JPH01281072A - Cell culture method and unit therefor - Google Patents

Abstract

PURPOSE:To provide the title unit designed so that a hydrophobic filtration membrane is used as a filtration membrane for a culture solution where cells are being put to liquid culture, and the membrane is intermittently backwashed with fresh culture solution to protect the cells from damage along with preventing the membrane from clogging. CONSTITUTION:Part of a culture solution where cells are being put to liquid culture is taken, and a hydrophobic filtration membrane is used as a filtration membrane for separating the cells from the liquid. Also this membrane is intermittently backwashed with fresh culture solution. Preferably, the membrane is a precoated one with a hydrophobic filtration medium layer on the surface of the hydrophobic filtration membrane. Furthermore, the hydrophobic membrane is pref. a hollow fiber membrane.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for cultivating biological cells, and in particular, a method for cultivating biological cells in a liquid medium with aeration and efficiently removing byproducts. Concerning large-scale and high-density cell culture methods.

[Conventional technology]

Recently, in addition to the conventional culture of microbial cells, that is, bacterial cells, the production of pharmaceuticals such as interferon has begun to be carried out by culturing animal cells.

In order to industrially produce such pharmaceuticals, a large-scale, high-concentration, long-term stable culture method is required. For this reason, two technologies were developed: ■Technology to separate biological cells and culture fluid and supply fresh culture fluid; and ■Technology to supply oxygen necessary for the growth of biological cells.

It is desired to develop a cell culture method that has the following features.

In order to achieve the above requirement (2), it is necessary to aseptically separate and concentrate the cells floating in the culture solution.

When cells are cultured without replacing the culture medium, most cell lines have a maximum cell concentration of around 2 x 10' cells/mQ. On the other hand, if the cells in the culture solution were separated and concentrated and the culture was continued in fresh culture solution, 107 cells/
The cell concentration can be increased to more than m 0, and the size of the culture tank and culture time can be reduced.

Centrifugation is generally used to separate and concentrate cells for small-scale tests, but performing this aseptically on a large scale is difficult due to the time required to exchange the culture medium and the complexity of the process. It is. Therefore, a method has been proposed in which the supply of a fresh culture solution into a culture tank and the removal of waste products produced by cells can be performed without taking the time required to separate and concentrate cells. For example, the methods described in Japanese Patent Publication No. 55-16635 and Japanese Patent Application Laid-open No. 62-265.

A cell sedimentation zone is provided in the culture tank, and fresh culture medium is supplied while draining the supernatant liquid from the sedimentation zone.

Also, JP-A No. 61-257181, JP-A No. 62-12
989 and Japanese Patent Publication No. 62-12990, a hollow fiber membrane having a wall membrane through which the culture solution permeates but not the cells is provided in the culture tank, and fresh culture solution is supplied and by-products are supplied through the hollow fiber membrane. Perform material removal.

Regarding the oxygen supply technique (2) above, in the cultivation of microorganisms and the like, conventional methods have been used to aerate air or oxygen-enriched air directly into the culture solution. However, the liquid medium or culture solution containing the cells used when culturing these cells is often foamy. Particularly in the case of culturing animal cells with the addition of serum, foaming of the culture solution is significant due to biopolymers contained in the serum and biopolymers secreted during the culture. These bubbles are difficult to burst even if left alone or mechanically subjected to shearing force, and the bubbles fill the gas phase of the culture tank and eventually overflow to the outside of the system.

In these foaming culture solutions, oxygen is supplied into the solution by dissolving the oxygen in the solution without creating bubbles, or by dissolving oxygen in the solution without creating bubbles.
It is necessary to eliminate bubbles generated by aeration in the liquid.

Conventionally, the former method has been mainly oriented.

Specifically, this is a method of dissolving oxygen from the liquid surface by aerating the gas phase of the culture tank. Usually, in order to improve contact with oxygen, the liquid is stirred or the amount of ventilation is increased to lower the liquid level. increasing contact.

Regarding the above, JP-A-61-74574, JP-A-61-36915, JP-A-60-259179
It is stated in the No.

[Problem to be solved by the invention]

Among the conventional techniques for separating and concentrating cells from a culture solution, in the method of separating and concentrating cells by sedimentation, the sedimentation rate of cells is slow, so it is necessary to increase the volume of the sedimentation tank in the culture tank. did not become. When the culture medium causes convection due to vibration or temperature difference.

There is a problem in that cells are mixed into the supernatant, reducing separation efficiency.

In addition, methods that separate and concentrate cells using hollow fiber membranes or filters cannot completely prevent cells from adhering to the membranes or filters, which can lead to clogging of the membranes or filters during culture. If pressure is applied to separate and concentrate the cells, there are problems such as damage to the cells.

On the other hand, in the oxygen supply method using the liquid level aeration method, if the liquid is strongly agitated, the cells will be crushed, so for animal cells, the oxygen supply method is practically limited to 50 rp- or less, and the aeration amount is also 1 cys.
/see is the upper limit. In order to maintain the above-mentioned aeration amount, a large volume of sterile air conditioning equipment is required in the culture equipment, and in addition, in long-term culture, there is a large loss of culture solution that is dissipated along with the gas.

On the other hand, the oxygen supply method using the submerged aeration force method does not have an effective defoaming technology. In culture methods targeting some microorganisms with strong cell walls and high growth rates, only a method (% Formula % Publication No. 108) is used in which air bubbles are mechanically destroyed using high-speed rotary blades. This method requires a high-speed drive unit to be installed inside the tank.

The structure becomes complicated. This method cannot be applied to animal cells because they are extremely weak against such shearing forces.

The purpose of the present invention is to:

■Prevents cell damage and filter clogging, and performs cell separation and concentration and culture medium exchange aseptically and efficiently;■Removes waste components dissolved in the culture medium from which cells have been separated; (■ Large capacity with high oxygen supply capacity without harming cell growth,
An object of the present invention is to provide a method and apparatus for culturing cells at high concentration.

[Means to solve the problem]

The above objective can be achieved by:

(1) In a cell culture method in which cells of an organism are cultured in liquid, a part of the culture solution is filtered out using a filtration membrane, and a new culture solution is replenished, wherein the filtration membrane is a hydrophobic filtration membrane; A cell culture method characterized by intermittently backwashing the filtration membrane with fresh culture solution.

(2) In a cell culture method in which biological cells are cultured in liquid, a part of the culture solution is filtered out using a filtration membrane, and new culture solution is replenished, in which the filtration membrane is hydrophilic to the surface of the hydrophobic filtration membrane. It is a pre-coated membrane having a filtration aid layer,
A cell culture method characterized in that the filtration membrane is intermittently backwashed with fresh culture solution.

(3) The cell culture method according to item 1 or 2 above, wherein the hydrophobic filtration membrane is one or more hollow fiber membranes.

(4) The cell culture method according to item 1 or 2 above, characterized in that the hydrophobic filtration membrane is formed of a material having a contact angle with the droplets of the culture solution of 80 degrees or more.

(5) The cell culture method according to item 1 or 2, characterized in that filtration is performed after filling the filtration pores of the hydrophobic filtration membrane with a culture solution.

(6) The cell culture method according to the preceding item 1 or 2, characterized in that backwashing is carried out at a pressure higher than the critical pressure of the hydrophobic filtration membrane.

(7) The cell culture method according to item 1 or 2, wherein the culture solution for backwashing the hydrophobic filtration membrane is a culture solution obtained by removing waste components from a filtered culture solution.

(8) In the preceding item 1 or 2, waste components in the filtered culture solution are removed by a waste component removal means, and the waste component removal liquid is used as part or all of the backwashing liquid for the hydrophobic filtration membrane. Characteristic cell culture method.

(9) The cell culture method according to item 8 above, wherein the means for removing waste components is a diffusion dialysis method, an ultrafiltration method, or a microfiltration method.

