JPS6331191B2 - - Google Patents

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a method for reproducing the culture physical properties of a culture solution in a culture tank of a large-scale industrial culture device in a culture tank of a small-scale experimental culture device. This invention relates to biological cell culture equipment for scale-up.

[Prior art and its problems] So-called scale-up, in which the culture results obtained in a small-scale culture device are reproduced in a large-scale culture device, has conventionally been achieved by changing the physical properties of the culture solution (medium composition, temperature, etc.) in both devices. This has been done on the assumption that the pH (dissolved oxygen, etc.) is equal.

However, in small-scale culture devices such as flasks and jar fermentors, the culture properties are uniform, whereas in large-scale culture devices on an industrial scale of tens or hundreds of tons, the liquid depth varies. The length ranges from several meters to several tens of meters, and a considerable pressure difference occurs between the liquid surface and the bottom of the culture tank, and as a result, some of the physical properties of the culture become non-uniform within the tank. For example, in a large tower-type culture device, the liquid flow is close to plug flow, so the concentration distribution of various dissolved gases occurs similar to the pressure distribution between the tank bottom and the liquid surface, and a completely mixed state is formed. It has been reported that in large-scale culture apparatuses, not only a pressure distribution occurs between the tank bottom and the liquid surface, but also a dissolved gas concentration distribution occurs in large-scale culture apparatuses, even in aeration-stirred culture apparatuses.
Biotechnol.Bioeng., Volume 25, Page 3115, 1983
Year).

As described above, in conventional scale-up, little consideration is given to the physical properties of culture, which are uniform in a small culture device but non-uniform in a large culture device. Among the non-uniform culture properties in a large-scale culture apparatus, the most important one is the concentration of dissolved gases, and among these, oxygen and carbon dioxide gas are considered to be the most important.

In particular, oxygen is the most important factor governing the culture results in aerobic culture, and carbon dioxide is a factor that has a large effect on the pH of the culture solution and the physiological activity of cells. Another non-uniform factor is pressure, and depending on the type of cell, pressure itself is thought to have a large effect on the physiological activity of the cell. Physiological cells are inoculated into the culture tank of the culture device,
Once culture is started, cells circulate within the culture tank at a certain average cycle, during which they proliferate and perform other physiological activities (hereinafter collectively referred to as life activities).

In other words, as mentioned earlier, large-scale culture equipment has uneven concentration distribution of dissolved gas within the tank,
A pressure distribution occurs, through which cells circulate at a certain average cycle, and cells carry out their life activities while being influenced by these heterogeneous factors. How these heterogeneous factors within the culture tank will affect the culture results cannot be easily predicted using conventional scale-up methods, and it is difficult to actually conduct experiments using large-scale culture equipment, which imposes a heavy economic burden. When I went there, it was revealed for the first time.

Therefore, the problem with conventional scale-up methods was that they were completely meaningless.

[Summary of the Invention] The present inventors have solved the above-mentioned problems, and the present inventors have solved the above-mentioned problems by cultivating biological cells (microorganisms, microorganisms,
plants, animals, etc.), such as pressure in the culture tank, periodic fluctuations in dissolved gas concentration,
We have developed a biological cell culture device for scale-up that can reproduce these conditions in a small experimental biological cell culture device in response to the shear stress, their fluctuation range, cycle time, etc. ) using gas or a stirring device such as a stirring blade,
A regulator and a solenoid valve are interposed in the culture tank in which the culture solution is stirred, and a gas introduction pipe opened into the culture solution, a dissolved gas concentration detector, and a gas discharge pipe with an intervening solenoid valve are provided. A gas mixer is connected to the solenoid valve of the gas inlet pipe and the gas inlet pipe between the culture tank through the solenoid valve, and this gas mixer has an oxygen gas supply pipe, a nitrogen gas supply pipe, and a carbon dioxide gas supply pipe. An appropriate number of pipes among the pipes and other gas supply pipes are connected via regulators and solenoid valves, and the detected value by the dissolved gas concentration detector in the culture tank fluctuates in a preset periodic manner. The operation of each of the regulators and each electromagnetic valve is controlled by an automatic control device so as to correspond to and match the set value of the gas concentration.

