JPS62289173A - Culture apparatus having gas atmosphere controlling means - Google Patents

Abstract

PURPOSE:To carry out culture in the same apparatus consistently from a material to be cultivated to histodifferentiation, by adjusting an angle of inclination of a culture medium, the intensity of irradiation, etc., and a gas atmosphere composition by a gas atmosphere controlling means to optimum conditions depending upon each process of culture. CONSTITUTION:A culture device 13 having plural culture tubes 14 is set rotatably by a rotary drive motor 15 and can be laid at any angle of inclination from a horizontal state to a slant state on a rotary table 12 in a constant temperature tank 11 laid in a culture apparatus 10 and an optical system 16 equipped with plural lamps 17 which are fixed in each group unit to control the intensity of irradiation is set. Then a material to be cultivated and a culture solution are put in the culture tubes 14, temperature, illuminance, an angle of inclina tion of the culture medium 13 and number of revolutions are adjusted to optimum states depending upon each process of culture. An air feed source 22, an N2 gas feed source 23 and a CO2 gas fed source 24 are connected through lines L1-3, respectively, to a piping storing part 21b of a control device 21, the air feed source is connected to a joint terminal 26, the N2 gas feed source and the CO2 gas feed source are connected to solenoid valves 28 and 29 and from a common line L4 to a joint terminal 27. On the other hand, the solenoid valves 28 and 29 are connected through a common line L5 to a control part 21a and feed of gases are controlled through a gas sensor in the constant temperature bath tank 11 to adjust a gas atmosphere composition to optimum condition.

Description

[Detailed Description of the Invention] 3. Detailed Description of the Invention [Object of the Invention] (Field of Industrial Application) This invention provides a method for culturing cultured materials such as cells (including 7” rotograsts) and tissues. Regarding equipment.

(Prior Art) Cultivation and propagation of cells, tissues, etc. are not only important for basic research in biotechnology, but are also known to be useful for mass propagation of superior plants.

Taking two examples of plant cells, when the protogellasts are immersed in a culture solution and allowed to proliferate, a callus (amorphous cell mass) is formed, and by changing the cell composition of the culture solution, various organs W are 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 may be performed until cell walls are formed, and after cell wall formation, tilted rotation culture may be 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.

In particular, with conventional culture equipment, it is not possible to control the culture atmosphere according to the growth process of the cultured object. 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. In other words, since the culture conditions vary considerably during the growth process of the cultured object, when using conventional culture equipment, it is difficult to achieve consistent culture according to the growth process of the cultured object. It cannot be done.

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) In the culture apparatus of the present invention, a culture tube is placed in a constant temperature bath whose internal temperature can be arbitrarily set. I had to store it.

In addition to installing a rotatable incubator and an optical system that can adjust the amount and wavelength of irradiation light to irradiate the light necessary for the growth of the cultured material, the incubator can be tilted from a horizontal state to an inclined state. The angle can be set arbitrarily.

In addition, means for controlling the gas atmosphere composition (oxygen concentration, carbon dioxide concentration, and humidity if necessary) in the thermostatic chamber is provided.

(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 can set brightness and darkness conditions according to each stage of culture, and irradiates the culture tube containing the cultured material together with the culture solution with a necessary amount of light.

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

As shown in FIG. 1, a culture device 10 according to the present invention includes:
It is equipped with a constant temperature bath 11 and a control device 2 attached thereto. The thermostatic chamber 11 can arbitrarily set and maintain its internal temperature according to each stage of culturing the cultured material.

A support stand 12 and a rotary incubator 13 supported on the support stand 12 are installed inside the constant temperature bath 11. This rotary incubator 13 is made up of a plurality of tubes, for example, which accommodate a culture material to be cultured together with a culture solution. It accommodates up to 26 culture tubes 14. 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 rotating incubator 13 can be moved from a horizontal state to an inclined state (for example, up to 85 degrees) depending on the growth process of the cultured object.
The inclination angle can be arbitrarily set up to 100 degrees, and it can be rotated by the rotary drive motor 15.

The rotation speed can be adjusted up to 10 rpm, and the rotation direction can be either left or right. Inclined rotation culture is sometimes necessary when cells, callus, etc. are placed in a nonpolar state to differentiate organs, but in the culture device of the present invention, the incubator 13 is rotatable and the inclination angle is adjustable. The reason for this is to accommodate this.

