JPH01289483A - Cell culture method - Google Patents

Abstract

PURPOSE:To avoid generation of air bubble, remove obstruction by the bubble and make possible to sterilize an oxygen enrichment membrane consisted of a specific polymer by high-pressure steam, by diffusing oxygen enriched air into a cell culture solution through said membrane. CONSTITUTION:Oxygen enriched air is diffused into a cell culture solution through an oxygen enrichment membrane constituted of polyamide expressed by formula I (R is one of H, CH3 or C2H5; n is number of repeating unit) or polyarylate expressed by formula II (R is one of H, CH3 or C2H5; n is number of repeating unit) as a raw material. Said polymers are excellent in heat resistance and hydrophilic nature.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for cultivating animal cells, plant cells, aerobic microorganisms, etc. In particular, the present invention relates to a method for culturing animal cells, plant cells, aerobic microorganisms, etc. The present invention relates to a cell culture method that enables large-scale, high-density culture of cells by directly diffusing enriched air into a culture medium.

BACKGROUND OF THE INVENTION In order for animal cells, plant cells, and aerobic microorganisms to grow and multiply in liquid or suspension cell cultures, it is necessary to provide a solid surface that meets the needs of the cells, as well as to provide nutrients and nutrients. Removal of harmful metabolites.

and oxygen supply, which are limiting factors for cell culture. Therefore, in order to industrially achieve large-scale and high-density culture of cells and microorganisms, the obstacles of these culture-limiting factors must be solved, and among these limiting factors, oxygen supply is the most improved. This is what is expected.

For the oxygen concentration in the culture solution, that is, the dissolved oxygen concentration, given the temperature and culture solution composition, the maximum value is the equilibrium solubility of oxygen determined by the oxygen partial pressure in the gas phase. However, since cells in a culture solution constantly consume oxygen, the dissolved oxygen concentration is lower than this equilibrium solubility, and a stable supply of oxygen is required for cell growth and proliferation.

On the other hand, cell culture methods are aimed at the growth and proliferation of only a specific cell type, and the contamination and proliferation of foreign cells or bacteria must be reliably eliminated. The culture device and culture medium must be completely sterile and non-living. As such sterilization methods, sterilization with ethylene oxide, chemical sterilization with an aqueous hypochlorite solution, and high-pressure steam sterilization are known. Among these sterilization methods, the high-pressure steam sterilization method is the most industrially preferable because it does not require cleaning after the sterilization operation or waste management. Therefore, the method of supplying oxygen in cell culture must be one of these sterilization methods, preferably a method that allows high-pressure steam sterilization.

As a method of supplying oxygen to the culture solution, bubble aeration of air that has been sterilized using a filter or the like has been used for a long time. In such bubble aeration, the dissolution of oxygen occurs on the surface of the bubbles rising in the sterile body, so in order to increase the rate of oxygen dissolution, it is necessary to devise ways to increase the amount of aeration and generate fine bubbles. The dissolved oxygen concentration can also be increased by bubbling separately adjusted oxygen-enriched air with a higher oxygen concentration than normal air, but most of the enriched air is released without being dissolved.
Difficult to adopt industrially.

In cell culture, bubble aeration is the simplest oxygen supply method, and high-pressure steam sterilization is the most commonly used method, but there are some problems that need to be solved. The first is intense foaming due to bubble ventilation. In general, cell culture solution is a high viscosity liquid, and air bubbles introduced from the bottom of the culture container remain on the surface of the culture solution for a long time without bursting, resulting in a larger culture container volume compared to the culture solution volume. I need.

The second problem to be solved is that cells that come into contact with the bubbles are damaged during the process in which the bubbles generated at the bottom of the culture container rise through the liquid until they finally burst. In #, it has been pointed out that animal cells, whose cell membranes have poor mechanical strength, suffer significant damage due to contact with air bubbles.

The third problem to be solved with bubble aeration is evaporation loss of the culture solution. This loss of medium is noticeable in animal and plant cell cultures with long culture periods;
In order to correct the concentration of non-volatile components contained in the culture solution and the drop in the liquid level, it is necessary to replenish water and culture solution as appropriate.

As a means of suppressing the above-mentioned problems associated with bubble ventilation,
Various methods have been proposed in the past. For example, Japanese Patent Laid-Open No. 82-FI35278 proposes a method of culturing while blowing oxygen-containing gas onto the surface of the culture solution. Although this air supply method has the advantage of eliminating problems caused by air bubble generation, the contact surface between the gas and the culture medium is limited to the surface of the culture medium, and the increase in the depth direction of the culture container reduces the amount of air supply. Scaling up cell culture is difficult because of the shortage.