(b) and a gas introduction pipe opened into the culture solution with a regulator and a solenoid valve interposed in the culture tank, and a gas discharge pipe with a pressure detector, a dissolved gas concentration detector, and a solenoid valve interposed therein. and,
A stirring blade connected to a motor is provided,
An oxygen gas supply pipe, a nitrogen gas supply pipe, a carbon dioxide gas supply pipe, and other gas supply pipes are connected to the solenoid valve of the gas introduction pipe and the gas introduction pipe between the culture tank via a regulator and a solenoid valve, respectively. At the same time, the detected values of the culture solution in the culture tank by the pressure detector and the dissolved gas concentration detector, as well as the shear stress value, are made to match in accordance with these periodically fluctuating set values set in advance. , the operation of each of the regulators, solenoid valves, and motors is controlled by an automatic control device;

A gas mixer is connected via a solenoid valve to the downstream side of the solenoid valve of the gas introduction pipe in the device (c) and (b) above, and this gas mixer is connected to an oxygen gas supply pipe, a nitrogen gas supply pipe, Among the carbon dioxide gas supply pipe and other gas supply pipes, at least the former three pipes are each connected via a regulator and a solenoid valve.

[Example] An example of the biological cell culture device for scale-up according to the present invention will be described with reference to the attached drawings.

In a known culture tank 1 configured to be airtight,
A gas introduction pipe 3 of a pressurizing device 2 consisting of a compressor and an air transformer (not shown) is opened to the inner bottom surface of the culture tank 1 via a regulator 4 and a solenoid valve 5. Gas introduction pipe 3 between tanks 1
A gas mixer 7 is connected by a mixed gas supply pipe 8 via a solenoid valve 6, and an oxygen gas cylinder 9 and a nitrogen gas cylinder 1 are connected to the gas mixer 7.
0, a carbon dioxide gas cylinder 11 is connected to an oxygen gas supply pipe 18, a nitrogen gas supply pipe 19, and a carbon dioxide gas supply pipe 20 via regulators 12, 13, 14 and solenoid valves 15, 16, 17, respectively, and the culture tank 1 A stirring shaft 22 having a stirring blade 21 at its lower end is suspended from the center of the stirring shaft 22 , and the stirring shaft 22 is connected to a motor 23 .

An oxygen electrode 25 for detecting dissolved gas concentration is attached to the side wall of the culture tank 1 at a position where it is immersed in the culture solution A, and a pressure transmitter 24 and a pressure transmitter are installed at a position on the top surface of the culture tank 1 that is not in contact with the culture solution A. A gas exhaust pipe 27 via a solenoid valve 26 is attached.

The pressure transmitter 24 and the oxygen electrode 25 are connected to an input 29 of the automatic control device 28 . Automatic control device 2
The input section 29 and setting section 31 of 8 are connected to the calculation section 32, and the calculation section 32 outputs the output section 3 via the conversion section 33.
Connected to 0. The output section 30 of the automatic control device 28 includes the aforementioned solenoid valves 5, 6, 15, 16, 1.
7, 26, and each regulator 4, 12, 13,
14 and motor 23 are connected to each other.

Next, a method of controlling the pressure, dissolved gas concentration, and shear stress on the culture solution A in the culture tank 1 using this device will be explained.

Pressure control: To control the pressure inside the culture tank 1, a pressurizing device 2 is used.
This is done by supplying pressurized air into the culture tank 1, or by discharging this air from the culture tank 1 through the gas exhaust pipe 27, and in the first half of the set cycle, the regulator 4 and the solenoid valve 5 are opened. ,
The solenoid valve 6 and the solenoid valve 26 are closed to increase the pressure to the set pressure, and in the second half, the regulator 4 and the solenoid valve 5 are closed and the solenoid valve 26 is opened to reduce the pressure in the culture tank 1 to normal pressure. This process is repeated to recreate the pressure fluctuations experienced by biological cells as they circulate through a large culture tank.

