Cell detachment device and cell detachment method

Abstract

To provide a cell peeling device and a cell peeling method in which a cell having an uneven adhesion state corresponding to a culture state of a cell can be efficiently peeled from a culture vessel in a non-contact manner.SOLUTION: A cell peeling device 10 according to the present invention peels a cell 1 from a culture vessel 2 on which the cultured cell 1 is adhered, and the device comprises: an ultrasonic probe 12 having a plurality of piezoelectric elements 11 which generate ultrasonic wave by an applied voltage into an ultrasonic beam 20; a moving mechanism 14 which moves the ultrasonic probe 12, and makes a relative position between the ultrasonic probe 12 and the culture vessel 2 changeable; a voltage application part 13 which is connected to the plurality of piezoelectric elements 11, gives delay time to the plurality of piezoelectric elements 11, and applies a voltage; a cell state detection part 18 which detects a cell state in the culture vessel 2; and a peel planning part 30 which refers to the cell state obtained by the cell state detection part 18, and determines a transmission condition of the ultrasonic beam 20.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell detachment device and a cell detachment method for detaching specific cells from a culture surface in a culture vessel using ultrasound. [Background technology]

[0002] Treatments using cultured cells have been put to practical use in the application of regenerative medicine. Cells are grown in specific cell culture vessels (hereinafter sometimes simply referred to as "culture vessels"), including cell culture dishes and plates, multi-well plates, microtiter plates, flasks, multi-cell culture flasks, and multi-layer cell culture vessels. During culture, cells attach (adhere) to the bottom of the culture vessel and grow. The grown cells form cell sheets or colonies, which are used for treatment, etc.

[0003] As a method for detaching cells adhered to a culture vessel from the vessel, for example, techniques described in Patent Documents 1 and 2 are known. Patent Document 1 describes a technology that uses dispase, an enzyme that selectively cuts adhesion between cells and a cell culture substrate. Patent Document 2 describes a technology for recovering cell sheets using a temperature-responsive polymer.

[0004] However, the techniques described in Patent Documents 1 and 2 each have the following drawbacks. The technology described in Patent Document 1 also degrades the extracellular matrix used to adhere cells to the cell culture surface, potentially reducing cell function. Furthermore, dispase, which is used to recover cell sheets, is expensive, increasing costs.

[0005] Furthermore, the technology described in Patent Document 2 requires special processing using a temperature-responsive polymer on the cell culture surface, which increases costs. Furthermore, the technology described in Patent Document 2 detaches cells by lowering the temperature. Therefore, with this technology, some cells may detach when exposed to room temperatures lower than the temperature suitable for culture during tasks such as changing the medium or observing under a microscope. Therefore, this technology may make culture operations more difficult than when using a general culture dish. Furthermore, this technology may also result in a decrease in cell activity due to a decrease in temperature, which may lead to a decrease in the quality of the cells produced.

[0006] Under these circumstances, the technology described in Patent Document 3 has been proposed in recent years. Patent Document 3 describes a technology for detaching cells by mechanical vibrations caused by a piezoelectric element attached to a cell culture vessel. According to this technology, by applying uniform mechanical vibrations to the cultured cells, the entire cell sheet can be detached from the culture vessel without contact. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-157624 [Patent Document 2] International Publication No. 2012 / 029882 [Patent Document 3] International Publication No. 2019 / 044804 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the adhesion state of cells to the culture vessel varies depending on the proliferation of the cells, and therefore the technique described in Patent Document 3 has the problem that it takes time to evenly detach the entire cell sheet, making it inefficient.

[0009] The present invention has been made in consideration of the above-mentioned problems, and its purpose is to provide a cell detachment device and a cell detachment method that can efficiently detach cells that have uneven adhesion depending on the cell culture state from a culture vessel in a non-contact manner. [Means for solving the problem]

[0010] In order to solve the above problems, the cell detachment device according to the present invention is a cell detachment device for detaching cultured cells from a culture vessel to which the cells are adhered, and includes an ultrasonic probe having a plurality of piezoelectric elements that generate ultrasonic waves in response to an applied voltage to form an ultrasonic beam, a movement mechanism that moves the ultrasonic probe to change the relative position of the ultrasonic probe and the culture vessel, a voltage application unit that is connected to the plurality of piezoelectric elements and applies a voltage to the plurality of piezoelectric elements with a delay time, a cell state detection unit that detects the state of the cells in the culture vessel, and a detachment planning unit that refers to the cell state obtained by the cell state detection unit and determines transmission conditions for the ultrasonic beam. The cell state detection unit transmits a detection ultrasonic beam from the ultrasonic probe and detects the detachment state of the cell by measuring whether the cell moves or not by the detection ultrasonic beam, and includes a system synchronization unit that synchronizes the time of the voltage application unit and the cell state detection unit. do. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a cell detachment device and a cell detachment method that can efficiently detach cells, which have uneven adhesion depending on the cell culture state, from a culture vessel in a non-contact manner. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a schematic diagram illustrating the configuration of a cell detachment device according to a first embodiment. [Figure 2] FIG. 2 is a schematic diagram illustrating an example of a moving path of an ultrasonic beam. [Figure 3] FIG. 2 is an explanatory diagram illustrating an example of an electrical signal. [Figure 4] 1 is a flowchart illustrating the details of a cell detachment method according to the first embodiment. [Figure 5] FIG. 10 is a schematic diagram of a cell detachment device according to a second embodiment. [Figure 6]10 is a flowchart illustrating the details of a cell detachment method according to a second embodiment. [Figure 7] FIG. 10 is a schematic diagram illustrating the configuration of a cell detachment device according to a third embodiment. [Figure 8] FIG. 10 is a schematic diagram illustrating the configuration of a cell detachment device according to a fourth embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. In this embodiment, cells adhered to the inner wall surface of a culture vessel are detached. The cell detachment device according to this embodiment can be used as part of a cell culture device. Furthermore, in the cell detachment method according to this embodiment, a method of culturing adherent cells by forming a cell culture medium in a commonly used cell culture dish is described as an example, but the scope of application of the present invention is not limited to this. Furthermore, this embodiment can be used with various culturable adherent cells and is not limited to specific cells. Furthermore, the medium used for cell culture in this embodiment can be either a serum-free medium or a medium containing serum.

