JPH0427833B2 - - 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 13 placed in the thermostatic chamber 11, holding culture tubes 14 each containing a material to be cultured and a culture liquid and having arbitrarily adjustable inclination angle from horizontal state to inclined state according to the growth of the material to be cultured. An optical system 21 for irradiating the material with light necessary for the growth of the material and capable of adjusting the intensity and wavelength of the light. Means (17, 18, 16, 19, 20) for controlling the composition of gaseous atmosphere in the thermostatic chamber 11. Said optical system 21 is furnished with a plurality of light sources 22 attached detachably to the top of the thermostatic chamber 11, a wavelength-selection filter 24 to control the wavelength of light to be radiated to the culture tube 14 and a light-source controlling device 25 to control the radiation of the light source 22.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Industrial Application Field) The present invention relates to an apparatus for culturing cultured materials such as cells (including protoplasts) and tissues.

(Prior Art) Cultivation and propagation of cells, tissues, etc. are not only important for basic research in biotechnology, but also are 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 can be differentiated from the callus. By the way, culture until callus formation and subsequent culture require not only changing the composition of the culture solution but also changing the physical conditions. For example, static culture is performed until cell walls are formed, and tilted rotation culture is performed after cell wall regeneration. Furthermore, it may be necessary to change the culture atmosphere gas composition during the growth process of the cultured material. Furthermore, it is also necessary to change the amount of irradiation light.

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 deal with this, 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 a conventional culture device 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 perform 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 amount and wavelength of irradiation light to irradiate the light necessary for the growth of the cultured object, a means for controlling the composition of the gas atmosphere within the thermostatic chamber is installed. . 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, the incubator is tilted in the culture stage where tilted culture is required. The optical system irradiates the culture tube containing the culture medium with the culture medium with light having an irradiation amount and a wavelength depending on 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 the accompanying drawings, a culture device 10 according to the present invention includes a constant temperature bath 11. This constant temperature bath 11 can arbitrarily set and maintain its internal temperature according to each stage of culturing the cultured object.

A rotatable incubator 13 supported on a rotating table 12 is installed inside the constant temperature bath 11 . This rotary incubator 13 accommodates a plurality of culture tubes 14, for example up to 25 tubes, each containing a culture material together with a culture solution. The rotating culture vessel 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 incubator 13 can be arbitrarily set from a horizontal state to an inclined state depending on the growth process of the cultured object, and it can be rotated by a rotary drive motor 15. The inclination angle of the rotating incubator 13 can be adjusted up to, for example, 85 degrees with respect to the horizontal, and the rotation speed can be controlled within a range of, for example, up to 10 rpm. Furthermore, the direction of rotation can be changed to either the left or right. Inclined rotational culture may be necessary when cells, callus, etc. are placed in a nonpolar state to differentiate organs, but in the culture apparatus of the present invention, the rotational incubator 13
This is why it is made rotatable and its inclination angle adjustable.

Further, as a means for controlling the gas atmosphere composition (oxygen concentration, carbon dioxide concentration, humidity, etc.) in the thermostatic oven 11, a gas supply source 17 is provided connected to the gas introduction pipe 16 communicating with the interior of the thermostatic oven 11. It is being The amount of gas supplied is regulated by a valve 18, and the gas is discharged through a gas exhaust pipe 19 connected to the constant temperature bath 11 and a valve 20 installed therein.
Adjusted by. The gas supply source 17 can be switched as appropriate depending on the type of gas whose composition needs to be changed in the thermostatic chamber 11. Cultivation can be carried out under normal atmospheric conditions, but for example, the oxygen concentration, carbon dioxide concentration,
It is preferable to adjust the humidity and the like as appropriate by introducing nitrogen gas, carbon dioxide gas, and water vapor into the constant temperature bath 11 according to each stage of culture. For example, when lowering the oxygen concentration in the constant temperature bath 11, the oxygen concentration is diluted using a nitrogen source. When increasing the carbon dioxide concentration in the thermostatic chamber 11, use a carbon dioxide source and
to be introduced within. Further, when adjusting the humidity inside the constant temperature bath 11, a water vapor source is used.

In addition, regarding the control of the gas atmosphere composition in the thermostatic chamber 11, a gas sensor or the like for detecting the gas concentration can be installed in the thermostatic chamber 11, and these sensors can be linked with the gas atmosphere control system. Each indicator may also be provided to issue an alarm if the gas concentration varies from a predetermined set value. These controls can be performed by a control device 25 attached to the constant temperature bath 11.

