JPS63202371A - Spin culture apparatus - Google Patents

Abstract

PURPOSE:To provide the titled apparatus effective in preventing the blocking of exhaust pipe with culture liquid, etc., and keeping air-transmitting state, by constructing the apparatus in a manner that the culture liquid, etc., in an exhaust pipe flows to the culture liquid side when one of the exhaustion ports is positioned above the surface of the culture liquid to keep the exhaust pipe and the exhaustion channel in air-transmitting state. CONSTITUTION:Culture liquid 15, etc., in a culture tank 1 is agitated by the agitation caused by the rotation of the tank 1 and the aeration caused by air bubbles ejected from nozzles 14. A pair of exhaustion ports 8 are alternately dipped into the liquid in the tank 1 and lifted above the liquid surface according to the rotation of the tank 1 and the air floated to the liquid surface is exhausted through the exhaustion port 8 above the liquid level. When the level of the culture liquid 15, etc., is in the state shown in the figure, both exhaustion ports are temporarily dripped in the culture liquid 15, etc., when the exhaust pipe 7 is turned to horizontal state. However, the period of dipping both exhaustion ports 8 in the liquid is extremely short and there is no particular problem in the exhaustion of air. In the figure, 6 is liquid reservoir, 9 is air chamber, 11 is exhaust pipe and 12, 13 are air-introduction pipe.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates in particular to a rotary culture device suitable for efficiently culturing a large amount of plant cells and tissues (hereinafter simply referred to as cells).

Conventional technology When cultivating microorganisms in large quantities as in the field of fermentation, aerated culture tanks equipped with stirring blades are generally used, but such aerated culture tanks are also used when cultivating plant cells, etc. Attempts are being made. Stirring in cell culturing equipment is mainly done to prevent cells from settling and to bring them into sufficient contact with the nutrients and oxygen necessary for cell proliferation, but when culturing plant cells, etc. ,
Unlike microorganisms, they are relatively susceptible to the influence of agitation intensity, and if agitation intensity is too strong, growth may decrease or stop.
As a result, the production of secondary metabolites often cannot be carried out efficiently.For this reason, in culture equipment that uses stirring blades, strong stirring force is applied locally at the time of loading, and the culture tank is located diagonally below the stirring blades. It is not suitable for use as a cell culture device because the cells that have settled near the bottom tend to aggregate.

In order to solve these problems, attempts have been made to culture plant cells and the like using a rotating cylindrical culture device in which the culture tank itself rotates. According to this type of culture device, it is possible to perform uniform stirring, and the stirring intensity can be freely changed by appropriately selecting the rotation speed.
It also has the advantage of being able to reduce sedimentation and aggregation of cells, etc., and several improvement proposals have been made for this type of culture device in recent years.0 Japanese Society of Fermentation Engineering Abstracts, 1981 247 pages, JP-A-58-134
This is the case as shown in No. 989.

Problems to be Solved by the Invention In the above-mentioned rotary cylindrical culture device, it is difficult to supply a culture medium containing cells, etc. and nutrients to the axial center of the culture tank, and to discharge a culture solution containing products to the outside. The culture tank is rotated around it through a supply and exhaust pipe, an air supply pipe for supplying oxygen to the culture solution, an air exhaust pipe with an exhaust port located above the culture solution, etc. As the culture tank rotates, the culture solution (hereinafter simply referred to as culture solution) containing cells, etc. that adheres to the inner wall surface drips and adheres to the exhaust port, which gradually increases in volume and solidifies, causing the exhaust port to It may get blocked. In particular, if the exhaust port is directed upward, culture fluid or the like may enter the exhaust pipe and cause clogging.

This invention aims to solve the above problems and prevent clogging of the exhaust pipe.

