Patent Application
20120244602
The present invention relates to a culture method for culturing cells or microorganisms in a culture solution containing a nutrient by dissolving oxygen or carbon dioxide in the culture solution.
As a method for culturing, for example, animal or plant cells or microorganisms, a method is known in which cells or microorganisms are cultured by supplying gas containing oxygen or carbon dioxide, e.g., air, to a culture solution containing a nutrient. This nutrient is, for example, mixed in advance in the culture solution in a predetermined amount, or appropriately supplied into the culture solution during culturing. However, it is necessary that air be continued to be supplied into the culture solution during culturing. In a known method for supplying air into a culture solution, for example, a tubular sparger (e.g., U-shaped sparger) having a large number of gas discharge openings on the leading end side thereof is immersed in the culture solution and bubbles having a diameter of, for example, about several millimeters are supplied into the culture solution.
In this method, in order to further increase the amount of air dissolved in the culture solution, air is made to be easily dissolved in the culture solution by, for example, reducing the size of (breaking up) air bubbles supplied from the sparger by stirring the culture solution with a turbine-type stirring blade so as to increase the contact area between gas and liquid (air and the culture solution). Therefore, in order to further increase the rate (amount) of oxygen dissolution, it is necessary to more vigorously stir the culture solution. Consequently, a large amount of energy is necessary for stirring, and the size and the cost of an apparatus (stirring apparatus) for conducting the culture are also increased. In addition, in the case where the temperature of the culture solution is increased by stirring, cooling energy for decreasing the temperature of the culture solution is necessary. Furthermore, in the case of culturing cells, the cells may be physically damaged by stirring with the stirring blade. Furthermore, the cells may be damaged by an impact caused when the bubbles are broken up (popped) by stirring.
Patent Document 1 describes a technology in which fine bubbles are generated in a liquid through a porous body and these bubbles are utilized in hydroponics, aquaculture of fish and shellfish, foods, microcapsules, pharmaceutical preparations, cosmetics, and the like, and a technology for suppressing proliferation of microorganisms utilizing the fine bubbles, etc. However, the culture of cells or microorganisms has not been studied.
[Patent Document 1] Japanese Laid-open Patent Publication No. 2005-169359 (paragraph 0045)
The present invention has been made under the above circumstances, and an object of the present invention is to provide a method for culturing cells or microorganisms wherein when cells or microorganisms are cultured by dissolving oxygen or carbon dioxide in a culture solution containing a nutrient, the oxygen or carbon dioxide can be rapidly dissolved in the culture solution while suppressing stirring of the culture solution to mild or without conducting stirring.
A method for culturing cells or microorganisms by dissolving oxygen or carbon dioxide in a culture solution containing a nutrient, the method including: a step of culturing cells or microorganisms by supplying gas containing oxygen or carbon dioxide to a porous body to generate, in a culture solution, bubbles that have a 50% diameter of 200 μm or less in a volume-based particle size distribution, thereby dissolving oxygen or carbon dioxide in the culture solution, wherein the culture solution contains at least one of a cell-protecting agent for protecting the cells and a protein hydrolysate.
The method for culturing cells or microorganisms according to claim 1, wherein the culture solution has a surface tension of 51.5 dyne/cm or less.
According to the present invention, in culturing cells or microorganisms such as yeast, mold, bacteria, or microalgae by supplying gas containing oxygen or carbon dioxide in a culture solution containing a nutrient, gas containing oxygen or carbon dioxide is supplied to a porous body to generate, in a culture solution, bubbles that have a 50% diameter of 200 μm or less in a volume-based particle size distribution, and the culture solution contains at least one of a cell-protecting agent for protecting cells and a protein hydrolysate. Accordingly, coalescence (aggregation) of bubbles in the culture solution is suppressed by a surface-active action of the protein hydrolysate or the cell-protecting agent, and bubbles having a very small diameter can be obtained. Thus, the contact area between gas and liquid (air and the culture solution) can be further increased. In addition, since the buoyant force of the bubbles can be suppressed so as to be very small, the bubbles can be maintained in the culture solution in a so-called stationary state, as compared with bubbles that have a diameter of, for example, 300 μm or more and that move upwards upon receiving the buoyant force in the related art. Accordingly, since the gas and the culture solution can be brought into contact with each other over a long period of time, oxygen or carbon dioxide can be rapidly dissolved in the culture solution. Furthermore, it is not necessary to conduct stirring vigorously to such an extent that the bubbles are broken up, and thus stirring can be suppressed to be slow or stirring need not be conducted. Accordingly, the size of the entire bio reactor and consumption energy for the culture can be reduced. In particular, in the case where cells are cultured, physical damage to the cells by stirring can be suppressed.
