JP6956956B2 - Cell production method - Google Patents

Description

The present invention relates to a cell production method and a cell production apparatus.

In recent years, there are increasing expectations for the application of regenerative medicine to treatment, including iPS cells. Since a large amount of cells are required in the treatment applying regenerative medicine, it is desired to efficiently and stably supply a large amount of cells. In addition, cells can be roughly classified into floating cells and adhesive cells, but since most of the living tissue is composed of adhesive cells, a particularly large amount of adhesive cells is required.
In the conventional cell culture, the target cells are obtained through the steps of culturing the cells in the dish, exfoliating the cells from the dish, collecting the cells, and washing the cells.
In such a cell culture method, in order to mass-produce cells, it is necessary to shorten the time required for cell production, simplify the cell production process, and ensure the quality of the cells produced. Is required.
The technique described in Patent Document 1 is known as a method for producing cells capable of suppressing deterioration of cell quality.
In the technique described in Patent Document 1, a method of exfoliating cells by temperature stimulation is realized, and cells are exfoliated without using trypsin. Therefore, according to the technique described in Patent Document 1, it is considered that it is possible to suppress a decrease in cell proliferation and activity due to lysis of a protein by trypsin.

Japanese Unexamined Patent Publication No. 2015-216868

However, in the method of exfoliating cells by temperature stimulation, it is necessary to lower the temperature around the cells at the time of exfoliating the cells, and the decrease in temperature may affect the proliferative property of the cells. In addition, the method of exfoliating cells without using trypsin tends to be more costly and technically difficult than the method using trypsin.
Therefore, in the conventional technique, it has been difficult to mass-produce cells efficiently and with high quality.

An object of the present invention is to mass-produce cells more efficiently and with high quality.

In order to achieve the above object, the cell production method of one aspect of the present invention is:
The first step of preparing a culture substrate having an amorphous carbon coating, and
A second step of culturing cells on the culture substrate prepared in the first step, and
The third step of exfoliating the cultured cells by applying acoustic radiation pressure to the cells in the culture substrate cultured in the second step, and
including.

According to the present invention, cells can be mass-produced more efficiently and with high quality.

It is a schematic diagram which shows the structure of the DLC coating dish 100 used in this embodiment. It is a schematic diagram which shows the whole structure of the cell production apparatus 1 which concerns on 1st Embodiment of this invention. It is a schematic diagram which shows the example of use form of the cell production apparatus 1 which concerns on 1st Embodiment of this invention. It is a figure which shows typically the state transition in the DLC coating dish 100 at the time of operation of a cell production apparatus 1. It is a schematic diagram which compares and shows the procedure of the existing cell production method, and the procedure of the cell production method in this invention. It is a schematic diagram which shows the result of having performed the cell recovery experiment in the case of using the cell production method which concerns on this invention, and the case of using trypsin. It is a schematic diagram which shows the result of having performed the cell colonization experiment in the case of using the trypsin and the cell production method which concerns on this invention. It is a micrograph which photographed the surface of the cell in the case of using the cell production method and trypsin which concerns on this invention. It is a schematic diagram which shows the structural example of the vibrating member 13 which consists of a Langevin type ultrasonic vibrator.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[Basic Concept of the Present Invention]
In the cell production method according to the present invention, a predetermined amorphous carbon film is formed on the surface of a culture dish, a cell medium is formed on the culture dish, and adhesive cells are cultured. The predetermined amorphous carbon film formed here includes a film of DLC (Diamond-Like Carbon) or PLC (Polymer-Like Carbon), but in the present embodiment, DLC is an example of the amorphous carbon film. A film shall be formed on the surface of the culture dish. Hereinafter, the culture dish having this structure is appropriately referred to as "DLC coating dish". Then, ultrasonic vibration is input from the outer bottom surface (back surface) of the DLC coating dish by an oscillator, and the cultured cells are separated by acoustic radiation pressure.
At this time, as a device for generating acoustic radiation pressure (ultrasonic vibration), for example, a vibration generator made of an ultrasonic vibrator or the like using a piezoelectric element can be used.
In the DLC coating dish used in the present embodiment, the cell colonization property is a surface property suitable for culturing, and the cell detachability when acoustic radiation pressure is applied is at least a certain level with respect to the non-deposition of DLC. It has surface characteristics.
Therefore, in the production of cells, by inputting ultrasonic vibration to the DLC coating dish, the cells are easily separated from the DLC coating by acoustic radiation pressure, and the cultured cells are detached without using an enzyme such as trypsin.・ Can be collected.
Therefore, according to the present invention, in the cell detachment step and the recovery step, it is not necessary to soak or wash with an enzyme such as trypsin, so that the step is simplified, the production time is shortened, and the produced cells are highly activated. Effects such as high quality can be realized.
That is, according to the present invention, cells can be mass-produced more efficiently and with high quality.
Hereinafter, the case where the cell to be cultured is CHO (Chinese Hamster Ovary) will be described, but various adhesive cells such as iPS cells can be targeted for culture. Further, in the present invention, as the medium used for culturing cells, either a serum-free medium or a medium to which serum is added can be adopted.

