Cell processing apparatus and cell processing method

Abstract

To enable cell culture without immersing cells in a culture medium.SOLUTION: A cell processing apparatus 1 comprises an ultrasonic transducer 13 and a control unit 16. The ultrasonic transducer 13 irradiates the culture medium in a flask 15 having cells adhered inside, in which the cells are exposed to the atmosphere, with ultrasonic waves. The control unit 16 controls the irradiation of ultrasonic waves by the ultrasonic vibrator 13.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to a cell processing apparatus and a cell processing method.

Background Art

[0002] Cell culture is roughly classified into two-dimensional culture in which cells are adhered to a culture surface such as the bottom surface of a dish and three-dimensional culture in which cells are suspended in a culture solution. Two-dimensional culture is easy to make the culture environment uniform and is used for growth culture and the like. On the other hand, in three-dimensional culture, since cells adhere to each other, it is often used for the generation of cell tissues in vitro in terms of cell-cell interaction and the like. Such two-dimensional and three-dimensional cultures of cells are widely used in cell engineering and medical research (for example, see Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In recent years, research on drug discovery and drug screening using cultured cells has been active, and for example, expectations for an experimental system that can verify in vitro the effects of drug administration to the lungs in a spray form have been increasing. However, in the above-described conventional culture methods that have been used, cells are cultured in a state of being immersed in a culture solution, and it cannot necessarily be said that the in-vivo situation is reproduced. Therefore, it has been difficult to construct an in-vitro experimental system for spray-like drug administration.

[0005] An object of the present invention is to enable cells to be cultured without being immersed in a culture solution.

Means for Solving the Problems

[0006] To achieve the above object, a cell processing apparatus according to one aspect of the present invention includes ultrasonic irradiation means for irradiating ultrasonic waves to a culture medium in a culture vessel in which cells are adhered and the cells are exposed to the atmosphere, and control means for controlling the irradiation of ultrasonic waves by the ultrasonic irradiation means, and is characterized by comprising the above.

Advantages of the Invention

[0007] According to the present invention, it becomes possible to culture cells without immersing them in a culture solution.

Brief Description of the Drawings

[0008]

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Mode for Carrying Out the Invention

[0009] Hereinafter, embodiments of the present invention will be described with reference to the drawings. [Basic Concept of the Present Invention] In the present invention, for cells adhered to a substrate such as a culture vessel, the medium is atomized by ultrasonic vibration to supply minute droplets of the medium. At this time, in order to suppress the rise in the temperature of the medium due to the irradiation of ultrasonic waves, the generation pattern of the ultrasonic vibration is controlled. Thereby, it becomes possible to culture cells without immersing them in a culture solution. That is, according to the present invention, since cells adhered to a substrate such as a culture vessel can be cultured in an environment exposed to the atmosphere, cells (such as mucosal epithelial cells) of organs exposed to the atmosphere such as alveoli, bronchi, and corneas can be cultured by more appropriately reproducing the in-vivo situation. Hereinafter, specific embodiments of the present invention will be described.

[0010] [Cell Processing Method] FIG. 1 is a diagram showing the procedure of a cell processing method according to an embodiment of the present invention. As shown in FIG. 1, in the cell processing method according to the present embodiment, first, on the culture surface of a culture vessel such as a flask, cells are cultured in a medium by various existing culture methods (adherent cell culture step). The cells cultured in the adherent cell culture step are in a state of adhering to the culture surface of a culture vessel such as a flask.

[0011] Next, in the space where the adherent cells cultured in the adherent cell culture step are present, ultrasonic waves are irradiated onto the culture medium by an ultrasonic oscillator, and the culture medium is sprayed into the air in the culture vessel (culture medium spraying step). The minute droplets of the culture medium (atomized culture medium) sprayed in the culture medium spraying step are supplied to the cells adhered to the culture surface.

[0012] Figure 2 is a schematic diagram showing the concept of the culture medium spraying step. As shown in Figure 2, in the culture medium spraying step, ultrasonic waves are irradiated onto the liquid culture medium stored in a culture vessel such as a flask by an ultrasonic oscillator. Then, the culture medium is sprayed into the air in the vessel, and the atomized culture medium is supplied to the cells adhered to the culture surface. Thereby, the atomized culture medium functions as a culture medium for the cells adhered to the culture surface, and it becomes possible to supply the culture medium to the cells without immersing them in the culture medium.