Further, in the constant temperature bath 11, an optical system 16 is installed above the rotating incubator 13 to irradiate the culture tube 14 with light necessary for the growth of the cultured object. This optical system 16 is composed of, for example, a plurality of lamps 17, and these lamps 17 have a configuration in which the amount of irradiation light can be adjusted individually or in units of several groups. This results in
Is the illuminance equalized over the entire surface of the rotating incubator 13 according to each stage of culture, is the illuminance changed depending on the position, or is it bright and dark during the day and night? can be made to appear. Moreover, when the rotary incubator 13 is tilted, the upper and lower parts of the rotary incubator 13 are 1
It can also be combined with an illuminance difference of 10,000 look steps.

Regarding the control of the amount of light irradiated by the optical system 16, an illuminance sensor for detecting the illuminance on the rotating incubator I3 can be installed in the constant temperature bath 11, and this sensor can be linked with the optical system. Additionally, an indicator may be provided to issue an alarm if the illuminance varies from a predetermined set value. These controls are performed by a control unit M attached to the constant temperature bath 11.
21.

Now, the culture gas atmosphere in the thermostatic chamber 11 is based on the atmospheric atmosphere, and is normally under the atmospheric atmosphere, but the composition of this gas atmosphere (oxygen concentration, carbon dioxide concentration, etc.), an air supply source 22, a nitrogen gas supply source 23, and a carbon dioxide gas supply source 24 are installed outside the constant temperature bath 11. The air supply source 22 is connected to the piping housing portion 21b of the control device 21 at a connecting terminal 26 via an air purifier 25 and a line L1. Further, the nitrogen gas supply source 23 and the carbon dioxide gas supply source 24 are connected to solenoid valves 28 and 29 through lines L2 and L3, respectively, and these lines are connected to a common line L.
The connecting terminal 22 is connected to the pipe accommodating portion 21b via the connecting terminal 22. Solenoid valves 28 and 29 are connected to the common control line L
It is connected to the control section 21& of the control device 2 through the terminal 5'fC.

Next, the control system for the gas atmosphere composition will be explained in more detail with reference to FIG. The illustrated example is Tsunetan Hinoki lj! :4 units (
1z-1, xz-z, 11-s and 1l-4) are installed in series and are controlled by a control device 21.

The air supply source 22 is constituted by an air ingressor, and the nitrogen supply source 23 and the carbon dioxide supply source 24 are composed of these gases. Each house is made up of Sugonpe. Also,
The air cleaner 25 is composed of an air filter 25&, a mist separator 25b, and a micro-mist/4'lator 25c.

The air supply line L1 is connected to a line L6 inside the pipe housing section 2Ib. A pressure gauge 32 is connected between the pressure regulating valve 30 and the solenoid valve 31. Further, the common connection line L4 of the nitrogen gas supply source 23 and the carbon dioxide gas supply source 24 is connected to the pressure regulating valve 33 and the electromagnetic valve 34 in the piping accommodation portion 21b.
The solenoid valve 31 and the three-way solenoid valve 36 are connected to the air supply line LI through the solenoid valve 31 and the three-way solenoid valve 36. A pressure gauge 35 is connected between the pressure regulating valve 33 and the solenoid valve 34.

The common supply line L6 each has a solenoid valve 37a.

37b, 37a and ? Temperature bath 11-1 via 7d
゜11-2. Connected to 11-3 and 11-4.

A control line L5 connecting the electromagnetic valves 28 and 295 is connected to a gas concentration control section 38 installed in the control section 21b. The X solenoid valve 31 is connected to the control unit 38 via line L7i, and the solenoid valve 34 is connected to line L8? Control unit 3 through
It is connected to 8. Furthermore, the three-way solenoid valve 36 is connected to the line L.
It is connected to line L8 via 9f. Note that the control unit 3
8 is each constant temperature bath 11-1.11-2.11-3゜11-
A gas sensor is installed to detect the concentration of the sampling gas from gas sensor 4, and the opening and closing of each electromagnetic valve is controlled by signals from a controller driven based on information from each gas sensor.