(10) The cell culture method according to item 1 or 2 above, characterized in that a stirring blade is provided in the culture solution, and a hydrophobic filtration membrane is provided around the rotating surface of the stirring blade.

(11) In the preceding item 1 or 2, the foam of the culture solution generated by the culture is broken by providing an anti-foaming layer made of a water-repellent material placed on the liquid surface and bringing the foam into contact with this. A cell culture method characterized by culturing while

(12) In the previous item 10, the antifoaming layer has a viscosity of 1×1×
A cell culture method characterized in that a base material is coated or impregnated with a water repellent of 104 centipoise or more.

In addition, by providing a foam-breaking layer made of the above-mentioned water-mixed material,
The generated foam can be efficiently broken, increasing the amount of gas injected, and enabling highly efficient cell culture.

(13) The cell culture method according to item 2, wherein the hydrophilic filter aid has a sedimentation rate higher than that of the cells.

(14) In the preceding item 2, the filter containing the filtration membrane is connected to the culture tank with piping and installed outside the culture tank, and the hydrophobic filtration membrane is placed in the upper part of the filter, so that the density is the same as that of the culture solution. A cell culture method characterized by using a hydrophilic filter aid smaller than .

(15) A liquid culture device for biological cells having a means for supplying a useful gas to a thoracotomy, a means for supplying a culture solution, and a means for discharging a waste culture solution, wherein the means for discharging the waste culture solution is a hydrophobic filtration membrane. provided via liquid filtration means;
and a liquid culture device for biological cells, comprising means for backwashing the hydrophobic filtration membrane with a culture solution.

(16) The liquid culture device for biological cells according to item 15, characterized in that the culture solution filtration means comprises a precoated membrane having a hydrophilic filter aid layer on the surface of a hydrophobic hollow fiber membrane.

(16) Both ends of the hydrophobic hollow fiber membrane are formed with a slope so that the outer diameter is larger than the inner diameter of the hollow fiber membrane, and the outer diameter at the tip part is such that it can be inserted into the hollow fiber membrane. , and a membrane expansion member provided with a protrusion for preventing detachment of the hollow fiber membrane is inserted, and the insertion part is fixed to the connection part so that it can be connected to the piping via the connection part. A filter for separating biological cells, which is characterized by:

The cell culture method of the present invention is used to propagate and culture biological cells, and includes a culture container, a means for separating cells and a culture solution, a means for removing waste components, and a means for returning the culture solution from which waste components have been removed to the culture container. , and the piping that connects them.

In a culture container, ie, a culture tank, cells of an organism are suspended in a culture solution. Inorganic salts, glucose, amino acids, antibiotics, etc. are added to the culture solution, and serum is added as needed.

The culture tank used in the culture method of the present invention is characterized by a container for storing the culture solution, a means for stirring the culture solution, a means for separating the cells from the culture solution, and a container for storing the culture solution from near the bottom of the container. a means for supplying gas to the culture solution; a water-repellent defoaming means located above the surface of the culture solution and having a number of openings through which gas can pass; and an aeration device having means for detecting the progress of the culture. It's in the culture tank.

In the present invention, a hydrophobic filtration membrane is used as a means for separating cells and culture medium. Particularly preferred are hydrophobic hollow fiber membranes. The hydrophobic filtration membrane is desirably made of a material that has a contact angle with the culture droplet of 80 degrees or more, preferably 95 degrees or more.

The opening degree is 5 μm to 0.5 μm in order to substantially prevent biological cells from passing through and to allow the required amount of culture solution to pass through.
m is preferred.

FIG. 15 shows the permeation characteristics of a hydrophilic membrane and a hydrophobic membrane. In the figure, the broken line is the case where a hydrophilic membrane is used, and the solid line is the case where a hydrophobic membrane is used.

The permeability properties of hydrophilic membranes are proportional to the filtration pressure. On the other hand, when a gas exists in the pores of a hydrophobic membrane, that is, before operation, the critical pressure (degree of hydrophobicity, pore diameter,
At a filtration pressure below (depending on membrane thickness, membrane porosity, etc.), the liquid will not pass through the filter, and the liquid will pass through only when the critical pressure is reached. - When a hydrophilic liquid permeates through the membrane, that is, when the pores of the membrane are filled with liquid, its permeation characteristics become equivalent to those of a hydrophilic membrane, regardless of the filtration pressure. It changes as shown by the solid line arrow in the figure.

The critical pressure of a polytetrafluoroethylene hollow fiber with a pore diameter of 0° and 8 μm (outer diameter 211 mm, inner diameter 1 mm) is:
1.5 kg/cm", and the permeation characteristics after filling with liquid are:

Habo 100s+l/mdg, m2. It is hr. This amount of permeation approximately corresponds to the amount of permeation through the hydrophilic membrane.

Therefore, when using the hollow fiber for filtration or backwashing, when it is used for the first time, it is necessary to operate it at a pressure above the above-mentioned limit pressure to fill the membrane pores with liquid.

The hydrophobic hollow fiber membrane is not particularly limited, but it is necessary to use one made of a hydrophobic material that maintains sufficient strength even in an atmosphere of about 130°C and does not change the small pore size for filtration. be.

In particular, those made of tetrafluoroethylene resin are preferable from this point of view.

A single long hollow fiber membrane may be used as the hydrophobic filtration membrane, but
In order to effectively utilize the membrane surface, it is desirable to use a plurality of membranes in a bundle.

Particularly when using hollow fiber membranes made of tetrafluoroethylene resin, which are difficult to connect by welding or adhesion, a good membrane unit can be obtained by the following connection method.

As shown in FIG. 9, membrane expansion members 52 are inserted into both ends of the hollow fiber membrane 10. The membrane expansion member 52 has a hollow tubular shape with an outer diameter larger than the inner diameter of the hollow fiber membrane 10, and its tip is cut off toward the tip to facilitate insertion into the hollow fiber membrane 10. It has a shape whose area gradually decreases,
In addition, a portion of the surface has locally large protrusions with a large outer diameter. After the end of the hollow fiber membrane 10 into which the membrane expansion member 52 has been inserted is covered with a room temperature curable resin face, this is cured.

If necessary, the membrane connecting member 51 is formed by shaping the outer shape, and is used as a connecting end to the filtration pipe. By bundling the ends of a plurality of hollow fiber membranes 10 and attaching them to the same membrane connecting member 51, a filtration unit consisting of a plurality of membranes can be produced. An example of a hollow fiber membrane filtration unit is shown in FIG. 8 and FIGS. 10 to 13.

By using a hydrophobic membrane, the adhesion of cells and dirt to the membrane surface can be greatly reduced, making it possible to use it for long-term filtration.

After a predetermined amount of the culture solution is removed, fresh culture solution is injected into the hydrophobic filtration membrane under pressure to release cells and dirt attached to the surface of the filtration membrane. The backwashing liquid may be an unused fresh culture solution, a used culture solution from which waste components have been removed, or a mixture thereof.

When the hydrophobic filtration membrane 10 is provided in the culture solution in the culture tank 2 (FIG. 5), a stagnation part is generated in the culture tank 2.

It is necessary to consider a shape and installation position that can maintain smooth flow of the culture solution 1 without causing cell sedimentation and that does not apply unnecessary shearing force to the cells. Note that in this case, it is desirable to draw out the culture solution at a flow rate as low as possible.

When the hydrophobic filtration membrane 10 is provided outside the culture tank 2 (FIG. 2), it is accommodated within the filter 8. The filter 8 is connected to the culture tank 2 via piping or the like. In addition.

When the hydrophobic filtration membrane 10 is installed outside the culture tank 2 in this way, the culture solution is withdrawn at a high flow rate as possible, and the cells are quickly removed from the culture tank 2 when a predetermined amount of liquid is reached.
It is necessary to operate the vehicle to return it to normal.