[0013] In this embodiment, in the culture medium spraying step, intermittent irradiation of ultrasonic waves onto the culture medium is continued for a set time (for example, 24 hours) until the adherent cells reach the target state and are cultured. The reason for intermittently irradiating the culture medium with ultrasonic waves is that when ultrasonic waves are continuously supplied to the culture medium, the temperature of the culture medium rises and deviates from the temperature suitable for cell culture.

[0014] Figure 3 is a diagram showing the temperature change of the liquid when ultrasonic waves are continuously irradiated onto the liquid and when ultrasonic waves are intermittently irradiated onto the liquid. In Figure 3, an example is shown in which water is used as the liquid and pulsed ultrasonic waves are used as the intermittent ultrasonic waves.

[0015] As shown in Figure 3, when ultrasonic waves are continuously irradiated onto the liquid, the temperature of the liquid rises with the increase in the irradiation time, and exceeds 60[℃] in about 3 minutes of irradiation from the initial 20[℃]. On the other hand, when ultrasonic waves are intermittently irradiated onto the liquid, it can be seen that even when the irradiation time increases, the temperature of the liquid is suppressed to rise from the initial 20[℃] to about 35℃, and the temperature suitable for cell culture is maintained.

[0016] As described above, in the medium spraying step, by intermittently irradiating the medium with ultrasonic waves, it is possible to continuously supply fresh medium (atomized medium) to the adherent cells while suppressing the rise in the temperature of the medium. Therefore, it is possible to perform culturing for a necessary period of time while appropriately controlling the temperature with the cells exposed to the air, so that the in-vivo conditions can be more appropriately reproduced, and cells of organs exposed to the air (such as mucosal epithelial cells) can be easily produced.

[0017] [Cell Processing Apparatus] Next, the cell processing apparatus used in this embodiment will be described. [Configuration] FIG. 4 is a schematic diagram showing the configuration of a cell processing apparatus 1 according to an embodiment of the present invention. As shown in FIG. 4, the cell processing apparatus 1 includes a pool 11, a base 12, an ultrasonic vibrator 13, a conducting wire 14, a flask 15, and a control unit 16. The pool 11 is constituted by a tray-shaped container having a bottom plate and side walls, and stores a medium (such as water) for transmitting ultrasonic waves.

[0018] The base 12 is a rectangular parallelepiped-shaped member on which the flask 15 is placed. In this embodiment, two bases 12 are installed at positions supporting one end and the other end on the bottom surface of the flask 15. The ultrasonic vibrator 13 generates ultrasonic waves when a voltage is applied through the conducting wire 14. The ultrasonic vibrator 13 is installed in a space formed between the two bases 12 on the bottom surface of the pool 11. One end of the conducting wire 14 is connected to the electrode of the ultrasonic vibrator 13, and the other end is connected to the control unit 16 (more specifically, a driver). Note that the conducting wire 14 is installed for each electrode of the ultrasonic vibrator 13.

[0019] The flask 15 stores a medium inside, and cells pre-cultured adhere to the inner side surface. Note that the inside of the flask 15 is filled with air, and it is in an environment close to that in the living body where cells of organs exposed to the air survive. The control unit 16 is composed of an information processing device or a signal processing device such as a PC (Personal Computer), a microcomputer, or a function generator, and controls the driving of the ultrasonic vibrator 13 according to preset experimental conditions. Specifically, the control unit 16 controls the pattern when irradiating ultrasonic waves intermittently, the duration for continuing the intermittent ultrasonic irradiation, the voltage applied to the ultrasonic vibrator 13, and the like.

[0020] [Cell culture process] Next, a process of culturing cells (cell culture process) using the cell processing device 1 will be described. FIG. 5 is a flowchart showing the steps of the cell culture process. As shown in FIG. 5, when the cell culture process is performed, first, as an adherent cell culture step, the cells are statically cultured in the medium on the side surface inside the flask 15 (step S1). In this embodiment, the flask 15 is placed horizontally, and 1.8×10 5 [cells] are seeded in 3.0 [mL] of the medium and statically cultured for 24 hours. As a result, the cells adhere to the side surface inside the flask 15.

