JPS6234394B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a perfusion culture method for studying the metabolism of cells or organ tissues from an academic standpoint, and from an industrial or practical standpoint, for example, to a perfusion culture method for studying the metabolism of cells or organ tissues. The present invention relates to a method that can be applied to culture cells that produce substances effective in treating cancer, diabetes, etc., or to produce and extract useful physiologically active substances directly as a cytoreactor.

Conventional technology Conventionally, as a technically basic cell culture system, the gas phase and the liquid or gel-like medium are placed in a static equilibrium state, and _O2 , _CO2 , etc. in the gas phase are By controlling the gas partial pressure, it is suitable for cells in liquid medium or cells on the surface layer of gel-like medium.
Methods include methods that provide environmental conditions such as DO and PH, and methods that increase the ability to supply gas into the culture medium by rotating or vibrating the container containing the gas phase and liquid medium or gel medium. However, these conventional methods have problems such as a gradient in the dissolved gas concentration in the medium depending on the distance from the boundary between the medium and the gas phase, and a small ratio of the gas contact surface area to the medium volume. Therefore, there was a drawback that it was difficult to sufficiently supply and control O ₂ and PH adjustment gas to the culture medium or cells in the culture medium.

Conventional advanced cell culture techniques include the tank culture method, which is the mainstream cell culture method that has become popular with the advancement of biotechnology in recent years. There are those that insert a trachea and aerate and bubble air, O ₂ or CO ₂ into the culture medium, and those that bubble air or O 2 or CO 2 into the culture medium.
In order to achieve uniform distribution of dissolved gases such as O ₂ and CO ₂ , there are methods that add agitation using blades or sails of various shapes. This conventional method can solve the problems caused by the rudimentary conventional method described above, but it also causes contact between cells and air bubbles due to bubbles created by aeration bubbling into the medium, and mechanical stress caused by agitation on cells. Because of this, cells, especially fragile cells such as animal cells, are killed or damaged, and initial culture results are often not obtained, and extremely serious problems remain in cell culture.

Problems to be Solved by the Invention Therefore, the present invention eliminates the drawbacks of the above-mentioned prior art, does not apply foaming due to aeration bubbling or mechanical stress due to agitation to cells, and also reduces the medium volume or the number of cultured cells. This ensures that the gas contact area is secured, thereby providing a sufficient amount of DO and PH adjustment gas to the culture medium, which in turn provides a sufficient amount and precision of O ₂ and PH values for the cells. The aim is to provide a perfusion culture method that allows cells to be cultured and maintained in an environment extremely similar to in vivo by supplying and controlling important environmental factors for culture.

Furthermore, the present invention provides devices for research and experiments related to the long-term maintenance of cells and organ tissues and basic metabolism, devices for mass culture of animal cells, which have been considered extremely difficult in the past, and even devices such as cytoreactors. The purpose of this study is to provide a perfusion culture method that will contribute to the realization of this development.

Means for Solving the Problems The method of the present invention for achieving the above object is as follows:
Perfusion culture in which the perfusate is passed through a gas exchange column having an O ₂ supply means and a PH adjustment gas supply means to control the DO value and PH value in the perfusate, and then the perfusate is placed in a culture column for cultivation. It is a method,
O ₂ and perfusate in the gas exchange column
It is characterized in that the PH adjustment gas is supplied while the perfusion liquid is flowing through a flow path made of gas-permeable hollow fibers under the gas permeability of the hollow fibers.

Effect According to the invention, the perfusate flows through the gas exchange column.
Under the supply of O ₂ through the O ₂ supply means and the PH adjustment gas through the PH adjustment gas supply means, the perfusate is flowing through the gas-permeable hollow fiber that is the perfusate flow path of the column. Under gas permeation through a gas-permeable hollow fiber, O ₂ and PH adjustment gas are supplied, gas exchange necessary for perfusion culture is performed, and O ₂ and PH adjustment gas is supplied to the gas exchange column. By regulating the supply to an appropriate amount, the DO and PH values of the perfusate are controlled to predetermined values, and the dissolved gas is evenly distributed as the perfusate flows through the flow path made of gas-permeable hollow fibers. .