Furthermore, in the constant temperature bath 11, light necessary for the growth of the cultured material is provided in the culture tube 14 above the rotary incubator 13.
An optical system 21 for irradiating light is removably installed. This optical system 21 has, for example, a plurality of lamps 22, and these lamps 22 are configured to be able to adjust their irradiation light amount individually or for each group. This makes it possible to equalize the illuminance over the entire surface of the rotating incubator 13 according to each stage of culture, to change the illuminance depending on the position, and to make wavelengths or day and night light and dark conditions appear according to the growth process. Also,
When the rotary incubator 13 is tilted, a difference in illuminance of about 10,000 lux can be created between the upper and lower parts.

That is, a wavelength selection filter 24 is replaceably installed below the optical system 21, and when obtaining the wavelength necessary for culturing, the filter that selectively transmits only that wavelength can be replaced as appropriate. I can deal with it. Furthermore, an illuminance sensor 23 is installed at an appropriate position around the rotating incubator 13, and this illuminance sensor 23 is connected to an illuminance meter 26 of a control device 25. While observing the illuminance detected by the illuminance sensor 23 with the illuminance meter 26, the optical system 21 is controlled as a whole, individually, or in groups. This control can be performed by a controller 27 consisting of all control sliders of the control device 25 and a controller 28 consisting of group unit control sliders. The group control slider can also control the lamps 22 individually.

FIG. 2 shows another embodiment of the culture device according to the invention. In this culture apparatus, the inner wall of the constant temperature bath 11 is made of a highly reflective material such as aluminum vapor deposition film, stainless steel, or chrome plating film. Further, a shutter 31 is installed to cover the entire surface of the transparent window member 11a of the thermostatic oven 11 and to block light from outside. By covering the window member 11a with the shutter 31, the optical system 21 can be turned off and the inside of the thermostatic chamber 11 can be kept at night, or external light can be blocked, so that the cultivation can be performed from the optical system 21. It can also be done using light alone. The surface (back surface) of the shutter 31 facing the constant temperature bath 11 is made of the above-mentioned highly reflective material. Constant temperature bath 11
If the inner wall of the shutter 31 and the back surface of the shutter 31 are made of a highly reflective material, the light from the lamp 22 will be reflected,
Since the reflected light is irradiated onto the culture tube 14 again,
The output of lamp 22 can be reduced. Note that the shutter 31 may have a double structure for the window member 11a and be installed in the space between them. Furthermore, when the rotary incubator 13 is tilted, the spherical light condensing plate 32 may be provided in the constant temperature bath 11 so as to face the rotating incubator 13 at any angle. This makes it possible to adjust the irradiation light.

Furthermore, it is also possible to install a blind 33 below the wavelength selection filter 24 and to control the irradiation angle of the light from the optical system 21 to the rotating incubator 13 by opening and closing the blind 33.

Furthermore, in any of the culture apparatuses shown in FIGS. 1 and 2, an ultraviolet lamp can be installed in the optical system 21, and the irradiation can be controlled to induce mutations. In addition, rotating incubator 1
It is also possible to generate a flash in synchronization with the rotation of No. 3. Furthermore, the optical system 21 and the rotating incubator 1
It is also possible to relatively adjust the distance to 3. The optical system 21 may generate pulsed light to irradiate the cultured object with light while periodically changing the amount of irradiation light.

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, Haplopatpus, tobacco, petiunia, rice, corn, carrot, potato, wheat, barley,
Herbaceous plants such as sugarcane and soybean, and pine,
Examples include cells and tissues of woody plants such as eucalyptus, poplar, coffee, paragum, grape, elm, and apple. In addition, cells (including protoplasts), tissues, and organs to be cultured include:
Cells such as mesophyll cells, cultured cells derived from various tissues, tissues such as scutellum, embryo, mesophyll, cortex, and pith;
Meristem tissues such as the shoot apex and root tip, as well as stems, leaves, anthers,
Examples include organs such as roots and flowers.

For example, in order to culture plant cells using the culturing apparatus of the present invention, the temperature of 0 to 30°C and the temperature of 0 to 30° C.
Illuminance of 20000 lux, oxygen concentration of 2-20%, 0
~50% carbon dioxide concentration, rotating incubator tilt angle 0
Plant cells and tissues are suspended in a liquid medium such as Gamborg B5's basic medium (Gamborg et al. 1968) or Murashige and Skoog's MS basic medium (Murashige and Skoog 1962) at ~85°, and cultured statically and rotated. It can be cultured, or it can be placed in a medium solidified with agar or the like to perform static culture or rotational culture. The plant growth hormones added to the medium used include naphthalene acetic acid (NAA),
2,4-dichlorophenoxyacetic acid (2,4-D),
Indole-3-acetic acid (IAA), indole-3
-Propionic acid (IPA), indole-3-butyric acid (IBA), phenyl acetic acid (PAA), benzofuran-
3-Auxins such as acetic acid (BFA) and phenylbutyric acid (PBA) and KT-30 (manufactured by Kyowa Hakko Co., Ltd.), 6
- Cytokinins such as benzylaminopurine (BA), zeatin (Z), kinetin, etc. may be used.