These and other objects and features of the present invention will become clearer from the following description of the preferred embodiments given with reference to the attached drawings, wherein:
A culture method for culturing cells or microorganisms such as yeast, mold, bacteria, or microalgae according to an embodiment of the present invention is described with reference to
The sparger 22 includes, for example, a porous body (porous membrane) 11 configured to have a substantially cylindrical shape so that an inner region 11a thereof is hollow, and is immersed in the culture solution 13. The upper end of the porous body 11 is hermetically connected to the oxygen supply path 24, and the lower end of the porous body 11 is sealed with, for example, a sealing member (not illustrated). As illustrated in the enlarged view on the lower side of
The culture solution 13 in the culture tank 21 contains cells 2 or microorganisms, cells 2 in this example, to be cultured and a nutrient serving as nutrition of the cells 2. This nutrient is a basal medium prepared by mixing plural types of amino acids, vitamins, inorganic salts, sugars, etc. in a predetermined ratio. In addition, the culture solution 13 contains, as an additive, at least one of a protein hydrolysate and a cell-protecting agent for protecting the cells 2. Each of these additives has a surface-active action and suppresses coalescence (aggregation) of the fine bubbles 12 supplied from the sparger 22 into the culture solution 13 by the surface-active action. Specific components of these additives are described in detail below.
The protein hydrolysate is a product obtained by hydrolyzing a protein to amino acids and low-molecular-weight peptides. Examples thereof include a hydrolysate of casein, which is a protein derived from cow's milk, polypeptone, peptone, yeast extract, meat extract, and casamino acids. Examples of the method of this hydrolysis include acidolysis, enzymolysis, and self-digestion. Peptone is a generic name of a compound obtained by hydrolyzing an animal protein or a vegetable protein to amino acids and low-molecular-weight peptides. Polypeptone, which is an example of peptone, is a product manufactured by Nihon Pharmaceutical Co., Ltd. and is a powder obtained by decomposing cow's milk casein with an enzyme derived from an animal, followed by purification and drying. Yeast extract is a powder obtained by extracting a water-soluble component of brewer's yeast (Saccharomyces Cerevisiae Meyen), followed by drying. An example of yeast extract is a product (product name: Dried yeast extract D-3) manufactured by Nihon Pharmaceutical Co., Ltd. Casamino acids are products obtained by hydrolyzing a protein to only amino acids using hydrochloric acid, the products being other than peptides. Note that this protein hydrolysate may be used instead of the nutrient described above.
Examples of the cell-protecting agent include Pluronic F68, Daigo's GF21 (growth promoting factor), and serum. Pluronic F68 is a product (CAS No. 9003-11-6) manufactured by BASF Japan Ltd., and is a surfactant that does not have a function as a nutrient component or a cell growth factor but that have a function of protecting the cells 2. Daigo's GF21 is a product manufactured by Nihon Pharmaceutical Co., Ltd. and is a cell growth-promoting factor containing, as a main component, a growth factor in serum (GFS) obtained by purifying bovine serum to remove γ-globulin. The serum is, for example, fetal calf serum or calf serum, and has not only a function of supplying a nutrient component and a cell growth factor but also a function of a cell-protecting agent that protects the cells 2 from physical stress due to stirring of the culture solution 13 and aeration during the culture of the cells. The amounts of additives for obtaining fine bubbles 12 in the culture solution 13 by the surface-active action are described in Examples below.
Next, a method for culturing cells 2 of the present invention is described. First, cells 2, a nutrient, and at least one of the protein hydrolysate and the cell-protecting agent are charged in the culture tank 21 together with the culture solution 13. Specifically, in the case of serum culture, in addition to the cells 2, for example, the above-described basal medium and either serum or Daigo's GF21 are charged in the culture solution 13. In the case of serum-free culture, in addition to the cells 2, for example, the basal medium, a cell growth factor, and Pluronic F68 are charged. The amount of protein hydrolysate or cell-protecting agent added to the culture solution 13 is such that coalescence (aggregation) of bubbles 12 can be suppressed by the surface-active action of the protein hydrolysate or the cell-protecting agent. Specifically, the amount added is determined so that the surface tension of the culture solution 13 is 51.5 dyne/cm or less. Note that, as described above, the protein hydrolysate may be used instead of the nutrient.