When the hydrophobic filtration membrane 10 is housed in the filter 8 provided outside the culture tank 2, the membrane is preferably a pre-coated membrane having a hydrophilic filtration aid on its surface. In the filter 8, a filter 11 for holding the filter aid 9 in the vicinity of the hydrophobic filtration membrane 10 is provided at the bottom or at both the bottom and the top.

Here, the filter 11 is made of an organic polymer material and glass.

It is made of a porous material such as ceramics or sintered metal, and the degree of pore opening is selected so that particles of the filter aid 9 do not substantially pass through.

When the culture solution is introduced from the culture tank 2 into the filter 8, cells in the culture solution are captured by particles of the filter aid 9. The filter aid 9 is a mixture of one or more organic polymers and inorganic compounds that are not toxic to cells, and the material is selected depending on the adhesion to cells, particle size of cells, etc.

Select particle size, shape, and loading amount.

For example, for cells with a particle size of 5 to 50 μm, if the cells are captured only by the so-called "intercepting effect" between the particles of the filter aid 9, the particle size of the filter aid should be about the same as the particle size of the cells. do. On the other hand, when cells exhibit adhesion to the filter aid 9, it is preferable to use a filter aid 9 having a particle size of approximately several tens to several hundreds of micrometers. Adherent culture is possible using microcarrier beads. For many cells, the filter aid 9 is selected in consideration of both of the above effects, and the filling amount is selected so that the filter aid 9 captures most of the cells in the culture solution.

Therefore, when the culture solution introduced into the filter 8 passes through the filter aid 9 and reaches the hydrophobic filtration membrane 10, there are almost no cells in the culture solution, and it is difficult to operate for a long period of time. However, clogging of the hydrophobic filtration membrane 10 can be substantially prevented.

The flow rate of the culture solution to the filter 8 is 0.1 to 0.1 per unit surface area based on the representative inner diameter of the filter 8. When the value is about 10 rn" / rrl'-h, it is possible to prevent cell damage due to shear force when the culture solution passes between the particles of the filter aid 9. However, this value depends on the type of cells and the filter aid 9. It varies depending on the particle size of the filter, the structure of the filter 8, etc.

Since the hydrophobic filtration membrane 10 can permeate byproducts in the culture solution, the cells in the culture solution can be separated and concentrated, and byproducts such as waste products can be discharged.

After the required amount of culture solution has been discharged from the culture tank, fresh culture solution is introduced into the filter 8. Here, the fresh culture solution may be an unused culture solution or a used culture solution that has been treated to remove cell byproducts. Further, the composition of the culture solution may also be different from that in the culture tank. When such fresh culture fluid is introduced back into the filter 8, this causes the particles of the filter aid 9 to rise (or fall) and spread out together with the cells captured in the previous step.

The material 2 particle size, shape, etc. required for the filter aid 9 are as follows:
In order to carry out the above steps efficiently, particles must settle faster than cells (or particles must float in the culture solution). When a particle group that settles faster than cells is used as the filter aid 9, the flow rate of the fresh culture solution to the filter 8 is limited to the particle group with the fastest sedimentation rate among the filter aids 9 to be expanded. It is desirable to make it so that it floats up to the surface.

At this time, since substantially all of the filter aid material 9 to be developed is developed, the cells are separated from the filter aid material 9 and float at least to the -E part of the particle group, which has a higher sedimentation speed than the cells. Since the piping is arranged so that the fresh culture solution that has passed through the filter aid 9 can be transferred to the culture tank 2, the cells floating on the upper part of the filter 8 are returned to the culture tank 2 together with the fresh culture solution.

Here, when the filter aid 9 contains particles larger than the particle size of the cells, a screen or the like having a degree of aperture that allows the cells to pass through but not the particles to pass through is placed above the filter 8. When installed in the culture tank 2, it is possible to prevent the filter aid 9 from floating up and mixing into the culture tank 2. Furthermore, since the hydrophobic filtration membrane 10 is backwashed with fresh culture solution, the service life of the membrane can be further extended.

In addition, in the case shown in FIG. 2 where a group of particles floating in the culture solution is used as the filter aid 9, the amount of fresh culture solution passed through the filter 8 depends on the amount of the filter aid 9 to be expanded. It is desirable that the particle group with the highest floating speed descends and the filter aid layer shows a fluid state.As a result, the cells separate from the filter aid 9 and settle, causing the filter 8 and the culture tank to separate. 2 is conveyed together with the backwashing liquid through the pipe connecting the two, and returned to the culture tank 2. Here, it is preferable to install a filter 11 at the bottom of the filter 8, which has a pore size that allows cells to pass through but does not allow the filter aid 9 to pass through.

The amount of fresh culture solution introduced in one operation in this step is:
Although not particularly limited, it is preferable that the amount of culture solution discharged is approximately the same as the amount of culture solution discharged in the culture solution discharge step, and furthermore, most of the cells captured by the filter aid 9 are returned to the culture tank 2 together with the fresh culture solution. It is desirable that the flow rate be as high as possible.

Filter aid 9 that had expanded when the flow of fresh culture solution was stopped
The particles settle on or around the filter 1o, but since some particles settle faster than the settling speed of the cells, cells that could not be transferred to the culture tank may float in the fresh culture solution. Even if the cells are present, the amount of these cells reaching the filter 10 is small.

Water-repellent defoaming means is intended for defoaming the foam generated on the liquid surface when aerating into a liquid medium containing biological cells, and has a large number of openings through which gas can pass. Installed above the liquid level of the culture solution in the tank.

The aperture ratio is desirably 50% or more, and the aperture diameter is desirably 50 to 2 m. Furthermore, at least the surface layer of the defoaming means is made of a water-repellent material.

In particular, it has a great antifoaming effect when culturing animal cells using a serum-added medium with marked foaming properties, making it possible to supply oxygen with high efficiency using the submerged aeration method.

The water-repellent material is a water-repellent material that forms a contact angle of 30 degrees or more with the surface of the water-repellent material with the droplets of the culture solution, is substantially insoluble or non-dispersible in the culture solution, and is not suitable for the target. Any material may be used as long as it is not substantially toxic to the cells to be treated. The antifoaming layer itself may be composed of the above-mentioned water-repellent material, or may be coated, coated, or impregnated with the water-repellent material.

For example, silane or siloxane having 10 or more carbon atoms can be used. Particularly suitable are those having a viscosity of I x 10' centivoise or higher.

A water-repellent material with a lower viscosity than centiboise covers the surface of the culture solution, or forms an emulsion in the solution that adheres to cells and inhibits their growth. Also,
This makes it difficult to separate the product from the culture solution after culturing.

The upper limit of the number of carbon atoms and the viscosity of the polysiloxane are not particularly limited, but the upper limit of the number of carbon atoms to become a substantially solid is 1 x 104, and the upper limit of the viscosity is I x 10' centiboise.

If the opening ratio of the antifoaming layer is less than 50%, when the bubbles on the liquid surface break, gas cannot pass through the antifoaming layer smoothly, and secondary bubbles are likely to occur. The diameter of the part is preferably 2 to 5
It is 0m. If the length exceeds 5001 m, if the bubbles are small, contact with the defoaming layer will be incomplete and defoaming will be difficult. On the other hand, if the length is less than 2 m, gas and liquid will not flow smoothly in and out of the opening.

The structure of the defoaming layer may be a single layer or a layered structure, and furthermore, these may be integrated or separable in the vertical and horizontal directions. The layer structure is appropriately selected depending on the liquid permeability and the bubble diameter.