[0021] Next, the bottom surface of the flask 15 obtained in step S1 is placed on the pedestal 12 in the pool 11 (step S2). Note that, at the bottom surface of the pool 11, an ultrasonic vibrator 13 is installed between the pedestals 12 (at the central position). Also, the pool 11 is filled with water up to a position higher than the upper surface of the pedestal 12, and the bottom surface of the flask 15 placed on the pedestal 12 is in contact with the water.

[0022] Next, the ultrasonic vibrator 13 intermittently irradiates ultrasonic waves toward the bottom surface of the flask 15 (step S3). FIG. 6 is a schematic diagram showing a state in which ultrasonic waves are transmitted from the ultrasonic vibrator 13 to the medium inside the flask 15. As shown in FIG. 6, since the space between the bottom surface of the flask 15 and the ultrasonic vibrator 13 is filled with water, the ultrasonic waves irradiated from the ultrasonic vibrator 13 can reach the bottom surface of the flask 15 without significant attenuation, pass through the flask 15, and vibrate the culture medium. In step S3, for example, a pattern is set in which ultrasonic waves are irradiated for 0.1 [s] and the irradiation is stopped for 4.9 [s], and this is continued for a set time (here, 24 hours). Also, the irradiation conditions of the ultrasonic waves can be, for example, a frequency of 2.08 [MHz], a voltage of 184.1 [Vpp], and a waveform of a sine wave.

[0023] FIG. 7 is a schematic diagram showing a culture mode (spray culture) in which a mist-like culture medium is supplied into the flask 15 to culture adherent cells. As shown in FIG. 7, as a result of step S3, an environment can be formed in which a mist-like culture medium is continuously supplied to the adherent cells exposed to the atmosphere. Next, the flask 15 is recovered from the base 12 (step S4). Optionally, the cultured cells may be detached from the culture surface of the flask 15 and recovered. After step S4, the cell culture process is completed.

[0024] [Verification 1] To verify the effect of the above cell culture process, instead of spray culture, a culture mode (static culture) in which cells are immersed in the culture medium for culture and a culture mode (vertical culture) in which adherent cells are located on the side surface inside the flask 15 were performed. FIG. 8 is a schematic diagram showing a culture mode (static culture) in which cells are immersed in the culture medium for culture. In static culture, the flask 15 was placed horizontally, and 1.8×10 5 [cells] were seeded in 3.0 [mL] of the culture medium and statically cultured for 24 hours, and then further statically cultured for 24 hours.

[0025] FIG. 9 is a schematic diagram showing a culture mode (vertical culture) in which adherent cells are located on the side surface inside the flask 15. In vertical culture, the flask 15 was placed horizontally, and 1.8×10 5[Cells] were seeded and statically cultured for 24 hours. Then, the flask 15 was placed vertically, and left for 24 hours with the adherent cells positioned on the side surface inside the flask 15. In addition, in the spray culture (see Fig. 7), as described above, the flask 15 was placed horizontally, and 1.8×10 5 [Cells] were seeded in 3.0 [mL] of the medium and statically cultured for 24 hours, and then intermittent ultrasonic irradiation was continued for 24 hours.

[0026] Fig. 10 is a diagram showing the number of cells in static culture, spray culture, and vertical culture. As shown in Fig. 10, the number of viable cells in the spray culture is significantly increased compared to the vertical culture. That is, it can be seen that the cells are not cultured only by placing the flask 15 vertically, and the cells are cultured by the atomized medium. Also, it can be seen that the number of viable cells in the spray culture is increased to about 3.4 times the seeding number. In the case of static culture, the number of cells becomes about twice in the first 24 hours and about twice in the next 24 hours, so it becomes about four times the seeding number. On the other hand, it can also be seen that in the spray culture, 70% or more viable cells are cultured compared to the static culture.

[0027] Thus, in the cell culture process according to this embodiment, the cells can be cultured without being immersed in the culture solution. That is, according to the cell culture process according to this embodiment, since the cells adhered to a base material such as a culture container can be cultured in an environment exposed to the air, cells (such as mucosal epithelial cells) of organs exposed to the air such as alveoli, bronchi, and corneas can be cultured by more appropriately reproducing the in-vivo situation.

