is variable is used as the culture vessel, as in the above-described embodiment.

[0056] The transport device 310 is a device on which the culture vessel to be treated is placed and transported, and is constituted by, for example, a roller conveyor, a belt conveyor, etc. When treatments such as supplying or removing a culture medium or a protease are performed on the transport path of the transport device 310, the culture vessel may be temporarily held by a mechanism such as an auxiliary arm at the position where such treatments are performed, and the treatments may be performed with the culture vessel stopped. The transfer device 320 transfers a culture vessel from a position outside the line of the transport device 310 to the transport device 310, or transfers a culture vessel placed on the transport device 310 to a position outside the line of the transport device 310. The transfer device 320 can be configured by a handling device such as a robot hand, for example.

[0057] The cultured cell supplying device 330 supplies a prescribed amount of medium in which cultured cells (primary generation) are suspended to the culture container placed on the transport device 310. The medium supplying device 340 supplies a necessary amount of medium for subculture to a predetermined position in the transporting device 310 (the position where the subculture is performed). The culture medium removal device 350 removes unnecessary culture medium from within the culture vessel at a predetermined position in the transport device 310 (the position where subculture is performed).

[0058] The protease supplying device 360 ​​supplies a protease (trypsin, etc.) to a predetermined position in the transport device 310 (the position where subculture is performed) for reducing the adhesive force of the cultured cells in the culture vessel. The protease removal device 370 removes unnecessary protease (trypsin, etc.) from within the culture vessel at a predetermined position in the transfer device 310 (the position where subculture is performed).

[0059] The vibration applying unit 380 applies a physical stimulus to the culture vessel holding the cultured cells whose adhesive strength has been reduced by the protease, thereby detaching the cultured cells. In this embodiment, the vibration applying unit 380 uses the vibration generated during transportation in the transport device 310 as the forced vibration. For this reason, the vibration applying unit 380 is configured such that a part of the transport section in the transport device 310 where the subculture is performed is allocated as an area for applying the forced vibration.

[0060] With this configuration, since there is no need to provide a vibration applying device or other vibration applying equipment separately, the cultured cells can be detached more efficiently at lower cost. Note that the transport section of the transport device 310 assigned as the vibration applying section 380 may be configured to have a larger vibration during transport than other sections (such as a configuration with a difference in height in the vertical direction). The culture volume management device 390 is a device that performs a process of expanding the culture region of the variable volume culture vessel 200, and can be configured by, for example, a handling device such as a robot hand.

[0061] The incubator 400 is placed at a predetermined position on the transport device 310 (a position where processes requiring environmental control, such as cell culture and treatment with proteolytic enzymes, are performed). The incubator 400 has a chamber that forms a set culture environment, and is capable of controlling the temperature and humidity in the chamber, and has a function of ventilating the chamber when necessary. The incubator 400 can be placed on the line of the transport device 310, or can be placed at a predetermined position outside the line of the transport device 310 (such as a position adjacent to the line). In this case, the transfer device 320 transfers the culture vessel between the line of the transport device 310 and the incubator 400.

[0062] The control device 410 is configured with an information processing device such as a PC or a PLC, and controls the entire cell processing system 300. That is, the control device 410 controls the operations of the transport device 310, the transfer device 320, the cultured cell supplying device 330, the culture medium supplying device 340, the culture medium removing device 350, the protease supplying device 360, the protease removing device 370, the vibration applying unit 380, the culture volume managing device 390, and the incubator 400.

[0063] One or more of the transfer device 320, cultured cell supply device 330, culture medium supply device 340, culture medium removal device 350, protease supply device 360, protease removal device 370, vibration applying unit 380, culture volume management device 390, and incubator 400 are installed at predetermined positions of the transport device 310 as necessary. Various sensors are installed in each part of the cell processing system 300 as necessary. For example, the cell processing system 300 may include a sensor for detecting the transport speed of the transport device 310, an imaging sensor for imaging each part of the transport device 310, and various sensors (such as a temperature sensor, a humidity sensor, and an imaging sensor) for measuring the inside of the chamber of the incubator 400. The cell processing system 300 may also be equipped with position sensors (encoders, etc.) that detect the positions of the nozzles provided on the cultured cell supplying device 330, the culture medium supplying device 340, the culture medium removal device 350, the protease supplying device 360 ​​and the protease removal device 370, and the positions of the moving parts (robot hands, etc.) of the transfer device 320 and the culture volume management device 390. In this embodiment, the control device 410 realizes the following functional configuration by executing a control program using a processor.

