JPH0257173A - Liquid immersion type oxygen enriching aerator - Google Patents

Abstract

PURPOSE:To obtain the subject apparatus, having a structure of many bundled hollow yarns with oxygen-enriching performance and capable of feeding air in a high oxygen concentration into a culture solution with low foaming and evaporation by dipping the bundle in the culture solution and feeding compressed air thereto. CONSTITUTION:Hollow yarns 2 having oxygen-enriching performance are arranged around a hollow pipe 3 for introducing air and both ends of the hollow yarns 2, together with the hollow pipe 3, are sealed in joining parts 5 and 9. The one joining part 5 is closed with a cap and the hollow pipe 3 and the hollow yarns 2 are communicated to connect the other joining part 9 through a cap 11 to an air conduit pipe 13. The air conduit pipe 13 and hollow pipe 3 for introducing air are respectively provided with pressure control valves 14 and 15. The hollow yarns are preferably formed from an aromatic polyamide or polyarylate having heat resistance, since thermal sterilization can be carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an aeration device for a culture solution of animal cells, plant cells, and aerobic microorganisms, and in particular, it has oxygen enrichment performance when immersed in the culture solution. The present invention relates to an immersion-type oxygen-enriched aeration device that enables large-scale, high-density culture of cells by aerating oxygen-enriched air into a culture medium through hollow fibers.

BACKGROUND OF THE INVENTION The growth and multiplication of animal cells, plant cells, and aerobic microorganisms in liquid or suspension cell cultures requires the provision of a solid surface that meets the needs of the cells, as well as the provision of nutrients and nutrients. Removal of harmful metabolites and supply of oxygen are necessary, and these are limiting factors for cell culture. Therefore, in order to industrially achieve large-scale, high-density culture of cells and microorganisms, the obstacles of these culture-limiting factors must be overcome. Furthermore, among these limiting factors, the supply of oxygen is the one that is most promising for improvement.

On the other hand, cell culture methods are aimed at growing and multiplying only specific cell types, and the contamination and proliferation of foreign cells or bacteria must be reliably excluded. That is, before introducing the target cell type, the culture device and the culture medium must be completely sterile and in an organism state. As such sterilization methods, sterilization using ethylene oxide, chemical sterilization using an aqueous hypochlorite solution, and high-pressure steam sterilization are known. Among these sterilization methods, the high-pressure steam sterilization method is industrially the most preferred because it does not require cleaning after the sterilization operation or waste management.

Therefore, an aeration device for the purpose of supplying oxygen in cell culture must be capable of one of these sterilization methods, preferably a device capable of high-pressure steam sterilization.

As a method of supplying oxygen to the culture solution, bubble aeration of air that has been sterilized using a filter or the like has been used for a long time. In such bubble aeration, the dissolution of oxygen occurs on the surface of the bubbles rising in the liquid, so in order to increase the rate of oxygen dissolution, it is necessary to increase the amount of aeration and to generate fine bubbles. The dissolved oxygen concentration can also be increased by bubbling separately adjusted oxygen-enriched air with a higher oxygen concentration than normal air, but most of the enriched air is released without being dissolved.
Difficult to adopt industrially.

In cell culture, bubble aeration is the simplest oxygen supply method and enables high-pressure steam sterilization, but several issues have been pointed out that need to be resolved.

The first is intense foaming due to bubble ventilation. In general, cell culture solution is a high viscosity liquid, and air bubbles introduced from the bottom of the culture container remain on the culture solution surface for a long time without bursting, resulting in a larger culture container compared to the culture solution volume. Requires volume.

The second problem to be solved is that cells that come into contact with the bubbles are damaged during the process in which the bubbles generated at the bottom of the culture container rise through the liquid until they finally burst. In particular, it has been pointed out that animal cells, whose cell membranes have poor mechanical strength, suffer significant damage due to contact with air bubbles.

The third problem to be solved with bubble aeration is evaporation loss of the culture solution. This loss of medium is noticeable in animal and plant cell cultures with long culture periods;
In order to correct the concentration of non-volatile components contained in the culture solution and the drop in the liquid level, it is necessary to replenish water and culture solution as appropriate.