[0028] [Verification 2] In the above-described cell processing apparatus 1, a cell staining experiment was conducted to verify whether chemicals or the like contained in the medium can act on the cells. Specifically, adherent cells were cultured in the same manner as in step S1 of the cell treatment method shown in FIG. 5. In the form of spray culture shown in FIG. 7, a medium with a calcein concentration of 0.1[%] was sprayed by ultrasonic waves for 1 hour. The irradiation conditions of the ultrasonic waves are the same as those in step S3 of the cell treatment method shown in FIG. 5.

[0029] FIG. 11 is a diagram showing the staining state of adherent cells. As shown in FIG. 11, living adherent cells (viable cells) are stained by calcein contained in the mist-like medium (shown as white in FIG. 11). That is, it can be seen that chemicals and the like contained in the medium can act on living adherent cells. Also, in FIG. 11, the lower adherent cells on the side surface (culture surface) of the flask 15 have a smaller degree of staining compared to the upper adherent cells. That is, when spray culture is performed, a gradient (change) is imparted to the amount of medium supplied per adherent cell. This is presumably because there is a gradient in the distribution of the medium atomized by the irradiation of ultrasonic waves.

[0030] FIG. 12 is a schematic diagram showing the distribution status of the mist-like medium in the flask 15. As shown in FIG. 12, in the flask 15, the mist-like medium diffuses upward from the liquid in the medium generated by ultrasonic vibration and fills the upper side in the flask 15, and has a tendency to be difficult to distribute in the lower part. Therefore, when chemicals and the like contained in the medium act on cells, a gradient of action can be given, and cell screening and the like can be appropriately performed. When giving a gradient of action of chemicals and the like in this way, if the mist-like medium is supplied excessively, the mist-like medium will be distributed throughout the flask 15. Therefore, it is necessary to control the supply amount of the mist-like medium to be within an appropriate range.

[0031] [Verification 3] In the above-described cell treatment method, a cell staining experiment was conducted to verify whether a medium containing a drug or the like can be locally supplied to the adherent cells on the culture surface and the drug or the like can be selectively allowed to act on the cells. FIG. 13 is a schematic diagram showing the experimental procedure in Verification 3. As shown in FIG. 13, in Verification 3, cells were seeded on a glass culture surface (slide glass G serving as a substrate) surrounded by a silicone wall, and static culture was performed for 24 hours. Then, the silicone wall was removed from the slide glass G, and in the cell treatment apparatus 100 having the configuration shown in FIG. 13, the slide glass G to which the cells had adhered was fixed to the stage, and the medium was supplied by permeating it from the centrifuge tube C serving as the medium supply source into the nonwoven fabric F. Furthermore, the medium supplied through the nonwoven fabric F was irradiated with ultrasonic waves by the ultrasonic vibrator 113 to spray the medium onto a local region of the slide glass G.

[0032] In Verification 3, 1.0 × 10 6 [cells] were seeded on the glass culture surface surrounded by the silicone wall, and static culture was performed for 24 hours to allow the cells to adhere to the glass culture surface. Then, 50.0 [mL] of a medium having a calcein concentration of 0.1 [%] was stored in the centrifuge tube C, and the medium permeated from the centrifuge tube C into the nonwoven fabric F was sprayed by ultrasonic waves. At this time, ultrasonic waves were irradiated for 0.5 [s], and a pattern of stopping the irradiation for 0.5 [s] was continued for 1 hour. The irradiation conditions of the ultrasonic waves were a frequency of 1.97 [MHz], a voltage of 368.2 [Vpp], and a waveform of a sine wave.

[0033] FIG. 14 is a diagram showing the staining state of the adherent cells. Further, FIG. 15 is a diagram showing the culture surface on which the medium was sprayed. Note that FIGS. 14 and 15 show the same region on the culture surface. As shown in FIG. 14, the cells (living adherent cells) present in the region where the medium was sprayed are stained with calcein contained in the mist-like medium (shown in white in FIG. 14), while the cells present in the region where the medium was not sprayed (inside the broken line region) are not stained. That is, by locally spraying a medium containing calcein, the cells are partially stained. From this, it can be understood that a medium containing a drug or the like can be locally supplied to the adherent cells, and the drug or the like can be selectively allowed to act on the cells.