[First Embodiment]
[Composition of culture dish]
The culture dish (DLC coated dish) used in the present embodiment has a structure in which a predetermined DLC film is formed on the surface of the dish.
FIG. 1 is a schematic view showing the configuration of the DLC coating dish 100 used in the present embodiment.
In FIG. 1, a vertical cross-sectional view of the DLC coating dish 100 is shown, and a state in which a DLC coating is formed on the inner peripheral surface and the inner bottom surface is schematically shown. The film thickness of the DLC film in FIG. 1 is exaggerated from the actual film thickness for convenience of explanation.

In FIG. 1, the DLC coating dish 100 is obtained by preparing, for example, a culture dish of a glass material and uniformly forming a DLC film by a coating method such as CVD (Chemical Vapor Deposition). If DLC coating is possible, it is also possible to use a culture dish other than the glass material.

The DLC coating dish 100 in the present embodiment has a film form in which, when ultrasonic vibration is input, the cultured cells can be easily peeled off by acoustic radiation pressure as compared with a culture dish having no DLC coating. It is necessary to have a film thickness, film type, etc.).
On the other hand, for convenience of cell culture work, it is desirable that the cell culture state in the DLC coating dish 100 can be visually recognized from the outside. For example, the lid of the DLC coating dish 100 does not form a DLC coating (or the DLC coating dish 100). It is preferable that the film thickness is made thinner than that of the DLC coating dish 100) and that the morphology (thickness, film type, etc.) of the film formed on the DLC coating dish 100 is such that the cell culture state can be visually recognized from the outside. ..

By forming the DLC film on the surface of the culture dish in this way, the characteristics are changed from the surface of the culture dish before the DLC film is formed, and the cells have appropriate fixing property and acoustic radiation pressure is applied. It is possible to construct a DLC coating dish 100 having higher peelability in the case.

In the present embodiment, the DLC coated dish 100 is used to culture CHO cells and then exfoliated by acoustic radiation pressure to collect the exfoliated cells. Then, the collected cells are reseeded and recultured in another DLC coated dish 100. By detaching the cells in this way, it is not necessary to use an enzyme such as trypsin, so that the activity of the detached cells becomes high, and the time required for adhesion or the like when the culture is repeated a plurality of times is shortened. The cells can be mass-produced more efficiently and with high quality.
Further, in the present embodiment, one DLC coated dish 100 is used to culture CHO cells and exfoliate them by acoustic radiation pressure once or multiple times, and collect the exfoliated cells to collect a large amount of cultured cells. Can also be produced without the use of enzymes such as trypsin. In this case, after culturing the CHO cells, the cells exfoliated by acoustic radiation pressure were collected from the DLC-coated dish 100, and the cells remaining in the DLC-coated dish 100 were further cultured to once prepare the culture environment. It is possible to reuse the DLC coating dish 100 and perform culturing a plurality of times.