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

Experimental example 1 Annual plant Haploppus
gracilis) was cut after sterilization, and added to Gamborg B5 basal medium with 2 mg/6-benziaminopurine (BA) and 30,000 mg/carbon source.
Suspended in a modified medium (PH5.6) containing sucrose,
Rotational culture was performed using the apparatus of the present invention at an inclined angle (rotary incubator inclination angle of 65°). The culture conditions were: temperature 28℃;
The illuminance was 2000 to 12000 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 extracted and added to Gamborg B5 basic medium at 2 mg/kg.
of naphthalene acetic acid (NAA), 4 mg of BA, and 30,000 mg of sucrose as a carbon source, and solidified with agar (agar concentration 0.8%; PH 5.6)
The cells were placed on a bed and cultured stationary using the culture apparatus of the present invention (tilt angle: 0°). The culture conditions were a temperature of 28°C, an illumination intensity of 3000 lux, and an oxygen concentration of 5% in the culture atmosphere. Under these conditions, the callus formed from the embryos is suspended in a liquid medium of the same composition (no agar added) and continuously cultured in the same apparatus with rotation (tilt angle of 60°). By this method, we succeeded in producing a large number of pale green cultured cells that were uniform and proliferated vigorously.

For comparison, we performed static culture for one month under the same conditions, subcultured them to the same medium, and continued static culture, but the proliferation rate in this area was about 50% of that in the area where we switched to rotary culture. Ta.

[Effects of the Invention] As described above, in the culture apparatus of the present invention, an incubator that is rotatable and whose tilt angle can be adjusted and an optical system that can adjust the amount and wavelength of irradiated light are installed in a constant temperature bath. Since a means for controlling the gas atmosphere composition in the constant temperature bath is provided, the inclination angle of the culture vessel, the amount of irradiation light, and the culture gas atmosphere composition can be adjusted to the optimum state according to each stage of culture. Therefore, for example, culturing before and after callus formation can be performed consistently in the same device.

[Brief explanation of drawings]

FIG. 1 is a front view showing one embodiment of the culture device of the present invention, and FIG. 2 is a front view showing another embodiment of the culture device of the present invention. DESCRIPTION OF SYMBOLS 11... Constant temperature bath, 13... Rotary incubator, 14... Culture tube, 21... Optical system, 22... Lamp, 16... Gas introduction pipe, 17... Gas supply source, 18, 20... Valve, 19
...Gas exhaust pipe, 23...Illuminance sensor, 24...Wavelength selection filter, 27, 28...Light source controller, 31...Shutter, 32...Reflector, 33...Blind.

Claims (1)

[Scope of Claims] 1. A thermostatic chamber capable of setting and maintaining an internal temperature at a desired temperature suitable for the growth process of a cultured material, and a cultured material placed in the thermostatic chamber and to be cultured together with a culture solution. A rotatable incubator for accommodating culture tubes, the inclination angle of which can be set arbitrarily from a horizontal state to an inclined state according to the growth process of the cultured material, and a culture vessel installed in the thermostatic chamber. an optical system capable of adjusting the amount of irradiation light for irradiating the culture tube with light necessary for the growth of the cultured material, and for controlling the gas atmosphere composition in the thermostatic chamber according to the growth process of the cultured material. A culture apparatus comprising a gas atmosphere control means, wherein the optical system includes a plurality of light sources removably provided above the constant temperature bath, and a plurality of light sources provided below the light sources for illuminating the culture tube. A culture apparatus having an optical system, comprising a wavelength selection filter that adjusts the wavelength of light, and a light source control section that controls light irradiation from the optical source. 2. A culture apparatus having an optical system according to claim 1, wherein the light source control unit controls the amount of irradiation based on a signal from an illuminance sensor installed around the rotating incubator. 3. A culture apparatus having an optical system according to claim 1 or 2, wherein a blind is installed below the wavelength selection filter to adjust the angle of light irradiation to the culture tube. 4. Any one of claims 1 to 3, comprising a reflecting plate that is arranged to face the rotating culture vessel when it is in an inclined state and reflects light from the optical system to the culture tube. A culture device having the optical system according to item 1. 5. A culture apparatus having an optical system according to any one of claims 1 to 4, wherein the light source control section independently controls the light source. 6. A culture device having an optical system according to any one of claims 1 to 5, wherein the light source includes an ultraviolet lamp for inducing mutations. 7. Any one of claims 1 to 6, wherein the thermostatic chamber has a transparent window member, and a light shutter is installed to cover the window member and block light from the outside. A culture device having the optical system described in .