[0064] FIG. 14 is a schematic diagram showing the functional configuration of the control device 410. As shown in FIG. As shown in FIG. 14, the control device 410 has, as its functional configuration, a parameter setting unit 451, a sensor information acquisition unit 452, an imaging control unit 453, a cell supply control unit 454, a culture medium supply control unit 455, a culture medium removal control unit 456, a protease supply control unit 457, a protease removal control unit 458, a culture volume control unit 459, a culture control unit 460, and a transport control unit 461.

[0065] The parameter setting unit 451 reads a setting file in which set values ​​of various parameters are stored in advance, and thereby sets various parameters for determining the operation of the cell processing system 300. For example, the parameter setting unit 451 sets a parameter representing the transport speed of the transport device 310, parameters determining the operation timing of the transfer device 320, the cultured cell supplying device 330, the culture medium supplying device 340, the culture medium removing device 350, the protease supplying device 360, the protease removing device 370, and the culture volume managing device 390, and parameters for controlling the culture environment in the incubator 400 (such as set values ​​for temperature, humidity, or ventilation).

[0066] The sensor information acquiring unit 452 acquires data representing detection results from various sensors installed in each part of the cell processing system 300. The imaging control unit 453 controls imaging by imaging sensors installed in each part of the cell processing system 300 , and causes data of captured images required for managing the operation of the cell processing system 300 to be acquired. The cell supply control unit 454 causes the cultured cell supplying device 330 to supply a medium in which cultured cells are suspended to the culture container to be treated.

[0067] The culture medium supply control unit 455 causes the culture medium supply device 340 to supply a necessary amount of culture medium for subculture to the culture vessel to be treated. The culture medium removal control unit 456 causes the culture medium removal device 350 to remove unnecessary culture medium from within the culture vessel to be treated. The protease supply control unit 457 controls the protease supply device 360 ​​to supply a protease (trypsin, etc.) for reducing the adhesive force of the cultured cells in the culture vessel.

[0068] The protease removal control unit 458 controls the protease removal device 370 to remove unnecessary protease (trypsin, etc.) in the culture vessel to be treated. The culture volume control unit 459 causes the culture volume management device 390 to execute a process for enlarging the culture region of the variable volume culture vessel 200 in which subculture is performed (for example, an operation for removing the inner partition member 204 that forms the small region). The culture control unit 460 controls the culture environment in the incubator 400 by operating a heater, a humidifier, or the like according to the parameters of the culture environment control set by the parameter setting unit 451. In this embodiment, since a plurality of incubators 400 are installed corresponding to the subculture of cells, the culture control unit 460 controls each incubator 400 individually according to the parameters of the culture environment control set for each incubator 400.

[0069] The transport control unit 461 controls the transport operation of the transport device 310, and controls the start and stop of transport and the transport speed of the transport device 310. If the transport device 310 is configured such that the transport speed can be changed for each section of the transport path, the transport control unit 461 can control the transport operation for each section of the transport device 310, and control the start and stop of transport and the transport speed of the transport device 310.

[0070] [Operation of cell processing system 300] Next, the operation of the cell processing system 300 will be described. FIG. 15 is a flow chart showing the flow of the cell culture control process executed by the cell processing system 300. The cell culture controlling process is started in response to an instruction to execute the cell culture controlling process being input via the input unit of the control device 410.

[0071] When the cell culture control process is started, in step S31, the parameter setting unit 451 reads a setting file in which the set values ​​of various parameters are stored in advance, thereby setting various parameters for determining the operation of the cell processing system 400. This defines the operation for subculture of cells in the entire cell processing system 300. In step S32, the imaging control unit 453 detects that the culture vessel to be processed has been placed at the first position (initial position for seeding cultured cells) in the transport device 310, based on data of the captured image captured by the imaging sensor.

[0072] In step S33, the cell supply control unit 454 supplies a medium in which cultured cells are suspended into a culture vessel installed at a first position by the cultured cell supply device 330. The culture vessel that has been subjected to the process of step S33 is transported by the transport operation of the transport device 310 to the position of the first incubator 400 for culturing the primary cultured cells. In step S34, the culture control unit 460 controls the culture environment in the first incubator 400 by operating a heater, a humidifier, or the like, according to the parameters of the culture environment control set by the parameter setting unit 451. At this time, the culture vessel transported by the transport device 310 is placed in the first incubator 400, and after the culture based on the parameters of the culture environment control set by the parameter setting unit 451 is performed, the culture vessel in which the primary cultured cells are cultured is transported from the first incubator 400 to the outside.