[0034] [Modification Example 1] In the above-described embodiment, the medium or the medium containing a drug or the like was sprayed by ultrasonic waves, but it is not limited to this. For example, cells may be mixed in the medium, and the medium in which the cells are mixed may be sprayed by irradiation with ultrasonic waves, and cell culture may be performed by utilizing the fact that the atomized medium adheres to the adherent cells or the culture surface of the culture vessel. In this case, it becomes possible to form a cell layer that stacks on the adherent cells with respect to the adherent cells that grow along the culture surface, or to form a heterogeneous cell layer by mixing a different type of cell than the adherent cells in the medium. Also, after performing cell culture by spraying the medium in which cells are mixed by irradiation with ultrasonic waves and utilizing the fact that the atomized medium adheres to the adherent cells or the culture surface of the culture vessel, the medium in which cells are mixed may be sprayed again by irradiation with ultrasonic waves to perform further cell culture. That is, cell culture by spraying the medium in which cells are mixed can be performed a plurality of times. Thereby, it becomes possible to stratify the cells to be cultured according to the purpose.

[0035] Note that the present invention can be appropriately modified, improved, etc. within the scope in which the effects of the present invention are achieved, and is not limited to the above-described embodiment. For example, in Verification 3 described above, it was described assuming that a drug or the like is locally allowed to act on cells with a medium containing a drug or the like, but by supplying the medium by the method shown in Verification 3, local cell culture may be promoted.

[0036] Also, in the above-described embodiment, the specific ultrasonic irradiation conditions can be made variously different depending on the amount of the medium, the depth of the medium layer, the thickness of the culture vessel, the size of the droplets of the medium to be sprayed, and the like. For example, in the above-described embodiment, when irradiating ultrasonic waves intermittently, it was found that when the irradiation time was 20 [ms] or less, vibration was difficult to be transmitted to the culture medium, and there was a lower limit value for the irradiation time of the ultrasonic waves. Further, in Verification 1 described above, when irradiating ultrasonic waves at 2.0 [MHz] or less, it was found that the droplets of the culture medium to be sprayed were large and detachment of adherent cells occurred. That is, it was found that the size of the droplets of the culture medium to be sprayed changes depending on the frequency of the ultrasonic waves to be irradiated. Since the thresholds at which these events occur vary depending on the device configuration to which the present invention is applied, etc., it is useful to verify specific thresholds through experiments, etc. and use them for control.

[0037] Also, in the above-described embodiment, the drug contained in the culture medium and sprayed can be a pharmaceutical (such as an asthma drug) that a patient inhales and uses. Thereby, an environment close to the case where the pharmaceutical is actually used can be formed, and drug screening can be performed.

[0038] Also, the present invention can be implemented by appropriately combining the above-described embodiment and modification examples. The processing for control in the above-described embodiment can be executed by either hardware or software. That is, it suffices that the cell processing apparatus 1 is provided with a function capable of executing the above-described processing, and the functional configuration and hardware configuration for realizing this function are not limited to the above examples.

[0039] Note that the above embodiment shows an example to which the present invention is applied, and does not limit the technical scope of the present invention. That is, the present invention can be variously changed, such as omission and substitution, without departing from the gist of the present invention, and it is possible to adopt various embodiments other than the above-described embodiment. Various embodiments and their modifications that the present invention can adopt are included in the invention described in the claims and the equivalent scope thereof.

[0040] The cell processing apparatus 1 configured as described above includes an ultrasonic vibrator 13 and a control unit 16. The ultrasonic vibrator 13 irradiates the culture medium in the flask 15 with ultrasonic waves in a state where cells are adhered inside and the cells are exposed to the atmosphere. The control unit 16 controls the irradiation of ultrasonic waves by the ultrasonic vibrator 13. This makes it possible to culture the cells without immersing them in the culture solution.

[0041] The control unit 16 intermittently generates ultrasonic waves in the ultrasonic vibrator 13. This makes it possible to continuously supply fresh culture medium (atomized culture medium) to the cells while suppressing an increase in the temperature of the culture medium.

[0042] The culture medium in the flask 15 may contain a drug. This makes it possible to act the drug or the like contained in the culture medium on the cells, and to perform cell screening or the like.

[0043] In the flask 15, a gradient is formed in the distribution of the atomized culture medium. This makes it possible to give a gradient of action when acting the drug or the like contained in the culture medium on the cells, and to appropriately perform cell screening or the like.

