JPH0775550A - Cell culture apparatus - Google Patents

Abstract

PURPOSE:To provide a cell culture tank which prevents the cells from being broken by a strong stress, can culture the cells growing in a fibrous form without pellet formation or lumping, and can prevent the cells immobilized to the support from being peeled off. CONSTITUTION:In a culture tank for mammalian or plant cells and/or cells of microorganisms, the culture tank is provided with a stirring mechanism which can arbitrarily vary the liquid flow rate and turn the flow direction. The mechanism is a cage type of rotor 2 in which the screw blades 10 fitted to the rotation shaft 3 for the rotor and the peripheral blades 9 have the structures to cause the flows reverse to each other. It is preferred that the cell culture installation is provided with a means for observation of the cell conditions and a system for controlling the flow rate and/or the flow direction of the stirring mechanism on the basis of the observation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for culturing animal and plant cells and / or microbial cells, and more particularly to an apparatus for culturing cells having weak cell strength and / or cells that grow into filaments.

[0002]

2. Description of the Related Art In a culture apparatus for the purpose of culturing floating animal and plant cells and / or microbial cells and / or culturing adherent animal and plant cells and / or microbial cells immobilized on a floating carrier, Oxygen or carbon dioxide gas or other gas required for the growth of the cells is supplied, and the culture medium and the cells are mixed to achieve a uniform suspension state so as to promote the mass transfer of the metabolic products of the cells. Aeration and stirring is performed.

[0003] Conventional culture devices include those that perform aeration and stirring only with gas, and those that use aeration and mechanical stirring together. Usually, air bubbles are finely dispersed and turbulent flow of the culture solution is used. Stirrer blades are provided to enhance the condition and are used together with ventilation. Since the mass transfer rate in the culture solution has a positive correlation with the stirring power, it is necessary to increase the stirring strength in order to increase the mass transfer rate.

However, especially when culturing cells having weak cell strength, if the stirring strength is unnecessarily increased, the shearing force applied to the cells is increased and the cells are crushed. For this reason, conventionally, the shape of the stirring blade has been devised so as to increase the mass transfer rate without giving shearing force to the cultured cells, but it has not been possible to finely respond to the state of the cells. On the other hand, in the case of filamentous cells such as molds and actinomycetes, the cells are often pelletized or agglomerated by aeration and stirring to inhibit mass transfer. When cells that adhere to the solid surface and proliferate are immobilized and cultivated on the surface of a floating carrier, if the stirring strength is unnecessarily increased, the shearing force applied to the carrier surface causes the cells to peel off.

[0005]

DISCLOSURE OF THE INVENTION In the present invention, when culturing cells having weak cell strength, a strong shearing force can be applied to the cells to prevent the cells from crushing, and the cells that grow into filaments are cultivated. In doing so, it is an object of the present invention to provide a cell culture device capable of preventing cell pelletization or lump formation and preventing detachment of cells immobilized on a floating carrier.

[0006]

In order to solve the above problems, in the present invention, in a culture device for culturing animal and plant cells and / or microbial cells, the flow velocity can be arbitrarily changed in the culture device and the flow direction can be changed. The mechanical stirring means having a structure capable of reversing the above is provided. In the above culturing device, the mechanical stirring means is a cage rotor, and it is preferable that the screw blades attached to the rotating shaft and the peripheral blades have a structure capable of forming a flow in the opposite direction. The stirring means preferably has a structure capable of repeating a state in which stirring is stopped at regular time intervals.

Further, the culture apparatus comprises means for observing the state of cells in the culture tank for controlling the flow velocity and / or flow direction of the mechanical stirring means, and a control system for controlling based on the means. Is good. The means for observing the state of cells in the culture tank is preferably performed by means for online measuring the amount of cells in the culture tank or means for measuring online the broken state of cells in the culture tank.

As described above, according to the present invention, while observing the state of cells in the culture tank, the flow velocity is appropriately changed by mechanical stirring, and / or the flow direction is appropriately reversed, and Animal and plant cells and / or microbial cells with weak cell strength and / or cells and / or microbial cells that grow in a filamentous form and / or
Alternatively, cells immobilized on a floating carrier can be cultured.

Next, the present invention will be described in detail. The mechanical stirring means in the culture tank is preferably a squirrel-cage rotor, and has a structure in which the screw blades attached to the rotating shaft and the blades in the peripheral portion can make flows in opposite directions. This allows
The culture solution can be sufficiently turbulent without giving a strong shearing force to the cells in the tank, and the mass transfer rate can be kept high. In addition, by appropriately changing the flow rate and / or by reversing the flow direction as appropriate and stopping the agitation as appropriate, the cells with weak cell strength are not applied with excessive shearing force, and are grown into filaments. The cells to be cultured can be cultured without pelleting or clumping, and without detaching the cells immobilized on the floating carrier.