As a means of suppressing the above-mentioned problems associated with bubble ventilation,
Conventionally, various methods have been proposed. For example, Japanese Patent Application Laid-Open No. 6:195278 proposes a method of culturing while blowing oxygen-containing gas onto the surface of the culture solution. Although this air supply method has the advantage of eliminating problems caused by air bubble generation, the contact surface between the gas and the culture medium is limited to the surface of the culture medium, and the increase in the depth direction of the culture container reduces the amount of air supply. Scaling up cell culture is difficult because of the shortage.

An oxygen supply method that provides a large contact area between the gas and the culture solution and can suppress the generation of bubbles is to diffuse oxygen-containing gas into the culture solution through a membrane immersed in the culture solution. For example, JP-A-61-100190 proposes a bubble-free gas swing method and device for supplying air into a culture solution through a porous hollow fiber membrane having no surface dense layer. However, such porous hollow fiber membranes do not have an oxygen enrichment function, so as long as normal air is introduced, the dissolved oxygen concentration cannot exceed the air equilibrium value. oxidized air must be supplied.

A method of supplying air into the culture medium using a material with an oxygen enrichment function has also been proposed. A method is disclosed. However, since a thick tube is used, a high gas permeation rate cannot be expected, and as a result, the gas supplied is limited to oxygen, and it is difficult to scale up cell culture.

On the other hand, as an oxygen enrichment membrane having excellent oxygen enrichment performance and high gas permeation rate, a composite membrane of a polysiloxane thin film and a porous carrier is known, as disclosed in Japanese Patent Publication No. 5851321. It has already been put into practical use as an oxygen enricher. Although it is theoretically possible to convert ordinary air into oxygen-enriched air by immersing such a polysiloxane-based oxygen-enriched membrane in a cell culture solution and to supply air under bubble-free conditions, polysiloxane Because of the low heat resistance of the thin film, the device and culture medium cannot be sterilized with steam under pressure in an autoclave, and it has not been used as a means for supplying oxygen-enriched air in cell culture.

The conventional oxygen supply means for cell culture methods have been described above, and it is an air supply method that increases the dissolved oxygen concentration in the culture medium, suppresses problems caused by bubble generation, and is also industrially possible. The present inventors proposed the general formula (A
) proposed a cell culture method in which oxygen-enriched air is aerated through an oxygen-enriched membrane made of a polyamide represented by the formula (B) and a polyarylate represented by the general formula (B).

Problems to be Solved by the Invention The purpose of the present invention is to diffuse oxygen-enriched air into a liquid or suspended cell culture medium through an oxygen-enriched membrane that has excellent heat resistance and is capable of high-pressure steam sterilization. To provide an immersion-type oxygen-enriching aeration device that reduces foaming and evaporation of a culture solution caused by culture fluid, and enables large-scale, high-density culture of animal cells, plant cells, and aerobic microorganisms in an environment with high dissolved oxygen concentration. That's true.

Means for Solving the Problems The present invention provides the following features: 1. A hollow fiber (2) having oxygen enrichment performance is arranged around a hollow pipe (3) for introducing air that is longer than the hollow fiber (2). ) are both ends of the hollow tube (3) and the resin body (4).
, 8) to the joints (5, 9), and at one of the sealed joints (5), the hollow fiber (2) and the hollow tube (3) open into the closed cavity (7). to form an air inlet to the hollow fiber (2), and at the other joint (9), the hollow fiber (2) connects to an air outlet (13) connected to the pressure regulating valve (14) and an air inlet to the hollow fiber (2). It consists of an assembly of hollow fiber oxygen enrichment membranes opening into an open cavity part (11) having a gland seal part (12) that fixes the side surface of a hollow tube (3), and is immersed in a cell culture solution. A liquid immersion type oxygen-enriching aeration device characterized by being able to aerate oxygen-enriched air into a culture solution.

2. A hollow fiber (2) having oxygen enrichment performance and an air introduction hollow tube (3) are sealed to a joint (5, 9) by a resin body (4, 8) at both ends of the hollow fiber, and further, The entire hollow fiber of the oxygen-enriched air permeation section (1) between both joints weighs 0.05 g.
located in a porous tubular body having a pore diameter of m or more to protect the hollow fibers from direct contact with the culture fluid;
2. The immersion type oxygen-enriching aeration device according to item 1 above, wherein oxygen-enriched air can be aerated into the culture medium through the pores of the porous tubular body.