[0044] The culture medium is mixed with cells of the same or different types as the cells adhered in the flask 15. This makes it possible to form a cell layer on top of the cell layer adhered in the flask 15.

[0045] After the first culture step of forming a cell layer cultured by the culture medium sprayed by the irradiation of ultrasonic waves, the control unit 16 executes a second culture step of forming a further cell layer cultured by the culture medium sprayed by the irradiation of ultrasonic waves. This makes it possible to stack layers of cells of the same or different types.

[0046] The cell processing apparatus 100 also includes a nonwoven fabric F, an ultrasonic vibrator 113, and a control unit 116. The nonwoven fabric F supplies the culture medium from a supply source (centrifuge tube C) of the culture medium. The ultrasonic vibrator 113 sprays the culture medium onto a specific region of a substrate (slide glass G) to which cells are adhered on the culture surface by irradiating the culture medium supplied by the nonwoven fabric F with ultrasonic waves. The control unit 116 controls the irradiation of ultrasonic waves by the ultrasonic vibrator 113. Thereby, it becomes possible to locally supply a culture medium containing a drug or the like to cells on the culture surface and to selectively cause the drug or the like to act on the cells.

Explanation of reference numerals

[0047] 1,100 cell processing apparatus, 11 pool, 12 base, 13,113 ultrasonic vibrator, 14 conducting wire, 15 flask, 16,116 control unit, G slide glass, C centrifuge tube, F nonwoven fabric

Claims

1. Ultrasonic irradiation means for irradiating ultrasonic waves to a culture medium in a culture vessel in which cells are adhered inside and the cells are exposed to the atmosphere, Control means for controlling the irradiation of ultrasonic waves by the ultrasonic irradiation means, Comprising, The control means intermittently generates ultrasonic waves in the ultrasonic irradiation means at an irradiation time and a stop time such that an atomized culture medium is continuously supplied into the culture vessel and the temperature of the culture medium does not deviate from a set temperature range, and controls the temperature of the culture medium. A cell processing apparatus characterized by the above.

2. The cell processing apparatus according to claim 1, characterized in that the culture medium in the culture vessel contains a drug.

3. The cell processing apparatus according to claim 1 or 2, characterized in that a gradient is formed in the distribution of the atomized culture medium in the culture vessel.

4. The culture medium is mixed with cells of the same or different types as the cells adhered in the culture vessel, and by the irradiation of the ultrasonic waves, in addition to the cells adhered in the culture vessel, the cells of the same or different types are cultured in the culture vessel. The cell processing apparatus according to any one of claims 1 to 3, characterized by the above.

5. After the first culture step of forming a layer of cells cultured by the culture medium sprayed by the irradiation of ultrasonic waves by the control means, a second culture step of forming a further layer of cells cultured by the culture medium sprayed by the irradiation of ultrasonic waves is performed. The cell processing apparatus according to claim 4, characterized in that the cells to be cultured are stratified.

6. Culture medium supply means for supplying a culture medium containing a drug from a culture medium supply source, Ultrasonic irradiation means for supplying the culture medium by locally irradiating ultrasonic waves for spraying the culture medium to a specific region where the drug contained in the culture medium supplied by the culture medium supply means acts, Control means for controlling the irradiation of ultrasonic waves by the ultrasonic irradiation means so that the drug acts locally on the cells in the specific region in a substrate having cells adhered to the culture surface, A cell processing apparatus characterized by comprising the above.

7. A culture step of culturing cells inside a culture vessel, An ultrasonic irradiation step of irradiating ultrasonic waves to a culture medium in the culture vessel in which cells are adhered inside and the cells are exposed to the atmosphere by the culture step, Including, In the ultrasonic irradiation step, an atomized culture medium is continuously supplied into the culture vessel, and ultrasonic waves are intermittently generated for an irradiation time and a stop time such that the temperature of the culture medium does not deviate from a set temperature range, and the temperature of the culture medium is controlled. A cell treatment method characterized by the above.

8. A culture medium supply step of supplying a culture medium containing a drug from a culture medium supply source; The culture medium is supplied by locally irradiating ultrasonic waves for spraying the culture medium onto a specific region where the drug contained in the culture medium supplied by the culture medium supply step acts, and in a substrate having cells adhered to the culture surface, the ultrasonic irradiation step of locally causing the drug to act on the cells in the specific region; A cell treatment method characterized by including the above.

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