Furthermore, each thermostatic oven has a control unit 3 that controls the atmospheric gas inside the thermostatic oven.
It has a common line L12 for sampling 8 times.

In addition to these, a humidity control mechanism may be installed to control the humidity of the atmosphere within the thermostatic chamber. As shown in FIG. 2, this humidity control mechanism has a water storage container (steam source) 39, which is connected to a three-way solenoid valve 36 and blows air from the near compressor 22 into the water contained therein. Line LIO is inserted. Air containing a predetermined amount of water vapor by this air blowing is supplied to the line L6 via the drain generator 41 and the line LllfJ:.

Water is supplied to the bottom of the water storage container 39 via a pump 7'40.
42 is installed, and a drain water storage container 43 is installed at the bottom of the drain sensor or rater 41.

Each gas supply source and water vapor source 39 can be switched as appropriate depending on the type of gas whose composition needs to be changed in the thermostatic chamber 11.

Cultivation can also be carried out under normal atmospheric conditions,
For example, nitrogen gas, carbon dioxide gas, and water vapor are added to the constant temperature chamber 11 according to each stage of culture, with equal amounts of oxygen concentration, carbon dioxide concentration, and humidity.
It is preferable to introduce it into the interior and adjust it appropriately.

For example, when lowering the oxygen concentration in the constant temperature bath 11, the oxygen concentration is diluted using a nitrogen source.

That is, the nitrogen gas from the nitrogen gas pump 23.

The pressure is reduced by the pressure regulating valve 33, and the solenoid valves 2g, 34.3B, 37 and 44 (44&
, 44b, 44e, 44ti)f from inside the thermostatic chamber 11 to the outside. At this time, the oxygen gas concentration in the thermostatic chamber 11 is detected by an oxygen gas sensor in the control section 38, and the pW magnetic valve 34 is controlled in response to a signal from the controller in the control section 38. When the oxygen gas concentration approaches the set value, the opening degree of the electromagnetic valve 34 is automatically reduced, and the electromagnetic valves 37 and 44 are also closed to prevent excessive supply of nitrogen gas.

When increasing the oxygen gas concentration, near compressor 2
2 is purified by an air purifier 25, and is supplied into each thermostatic chamber 11 via line L1 in the same manner as the supply of nitrogen gas.

When increasing the carbon dioxide concentration, the carbon dioxide gas from the carbon dioxide gas pump 24 is passed through the line L3, the solenoid valve 29, the pressure regulating valve 33, the solenoid valve 34, and the three-way solenoid valve 36, and then through the line L6 from the solenoid valve 37 to each constant temperature bath 11. Supply carbon dioxide gas inside. As another method for increasing the carbon dioxide concentration, since it is sufficient for the carbon dioxide concentration to be less than s o o o ppm, after setting the oxygen gas concentration to a predetermined concentration using the above method, the adapter 45 & ,45be4
5c and 45tl, even if a certain amount of carbon dioxide gas is injected using a sushiri 7, the three-way solenoid valve 36 should be moved in the direction of the water vapor source 39 in order to increase the humidity in the thermostatic chamber. After changing the cut and removing the water droplets entrained with humidified air using a drain sensor or a rotor 41, the water is supplied into each thermostatic chamber 11 via line Lll and line L6.

As described above, the composition of the gas surrounding the thermostatic chamber is controlled according to the atmospheric composition required for the growth process of the cultured specimen.

Examples of the disrupted culture that can be cultured using the culture apparatus of the present invention include animal and plant cells and tissues, and microorganisms. Preferably, herbaceous plants such as habropagus, tobacco, petunia, rice, corn, carrot, potato, wheat, barley, sugar cane, and maize, as well as pine, eucalyptus, poplar, coffee, grape, elm, apple, etc. Examples include cells and tissues of woody plants. In addition, the cells to be cultured (76
Tissues and organs include mesophyll cells, cells such as cultured cells derived from various tissues, platens, embryos, mesophyll, cortex, tissues such as pith, and shoot tips.

Examples include meristems such as the end of the body, and organs such as stems, leaves, pieces, roots, and flowers.