3. The hollow fiber having the above-mentioned oxygen enrichment performance has the general formula (A)
The above-mentioned first material is made of a polyamide represented by the formula (B) or a polyarylate represented by the general formula (B).
This is a liquid immersion type oxygen enriched aeration device as described in Section 1.

Here, n indicates the number of repeating units.

EMBODIMENT OF THE INVENTION The liquid immersion type oxygen-enriched aeration device of the present invention will be explained in more detail below based on the drawings.

The hollow fibers having oxygen enrichment performance used in the present invention are made of polyamide represented by the above-mentioned general formula (A) or polyarylate represented by general formula (B), and are produced by a wet method with an outer diameter of 3 to 3. 0.1 l1rs, inner diameter 2.8~0.0
It is spun to 5 mm and has excellent heat resistance. 12
It is characterized by high heat resistance, with its oxygen enrichment performance not deteriorating even after steam sterilization at 7°C.

In this device, the hollow tube for introducing air also functions as the core, and the material must be specially specified as long as it has mechanical strength and heat resistance that can maintain the structure of this device, and that it will come into close contact with the resin body described below. However, non-corrosive metal tubes such as stainless steel tubes are preferably used. Thin stainless steel tubes with an outer diameter of 1/4 inch or less are expected to have sufficient mechanical strength to maintain the structure and are suitable for cultivating this device. Since it can be deformed into any shape, such as a spiral shape, inside the tank, it is optimally used as a hollow tube.

Several to thousands of oxygen-enriched hollow systems of water are placed around a longer hollow tube to form a joint by sealing the ends of the hollow fibers and the hollow tube. However, the sealing resin body is made of an embedding material with excellent mechanical hardness and heat resistance, such as epoxy-based hardened resin, and the joining member is made of the sealing resin itself, another heat-resistant resin, or a metal joining member. is used.

The hollow fiber opens at the joint that becomes the air inlet to the hollow fiber, that is, at the end of the joint (5) in Figure 1, and the opening surface (
6), the opening surface of the hollow tube may be the same as this, or it may be uneven in several layers.

The joining method between the joint (5) and the closed cavity (7) and between the joint (9) and the open cavity (11) is 1.
Although it is not specified as long as it has a pressure resistance of 0 kg/cta2 and is easy to remove, a beveled screw is most conveniently used. Further, an O-ring or the like is used to connect the hollow tube passing through the hollow fiber opening surface (10) of the joint part (9) and the open cavity part (11), and the hollow part (11) and the hollow fiber part This is a separable joining method.

The hollow fibers of the oxygen-enriched air permeation section (1) between the front joints of the hollow fibers come into contact with the culture solution and can aerate oxygen-enriched air into the liquid, but the entire hollow fiber of the permeation section (1) The hollow fibers may be located within the porous tubular body to protect them from direct contact with the medium fluid in which they are being squeezed and vented through the pores of the porous tubular body.

For such a porous tubular body, a porous tube made of fluororesin is most suitable due to its light weight and shape deformability, but a metal tube or a ceramic tube may also be used, and the weight of either material is 0.05 g.
11 with a pore size of 0.2 g m or more, preferably 0.2 g m or more
A porous tubular body having a pore diameter of 0.01 L or less and a bubbling pressure at which oxygen-enriched air passes through the liquid is 1 atm or less, preferably 0.5 atm or less is used.

The immersion-type oxygen-enriching aeration device of the present invention immerses hollow fibers with oxygen-enriching performance in a culture solution, increases the dissolved oxygen concentration in the culture solution, and enables high-density culture of cells. ,
It is characterized by being able to reduce problems associated with bubble generation. When this device is fixed to a culture tank and used for aeration of oxygen-enriched air into the culture medium, the joint (9) shown in Figure 1 is used.
) and the cavity (7) are located within the culture tank.