[Structure of cell production device]
Next, the configuration of the cell production device 1 used in the present embodiment will be described.
FIG. 2A is a schematic view showing the overall configuration of the cell production apparatus 1 according to the first embodiment of the present invention. Further, FIG. 2B is a schematic view showing an example of usage of the cell production apparatus 1 according to the first embodiment of the present invention.
As shown in FIGS. 2A and 2B, the cell production device 1 includes a vibration unit 10, a transmitter 20, an amplifier 30, and a controller 40.

The vibration unit 10 includes a lower acrylic plate 11, a felt member 12, a vibrating member 13, a glass plate 14, an upper silicone rubber member 15, an upper acrylic plate 16, bolts 17, and nuts 18. There is. In FIG. 2A, the vibration unit 10 is shown as an exploded view for convenience of explanation.

The lower acrylic plate 11 is made of a square acrylic resin and serves as a base member on which each member of the cell production apparatus 1 is installed. Instead of the lower acrylic plate 11, a plate-shaped member made of polycarbonate or the like may be used.
The felt member 12 is formed in a ring shape and is installed on the lower acrylic plate 11. Further, the felt member 12 supports the vibrating member 13 installed on the felt member 12 from below.
The vibrating member 13 is configured to include an ultrasonic vibrator or the like made of a piezoelectric element, and in the present embodiment, is configured as a ring-shaped member. Further, the lower surface of the vibrating member 13 is supported by the felt member 12. As will be described later, in the present embodiment, the vibrating frequency of the vibrating member 13 is swept so as to cover the range of the natural frequency of the vibrating member 13. As a result, even if there are differences in conditions such as individual differences in the DLC coating dish 100, these conditions can be absorbed and natural vibration (ultrasonic vibration) can be reliably generated in the vibrating member 13. ..

The glass plate 14 is installed on the vibrating member 13 and is composed of a circular member that serves as a stage on which the DLC coating dish 100 is placed. Further, the glass plate 14 is composed of a member having a transmissibility of ultrasonic vibration close to that of the material of the DLC coating dish 100. The reason why the glass plate 14 is composed of a member having an ultrasonic vibration transmission rate close to that of the material of the DLC coating dish 100 is that when the ultrasonic vibration transmission rate is significantly different, sound waves are greatly reflected at the boundary and the DLC coating is applied. This is because ultrasonic vibration cannot be efficiently transmitted to the dish 100.

Here, a liquid such as water or a gel-like substance such as glycerol may be interposed between the glass plate 14 and the DLC coating dish 100. By forming a layer of these liquid or gel-like substances, it is possible to prevent an air layer from being formed between the glass plate 14 and the outer bottom surface (back surface) of the DLC coating dish 100. Since these liquid or gel-like substances have a transmissibility of ultrasonic vibration close to that of the material of the DLC coating dish 100, the reflection of sound waves can be suppressed.

The upper silicone rubber member 15 is made of a square silicone resin and is installed on the glass plate 14. Further, the upper silicone rubber member 15 has a through hole in the center for accommodating the DLC coating dish 100, and supports the peripheral edge of the glass plate 14 from above.
The upper acrylic plate 16 is made of a rectangular acrylic resin and is installed on the upper silicone rubber member 15. Further, the upper acrylic plate 16 has a through hole in the center for accommodating the DLC coating dish 100 and supports the upper silicone rubber member 15 from above, similarly to the upper silicone rubber member 15. Instead of the upper acrylic plate 16, a plate-shaped member made of polycarbonate or the like may be used.