Aeration into the culture tank 2 is normally carried out by arranging the nozzle 4 at the bottom of the culture tank, but it may be arranged as appropriate if it is below the liquid level. Alternatively, it may be provided between the bottom and the liquid surface. In particular, when the draft tube 3 is provided, placing the nozzle 4 under the draft tube 3 allows air bubbles to remain in the solution for a longer period of time due to the downward flow of the culture solution, thereby increasing the oxygen utilization rate. can be improved.

In the present invention, the culture solution has a surface tension of 45
~90 dyn/a# is good. An example of a highly effective culture solution is a culture solution containing biopolymers such as serum. In addition to serum, a culture solution containing or adding proteins such as albumin or nucleic acids is also extremely effective. As well as ingredients added from outside.

Culture fluids containing effervescent components secreted during culture are also targeted.

Waste components include lactic acid, ammonia, etc. contained in the culture filtrate using a hydrophobic membrane.

The means for removing waste components is not particularly limited, but can be carried out by known methods such as a diffusion dialysis device, an ultrafiltration device, an electrodialysis device, and the like.

However, since the culture filtrate contains a large amount of fine solid matter such as serum-derived precipitates and cell debris, it is desirable that the culture filtrate has a shape that prevents clogging. Among waste components, especially lactic acid and ammonia, inhibit cell proliferation.

Therefore, it is best to remove waste components using the above-mentioned ammonia and lactic acid as indicators.Furthermore, the culture filtrate also contains large amounts of components necessary for cell growth, such as various hormones and proteins. It is desirable to recover and reuse these components as much as possible. Based on these facts, the 2nd molecular weight cutoff for diffusion dialysis membranes and ultrafiltration membranes is 1,000 to 1.
．． It is desirable to use a value of about 0,000. Membranes with a molecular weight cut-off of less than 1,000 have a small permeation rate, require a large membrane area, and can become clogged significantly, so they may not be able to withstand long-term use. on the other hand,
If the membrane has a molecular weight cut-off of more than 10.000, even substances beneficial to cell proliferation, such as hormones, will permeate therethrough, resulting in a decrease in the recovery rate of these substances.

Note that for the separation of useful substances such as proteins and antibodies with large molecular weights, 0. It is preferable to use a microfiltration membrane having a pore size of O1μ. Furthermore, it is more advantageous to perform the separation of useful substances in multiple stages by combining these methods.

When removing waste components using a diffusion dialysis device, it is possible to use a solution with the same concentration and composition as the various solutions contained in the fresh culture solution, various amino acids, vitamins, and other low-molecular components as the dialysis solution. desirable.

The liquid after waste components have been removed is used as a backwash liquid for the filter and returned to the culture tank, where it is effectively utilized for cell culture.

Biological cells applicable to the present invention are not particularly limited, and include animal cells and microbial cells.

Contains plant cells. Animal cells include, for example, various cells of living animals, various cells of living animals, and various cells of living animals. Cells include not only single cells but also cell aggregates.

Microbial cells include various microorganisms such as bacteria, yeast, filamentous fungi, and actinomycetes.

Plant cells include cells and cell aggregates of higher plants, cells and cell aggregates of algae.

The ventilation gas that can be applied to the present invention includes, depending on the use,
Air, oxygen, carbon dioxide gas alone or a mixture of any composition, or a mixture of an inert gas may be used. Generally, oxygen-containing gas is used for animal cell culture, and carbon dioxide-containing gas is used for plant culture.

[Effect]

Filtration membranes made of hydrophobic materials have less adhesion of cells, etc. to the membrane surface compared to conventionally used hydrophilic filtration membranes, and backwashing also has the effect of easily removing adhering cells, etc. , clogging is less likely to occur, and therefore cells can be cultured with high efficiency by performing backwashing intermittently.

〔Example〕

The present invention will be explained below based on five examples.

FIG. 1 is a diagram showing the configuration of an embodiment of the cell culture system according to the present invention.

In the culture tank 2, cells are cultured in a dispersed suspension state in the culture solution 1. There is a nozzle 4 at the bottom of the culture tank for aerating oxygen-containing gas into the liquid, which supplies oxygen and gas necessary for cell growth.
Supply carbon dioxide gas to adjust pH. In other words, the air compressor 1 is adjusted so that the dissolved oxygen concentration is constant.
After controlling the flow rate of air from 6 and oxygen-containing gas from cylinder 17, and controlling the flow rate of carbon dioxide gas from cylinder 18 so that the pH becomes 7.0 to 7.6, and passing through sterilization filter 7, It is blown from nozzle 4.

The gas blown into the liquid becomes bubbles and floats toward the top of the culture liquid, causing the culture liquid to flow. A trough 1 and a hetube 3 are installed in order to efficiently flow the culture solution within the tank by the blown gas. The bubbles that reach the liquid surface do not break and form a foam layer, so
The bubbles are efficiently broken by contacting the antifoaming layer 5 made of a water-repellent material on the liquid surface.

The gas passes through the mist separator 6 and the filter 7 to the exhaust gas 2.
4 and is discharged outside the culture tank. In addition.

The culture tank 2 includes a means for keeping the dissolved oxygen concentration and pH constant, a means for sterilizing the inside of the tank and piping, a means for injecting and discharging the culture solution, and a means for keeping the temperature constant. Means, dissolved oxygen concentration? PHI temperature.

Various devices are installed, such as sensors for the internal pressure of the tank and sensors for detecting the level of the culture solution in the tank, but these are omitted in Figure 16.A filter 8 is provided at the bottom of the culture tank 2. It is being

A porous hollow fiber membrane filter 10 is installed in the filter 8, and a filter aid 9 is filled above or around the filter. The filter aid 9 is held by a holding filter 11 .

Even when the liquid moves rapidly during sterilization and backwashing, the hollow fiber membrane filter 10 does not deform in shape or the filter aid 9
Care must be taken not to expose the layer, but its shape is not particularly limited.

FIGS. 8A and 8B illustrate the hydrophobic hollow fiber membrane unit used in this example. The temperature of the membrane support member 50 is 130°C.
However, it is made of a material that has rigidity and is not likely to be corroded by the culture solution, and its external shape is made to match the internal shape of the filter 8 in Fig. 1, so that it can withstand some external force. It is provided so that its position within the filter does not move even if the filter is actuated. As shown in FIG. 8, the membrane support member 50 is provided with a large number of small holes 55 for holding hollow fiber membranes.

The size of the hole should be such that the hollow fiber membrane can freely pass through it. FIG. 8B shows a filtration unit in which the hollow fiber membrane 10 is attached to the membrane support member 5o. Hollow fiber membrane 1
Although the filtration unit may be composed of one hollow fiber membrane, it is usually preferable that the filtration unit is composed of two or more hollow fiber membranes. When used as a filter, the filtration unit can be easily attached to the filter.

9A and 9B are diagrams showing the concept of the membrane connecting member 51. FIG. Among hydrophobic hollow fiber membranes, this membrane connecting member is used to connect hollow fiber membranes made of materials in which membranes can be joined in a rotational manner, especially by adhesive or welding methods, such as hollow fiber membranes made of tetrafluoroethylene resin. It is effective for

FIG. 9A is a diagram showing the surface of the membrane connecting member 51 opposite to the surface to which the hollow fiber membrane is connected. It is desirable that the membrane connecting member 51 includes a membrane expanding member 52, and that the outer surface of the membrane connecting member 51 is shaped into a circle.

FIG. 9B is a cross-sectional view taken along line A-A' in FIG. 9A.

The hollow fiber membrane 10 has an inner diameter comparable to the inner diameter of the hollow fiber membrane, an outer diameter larger than the inner diameter of the hollow fiber membrane, and a tapered tip so that it can be easily inserted into the inner surface of the hollow fiber membrane. It is molded and locally has a protruding portion with a large outer diameter to prevent the hollow fiber membrane from detaching. Next, the portion into which the membrane expansion member 52 has been inserted is covered with the membrane connection member 51 and cured, thereby making it possible to firmly bind the hollow fiber membranes. The membrane connecting member is not particularly limited, but does not have an adverse effect on cells, does not corrode in the culture solution, and].