[0014] To explain this embodiment, a system will be described in which ultrasonic waves are transmitted upward from the outside of the bottom surface of a culture vessel while cells immersed in a culture medium inside the culture vessel are attached to the bottom surface. However, this embodiment is not limited to this system and can also be applied to a system in which ultrasonic waves are transmitted from the side or top surface of the culture vessel through the culture medium, etc.

[0015] In the cell detachment device and method according to this embodiment, an ultrasonic probe having a piezoelectric element is attached (contacted or adhered) to the outer surface, e.g., the bottom surface, of a culture vessel, and arbitrarily controlled ultrasonic waves are transmitted to the cells in the culture vessel. The ultrasonic probe and the outer surface of the culture vessel may be in contact with each other via an acoustic coupler for efficient transmission of ultrasonic waves, or a matching layer formed of water, silicone rubber, fluororubber, or the like. In the cell detachment device and method according to this embodiment, ultrasonic waves having a certain intensity or higher are transmitted from the ultrasonic probe, thereby applying acoustic radiation pressure to the cells. Acoustic streaming may also occur along the ultrasonic wave path due to the transmitted ultrasonic waves. Cells are detached by applying an upward force to the cells due to the acoustic radiation pressure and / or acoustic streaming. In this embodiment, arbitrarily controlled ultrasonic waves include those in which the size of the ultrasonic beam at the interface between the cells and the culture vessel, the intensity of the ultrasonic beam, and the transmission time of the ultrasonic beam (duty ratio, repetition interval) are electronically controlled.

[0016] The size of the ultrasonic beam can be controlled, for example, by the following methods: In an ultrasonic probe having multiple piezoelectric elements, all or some of the piezoelectric elements to which a voltage is applied are selected; or the position of the focal point is changed by applying an arbitrary delay time to the voltage applied to each piezoelectric element of the ultrasonic probe; or the frequency of the applied voltage is changed; or the distance of the ultrasonic probe from the adhesion surface between the culture vessel and the cells may be changed.

[0017] The intensity of the ultrasonic beam can be controlled by, for example, the following methods: Changing the intensity of the voltage applied to the plurality of piezoelectric elements of the ultrasonic probe Changing the wave number of the voltage waveform to be applied Selecting all or some of the piezoelectric elements to which the voltage is applied

[0018] The transmission time of the ultrasonic beam can be controlled, for example, by changing the wave number or application time of the voltage applied to the multiple piezoelectric elements of the ultrasonic probe at a specific frequency. Note that the specific frequency is determined by the dimensions and material of the piezoelectric elements, and is determined arbitrarily depending on the piezoelectric elements.

[0019] Since the ultrasonic probe has multiple piezoelectric elements, applying voltage to each piezoelectric element with a time lag creates an ultrasonic beam that is focused at a predetermined location. At the focused position, the ultrasonic waves converge, resulting in a strong ultrasonic beam, while at unfocused positions, the ultrasonic waves do not converge, resulting in a weak ultrasonic beam. By increasing the wave number of the voltage or the application time of the focused and strong ultrasonic beam, a strong ultrasonic beam can be transmitted to the predetermined location for a long period of time. This can enhance the effectiveness of detaching cells adhering to the inner wall surface of the culture vessel.

[0020] Although the control methods for the size, intensity, and transmission time of the ultrasonic beam have been described above, these control methods are not limited to those described above.

[0021] To detect the state of cells during culture, a cell state detection unit such as an optical camera or optical microscope, which is commonly used for general cell observation, can be provided. The observed cell states, such as shape and density, are stored in a cell state database. The detachment planning unit then refers to this cell state database to calculate the ultrasound transmission position and the size, intensity, and transmission time of the ultrasound beam, thereby determining the ultrasound beam transmission conditions. The movement mechanism moves the ultrasound probe to any position according to the ultrasound transmission conditions determined by the detachment planning unit. The voltage application unit transmits ultrasound by applying voltage to each piezoelectric element, thereby detaching cells adhering to the inner wall surface of the culture vessel.