That is, the air pressurized by the pressurizing device 2 is introduced into the regulator 4 after moisture, dust, etc. are removed by the air transformer, and the regulator 4 sets a constant pressure.

When the solenoid valve 26 on the discharge side of the culture tank 1 and the solenoid valve 6 of the mixed gas supply pipe 8 which also serves as backflow prevention are closed, and the solenoid valve 5 on the suction side is opened, the pressure inside the culture tank 1 gradually increases. Therefore, this pressure change is caused by culture tank 1.
The pressure signal is detected by a pressure transmitter 24 provided at the automatic control device 28 and enters the input section 29 of the automatic control device 28.

When the pressure within the culture tank 1 reaches the pressure set by the setting unit 31, the solenoid valve 5 is closed by a signal from the output unit 30, and after an arbitrary time set by the setting unit 31 has elapsed, the solenoid valve 26 is opened. The pressurized air inside the culture tank 1 is discharged, and the pressure inside the culture tank 1 is lowered to normal pressure. By adjusting the set pressure of the regulator 4 and the time between closing the solenoid valve 5 and opening the solenoid valve 26, the set value of the pressure and the slope of the rise and fall of the pressure can be freely set.

By repeating the above series of operations, the pressure fluctuations that the biological cells undergo while circulating in the large culture tank are reproduced.

Control of gas concentration: As mentioned above, a factor that changes with pressurization is the concentration of dissolved gas in the liquid, which can be roughly divided into dissolved oxygen concentration and dissolved carbon dioxide concentration. Since the dissolved oxygen concentration is proportional to the pressure acting on the system, the dissolved oxygen concentration in the large culture tank differs between the upper and lower parts of the culture tank 1 even if pressurized air is supplied at a constant pressure.

Therefore, it is necessary to change the dissolved oxygen concentration in the culture tank 1 in accordance with the cycle in which biological cells circulate within the large culture tank.

For this purpose, a mixture of oxygen and nitrogen gas is supplied to the culture tank 1 through the air supply line, and the amount of oxygen or nitrogen gas is periodically adjusted to change the dissolved oxygen concentration so that the biological cells can move through the large culture tank. Reproduces the changes in dissolved oxygen concentration that occur during circulation.

Similarly, changes in dissolved carbon dioxide are also reproduced.

That is, first, the dissolved oxygen concentration is controlled by pressure by changing oxygen or by mixing nitrogen gas with oxygen.

Once the reference dissolved oxygen concentration is set, the dissolved oxygen concentration under a certain pressure is proportional to the pressure acting on the system, so it can be determined by calculation. The value is set in the setting section 31.

Then, when the regulator 4 and solenoid valve 5 of the pressurizing device 2 are closed, the regulator 12 and solenoid valve 15 of the oxygen cylinder 9 are opened, and the solenoid valve 6 is opened, the oxygen from the oxygen cylinder 9 is set at a constant pressure by the regulator 12. and is supplied into the culture tank 1 via the gas mixer 7. The dissolved oxygen concentration in the culture tank 1 is detected by the oxygen electrode 25 installed in the culture tank 1, and when the amount of oxygen reaches the set value set in the setting section 31, the regulator 12 and the solenoid valve 15 are activated.
is closed.

In this way, the regulator 12 and the solenoid valve 15 are opened in accordance with the cycle in which biological cells circulate within the large culture tank, and the amount of oxygen supplied into the culture tank 1 increases the dissolved oxygen concentration to a certain pressure. The dissolved oxygen concentration below can be reproduced.

The process up to this point reproduces the state in which cells move from the top to the bottom of the culture tank 1.