There are various ways to fix the culture tank and the joint (9) depending on the capacity and shape of the culture tank, and it is necessary to process the joint according to the requirements, but research and development with a capacity of 10 liters or less In the case of a small culture tank for commercial or intermediate plant level, it can be easily fixed to the equipment installation opening at the top of the culture tank with silicone rubber banking without any special processing. Second
The figure illustrates a method of fixing a small culture tank to a branch pipe (18) for equipment installation using a silicone rubber backing (17).

J8 As the amount of nutrient solution increases, the number of hollow fibers required for oxygen-enriched aeration inevitably increases, and as a result, the part that connects it to the culture tank needs to be strong, but it is necessary to determine the method of fixing the culture tank in advance. This can be easily solved by adding to the structure of the joint (9). For example, as shown in Figure 3, when using the O-ring tightening method, use a liquid immersion type oxygen-enriching aeration device with an opening diameter of 50+s+* or more, in which several thousand hollow fibers are bundled. Can be done.

Function: The process of aerating oxygen-enriched air into the culture medium using the immersion-type oxygen-enriched aeration device of the present invention involves passing the pressure regulating valve (15) to the hollow tube (3) in FIG. A small portion of the normal air introduced into the hollow fiber (2) from the opening surface (6) via the hollow part (7) that is Most of the air introduced into the chamber passes through the opening surface (10) and passes through the pressure regulating valve (10).
4) is discharged from the device.

Under such operating conditions of this device, the pressure inside the hollow fiber is 1
The amount of oxygen-enriched air that permeates through the hollow fibers relative to the total amount of supplied air is 0 atm or less, preferably 5 to 2 atm, and is effective for oxygen enrichment and reduces the power cost required for air supply. It is preferable that the pressure regulating valves (14, 15) be used to adjust the ratio, that is, the flow rate ratio, to be 115 to 1/20.

In addition, this flow rate ratio and the surface area of the hollow fiber effective for oxygen enrichment,
The inner diameter of the hollow tube for air supply is determined in advance according to the oxygen enrichment performance of the hollow fiber. For example, 40℃, 3
When introducing atmospheric pressure air at a flow rate ratio of 1/20, the general formula (
In the hollow fiber made of polyamide represented by A), oxygen-enriched air with an oxygen concentration of 34% can be passed through at an oxygen supply rate of 1.9 x 1 O-3 NrrI''/hour per 1 meter of membrane area. , 1.2X 10 to be supplied into the hollow fiber at this time
-''Nd/hr air volume can be adequately handled by a 1/8 inch hollow tube.

Such an oxygen-enriching aeration device having a surface area of 1 m'' may be constructed by bundling approximately 650 hollow fibers with a diameter of 0.5 mm to an effective length of 1 m and having an opening surface diameter of 1c11 as shown in FIG. 1. Oxygen demand 3
Oxygen can be supplied to the culture solution of 0.1rn' as 07/Cll3.

The immersion type oxygen-enriched aeration device obtained by the present invention immerses hollow fibers with oxygen-enriching performance in a culture solution and supplies oxygen-enriched air into the culture solution to create a high dissolved oxygen environment. The present invention provides an aeration device that enables large-scale, high-density culture of animal cells, plant cells, and aerobic microorganisms by solving the problems of cell damage and culture solution evaporation caused by bubble aeration. Furthermore, by using a hollow fiber material with excellent heat resistance, it is also possible to sterilize the entire culture container including the culture solution and the oxygen-enriched membrane with high-pressure steam.

EXAMPLES Examples of the present invention are listed below, but the present invention is not limited thereto.

Example 1 Outer diameter 0 made of polyamide represented by general formula (A)
．． 20 7-layer hollow fibers with an inner diameter of 0.5 shoulder layer are arranged around a 1/16 inch stainless steel hollow tube and fixed with thermosetting epoxy resin within the stainless steel joint, making it effective for oxygen enrichment. An immersion-type oxygen-enriching aeration device with a length of 1.2 m deformed into a spiral shape was fabricated, and a second glass incubator containing 2 liters of pure water was equipped with a stirrer and a dissolved oxygen concentration meter.
Assemble the simulated culture device by fixing it as shown in the figure 40
The temperature was maintained constant at ℃.