[0073] In step S35, the culture medium removal control unit 456 controls the culture medium removal device 350 to remove unnecessary culture medium in the culture vessel that has been transported from the first incubator 400 to the outside. In step S36, the protease supply control unit 457 supplies a protease (trypsin, etc.) for reducing the adhesive force of the cultured cells to the culture vessel from which unnecessary medium has been removed by the protease supply device 360. The culture vessel that has been subjected to the process of step S35 is transported by the transport operation of the transport device 310 to the position of the second incubator 400 for reducing the adhesive force of the cells.

[0074] In step S37, the culture control unit 460 controls the culture environment in the second incubator 400 by operating a heater, a humidifier, or the like, according to the parameters of the culture environment control set by the parameter setting unit 451. At this time, the culture vessel transported by the transport device 310 is placed in the second incubator 400, and after a protein decomposition process based on the parameters of the culture environment control set by the parameter setting unit 451 is performed, the culture vessel containing the cultured cells with reduced adhesive force is transported from the second incubator 400 to the outside. In step S38, the protease removal control unit 458 causes the protease removal device 370 to remove unnecessary proteases (trypsin, etc.) from within the culture vessel containing the cultured cells whose adhesive strength has been reduced.

[0075] In step S39, the culture medium supply control unit 455 controls the culture medium supply device 340 to supply a necessary amount of culture medium for subculture into the culture vessel containing the cultured cells whose adhesive force has been reduced. In step S40, the transport control unit 461 applies forced vibration to the culture vessel containing the cultured cells with reduced adhesive strength by the vibration applying unit 380. In this embodiment, as described above, in the transport device 310, a part of the transport section where the subculture is performed is allocated as an area for applying forced vibration, so that the culture vessel is transported to the transport device 310, whereby the cultured cells with reduced adhesive strength are detached.

[0076] In step S41, the culture volume control unit 459 executes a process of expanding the culture area of ​​the culture vessel (variable-capacity culture vessel 200) in which subculture is performed by the culture volume management device 390. As a result, the inner partition member 204 of the variable-capacity culture vessel 200 is removed, and the culture area is expanded from a small area to a medium area. The culture vessel that has been subjected to the process of step S41 is transported by the transport operation of the transport device 310 to the position of the third incubator 400 for the first subculture. In step S42, the culture control unit 460 controls the culture environment in the third incubator 400 by operating a heater, a humidifier, or the like according to the parameters of the culture environment control set by the parameter setting unit 451. At this time, the culture vessel transported by the transport device 310 is placed in the third incubator 400, and after the culture based on the parameters of the culture environment control set by the parameter setting unit 451 is performed, the culture vessel in which the first subculture has been performed is transported from the third incubator 400 to the outside.

[0077] In step S43, the culture medium removal control unit 456 controls the culture medium removal device 350 to remove unnecessary culture medium in the culture vessel that has been transported from the third incubator 400 to the outside. In step S44, the protease supply control unit 457 supplies a protease (trypsin, etc.) for reducing the adhesive force of the cultured cells into the culture vessel from which unnecessary medium has been removed by the protease supply device 360. The culture vessel that has been subjected to the process of step S44 is transported by the transport operation of the transport device 310 to the position of the fourth incubator 400 for reducing the adhesive force of the cells.

[0078] In step S45, the culture control unit 460 controls the culture environment in the fourth incubator 400 by operating a heater, a humidifier, or the like, according to the parameters of the culture environment control set by the parameter setting unit 451. At this time, the culture vessel transported by the transport device 310 is placed in the fourth incubator 400, and after a protein decomposition process based on the parameters of the culture environment control set by the parameter setting unit 451 is performed, the culture vessel containing the cultured cells with reduced adhesive force is transported from the fourth incubator 400 to the outside.

[0079] In step S46, the protease removal control unit 458 causes the protease remover 370 to remove unnecessary proteases (trypsin, etc.) from within the culture vessel containing the cultured cells whose adhesive strength has been reduced. In step S47, the culture medium supply control unit 455 controls the culture medium supply device 340 to supply a necessary amount of culture medium for subculture into the culture vessel containing the cultured cells whose adhesive force has been reduced. In step S48, the transport control unit 461 applies forced vibration to the culture vessel containing the cultured cells with reduced adhesive strength by the vibration applying unit 380. In this embodiment, as described above, in the transport device 310, a part of the transport section where the subculture is performed is allocated as an area for applying forced vibration, so that the culture vessel is transported to the transport device 310, whereby the cultured cells with reduced adhesive strength are detached.

