JP2018113907A - Culture method for animal cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To maintain the activity of cultured animal cells over a long period and produce many substances.SOLUTION: The culture method for animal cells involves adding to a culture medium before culturing and/or during culturing one or more bases or base derivatives selected from a purine base, a purine base derivative, a pyrimidine base, and a pyrimidine base derivative.SELECTED DRAWING: None

Description

  The present invention relates to a method for culturing animal cells, a system for culturing animal cells, and a method for producing a medium for culturing animal cells.

  Useful substances such as interferon, interleukin, chemokine, erythropoietin, hematopoietic factor, cytotoxic factor, various growth factors, and antibodies are produced using animal cells. Since the medium for culturing animal cells is expensive, the cost of useful substances increases when continuous culture is performed. Therefore, batch culture is mainly used for production of useful substances using animal cells. However, in batch culture, as the cells grow, the nutrients in the medium decrease and harmful metabolites accumulate, so that the cells can grow stably and densely for a long time to produce useful substances. It is difficult.

  In order to solve the above problems, it has been studied to control the concentration of carbon source and amino acid in the medium (Patent Documents 1 and 2). Moreover, in order to supplement a nutrient source during culture | cultivation, flow-down culture is also performed (patent documents 1-3). However, it is still difficult to produce useful substances in large quantities at low cost using animal cells.

JP-T-2004-532642 JP 2008-43301 A International publication WO2011 / 093426A1

  It was a problem to be solved by the present invention to proliferate animal cells stably for a long period of time at a high density and to produce useful substances in large quantities at low cost.

  As a result of intensive studies to solve the above problems, the present inventors have cultured one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives. It has been found that the above-mentioned problems can be solved by adding to the medium before and / or during culture, and the present invention has been completed.

That is, the present invention provides the following.
(1) One or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives are added to the medium before and / or during the culture, Animal cell culture method.
(2) The method according to (1), wherein the base or base derivative is one or more selected from purine nucleosides, purine nucleoside derivatives, hypoxanthine, pyrimidine nucleosides, and pyrimidine nucleoside derivatives.
(3) the base or base derivative is one or more selected from adenosine, deoxyadenosine, cytidine, deoxycytidine, guanosine, deoxyguanosine, thymidine, deoxythymidine, uridine, deoxyuridine, and hypoxanthine; (1) The method as described.
(4) The method according to any one of (1) to (3), wherein the base or the base derivative is added so as to be about 1 mg / L to about 100 mg / L in the medium.
(5) The method according to any one of (1) to (4), which is performed by fed-batch culture.
(6) An animal cell culture system for carrying out the method according to (5), wherein one or more bases or bases selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives A system comprising a vessel containing a feed solution containing a derivative, a culture vessel, and a pump.
(7) A feed solution added to a liquid medium of animal cells containing one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives.
(8) A method for producing an animal cell culture medium, comprising adding one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives.
(9) The method according to (8), wherein the base or base derivative is one or more selected from purine nucleosides, purine nucleoside derivatives, pyrimidine nucleosides, and pyrimidine nucleoside derivatives.
(10) The base or base derivative is one or more selected from adenosine, deoxyadenosine, cytidine, deoxycytidine, guanosine, deoxyguanosine, thymidine, deoxythymidine, uridine, deoxyuridine, and hypoxanthine. (8) The method described.
(11) About 1 mg / L of base or base derivative in the medium
The method according to any one of (8) to (10), which is added so as to be about 100 mg / L.
(12) Animal cells are cultured by adding one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives to the medium before and / or during the culture. A method for producing a useful substance.

  According to the present invention, animal cells can be stably grown at a high density for a long period of time, and useful substances can be produced in large quantities at low cost. Therefore, useful substances such as interferon, interleukin, chemokine, erythropoietin, hematopoietic factor, growth factor, hormone, and antibody can be produced in large quantities and at low cost.