As the cells move from the bottom to the top, the dissolved oxygen concentration decreases, but in order to cope with this, the regulator 13 and solenoid valve 16 of the nitrogen gas cylinder 10 are opened, and the nitrogen gas is kept at a constant pressure by the regulator 13 and mixed. When introduced into the vessel 7, nitrogen gas and oxygen gas are mixed in the gas mixer 7, and this mixed gas is supplied into the culture tank 1 to dilute the dissolved oxygen concentration, so that the value detected by the oxygen electrode 25 is When the standard oxygen amount set by the setting unit 31 is reached, the regulator 13 and the solenoid valve 16 of the nitrogen gas cylinder 10 are closed.

By repeating this series of actions and adjusting the amount and time of adding oxygen and adjusting the amount and time of adding nitrogen gas to reach the standard amount of oxygen, the cells circulate through the large culture tank. This reproduces the fluctuations in dissolved oxygen concentration that occur when Carbon dioxide gas is also controlled in the same way.

Control of shear stress: A stirring blade is rotated to mix and stir the culture solution, but the force applied to biological cells is not constant depending on the position of the tip of the rotating stirring blade and the position away from the stirring blade. Cells are not subject to constant shear stress, and the shear stress they receive when they are close to the stirring blade is not equivalent to the shear stress they receive when they are close to the liquid surface and far from the stirring blade.

Therefore, by changing the stirring speed, changes in shear stress that biological cells undergo while circulating in a large culture tank can be reproduced.

That is, the shear stress is determined by the stress of the stirring blade 21, and the number of rotations per unit time and the stirring blade 21.
Let it be the product of the distance from the center of .

The standard stirring blade rotation speed is determined from the set rotation speed of the large culture tank.

The rotational speed is changed in accordance with the cycle of biological cells circulating in the large culture tank, and at this time the minimum shear stress value is determined. This determination is made by measuring the flow velocity in the upper part of the large culture tank in advance, determining the rotation speed of the stirring blade 21 from the measured velocity, and determining the lowest shear stress value.
By decelerating or accelerating the rotational speed of the stirring blade 21 to the lowest rotational speed in accordance with the circulation periodic speed of the biological cells, the shear stress experienced by the biological cells circulating in the large culture tank is reproduced.

All of the above controls are performed by the automatic control device 28.

That is, the pressure inside the culture tank 1 and the culture solution A
Setting of maximum, minimum value, variation time, etc. of dissolved oxygen concentration and shear stress, etc., and each solenoid valve 5, 6, 15, 16, 17 based on these set values and input signals of pressure transmitter 24 and oxygen electrode 25. , 26 and each regulator 4, 12, 13, 14, and the rotation speed control of the motor 23 is performed by a known automatic control device 2.
8, by incorporating a microcomputer into the automatic control device 28, the control values of the reproduction system are determined by inputting the shape of the large-scale culture device, the amount of culture solution, etc.

In the explanation of the control method above, we have described pressure control, dissolved gas concentration control, and shear stress control, but depending on the bacterial species, pressure control and shear stress control may not be necessary. It is obvious that these controls may not be necessary in some cases.

Although the explanation is omitted in this embodiment, the temperature inside the culture tank 1 can also be controlled by the automatic control device 28, and the means for stirring the culture solution is not limited to a stirring blade, but may also be an air lift method or a magnetic one. It is possible to use known stirring means such as rotation of a rotor using a stirrer.

[Effect] According to the biological cell culture device for scale-up according to the present invention, it is possible to reproduce the culture physical properties of the culture solution in a large-scale industrial culture device in a small-scale experimental culture device, and it is also possible to reproduce the culture properties of the culture solution in a large-scale industrial culture device. It is possible to easily check the influence of the culture results due to the varying conditions of the culture medium in a small amount of experimental culture medium, and it is possible to eliminate the waste of culture medium when experimentally culturing biological cells, which will contribute to the industrialization of biological cell culture. There are great benefits.