[0080] In step S49, the culture volume control unit 459 executes a process of expanding the culture area of ​​the culture vessel (variable capacity culture vessel 200) in which subculture is performed by the culture volume management device 390. As a result, the outer partition member 203 of the variable capacity culture vessel 200 is removed, and the culture area is expanded from a medium area to a large area. The culture vessel that has been subjected to the process of step S49 is transported by the transport operation of the transport device 310 to the position of the fifth incubator 400 for the second subculture. In step S50, the culture control unit 460 controls the culture environment in the fifth incubator 400 by operating a heater, a humidifier, or the like, according to the parameters of the culture environment control set by the parameter setting unit 451. At this time, the culture vessel transported by the transport device 310 is placed in the fifth incubator 400, and after the culture based on the parameters of the culture environment control set by the parameter setting unit 451 is performed, the culture vessel in which the second subculture has been performed is transported from the fifth incubator 400 to the outside.

[0081] In step S51, the process for cell culture in the cell processing system 300 is completed, and the culture vessel is transferred from the line of the transfer device 310 to a predetermined storage position. Although the cell culture control process has been described here by taking as an example a case where subculture is performed twice, subculture may be performed once or three or more times.

[0082] As described above, when subculturing cells, the cell processing system 300 in this embodiment cultures primary cells, then performs a process to reduce the adhesive strength of the cultured cells in the culture vessel using a protease (trypsin, etc.), adds medium to the cultured cells with reduced adhesive strength, and then detaches the cultured cells by applying a physical stimulus (forced vibration, etc.).Then, the cell processing device 100 expands the culture volume and performs subculturing in the same culture vessel with the expanded culture area. Furthermore, the cell processing system 300 automates each step in the cell processing method, making it possible to carry out major steps, including the step of culturing the primary cultured cells and the step of subculture, without the need for manual labor by an operator. This type of treatment can suppress damage to cells caused by the action of proteases, compared to when cells are detached using proteases. Also, compared to when cultured cells are removed by centrifugation or the like, the workload required for subculture can be reduced and efficiency can be increased. Furthermore, each step of the cell treatment method can be automated, and mass production of cultured cells can be facilitated. Therefore, according to the cell processing system 300 of the present embodiment, it is possible to realize a cell subculture technique that can more efficiently obtain higher quality cells.

[0083] [Variations] Next, various modifications of the present invention will be described. The cell processing method described in the above embodiment shows a basic example of the present invention, and the present invention can be implemented in the following modified forms.

[0084] [Variation 1] In the above-described embodiment, an example was given in which one outer partition member 203 and one inner partition member 204 are installed in the variable capacity culture vessel 200, and one small region is expanded to form one medium region, but this is not limited to this. For example, a configuration may be adopted in which one middle region is defined by an outer partition member 203 within the variable volume culture vessel 200, and multiple small regions are defined within the frame of the outer partition member 203 (within the middle region) by an inner partition member 204, and the multiple small regions are expanded to form one middle region.

[0085] FIG. 16 is a schematic diagram showing an example of steps of a cell culture method in which one medium region is provided in a variable-volume culture vessel 200, and a plurality of small regions are provided within the medium region. As shown in FIG. 16(A), multiple small regions (four in FIG. 16) can be set within the frame (middle region) of the outer partition member 203, and primary cultured cells can be cultured in each small region. Then, as shown in FIG. 16(B), the adherent cells are cultured in the small regions, and the cell number increases.

[0086] In each of the small regions in which the primary cultured cells are cultured in this manner, the adhesive strength of the cultured cells is reduced using a proteolytic enzyme (trypsin, etc.), and after the proteolytic enzyme is removed, medium is supplied and forced vibration is applied to the culture vessel. Then, as shown in FIG. 16(C), in each small region, the detached cultured cells are suspended in the medium. In this state, when the inner partition member 204 is removed, the medium in which the cultured cells are suspended flows out into the middle region, as shown in FIG. 16(D).

[0087] Furthermore, by culturing the culture vessel in a set culture environment, the number of cultured cells increases in the medium region, and subculture from the small region to the medium region is realized, as shown in FIG. 16(E). By using such a cell processing method, small regions are placed at multiple positions in the middle region, so that the distribution of cultured cells can be made more uniform in the initial state when the culture region is expanded into the middle region. Therefore, when subculture is performed, conditions conducive to proliferation of cultured cells can be created throughout the entire middle region. In this modified example, a case where a plurality of small regions are formed inside a medium region has been described as an example, but a plurality of medium regions may be formed inside a large region.

[0088] [Variation 2] In variant example 1, a configuration example was described in which one middle region is formed within a variable volume culture vessel 200 by an outer partition member 203, and multiple small regions are placed within the frame of the outer partition member 203 (within the middle region) by an inner partition member 204, and the multiple small regions are expanded to form one middle region.In this case, the cells cultured in the multiple small regions can be of different species. FIG. 17 is a schematic diagram showing an example of steps of a cell culture method in which one medium region is provided in a variable-volume culture vessel 200, and multiple small regions are provided within the medium region, and different types of cells are cultured in the multiple small regions.