Any material may be used as long as it can withstand steam sterilization at around 30°C. For example, a two-component curing type epoxy resin is used.

The culture solution is replaced as follows. Valve 3 in Figure 1
1 and 32 are closed, valve 33 is opened, and pump 22 is operated, culture solution 1 is introduced from culture tank 2 to filter 8. Cells in the culture solution are filtered through a filter aid 9 and a porous hollow fiber filter 1
The six-porous hollow fiber filter 10, which is captured on each surface during the process of passing through zero, was selected to have a pore size that does not allow particles and cells of the filter aid 9 to pass through. The culture filtrate obtained by filtering the cells is stored in the culture filtrate storage tank 15.

After discharging a predetermined amount of culture solution from the culture tank 2, the pump 22
The pump 19.20 supplies the fresh culture solution from the fresh culture solution storage tank 12 to the filter. At this time, the pump 19 supplies fresh culture solution from the bottom of the filter 8, and sets the flow rate so that the packed layer of the filter aid 9 floats and expands. In addition, the pump 20 reversely pressurizes the fresh culture solution into the porous hollow fiber membrane filter 10 for backwashing.
Solid matter such as cells adhering to the surface of the filter 10 is peeled off. Note that the total flow rate of the pumps 19 and 2o is adjusted so that the filter aid 9 does not dissipate from the surroundings of the filter.

In the above-mentioned filtration process, the filter aid 9 and the hollow fiber membrane filter 10 are
The cells trapped above are released from the packed bed by the expansion of the filter aid 9. The particles of the filter aid 9 have a specific gravity 2 particle size and shape selected so that the sedimentation rate is higher than that of the cells, so the cells float to the top of the expanded packed bed.
It is returned to the culture tank 2 together with the fresh culture solution.

FIG. 2 shows the configuration of another embodiment of the cell culture system according to the present invention. The feature of this embodiment is that the filter aid 9 used in the filter 8 is made of particles smaller than the specific gravity of the culture solution. As the filter aid 9, hollow glass beads, hollow carbon beads, etc. are used. Filter aid 9
is held by two holding filters 11 provided at the top and bottom of the filter 8. The opening degree of the holding filter 11 is selected so that particles of the filter aid 9 do not pass therethrough, but cells freely pass therethrough.

According to this embodiment, the cells trapped in the filter aid 9 packed layer and the hollow fiber membrane filter 10 can be more completely returned to the culture tank during backwashing of the filter.

FIG. 3 shows the structure of another embodiment of the cell culture system according to the present invention. The feature of this embodiment is that a means for removing waste components is added to the embodiment shown in FIG. In addition,
In this embodiment, an ultrafiltration method is used to remove waste components.

An ultrafilter 14 is connected to the culture filtrate storage tank 15 via piping. The ultrafilter 14 contains all the molecules 1tto,o
oo ultrafiltration membrane is used, which allows waste components such as lactic acid and ammonia to pass through, but does not allow substances useful for cell growth such as hormones to pass through. The culture filtrate is forced into the ultrafilter 141 by the pump 23, and while in contact with the ultrafiltration membrane, low-molecular substances such as waste components pass through the membrane and are discharged from the system as waste components 26.

The liquid from which waste components have been removed by the ultrafilter 14 is stored in a waste component removal liquid storage tank 13. Note that since the amount of liquid to be filtered by ultrafiltration is small, the pump 23 circulates the liquid between the ultrafilter 14 and the waste component removal liquid storage tank to perform filtration and remove waste components. Normally, when the waste component removal solution is reused as part of a fresh culture solution, the concentration of waste components as lactic acid is 100ppm or less, preferably 100P.
It is necessary to keep the content below PI, and below 2Q ppm as ammonia, preferably below 5 ppm.

The waste component removal liquid is added to the fresh culture liquid as part of the backwashing liquid of the filter 8 by operating the pump 21,
The cells are returned to the culture tank 2 and used again for cell proliferation. The waste component removal solution contains large amounts of active ingredients such as hormones and growth factors that remained in the culture filtrate, as well as growth growth factors and useful products secreted by the cells themselves. So.

Using this method gives good results for cell growth.6 FIG. 4 shows the structure of another embodiment of the cell culture system according to the present invention.

The feature of this embodiment is that, in the embodiment shown in FIG. 3, particles having a specific gravity smaller than the specific gravity of the culture solution are used as the filter aid 9 used in the filter 8.

As the filter aid 9, hollow glass pieces, hollow carbon beads, etc. are used. The filter aid 99 is held by two holding filters 11 provided at the top and bottom of the filter 8. The opening degree of the holding filter 11 does not allow particles of the filter aid 9 to pass through.

Those that freely permeate cells are selected.

Other details are the same as in Figure 3.

According to this embodiment, the cells trapped in the filter aid 9 packed layer and the hollow fiber membrane filter 10 can be more completely returned to the culture tank during backwashing of the filter.

FIG. 5 shows the structure of another embodiment of the cell culture system according to the present invention.

The feature of this embodiment is that, in the embodiment shown in FIG. 3, a hydrophobic membrane 10 was provided in the upper layer of the culture tank 2.

and a stirrer was installed. Exchange of the culture medium in this example is carried out as follows. Open the valve 33,
When the valve 32 is closed and the pump 22 is operated, the pressure inside the hollow fiber membrane is reduced. As a result, the cells floating in the culture solution are captured on the surface of the hollow fiber membrane, and the culture filtrate containing no cells is extracted from the membrane and stored in the culture filtrate storage tank 15.

After discharging a predetermined amount of culture filtrate from the culture tank 2, the pump 2
2 and close the valve 33. Valve 32 is then opened and pumps 20 and 21 are activated.

The pump 20 supplies fresh culture solution from the fresh culture solution storage tank 12, and the pump 21 supplies waste component removal solution from the waste component removal solution storage tank 13. The fresh culture solution and the waste component-removed solution are pressurized into the hollow fiber membrane and flow into the culture tank 2 through the pores on the membrane surface.

At this time, solid matter such as cells adsorbed on the outer surface or inside the pores of the hollow fiber membrane is peeled off to regenerate the filtration surface. Hydrophobic membrane 10
In order to prevent cells, dirt, etc. from sticking to the membrane surface, it is preferable to provide the membrane at a location where the movement speed of the culture solution is as high as possible. As in this embodiment, stirring blades 29 are provided.

If a hydrophobic membrane is installed around the membrane, the flow of the culture liquid discharged by the rotation of the stirring blades will move the solids on the membrane surface without stagnation, effectively preventing deposition on the membrane surface. I can do it.

Although the shape of the filtration unit used in this example is not particularly limited, it is desirable that the shape does not obstruct the flow of the culture solution and does not apply unnecessary external force to the cells. Each of the filtration units shown in FIGS. 10 to 13 has a particularly preferable shape for this embodiment.

According to this example, filtration is performed in a culture tank.

No oxygen deficiency or temperature drop occurs during filtration, and cell growth can be maintained at a good level.

FIG. 6 shows the structure of another embodiment of the cell culture system according to the present invention. The feature of this embodiment is that a culture solution tank 41 is added to the embodiment shown in FIG.

The backwash liquid used for backwashing the hydrophobic hollow fiber membrane provided in the culture tank is prepared by mixing a fresh culture liquid and a waste component removal liquid in advance in the culture liquid W4 manufacturing tank 41.

Prepare. The culture solution preparation tank 41 contains lactic acid, ammonia,
A means for measuring the concentration of protein is attached, and the mixing ratio of the fresh culture solution and the waste component removed solution is adjusted.