An oxygen supply method that allows a large contact area between the gas and the culture solution and suppresses the generation of bubbles is to diffuse oxygen-containing gas into the culture solution through a membrane immersed in the culture solution. For example, JP-A-81-100190 proposes a bubble-free gas swing method and device for supplying air into a culture solution through a porous hollow fiber membrane having no surface dense layer. However, since such porous hollow fiber membranes do not have an oxygen enrichment function, separately adjusted oxygen enriched air must be supplied to increase dissolved oxygen.

As a method for supplying air into the culture solution using a material with an oxygen enrichment function, Japanese Patent Publication No. 80-23834 discloses a culture method in which gas is diffused into the culture solution via a silicone rubber tube. has been done. However, since a thick tube is used, a high gas permeation rate cannot be expected, and as a result, the gas supplied is limited to oxygen, and it is difficult to scale up cell culture.

On the other hand, as an oxygen enrichment membrane having excellent oxygen enrichment performance and high gas permeation rate, for example, Japanese Patent Publication No. 58-51321
As disclosed in the publication, a composite membrane of a polysiloxane thin film and a porous carrier is known and has already been put into practical use as an oxygen enricher. The Volisia chitin-based oxygen-enriched membrane was immersed in a cell culture solution to convert normal air into oxygen-enriched air.

Although it is theoretically possible to supply air under bubble-free conditions, the low heat resistance of the polysiloxane thin film makes it impossible to autoclave the apparatus and culture medium with steam sterilization, which results in oxygen-rich cell culture. It is difficult to practically utilize this method as a method of supplying converted air.

The conventional oxygen supply means for cell culture methods have been described above, and it is an air supply method that increases the dissolved oxygen concentration in the culture medium, suppresses problems caused by bubble generation, and is also industrially possible. Water vapor; nothing capable of mtx has yet been proposed.

Problems to be Solved by the Invention The purpose of the present invention is to diffuse oxygen-enriched air into a liquid or suspended cell culture solution through an oxygen-enriched membrane that has excellent heat resistance and is capable of high-pressure steam sterilization. An object of the present invention is to provide a cell culture method that reduces foaming and evaporation of a culture solution due to oxygen supply, and enables large-scale, high-density culture of animal cells, plant cells, and aerobic microorganisms in an environment with a high dissolved oxygen concentration.

Means for Solving the Problems The present invention provides an oxygen-enriching membrane made of a polyamide having excellent heat resistance and high hydrophilicity represented by the general formula (A), or an oxygen-enriching membrane represented by the general formula (B). A cell culture method characterized by diffusing oxygen-enriched air into a liquid or suspended cell culture medium through an oxygen-enriched membrane made of polyarylate, which has excellent heat resistance and water repellency. It is.

(However, R indicates H, CH3, C2H, or QL1, and n indicates the number of repeating units.) In the present invention, the cell culture solution is immersed in a liquid or suspension cell culture medium and then exposed to oxygen-enriched air. The oxygen-enriching membrane that supplies oxygen is a sheet-like dense uniform layer that has an oxygen-enriching function and is made of polyamide represented by the above general formula (A) or polyarylate represented by the general formula (B). It is characterized by being a membrane.

In addition, the oxygen-enriching membrane in the present invention is made of polyamide represented by the general formula (A) or polyarylate represented by the general formula CB), and the structure of the oxygen-enriching membrane is such that the membrane surface layer is oxygen-enriched. It is characterized by being a sheet-like or hollow-fiber-like membrane with a multilayer structure, which is a dense layer with a oxidizing function and a porous layer inside.

Among the materials of the oxygen-enriched membrane used in the cell culture method of the present invention, the polyamide represented by the general formula (A) has one formula (wherein R is H, c) Is, C2H5. , ) and terephthalic acid chloride or isophthalic acid chloride, or a mixture of both phthalic acid chlorides represented by the formula,
It can be obtained by reacting for several hours under cooling in a solvent such as N,N-dimethylacetamide or tomethyl-2-pyrrolidone. The heat resistance of the obtained polyamide is high, for example, the glass transition temperature of polyamide in which R is H is 38
0°C, and has sufficient heat resistance to withstand high-pressure steam sterilization at 127°C. In addition, this polyamide has a high water absorption rate and is highly hydrophilic, so it has good cell adhesion.

On the other hand, among the materials for the oxygen-enriched membrane used in the cell culture method of the present invention, polyarylates represented by the general formula CB) are of the formula (where R is any one of H%CH3, C, and Hs). Triethylamine is added as a dehydrochlorination agent to a mixture of 8,3-bis(4-hydroxyphenyl)fluorenes represented by ) and terephthalic acid chloride, or isophthalic acid chloride, or both phthalic acid chlorides represented by the formula. It can be obtained by reacting it in a solvent such as 1,2-dichloroethane. The obtained polyarylate has high heat resistance and can sufficiently withstand high-pressure steam sterilization at 127° C. like the above-mentioned polyamide.