The perfusate, which has reached the required DO and PH values, flows into the culture column, where it can be used as a culture solution.

Effects of the Invention As described above, according to the present invention, a perfusate is caused to flow through a channel made of a gas exchange column permeable hollow fiber, thereby controlling the DO and PH values in the perfusate, gas exchange necessary for perfusion culture, and Since uniform distribution of dissolved gases is achieved, this is achieved without subjecting the cells to foaming due to aeration bubbling or mechanical stress due to agitation.

Moreover, according to the present invention, by bundling a large number of gas-permeable hollow fibers that serve as the perfusate flow path of the gas exchange column, it is guaranteed that a large gas contact area can be secured for the medium volume or the number of cultured cells. , therefore a sufficient amount of
Supplying and controlling important environmental factors for cell culture, providing DO and PH regulating gases and thus providing cells with sufficient O ₂ and PH values in quantity and precision, thereby making it highly in vivo. It is possible to culture and maintain cells in a similar environment.

Furthermore, as a result of the above, the present invention provides an apparatus for research and experiments related to long-term maintenance of cells and organ tissues and basic metabolism, an apparatus for mass culturing of animal cells, which has been considered extremely difficult in the past, and furthermore, a cytoreactor. Contribute to the realization of the development of devices such as

Embodiments Examples of the present invention will be described below with reference to the drawings.

In the figure showing an example of the apparatus used to carry out the method of the present invention, 1 is a gas exchange column, and the gas exchange column 1 is a tube made of gas-permeable hollow fibers, such as silicone hollow fibers used for ultrafiltration of polymers. A large number of these are bundled together as a flow path for irrigation fluid. The gas permeable hollow fiber is designated by the numeral 2. The gas permeable hollow fiber 2 has a suitable diameter, for example, an inner diameter of 0.3 mm.
The outer diameter can be approximately 0.6 mm, and the length of the gas permeable hollow fiber of the relevant diameter is generally 60 mm.
It is sufficient to make one bundle of about 120 mm ones and apply several bundles. The gas exchange column 1 has an O ₂ supply port 3 as an O ₂ supply means and a CO ₂ supply port 4 as a CO ₂ supply means, which is a PH adjustment gas.
An O ₂ line 5 and a CO ₂ line 6 are connected to the supply port 4, respectively, and control valves (not shown) are inserted into the lines 5 and 6. The control valve controls the O ₂ supply amount and CO ₂ based on the PH value detection of the perfusate by the sensor cell 13 described below, as well as the CO ₂ value detection and the O ₂ value detection.
The supply amount is controlled to the required value.

The perfusate in the gas-permeable hollow fibers 2 of the gas exchange column 1 is circulated through a circulation circuit 7 by means of a suitable pump, for example a peristaltic pump (not shown).

A bypass 8 is connected to the circulation circuit 7, and the bypass 8 is equipped with a culture column 9. In some cases, the culture column 9 may be provided in the circulation circuit 7 without providing the bypass 8. Further, in the circulation circuit 7, a PH sensor 10, a CO ₂ sensor 11 and an O ₂ sensor 1 are installed between the gas exchange column 1 and the culture column 9.
A sensor cell 13 , which includes 2 as a component, is provided, and a sensor cell 13 ′ similar to the sensor cell 13 is also connected to the secondary side of the bypass 8 , that is, to the downstream side of the culture column 9 . sensor cell 13,
13' has inlets 14, 14' for introducing the irrigation fluid and outlets 15, 15' for leading out the irrigation fluid.
PH sensor of the sensor cell 13, 13', CO ₂
As the sensor and O ₂ sensor, those that are known per se may be used.

Reference numeral 16 denotes a 4-port, 2-position switching valve that is interposed at a convergence point of a perfusate outlet pipe 17 from the sensor cell 13', a pipe 18 connected to the circulation circuit 7, and a pipe 20 connected to the perfusion liquid tank 19. is the connection between pipe 17 and the outside of the system, the connection between pipes 18 and 20, and the connection between pipe 1
It is possible to change the way in which the pipe 20 is connected to the outside of the system under the connection between the pipes 7 and 18.