The mechanical stirring means of the culture apparatus of the present invention is equipped with a control system for observing the state of cells in the culture tank and judging the state of the cells to determine the flow velocity and / or the reversal frequency of the flow direction. . As means for observing the state of cells in the culture tank,
There is a method of monitoring the amount of cells in the culture tank and / or a method of monitoring the damage state of the cells in the culture tank. In both cases, it is desirable to import the data measured online into the measurement control computer.

As a method of monitoring the amount of cells in the culture tank, a method of measuring the absorbance of the culture solution, a method of quantifying the concentration of adenosine triphosphate (ATP) in the culture solution, and counting the number of cells in the culture solution Any of the method, the method of measuring the cell weight in the culture solution, the method of quantifying the fluorescent substance in the culture solution and the like may be used, but it is desirable to adopt the method most suitable for the cells to be cultured. In addition, as a method of monitoring the damage state of cells in the culture tank, a method of image analysis of a microscopic field of the culture solution, a method of measuring the amount of nucleic acid (DNA) in the filtrate of the culture solution, and a part of the culture solution are used. Any method such as measuring the turbidity or the absorbance of the supernatant by allowing it to stand still may be used.

The data indicating the state of the cells in the culture tank measured by the above method is loaded into a computer for measurement control. The computer determines the state of the cells based on the data, determines the conditions of flow velocity fluctuation and / or flow direction fluctuation, and changes the operating state of the stirring device. The operation factors in the flow velocity fluctuation are the flow velocity, that is, the rotation speed of the stirring intensity, the duration at each flow velocity, and the stirring stop time, and the operation factors in the variation in the flow direction are the flow direction change frequency, the duration of each flow direction, And the stirring stop time.

[0013]

[Effect] Among floating animal and plant cells and / or microbial cells, particularly in the culture of cells having weak cell strength, aeration stirring for maintaining a high mass transfer rate, particularly mechanical stirring is strong for the cells. A major obstacle to practical use is applying a shearing force to destroy cells and inhibit cell growth. In the case of cells that grow in the form of filaments, the cells are often pelleted or agglomerated by aeration and agitation to prevent normal growth.

In the present invention, in mechanical agitation, when the agitation speed and direction are kept constant for a long time, cell destruction and pelletization of filamentous cells occur, but the flow rate can be appropriately changed or the flow rate can be changed. By changing the direction and stopping the stirring as appropriate, it is possible to prevent the destruction of cells, and / or to prevent the pelletization of filamentous cells, and / or to prevent the detachment of cells immobilized on a floating carrier. I found it and came to complete it.

Further, in the present invention, as a mechanical stirring method, the cage rotor gives the least shearing force to the cells,
In particular, if a screw blade is provided on the rotating shaft and the blade and the peripheral blades make a flow in the opposite direction, it is possible to maintain the culture solution in a sufficiently turbulent state at an extremely low rotational speed. Found and completed. Generally, since the mass transfer rate has a positive correlation with the stirring power, it is necessary to increase the stirring strength in order to increase the mass transfer rate, but since it may cause cell destruction, the stirring strength should be small on the safe side. That is, the mass transfer rate must be kept low. On the other hand, in the present invention, since the stirring conditions are changed while grasping the state of the cells, the state in which the cells are actively growing can be always maintained.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below with reference to the drawings, but the present invention is not limited thereto. Example 1 FIG. 1 shows a schematic cross-sectional view of a culture apparatus which is an example of the present invention. FIG. 1 is a schematic view of a culturing apparatus provided with a squirrel-cage rotor 2 in a culturing tank 1, 3 is a rotating shaft, 4 is an air diffuser, 5 is a magnet coupling, 6 is a gas introducing tube for air culture, and 7
Is a sensor insertion port.

Since the rotor 2 can arbitrarily change the rotation speed, can reverse the rotation direction as appropriate, and can appropriately stop the rotation, the animal and plant cells and / or microorganisms having weak cell strength can be obtained. It can be cultured without destroying cells. Further, in the case of cells that grow in the form of filaments, it is possible to prevent the cells from being pelletized or clumped, and to perform stable culture. Further, in the case of cells that grow by adhering to a solid surface, they can be stably cultured by preventing them from peeling off while being immobilized on the surface of a floating carrier.