In addition, from the viewpoint of the structure of the apparatus, in the first invention, the numerical conditions of the culture physical properties of the culture medium in the large-scale culture apparatus to be reproduced are inputted into the input section of the automatic control apparatus, and the small-scale culture apparatus It is possible to reproduce the culture physical properties of the culture medium in a large culture device,
In addition to being easy to operate, the gas introduction pipe, oxygen gas supply pipe, nitrogen gas supply pipe, and carbon dioxide gas supply pipe are equipped with regulators or gas mixers, so that the pressurized air or mixed gas is uniform. In the second invention, a pressure detector is provided in the culture tank and connected to the motor. Since it has a stirring blade, by controlling the pressure inside the culture tank and controlling the rotation of the stirring blade, it is possible to adjust the pressure inside the culture tank and reproduce the shear stress according to the bacterial species, and it has versatility. The third invention has both the effects of the first invention and the second invention.

[Brief explanation of the drawing]

The drawing is an explanatory view of the biological cell culture device for scale-up according to the present invention. 1...Culture tank, 3...Gas introduction pipe, 5,
6, 15, 16, 17, 26... solenoid valve, 7...
Gas mixer, 18...Oxygen gas supply pipe, 19
... Nitrogen gas supply pipe, 20 ... Carbon dioxide gas supply pipe, 21 ... Stirring blade, 23 ... Motor, 2
4...Pressure transmitter, 25...Oxygen electrode, 27...
Gas discharge pipe, 28... automatic control device.

Claims (1)

[Claims] 1. By a gas or a stirring device such as a stirring blade,
A regulator and a solenoid valve are interposed in the culture tank in which the culture solution is stirred, and a gas introduction pipe opened into the culture solution, a dissolved gas concentration detector, and a gas discharge pipe with an intervening solenoid valve are provided. A gas mixer is connected to the gas inlet pipe between the solenoid valve of the gas inlet pipe and the culture tank via the solenoid valve, and this gas mixer has an oxygen gas supply pipe, a nitrogen gas supply pipe, and a carbon dioxide gas supply pipe. An appropriate number of pipes among the pipes and other gas supply pipes are connected via regulators and solenoid valves, and the detected value by the dissolved gas concentration detector in the culture tank fluctuates in a preset periodic manner. to match the set value of the gas concentration.
A biological cell culture device for scale-up, characterized in that the operation of each of the regulators and each electromagnetic valve is controlled by an automatic control device. 2. In the culture tank, a regulator and a solenoid valve are interposed, a gas introduction pipe opened into the culture medium, a pressure detector, a dissolved gas concentration detector, a gas discharge pipe with a solenoid valve interposed, and a motor. The gas introduction pipe between the electromagnetic valve of the gas introduction pipe and the culture tank is provided with an oxygen gas supply pipe, a nitrogen gas supply pipe, a carbon dioxide gas supply pipe, and other gas supply pipes. They are connected via a regulator and a solenoid valve, respectively, and the values detected by the pressure detector and dissolved gas concentration detector of the culture solution in the culture tank, as well as the shear stress value, fluctuate periodically in a preset manner. 1. A biological cell culture device for scale-up, characterized in that the operation of each of the regulators, each solenoid valve, and the motor is controlled by an automatic control device so as to correspond to and match a set value. 3 In the culture tank, a regulator and a solenoid valve are interposed, a gas introduction pipe opened into the culture medium, a pressure detector, a dissolved gas concentration detector, a gas discharge pipe with a solenoid valve interposed, and a motor. A gas mixer is connected to the gas introduction pipe between the solenoid valve of the gas introduction pipe and the culture tank via the solenoid valve, and oxygen gas is supplied to the gas mixer. pipes, nitrogen gas supply pipes, carbon dioxide gas supply pipes, and other gas supply pipes, at least the former three pipes are connected via regulators and solenoid valves, respectively, and the pressure of the culture solution in the culture tank is detected. so that the values detected by the detector and the dissolved gas concentration detector, as well as the shear stress values, correspond to and match these periodically fluctuating set values set in advance.
A biological cell culture device for scale-up, characterized in that the operation of each of the regulators, each solenoid valve, and the motor is controlled by an automatic control device.