JPH01211484A - Method for germ-free mass culture and apparatus therefor - Google Patents

Abstract

PURPOSE:To make it possible to provide a larger spatial scale and efficiently cultivate cells at a high speed, by irradiating the whole area of the liquid surface in the interior of a culture vessel with light, aseptically inoculating algae and bacteria thereinto and continuously carrying out pure germ-free culture and collection. CONSTITUTION:The whole area of the liquid surface in the interior of a culture vessel 4 is irradiated with light and algae and bacteria are aseptically inoculated thereinto to continuously carry out pure germ-free culture and collection. That is a light source 15 for irradiating the whole area of the liquid surface in the interior of the culture vessel 4 is provided and a xenon lamp or natural light sent by an optical fiber is cited as the light source. A glass window 13 is mounted on the top surface of the culture vessel 4 and the light source 15 and a whole reflecting mirror 16 behind the light source are provided above the glass window 13. Furthermore, feed passages 10 and 9 for air and a culture solution to the culture vessel 14 are provided and equipped with sterile filters 6 and 3. Pipes between the filters and the culture vessel are sterilized with steam. As a result, a larger spatial scale can be provided and cells can be efficiently cultured at a high speed.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a sterile mass culture method and an apparatus therefor. More specifically, the present invention relates to a device and method that enable mass culture of cells at high speed, and a highly efficient pure culture device useful as experimental equipment or production equipment in research institutions, factories, etc., and a method using the same. The present invention provides a sterile culture method.

(Background Art) With the development of bioengineering, experimental biology, and cell culture technology, there is a need to establish a technology for culturing biological cells in large quantities with high efficiency.

Until now, it has been considered extremely difficult to stably maintain the conditions for the survival and growth of organisms on a relatively large scale when it comes to mass culture.

The reason for this is that the mechanisms of survival and proliferation of living organisms and cells themselves have not been elucidated, and progress has been made in the development of devices and systems to study these mechanisms stably and on a relatively large scale under optimal conditions. There was something I hadn't done.

For example, regarding the biological II mechanism of red tide generation, which the inventor of this invention is currently investigating, until now there has been a lack of equipment systems for high-speed, large-scale cultivation. It was difficult to make accurate predictions and defenses. Research on red tide focuses on Heterosigia akasiwo, which frequently occurs in the Seto Inland Sea, and Ch.
This is due to the abnormal growth of Attonella antigua, but based on the results of laboratory tests, flasks, and even benthic gales, it is difficult to predict this abnormal growth in the actual sea area. This is because temperature, nutrient concentration, etc. change on a large scale, and vertical movement of algae, which causes red tide, occurs. In order to elucidate the mechanism of red tide occurrence along with these changes and movements, it is essential to establish a culture system that has an appropriate spatial size and can create a field closer to natural conditions for environmental factors. Ta.

This situation is not limited to the case of red tide research, but devices and systems for culturing biological cells on a larger scale and efficiently are needed for various experimental research and production of useful substances. It was needed.

(Purpose of the Invention) This invention was made in view of the above-mentioned circumstances, and provides an efficient and high-speed culture that overcomes the problems of conventional culture systems, has a larger scale spatially, and The purpose of this study is to provide a new high-speed, large-scale culturing device and a sterile culturing method using the same.

(Disclosure of the Invention) In order to achieve the above object, the aseptic mass culture method of the present invention irradiates the entire liquid surface inside the culture tank with light, inoculates algae and bacteria aseptically, and continuously produces pure aseptic culture. It is characterized by sterile collection.

That is, the method of this invention and the device for this method have a major feature of irradiating light over the entire liquid surface inside the culture tank, and by making this feature a part of the entire system of the device, it is possible to It enables high-speed, large-scale culture of cells.

The culturing method and apparatus of the present invention will be explained with reference to the attached drawings.

FIG. 1 is a block diagram showing one embodiment of the present invention. In this example, the culturing apparatus includes a storage tank (1) for storing mother liquor for culturing, a mixing tank (2) for adding additional ingredients, a sterilization filter (3), and a culture tank (4) and a light illumination unit (5). ).

The culture tank (4) also includes a sterilization filter (6) for air supply, a liquid temperature adjustment jacket (7), and a liquid temperature adjustment bath (
8) etc. are installed. It is important to provide a sterile filter (6) device and to perform steam sterilization between this filter (6) and the culture tank (4).

FIG. 2 shows an enlarged view of the culture tank (4) and the light illumination section (5). As shown in Figure 2, a liquid temperature adjustment jacket (7) is attached to the culture tank (4).
Culture solution inlet (9), air inlet (10), culture solution circulation outlet (11), culture solution overflow (1)
2), and is also equipped with other means for measuring the culture state, such as a flow meter, thermometer, pressure gauge, and biological cell sampling device.

A glass window (13) is attached to the top opening of the culture tank, and the entire surface of the culture solution (14) is illuminated with light through this glass window (13). This lighting uses a light source (15) installed above the glass window (13), a total reflection mirror (16) behind it, and a concave lens (17) for expanding the light if necessary. conduct.

A light source (15), a total reflection mirror (16), and a concave lens (
17) and the distance between these glass windows (13), the entire liquid surface can be illuminated at all times depending on the size of the glass windows (13).

Depending on the case, this adjustment can also partially illuminate the liquid surface.

Of course, the culture apparatus of the present invention is not limited to the above examples; the shape and structure of the culture tank, and the overall configuration of the culture system can be selected as appropriate depending on the purpose of the culture system and the target organism. can do. Although a light source and a total reflection mirror behind it are not necessarily required, the culture method and apparatus of the present invention do not lack a light illumination section to irradiate the entire surface of the liquid with light in order to achieve high-speed, large-scale culture. Can not.