[0089] As shown in Figure 17(A), multiple small areas (two in Figure 17) are set up within the frame (middle area) of the outer partition member 203, and different types of primary cultured cells (cell types C1 and C2) can be cultured in each small area. Then, as shown in FIG. 17(B), different types of adherent cells are cultured in each small region, and the number of each type of cell increases. In each of the small regions in which the primary cultured cells are cultured in this manner, the adhesive strength of the cultured cells is reduced using a proteolytic enzyme (trypsin, etc.), and after the proteolytic enzyme is removed, medium is supplied and forced vibration is applied to the culture vessel.

[0090] Then, as shown in FIG. 17(C), in each small region, the detached different types of cultured cells are suspended in the medium. In this state, when the inner partition member 204 is removed, the culture medium in which the different types of cultured cells are suspended flows out into the middle region, as shown in Figure 17 (D), and a culture medium in which the different types of cultured cells are mixed is formed in the middle region. Furthermore, by culturing the culture vessel in a set culture environment, the number of different types of cultured cells increases in the medium region, as shown in Figure 17(E), and subculture of different types of cultured cells from the small region to the medium region is achieved.

[0091] By using such a cell processing method, it is possible to culture heterogeneous cultured cells at a constant density in a small region, and then expand the heterogeneous cultured cells with an increased cell number to a medium region, thereby performing subculture of a mixture of heterogeneous cells. Therefore, when subculture is performed, cell culture results in which different types of cultured cells are mixed can be easily obtained. In this modified example, an example has been described in which multiple small regions are formed inside a medium region and different types of cultured cells are cultured in each small region, but it is also possible to form multiple medium regions within a large region and culture different types of cultured cells in each medium region.

[0092] [Variation 3] In the above-described embodiment and modified examples, the case where the small, medium and large regions all have the same bottom shape (flat surface) and culture cells is cultured has been described as an example, but the present invention is not limited to this. For example, the shape of the bottom surface of the culture region can be different between the small region and the middle region. As an example, the bottom surface of the small region can be made flat, and a recess (such as a hemispherical depression) can be formed in the bottom surface of the middle region to culture the cultured cells. FIG. 18 is a schematic diagram showing an example of steps of a cell treatment method in which the bottom surface of the small region is flat and a recess (semispherical depression) is formed in the bottom surface of the middle region to culture culture cells.

[0093] As shown in Figure 18(A), first, primary cultured cells are cultured in a small area with a flat bottom surface, and then the adhesive strength of the cultured cells is reduced in the small area using a proteolytic enzyme (trypsin, etc.). After the proteolytic enzyme is removed, medium is supplied and forced vibration is applied to the culture vessel. Then, as shown in FIG. 18(B), in the small region, the detached cultured cells are suspended in the medium. In this state, when the inner partition member 204 is removed, the medium with the cultured cells suspended therein flows out into the middle region as shown in Fig. 18(C). At this time, since a recess is formed on the bottom surface of the middle region, some of the detached cultured cells are concentrated in the recess.

[0094] In this state, when the culture vessel is further cultured in a set culture environment, the cultured cells aggregated in the recesses of the middle region form cell clusters (spheroids). In other words, the recesses on the bottom surface of the middle region provide an environment in which cells can be cultured three-dimensionally.

[0095] By using this cell processing method, it is possible to culture adherent cells two-dimensionally in a small area, and then expand them to a medium area and form three-dimensional cell masses (spheroids) when the time comes to perform subculture. Therefore, it is possible to simultaneously carry out two-dimensional cell culture and three-dimensional cell culture while performing subculture. In this modified example, an example has been described in which a recess is formed on the bottom surface of the medium region, but it is also possible to form a recess on the bottom surface of the large region or the small region and form cell clusters (spheroids) in these recesses.

[0096] The present invention can be modified and improved as appropriate within the scope of the effects of the present invention, and is not limited to the above-described embodiment. For example, when carrying out the cell treatment method of the present invention, a step of washing the cultured cells with a washing solution can be added as appropriate at a necessary timing in the subculture step. This makes it possible to increase the number of cells obtained by culture and improve the quality of the cultured cells.

[0097] In the above-mentioned embodiment, the material of the culture vessel is described as glass or PDMS, but the material is not limited to this. For example, the culture vessel may be made of silicone (silicone rubber, silicone resin, etc.) in part or in whole. In the cell processing method of the present invention, cultured cells with reduced adhesive strength can be detached by forced vibration rather than ultrasonic resonance, etc., so that cultured cells can be easily detached even from flexible materials such as silicone.