[0022] According to this embodiment, the culture state of cells with uneven adhesion is detected depending on the culture state of the cells, and based on the detection data, the beam intensity and beam diameter are adjusted to locally transmit ultrasound from outside the culture vessel, promoting cell detachment. Therefore, according to this embodiment, cells can be efficiently detached from the culture vessel without contact. Furthermore, as a result, a cell culture device equipped with the cell detachment device according to this embodiment can automate the cell detachment process and achieve high throughput.

[0023] In this embodiment, for example, the following configuration is disclosed. The cell detachment device according to this embodiment detaches cells that are being cultured and adhered to a culture vessel by transmitting ultrasound waves to the cells. The device includes an ultrasonic probe, a movement mechanism, a voltage application unit, a cell state detection unit, and a detachment planning unit. The ultrasonic probe has multiple piezoelectric elements that generate ultrasonic waves in response to an applied voltage to produce an ultrasonic beam. The movement mechanism moves the ultrasonic probe, enabling the relative position of the ultrasonic probe and the culture vessel to be changed. The voltage application unit is connected to the multiple piezoelectric elements and applies voltage to the multiple piezoelectric elements with a delay time. The cell state detection unit detects the state of the cells in the culture vessel. The detachment planning unit determines the transmission conditions for the ultrasonic beam by referring to the state of the cells obtained by the cell state detection unit. Here, the transmission conditions for the ultrasonic beam include at least the range and intensity of ultrasound irradiation from the ultrasonic probe. The cell detachment device according to this embodiment may include a plurality of ultrasonic probes, which may be controlled independently.

[0024] (First embodiment) A cell detachment device 10 and a cell detachment method according to a first embodiment will be described with reference to Figures 1 to 4. Figure 1 is a schematic diagram of the cell detachment device 10 according to the first embodiment. Figure 2 is a schematic diagram illustrating an example of the movement path of an ultrasonic beam 20. Figure 3 is an explanatory diagram showing an example of an electrical signal. Figure 4 is a flowchart illustrating the details of the cell detachment method according to the first embodiment.

[0025] 1, a cell detachment device 10 transmits an ultrasonic beam 20 to cells 1 immersed in a culture solution 3 such as a medium inside a culture vessel 2, and detaches the cells from the culture vessel 2. The cells 1 are adhered to the bottom surface of the culture vessel 2 in the form of, for example, a single cell, a cell colony, or a cell sheet.

[0026] The cell detachment device 10 includes an ultrasonic probe 12 attached to the outside of the culture vessel 2 and a voltage application unit 13 that applies voltage to multiple piezoelectric elements 11 of the ultrasonic probe 12. The cell detachment device 10 also includes a movement mechanism 14 that moves the relative position of the ultrasonic probe 12 and the culture vessel 2 along the contact surface, and a detachment planning unit 30 that calculates the conditions of the voltage to be applied to the voltage application unit 13 and the position of the ultrasonic probe 12. The detachment planning unit 30 also includes a beam adjustment calculation unit 15 that calculates the conditions of the voltage to be applied to the voltage application unit 13 and adjusts the ultrasonic beam 20, and a probe trajectory calculation unit 16 that calculates the amount of movement of the ultrasonic probe 12, i.e., the trajectory. The cell detachment device 10 also includes a cell state detection unit 18 that detects the state of the cells and a cell state database (DB) 17 that stores the cell states detected by the cell state detection unit 18.

[0027] As an example, the ultrasonic probe 12 has a plurality of flat piezoelectric elements 11. The ultrasonic probe 12 has a ground electrode on the upper surface side of the piezoelectric element 11, i.e., on the side facing the culture vessel 2, and has a protective film adhered to the upper surface side of this ground electrode. The protective film of the ultrasonic probe 12 comes into contact with the outer surface of the culture vessel 2. The protective film is a thin film made of metal or resin.

[0028] The ultrasonic probe 12 has a plurality of signal electrodes arranged concentrically on the underside of the piezoelectric element 11, i.e., on the side of the voltage application unit 13. The concentric arrangement can be achieved, for example, by using a plurality of annular signal electrodes with different diameters, but is not limited to this. For example, the concentric arrangement can also be achieved by arranging a plurality of dot-shaped signal electrodes concentrically. The signal electrodes are connected to the voltage application unit 13 via a plurality of signal lines on their undersides, i.e., on the side of the voltage application unit 13. The connection portions between the signal electrodes and the signal lines are covered and fixed with resin.

[0029] For example, ceramics such as lead titanate zirconate or lead titanate, materials such as zinc oxide or lithium niobate, or composites are used for the piezoelectric element 11. Materials such as gold, silver, copper, platinum, titanium, and aluminum are used for the ground electrode and the signal electrode.

[0030] The trajectory of the ultrasonic beam 20 by the probe trajectory calculation unit 16 can be set arbitrarily. The probe trajectory calculation unit 16 calculates the amount of movement of the ultrasonic beam 20 so that the trajectory becomes, for example, the trajectory shown in FIG. 2. In FIG. 2, the calculation is first performed so that the ultrasonic beam 20 becomes a trajectory that detaches the outer edge of the cell 1 from the culture vessel 2. Next, the calculation is performed so that the ultrasonic beam 20 becomes a spiral trajectory that spirals toward the center of the cell 1. The spiral trajectory may also be calculated so that a part of the outer trajectory of the previous portion overlaps with a part of the inner trajectory of the current portion. In this way, it is possible to prevent the cell 1 from being left undetached.