According to this example, culture can be carried out using a culture solution suitable for cell proliferation at all times.

FIG. 7 shows the structure of another embodiment of the cell culture system according to the present invention. The feature of this embodiment is that in the embodiment shown in FIG. 6, a diffusion dialysis device was used instead of the ultrafilter as the waste component removal device.

The culture filtrate is led to the diffusion dialyzer 44 by piping, and is transferred to the waste component removal liquid storage tank 13 and the diffusion dialyzer 44 by the pump 23.
Cycle between.

Further, the low molecular component liquid storage tank 42 stores a low molecular component liquid in which low molecular components such as various amino acids, vitamins, inorganic salts, and glucose are dissolved, and is sent to the diffusion dialyzer by a pump 47. .

In the antidialyser 44, the culture filtrate comes into contact with the low-molecular component liquid through the dialysis membrane, and during this time, the low-molecular components that can pass through the dialysis membrane are dialyzed. That is, waste components such as lactic acid and ammonia contained in the culture filtrate diffuse into the low molecular component liquid, and various amino acids are dissolved in the low molecular component liquid.

Glucose, various inorganic salts, and vitamins diffuse into the culture filtrate. Polymer components such as various hormones and growth factors useful for cell proliferation remain in the waste component removal solution without being dialyzed. Dialysis has a lactic acid concentration of 11,000
ppm or less, preferably 1100 ppm or less, and ammonia to 20 ppm or less, preferably 5 ppm or less.

The waste component removal solution that removes waste components by diffusion dialysis is
It is sent to the culture solution preparation tank 41 by the pump 46. A polymer component liquid is supplied to the culture liquid preparation tank from a polymer component liquid storage tank 42 by a pump 45, and is mixed and adjusted with the waste component removal liquid to obtain a fresh culture liquid. Polymer components that cannot pass through the dialysis membrane, such as serum and hormones, are dissolved in the polymer component liquid. In addition, in the medium 1 manufacturing tank 42,
A means for measuring the concentration of ammonia lactate and protein is attached, and the mixing ratio of the polymer component liquid and waste component removal liquid is adjusted based on the measured values.

According to this example, it is possible to always prepare a culture solution suitable for cell proliferation, and therefore cells can be cultured at high concentration.

FIG. 10 shows an example in which the waste component removing means in FIG. 6 is replaced with an adsorption cylinder.

Zeolite was used to adsorb ammonia, and lactate dehydrogenase was immobilized on an insoluble carrier to adsorb lactic acid.

In addition, by providing several subadsorption cylinders and switching the piping as appropriate, adsorption and regeneration can be performed without affecting the culture process.According to this embodiment, only waste components can be selectively removed. , there is little loss of culture solution.

Next, more specific examples will be shown and explained.

Example 1 Outer diameter 2mm, inner diameter 1mm, pore diameter 0.8μm.

A maximum outer diameter of 2.5 mm was attached to each end of a hydrophobic hollow fiber membrane (manufactured by Sumitomo Electric Chemical Industries, Ltd., TB-21) made of tetrafluoroethylene resin and having a length of 1 m.

A SUS3], which has a cross section shown in FIG. 9 with an inner diameter Q of 91 nm and a length of 30 mm, was inserted with a raw wave tension member 52 made of 6.Next, the four hollow fiber membranes were aligned and the inner diameter was 15 mm.
It was inserted into a cylindrical curing mold with a depth of 30 mm and a depth of 30 mm. A two-component thermal curing type epoxy adhesive (manufactured by Ciba Geigy, Araldite Travid) was mixed with the base resin and curing agent, and then immediately poured into the above curing mold, and was heated using a centrifugal separator to form a 1;0OOG A centrifugal force was applied during which time the material was cured. After being completely cured, the cured product was taken out from the curing mold and its end was cut by 5 mm.

For the hollow fiber unit manufactured above, steam at 130°C and a gauge pressure of 1.8 kg/cm" was passed from both ends of the unit.
It was held for 1 hour. After cooling to room temperature, even when a tensile load of 1 kg was applied to each hollow fiber membrane, the hollow fiber membrane did not come off from the membrane connecting member 51.

Comparative Example 1 In Example 1, the raw wave tension members 52 were not inserted at both ends of the hollow fiber membrane, but were fixed with an epoxy adhesive.

After manufacturing the hollow fiber membrane unit, 1 from both ends.
1 at 30°C and through steam at a gauge pressure of 1.8 kg/am.
Holds time. After cooling to room temperature, a tensile load of 1 kg was applied to each hollow fiber membrane, and the hollow fiber membrane came off from the membrane connecting member 51.

Example 2 Eagle MEM medium (Nissui Pharmaceutical Eagle MEM Nissui ■) 9.4 g/Q, glutamine 0.292 g/Q, 7
．． 5% sodium bicarbonate aqueous solution 29 m Q/Q
, glucose 20 g/Q, and fetal bovine serum 1
Rat liver cancer cell line JTC-1 (Japan Ti5
15 flat flasks inoculated with sueCulture No. 1) were statically cultured. The culture temperature was 37°C, and the gas phase was air.

The cells that had adhered to the surface of the flask that had been cultured for 3 days were detached, and the cell concentration was 5. 75ml of culture solution with lXl0' cells/mQ
The obtained 0-cell culture solution was centrifuged, the supernatant was discarded, and the suspension was suspended in the same volume of fresh culture solution.

Next, the diameter is 110 mm, the length is 171 mm, and the volumes are 1 and 2.
Add 75 m of the above cell suspension to a 50 m Q roller bottle.
Add Q and the above fresh culture solution 175+nQ,
℃, the gas phase gas was air, 2 ppm, and cultured for 3 days. The culture solution was then centrifuged.

The supernatant was discarded and suspended in the same volume of fresh culture medium.

Then, the cells were cultured again under the above conditions for 3 days. The culture solution was centrifuged, the supernatant was discarded, and the suspension was suspended in twice the volume of fresh culture solution, divided into twice the number of roller bottles, and cultured again under the above conditions for 3 days.

Repeat the above operation two more times, 1.7X10' pieces/
m Q seed culture solution 2. OOOmQ was obtained. The main culture solution was centrifuged, the supernatant was discarded, and the suspension was suspended in the same volume of fresh culture solution.

Next, the diameter of the cell culture system shown in Fig. 1 is 1.65 m.
m, height 350mm, capacity 7. The above seed cell suspension 2. was placed in an OOOmQ cylindrical stainless steel culture tank. OOOmQ
and culture solution 3. Add OOO+nQ (total 5.OOO
mQ) and kept at 37°C. A silane-based organosilicon polymer (viscosity I x 10'
A square-mesh stainless steel mesh (length of one side of the mesh: 3 mm) coated with a thin layer of Centiboise) was placed, and a ring sparger with a diameter of 60 mm (pore diameter 1 mm, 110 mm) was placed at the bottom of the tank.
The culture was carried out by aerating oxygen-containing gas from (vii). The amount of aeration of oxygen-containing gas is such that the dissolved oxygen concentration (DO) of the culture solution is 2.5p.
pm using a dissolved oxygen concentration regulator that automatically adjusts the amount of air and oxygen. Further, carbon dioxide gas was added to the oxygen-containing gas using a pH controller and aerated so that the pH of the culture solution was in the range of 7.0 to 7.6.

A filter is connected to the bottom of the culture tank via piping.

At the bottom of the filter is a support filter that holds a layer of hydrophilic filter aid. A hydrophobic hollow fiber filter unit is embedded in the hydrophilic filter aid layer. A hydrophobic hollow fiber filter unit was produced by the method of Example 1 using four hydrophobic hollow fiber filters each having a length of 1.2 m and having the shape shown in FIG. 8. As the tA aqueous filter aid, glass beads with a diameter of 70 μm made hydrophilic by treatment with ethyl alcohol were used.

