JP4883666B2 - Cultivation container and culturing unit - Google Patents

Description

  The present invention relates to a culture container and a culture unit for culturing biological samples such as cells.

In general, as a method for culturing a biological sample such as cells using a petri dish made of glass or plastic as a culture container, a biological sample is placed in the petri dish, and the petri dish is sufficient for the biological sample to grow. Some cultivate target biological samples by storing and maintaining them in a culture environment. Here, a method of proliferating a biological sample in a simulated environment closer to the biological environment by applying pressure or temperature has been proposed (see, for example, Patent Document 1).
JP 2001-238663 A

However, the conventional culture vessel has the following problems. That is, in the conventional culture container, the cultured biological sample is used when it is necessary to observe the cultured sample after culturing the biological sample or to stimulate the biological sample during observation. It is necessary to transfer to another container having a function of giving a stimulus or to give a stimulus from the outside. At this time, the physical sample may be subjected to a physical and heavy load accompanied by separation due to movement from the culture environment, and the biological sample may be destroyed. Moreover, there is a problem that rapid observation in a more natural environment is difficult because a load is applied by changing the temperature, PH environment, surrounding ion state, and the like significantly from the culture environment.
In addition, for example, when stimulating cell division by accelerating cell division by giving a physical stimulus to the biological sample, a stimulus such as a vibration stimulus is given from the outside through the vessel, or the vessel is This is done by vibrating the stored device itself. However, these methods cannot independently control the vibrational stimulus applied to the biological sample, and it is impossible to promote the culture diversified at once.

  The present invention has been made in view of the above-described problems, and can perform culture to observation without transferring the biological sample after culturing, and separately gives vibration stimulation to the biological sample in the container. It is an object to provide a culture container and a culture unit that can be used.

The present invention employs the following configuration in order to solve the above problems. That is, the culture container according to the present invention is a culture container provided with a sample mounting part for mounting a sample to be cultured on the upper surface, and a concave part in which the sample can be placed at a plurality of locations of the sample mounting part. And a vibration stimulus applying unit capable of independently applying a vibration stimulus to the sample is provided for each of the recesses .

According to this invention, since the vibration stimulus is applied from the vibration stimulus application unit to the bottom surface of the sample by placing the sample on the vibration stimulus application unit, the sample is transferred to another container after the sample is cultured. Observation can be performed without changing. Therefore, it is possible to perform observation after culturing in a natural environment without giving a physical load or a load due to environmental changes to the sample. In addition, different vibration stimuli can be applied even when each sample is placed in the same environment, which is the same medium, and the culture state of each sample changes only by the intensity of the vibration stimulus, so the error factor is reduced. A more efficient culture test can be performed.
Here, when the vibration stimulation application unit is formed so that a plurality of samples to be cultured are placed on different vibration stimulation application units, vibration stimulation can be applied to the samples independently. It is possible to apply vibration stimuli with different intensities to these samples, or to apply vibration stimuli only to a desired sample among a plurality of samples.
In addition, when the vibration stimulation application unit is formed so that the sample is placed on the plurality of vibration stimulation application units, for example, vibration stimulation is applied from a part of the plurality of vibration stimulation application units in contact with the sample. As a result, it is possible to partially apply a vibration stimulus to the sample. Thereby, the local vibration stimulation for the culture | cultivation promotion of a sample can be given. Further, by applying a vibration stimulus having a desired intensity to each of the plurality of vibration stimulus application units, a vibration stimulus having a desired intensity distribution can be applied to the sample.

Further, the culture container according to the present invention includes a bottom portion and a peripheral wall portion provided substantially vertically upward from a peripheral portion of the bottom portion, and the sample placement portion is integrally formed on the upper surface of the bottom portion. It is preferable.
According to the present invention, the wiring from the vibration stimulation application unit or the like is provided on the outer surface of the container, and the intensity of stimulation by the vibration stimulation application unit can be controlled from the outside of the culture container.