In this way, in the vibration unit 10, the lower acrylic plate 11, the felt member 12, the vibrating member 13, the glass plate 14, the upper silicone rubber member 15, and the upper acrylic plate 16 are laminated and arranged, and are square. Through holes through which bolts 17 are inserted are formed at the four corners of the lower acrylic plate 11, the felt member 12, the upper silicone rubber member 15, and the upper acrylic plate 16. Then, bolts 17 are inserted through these through holes from the upper side of the vibration unit 10, and the lower acrylic plate 11 and the upper acrylic plate are tightened with nuts 18 from the lower side of the lower acrylic plate 11 located at the lowermost portion. Each member is sandwiched and supported between 16 and 16.
Further, in the vibration unit 10 configured in this way, as shown in FIG. 2B, the DLC coating dish 100 is arranged in the through holes formed in the upper silicone rubber member 15 and the upper acrylic plate 16 and is placed on the glass plate 14. The DLC coating dish 100 is placed on the DLC coating dish 100, and the ultrasonic vibration is input with the weight W placed on the lid of the DLC coating dish 100.

The transmitter 20 generates an AC voltage signal (AC signal) at a frequency indicated by an instruction signal from the controller 40, and applies the generated AC signal to the piezoelectric element of the vibrating member 13.
The amplifier 30 amplifies the AC signal generated by the transmitter 20 to a target level. The output of the amplifier 30 is applied as a driving voltage of the piezoelectric element in the vibrating member 13.
The controller 40 is composed of a control device such as a PC (Personal Computer) or a PLC (Programmable Logical Controller). The controller 40 is set with a range of vibration frequencies applied to the vibrating member 13. The range of the vibrating frequency is set so as to cover the range of the natural frequency of the vibrating member 13. Then, the controller 40 outputs an instruction signal to the transmitter 20 to sweep the set vibration frequency range in a predetermined time.

[motion]
Next, the operation of the cell production device 1 will be described.
FIG. 3 is a diagram schematically showing a state transition in the DLC coating dish 100 during operation of the cell production apparatus 1.
Hereinafter, the operation of the cell production apparatus 1 will be described with reference to FIG. 3 as appropriate.
[Basic operation of the device]
Prior to driving the cell production apparatus 1, a medium is formed on the inner bottom surface of the DLC coating dish 100 as a culturing step, and cells are cultured in this medium (state (a) in FIG. 3).
Then, as a peeling step, the cell production apparatus 1 applies acoustic radiation pressure to the cells in the cultured DLC coating dish 100 to peel off the adhesive cells.
Specifically, as the peeling step, the following processing by the cell production apparatus 1 is executed.
The DLC coating dish 100 in which the cells are cultured is placed on the stage (glass plate 14) of the cell production apparatus 1.
In this state, the transmitter 20 of the cell production device 1 generates an AC signal at a set frequency, and the amplifier 30 amplifies the generated AC signal and applies it to the piezoelectric element of the vibrating member 13. At this time, the range of the vibration frequency set in the controller 40 is swept, and an AC signal is generated from the transmitter 20.
Then, the AC signal generated by the transmitter 20 is amplified by the amplifier 30 and applied to the piezoelectric element of the vibrating member 13.

In the present embodiment, the vibrating member 13 generates ultrasonic vibration in the thickness direction by inputting an AC signal.
As a result, ultrasonic vibration is input to the outer bottom surface of the DLC coated dish 100, and acoustic radiation pressure is applied to the cells in the DLC coated dish 100 (state (b) in FIG. 3).
At this time, since the range of the vibration frequency set in the controller 40 is swept, natural vibration is surely generated in the vibrating member 13 installed in the cell production device 1, and the adhesive cells are generated by the acoustic radiation pressure. It is in a state of being peeled off (state (c) in FIG. 3).