Means for Solving the Problems The present invention therefore provides at least two exhaust ports of mutually communicating exhaust pipes connected to an exhaust path, which are provided in the culture tank at appropriate intervals in the direction of rotation, and are rotated integrally with the culture tank. By doing this, each exhaust port is alternately immersed and floated in the culture solution to prevent the culture solution, etc. in the exhaust pipe from solidifying and maintain fluidity. The culture solution in the tube flows down to the culture solution side, and the exhaust pipe and discharge path are maintained in an aerated state. That is, the present invention provides a rotary culture device in which the culture tank itself rotates to agitate the culture solution.
Two or more exhaust ports are provided in the culture tank at appropriate intervals in the rotational direction, and each of them alternately immerses in the culture solution and floats up from the culture solution as the culture tank rotates, and the exhaust port is provided at one end. It is equipped with a vent that communicates with each other, rotates together with the culture tank, and has a fixed exhaust passage and an exhaust pipe that vents, and the culture solution, etc. that enters the exhaust pipe through the vent that is immersed in the culture solution, etc. It is characterized in that as the culture tank rotates, the liquid floats above the liquid surface at the vent and flows down the inside of the tube in the opposite direction to the exhaust port, thereby bringing the exhaust pipe and the exhaust path into a ventilated state. be.

Although various shapes such as cylindrical, spherical, elliptical, etc. can be used as the culture tank, it is preferable to use a spherical culture tank.

The culture tank may also rotate around a horizontal axis, but it is preferable that the axis of rotation be inclined with respect to the horizontal plane so that the shaft seal provided at the connection with the exhaust path faces upward. , particularly preferably from 5″″ to 4
It is desirable that the inclination angle can be adjusted within a range of 5° depending on the amount of culture solution put into the culture tank.
When a culture solution or the like comes into contact with the shaft seal, cells, etc. tend to get caught between the sliding surfaces and get damaged or contaminated with germs, but by tilting the shaft seal upward, rotation can be prevented. Without sacrificing the advantages of a type culture device, it is possible to prevent culture fluid from coming into contact with the shaft seal, thus preventing damage to cells and contamination by bacteria, allowing smooth and efficient culture for a long time. It can be carried out.

A typical example of the oxygen-containing gas mentioned here is sterilized air.

The air supply pipe may be simply immersed in the culture solution at its air supply port, but preferably, a porous hollow body for blowing out gas, such as a sparger, is connected to the tube and immersed in the culture solution, from which gas is blown out. This makes it easy to increase the oxygen transfer visibility coefficient (k,) to the required value, and also allows for aeration agitation in addition to ffl agitation by rotating the culture tank itself. By appropriately selecting the amount of aeration and the rotational speed of the culture tank, it is possible to perform cell culture under agitation conditions that are favorable for cell culture, ie, in a state in which the cells are not damaged and there is a sufficient amount of dissolved oxygen.

The exhaust port, which alternately immerses and floats in the culture solution as the culture tank rotates, may be such that the exhaust port of the rotating holder of the culture tank is temporarily immersed in the culture solution, but it is preferable that the exhaust port is temporarily immersed in the culture solution. One of the exhaust ports is always positioned above the surface of the culture solution and exposed.

Each exhaust pipe may be directly connected to each other, and a branch pipe may be provided that protrudes axially from the connecting portion (in this case, the branch pipe is rotatably connected to the exhaust pipe), but preferably the branch pipe is rotatably connected to the exhaust pipe. connected to a liquid reservoir connected to. This liquid reservoir always stores a culture liquid at the same liquid level as the liquid level in the culture tank, and is ventilated above the liquid level to the ventilation pipe and the ventilation path, respectively.

Embodiment A cylindrical culture tank 1 is tilted in the vertical direction, and a cylindrical portion 2 protruding diagonally upward at one end is connected to a pedestal 3, and a shaft portion 4 protruding diagonally downward at the other end is connected to a pedestal 5. Rotatably supported. A liquid reservoir 6 with an inverted T-shaped cross section is attached to one side of the cylindrical portion 2, from which a pair of exhaust pipes 7 extend in opposite directions in the diametrical direction, and an exhaust port 8 is attached to one side of the culture tank 1. are opened at symmetrical positions. The end of the liquid reservoir 6 has a double pipe structure, with a small diameter part 6b protruding from a large diameter part 6a, and a ventilation chamber 9.
The air chamber 9 (fixed exhaust path) consisting of the air chamber 9a and the exhaust chamber 9b has the large diameter portion 6a in the ventilation chamber 9a, and the small diameter portion 6b in the exhaust chamber 9b.
are rotatably connected to each other, and the joints between the two are sealed by a known method such as a mechanical seal. The air chamber 9 also has an exhaust pipe 1) in the exhaust chamber 9b, and an exhaust pipe 1) in the ventilation chamber 9a.
A vent pipe 12 is connected to each of the vent pipes 12, and a vent pipe 13 that communicates with the vent chamber 9a is connected to the large diameter portion 6a. A sparger that protrudes inward and has a porous hollow body at the protruding end.
14 are connected adjacent to the sides. Thus, the sterilized air is transferred to the ventilation pipe 12, the ventilation chamber 9a and the ventilation pipe 1.
3 and is ejected from the sparger 14. The rotation of the culture tank 1 is carried out, for example, by providing a gear transmission device including a speed reducer capable of adjusting the rotation speed on an extension of the shaft portion 4, and connecting a motor to the device. Culture tank 1 also contains culture solution etc.
The culture solution, etc. enters the liquid reservoir 6 and is maintained at the same level, but the maximum amount of culture solution, etc. that can be put in is determined by maintaining ventilation between the exhaust port 8 and the exhaust chamber 9b, and by ensuring that the shaft seal part is kept at the same level. It is kept to the extent that it is not immersed in the culture solution, that is, to the extent shown in the figure.