Subsequently, gas containing oxygen, specifically, air in this example, is supplied from the oxygen supply path 24 to the sparger 22 while controlling the temperature of the culture solution 13 in the culture tank 21 to a predetermined temperature using a heater, a jacket, or the like (not illustrated). The stirring blades 26 are slowly rotated by the motor 27 to disperse bubbles 12 supplied from the sparger 22 to the culture solution 13 in the culture solution 13.
As illustrated in
The coalescence of the bubbles 12 is also similarly suppressed in the culture solution 13 by the surface-active action of the additive. Accordingly, for example, as illustrated in
In this case, since the diameters of the bubbles 12 are very small, for example, 200 μm or less, the bubbles 12 are hardly affected by the buoyant force and are substantially in a so-called stationary state in the culture solution 13. Accordingly, the bubbles 12 move upward very slowly in the culture solution 13, and thus the contact time with the culture solution 13 becomes long, as compared with the case where the diameters of the bubbles are large. In addition, since the diameters of the bubbles 12 are very small as described above, the inner pressure of the bubbles 12 (the force of the inner air to dissolve in the culture solution 13) is higher than that of bubbles each having a diameter of 300 μm or more. Consequently, the bubbles 12 generated in the culture solution 13 are rapidly dissolved in the culture solution 13.
Here, the cells 2 in the culture solution 13 consume oxygen in the culture solution 13 together with the nutrient, and produce, for example, a product and carbon dioxide. Since the amount of cells 2 (the number of individuals) in the culture solution 13 increases with the lapse of time, the amount of oxygen consumed by the cells 2 increases with the continuation of the culture of the cells 2. Accordingly, the amount of oxygen dissolved in the culture solution 13 (dissolved oxygen) may be decreased with the lapse of time. However, since the bubbles 12 are supplied from the sparger 22 into the culture solution 13 as described above, and the bubbles 12 dissolve in the culture solution 13 as described above, oxygen consumed by the cells 2 is compensated for. That is, by supplying the fine bubbles 12 into the culture solution 13, the rate of decrease in the dissolved oxygen concentration in the culture solution 13 becomes slow or the decrease in the dissolved oxygen is suppressed, as compared with the case where bubbles having a large diameter are supplied. Carbon dioxide produced in the culture solution 13 is then discharged from the discharge path 25. At the time when the consumption of the nutrient and oxygen by the cells 2 and the increase in the cells 2 (culture) are conducted for a predetermined time and the nutrient is used up, the cells 2 no longer consume oxygen. Consequently, the dissolved oxygen concentration in the culture solution 13 rapidly increases.
According to the above embodiment, in conducting the culture of the cells 2 in the culture solution 13, very small bubbles 12 having a 50% diameter of 200 μm or less in a volume-based particle size distribution are generated by supplying air to the porous body 11, and at least one of the protein hydrolysate and the cell-protecting agent is incorporated as an additive in the culture solution 13. Accordingly, coalescence (aggregation) of the bubbles 12 in the culture solution 13 is suppressed by a surface-active action of the additive and the bubbles 12 having a very small diameter can be obtained. Thus, the contact area between gas and liquid (the bubbles 12 and the culture solution 13) can be further increased, as compared with bubbles having a diameter of, for example, 300 μm or more. In addition, since the buoyant force of the bubbles 12 can be suppressed so as to be very small, the bubbles 12 can be maintained in the culture solution 13 in a so-called stationary state, as compared with bubbles having the above large diameter. Accordingly, the bubbles 12 and the culture solution 13 can be brought into contact with each other for a long time, and thus oxygen can be rapidly dissolved in the culture solution 13. In addition, the pressure of the inner air of the fine bubbles 12 to dissolve outside the bubbles 12 is higher than that of bubbles having a large diameter. Consequently, oxygen can be more rapidly dissolved in the culture solution 13.