As described above, according to the cell production apparatus 1, the adhesive cells can be exfoliated from the DLC coating dish 100 without using an enzyme such as trypsin. Further, since the vibration frequency applied to the vibrating member 13 is swept, even if there are differences in conditions such as individual differences of the DLC coating dish 100, the vibration frequency can be swept. By absorbing these conditions, it is possible to reliably generate natural vibration in the vibrating member 13.
That is, according to the cell production apparatus 1, cells can be mass-produced more efficiently and with high quality.

[Specific example of cell production method]
Next, a specific example of the cell production method using the cell production apparatus 1 will be described.
The following steps are performed while maintaining a sterilized environment.

First, as a culturing step, a medium is formed on the inner bottom surface of the DLC coated dish 100, and cells are cultivated in this medium (step 1).
Specifically, step 1 includes the following steps (1) to (4).
(1) The cells are suspended in a medium and seeded on a DLC coated dish 100.
(2) incubator _{(37 ℃, CO 2 5%} , 100% humidity) for several days of culture in the.
(3) Observe the cells, confirm that the cells are adhered to about 80 to 90% of the inner bottom surface of the DLC coating dish 100, and remove the medium.
(4) The surface of the DLC coated dish 100 is washed 1 to 3 times with physiological saline (PBS) to completely remove the medium.
Then, as a peeling step, the cell production apparatus 1 applies acoustic radiation pressure to the cells in the DLC coating dish 100 to peel off the adhesive cells (step 2).
Specifically, the step 2 includes the following procedures (5) and (6).
(5) was added PBS, incubator _{(37 ℃, CO 2 5%} , 100% humidity) and allowed to stand for 5 minutes in a.
(6) Acoustic radiation pressure (29 to 31 kHz, 200 V) is applied from the outer bottom surface of the DLC coating dish 100 (that is, ultrasonic vibration is input).
Further, as a recovery step, the cells exfoliated in step 2 are recovered from the DLC coating dish 100 (step 3).
Specifically, the step 3 includes the following procedure (7).
(7) The cell suspension is collected from the DLC coating dish 100 in a centrifuge tube and suspended in a medium.

Then, the cells acquired in step 3 are reseeded into a new DLC-coated dish 100 and cultured (recursion to step 1).
At this time, a part of the sample obtained in step 3 is re-seeded in a new DLC-coated dish 100 filled with a medium, and the culture is performed again.
By implementing such a cell production method, the cells exfoliated and recovered without using an enzyme such as trypsin are recultured, so that the activity of the cultured cells becomes high, and the culture is repeated a plurality of times. The time required for adhesion and the like is shortened, and cells can be mass-produced more efficiently and with high quality.

In the step of recurring to step 1, it is also possible to continue culturing the cells remaining in the DLC coating dish 100 after step 3.
In this case, the medium in the DLC-coated dish 100 is replaced with a new medium, but the protein (extracellular matrix) produced by the cells remains on the inner bottom surface of the DLC-coated dish 100. Therefore, compared to the case where a new medium is formed in a new DLC coating dish 100 and the cells are cultured, the cells are more likely to proliferate and the activated cells are more activated, so that a large amount of cells are cultured. It is advantageous on.
By carrying out such a cell production method, cells can be cultured, detached and recovered a plurality of times in the same DLC coated dish 100 without using an enzyme such as trypsin. It is possible to increase the automation rate and improve the cell production efficiency.
Hereinafter, steps 1 to 3 are repeated as many times as desired.

Incidentally, in the existing cell production method, the steps (5) and (6) of the above step 2 and the procedure (7) of the step 3 are significantly different from the cell production method in the present invention.
FIG. 4 is a schematic diagram showing a comparison between the procedure of the existing cell production method and the procedure of the cell production method in the present invention.
As shown in FIG. 4, in the existing cell production method, cells are produced through the following steps (5) to (9).
(5) adding trypsin-EDTA, incubator _{(37 ℃, CO 2 5%} , 100% humidity) and allowed to stand 3-10 minutes in.
(6) The cells are detached by pipetting or shaking.
(7) Add medium to inhibit the effect of trypsin.
(8) Collect the cells in a centrifuge tube and centrifuge for 2 to 5 minutes to separate the cells and the solution.
(9) The overlay liquid is collected and suspended again in the medium.
In the case of the existing cell production method, after the procedure (9), the cells obtained in the procedure (9) are re-seeded in a new culture dish filled with a medium, and the cells are cultured again.

