JP7704512B2 - Cell processing device and cell processing method - Google Patents

Description

Applicable under Article 30, Paragraph 2 of the Patent Act. Announced on August 20, 2018 in the Program Book of the EMBS Micro and Nanoengineering in Medicine Conference 2018 at the following address: http://mnm. embs. org/2018/wp-content/uploads/sites/15/2018/11/MNM-18-Final-Program.pdf

The present invention relates to a cell processing device and a cell processing method.

Cell culture can be broadly divided into two-dimensional culture, in which cells are attached to a culture surface such as the bottom of a dish, and three-dimensional culture, in which cells are suspended in a culture solution. Two-dimensional culture is used for proliferation culture, etc., because it is easy to make the culture environment uniform. On the other hand, in three-dimensional culture, cells adhere to each other, so it is often used to generate cell tissues in vitro due to cell-cell interactions, etc. Such two-dimensional and three-dimensional cell culture is widely used in cell engineering and medical research (see, for example, Patent Document 1).

JP 2016-077229 A

In recent years, research into drug discovery and screening using cultured cells has become more active, and there is growing expectation for experimental systems that can verify in vitro the effects of administering a nebulized drug to the lungs, for example.
However, in the above-mentioned conventional culture methods, cells are cultured in a state immersed in a culture solution, which does not necessarily reproduce the in vivo condition, making it difficult to construct an in vitro experimental system for aerosolized medication.

The purpose of the present invention is to enable cells to be cultured without being immersed in culture medium.

In order to achieve the above object, a cell processing device according to one embodiment of the present invention comprises:
an ultrasonic irradiation means for irradiating ultrasonic waves to a culture medium in a culture vessel to which cells are attached and which is exposed to the atmosphere;
A control means for controlling the irradiation of ultrasonic waves by the ultrasonic irradiation means;
The present invention is characterized by comprising:

According to the present invention, it is possible to culture cells without immersing them in a culture medium.

FIG. 1 is a diagram showing the procedure of a cell treatment method according to one embodiment of the present invention. FIG. 1 is a schematic diagram showing the concept of a culture medium spraying process. 1 is a diagram showing the temperature change of a liquid when ultrasonic waves are continuously irradiated to the liquid and when ultrasonic waves are intermittently irradiated to the liquid. FIG. 1 is a schematic diagram showing a configuration of a cell processing apparatus 1 according to one embodiment of the present invention. 1 is a flow chart showing steps of a cell culture treatment. 1 is a schematic diagram showing a state in which ultrasonic waves are transmitted from an ultrasonic transducer 13 to a culture medium in a flask 15. FIG. FIG. 1 is a schematic diagram showing a culture mode (spray culture) in which adherent cells are cultured by supplying mist-like medium into a flask 15. FIG. 1 is a schematic diagram showing a culture form (static culture) in which cells are cultured by immersing them in a medium. FIG. 1 is a schematic diagram showing a culture form (vertical culture) in which adherent cells are located on the side of a flask 15. FIG. 1 shows the cell counts in static culture, spray culture, and vertical culture. FIG. 1 shows the staining state of adherent cells. 1 is a schematic diagram showing the distribution of atomized medium in a flask 15. FIG. FIG. 13 is a schematic diagram showing an experimental procedure in Verification 3. FIG. 1 shows the staining state of adherent cells. FIG. 1 is a diagram showing a culture surface onto which a culture medium is sprayed.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
[Basic Concept of the Invention]
In the present invention, the medium is atomized by ultrasonic vibration to supply minute droplets of the medium to cells attached to a substrate such as a culture vessel. At this time, the generation pattern of the ultrasonic vibration is controlled to prevent the temperature of the medium from increasing due to the irradiation of ultrasonic waves.
This makes it possible to culture the cells without immersing them in a culture solution.
That is, according to the present invention, cells adhered to a substrate such as a culture vessel can be cultured in an environment exposed to the atmosphere, making it possible to culture cells of organs exposed to the atmosphere, such as the alveoli, bronchi, and cornea (mucosal epithelial cells, etc.), by more appropriately reproducing the conditions within the body.
Specific embodiments of the present invention will now be described.