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 degrees, and the carbon dioxide gas is 0 to 50%. Plant cells and tissues were grown in Gamborg B5 basal medium (Gamborg
@t ml, 1968), Murashida Skoog's M
S basal medium (Murashlge, Skoog 196
2) It is also possible to perform static culture or rotational culture by suspending it in a liquid medium such as the above, or it is also possible to perform static culture or rotational culture by placing the above medium in a medium solidified with agar or the like. . The plant growth hormones added to the medium used include naphthalene acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and indole-3-globionic acid. (I.P.A.
), indole-3-butyric acid (IBA)% phenylacetic acid (FAA), benzofuran-3-acetic acid (BFA)
), auxins such as phenylbutyric acid (PBA), and cytokinins such as KT-30 (manufactured by Kyowa Hakko Co., Ltd.), 6-benzylaminopurine (BA), zeatin (2), and kinetin.

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

Experimental Example 1 Annual plant Haplopappusg
racilm) was sterilized, cut out, and added to Ganshiki B5 basal medium with 2 mg/L of 6-benzylaminopurine (BA) and 30,000 ra as a carbon source.
t/t of soda sugar? The cells were suspended in a modified medium (pH 5, 6) and cultured in a tilted manner (rotating incubator tilt angle 65°) using the apparatus of the present invention. The culture conditions were a temperature of 28° C., an illuminance of 2,000 to 12,000 lux, an oxygen concentration of 15% in the culture atmosphere, and a rotation speed of 3 rpm.

As a result, 5 weeks after the start of culture, light green shoot primordium aggregates, which could not be obtained conventionally with the tilt angle o0, were obtained.

Experimental Example 2 After sterilizing the seeds of Japanese red pine (Pinus d@naiflora), the seeds were removed and placed in the basal medium of Gunborg B5.
mg/l naphthaleneacetic acid (NAA), 4 mvt BA and 30.0 mg/l as carbon source. OOOmL/l of shi! $
1 and a medium solidified with agar (agar concentration 0.8%; p
H5, 6) and cultured stationary using the culture apparatus of the present invention (tilt angle 0°). The culture conditions were a temperature of 28°C;
The illuminance was 3000 lux, and the oxygen concentration in the culture atmosphere was 5%. After culturing for one month under these conditions, the callus formed by 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: 600). By this method, they 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 then static culture continued for 20 minutes. In this group, the proliferation rate was 50% of that in the group switched to rotary culture.
It was about.

[Effects of the Invention] As described above, in the culture apparatus of the present invention, a rotatable incubator and an optical system capable of adjusting the amount of irradiation light are installed in a thermostatic chamber, and the gas atmosphere composition within the thermostatic chamber is controlled. 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 irradiation light, and the culture gas atmosphere composition can be adjusted according to each stage of culture. It can be adjusted to the optimum condition. Therefore, the culture conditions during the entire growth process of cells etc. can be consistently controlled within the same apparatus.

[Brief explanation of drawings]

FIG. 1 is a front view showing an embodiment of the culture device of the present invention, and FIG. 2 is a circuit diagram of a gas-based concentration controller of the culture device of the present invention. 11... Constant temperature bath, 13... Rotary incubator, 14...
Culture tube, 16... Optical system, 17... Lamp, 21.
. . . Control device, 22, 23, 24, 39 . . . Gas supply source, 38 . . . Gas concentration control unit.

Claims (3)

[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 culture medium, 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; An optical system capable of adjusting the amount of irradiation light for irradiating the culture tube with the light necessary for the growth of the object, and a gas atmosphere control for controlling the gas atmosphere composition in the thermostatic chamber according to the growth process of the object to be cultured. A culture device comprising means,
The gas atmosphere control means has a gas supply source including a supply source of oxygen gas, nitrogen gas, and carbon dioxide gas to be supplied to the thermostatic oven, and controls gas supply from the gas supply source to the thermostatic oven. A culture apparatus having a gas atmosphere control means, characterized in that it is equipped with a control mechanism. (2) A culture apparatus having a gas atmosphere atmosphere means according to claim 1, wherein the gas supply source includes a water vapor supply source. (3) The gas atmosphere according to claim 1 or 2, wherein the control mechanism has a gas sensor that detects the gas concentration in the thermostatic chamber, and controls gas supply based on a signal from the gas sensor. A culture device having control means.