FIG. 1 is a graph showing changes over time in the concentrations of nucleosides, amino acids, organic acids and sugars in a medium. Numbers 3-8 on the right of each figure are cultured in a medium containing 5% fetal bovine serum not virus-cleared, 9-14 are cultured in a medium containing 5% fetal bovine serum cleared by γ-rays, 15- 20 is cultured in a medium containing 15% fetal bovine serum that has been virus-cleared by gamma rays, 21-26 is cultured in a medium that contains 5% fetal bovine serum that has been virus-cleared by electron beams, and 27-32 is an electron beam. It shows that it culture | cultivated in the culture medium containing the fetal calf serum 15% which carried out virus clearance. In each figure, the horizontal axis represents the number of culture days, and the vertical axis represents the relative amount.

  In one aspect, the present invention adds one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives to a medium before and / or during culture. A method for culturing animal cells is provided. Although it has been conventionally performed to supplement a medium with a carbon source or amino acids during culture of animal cells, the present inventors have added purine base, purine base derivative, pyrimidine base, and / or pyrimidine base derivative to the medium. It has been found for the first time that animal cells can be activated and proliferated for a long time by supplementation.

  Any animal cell can be cultured in the present invention. Preferred animal cells include, but are not limited to, cells derived from mammals such as humans, monkeys, dogs, cats, horses, cows, rabbits, guinea pigs, rats, mice, hamsters and the like. More preferred animal cells include human-derived cells. Insect cells are also encompassed by animal cells. Examples of animal cells often used for production of useful substances include, but are not limited to, CHO, COS, CV-1, Sp20, NS0, Vero and the like. Animal cells may be wild-type or genetically engineered, mutated or cell-fused. The animal cell may be, for example, a human cell into which a gene encoding a desired polypeptide has been introduced, or a hybridoma that produces a desired monoclonal antibody. The cell may be a primary cell or a passage cell. Furthermore, in the present invention, stem cells such as iPS cells and ES cells, and progenitor cells such as ADPMC (adipose tissue-derived multilineage progenitor cells) may be cultured.

  Any purine base or purine base derivative can be used in the present invention. A base is a substance constituting a nucleic acid, and includes purine bases, pyrimidine bases, and the like. Purine base is a basic substance having a purine skeleton and is a constituent element of nucleic acid. Examples of purine bases include, but are not limited to, purine, adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine and the like. In this specification, a derivative is a compound obtained by introducing a functional group into an original compound, oxidation, reduction, atom substitution, etc., and maintaining the basic structure and properties of the original compound. Say. Examples of purine base derivatives include, but are not limited to, purine nucleosides, purine nucleotides, and derivatives thereof. Examples of preferred purine bases or purine base derivatives in the present invention include, but are not limited to, purine nucleosides, purine nucleoside derivatives, hypoxanthine and the like. Examples of preferred purine nucleosides or purine nucleoside derivatives in the present invention include, but are not limited to, adenosine, deoxyadenosine, guanosine, deoxyguanosine, and derivatives thereof.

  Any pyrimidine base or pyrimidine base derivative can be used in the present invention. A pyrimidine base is a basic substance having a pyrimidine skeleton and is a constituent element of a nucleic acid. Examples of pyrimidine bases include, but are not limited to, thymine, cytosine, uracil, 5-methyylcytosine, 5- (hydroxymethyl) cytosine and the like. Examples of pyrimidine base derivatives include, but are not limited to, pyrimidine nucleosides, pyrimidine nucleotides, and derivatives thereof. Examples of preferred pyrimidine bases or pyrimidine base derivatives in the present invention include, but are not limited to, pyrimidine nucleosides and pyrimidine nucleoside derivatives. Examples of preferred pyrimidine nucleosides or pyrimidine nucleoside derivatives in the present invention include, but are not limited to, cytidine, deoxycytidine, thymidine, deoxythymidine, uridine, deoxyuridine, and derivatives thereof.