Further, the culture container according to the present invention includes a bottom, a peripheral wall provided substantially vertically upward from a peripheral edge of the bottom, and a sample placement member provided with the sample placement portion on an upper surface. It is preferable that the sample mounting member is provided detachably with respect to the bottom portion.
According to this invention, the application range of the culture vessel is expanded by making the sample mounting member on which the vibration stimulus applying unit is formed detachable from the bottom.

In the culture container according to the present invention, it is preferable that the vibration stimulus applying unit is arranged on the sample mounting unit so as to form a matrix at equal intervals in two different directions.
According to the present invention, when the vibration stimulus application unit is formed so that the plurality of samples are placed on the plurality of different vibration stimulus application units, respectively, and vibration stimuli having different intensities are applied to the respective samples. This makes it easy to identify the location where vibration stimulation is applied. As described above, the control for applying the vibration stimulus to the sample becomes easy.

In the culture container according to the present invention, it is preferable that the vibration stimulation application unit includes an activity sensor that acquires biological information of the sample placed on an upper surface.
According to the present invention, when a sample is placed on the vibration stimulation application unit, biological information of the sample can be acquired, and vibration stimulation can be applied to the sample while confirming the weak state of the sample. Thereby, a sample can be cultured more efficiently.

In addition, a culture unit according to the present invention includes the culture container described above and drive means for driving the vibration stimulus applying unit to give a stimulus to the sample.
According to the present invention, the driving means drives the respective vibration stimulation application units, so that the vibration stimulation is directly applied to the sample placed on the vibration stimulation application unit. Here, the intensity distribution of the vibration stimulus to be applied can be formed by appropriately setting the intensity of the vibration stimulus for each vibration stimulus application unit by the driving means.

  According to the culture container and the culture unit according to the present invention, vibration stimulation can be directly applied to the sample, and observation can be performed without transferring the sample to another container after the sample is cultured. Therefore, observation after culturing can be performed in a natural environment without giving a physical load or a load due to environmental changes to the sample. In addition, by configuring the plurality of samples to be placed on different vibration stimulation application units, it is possible to apply vibration stimulation of different strengths to each of the plurality of samples, The vibration stimulus can be applied only to the desired sample. Then, by configuring the sample so as to be placed on a plurality of vibration stimulation application units, it is possible to give vibration stimulation locally to the sample or vibration stimulation having a desired intensity distribution to the sample. Can be given.

  Hereinafter, a first embodiment of a culture container and a culture unit according to the present invention will be described with reference to FIGS. 1 and 2. In the present embodiment, the petri dish (culture container) 1 is a culture container used for culturing biological samples such as cells.

As shown in FIGS. 1 and 2, the petri dish 1 according to the present embodiment is provided on the surface on which a sample S to be cultured can be placed, and from the peripheral edge of the bottom 11 toward substantially vertically upward. And a peripheral wall portion 12.
The bottom portion 11 is made of glass, and the upper surface is a sample placement portion 15 on which the sample S to be observed is placed, and the recess portions 16 are formed so as to form a matrix at equal intervals in the row direction and the column direction. Has been. The recess 16 is formed so that the area of the upper surface thereof is substantially equal to that of the sample S. The bottom portion 11 includes a vibrator (vibration stimulus application section) 17 that applies vibration stimulation to the sample S at a plurality of locations on the sample placement section 15, a conductive pattern 18 that causes a current to flow through the vibrator 17, and And an activity sensor 19 for detecting biological information of the sample S.

  The vibrator 17 is made of PZT (lead zirconate titanate), which is a piezoelectric material, and is disposed in a recess 16 formed in the bottom 11. Further, a sample is placed on each transducer 17, and the transducer 17 is configured to give a vibration stimulus having a vibration amplitude of 10 μm to 20 μm and a vibration frequency of 10 Hz to 10 MHz to the sample S, for example. Has been. The vibrator 17 is formed integrally with the bottom 11 by patterning. The vibrator 17 is not limited to PZT, and other piezoelectric materials such as ZnO (zinc oxide) may be used.

  The conductive pattern 18 is made of ITO (Indium Tin Oxide), which is a transparent electrode, and is formed integrally with the bottom 11 by patterning in the same manner as the vibrator 17. One end of the conductive pattern 18 is electrically connected to a connector 21 provided on the peripheral edge of the bottom 11. The connector 21 is configured to be electrically conductive and vibrate the vibrator 17 by being connected to a connector 33 of a driving unit 31 described later. An insulating layer 22 for electrically insulating the vibrator 17 and the sample S is formed on the surface of the sample mounting portion 15. The conductive pattern 18 is not limited to ITO, and other conductive materials may be used.

The activity sensor 19 is formed by an electrode in contact with the sample S, and is configured to detect the activity state of the sample S as an electrical signal. The activity sensor 19 is electrically connected to the connector 21 through the signal transmission pattern 23 and is configured to output an electrical signal obtained from the sample S from the connector 18.
The signal transmission pattern 23 is formed integrally with the bottom portion 11 by patterning in the same manner as the conductive pattern 18.

As shown in FIG. 1, the petri dish 1 is connected to driving means 31 that drives the vibrator 17. The petri dish 1 and the drive means 31 constitute a culture unit 32.
The driving unit 31 includes a connector 33 that can be connected to the connector 21 and a control unit 34 that controls the amount of current flowing through the conductive pattern 18. By connecting the connector 33 and the connector 21, current is passed through the conductive pattern 18, and when current is passed through the conductive pattern 18, the vibrator 17 vibrates due to piezoelectric conversion. The driving means 31 is connected to each transducer 17 independently, and is configured to be able to generate vibration stimuli with different intensities in each transducer 17.

Next, the manufacturing method of this petri dish 1 is demonstrated, referring a figure.
First, a PZT film 42 that is a piezoelectric material and serves as a vibrating electrode film is formed by sputtering on the upper surface of a glass substrate 41 (see FIG. 3A) (see FIG. 3B). Then, a resist is applied to the upper surface of the PZT film 42 to form a resist film 43 (see FIG. 3C), and patterning is performed using a photolithography technique (see FIG. 3D). Thereafter, the PZT film 42 is etched and patterned using the resist film 43 as a mask, thereby forming the vibrator 17 shown in FIG. 2 (see FIG. 4A). The resist used as the resist film 43 may be positive or negative. Thereafter, the resist film 43 used as a mask is removed.

  Subsequently, a resist is applied to the upper surfaces of the glass substrate 41 and the PZT film 42 to form a resist film 44 (see FIG. 4B), and patterning is performed using a photolithography technique, whereby the resist film 44 is formed on the glass substrate. A through hole 44a reaching 41 or the PZT film 42 is formed (see FIG. 4C). Then, an ITO film 45, which is a conductive material, is formed by sputtering on the upper surface of the resist film 44 so as to fill the through hole 44a (see FIG. 4D). Thereafter, by performing lift-off, the resist film 44 and the ITO film 45 formed on the upper surface of the resist film 44 are removed, and the conductive pattern 18 and the signal transmission pattern 23 shown in FIG. 2 and the connector 21 shown in FIG. And an extraction electrode connected to each other (see FIG. 5A). The ITO film 45 may be formed by vapor deposition. The resist used as the resist film 44 may be positive or negative as described above.

Next, a silicon dioxide (SiO ₂ ) film 46, which is an insulating material, is formed by sputtering on the glass substrate 41, the PZT film 42, and the ITO film 45 (see FIG. 5B). Further, a resist is applied on the upper surface of the SiO ₂ film 46 to form a resist film 47 (see FIG. 5C), and patterning is performed using a photolithography technique (see FIG. 5D). Then, the insulating layer 22 shown in FIG. 2 is formed by etching and patterning the SiO ₂ film 46 as the resist film 47 mask (see FIG. 6A). The SiO ₂ film 46 may be formed by vapor deposition or CVD (Chemical Vapor Deposition). The resist used as the resist film 47 may be positive or negative as described above. Thereafter, the resist film 47 is removed (see FIG. 6B).

Subsequently, a resist is applied to the upper surfaces of the glass substrate 41, the ITO film 45, and the SiO ₂ film 46 to form a resist film 48 (see FIG. 6C), and patterning is performed using a photolithography technique. A through hole 48a reaching the glass substrate 41 or the ITO film 45 is formed in the resist film 48 (see FIG. 6D). Then, a Pt (platinum) film 49, which is a conductive material, is formed by sputtering on the upper surface of the resist film 48 so as to fill the through hole 48a (see FIG. 7A), and lift-off is performed. The Pt film 49 formed on the upper surface of the film 48 and the resist film 48 is removed, and the active sensor 19 shown in FIG. 2 and the extraction electrode electrically connected to the connector 21 shown in FIG. 1 are formed (FIG. 7). (See (b)). Note that the Pt film 49 may be formed by vapor deposition. The resist used as the resist film 48 may be positive or negative as described above.

Next, a protective layer 50 formed by applying a resist such as polyimide is provided on the upper surface of the glass substrate 41, the SiO ₂ film 46, and the Pt film 49 (see FIG. 7C), and a photolithography technique is used. Then, the recess 16 shown in FIGS. 1 and 2 is formed (see FIG. 7D). The petri dish 1 is manufactured as described above.

A method for culturing the sample S using the petri dish 1 and the culture unit 32 configured as described above will be described.
First, cells are placed on the sample placement unit 15 and kept active in the culture medium. In a state where the connector 21 is connected to the connector 33 of the driving means 31, a vibration stimulus having a desired strength is applied to the sample S by causing the control means 34 to pass a current through the vibrator 17. At this time, the intensity of the vibration stimulus applied while checking the activity state of the sample S with the activity sensor 19 is adjusted, or the application of the vibration stimulus to the weakened sample S is stopped.
When observing the sample S cultured after culturing with the petri dish 1, the petri dish 1 is placed on a stage of a known optical microscope for observation. Here, observation with an optical microscope may be performed while applying vibration stimulation to the sample S.

  According to the petri dish 1 and the culture unit 32 configured as described above, vibration stimulation can be directly applied to the sample S from the back surface of the sample S, and the sample S is transferred to another container after the sample S is cultured. Observation can be performed without changing. Therefore, observation after culture can be performed in a natural environment without applying a physical load or a load due to environmental changes to the sample S.

  In addition, since the plurality of samples S are placed on different vibrators 17, vibration stimuli having different strengths are applied to the respective samples S, or vibration stimuli are applied only to the desired sample S. be able to. Here, since each sample S can be applied with different vibration stimuli while being placed in the same environment as the same culture solution, the culture state of each sample S changes only depending on the intensity of the vibration stimulus. Factors are reduced, and more efficient culture tests can be performed.

Next, a second embodiment of the measurement container according to the present invention will be described. In the following description, the same reference numerals are given to the components described in the above embodiment, and the description thereof is omitted.
The difference between the second embodiment and the first embodiment is that, in the first embodiment, the sample mounting portion 15 is provided on the bottom portion 11, whereas the petri dish 51 of the second embodiment is different. Then, the sample mounting part 15 is provided in the sample mounting board (sample mounting member) 53 provided in the bottom part 52 so that attachment or detachment was possible.

That is, as shown in FIGS. 8 and 9, the petri dish 51 of the present embodiment includes a petri dish body 55 having a bottom part 52 and a peripheral wall part 54 provided substantially vertically upward from the peripheral part. And a sample mounting plate 53 provided detachably inside.
Similar to the first embodiment described above, a wiring cable 56 connected to each transducer 17 via the conductive pattern 18 is provided on the sample mounting plate 53 so as to be exposed to the outside. The distal end of the wiring cable 56 can be connected to the connector 33 described above.

  When culturing the sample S using the petri dish 51 of the present embodiment, the sample mounting plate 53 is first settled in the petri dish body 55. Thereafter, the tip of the wiring cable 56 is connected to the connector 33 of the culture unit 32, and the sample S is cultured in the same manner as in the first embodiment described above. And also when observing after culture | cultivation, it carries out by carrying out similarly to the above-mentioned.

  The petri dish 51 configured as described above has the same operations and effects as those of the first embodiment described above, but the petri dish body is made detachable from the petri dish body 55. 55 can be made disposable.

The technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.
For example, in the above embodiment, as shown in FIG. 10, the area of the upper surface of the vibrator 61 is formed so as to be smaller than the sample S, and the sample S is placed on the plurality of vibrators 61. The petri dish 62 configured as described above may be used. In this way, for example, a vibration stimulus can be partially applied to the sample S by giving a vibration stimulus from a part of the plurality of vibrators 61. Thereby, the local vibration stimulation for culture | cultivation promotion of the sample S can be given. Moreover, by applying a vibration stimulus having a desired intensity to each of the vibrators 61 to the sample S, a vibration stimulus having a desired intensity distribution can be applied to the sample S.

  Further, in order to place the sample at a desired position in the sample placing portion, a base made of, for example, a gold thin film may be provided at a desired position where the sample is placed. By doing in this way, a coupling | bonding with the SH group of a sample and a base is promoted by gold-thiol reaction between a sample and a base, and it becomes easy to mount a sample in the desired position which provided the base. Further, a lyophobic film that is difficult to bond with the sample may be formed except for a desired position where the sample is placed. By doing in this way, it suppresses that a sample is mounted in places other than the desired position of a sample mounting part.

It is a schematic plan view which shows the unit for observation concerning the 1st Embodiment of this invention. It is a schematic sectional drawing which shows the petri dish of FIG. It is a schematic sectional drawing explaining the manufacturing process of the petri dish of FIG. FIG. 4 is a schematic cross-sectional view illustrating a manufacturing process of the petri dish of FIG. 1 following FIG. 3. FIG. 5 is a schematic cross-sectional view illustrating the manufacturing process of the petri dish of FIG. 1 following FIG. 4. FIG. 6 is a schematic cross-sectional view illustrating the manufacturing process of the petri dish of FIG. 1 following FIG. 5. FIG. 7 is a schematic cross-sectional view illustrating the manufacturing process of the petri dish of FIG. 1 following FIG. 6. It is a schematic plan view which shows the unit for observation concerning the 2nd Embodiment of this invention. It is a general | schematic fragmentary sectional view which shows the petri dish of FIG. It is the elements on larger scale which show the petri dish which can apply this invention other than embodiment of this invention.

Explanation of symbols

1, 51, 62 Petri dish (culture container)
11, 52 Bottom portion 12, 54 Peripheral wall portion 15 Sample placement portion 17, 61 Vibrator (stimulation application portion)
19 activity sensor 31 driving means 32 culture unit 53 sample mounting plate (sample mounting member)
S sample

Claims (6)

In a culture vessel provided with a sample placement part for placing a sample to be cultured on the upper surface,
Concave portions in which the sample can be placed are formed at a plurality of locations on the sample placement portion, and a vibration stimulus applying unit capable of independently applying a vibration stimulus to the sample is provided for each of the recess portions . A culture vessel characterized by that. A bottom portion and a peripheral wall portion provided substantially vertically upward from a peripheral portion of the bottom portion;
2. The culture container according to claim 1, wherein the sample mounting portion is integrally formed on the upper surface of the bottom portion. A bottom portion, a peripheral wall portion provided substantially vertically upward from a peripheral portion of the bottom portion, and a sample placement member provided with the sample placement portion on an upper surface,
2. The culture container according to claim 1, wherein the sample mounting member is detachably provided on the bottom.   The said vibration stimulation application part is arrange | positioned at the said sample mounting part so that it may become a matrix form at equal intervals in two mutually different directions. Culture container.   The culture vessel according to any one of claims 1 to 4, wherein the vibration stimulation application unit includes an activity sensor that acquires biological information of the sample placed on an upper surface.   A culture unit comprising: the culture container according to any one of claims 1 to 5; and a driving unit that drives the vibration stimulation application unit to apply vibration stimulation to the sample.

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