[0031] Conversely, the probe trajectory calculation unit 16 may calculate a trajectory such that the ultrasonic beam 20 spirals from the center of the cell 1 outward (not shown). Alternatively, the probe trajectory calculation unit 16 may calculate a trajectory such that the ultrasonic beam 20 moves linearly from any end of the cell 1, and when it reaches the outer edge of the cell 1, moves to the next row, turns back, and continues moving linearly (not shown). Furthermore, the probe trajectory calculation unit 16 may calculate a trajectory such that the cells 1 are peeled off in order from areas with high cell density to areas with low cell density (not shown). By doing these, the cells 1 can be peeled off efficiently from the culture vessel 2.

[0032] The voltage application unit 13 can apply voltage to the plurality of piezoelectric elements 11 using, for example, an electrical signal as shown in Fig. 3. Φi is an example of a time waveform of the voltage applied to the i-th piezoelectric element. As shown in Fig. 3, the voltage application unit 13 applies a voltage Δt to each piezoelectric element 11 at a predetermined period Ts and a predetermined time T. j The voltage is applied with a delay time Δt jis calculated as the delay time that realizes the specific focal depth determined in the beam adjustment calculation unit 15.

[0033] The moving mechanism 14 is a mechanism for moving the ultrasonic probe 12 along a trajectory calculated by the probe trajectory calculation unit 16 and along the contact surface with the culture vessel 2 . The cell state database 17 saves (stores) data on the cell state and culture state, such as the shape and density of the measured cells 1. The cell state database 17 also stores control data for the ultrasound required for detachment depending on the type of cultured cell and the culture state, such as the size, intensity, and transmission time of the ultrasound beam 20.

[0034] A general cell observation device such as an optical camera or an optical microscope can be used as the cell state detection unit 18. The cell state detection unit 18 measures the culture state of the cells 1, such as the shape and density, and stores the measured results in the cell state database 17.

[0035] The detachment planning unit 30 reads, for example, the shape and spatial distribution of density of the desired cells 1 being cultured from the cell state data stored in the cell state database 17. Then, the beam adjustment calculation unit 15 of the detachment planning unit 30 calculates the output of the ultrasonic beam 20, i.e., the signal and intensity of the voltage to be applied to the ultrasonic probe 12, from the adhesion state of the cells 1 being cultured. Furthermore, the probe trajectory calculation unit 16 of the detachment planning unit 30 calculates the position of the ultrasonic probe 12 relative to the culture vessel 2. The detachment planning unit 30 provides the voltage application unit 13 with conditions for the signal of the voltage to be applied to the ultrasonic probe 12, and also provides the movement amount conditions to the movement mechanism 14. The calculation method in the detachment planning unit 30 will be described later.

[0036] The peeling planning unit 30 can be configured by, for example, a computer or the like equipped with a processor such as a CPU (Central Processing Unit) or a GPU (Graphics Processing Unit) and a memory.

[0037] The CPU reads and executes computer programs stored in the memory, thereby implementing the calculation functions, i.e., the functions of the beam adjustment calculation unit 15 and the probe trajectory calculation unit 16, by software. The peel planning unit 30 may implement some or all of its functions by hardware. For example, the peel planning unit 30 may be designed to implement its functions using a custom IC such as an ASIC (Application Specific Integrated Circuit) or a programmable IC such as an FPGA (Field-Programmable Gate Array).

[0038] Next, the cell detachment method according to this embodiment will be described using the flowchart in FIG. 4. Note that the cell detachment method according to this embodiment will be described taking as an example a method of sequentially detaching a cell sheet, which is made up of cells 1, from the edge toward the inside of the cell sheet, but is not limited to this. Alternatively, the cell sheet may be detached from the inside toward the edge. Alternatively, the cell sheet may be detached sequentially from areas with high cell density toward areas with low cell density. Alternatively, the target may be a cell colony.

[0039] As shown in Figure 4, first, the cell state detection unit 18 detects the state of the cells during culture (cell state detection step), measures the shape and density distribution of the cell sheet, the number of cells, etc., and stores them in the cell state database 17 (S41).

[0040] The probe trajectory calculation unit 16 reads out the coordinates of the edge of the cell sheet relative to the origin of the cell sheet (the origin is, for example, the center of the culture vessel 2) stored in the cell state database 17. Next, the probe trajectory calculation unit 16 calculates the trajectory of the transmission point of the ultrasonic beam 20 so that the edge is filled with the ultrasonic beam 20 and then spirals toward the center. Furthermore, immediately after or in parallel with the calculation by the probe trajectory calculation unit 16, the beam adjustment calculation unit 15 calculates the conditions (delay time, etc.) of the voltage to be applied to each piezoelectric element 11 of the ultrasonic probe 12 with reference to the size and intensity of the ultrasonic beam 20 set according to the type of cell, cell density, etc., for the read-out coordinates of the edge of the cell sheet (S42, detachment planning step).