By reducing the pressure inside the hollow fiber membrane filter using a peristaltic pump for drawing, 20 m Q / m
The culture filtrate was withdrawn at a flow rate of i. No contamination of cells into the culture filtrate was observed.6 The amount of withdrawal was 5.
When it reached 00 mm, the peristaltic pump for withdrawal was stopped. Next, the hollow fiber polishing filter was backwashed and the filter aid layer was developed using a peristaltic pump for backwashing the hollow fiber membrane filter and a peristaltic pump for developing the filter aid layer.

Hollow fiber membrane filter backwashing is 80 m Q/m i
It was run for 2 minutes at a flow rate of n. The filtration aid layer development is 70 mm/min + during backwashing of the hollow fiber membrane filter.
After backwashing of the hollow fiber membrane filter was completed, it was carried out at a flow rate of 150 mQ/min, and was controlled to end when the same amount as the amount withdrawn was injected into the culture tank. A fresh culture solution prepared in a fresh culture solution storage tank was used for backwashing the hollow fiber membrane filter and developing the filter aid layer. Filtration of the culture solution, backwashing of the hollow fiber membrane filter, and development of the filter aid layer were performed 10 times per day so that the culture solution in the culture tank was replaced once per day.

The results of this example are shown in FIG. After 12 days from the start of culture, IX10' cells/mQ or higher and survival rate of 88% or higher were maintained. Maximum cell concentration is 1.6 x 107 cells/
It was mQ.

Comparative Example 2 The same culture as in Example 2 was carried out using the culture system shown in FIG. 1 except that the filter, hollow fiber membrane filter, and filter aid were removed. The results are shown in FIG. Due to the removal of the filter, hollow fiber membrane filter, and filter aid, the culture medium could not be replaced, and nutrients were depleted and the cell concentration reached 2.1 × 101 cells/mn and then rapidly decreased. .

This shows that it is essential to replace the culture solution.

Comparative Example 3 In the culture system shown in FIG. 1, the same culture as in Example 2 was carried out using a culture system in which oxygen was supplied by aeration of the liquid surface. The results are shown in FIG. Cell concentration up to 5
．． 6X10' cells/mQ, with a gradual decrease in viability. This result indicates that there is a lack of oxygen necessary for cell growth. That is, it can be seen that oxygen supply is not sufficient in the liquid level aeration method.

Comparative Example 4 The same culture as in Example 2 was carried out using the culture system shown in FIG. 1 except that the antifoaming layer was removed.

As a result, bubbles flowed out from the culture waste gas pipe. Than this,
It can be seen that defoaming means is required in the culture tank in order to supply oxygen through submerged aeration.

Example 3 Using the method of Example 2, the cell concentration was 1.8 x 10' cells/
A seed cell suspension of mQ was prepared.Next, it was cultured using the cell culture system shown in FIG.

The culture tank of this system has an internal volume of 7. OOOmQ's 5US3
It was made in the shape of a cylindrical tube made of No. 16, and was equipped with a stirrer 29 to cause the culture solution to flow. The hydrophobic hollow fiber membrane filter unit 10 for separating cells and culture medium is the 13th
It has the shape shown in the figure and is placed around the rotating surface of the stirring blade. Note that the hydrophobic hollow fiber membrane filter unit was created using four hollow fiber membrane filters that were the same as those used in Example 1 and each having a length of 1.5 m. The oxygen supply means, defoaming means, and culture conditions were the same as in Example 2.

The above seed cell suspension 2, OOOm Q, and the same culture solution as in Example 2 were placed in a culture tank. '0OOrnQ was added (5.OOOmQ in total) and kept at 37°C. Batch culture was performed for two days after the start of culture, and the culture solution was replaced from the third day. The pressure inside the hollow fiber membrane filter was reduced using a peristaltic pump, and the culture filtrate was drawn out at a flow rate of lomQ/min. No contamination of cells into the culture filtrate was observed.

When the pulling amount reached 500 mu, the pulling riverside staric pump was stopped. Next, a fresh culture solution was pressurized into the hollow fiber membrane filter 10 at a flow rate of 200 mQ/min using a hollow fiber membrane filter backwashing Belisteric pump to backwash the hollow fiber extended filter 10. The culture solution used for backwashing was Eagle MEM medium 9.4g/Q.
, glutamine 0°292g/Q, 7.5% sodium bicarbonate aqueous solution 29ml, and fetal bovine serum 50mQ
/Q, and was stored in the fresh culture solution reservoir 12. Backwashing was controlled so that it ended when the same amount of culture filtrate was injected into the culture tank. Filtration and backwashing.

The experiment was carried out 10 times a day so that the culture solution in the culture tank was replaced once a day.

The drawn culture filtrate was stored in the culture source liquid storage layer 15. Thereafter, low-molecular components including waste components were filtered by the ultrafilter 14, and high-molecular components were concentrated and stored in the waste component removal liquid storage tank 13. The volume of the waste component removal solution is 1 of the culture filtrate.
The liquid was circulated between the ultrafilter 14 and the waste component removal liquid storage tank 13 by a peristaltic pump until the liquid became 710 ml. The ultrafiltration membrane used had a molecular weight cutoff of 10,000.

From the 8th day after the start of culture, a waste component removal solution was mixed in a ratio of 100 mQ to 900 mQ of fresh culture solution in a culture solution preparation tank, and this was used for backwashing the hollow fiber membrane filter IO.

On the 188th day after the start of culture, the culture solution in the culture tank was reduced to 2 times a day.
The number of filtration and backwashing was increased to 20 times per day.

The results of this example are shown in FIG. 19. As a result of removing waste components by ultrafiltration, the concentrations of lactic acid and ammonia are lower than in Example 2. Furthermore, the maximum cell concentration reached 2.8 x 10' cells/mQ, which confirms the effect of this example.

Example 4 Using the method of Example 2, a cell concentration of 1. I x 10″ pieces/
m Q's seed cell lI! l? IS solution was prepared. Next, culture was performed using the cell culture system shown in FIG.

In this system, only the means for removing waste components of the culture system shown in FIG. 6 used in Example 3 was replaced with diffusion dialysis 44. Oxygen supply means, defoaming means.

The culture solution exchange means and culture conditions were the same as in Example 3.

The above seed cell suspension 2,000 m'n and the same culture solution as in Example 2 were placed in a culture tank. Add OOOmQ (total 5
．． OOOmQ) and kept at 37°C. Batch culture was performed for two days after the start of culture, and the culture solution was replaced from the third day. The pressure inside the hollow fiber membrane filter 10 was reduced using a peristaltic pump, and the culture filtrate was drawn out at a flow rate of 15 mQ/min and stored in the culture source liquid storage M15. No contamination of cells into the culture filtrate was observed.

When the pulling amount reached 500 mQ, the pulling riverside staric pump was stopped.

The culture filtrate was sent from the culture filtrate reservoir 15 to the diffusion dialysis machine 44 using a peristaltic pump, and low molecular weight components including waste components were removed by diffusion dialysis. The diffusion dialysis membrane used was one that allowed substances with a molecular weight of 6,000 or less to pass through, but did not allow substances with a molecular weight larger than 6,000 to pass therethrough.

In diffusion dialysis, Eagle MEM medium 9.
4 g/R, glutami yo, 292 g/n.

7.5% sodium hydrogen carbonate aqueous solution 29 m Q /
A low molecular component solution consisting of Q and glucose at 20 g/12 was used at a flow rate twice that of the culture filtrate. The waste component removal solution from which low molecular weight components including waste components had been removed was stored in a waste component removal solution storage tank, and fetal bovine serum was added to the waste component removal solution to a concentration of 1% in 6 culture solution preparation tanks. This was pressurized into the hollow fiber membrane filter 10 using a peristaltic pump at a flow rate of 200 mQ/min to backwash the hollow fiber membrane filter 10. Backwashing was controlled so that it ended when the same amount of culture filtrate was injected into the culture tank. Filtration and backwashing are carried out at a rate of 30% per day so that the culture solution in the culture tank is replaced 3 times per day.
Conducted twice.