FIG. 2 is a sectional view showing an example of the structure of the stirring rotor. By the screw blades 10 attached to the rotating shaft 3 and the blades 9 attached to the outer frame of the rotor, the forward flow along the rotating shaft inside the rotor and the inner wall of the culture tank outside the rotor at the time of normal rotation. Downflow occurs. Further, a centrifugal flow is generated at the time of normal rotation by the turbine blades 8 attached to the upper part of the rotating shaft. On the other hand, at the time of reverse rotation, a descending flow along the rotation inside the rotor, an ascending flow along the inner wall of the culture tank outside the rotor, and a centripetal flow due to the turbine blades 8 attached to the upper part of the rotating shaft.

FIG. 3 shows the disrupted state of cells in DN of the supernatant of the culture solution.
It is a schematic diagram of an example of a device which observes by measuring the amount of A and controls the number of rotations and direction of a rotor. A settling cell 13 is provided in a part of a piping system 12 for collecting a part of the culture solution 11, and the culture solution is flowed by a pump 20 for a certain period of time.
Close 9 and leave for a certain time. After measuring the absorbance at a wavelength of 254 nm of the supernatant portion in which the cells are precipitated by the absorptiometer 14, the electromagnetic valve is opened and the solution is pumped again, and this operation is repeated.

The data measured by the absorptiometer is cable 1
5 is sent to the measurement control computer 16 for data analysis, and a control signal for the rotation motor 18 is sent to the motor through the cable 17 to appropriately change the rotation speed, rotation direction, time, etc. of the rotor. For cells having a very poor sedimentation property, the supernatant may be squeezed by mechanical sedimentation such as centrifugation or filtration by a filter instead of spontaneous sedimentation.

As a method of observing the state of cells, FIG.
3 is a method for observing the cell amount by measuring the absorbance by providing a flow cell instead of the sedimentation cell 13 in FIG.
There is a method in which an automatic slide preparation device is used instead of 3, and a microscope is provided instead of the absorptiometer 14, and the microscope visual field is sent to a computer to analyze the image to observe the state of cell destruction, and any method may be used.

[0022]

INDUSTRIAL APPLICABILITY According to the present invention, animal and plant cells and / or microbial cells having weak cell strength can be cultured without destroying them, and in the case of cells that grow in a filamentous form, the cells are pelleted or aggregated. In the case of cells that can be grown stably by adhering to a solid surface while preventing them from becoming loose, in the state of being immobilized on the floating carrier surface,
Prevents peeling and enables stable culture.

[Brief description of drawings]

FIG. 1 is a schematic sectional view of a culture tank of the present invention.

FIG. 2 is an enlarged sectional view of the stirring rotor of FIG.

FIG. 3 is a schematic view of a culture device equipped with a cell state observation device and a stirring control device of the present invention.

[Explanation of symbols]

1: culture tank, 2: stirring rotor, 3: rotating shaft, 4: air diffuser, 5: magnet coupling, 6: air / culture gas, 7: sensor insertion port, 8: rotating shaft turbine blade,
9: Outer frame stirring blade, 10: Rotating shaft screw blade, 1
1: culture solution, 12: sampling pipe, 13: sedimentation cell,
14: Absorbance meter, 15: Data transmission cable, 16: Computer, 17: Signal cable, 18: Stirring motor, 19: Solenoid valve, 20: Pump,

Claims (6)

[Claims] 1. A culturing apparatus for culturing animal and plant cells and / or microbial cells, comprising a mechanical stirring means having a structure capable of arbitrarily changing the flow velocity and reversing the flow direction in the culturing apparatus. A cell culture device characterized in that 2. The mechanical stirring means is a squirrel-cage rotor, and the structure is such that the screw blades attached to the rotary shaft of the rotor and the peripheral blades make a flow in the opposite direction. Item 2. The cell culture device according to item 1. 3. The culture device comprises means for observing the state of cells in the culture tank for controlling the flow velocity and / or flow direction of the mechanical stirring means, and a control system for controlling based on the means. The cell culture device according to claim 1, wherein the cell culture device is a cell culture device. 4. The cell culture device according to claim 1, 2 or 3, wherein the mechanical stirring means has a structure capable of repeating a state in which stirring is stopped at regular time intervals. . 5. The cell culture device according to claim 3, wherein the means for observing the state of the cells in the culture tank is performed by means for measuring the amount of cells in the culture tank online. 6. The cell culture device according to claim 3, wherein the means for observing the state of the cells in the culture tank is a means for online measuring the state of damage of the cells in the culture tank.