21 connects the starting end side of the circulation circuit 7 to the gas exchange column 1
A connection port connected to the outlet inner space 22 of the is shown.
The connection between the perfusion liquid inlet side of the gas exchange column 1 and the end side of the circulation circuit 7 can also be configured in the same way as the connection between the outlet side of the column 1 and the starting end side of the circuit 7.

The perfusate is transferred to the gas permeable hollow fibers 2, which are the perfusate flow path of the gas exchange column 1 → the outlet inner space 22 of the column 1 → the circuit 7 → the inlet side of the column 1 → the gas permeable hollow fibers 2... group While circulating the gas permeable hollow fibers 2 of the gas exchange column 1 with a pump, the O _{2 line 5 is circulated through the O 2} line 5.
Ventilation of O ₂ and CO ₂ from the CO ₂ line 6 and the gas exchange necessary for perfusion culture are performed under the gas permeability of the gas permeable hollow fiber 2.

The PH sensor 10 in the sensor cell 13 in the circulation circuit 7 determines the PH of the perfusate circulating through the above path.
The CO 2 value is detected by the CO ₂ sensor 11 of the same cell, and _the O ₂ value is detected by the O ₂ sensor 12 of the same cell, and based on these detections, the control valve of the O ₂ line 5 and the CO ₂ line 6 are controlled. valve, perfusate PH value,
The O ₂ supply amount and CO ₂ supply amount are controlled so that they immediately correspond to the DO value reaching the required value.

Uniform distribution of dissolved gas in the perfusate is achieved by the perfusate flowing through the gas-permeable hollow fibers 2.

The PH and DO values are controlled to the required values and the circulation circuit 7
A part of the perfusate flowing through the tube is passed through the bypass 8 to the culture column 9, and cultured in the culture column 9.

The perfusate from the culture column 9 is returned to the circulation circuit 7. When the perfusate is refluxed, the PH value of the perfusate is measured by the PH sensor in the sensor cell 13' connected to the downstream side of the culture column 9 of the bypass 8, and the CO2 value is measured by the _CO2 sensor of the _same cell. Each O ₂ value is detected by an O ₂ sensor,
Based on these detections, it is monitored whether the PH value and DO value of the perfusate are suitable for reusing the perfusate by returning it to the circuit 7, and if appropriate, the perfusate from the sensor 13' is also transferred to the switching valve. With the predetermined way change of 16, the pipe 17,
If it is inappropriate, the switching valve 16 is switched to connect the pipes 18 and 20 while the pipe 17 is connected to the outside of the system.
9. While supplying an appropriate amount of fresh perfusion fluid, a corresponding amount of waste fluid is discharged from the system.

[Brief explanation of the drawing]

FIG. 1 is an overall circuit diagram showing an example of a perfusion culture apparatus used in carrying out the method of the present invention, FIG. 2 is an enlarged longitudinal view showing a part of the gas exchange column in the apparatus of FIG. 1, and FIG. The figure is an enlarged detailed longitudinal cross-sectional view of the irrigant outlet part of the same column, Figure 4 is an end view of the line cut section from Figure 2, and Figure 5 is an enlarged longitudinal cross-section of the sensor cell in the device shown in Figure 1. It is a detailed view. 1 is a gas exchange column, 2 is a gas permeable hollow fiber, 3 is an O ₂ supply port, 4 is a CO ₂ supply port, 5 is a
O ₂ line, 6 is CO ₂ line, 7 is circulation circuit, 8 is bypass, 9 is culture column, 10 is PH sensor,
11 is a CO ₂ sensor, 12 is an O ₂ sensor, 13,
13' is a sensor cell, 14, 14' is an inlet, 1
5 and 15' are outlet ports.

Claims (1)

[Claims] 1 Perfusion in which the perfusate is passed through a gas exchange column having an O ₂ supply means and a PH adjustment gas supply means to control the DO value and PH value in the perfusate, and then the perfusate is placed in a culture column for cultivation. A culture method comprising: O ₂ to the perfusate in the gas exchange column;
and a perfusion culture method characterized in that the PH adjustment gas is supplied under the gas permeability of the hollow fibers while the perfusate is flowing through a flow path made of gas-permeable hollow fibers.