[effect]
Next, the effect when the cell production method according to the present invention is applied will be described.
[Cell exfoliation]
FIG. 5 is a schematic diagram showing the results of a cell recovery experiment between the cell production method according to the present invention and the case where trypsin is used.
In the cell recovery experiment shown in FIG. 5, cells were recovered by the following procedure. In the following description, the notation of PBS (− / −) indicates that calcium and magnesium are not contained as components of PBS.
(1) CHO cells are seeded on a φ35 dish (DLC, Cellculture treated dish) (4.0 × 10 ⁵ cells).
(2) After 24 hours, the cells were washed with PBS (− / −), immersed in PBS (− / −), and swept with vibration excitation (29-31 kHz, 10 ms, 200 V) for 5 minutes to collect cells.
As a control, a series was prepared in which the mixture was immersed in PBS (− / −) for 5 minutes and then immersed in trypsin for 5 minutes.
After (2), the number of cells was measured with a blood cell counter.
As a result, as shown in FIG. 5, no significant difference was observed between the cell production method according to the present invention and the case of using trypsin (Control), and it was found that the same number of cells were recovered. ..
That is, it can be seen that the cell production method according to the present invention and the case where trypsin is used have the same cell exfoliation property.

[Cell colonization]
FIG. 6 is a schematic diagram showing the results of cell colonization experiments between the cell production method according to the present invention and the case where trypsin is used.
In the cell colonization experiment shown in FIG. 6, cells were collected by the following procedure.
(1) CHO cells are seeded on a φ35 dish (DLC, Cellculture treated dish) (4.0 × 10 ⁵ cells).
(2) After 24 hours, the cells were washed with PBS (− / −), immersed in PBS (− / −), and swept with vibration excitation (29-31 kHz, 10 ms, 200 V) for 5 minutes to collect cells.
As a control, a series was prepared in which the mixture was immersed in PBS (− / −) for 5 minutes and then immersed in trypsin for 5 minutes.
(3) The recovered cells were re-seeded in culture dishes (2.0 × ¹⁰ 5 cells), after 10 minutes, the cells were collected by immersing 5 minutes trypsin. At this time, the supernatant was washed and the floating cells were removed in advance.
As a result, as shown in FIG. 6, a significant difference was observed in the number of recovered cells between the cell production method according to the present invention and the case where trypsin was used (Control), and the cell production method according to the present invention showed a significant difference. , It was found that the number of colonized cells was large.
That is, it can be seen that the cell production method according to the present invention has higher cell colonization than the case of using trypsin.

[Observation results of cell surface]
FIG. 7 is a photomicrograph of the surface of cells in the case of using the cell production method according to the present invention and trypsin.
In the surface observation of the cells shown in FIG. 7, the cells were observed by the following procedure.
(1) CHO cells are seeded on a φ35 dish (DLC, Cellculture treated dish) (4.0 × 10 ⁵ cells).
(2) After 24 hours, the cells were washed with PBS (− / −), immersed in PBS (− / −), and swept with vibration excitation (29-31 kHz, 10 ms, 200 V) for 5 minutes to collect cells.
As a control, a series was prepared in which the mixture was immersed in PBS (− / −) for 5 minutes and then immersed in trypsin for 5 minutes.
(3) The recovered cells were re-seeded in culture dishes (2.0 × ¹⁰ 5 cells), After incubation for 10 min, fixed with glutaraldehyde, and observed by SEM (Scanning Electron Microscope).
As a result, as shown in the whole cell photograph of FIG. 7, in the cell production method according to the present invention, pseudopodia are extended from the cells and colonization is progressing, whereas in the case of using trypsin, the cells are in a state of progressing. It can be seen that the surface is decomposed and the entire cell becomes spherical, and the colonization has not progressed. Further, as shown in the enlarged photograph of the cell surface in FIG. 7, in the cell production method according to the present invention, the extracellular matrix remains on the cell surface and is in a state where it is easy to settle, whereas when trypsin is used. Then, it can be seen that the extracellular matrix on the cell surface is reduced by decomposition.
That is, it can be seen that the cell production method according to the present invention has a higher cell surface structure than the case where trypsin is used.

[Modification 1]
In the above-described embodiment, the vibrating member 13 has been described as including an ultrasonic vibrator made of a piezoelectric element, but the vibrating member 13 can be configured by a Langevin type ultrasonic vibrator.
FIG. 8 is a schematic view showing a configuration example of a vibrating member 13 made of a Langevin type ultrasonic vibrator.
As shown in FIG. 8, when the vibrating member 13 is configured by the Langevin type ultrasonic vibrator, the two piezoelectric elements P1 and P2 arranged so as to sandwich the electrode E are further sandwiched between the two metal blocks M1 and M2. It can be configured as a bolt-fastened Langevin-type ultrasonic vibrator bolt-fastened in the axial direction.
In the case of the Langevin type ultrasonic vibrator, since the end face of the metal block vibrates uniformly in the axial direction, the vibrating member 13 applies uniform acoustic radiation pressure to the cells in the DLC coating dish 100. Is possible.

(1) As described above, in the cell production method according to the present embodiment, the cells are mixed in the first step of preparing the culture substrate having the amorphous carbon coating and the culture substrate prepared in the first step. The second step of culturing and the third step of exfoliating the cultured cells by applying acoustic radiation pressure to the cells in the cultured substrate cultured in the second step are included.
Thereby, in the production of cells, by applying acoustic radiation pressure to the cells in a culture substrate having an amorphous carbon coating (for example, DLC coating dish 100), the cells are easily separated from the DLC coating and trypsin. The cultured cells can be detached and recovered without using an enzyme such as the above.
Therefore, in the cell exfoliation step and the recovery step, it is not necessary to soak or wash with an enzyme such as trypsin, which has the effects of simplifying the step, shortening the production time, and improving the activation and quality of the produced cells. Can be realized.
That is, it becomes possible to mass-produce cells more efficiently and with high quality.

(2) In the third step, the vibration frequency for generating the acoustic radiation pressure is swept.
As a result, even if there are differences in conditions such as individual differences in the culture medium having an amorphous carbon coating, these conditions are absorbed and natural vibration is generated in the vibrating member that is the source of acoustic radiation pressure. It can be generated reliably.

(3) Further, in the cell production method in the present embodiment, the exfoliated cells are recultured in another culture base material, and the cells in the culture base material cultured in the fourth step are acoustically subjected to the fourth step. It further comprises a fifth step of exfoliating the cultured cells by applying radiation pressure.
As a result, the cells exfoliated and recovered without using an enzyme such as trypsin are recultured, so that the activity of the cultured cells becomes high, and the time required for adhesion or the like when the culture is repeated a plurality of times is shortened. , Cells can be mass-produced more efficiently and with high quality.

(4) Further, in the cell production method in the present embodiment, after the third step, the cells remaining in the culture base material are recultured in the sixth step, and in the culture base material cultured in the sixth step. A seventh step of exfoliating the cultured cells by applying acoustic radiation pressure to the cells of the cell is further included.
As a result, cells can be cultured, detached, and recovered multiple times on the same culture substrate without using an enzyme such as trypsin, so that the automation rate of the cell production process can be increased and the cell production efficiency can be improved. It is possible to improve.