The polyamide represented by the general formula (A) and the polyarylate represented by the general formula (B) are characterized in that they are soluble in organic solvents. For example, this polyamide is soluble in polar solvents such as N,N-dimethylacetamide and tomethyl-2-pyrrolidone, and polyarylate is soluble in 1,2-dichloroethane and chloroform in addition to the above polar solvents. It is.

Therefore, by dry desolvation of polyamide and polyarylate solutions, it is possible to create a sheet-like, dense, uniform layer film with an oxygen enrichment function.On the other hand, by desolvation in a liquid such as water, a surface dense layer and a porous layer can be created. A multilayer structure film consisting of layers can be created. The ability of such polyamides and polyarylates to provide excellent oxygen-enriching membranes was proposed by the present inventors in Japanese Patent Applications No. 82-18237 and No. 83-58241.

The method for producing the sheet-like, dense, uniform layer membrane with oxygen enrichment function used in the cell culture method of the present invention is not specified, but a membrane-forming solution in which polyamide or polyarylate is dissolved in a solvent is used to form a smooth substrate. A dense sheet-like film having a thickness of 10 to 100 ILm can be obtained by casting a thin film using a doctor blade or the like and then evaporating the solvent by heating.

To use a sheet-like membrane as an oxygen enricher for cell culture, for example, two sheets are sealed on four sides with a thin spacer that allows gas to freely pass through, forming a thin box shape, and It is advisable to provide an air inlet and an air outlet.Sheet-like membranes have a higher oxygen to nitrogen permeation rate ratio, that is, a separation rate of approximately 5, compared to the hollow fiber porous membranes described below, and are highly enriched with oxygen. Although air can be obtained, the permeation rate of oxygen is low.

That is, in the cell culture method of the present invention, the sheet-like oxygen-enriched membrane can be used more effectively when a high dissolved oxygen concentration is required than when a high oxygen supply rate is required.

On the other hand, the method for manufacturing the multilayer structure membrane used in the cell culture method of the present invention is not particularly specified, but among the multilayer structure membranes, the hollow fiber membrane is manufactured by pushing out the membrane forming solution through the annular opening of a double-tubular spinning nozzle, and simultaneously It can be produced using a general wet manufacturing method for hollow fibers, in which the coagulated core liquid is extruded from a central circular opening. Such a hollow fiber membrane is characterized by a multilayer structure having a thin and dense oxygen-enriching functional layer with a thickness of 10 to 50 nm on one surface and a porous layer inside.

In order to use a hollow fiber multilayer oxygen enrichment membrane as an oxygen enrichment device for cell culture, it is recommended to bundle the hollow fibers, embed both ends in a tubular material, and open the hollow fibers at both ends. By introducing pressurized air into the hollow fibers of the hollow fiber oxygen enricher immersed in the solution, oxygen-enriched air is diffused into the culture solution, resulting in a higher concentration of dissolved oxygen than when normal air is diffused. can be obtained.

The feature of this hollow fiber multilayer oxygen-enriched membrane is that compared to the sheet-like membrane described above, the permeation rate ratio of oxygen and nitrogen, that is, the separation rate, is small at 3 to 4, but the permeation rate of oxygen is high. . In addition, since hollow fiber membranes have excellent pressure resistance and can easily increase the membrane area, they can be effectively used in cell culture where a high oxygen supply rate and a large container are required.

On the other hand, sheet-like multilayer structure membranes are created by casting the film-forming stock solution onto a smooth substrate and then removing the solvent in a liquid such as water. It can be done.

Furthermore, the oxygen enrichment membrane used in the cell culture method of the present invention is characterized in that its material is polyamide with high hydrophilicity or polyarylate with high water repellency.

Among these, polyamide oxygen-enriching membranes, which are highly hydrophilic, have excellent cell adhesion and can supply oxygen to floating cells in cell culture medium, as well as directly to cells attached to the membrane. It is. On the other hand, cells do not easily adhere to polyarylate oxygen-enriching membranes that are highly water-repellent, and therefore have the effect of supplying oxygen to cells floating in the culture medium by diffusion and avoiding a decrease in oxygen supply rate due to cell adhesion. These membrane materials can be selected depending on the properties of the cells intended for cell culture.

Effect The cell culture method obtained in the present invention supplies oxygen-enriched air into the culture medium through an oxygen-enriched membrane with excellent heat resistance, and enables cell culture in a high dissolved oxygen environment. , which solves the problems associated with bubble aeration and provides a method for culturing animal cells, plant cells, and aerobic microorganisms in large quantities and at high density.Furthermore, by using an oxygen-enriching membrane material with excellent heat resistance, the culture medium It also makes it possible to sterilize the entire culture vessel, including the oxygen enrichment membrane, by high-pressure steam sterilization.