[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, in the cell processing method according to the present embodiment, first, cells are cultured in a medium on a culture surface of a culture vessel such as a flask by various existing culture methods (adherent cell culture step). The cells cultured in the adherent cell culture step are in a state of being adhered to the culture surface of the culture vessel such as a flask.

Next, in the space where the adherent cells cultured in the adherent cell culture process are present, ultrasonic waves are irradiated onto the medium by an ultrasonic transducer, and the medium is sprayed into the air inside the culture vessel (medium spraying process). The tiny droplets of medium sprayed in the medium spraying process (mist-like medium) are supplied to the cells adhered to the culture surface.

FIG. 2 is a schematic diagram showing the concept of the culture medium spraying process.
As shown in Fig. 2, in the medium spraying process, ultrasonic waves are applied to a liquid medium stored in a culture vessel such as a flask by an ultrasonic transducer. The medium is then sprayed into the air in the vessel, and the mist-like medium is supplied to the cells attached to the culture surface. This allows the mist-like medium to function as a medium for the cells attached to the culture surface, making it possible to supply the medium to the cells without immersing them in the medium.

In this embodiment, in the culture medium spraying step, intermittent irradiation of the culture medium with ultrasonic waves is continued for a set period of time (e.g., 24 hours), and the adherent cells are cultured until they reach the desired state.
The reason why ultrasound is irradiated to the medium intermittently is that if ultrasound is continuously supplied to the medium, the temperature of the medium will rise and deviate from the temperature suitable for cell culture.

FIG. 3 is a diagram showing the temperature change of a liquid when ultrasonic waves are continuously irradiated to the liquid and when ultrasonic waves are intermittently irradiated to the liquid.
In addition, FIG. 3 shows an example in which water is used as the liquid and pulsed ultrasonic waves are irradiated as the intermittent ultrasonic waves.

As shown in FIG. 3, when ultrasonic waves are continuously irradiated onto a liquid, the temperature of the liquid increases with increasing irradiation time, from an initial temperature of 20° C. to over 60° C. after about 3 minutes of irradiation.
In contrast, when ultrasound is irradiated intermittently to the liquid, even if the irradiation time is increased, the temperature of the liquid is limited to a rise from the initial 20°C to approximately 35°C, and it is found that a temperature suitable for cell cultivation is maintained.

In this way, in the culture medium spraying process, by intermittently irradiating the culture medium with ultrasound, it is possible to continuously supply fresh culture medium (atomized culture medium) to the adherent cells while suppressing an increase in the temperature of the culture medium.
Therefore, the cells can be cultured for the required time while being exposed to the atmosphere and with appropriate temperature control, which more appropriately reproduces the conditions inside the body and makes it easy to produce cells of organs exposed to the atmosphere (such as mucosal epithelial cells).

[Cell Processing Device]
Next, the cell processing apparatus used in this embodiment will be described.
[composition]
FIG. 4 is a schematic diagram showing the configuration of a cell processing apparatus 1 according to one embodiment of the present invention.
As shown in FIG. 4, the cell processing apparatus 1 includes a pool 11 , a base 12 , an ultrasonic transducer 13 , a lead 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) that transmits ultrasonic waves.

The base 12 is a rectangular parallelepiped member on which the flask 15 is placed, and in this embodiment, two bases 12 are installed at positions that support one end and the other end of the bottom surface of the flask 15.
The ultrasonic transducer 13 generates ultrasonic waves when a voltage is applied via a conductor 14. The ultrasonic transducer 13 is installed in a space formed between two bases 12 on the bottom surface of the pool 11.
One end of the conductor 14 is connected to the electrode of the ultrasonic transducer 13, and the other end is connected to the control unit 16 (more specifically, a driver). The conductor 14 is provided for each electrode of the ultrasonic transducer 13.

The flask 15 stores a culture medium inside, and has pre-cultured cells attached to the inside side of the flask 15. The flask 15 is filled with air, creating an environment similar to that in the living body where cells of an organ exposed to air survive.
The control unit 16 is configured by an information processing device or signal processing device such as a PC (Personal Computer), a microcomputer, or a function generator, and controls the driving of the ultrasonic transducer 13 according to preset experimental conditions. Specifically, the control unit 16 controls the pattern of intermittent ultrasonic irradiation, the duration of intermittent ultrasonic irradiation, the voltage applied to the ultrasonic transducer 13, etc.

[Cell culture treatment]
Next, a process for culturing cells using the cell processing apparatus 1 (cell culture process) will be described.
FIG. 5 is a flow chart showing the steps of the cell culture treatment.
As shown in FIG. 5, when a cell culture process is performed, first, as an adherent cell culture step, cells are statically cultured in a medium on the side surface of a 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 medium and cultured statically for 24 hours.
This results in the cells being attached to the inner side of the flask 15 .

Next, the bottom surface of the flask 15 obtained in step S1 is placed on the base 12 in the pool 11 (step S2).
An ultrasonic transducer 13 is installed between the bases 12 (at the center position) on the bottom surface of the pool 11. The pool 11 is filled with water up to a position higher than the top surface of the bases 12, and the bottom surface of the flask 15 placed on the bases 12 is in contact with the water.

Next, ultrasonic waves are intermittently applied to the bottom surface of the flask 15 by the ultrasonic transducer 13 (step S3).
FIG. 6 is a schematic diagram showing a state in which ultrasonic waves are transmitted from the ultrasonic transducer 13 to the medium in the flask 15.
As shown in FIG. 6, the space between the bottom of flask 15 and ultrasonic transducer 13 is filled with water, so that the ultrasonic waves irradiated from ultrasonic transducer 13 reach the bottom of flask 15 without significant attenuation, pass through flask 15, and vibrate the culture medium.
In step S3, for example, the pattern of irradiating ultrasonic waves for 0.1 [s] and stopping the irradiation for 4.9 [s] is continued for a set time (here, 24 hours). In addition, 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.

FIG. 7 is a schematic diagram showing a culture mode (spray culture) in which a mist of 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 created in which misted 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). If necessary, 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.

[Verification 1]
In order to verify the effects of the above-mentioned cell culture treatment, instead of spray culture, a culture form in which cells were immersed in the medium and cultured (static culture) and a culture form in which adherent cells were positioned on the side of flask 15 (vertical culture) were performed.
FIG. 8 is a schematic diagram showing a culture form (static culture) in which cells are cultured by immersing them in a medium.
For static culture, the flask 15 was placed horizontally, 1.8×10 ⁵ cells were seeded in 3.0 mL of medium, and static culture was carried out for 24 hours, followed by another static culture for 24 hours.

FIG. 9 is a schematic diagram showing a culture form (vertical culture) in which adherent cells are located on the side surface of a flask 15.
For vertical culture, flask 15 was placed horizontally, 1.8 × ¹⁰ cells were seeded in 3.0 mL of medium, and static culture was performed for 24 hours. After that, flask 15 was placed vertically with the adherent cells positioned on the side of flask 15, and left for 24 hours.
In the spray culture (see FIG. 7 ), as described above, the flask 15 was placed horizontally, 1.8× ¹⁰ cells were seeded in 3.0 mL of medium, and static culture was performed for 24 hours, after which intermittent ultrasonic irradiation was continued for 24 hours.

FIG. 10 shows the cell counts 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 is understood that cells are not cultured by simply placing the flask 15 vertically, but are cultured by the atomized medium.
It can also be seen that the number of live cells in spray culture is increased to about 3.4 times the number of cells seeded. In the case of static culture, the number of cells is about doubled in the first 24 hours, and then doubled again in the next 24 hours, resulting in about four times the number of cells seeded. In contrast, it can be seen that more than 70% of the live cells are cultured in spray culture compared to static culture.

In this manner, in the cell culture treatment according to this embodiment, the cells can be cultured without being immersed in a culture solution.
That is, according to the cell culture treatment of this embodiment, cells adhered to a substrate such as a culture vessel can be cultured in an environment exposed to the atmosphere, so that cells of organs exposed to the atmosphere, such as the alveoli, bronchi, and cornea (mucosal epithelial cells, etc.) can be cultured by more appropriately reproducing the conditions in the living body.

[Verification 2]
In order to verify whether or not the above-described cell processing apparatus 1 can act on cells with chemicals and the like contained in the culture medium, a cell staining experiment was carried out.
Specifically, adherent cells were cultured in the same manner as in step S1 of the cell treatment method shown in Fig. 5, and a medium with a calcein concentration of 0.1% was ultrasonically sprayed for 1 hour in the form of spray culture shown in Fig. 7. The ultrasonic irradiation conditions were the same as in step S3 of the cell treatment method shown in Fig. 5.

FIG. 11 shows the staining state of adherent cells.
As shown in Fig. 11, the surviving adherent cells (viable cells) are stained by the calcein contained in the misted medium (white in Fig. 11). That is, it is understood that the chemicals contained in the medium can be made to act on the surviving adherent cells.
In addition, in FIG. 11, the adherent cells in the lower part of the side surface (culture surface) of the flask 15 are stained less than the adherent cells in the upper part.
That is, when spray culture is performed, a gradient (change) is imparted to the amount of medium supplied per adherent cell.
This is believed to be due to the presence of a gradient in the distribution of the medium atomized by ultrasonic irradiation.

FIG. 12 is a schematic diagram showing the distribution of the atomized medium in the flask 15. As shown in FIG.
As shown in FIG. 12, in flask 15, the mist of culture medium diffuses upward from the liquid culture medium generated by ultrasonic vibration, filling the upper part of flask 15 and having difficulty distributing to the lower part.
Therefore, when a chemical or the like contained in the medium is allowed to act on cells, a gradient of action can be provided, making it possible to appropriately screen cells, etc.
In addition, when providing a gradient of the effect of a chemical or the like in this manner, if an excessive amount of mist medium is supplied, the mist medium will be distributed throughout the flask 15, so it is necessary to control the amount of mist medium supplied so that it is within an appropriate range.

[Verification 3]
In the above-mentioned cell treatment method, a cell staining experiment was carried out to verify whether a medium containing a chemical or the like can be locally supplied to adherent cells on the culture surface and the chemical or the like can be selectively made 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 (glass slide G serving as a substrate) surrounded by a silicone wall, and static culture was carried out for 24 hours.
Then, the silicone wall was removed from the slide glass G, and in the cell processing device 100 having the configuration shown in Figure 13, the slide glass G with the cells adhered thereto was fixed to the stage, and the culture medium was supplied by permeating the nonwoven fabric F from the centrifuge tube C, which serves as a culture medium supply source.
Furthermore, the culture medium supplied through the nonwoven fabric F was irradiated with ultrasonic waves by the ultrasonic transducer 113, so that the culture medium was sprayed onto a localized area of the slide glass G.

In addition, in verification 3, 1.0×10 ⁶ cells were seeded on a glass culture surface surrounded by a silicone wall together with 5.0 mL of culture medium, and the cells were attached to the glass culture surface by static culture for 24 hours. Then, 50.0 mL of culture medium with a calcein concentration of 0.1% was stored in centrifuge tube C, and the culture medium permeated from centrifuge tube C to nonwoven fabric F was sprayed by ultrasonic waves. At this time, the pattern of irradiating ultrasonic waves for 0.5 s and stopping the irradiation for 0.5 s was continued for 1 hour. The ultrasonic irradiation conditions were a frequency of 1.97 MHz, a voltage of 368.2 Vpp, and a waveform of sine wave.

FIG. 14 shows the staining state of adherent cells.
FIG. 15 is a diagram showing the culture surface onto which the medium has been sprayed.
It should be noted that FIG. 14 and FIG. 15 show the same region on the culture surface.
As shown in FIG. 14, cells present in the area where the medium was sprayed (viable adherent cells) are stained by the calcein contained in the mist of medium (white in FIG. 14), whereas cells present in the area where the medium was not sprayed (within the dashed area) were not stained.
That is, by locally spraying the medium containing calcein, the cells are partially stained.
This shows that a medium containing a drug or the like can be locally supplied to adherent cells, allowing the drug or the like to act selectively on the cells.

[Modification 1]
In the above-described embodiment, the culture medium or the culture medium containing the chemicals or the like is sprayed by ultrasonic waves, but the present invention is not limited to this.
For example, cells may be mixed into a culture medium, and the medium containing the cells may be sprayed with ultrasonic waves, and the mist of medium may adhere to the adherent cells or the culture surface of a culture vessel, thereby culturing the cells.
In this case, it is possible to form a cell layer that is layered on the adherent cells that grow along the culture surface, or to mix cells of a different type than the adherent cells into the culture medium to form a heterogeneous cell layer.
In addition, the culture medium containing cells may be sprayed by irradiating ultrasonic waves, and the atomized culture medium may adhere to the adherent cells or the culture surface of the culture vessel. After culturing the cells, the culture medium containing cells may be sprayed by irradiating ultrasonic waves again to further culture the cells. In other words, the culture of the cells by spraying the culture medium containing cells may be performed multiple times.
This allows the cultured cells to be layered according to purpose.

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, in the above-mentioned Verification 3, it was assumed that a culture medium containing a chemical or the like would be used to locally act on cells, but it is also possible to promote local cell culture by supplying culture medium using the method shown in Verification 3.

In addition, in the above-mentioned embodiment, the specific ultrasonic irradiation conditions can vary depending on the amount of culture medium, the depth of the culture medium layer, the thickness of the culture vessel, the size of the culture medium droplets to be sprayed, etc.
For example, in the above embodiment, when ultrasonic waves are irradiated intermittently, it was found that the vibration is difficult to transmit to the culture medium when the irradiation time is 20 [ms] or less, and that there is a lower limit to the ultrasonic irradiation time. Also, in the above-mentioned verification 1, it was found that when ultrasonic waves are irradiated at 2.0 [MHz] or less, the droplets of the sprayed culture medium are large, and the adherent cells are peeled off. In other words, it was found that the size of the droplets of the sprayed culture medium changes depending on the frequency of the irradiated ultrasonic waves.
The thresholds at which these events occur vary depending on the configuration of the device to which the present invention is applied, and so it is useful to verify specific thresholds through experiments or the like before using them for control.

In the above-described embodiment, the drug contained in the culture medium and sprayed can be a medicine (such as an asthma drug) that is inhaled by the patient.
This makes it possible to perform drug screening in an environment similar to that in which pharmaceuticals are actually used.

Furthermore, the present invention can be implemented by appropriately combining the above-described embodiments and modified examples.
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 1 is provided with a function capable of executing the above-mentioned processing, and the functional and hardware configurations for realizing this function are not limited to the above-mentioned examples.

The above embodiment shows an example of the 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 by omission or substitution, 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.

The cell processing device 1 configured as above includes an ultrasonic transducer 13 and a control unit 16 .
The ultrasonic transducer 13 irradiates ultrasonic waves to the culture medium in the flask 15 to which the cells are attached and which is exposed to the atmosphere.
The control unit 16 controls the irradiation of ultrasonic waves by the ultrasonic transducer 13 .
This makes it possible to culture the cells without immersing them in a culture solution.

The control unit 16 causes the ultrasonic transducer 13 to intermittently generate ultrasonic waves.
This makes it possible to continuously supply fresh medium (atomized medium) to the cells while suppressing an increase in the temperature of the medium.

The medium in the flask 15 may contain a drug.
This allows the chemicals contained in the medium to act on the cells, making it possible to screen the cells.

Within the flask 15, a gradient is formed in the distribution of the atomized medium.
This makes it possible to provide a gradient of action when a chemical or the like contained in the medium is allowed to act on cells, thereby making it possible to appropriately screen cells, etc.

The medium contains cells of the same type or a different type to the cells adhered to the flask 15 .
This allows a cell layer to be formed over the layer of cells adhered within the flask 15 .

After a first culture step of forming a layer of cells cultured by the culture medium sprayed by ultrasonic irradiation, the control unit 16 executes a second culture step of forming a further layer of cells cultured by the culture medium sprayed by ultrasonic irradiation.
This allows for the formation of layers of homogeneous or heterogeneous cells.

The cell processing device 100 also includes a nonwoven fabric F, an ultrasonic transducer 113, and a control unit 116.
The nonwoven fabric F supplies the medium from a medium supply source (centrifuge tube C).
The ultrasonic transducer 113 irradiates the culture medium supplied by the nonwoven fabric F with ultrasonic waves, thereby spraying the culture medium onto a specific area of the substrate (glass slide G) having cells adhered to its culture surface.
The control unit 116 controls the irradiation of ultrasonic waves by the ultrasonic transducer 113 .
This makes it possible to locally supply a medium containing a drug or the like to cells on the culture surface, allowing the drug or the like to act selectively on the cells.

1,100 Cell processing device, 11 Pool, 12 Base, 13,113 Ultrasonic transducer, 14 Wire, 15 Flask, 16,116 Control unit, G Slide glass, C Centrifuge tube, F Nonwoven fabric

Claims (8)

an ultrasonic irradiation means for irradiating ultrasonic waves to a culture medium in a culture vessel to which cells are attached and which is exposed to the atmosphere;
A control means for controlling the irradiation of ultrasonic waves by the ultrasonic irradiation means;
Equipped with
The control means controls the temperature of the culture medium by causing the ultrasonic irradiation means to intermittently generate ultrasonic waves with irradiation time and stop time such that a mist of culture medium is continuously supplied into the culture vessel and the temperature of the culture medium does not deviate from a set temperature range. The cell processing device according to claim 1, characterized in that the medium in the culture vessel contains a chemical. The cell processing device 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. The cell processing device according to any one of claims 1 to 3, characterized in that the medium is mixed with cells of the same or different species as the cells adhered to the culture vessel, and the cells of the same or different species are cultured in the culture vessel in addition to the cells adhered to the culture vessel by irradiating the ultrasonic waves. The cell processing device according to claim 4, characterized in that the control means performs a second culture process to form a further layer of cells cultured in the culture medium sprayed by ultrasonic irradiation after a first culture process to form a layer of cells cultured in the culture medium sprayed by ultrasonic irradiation, thereby stratifying the cultured cells. a medium supply means for supplying a medium containing a drug from a medium source;
an ultrasonic irradiation means for locally irradiating ultrasonic waves for spraying the medium to a specific region where a chemical contained in the medium supplied by the medium supply means is to act, thereby supplying the medium;
a control means for controlling the irradiation of ultrasound by the ultrasound irradiation means so that the drug acts locally on the cells in the specific region of the substrate having the cells adhered to the culture surface ;
A cell processing device comprising: A culture step of culturing cells inside the culture vessel;
an ultrasonic irradiation step of irradiating ultrasonic waves to the medium in the culture vessel to which the cells have been attached by the culture step and in which the cells are exposed to the atmosphere;
Including,
The cell treatment method is characterized in that, in the ultrasonic irradiation process, mist-like culture medium is continuously supplied into the culture vessel, and ultrasonic waves are intermittently generated with irradiation times and stop times that do not cause the temperature of the culture medium to deviate from a set temperature range, thereby controlling the temperature of the culture medium. A medium supplying step of supplying a medium containing a drug from a medium supply source;
an ultrasonic irradiation step of locally irradiating ultrasonic waves for spraying the medium to a specific region where a chemical contained in the medium supplied in the medium supplying step is to act, and locally acting the chemical on the cells in the specific region of the substrate having cells adhered to the culture surface;
A cell treatment method comprising the steps of:

Images