When air pressurized to 3 atm (gauge pressure) was introduced into a wood liquid immersion type oxygen enrichment aeration device at 6 x 10-3 Nrn'' at 7 o'clock, a dissolved oxygen concentration of 10 ppm was obtained.The liquid of the present invention By using an immersion-type oxygen enrichment aeration device, a higher dissolved oxygen concentration (8.5 ppm at 40° C.) was obtained than normally achieved with air bubble aeration.

Example 2 The hollow fiber part of the oxygen enrichment aeration device shown in Example 1 was protected with a fluororesin porous tube with a maximum pore diameter of 3.5 μm, and the first
An immersion-type oxygen-enriching aeration device having the same configuration as shown in the figure was fixed in a glass culture tank containing 2 liters of MS medium used for plant callus culture in the same manner as in Example 1, and heated at 123°C for 1 hour. After high-pressure steam sterilization, carrot callus was introduced, and culture was continued for one week at 25° C., an air pressure of 3 atm (gauge pressure), and an air flow rate of 6×10 −3 Nm′/hour. The dissolved oxygen concentration in the culture solution was maintained at 12 ppm, and the amount of castor increased sixfold. In addition, the adhesion of callus to the hollow fibers was protected by the porous tube.

Effects of the Invention In cell culture of animal cells, plant cells, and aerobic microorganisms, the cell culture method for supplying oxygen-enriched air into the culture solution using the immersion type oxygen-enriched aeration device of the present invention is In addition to increasing the dissolved oxygen concentration in the solution, it also solves the problems of cell damage and evaporation loss of the culture solution caused by conventional bubble aeration, making it possible to culture cells in large quantities and at high density. be.

[Brief explanation of the drawing]

FIG. 1 is an elevational view of a liquid immersion type oxygen enrichment aeration device, FIG. 2 is a sectional view showing an example of a method for fixing the device, and FIG. 3 is a sectional view showing another example of a method for fixing the device. DESCRIPTION OF SYMBOLS 1... Oxygen enrichment effective hollow fiber part, 2... Hollow fiber, 3...
...Hollow tube, 4...Resin body, 5...Joint part, 6...
・Opening surface, 7...Closed cavity, 8...Resin body, 9...Joint part, 1o...Opening surface, 11φ/φ open cavity, 12...Hollow Pipe seal part, 13・φ・
Air outlet, 14... Pressure adjustment valve, 15... Pressure adjustment valve, 16... e Porous tubular body, 17... Silicone rubber backing, 18... Culture tank equipment installation port, I9...・0 ring.

Claims (1)

[Claims] 1. A hollow fiber (2) having oxygen enrichment performance is arranged around a longer hollow pipe (3) for introducing air, and both ends of the hollow fiber (2) are Along with the hollow tube (3), the resin body (4
, 8) to the joints (5, 9), and at one of the sealed joints (5), the hollow fiber (2) and the hollow tube (3) open into the closed cavity (7). to form an air inlet to the hollow fiber (2), and at the other joint (9), the hollow fiber (2) connects to an air outlet (13) connected to the pressure regulating valve (14) and an air inlet to the hollow fiber (2). It consists of an assembly of hollow fiber oxygen enrichment membranes opening into an open cavity part (11) having a gland seal part (12) that fixes the side surface of a hollow tube (3), and is immersed in a cell culture solution. A liquid immersion type oxygen-enriching aeration device characterized by being able to aerate oxygen-enriched air into a culture solution. 2. A hollow fiber (2) having oxygen enrichment performance and an air introduction hollow tube (3) are sealed to a joint (5, 9) by a resin body (4, 8) at both ends of the hollow fiber, and further, The entire hollow fiber of the oxygen-enriched air permeation part (1) between both joints is 0.05μm
The hollow fibers are located in a porous tubular body having a pore size of 100 to 100 mL or more, and the hollow fibers are protected from direct contact with the culture medium fluid, and oxygen-enriched air is introduced into the culture medium through the pores of the porous tubular body. The immersion type oxygen-enriching aeration device according to claim 1, characterized in that it is capable of aeration. 3. The hollow fiber having the above-mentioned oxygen enrichment performance has the general formula (A)
Claim 1, characterized in that the material is polyamide represented by or polyarylate represented by general formula (B).
The immersion type oxygen enrichment aeration device described. ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(A) ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(B) Here, n indicates the number of repeating units.