The results of this example are shown in FIG. By removing waste components by diffusion dialysis, the concentrations of lactic acid and ammonia can be lowered, resulting in a maximum cell concentration of 3. It reached OX 10' pieces/mQ.

Comparative Example 5 In the culture apparatus shown in FIG. 1, a hydrophilic filtration membrane (manufactured by Amicon, Diaflow Hollow Fiber System, DH-1, hollow fiber membrane: HIMP) was used instead of the hydrophobic filtration membrane.
Cell culture was carried out in the same manner as in Example 2 using O2-43 (pore size: 0.1 μm).

As a result, although the filtration membrane was backwashed, the filtration flow rate rapidly decreased and clogging occurred on the 6th day after the start of the culture, making it impossible to continue the culture.

〔Effect of the invention〕

According to the present invention, there are the following effects.

It is possible to effectively defoamer, which has been considered difficult in the past, and oxygen can be supplied using the submerged aeration method, which allows oxygen to be used with high efficiency, resulting in lower sterile oxygen consumption compared to the conventional surface aeration method. can be reduced to 1/10 to 115o. Furthermore, it is possible to supply 5 times or more oxygen to a culture solution using highly foamable serum or the like, thereby increasing the culture efficiency.

Furthermore, by filtering the culture solution using a hydrophobic membrane, a culture filtrate containing no cells can be extracted without causing damage to cells or clogging of the membrane due to shear force of the fluid. Thereby, the culture solution in the culture tank can be exchanged efficiently, and cells can be stably cultured at a high concentration for a long period of time.

The waste component removal solution, which removes waste components such as lactic acid and ammonia contained in the culture solution and recovers substances effective for cell proliferation such as hormones and growth factors, can be used again for culture, making it expensive. The amount of serum used can be reduced, and the cell concentration can be further increased.

[Brief explanation of the drawing]

1 to 7 and 14 are block diagrams showing an overview of an embodiment of the present invention, FIG. 8A is a perspective view of the membrane support member, and FIG. 8B is the membrane of FIG. 8A. FIG. 9 is a perspective view of a filtration unit in which a hollow fiber membrane is attached to a support member; FIG. 9 is a plan view and a sectional view showing the membrane support member; FIGS. FIG. 15 is a curve diagram showing the relationship between the filtration pressure of the hollow fiber membrane and the filtration flow rate, and FIGS. 16 to 20 are curve diagrams showing the relationship between the number of days of cell culture and cell concentration according to the present invention. 1...Culture solution, 2...Culture tank, 3...Draft tube. 4... Nozzle, 5... Defoaming layer, 6... Mist separator, 7... Filter, 8... Filter, 9...
Filtration aid, 1°... Porous hollow fiber membrane filter, 11.
...Retention filter, 12...Fresh culture solution storage tank, 13.
...Waste component removal liquid storage tank, 14...Ultrafilter, 15
...Culture filtrate storage tank. 16...Near compressor, 17.18...Gas cylinder, 19-23.45-47...Pump, 24...
Culture exhaust gas, 25... Culture filtrate, 26... Ultrafiltration membrane permeate, 27... Waste component removal liquid, 28... Magnetic coupling, 29... Stirring blade, 31-33.
··valve. 41...Culture solution! III tank, 42...Low molecular component liquid storage tank, 43°/high molecular component liquid storage tank, 44...Diffusion dialyzer, 49...Dialysis waste liquid, 5o...Membrane support member, 5
1. Membrane connection member, 52... Raw ore tension member, 55...
・Small hole, 56... Waste component adsorption cylinder. BozriJ 30th #4th Figure 5 Name Otsu Figure 7th ω OIS Exit High 16 Insenguchi Figure 4. ! - Pu paddy

Claims (1)

[Claims] 1. In a cell culture method in which biological cells are cultured in liquid, a part of the culture solution is filtered out using a filtration membrane, and a new culture solution is replenished, wherein the filtration membrane is a hydrophobic filtration method. 1. A cell culture method, wherein the filtration membrane is intermittently backwashed with fresh culture solution. 2. In a cell culture method in which biological cells are cultured in liquid, a part of the culture solution is filtered out using a filtration membrane, and a new culture solution is replenished. 1. A cell culture method comprising a pre-coated membrane having a filter aid layer, the filtration membrane being intermittently backwashed with fresh culture solution. 3. The cell culture method according to claim 1 or 2, wherein the hydrophobic filtration membrane is one or more hollow fiber membranes. 4. The cell culture method according to claim 1 or 2, wherein the hydrophobic filtration membrane is formed of a material having a contact angle with the droplets of the culture solution of 80 degrees or more. 5. The cell culture method according to claim 1 or 2, characterized in that filtration is performed after filling the filtration pores of the hydrophobic filtration membrane with a culture solution. 6. The cell culture method according to claim 1 or 2, characterized in that backwashing is carried out at a pressure higher than the critical pressure of the hydrophobic filtration membrane. 7. The cell culture method according to claim 1 or 2, wherein the culture solution for backwashing the hydrophobic filtration membrane is a culture solution obtained by removing waste components from a filtered culture solution. 8. In claim 1 or 2, waste components in the filtered culture solution are removed by a waste component removal means, and the waste component removal liquid is used as part or all of a backwashing liquid for the hydrophobic filtration membrane. Characteristic cell culture method. 9. The cell culture method according to claim 8, wherein the means for removing waste components is a diffusion dialysis method, an ultrafiltration method, or a microfiltration method. 10. The cell culture method according to claim 1 or 2, characterized in that a stirring blade is provided in the culture solution, and a hydrophobic filtration membrane is provided around the rotating surface of the stirring blade. 11. In claim 1 or 2, the foam of the culture solution generated by the culture is broken by providing an antifoaming layer made of a water-repellent material placed on the liquid surface and bringing the foam into contact with the foam. A cell culture method characterized by culturing while 12. In claim 10, the antifoaming layer has a viscosity of 1×10
A cell culture method characterized in that a base material is coated or impregnated with a water repellent of ^4 centipoise or more. 13. The cell culture method according to claim 2, wherein the hydrophilic filter aid has a sedimentation rate greater than that of the cells. 14. In claim 2, a filter containing a filtration membrane is connected to the culture tank via piping and provided outside the culture tank, and a hydrophobic filtration membrane is arranged in the upper part of the filter, and the density is equal to that of the culture liquid. A cell culture method characterized by using a hydrophilic filter aid smaller than . 15. A liquid culture device for biological cells having a means for supplying useful gas to cultured cells, a means for supplying a culture solution, and a means for discharging a waste culture solution, wherein the means for discharging the waste culture solution is a culture solution filtration comprising a hydrophobic filtration membrane. A liquid culture device for biological cells, the device being provided via a means and having a means for backwashing the hydrophobic filtration membrane with a culture solution. 16. A liquid culture device for biological cells according to claim 15, wherein the culture solution filtration means comprises a precoated membrane having a hydrophilic filter aid layer on the surface of a hydrophobic hollow fiber membrane. 17. Both ends of the hydrophobic hollow fiber membrane are formed with a gradient so that the outer diameter is larger than the inner diameter of the hollow fiber membrane and the tip part has an outer diameter that can be inserted into the hollow fiber membrane, and,
A membrane expansion member provided with a protruding portion for preventing separation of the hollow fiber membrane is inserted, and the inserted portion is fixed to a connecting portion so that it can be connected to piping via the connecting portion. A biological cell separation filter featuring: