JPS62289175A - Culture apparatus having rotary culture device - Google Patents

Abstract

PURPOSE:To enable integrated culture work from the preparation of a material to be cultured to the differentiation of tissue, by using a thermostatic chamber, a rotatable incubator having arbitrarily adjustable inclination angle, an optical system for irradiating the material with light having adjustable wavelength and intensity and a means for controlling the composition of gaseous atmosphere in the thermostatic chamber. CONSTITUTION:The culture apparatus is constructed from the following parts. A thermostatic chamber 11 having arbitrarily adjustable inner temperature. A rotatable incubator 30 placed in the thermostatic chamber 11 and holding culture tubes 14 each containing a material to be cultured and a culture liquid. An optical system 16 for irradiating the culture tube 14 with light necessary for the growth of the material and capable of adjusting the intensity of the light. Means (19, 20, 18, 21, 22) for controlling the composition of gaseous atmosphere in the thermostatic chamber 11. Said rotary incubator 30 is furnished with a culture tube holding member 13 to hold a plurality of culture tubes 14, a driving means 15 to rotate the culture tube holding member 13, an inclination angle controlling means 32 to arbitrarily adjust the inclination angle of the culture tube holding member 13 from the horizontal state to an inclined state according to the growth of the culture material and a supporting member 34 to detachably support the culture tube holding member 13.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Object of the Invention] (Field of Industrial Application) This invention relates to an apparatus for culturing cultured materials such as cells (including protoplasts) and tissues.

(Prior Art) Cultivation and propagation of one set of cells, such as la, is not only important for basic research in biotechnology, but is also known to be useful for mass propagation of superior plants. Taking plant cells as an example, when protoplasts are immersed in a culture solution and allowed to proliferate, a callus (amorphous cell mass) is formed, and by changing the composition of the culture solution, various organs are differentiated from the callus. By the way, culture until callus is formed and subsequent culture require not only changing the composition of the culture solution, but also changing the physical conditions. For example, until cell walls are formed, it is necessary to After cell wall formation, tilted rotation culture is performed.

Furthermore, it may be necessary to change the culture atmosphere gas composition during the growth process of the cultured material.

Conventionally, various devices such as incubators have been used for culturing cells, tissues, etc., but all of them can only be used under certain fixed culture conditions.

(Problems to be Solved by the Invention) Conventional culture equipment can only be used under fixed culture conditions, so it is not possible to set various culture conditions that occur during the growth process of the cultured material using these equipment alone. Can not,
Therefore, in order to cope with this problem, it is necessary to increase the number of devices used and furthermore to complicate the control of culture conditions at each stage of culture. That is, since the culture conditions vary considerably during the growth process of the cultured object, when conventional culturing equipment is used alone, it is not possible to perform consistent culture according to the growth process of the cultured object.

Therefore, an object of the present invention is to provide a culture device that can achieve consistent culture from the cultured material to tissue differentiation.

[Structure of the Invention] (Means for Solving the Problems) The culture apparatus of the present invention includes a rotatable incubator for storing a culture tube in a constant temperature bath whose internal temperature can be arbitrarily set. In addition to installing an optical system that can adjust the irradiation light amount and wavelength to irradiate the r# and length of the cultured material with the necessary light, a means for controlling the gas atmosphere composition in the thermostatic chamber is also installed. It is set up. In addition, the inclination angle of the incubator can be arbitrarily set from a horizontal state to an inclined state.

(Function) The inside of the constant temperature bath is set at a desired temperature suitable for each stage of culture, and the gas atmosphere control means provides a gas composition suitable for each stage of culture. In addition, at the culture stage where slant culture is required, the incubator is incised. The optical system is
A culture tube containing a culture medium and a culture medium is irradiated with light at an amount corresponding to each stage of culture.

(Example) Hereinafter, an example of the present invention will be described in detail with reference to the drawings.

As shown in FIG. 1, the culture device IO according to the present invention is
A constant temperature bath 11 is provided. This constant temperature bath! The internal temperature can be arbitrarily set and maintained throughout each stage of culturing the cultured material.

Inside the constant temperature bath 11, a rotary incubator 30 including a support stand 12 and a culture tube holder 13 supported on the support stand 12 is installed. This culture tube holder 13 accommodates a plurality of culture tubes 14, for example, up to 26 tubes, which accommodate the culture material to be cultured together with a culture solution.

The culture tube holder 13 supports the culture tube 14 almost vertically in a horizontal state (indicated by a broken line in the figure). Further, as shown in the figure, the inclination angle of the culture tube holder 13 can be arbitrarily set from a horizontal state to an inclined state according to the growth process of cells, and it can be rotated by a rotary drive motor 15. The rotation speed can be adjusted up to 10 rpm, and the rotation direction can be either left or right.

As shown in FIG. 2, the incubator support 13 has a circular shape, for example, and a culture container is provided on the concentric circle thereof so that the culture tube 14 can be accommodated therein.

As shown in detail in FIGS. 3 and 4, the rotating incubator 3
0 is equipped with a support stand 12 and a culture tube holder 13, and a fixing plate 31 is placed on the support stand 12. On the fixed plate 31, in the state shown in FIG. 3 (that is, the state in which the culture tube holder 13 is horizontal), the inclination angle adjustment member 32 of the culture tube holder and the turntable support member 33 are mounted.
is installed.

The turntable 3 is mounted on the turntable support member 33.
4 is supported, the culture tube holder 13 is removed and placed on this turntable 34, and the 0 rotation drive motor 15 is fixed to the turntable support member 33.
The turntable 34 and therefore the culture tube holder 13 placed thereon are rotated.

As best shown in FIG.
When performing tilted culture by tilting the
The turntable support member 33 is locked and supported by the angle adjustment member 32 by rotating around the angle. That is,
The angle adjustment member 32 is.

The guide slit 7) 32a is a plate-shaped body formed in the longitudinal direction, and communicates with the sled 7) 32a, and has locking portions 32 at predetermined intervals.
b is formed. By locking a locking member 37, such as a bottle or a nut, provided on the turntable support member 33 to the locking portion 32b, the inclination angle θ of the culture tube holder 13 can be adjusted stepwise, for example, by 5° up to 85°. It is structured so that it can contain 31 sections.

In addition, the angle adjustment member 32 may be a plate-shaped body having a guide slit in the longitudinal direction, and may be formed with a synonym angle index (angle scale). In this case, the locking member 37
By forming a convex indicating portion on the surface, the inclination angle can be continuously set in accordance with the angle scale.

Inclined rotational culture may be necessary when cells and calluses are placed equally in a nonpolar state to differentiate organs, but in the culture device of the present invention, the culture tube holder 13 is used as a rotating part and the inclination angle is adjusted. The reason why it is made portable is to accommodate this.

Returning to FIG. 1 again to explain the culture apparatus of this invention,
Inside the constant temperature bath II, an optical system 16 is provided above the culture tube holder 13 for irradiating the culture tube with light necessary for cell growth.
is installed. This optical system 16 includes a light source consisting of, for example, a plurality of lamps 17, and these lamps 17 are configured to be able to control the amount of light irradiated individually or in units of several groups. This results in
Depending on each stage of culture, the illuminance on the entire surface of the culture tube holder 13 can be made equal, the illuminance can be changed depending on the position t, or the brightness and darkness of day and night can be created.

Furthermore, when the culture tube holder 13 is tilted, a difference in illuminance of about 10,000 lux can be provided between the upper and lower parts.

Now, as a means for controlling the gas atmosphere set I& (oxygen concentration, carbon dioxide concentration, humidity, etc.) in the thermostatic oven 11, a gas supply source 19 is connected to the gas introduction pipe 18 communicating with the inside of the thermostatic oven 11. It is provided. Gas supply is regulated by a valve 20, and gas discharge is regulated by a gas discharge pipe 21 provided in communication with the thermostatic chamber 11 and a valve 22 provided therein. The gas supply source 19 is constant temperature #
e1! The composition can be changed as appropriate depending on the type of gas that requires a change in composition. The culture is
Although it can be carried out under normal atmospheric conditions, for example, the oxygen concentration, carbon dioxide concentration, humidity, etc. can be adjusted as appropriate by introducing nitrogen gas, carbon dioxide gas, or water vapor into a constant temperature bath according to each stage of culture. For example, thermostatic chamber 11
When lowering the oxygen concentration in the tank, a nitrogen source is used to dilute the oxygen concentration. When increasing the carbon dioxide concentration in the thermostatic chamber 11, a carbon dioxide gas source is used and introduced into the thermostatic chamber 11. Moreover, when adjusting the humidity in the constant temperature bath 11, an aquatic plant air source is used.

In addition, regarding the control of the irradiation light amount of the optical system 16 and the gas atmosphere composition in the thermostatic chamber 1, the culture tube holder 1 is placed inside the thermostatic chamber 1.
3. An illuminance sensor for detecting illuminance, a gas sensor for detecting gas concentration, etc. can be installed on the screen, and these sensors can be linked with an optical system and a gas atmosphere control system. For example, as shown in Fig. 2. As such, illuminance sensors 40 can be provided at predetermined intervals around the culture tube holder 13. As shown in FIGS. 3 and 4, this illuminance sensor 40 is mounted on a support member 39 that is rotatable about a support member 33 and a shaft 41.
It can be installed in This attachment allows the illumination meter 40 to maintain a horizontal state regardless of the inclination angle of the culture tube holder.

Further, indicators may be provided to issue an alarm when the illuminance or gas concentration varies from a predetermined set value. These controls are performed by a control device 2 attached to the constant temperature bath 11.
This can be done by 3.

Examples of objects to be cultured using the culturing apparatus of the present invention include cells and tissues of animals and plants, and microorganisms. Preferably, herbaceous plants such as habropagus, tobacco, petunia, rice, corn, carrot, potato, wheat, barley, sugar cane, and soybean, and woody plants such as pine, eucalyptus, poplar, coffee, rose gum, grape, elm, and apple. Examples include cells and tissues. In addition, the cells (including protoplasts), tissues, and organs to be cultured include mesophyll cells, cells such as cultured cells derived from various tissues, tissues such as cup disks, embryos, mesophyll, cortex, and pith, shoot tips, meristems such as plate edges,
In addition, organs such as stems, leaves, rot, roots, and flowers can be exemplified.

For example, in order to culture plant cells using the culturing apparatus of the present invention, the temperature is 0 to 30°C, the illuminance is 0 to 20,000 lux, the oxygen concentration is 2 to 20%, and the carbon dioxide gas is 0 to 50%. Plant cells and tissues were grown in Ganporg B5 (7) basal medium (Ganbor
g et at, 1968), Murashige Skoog (
7) MS basal medium (Murashlge aSkoog
It is also possible to suspend the cells in a liquid medium such as 1982) and perform static culture or rotational culture, or to perform static culture or rotational culture by placing the above-mentioned medium in a medium solidified with agar or the like. . The plant growth hormones added to the medium used include naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3.
-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-acetic acid (IBA), phenylacetic acid (FAA), benzofuran-3-acetic acid (BF
A), auxins such as phenyl acetic acid (PBA), and cytokinins such as KT-30 (manufactured by Kyowa Hakko Co., Ltd.), 6-benzylaminopurine (BA), zeatin (Z), and kinetin can be used.

During or after the culture described above, the culture solution in the culture tube 14 may be replaced. In this case, remove the culture tube holder 13 for single rotation culture from the turntable 34, attach it to the culture solution exchanger, and culture. For example, as shown in FIG.
A culture tube holder containing a culture solution is placed on a culture solution exchanger 50 equipped with a rotor pump 52 having a rotor pump 1 and a supply tube 53 and a discharge tube 54 which are squeezed by the rotor pump 52 to supply and discharge the culture solution, respectively. 13 is placed. One end of the discharge tube 54 is introduced into the drainage container 56, and the other end is connected to the culture tube 14 from which the culture solution is to be discharged.
be introduced within. Moreover, one end of the supply tube 53 is introduced into the supply container 55 that accommodates a new culture solution, and the other end is introduced into the culture tube 14 from which the culture solution has been discharged. By driving the rotor pump 52, the culture solution can be continuously supplied and discharged. 'Note that this supply and discharge can also be automated.

Examples of culture experiments using the culture apparatus of the present invention will be described below.

Experimental example 1 Annual plant Haplopappusg
racilis) was cut out after 19 bacteria, and 2 mg/z of 6-benzylaminopurine (BA) and 30.0 mg/z of 6-benzylaminopurine (BA) were added to Ganpaug B5 basal medium and carbon source.
Modified medium (pH 5,
6) and cultured in rotation using the apparatus of the present invention at an inclined angle (rotary incubator inclination angle of 65°). The culture conditions are temperature 2
The temperature was 8°C, the illumination intensity was 2000 to 12,000 lux, the oxygen concentration in the culture atmosphere was 15%, and the rotation speed was 3 rpm.

As a result, 5 weeks after the start of culture, pale green shoot primordium aggregates, which could not be obtained conventionally with an inclination angle of 0°, were obtained.

Experimental Example 2 After sterilizing the seeds of Japanese red pine (Pinus densiflora), the embryos were removed and placed in 2 m of Ganpaug B5 basic medium.
g/L naphthalene acetic acid (NAA), 4 mg/L BA, and 30,000 mg/L sucrose as a carbon source, and solidified with agar (agar concentration 0.8%).
, PH 5, 6) and cultured using the culture device of the present invention (tilt angle 0 @), the culture conditions were a temperature of 28
℃, illuminance 3000 lux, oxygen concentration in the culture atmosphere 5
%Met. Under these conditions, the callus formed from the embryo was cultured for one month in a liquid medium with the same composition. (no agar added), JJU and then tilted with the same device (tilt angle 60)
°) By rotating culture, we succeeded in producing a large number of pale green cultured cells that were uniform and proliferated vigorously.

For comparison, static culture was carried out for one month under the same conditions, subcultured on the same medium, and acid n culture was continued, but the proliferation rate in this section was about 50% of that in the section switched to rotational culture. Ta.

[Effects of the Invention] As described above, in the culture apparatus of the present invention, a rotatable incubator and a rotary optical system for adjusting the amount of irradiation light are designed in the thermostatic oven, and the gas atmosphere composition within the thermostatic oven is In addition, the inclination angle of the culture tube holder of the rotary incubator can be set arbitrarily, so the inclination angle of the incubator, the amount of light irradiation, and the composition of the culture gas atmosphere can be adjusted at each stage of culture. Accordingly, the rA order can be adjusted to the optimal state. Therefore, the culture conditions during the entire growth process of cells etc. can be consistently controlled within the same device.

[Brief explanation of drawings]

FIG. 1 is a front view showing an embodiment of the culture bag of the present invention, FIG. 2 is a plan view of a culture tube holder incorporated in the culture apparatus of the present invention, and FIG. 3 is a front view of a culture bag of the present invention. FIG. 4 is a side view showing a state in which the rotating incubator of the apparatus is arranged horizontally. FIG. Schematic diagram of the exchanger. 11--Thermostatic chamber, 12--Support stand, 13--Culture tube holder, 14-φΦ culture tube, 15-φ tube, drive motor, No. 16-φ optical system, 1711--Lamp 18-Oφ gas Inlet pipe, 19e... Gas supply source, 20, 22... Fist valve, 21... Gas discharge pipe. 32φφ・Angle adjustment member, 50mm・・Culture solution exchanger section Qtsujo C鵠”-ir-

Claims (5)

[Claims] (1) A constant temperature bath that can set and maintain the internal temperature at a desired temperature suitable for the growth process of the cultured material, and a culture tube installed in the constant temperature bath and containing the cultured material to be cultured together with the culture solution. a rotatable incubator for accommodating a rotatable incubator; an optical system installed in the constant temperature chamber and capable of adjusting the amount of irradiation light for irradiating the culture tube with light necessary for the growth of the cultured object; and the constant temperature chamber. A culture apparatus comprising a gas atmosphere control means for controlling the gas atmosphere composition in the culture medium according to the growth process of the cultured object, the rotary incubator having a culture tube holder for holding a plurality of culture tubes. a rotary drive unit for rotationally driving the culture tube holder, and an inclination for arbitrarily setting the inclination angle of the culture tube holder from a horizontal state to an inclined state according to the growth process of the cultured object. 1. A culture apparatus having a rotating culture vessel, comprising: an angle adjustment part; and a support body that removably supports the culture tube holder. (2) A culture apparatus having a rotating incubator according to claim 1, wherein the culture tube holder has an attachment part for an illuminance meter for measuring the amount of irradiated light. (3) A culture apparatus having a rotating incubator according to claim 2, wherein the illuminance meter mounting portion maintains a horizontal state regardless of the inclination angle of the culture tube holder. (4) The rotary incubator according to claim 1, wherein the culture tube holder has a circular shape, and a accommodating section for accommodating a plurality of culture tubes is arranged concentrically therein. A culture device with (5) The inclination angle adjusting member is made of a plate-shaped body with a slit for guiding a locking member provided on the culture tube holder, and the locking position of the locking member determines the position of the culture tube holder. A culture apparatus comprising a rotary culture vessel according to any one of claims 1 to 4, which adjusts the inclination angle.