[0098] Furthermore, the present invention can be implemented by appropriately combining the above-described embodiments and modifications. The control processes in the above-described embodiments can be executed by either hardware or software. In other words, it is sufficient that the cell processing device 100 or the cell processing system 300 has the functionality to execute the above-mentioned processing, and the functional and hardware configurations for realizing this functionality are not limited to the above-mentioned examples.

[0099] The above embodiment shows an example of application of the present invention, and does not limit the technical scope of the present invention. In other words, the present invention can be modified in various ways, such as omissions and substitutions, without departing from the gist of the present invention, and various embodiments other than the above embodiment can be adopted. The various embodiments and modifications that the present invention can adopt are included in the scope of the invention described in the claims and their equivalents.

[0100] The cell processing apparatus 100 configured as above includes a culture control unit 53 and a vibration control unit . The culture control unit 53 applies a protease to the cultured cells to reduce their adhesive strength. The vibration control unit 54 adds a medium to the culture vessel containing the cultured cells whose adhesive strength has been reduced, and applies a physical stimulus to the cultured cells, thereby detaching the cultured cells. The culture control unit 53 cultures the cultured cells detached by the vibration control unit 54 in a set culture environment, thereby subculturing the cells. This method can suppress damage to cells caused by the action of proteases compared to when cells are detached using proteases, and also reduces the workload required for subculture and increases efficiency compared to when cultured cells are removed by centrifugation or the like. Therefore, a cell subculture technique can be realized that can more efficiently obtain higher quality cells.

[0101] The culture vessel includes a first culture area for culturing primary cultured cells; and a second culture area that is formed by removing a partition member that separates the first culture area and has a culture volume larger than that of the first culture area. After culturing the primary cultured cells in the first culture area, the culture control unit 53 reduces the adhesive force of the cultured cells in the first culture area. The vibration control unit 54 detaches the cultured cells in the first culture region. The culture control unit 53 cultures the primary cultured cells in the first culture region in a set culture environment in the second culture region formed after the cells are detached by the vibration control unit . This allows subculture of cells to be performed whilst expanding into larger culture areas.

[0102] The culture vessel is formed by removing the partition member that separates the second culture region, and includes a third culture region having a culture volume larger than that of the second culture region. After culturing the passaged cells in the second culture area, the culture control unit 53 reduces the adhesive force of the cultured cells in the second culture area. The vibration control unit 54 detaches the cultured cells in the second culture region. The culture control unit 53 cultures the passaged cultured cells in the second culture region in a set culture environment in the third culture region formed after the cells are detached by the vibration control unit 54. This allows the cultured cells passaged from the primary cultured cells to be further passaged in the third culture area, which has a culture volume larger than that of the second culture area. Therefore, by using the cell treatment method of the present invention, the desired cultured cells can be appropriately obtained by repeated subculture.

[0103] The culture control unit 53 applies a physical stimulus such as forced vibration, impact force, or fluid stimulus to the cultured cells with reduced adhesive strength, thereby detaching the cultured cells. This allows the cells to be subcultured while reducing the time of immersion in the protease and reducing damage to the cells.

[0104] The cell processing system 300 of the present invention also includes a transport device 310, a culture control unit 460, and a vibration applying unit 380. The transport device 310 transports the culture vessel to be treated to a position where a process for culturing cells is carried out. The culture control unit 460 cultures the cultured cells to be treated, which have been seeded in the culture container, in a set culture environment. The culture control unit 460 applies a protease to the cultured cells to reduce their adhesive strength. The vibration applying unit 380 adds a medium to the culture vessel containing the cultured cells whose adhesive strength has been reduced, and applies a physical stimulus to the cultured cells, thereby detaching the cultured cells. The culture control unit 460 cultures the cultured cells detached by the vibration applying unit 380 in a set culture environment, thereby subculturing the cells. This makes it possible to suppress damage to cells caused by the action of proteases, compared to when cells are detached using proteases. Also, compared to when cultured cells are extracted by centrifugation or the like, the workload required for subculture can be reduced and efficiency can be increased. Furthermore, each step of the cell processing method can be automated, and mass production of cultured cells can be facilitated. Therefore, a cell subculture technique can be realized that can more efficiently obtain higher quality cells.

[0105] The vibration applying unit 380 applies vibrations, which are generated when the culture vessel is transported by the transport device 310, as a physical stimulus to the culture vessel, thereby detaching the cultured cells. This eliminates the need for a separate vibration-imparting device such as a vibrator, and allows the cultured cells to be detached more efficiently and at lower cost.