In the cell culture method of the present invention, when the culture solution is brought into contact with pressurized normal air through an oxygen-enriched membrane consisting of a sheet-like membrane, a hollow fiber-like membrane, or a sheet-like multilayer structure membrane, oxygen with a high oxygen concentration is produced. Enriched air diffuses into the culture medium.

The method of supplying air into the culture medium through an oxygen-enriching membrane as in the present invention has the effect of obtaining a high dissolved oxygen concentration and avoiding the generation of air bubbles.
i-Foam ventilation needs to be solved! ! This makes it possible to solve the problems of cell damage and evaporation of the culture medium at the same time.

EXAMPLES Examples of the present invention are listed below, but the present invention is not limited thereto.

Example 1 A 20 pm-thick sheet-like membrane with an oxygen enrichment function made of polyamide in which R is H in general formula (A) or polyarylate in which R is H in general formula (B) When an oxygen enricher with a membrane area of 20 cm2 is immersed in MS culture solution used for plant cell culture, and normal air pressurized to 2 atmospheres is introduced into the oxygen enricher, the concentration of the culture solution increases. The dissolved oxygen concentration was 18 ppm (polyamide membrane) and 14 ppm (polyarylate membrane) at 25°C.

Examples 2 to 5 A hollow fiber membrane with an outer diameter of 0.7 mm and an inner diameter of 0.551 mm made of the same polyamide or polyarylate as in Example 1,
Approximately 50 inner and outer surface layers! An oxygen enricher in which 15 hollow fiber multilayer structure membranes, in which III is the dense layer, is bundled in 75 axes and lengths was placed on a liquid surface fi38 containing the same MS culture solution 50G as in Example 1.
After being immersed in a cm2 culture container and sterilized with high-pressure steam at 127°C for 30 minutes, normal air was aerated at 25°C for 2 weeks, and the dissolved oxygen concentration was examined. Table 1 shows the air pressure introduced into the hollow fiber membrane and the dissolved oxygen concentration. Also, the evaporation loss of the culture solution was less than 1% even after two weeks.

Table 1 Comparative Examples Air was bubbled through the same cell culture vessels as in Examples 2 to 5 at a rate of 39 cm" for 7 minutes. As a result, the steady dissolved oxygen concentration at 25°C was 8 PP■, and the culture solution was approximately 10 PP. It was lost by evaporation at a rate of 1/H.

Comparing Examples 1 to 5 with Comparative Examples, it was found that by supplying air through the oxygen enrichment membrane, it was possible to obtain a high dissolved oxygen concentration, and the effect of suppressing evaporation loss of the culture solution was also remarkable. be.

Example 6 Under the same conditions as in Example 3, 2 g of carrot callus was cultured. As a result, there was no contamination by microorganisms, and the callus weight increased approximately six times two weeks after the start of culture. In addition, the drop in liquid level due to evaporative loss of the culture medium, which is caused by air bubble ventilation, and the adhesion of cells at the interface between the liquid level and the container wall were also reduced.

Effects of the Invention In cell culture of animal cells, plant cells, and aerobic microorganisms, the cell culture method of supplying oxygen to the culture medium through the oxygen-enriching membrane of the present invention increases the dissolved oxygen concentration in the culture medium. In addition, it can solve the problems of cell damage and evaporation loss of culture medium caused by conventional bubble aeration.
This makes it possible to culture cells in large quantities and at high density.

Furthermore, since an oxygen-enriched membrane material with excellent heat resistance is used, the entire culture vessel can be sterilized with high-pressure steam, and furthermore, the hydrophilicity and water repellency of the oxygen-enriched membrane can be effectively utilized.

Claims (1)

[Claims] 1. Oxygen-enriched air in liquid or suspended form is passed through an oxygen-enriched membrane made of polyamide, which has excellent heat resistance and is highly hydrophilic, represented by the general formula (A). A cell culture method characterized by dispersing cells in a cell culture solution. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (A) (However, R represents one of H, CH_3, C_2H_5, and n represents the number of repeating units.) 2. Represented by general formula (B) A cell culture characterized in that oxygen-enriched air is diffused into a liquid or suspended cell culture medium through an oxygen-enriched membrane made of polyarylate, which has excellent heat resistance and water repellency. Method. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(B) (However, R indicates one of H, CH_3, C_2H_5, and n indicates the number of repeating units.) 3. Liquid or suspension cell culture solution The cell according to claim 1 or 2, wherein the oxygen-enriching membrane that is immersed therein and supplies oxygen-enriched air is a sheet-like, dense, uniform layer membrane that has an oxygen-enriching function. Culture method. 4. The structure of the oxygen-enriching membrane that supplies oxygen-enriched air by immersing it in a liquid or suspended cell culture solution is that the surface layer is a dense oxygen-enriching functional layer and the inside is a porous layer. 3. The cell culture method according to claim 1, wherein the cell culture method is a sheet-like or hollow fiber-like membrane having a multilayer structure.