[0041] Next, the moving mechanism 14 changes the relative positions of the ultrasonic probe 12 and the culture vessel 2. Specifically, the moving mechanism 14 moves the ultrasonic probe 12 to a specified position so that the center of the ultrasonic beam 20 is positioned at the initial ultrasonic transmission point (origin) (S43, moving step). Next, the voltage application unit 13 applies a voltage to each piezoelectric element 11 of the ultrasonic probe 12 with a delay time, thereby generating ultrasonic waves and transmitting the ultrasonic beam 20 (S44, transmission step).

[0042] After transmitting the ultrasonic beam 20, the cell state detection unit 18 checks whether the cell sheet has been detached from the culture vessel 2 (S45). If the cell sheet has detached from the culture vessel 2 (YES in S45), the movement mechanism 14 moves the ultrasonic probe 12 to the next designated position calculated by the probe trajectory calculation unit 16 (S47).

[0043] On the other hand, if the cell sheet has not detached from the culture vessel 2 (NO in S45), the beam adjustment calculation unit 15 recalculates the conditions of the ultrasonic beam 20 to be transmitted, such as the intensity and transmission time (S46). Thereafter, the voltage application unit 13 transmits the ultrasonic beam 20 again under the recalculated conditions (return to S44). When the entire cell sheet has detached from the culture vessel 2, the transmission of the ultrasonic beam 20 is terminated. In this way, the cell sheet can be reliably detached from the culture vessel 2 even if there is unevenness in the adhesion state depending on the culture state of the cells 1.

[0044] That is, in this embodiment, immediately after transmitting the ultrasonic beam 20 toward the cell sheet, it is confirmed whether the cell sheet has detached from the culture vessel 2, and if it has not detached, the conditions for the ultrasonic beam 20 are immediately recalculated and the ultrasonic beam 20 based on the recalculated conditions is transmitted again to the same location. Examples of the recalculation of the conditions for the ultrasonic beam 20 include lengthening the transmission time of the ultrasonic beam 20, narrowing down the size of the ultrasonic beam 20, or increasing the transmission output.

[0045] The ultrasonic beam 20 is transmitted to the entire cell sheet, and when the cell state detection unit 18 confirms that the entire cell sheet has been detached, the cell detachment process for one cell sheet is completed.

[0046] Thereafter, if there are multiple cell sheets in one culture vessel 2 or if cell sheets are being cultured in other wells of a multi-well plate, the ultrasonic probe 12 is moved to the next position by the moving mechanism 14, i.e., to another cell sheet or another well in one culture vessel 2, and the above series of steps S41 to S47 are performed to detach the cell sheet from the culture vessel 2.

[0047] In this way, the cell detachment device 10 and cell detachment method of this embodiment can efficiently detach cells 1 that have uneven adhesion depending on the culture state of the cells 1 from the culture container 2 in a non-contact manner by transmitting an ultrasonic beam 20 to the cells 1 being cultured.

[0048] In this embodiment, the presence or absence of detachment of the cells 1 being cultured is confirmed at each transmission point of the ultrasonic beam 20, and if detachment is confirmed, the ultrasonic probe 12 is moved to the next position. However, the present invention is not limited to this. For example, the detachment planning unit 30 calculates the beam size, intensity, transmission time, and coordinates of the ultrasonic beam 20 according to the cell state (e.g., the shape of the cell sheet) read from the cell state database 17, and prepares ultrasonic transmission planning data in advance so that the ultrasonic beam 20 is transmitted over the entire cell sheet. Next, the voltage application unit 13 and the movement mechanism 14 transmit the desired ultrasonic beam 20 to the desired position in accordance with the planning data. After the transmission of the ultrasonic beam 20 has been completed in accordance with the planning data, the cell state detection unit 18 checks for the presence or absence of detachment of the cells 1. Next, ultrasonic transmission planning data may be prepared again for the adhered portion, and the entire cell 1 (cell sheet) may be detached by transmitting the ultrasonic beam 20.

[0049] (Second embodiment) Next, a cell detachment device 50 and a cell detachment method according to a second embodiment will be described with reference to Figures 5 and 6. Figure 5 is a schematic diagram of the cell detachment device 50 according to the second embodiment. Figure 6 is a flowchart illustrating the details of the cell detachment method according to the second embodiment. The following description will focus on the differences between this embodiment and the first embodiment.

[0050] In the first embodiment, cell state data, i.e., data related to the culture state, such as the shape and density of the cells 1, was acquired by a cell state detection unit 18 using an optical camera or optical microscope to measure the cell state. In contrast, in a cell detachment device 50 according to the second embodiment, an ultrasonic probe 12 transmits a detection ultrasonic beam (not shown) to detect whether the cells 1 have been detached from the culture vessel 2. The detection ultrasonic beam uses ultrasonic waves with a lower output and a larger beam size than the ultrasonic beam 20 for detachment. For example, the detection ultrasonic beam can be applied with a lower voltage than the ultrasonic beam 20 for detachment. Furthermore, the focus of the detection ultrasonic beam 20 may be located within the culture medium above the interface between the cells 1 and the culture vessel 2. When the detection ultrasonic beam is transmitted to a location where the cells 1 have already been detached, the cells 1 move in response to the ultrasonic output due to the driving force applied by acoustic radiation pressure and / or acoustic streaming. Locations where the cells 1 have not yet been detached do not move even when hit by the detection ultrasonic beam. Whether or not the cell 1 moves due to the detection ultrasonic beam is measured by the cell state detection unit 18, and whether or not the cell 1 has detached from the culture vessel 2 is confirmed.