Furthermore, vigorous stirring for breaking up large bubbles is unnecessary in order to obtain the bubbles 12 described above, and it is sufficient that stirring with the stirring blades 26 is gentle stirring for dispersing the bubbles 12 in the culture solution 13. Consequently, since the consumption energy and the size of the motor 27 can be reduced, the costs (operation cost and the cost of the bio reactor) for the culture of the cells 2 can be suppressed. In addition, heat generated by stirring can be suppressed, and thus, for example, the size of equipment for cooling the culture tank 21 and the culture solution 13 can be reduced. Furthermore, since even gentle stirring is sufficient, physical damage to the cells 2 by stirring can be suppressed. In addition, since the bubbles 12 need not be broken up, it is possible to suppress damage to the cells 2 due to an impact caused when the bubbles 12 are popped.
In the case where an additive is added to the culture solution 13, since the culture solution 13 is liquid used for culturing the cells 2, substances other than the protein hydrolysate and the cell-protecting agent, for example, substances harmful to the cells 2 or the culture of the cells 2 cannot be added to the culture solution 13. However, in the present invention, additives beneficial to the culture of the cells 2 can be used. Therefore, oxygen can be rapidly supplied to the culture solution 13 without adversely affecting the culture of the cells 2.
In the case of bubbles in the related art having a diameter of, for example, 300 μm or more, the bubbles receive a large buoyant force in the culture solution 13 and move rapidly upward, and thus a long contact time with the culture solution 13 cannot be secured. Furthermore, in the case where the above additive is not contained in the culture solution 13, even when fine bubbles 12 are generated by using the sparger 22, as illustrated in
In the above example, the bubbles 12 are dispersed in the culture solution 13 by stirring with the stirring blades 26. Alternatively, for example, in the case where the bubbles 12 are dissolved immediately after being released from the porous body 11, stirring may not be conducted.
In the above example, the cells 2 are cultured by supplying gas containing oxygen, e.g., air. Alternatively, the present invention may be applied when a plant such as plant cells or microalgae is cultured by supplying gas containing carbon dioxide. In this case, since fine bubbles 12 of the gas containing carbon dioxide are generated in the culture solution 13 through the sparger 22, carbon dioxide can be rapidly dissolved in the culture solution 13 as in the example described above. In such a case, a protein hydrolysate and a cell-protecting agent are used as additives added in order to reduce the diameter of the bubbles 12 (in order to reduce the surface tension of the culture solution 13). The amounts of additives added are appropriately determined on the basis of, for example, experiments.
Next, experiments conducted regarding fine bubbles 12 are described.
First, in the case where a cell-protecting agent (Daigo's GF21) was added to a culture solution 13 for culturing animal cells, a particle size distribution of bubbles 12 generated from the above-described sparger 22 (porous body 11 having a pore diameter d of 1 μm) was measured. The particle diameter was measured using a laser diffraction/scattering particle size distribution analyzer by continuously supplying the culture solution 13, in which the bubbles 12 were generated by the sparger 22, to a flow cell in the particle size distribution analyzer, irradiating the culture solution 13 with a laser beam, and by evaluating diffraction or scattering of the laser beam.
According to the results, in the case where the amount of cell-protecting agent added was 1% by volume, as illustrated in
To examine the correlation between the amount of additive added and the diameter of bubbles 12 generated, experiments were conducted as in Example 1 for various types and amounts of additive added.
As described above, the diameter of the bubbles 12 generated varies depending on the surface tension of the culture solution 13. Accordingly, first, the surface tension of the culture solution 13 for generating fine bubbles 12 having a diameter of 200 μm or less was examined. Specifically, bubbles 12 were generated using the sparger 22 described above in culture solutions 13 containing various amounts of Daigo's GF21 as an additive. The surface tension of each of the culture solutions 13 and the diameter of the bubbles 12 generated were measured. According to the results, as illustrated in
y=28.98x−1292 (1)
From this formula (1), it was found that, in order to generate bubbles 12 having a fine diameter of 200 μm or less as described above, it is necessary to control the surface tension of the culture solution 13 to be 51.5 dyne/cm or less.
Regarding the additives listed in Tables 1 to 3 below, the surface tension of culture solutions 13 was evaluated for various concentrations of each of the additives. In the case where such fine bubbles 12 were believed to be generated (the surface tension was 51.5 dyne/cm or less), the result was denoted by “A”. In the case where bubbles 12 having a diameter larger than the above were believed to be generated (the surface tension was more than 51.5 dyne/cm), the result was denoted by “B”. Tables 1 to 3 below include the results.
From these results, it was found that, in order to obtain bubbles 12 having a diameter of 200 μm or less, the bubbles 12 being believed to be less affected by the buoyant force, it is necessary to adjust the amount of additive added in accordance with the type of additive.
Next, in a culture medium (surface tension: 48.6 dyne/cm) for culturing microorganisms, the relationship between the bubble diameter of bubbles 12 and a pore diameter d of the porous body 11 was measured. The results illustrated in
y=3.4x+17.5 (2)
(where x represents the pore diameter of the porous body 11 and y represents the diameter (50% diameter) of the bubbles 12.) The R2 value in this case is 1.0. Thus, it is found that the pore diameter d of the porous body 11 can be calculated by the formula (2) from the diameter of the bubbles 12 in the culture solution 13 with a very high accuracy. Accordingly, the pore diameter d of the porous body 11 corresponding to the diameter (200 μm) of the bubbles 12, which are believed to be hardly affected by the buoyant force, was calculated. According to the result, the pore diameter d was 50 p.m. Thus, fine bubbles 12 which are hardly affected by the buoyant force can be obtained by using a porous body 11 having a pore diameter d of 50 μm or less.
Next, in this experiment, bubbles 12 were generated in a culture solution 13 using the sparger 22 of the present invention or an existing sparger (U-shaped sparger), and an oxygen supply performance in the culture solution 13 was examined. Specifically, a 3-L mini-jar fermenter manufactured by KK. Takasugi Seisakusho and having an inner diameter of 130 mm and a height of 260 mm (model: TSC-M3L) was used as a culture tank, and, as illustrated in
In this experiment, colon bacillus (Escherichia coli, NBRC3301), which is a microorganism, was actually cultured using the sparger 22 (porous body 11) of the present invention or the existing sparger (having a U-shape), and the cell concentration (OD600) was examined. The amount of air supplied was set to 150 mL/min in each case. According to the results, as illustrated in
Furthermore, colon bacillus was actually similarly cultured using the porous body 11 or the existing sparger, and the dissolved oxygen concentration was measured. According to the results, as illustrated in
In Example 6, the dissolved oxygen concentration in the culture solution 13 was measured. In Example 7, the oxygen concentration in gas released (discharged) from the culture solution 13 during culture of colon bacillus was measured. When oxygen in air supplied dissolves in the culture solution 13 and is consumed by colon bacillus, the amount of oxygen discharged decreases. On the other hand, in the case where oxygen does not dissolve in the culture solution 13, the oxygen is discharged directly from the culture solution 13. Thus, whether oxygen is effectively used or not was examined by measuring the oxygen concentration in the discharge gas.
According to the results, as illustrated in
Next, in this experiment, Chinese hamster ovary cells (Life Technologies Japan Ltd., Catalog No. 11619-012), which are animal cells, were actually cultured using the sparger 22 (porous body 11) of the present invention or an existing sparger (sintered metal), and the living cell concentration and the glucose concentration were examined. Specifically, a total 7-L animal cell culture tank (Model: BCP-07NP3) manufactured by ABLE Corporation was filled (charged) with 5 L of CHO-S-SFM II, which is a serum-free culture medium manufactured by Life Technologies Japan Ltd., and the Chinese hamster ovary cells were cultured. The living cell concentration was measured with a Thoma Hemocytometer (Model: A105) manufactured by Sunlead Glass Corp. using a solution prepared by adding a 0.05 wt (weight) % nigrosine solution, which is a reagent for determining life and death of cells, to a culture solution sampled from the culture tank. The glucose concentration was measured by a high-performance liquid chromatograph manufactured by Shimadzu Corporation (Column model No.: Shim-pack SPR-Pb 250 L×7.8). The amount of air supplied to the sparger 22 (sparger) was controlled so that the dissolved oxygen concentration of the culture solution was 6.31 mg/L in each of the case of the sparger 22 of the present invention and the case of the existing sparger. According to the results, regarding the living cell concentration, it was found that, as illustrated in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-280842 | Dec 2009 | JP | national |
The present application is a continuation of International Application No. PCT/JP2010/007202, filed on Dec. 10, 2010, which claims priority to JP Application 2009-280842, filed on Dec. 10, 2009, the contents of each of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2010/007202 | Dec 2010 | US |
| Child | 13485085 | US |