[0106] Moreover, the cell treatment method of the present invention includes a cell culture step, an adhesive force reducing step, and a cell detachment step. In the cell culture process, the cultured cells to be treated that have been seeded in a culture vessel are cultured in a set culture environment. In the adhesive strength reducing step, a protease is applied to the cultured cells to reduce their adhesive strength. In the physical stimulation step, a medium is added to the culture vessel containing the cultured cells whose adhesive strength has been reduced, and a physical stimulation is applied to detach the cultured cells. In the first subculture step, the cultured cells detached in the physical stimulus application step are cultured in a set culture environment, thereby subculture of the cells. This method can suppress damage to cells caused by the action of proteases compared to when cells are detached using proteases, and also reduces the workload required for subculture and increases efficiency compared to when cultured cells are removed by centrifugation or the like. Therefore, a cell subculture technique can be realized that can more efficiently obtain higher quality cells.

[0107] In the physical stimulus application step, a physical stimulus such as forced vibration, impact force or fluid is applied to the cultured cells whose adhesive strength has been reduced, thereby detaching the cultured cells. This allows the cells to be subcultured while reducing the time of immersion in the protease and reducing damage to the cells.

[0108] The method further includes a second subculture step in which the adhesive strength of the cultured cells cultured in the first subculture step is reduced by an adhesive strength reducing step, the cultured cells are detached by a physical stimulation applying step, and then the cells are cultured in a set culture environment. Using the cell treatment method of the present invention, the desired cultured cells can be appropriately obtained by repeated subculture.

[0109] When cultured cells are passaged, the culture volume of the culture region in which the cultured cells are cultured after passage is expanded compared to the culture region in which the cultured cells were cultured before passage. This allows the number of cells to be cultured appropriately increased while performing subculture, making it possible to obtain higher quality cells more efficiently.

[0110] When cultured cells are passaged, multiple culture areas are provided in which the cultured cells are cultured before passaging, and the culture area in which the cultured cells are cultured after passaging encompasses the multiple culture areas in which the cultured cells were cultured before passaging and is a culture area with a larger culture volume than the entire multiple culture areas. This allows multiple culture areas to be set up in which the cultured cells are cultured before passaging, making it possible to achieve a more even distribution of the cultured cells in the expanded culture area than in the initial state in which the culture area is expanded. Therefore, when subculture is performed, a condition favorable for proliferation of the cultured cells can be created in the entire culture region in which the cultured cells are cultured after subculture.

[0111] Different types of cultured cells are cultured in the multiple culture regions where the cultured cells are cultured before passaging, and the different types of cultured cells are mixed and cultured in the culture region where the cultured cells are cultured after passaging. This allows heterogeneous cultured cells to be cultured at a constant density in the culture area where the cultured cells are cultured before passaging, and the heterogeneous cultured cells with an increased cell number can be expanded to the culture area where the cultured cells are cultured after passaging, thereby performing a passaging culture of mixed heterogeneous cells. Therefore, when subculture is performed, cell culture results in which different types of cultured cells are mixed can be easily obtained.

[0112] When cultured cells are passaged, a recess is formed in the culture region in which the cultured cells are cultured after passage, and at least a portion of the cultured cells cultured after passage form a cell mass in the recess. This makes it possible to culture adherent cells two-dimensionally in the culture area where cultured cells are cultured before passaging, and then expand the cells to the culture area where cultured cells are cultured after passaging to form three-dimensional cell masses (spheroids) at the time of subculture. Therefore, it is possible to simultaneously carry out two-dimensional cell culture and three-dimensional cell culture while performing subculture. [Explanation of symbols]

[0113] 100 cell processing device, 110, 400 culture vessel (incubator), 120 vibration unit, 130 control unit, 200 variable volume culture vessel, 201 substrate, 202 side wall member, 203 outer partition member, 203a, 204a through hole, 204 inner partition member, 205 top plate, 205a protrusion, 51 parameter setting unit, 52 sensor information acquisition unit, 53, 460 culture control unit, 54 vibration control unit, 300 cell processing system, 310 transport device, 320 transfer device, 330 cultured cell supply device, 340 medium supply device, 350 medium removal device, 360 protease supply device, 370 protease removal device, 380 vibration applying unit, 390 culture volume management device, 410 control device, 451 parameter setting unit, 452 Sensor information acquisition unit, 453 imaging control unit, 454 cell supply control unit, 455 culture medium supply control unit, 456 culture medium removal control unit, 457 protease supply control unit, 458 protease removal control unit, 459 culture volume control unit, 461 transport control unit

Claims

1. A cell treatment device for culturing cells, comprising: A cell culture means for culturing the cultured cells to be treated, which are seeded in a culture vessel, in a set culture environment; an adhesive strength reducing means for reducing adhesive strength by applying a protease to the cultured cells; A physical stimulus applying means for adding a medium to the culture vessel containing the cultured cells with reduced adhesive strength, and applying a physical stimulus to the cultured cells to detach the cultured cells; Equipped with The cell culture means is characterized in that the cultured cells detached by the physical stimulus applying means are cultured in a set culture environment, thereby subculturing the cells.