[0051] As shown in FIG. 5, the cell detachment device 50 includes a system synchronization unit 19. To synchronize the time of the cell state detection unit 18 with the time of the voltage application unit 13, the system synchronization unit 19 can generate a time trigger signal for transmitting ultrasound from the voltage application unit 13 and input the time trigger signal to the cell state detection unit 18. The time trigger signal may be generated starting from the cell state detection unit 18. Alternatively, the system synchronization unit 19 may collect time information for voltage application in the voltage application unit 13 and time information for measurement in the cell state detection unit 18, and synchronize the system by referring to the measurement data of the cell state at the time the ultrasound is transmitted.

[0052] The voltage application unit 13 in this embodiment can switch between the ultrasonic beam for detection and the ultrasonic beam 20 for ablation by changing the applied voltage and transmission time.

[0053] When the system synchronization unit 19 synchronizes the time of the cell state detection unit 18 with the time of the voltage application unit 13, the cell state detection unit 18 can accurately measure the timing of the detection ultrasonic beam that caused the cell sheet to move or not move. Therefore, the cell detachment device 50 can accurately detect whether the cell sheet has been detached or not, and the locations (coordinates) of the undetached portions.

[0054] Next, the cell detachment method according to this embodiment will be described using the flowchart in FIG. 6. Note that the cell detachment method according to this embodiment will be described taking as an example a method of sequentially detaching a cell sheet, which is made up of cells 1, from the edge toward the inside of the cell sheet, but is not limited to this. Alternatively, the cell sheet may be detached from the inside toward the edge. Alternatively, the cell sheet may be detached sequentially from areas with high cell density toward areas with low cell density. Alternatively, the target may be a cell colony.

[0055] In addition, the system synchronization unit 19 inputs a time trigger signal to the cell state detection unit 18 at the timing when the voltage application unit 13 applies voltage to each piezoelectric element 11 on the ultrasonic probe 12, but this is not limited to this.

[0056] 6, first, a detection ultrasonic beam is transmitted from the ultrasonic probe 12 toward the cell sheet in the culture vessel 2 (S61). At this time, the system synchronization unit 19 transmits a time trigger signal to the cell state detection unit 18 at the timing when the voltage application unit 13 applies a voltage to each piezoelectric element 11 on the ultrasonic probe 12.

[0057] The cell state detection unit 18 detects the cell state while the detection ultrasonic beam is being transmitted based on the time trigger signal input from the system synchronization unit 19 (cell state detection step), and stores the presence or absence of cell sheet detachment and the detached location in the cell state database 17 (S62). The presence or absence of cell sheet detachment can be confirmed, for example, from the presence or absence of movement of the cell sheet during the transmission of the detection ultrasonic beam.

[0058] Next, the probe trajectory calculation unit 16 reads out the coordinates of the end of the cell sheet relative to the origin (the origin is, for example, the center of the culture vessel 2) stored in the cell state database 17. Next, the probe trajectory calculation unit 16 fills in the end with the ultrasonic beam 20 and then calculates the trajectory of the transmission point of the ultrasonic beam 20 so that it spirals toward the center. Also, immediately after or in parallel with the calculation by the probe trajectory calculation unit 16, the beam adjustment calculation unit 15 calculates the conditions of the voltage to be applied to each piezoelectric element 11 of the ultrasonic probe 12, including the size, intensity, transmission time, etc. of the ultrasonic beam 20, based on the location of detachment of the cell sheet confirmed in step S62 (S63, detachment planning step).

[0059] Next, the movement mechanism 14 changes the relative positions of the ultrasonic probe 12 and the culture vessel 2. Specifically, the movement mechanism 14 moves the ultrasonic probe 12 to a specified position so that the center of the ultrasonic beam 20 is positioned at the initial ultrasonic transmission point (origin) (S64, movement step).

[0060] Next, the voltage application unit 13 applies a voltage to each piezoelectric element 11 of the ultrasonic probe 12 with a delay time, thereby generating ultrasonic waves and transmitting the ultrasonic beam 20 (S65, transmission step).

[0061] After transmitting the ultrasonic beam 20, the cell state detection unit 18 checks whether the cell sheet has been detached from the culture vessel 2 (S66). If the cell sheet has detached from the culture vessel 2 (YES in S66), the movement mechanism 14 moves the ultrasonic probe 12 to the next designated position calculated by the probe trajectory calculation unit 16 (S68). After that, the process returns to step S61 and repeats the same steps.

[0062] On the other hand, if the cell sheet has not detached from the culture vessel 2 (NO in S66), the beam adjustment calculation unit 15 recalculates (adjusts) the conditions of the ultrasonic beam 20 to be transmitted, such as the intensity and transmission time (S67). Thereafter, the voltage application unit 13 transmits the ultrasonic beam 20 again under the recalculated conditions (return to S65). When the entire cell sheet has detached from the culture vessel 2, the transmission of the ultrasonic beam 20 is terminated. In this way, the cell detachment method according to the second embodiment, which uses a detection ultrasonic beam to check for cell detachment, can reliably detach the cell sheet from the culture vessel 2, just like the first embodiment, even if there is unevenness in the adhesion state depending on the culture state of the cells 1.

[0063] (Third embodiment) Next, a cell detachment device 70 according to a third embodiment will be described with reference to Fig. 7. Fig. 7 is a schematic diagram of the cell detachment device 70 according to the third embodiment. This embodiment will also be described mainly focusing on the differences from the first embodiment.

[0064] In the first embodiment, the relative positions of the culture vessel 2 and the ultrasonic probe 12 are changed using the moving mechanism 14 to change the transmission position of the ultrasonic beam 20. In contrast, in the cell detachment device 70 according to the third embodiment, a specific delay time is applied to the voltage applied to the plurality of piezoelectric elements 11 on the ultrasonic probe 12, so that the central axis of the ultrasonic beam 20 is electronically scanned, and the ultrasonic beam 20 is scanned (electronically scanned) so as to fill the entire cell 1, thereby detaching the entire cell 1 from the culture vessel 2. In this way, the cell 1 can be detached without moving the ultrasonic probe 12 with the moving mechanism 14.

[0065] FIG. 7 shows an example of the voltage application unit 13 and generation of the ultrasonic beam 20 of the cell detachment device 70 according to this embodiment. As shown in FIG. 7, the voltage application unit 13 applies a voltage to each piezoelectric element 11 with a specific delay time for electronic scanning. In this manner, the ultrasonic waves generated by each piezoelectric element 11 are focused at a specific position. Therefore, the central axis of the ultrasonic beam 20 can be tilted at a specific angle from the central axis of the piezoelectric element 11 on the ultrasonic probe 12. The cell detachment device 70 according to this embodiment can transmit the ultrasonic beam 20 generated in this manner toward a desired position, thereby detaching all of the cells 1 in the culture vessel 2 from the culture vessel 2 without driving the moving mechanism 14.

[0066] The third embodiment differs from the first embodiment only in that the delay time applied by the voltage application unit 13 is controlled to electronically scan the ultrasonic beam 20 to detach the cells 1, and the flow of the cell detachment method is the same as that of the first embodiment. Therefore, a description of the cell detachment method according to the third embodiment will be omitted here.

[0067] However, in the cell detachment method according to the third embodiment, in the detachment planning step (detachment planning section 30), the delay time provided by the voltage application section 13 is set under conditions suitable for electronic scanning. Furthermore, in the cell detachment method according to the third embodiment, in the transmission step, a voltage is applied to each piezoelectric element 11 used by the ultrasonic probe 12 with a delay time, and the multiple piezoelectric elements 11 generate ultrasonic waves by the applied voltage, causing the ultrasonic beam 20 to be electronically scanned.

[0068] (Fourth embodiment) Next, a cell detachment device 80 according to a fourth embodiment will be described with reference to Fig. 8. Fig. 8 is a schematic diagram of the cell detachment device 80 according to the fourth embodiment. This embodiment will also be described mainly focusing on the differences from the first embodiment.

[0069] As described above, the size of the ultrasonic beam 20 can be changed by changing the delay time of the voltage applied to the plurality of piezoelectric elements 11, by changing the number of piezoelectric elements 11 used, or the like. In contrast, a cell detachment device 80 according to a fourth embodiment is provided with an acoustic lens 21, such as a Fresnel lens or a concave lens, between the ultrasonic probe 12 and the culture vessel 2, as shown in Fig. 8. In the cell detachment device 80, by changing the frequency and delay time of the voltage applied to the plurality of piezoelectric elements 11 according to the characteristics of the acoustic lens 21 used, it is possible to focus the ultrasonic waves at a specific position and change the size of the ultrasonic beam 20.

[0070] The acoustic lens 21 also has the effect of focusing ultrasonic waves incident parallel to the acoustic lens 21 at a specific position. When a voltage is applied to each piezoelectric element 11, the generated ultrasonic waves pass through the acoustic lens 21, forming a focal point at the specific position. This allows ultrasonic waves to be focused at points that would be difficult to focus without the acoustic lens 21. Multiple acoustic lenses 21 can also be provided. This allows multiple focal positions to be set. In this case, the ablation planning unit 30 refers to the cell state obtained by the cell state detection unit 18 and determines the transmission conditions of the ultrasonic beam, including the conditions for focusing the ultrasonic waves by the acoustic lens 21. The transmission conditions are determined by, for example, the ultrasonic irradiation range and intensity of the ultrasonic probe 12 and the characteristics of the acoustic lens used.

[0071] Furthermore, when a Fresnel lens is used as the acoustic lens 21, the focus and size of the ultrasonic beam 20 can be easily changed by changing the frequency of the voltage applied to the piezoelectric elements 11. In this case, the voltage application unit 13 applies a voltage to the multiple piezoelectric elements 11 by modulating the frequency. Furthermore, the ablation planning unit 30 determines the transmission conditions of the ultrasonic beam, including the characteristics of the Fresnel lens and the modulated frequency, with reference to the cell state obtained by the cell state detection unit 18. The transmission conditions are determined to include, for example, the ultrasonic irradiation range and intensity of the ultrasonic probe 12, and the modulated frequency.

[0072] The fourth embodiment differs from the first embodiment only in that an ultrasonic beam 20 is transmitted to a specific position by focusing ultrasonic waves from a piezoelectric element 11 using an acoustic lens 21, and the flow of the cell detachment method is the same as that of the first embodiment. Therefore, a description of the cell detachment method according to the fourth embodiment will be omitted here.

[0073] However, in the cell detachment method according to the fourth embodiment, in the detachment planning step (detachment planning unit 30), the conditions for the voltage to be applied by the voltage application unit 13 are set taking into account the number and characteristics (e.g., how the ultrasonic beam is focused) of the acoustic lenses 21 to be used.

[0074] Furthermore, when a Fresnel lens is used as the acoustic lens 21 in the cell detachment method according to the fourth embodiment, the size of the ultrasonic beam 20 transmitted through the Fresnel lens is set in the detachment planning step (detachment planning unit 30) under conditions suitable for detaching the cell 1. In this case, the beam adjustment calculation unit 15 determines conditions including the modulation frequency in addition to the conditions (delay time, etc.) of the voltage applied to each piezoelectric element 11 of the ultrasonic probe 12.

[0075] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims. [Explanation of symbols]

[0076] 1 cell 2 Culture container 3 Culture solution 10, 50, 70, 80 Cell detachment device 11 Piezoelectric element 12 Ultrasound probe 13 Voltage application section 14 Moving mechanism 15 Beam adjustment calculation unit 16 Probe trajectory calculation unit 17 Cell State Database 18 Cell state detection unit 19 System Synchronization Section 20 Ultrasonic beam 21 Acoustic Lens 30 Peeling Planning Department

Claims

1. A cell detachment device for detaching cultured cells from a culture vessel to which the cells have adhered, an ultrasonic probe having a plurality of piezoelectric elements that generate ultrasonic waves as ultrasonic beams in response to an applied voltage; a movement mechanism that moves the ultrasonic probe to change the relative position of the ultrasonic probe and the culture vessel; a voltage application unit connected to the plurality of piezoelectric elements and applying a voltage to the plurality of piezoelectric elements with a delay time; a cell state detection unit that detects the state of cells in the culture vessel; an ablation planning unit that determines transmission conditions of the ultrasonic beam by referring to the cell state obtained by the cell state detection unit; Equipped with the cell state detection unit transmits a detection ultrasonic beam from the ultrasonic probe and detects a detached state of the cell by measuring whether the cell moves due to the detection ultrasonic beam; A system synchronization unit that synchronizes the time of the voltage application unit and the cell state detection unit is provided. A cell detachment device characterized by:

2. The cell detachment device according to claim 1, The peeling planning unit a beam adjustment calculation unit that calculates a voltage signal to be applied to the ultrasonic probe and adjusts the ultrasonic beam; a probe trajectory calculation unit that calculates a trajectory of the ultrasonic probe by the moving mechanism; A cell detachment device comprising:

3. The cell detachment device according to claim 1, A cell detachment device, characterized in that the cell state detection unit is equipped with at least one of an optical camera and an optical microscope.

4. The cell detachment device according to claim 1, the voltage application unit applies the voltage to each of the piezoelectric elements used by the ultrasonic probe while providing a delay time; The cell detachment device is characterized in that the plurality of piezoelectric elements generate the ultrasonic waves in response to the applied voltage and electronically scan the ultrasonic beam.

5. The cell detachment device according to claim 1, The cell detachment device further comprises a cell state database that stores cell state data detected by the cell state detection unit.

6. The cell detachment device according to claim 1, one or more acoustic lenses for focusing the ultrasonic beam; The cell detachment device is characterized in that the detachment planning unit refers to the cell state obtained by the cell state detection unit and determines the transmission conditions of the ultrasonic beam, including the focusing conditions of the ultrasonic beam by the acoustic lens.

7. The cell detachment device according to claim 1, a Fresnel lens for changing the focus of the ultrasonic beam; the voltage application unit applies a voltage to the plurality of piezoelectric elements by modulating the frequency, The cell detachment device is characterized in that the detachment planning unit refers to the cell state obtained by the cell state detection unit and determines the transmission conditions of the ultrasonic beam, including the Fresnel lens and the modulated frequency.

8. A cell detachment method for detaching cultured cells from a culture vessel to which the cells have adhered, comprising: a cell state detection step of detecting a cell state; an ablation planning step of determining ultrasonic beam transmission conditions by referring to the cell state obtained in the cell state detection step; a moving step of moving the ultrasonic probe to the designated position determined in the separation planning step; a transmitting step of applying voltages to a plurality of piezoelectric elements of the ultrasonic probe with delay times to generate ultrasonic waves and transmit ultrasonic beams; and The cell state detection step transmits a detection ultrasonic beam from the ultrasonic probe, and detects the detached state of the cell by measuring whether the cell moves or not using the detection ultrasonic beam, and synchronizes the time when a voltage is applied with the time when the detached state of the cell is detected. A cell detachment method characterized by:

9. The cell detachment method according to claim 8, The cell ablation method is characterized in that the transmission step applies a voltage to each of the piezoelectric elements used in the ultrasonic probe with a delay time, and the multiple piezoelectric elements generate the ultrasonic waves using the applied voltage, thereby electronically scanning the ultrasonic beam.

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