As a light source constituting the light illumination section, a suitable one such as a mercury lamp, a halogen lamp, or a xenon lamp can be used. Natural light transmitted through optical fibers may also be used.
The inner surface of the culture tank can be lined with a glass lining, an alloy, a metal such as titanium, or a resin such as Teflon, depending on the target culture system.

Next, a case will be described in which the culture apparatus of the present invention is applied to the culture of grandchildren in order to elucidate the mechanism of red tide occurrence.

(Culture Apparatus) Seawater is stored in a storage tank (manufactured by JilOm3, internal glass lining) (1) of an apparatus having the configuration shown in FIGS. 1 and 2. Seawater is stored in a mixing tank (capacity 1
0.2 m3) In step (2), mix with nutrients such as nitric acid, phosphoric acid, and vitamins.

The culture solution passes through a sterile filter (3) to a culture tank (4).
For example, the sterile filter (3) that leads to
．． czm Rogard rilter, 0.22
μxnHi1. Iigardfilter, 0.2
2) tm Hillidisk (formerly 11pora)
Co., Ltd.) etc. are used. The size of the culture tank (4) is
Height: 2m, inner diameter: 1m, culture medium capacity: 1m, with zero space above the tank.
．． It is possible to leave an air space of 4 m3.

Cultivate! Bi 1 between I tank (4) and sterile filter (3)
can be made of Teflon or inner surface made of Teflon. Withstands steam sterilization and does not corrode.

The culture tank (4), sterilization filter (3), air filter (6), and pipes can be steam sterilized for about 30 minutes to 1 hour before use. For example, a temperature of about 110° C. and a pressure of about 0.5 m/aJ are used.

The glass window (13) on the top surface of the culture tank (4) has a diameter of 30011, for example, and is illuminated from above with a 150A xenon lamp. The wavelength range is from 400 to 700 nm, which is similar to sunlight during the day in summer. In this case, the light intensity inside the tank is approximately 1
It will be about 75-173.

The water temperature in the culture tank (4) is controlled by a liquid temperature adjustment jacket attached to the outside (for example, height 35cm, thickness 5am).
By passing water at a rate of 2 to 6 j/min, the maximum temperature difference between the surface layer and the bottom layer can be stably maintained at about 15°C.

Measurements inside the culture tank can be automatically performed using a data logger controlled by a microcomputer and a sequence program. Water temperature can be measured using a platinum resistance thermometer or the like.

(Culture conditions) 1+et of species that often cause red tide in Osaka Bay
The zero conditions for culturing erO3il;lla akasiwo in the above culture tank are, for example, as follows.

・Aeration mixed system ・L/D: 12/12 cycles ・Culture temperature 20±2℃ ・F/2 medium (+P 1.5 μnol/J) (Culture results) When cultured under the above equipment and conditions Figure 3 shows the results of cell proliferation.

This Figure 3 shows that cells are rapidly proliferating in an extremely short period of time. Specific growth rate μ(d')
, that is, the cell concentration per measurement time (cell number/J-
1) was 1.06.

Conventionally, this specific growth rate .mu. is about 0.4 d-1 in a light-illuminated culture tank of approximately the same scale, which far exceeds the highest level to date. High-speed, large-scale culture has been achieved.

It can be seen that the light illumination method of the present invention works synergistically with the stability of the temperature gradient in the vertical direction of the culture tank to form an extremely excellent culture environment.

(Effects of the Invention) As explained in detail above, this invention enables high-speed, large-scale culture. A light illumination type high-speed, sterile mass culture method and its device are realized which are extremely useful as experimental equipment, production equipment, etc.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing one embodiment of the present invention. FIG. 2 is a cross-sectional view illustrating a culture tank and a light illumination unit. FIG. 3 is a correlation diagram between cell concentration and time showing the culture results of Heterosigna akasiwo. 1... Storage tank 2... Mixing tank 3.
... Sterilization filter 4 ... Culture tank 5 ... Light illumination section 6 ... Air sterilization filter 7 ... Flowing water adjustment jugget 8 ... Liquid temperature adjustment bath 9 ... Culture solution inlet 1
0...Air inlet 11...Circulating fluid outlet 12...Overflow port 13...Glass window 1
4...Culture solution 15...Light source 16...Total reflection mirror Patent attorney Toshihiro Nishizawa 1 Figure 2 Figure 3 Culture time (days) Procedural amendment (self-restraint)

Claims (7)

[Claims] (1) A sterile mass culture method characterized by irradiating the entire liquid surface inside a culture tank with light, aseptically inoculating algae and bacteria, and continuously performing pure sterile culture and sterile collection. (2) A sterile mass culture device characterized by being provided with a light source that irradiates the entire liquid surface inside the culture tank. (3) The sterile mass culturing apparatus according to claim (1), wherein the light source is a xenon lamp or natural light transmitted by an optical fiber. (4) A sterile mass culturing device characterized in that a glass window is attached to the top surface of the culture tank, and a light source and a total reflection mirror behind the light source are provided above the glass window. (5) Claim (2), comprising a supply path for air and culture solution to the culture tank, and a sterilization filter attached to the supply path;
Or the sterile mass culture device according to item (4). (6) The aseptic mass culturing apparatus according to claim (5), wherein the pipe line between the filter and the culture tank is steam sterilized. (7) Claim No. (2) wherein the inner surface of the device is lined.
) or the sterile mass culturing device described in (4).