(5) In the first step, a culture base material is prepared by forming an amorphous carbon film on the surface of a predetermined culture base material.
This makes it possible to more appropriately produce cells by using a culture medium having surface characteristics suitable for the cell production method.

(6) Further, the cell production device 1 in the present embodiment includes a glass plate 14 and a vibrating member 13.
The glass plate 14 has an amorphous carbon coating and transmits ultrasonic vibration to a culture substrate (for example, DLC coating dish 100) in which cells are cultured.
The vibrating member 13 generates ultrasonic vibration applied to the glass plate 14.
By transmitting ultrasonic vibration from the glass plate 14 to the culture substrate, the cells cultured in the culture substrate are separated by acoustic radiation pressure.
Thereby, in the production of cells, by inputting ultrasonic vibration into a culture substrate having an amorphous carbon coating (for example, DLC coating dish 100), the cells can be easily separated from the amorphous carbon coating by acoustic radiation pressure. However, the cultured cells can be detached and recovered without using an enzyme such as trypsin.
Therefore, in the cell exfoliation step and the recovery step, it is not necessary to soak or wash with an enzyme such as trypsin, which has the effects of simplifying the step, shortening the production time, and improving the activation and quality of the produced cells. Can be realized.
That is, it becomes possible to mass-produce cells more efficiently and with high quality.

The present invention can be appropriately modified, improved, and the like within the range in which the effects of the present invention are exhibited, and is not limited to the above-described embodiment.
For example, in the above-described embodiment, the target for forming the DLC coating has been described as being a culture dish, but the present invention is not limited to this. For example, it is possible to form a DLC coating on a substrate other than the culture dish, such as a cell culture substrate incorporated in the cell production apparatus 1, to culture and detach the cells.

In addition, it is possible to carry out the present invention by appropriately combining the examples described in the above-described embodiments.
The processing for control in the above-described embodiment can be executed by either hardware or software.
That is, it is sufficient that the cell production apparatus 1 is provided with a function capable of executing the above-mentioned processing, and what kind of functional configuration and hardware configuration are used to realize this function is not limited to the above-mentioned example.

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 modified in various ways such as omission and substitution without departing from the gist of the present invention, and various embodiments other than the above-described embodiment can be taken. Various embodiments and modifications thereof that can be taken by the present invention are included in the scope of the invention described in the claims and the equivalent range thereof.

1 cell production device, 10 vibration unit, 11 lower acrylic plate, 12 felt member, 13 vibrating member, 14 glass plate, 14a penetration part, 15 upper silicone rubber member, 16 upper acrylic plate, 16a cover, 17 bolts, 18 Nut, 20 transmitter, 30 amplifier, 40 controller, 100 DLC coated dish, W weight, E electrode, P1, P2 piezoelectric element, M1, M2 metal block

Claims (3)

The first step of preparing a culture substrate having an amorphous carbon coating, and
A second step of culturing cells on the culture substrate prepared in the first step, and
The third step of exfoliating the cultured cells by applying acoustic radiation pressure to the cells in the culture substrate cultured in the second step, and
After the third step, the exfoliated cells are recultured with another culture medium, and the fourth step.
The fifth step of exfoliating the cultured cells by applying acoustic radiation pressure to the cells in the culture substrate cultured in the fourth step, and
After the third step, the sixth step of culturing the cells remaining on the culture substrate again, and
In the seventh step, the cultured cells are exfoliated by applying acoustic radiation pressure to the cells in the culture substrate cultured in the sixth step.
Including
In the third step, the fifth step, and the seventh step, a cell production method is characterized in that acoustic radiation pressure is applied to the entire surface to which the cells of the culture substrate are adhered. The first aspect of claim 1, wherein in at least one of the third step, the fifth step, and the seventh step, the vibration frequency for generating the acoustic radiation pressure is swept. Cell production method. The cell production method according to claim 1 or 2, wherein in the first step, the culture medium is prepared by forming an amorphous carbon film on the surface of a predetermined culture medium.

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