According to this device, the culture liquid etc. 15 in the culture tank is agitated by the rotation of the culture tank l and the ventilation agitation by the air ejected from the sparger 14, and also by the pair of exhaust ports 8.
As the culture tank rotates, it alternately dips into the culture tank and floats up, and the air that floats to the liquid surface is discharged from the exhaust port that floats above the liquid surface.

Note that if the exhaust pipe 7 becomes horizontal when the liquid level of the culture solution, etc. 15 is at the level shown in the figure, both the re-exhaust ports 8 will be temporarily immersed in the culture solution, etc. 15; The time it takes to be immersed in the liquid is extremely short, and there is no particular problem with evacuation.

Effects of the Invention As described above, in the rotary culture apparatus of the present invention, the plurality of exhaust pipes communicating with each other rotate together with the culture tank, and thereby each exhaust port alternately immerses and floats in the culture solution, etc. The culture solution, etc. that has entered the exhaust gas etc. through the exhaust port, which is repeatedly immersed in the culture solution, etc., flows in the opposite direction of the exhaust port, that is, toward the center, as it floats above the liquid surface of the above-mentioned exhaust port, It is designed to ventilate with the exhaust passage, so even if some of the culture solution, etc. remains in the tube when the exhaust port floats to the surface and adheres to the inner wall, it will prevent coughing when the culture solution, etc. Since it comes into contact with liquid and becomes fluid, it does not solidify, thus preventing clogging in the pipe and maintaining breathability.

[Brief explanation of the drawing]

The figure is a schematic diagram of a rotary 1g feeding device according to the present invention. l...Culture tank 6...Liquid pool 7...Exhaust pipe 8...
Exhaust port 9...Air chamber 1)...Exhaust pipe 12.13
・・Vent pipe 14・・Sparger 15・・Culture solution

Claims (5)

[Claims] (1) In a rotary culture device in which the culture tank itself rotates and agitates the culture solution, an air supply pipe that supplies oxygen-containing gas to the culture solution and an air supply pipe that is installed at appropriate intervals in the rotation direction in the culture tank, and each is connected to the culture tank. Two or more exhaust ports that alternately repeat immersion in the culture solution and floating from the culture solution as the tank rotates, and each have the exhaust ports at one end, communicate with each other, and rotate as one with the culture tank. At the same time, it is equipped with a fixed exhaust passage and an exhaust pipe for ventilation, and the culture solution, etc. that enters the exhaust pipe through the vent, which is immersed in the culture solution, floats to the liquid surface of the vent due to the rotation of the culture tank. A rotary culture device characterized in that the flow flows down the inside of the pipe in a direction opposite to the exhaust port side, so that the exhaust pipe and the exhaust path are in a ventilated state. (2) The rotary culture device according to claim 1, wherein the culture tank is tilted with the exhaust port facing upward. (3) The rotary culture device according to claim 1, wherein the exhaust pipe is connected to a liquid reservoir rotatably connected to the exhaust path. (4) The rotary culture device according to claims 1 to 3, wherein at least one exhaust port is always located above the liquid level. (5) The rotary culture device according to claim 1, wherein the air supply pipe is connected to a sparger that is immersed in a culture solution or the like.