2. The culture vessel includes a first culture region for culturing the primary cultured cells; A second culture area having a larger culture volume than the first culture area, the second culture area being formed by removing a partition member that partitions the first culture area; Equipped with The adhesive force reducing means reduces the adhesive force of the cultured cells in the first culture area after the cell culture means cultures the primary cultured cells in the first culture area, The physical stimulus applying means detaches the cultured cells in the first culture region, The cell processing device according to claim 1, characterized in that the cell culture means cultures the primary cultured cells in the first culture area in a set culture environment in the second culture area formed after the physical stimulus applying means detaches the primary cultured cells in the first culture area.

3. The culture vessel includes a third culture region formed by removing a partition member that separates the second culture region and having a culture volume larger than that of the second culture region; The adhesive force reducing means reduces the adhesive force of the cultured cells in the second culture area after the cell culture means cultures the cultured cells passaged in the second culture area, The physical stimulus applying means detaches the cultured cells in the second culture region, The cell processing device according to claim 2, characterized in that the cell culture means cultures the passaged cultured cells in the second culture area in a set culture environment in the third culture area formed after the physical stimulus imparting means detaches the cultured cells.

4. The cell processing device according to claim 1, characterized in that the adhesive strength reducing means detaches the cultured cells by applying the physical stimulus of forced vibration, impact force or fluid to the cultured cells whose adhesive strength has been reduced.

5. A cell processing system for culturing cells, comprising: A transport means for transporting a culture vessel to be treated to a position where a process for culturing the cells is performed; A cell culture means for culturing the cultured cells to be treated seeded in the culture vessel in a set culture environment; an adhesive strength reducing means for reducing adhesive strength by applying a protease to the cultured cells; A physical stimulus applying means for adding a medium to the culture vessel containing the cultured cells with reduced adhesive strength, and applying a physical stimulus to the cultured cells to detach the cultured cells; Equipped with The cell processing system is characterized in that the cell culture means performs subculture of the cells by culturing the cultured cells detached by the physical stimulus applying means in a set culture environment.

6. The cell processing system according to claim 5, characterized in that the physical stimulus applying means detaches the cultured cells by applying vibrations generated during the process of transporting the culture vessel by the transport means as the physical stimulus to the culture vessel.

7. A cell culture process in which the cultured cells to be treated seeded in the culture vessel are cultured in a set culture environment; an adhesion reducing step of reducing the adhesion of the cultured cells by applying a protease to the cultured cells; A physical stimulation step of adding a medium to the culture vessel containing the cultured cells with reduced adhesive strength, and applying a physical stimulation to detach the cultured cells; a first subculture step of subculturing the cells by culturing the cultured cells detached in the physical stimulation step in a set culture environment; A cell treatment method comprising the steps of:

8. The cell treatment method according to claim 7, characterized in that in the physical stimulus application step, the cultured cells are detached by applying the physical stimulus, which is a forced vibration, an impact force or a fluid stimulus, to the cultured cells whose adhesive strength has been reduced.

9. The cell treatment method according to claim 7, further comprising a second subculture step of culturing the cultured cells in a set culture environment after the adhesive force of the cultured cells cultured in the first subculture step is reduced by the adhesive force reducing step and the cultured cells are detached by the physical stimulus applying step.

10. The cell treatment method according to claim 7 or 9, characterized in that, when the cultured cells are passaged, the culture volume of the culture region in which the cultured cells are cultured after passaging is enlarged compared to the culture region in which the cultured cells were cultured before passaging.

11. The cell treatment method according to claim 7, characterized in that, when the cultured cells are passaged, a plurality of culture areas in which the cultured cells are cultured before passaging are provided, and the culture area in which the cultured cells are cultured after passaging encompasses the plurality of culture areas in which the cultured cells were cultured before passaging and has a culture volume larger than the entirety of the plurality of culture areas.

12. The cell treatment method according to claim 11, characterized in that different types of cultured cells are cultured in the multiple culture regions in which the cultured cells are cultured before passaging, and the different types of cultured cells are mixed and cultured in the culture region in which the cultured cells are cultured after passaging.

13. The cell treatment method according to claim 7 or 9, characterized in that, when the cultured cells are passaged, a recess is formed in a culture region in which the cultured cells are cultured after the passage, and at least a portion of the cultured cells cultured after the passage form a cell mass in the recess.

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