  In the present invention, one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives are added to the medium before and / or during the cultivation of animal cells. By adding the above substances, animal cells can be stably grown at a high density for a long period of time, and useful substances can be produced in large quantities and at low cost. Therefore, useful substances such as interferon, interleukin, chemokine, erythropoietin, hematopoietic factor, growth factor, hormone, and antibody can be produced in large quantities and at low cost. In addition to the above substances, substances that improve cell growth and activity, for example, carbon sources such as glucose, nitrogen sources such as amino acids, vitamins, and trace metals may be added to the medium.

  The base or base derivative may be added to the medium before culturing animal cells, or may be added to the medium during the culture. Further, a base or a base derivative may be added to the medium before and during the cultivation of animal cells. The number of times of addition may be one time or a plurality of times. The base or base derivative may be added in the form of a solid, or may be added after making a solution. Preferably, the base or base derivative is added to the medium as a concentrated solution. The amount of these substances added, the number of additions, and the duration of addition are monitored in a timely or continuous manner, such as the concentration of these substances in the medium, the amount of cells, the viability of the cells, and the amount of the target substance produced. It can be determined in consideration.

  The amount of the base or base derivative added is determined so that the growth of animal cells, the enhancement of activity, or the increase in the yield of the target substance can be observed. Such an addition amount can be easily determined by a person skilled in the art by conducting a preliminary test. The amount of the base or base derivative added depends on the type of cell, the type of medium, other culture conditions, and the like. For example, the concentration in the medium may not be less than about 1000 mg / L. 1 mg / l to about 1000 mg / L, preferably about 1 mg / L to about 100 mg / L, such as about 2 mg / L to about 50 mg / L, about 5 mg / L to about 20 mg / L may be used. In this specification, the concentration of the base or base derivative in the medium is the concentration of each base or each base derivative to be added.

  The medium used in the present invention may be any medium as long as it is for animal cell culture. Examples of animal cell culture media include D-MEM, E-MEM, G-MEM, MEMα, RPMI-1640, Ham's F-12, IMEM, Leibovitz's L-15 media, and modifications of these media Examples thereof include, but are not limited to, a medium and a mixed medium of these mediums. Those skilled in the art can appropriately select and use a medium according to the cells to be used, desired useful substances, and the like. Those skilled in the art can also easily determine the culture conditions such as culture temperature, pH, culture time, concentration of each component in the medium, and stirring conditions. The medium may or may not be changed during the culture.

  The culture vessel is not particularly limited, and can be appropriately selected according to factors such as a desired amount of useful substance, useful substance type, cell type and the like. Various types and sizes of culture vessels are known, including but not limited to dishes, plates, tubes, flasks, bottles, tanks, bags, and the like.

  You may implement the culture | cultivation method of the animal cell of this invention by falling-down culture. Flow culture is a liquid culture method well known to those skilled in the art, and is a culture method in which a specific substrate is supplied to a culture container during culture, but the medium (including cells and medium) is not drawn. In the present invention, when animal cells are subjected to liquid culture, the base or base derivative described above is supplied to the culture vessel. Preferably, a feed solution containing a base or a base derivative is supplied to the medium. Usually, the feed liquid contains a high concentration of base or base derivative, and it is preferable to suppress changes in the medium volume when added. In the flow-down culture of the present invention, the concentration of the base or base derivative in the medium may be maintained so as not to fall below about 1000 mg / L, for example, from about 0.1 mg / l to about 1000 mg / L. Well, preferably it may be maintained at about 1 mg / L to about 100 mg / L, such as about 2 mg / L to about 50 mg / L, about 5 mg / L to about 20 mg / L, etc.

  In the flow-down culture embodiment of the present invention, the feed solution is connected to the culture vessel via a pump. The concentration of the base or base derivative in the medium and / or the number of cells, the desired useful substance concentration, etc. may be measured at appropriate times, and the pump may be operated as necessary to supply the base or base derivative to the medium. In addition, one or more sensors (connected to a detector and / or a computer) are provided in the culture vessel to determine the concentration of the base or base derivative in the culture medium and / or the number of cells, the concentration of desired useful substances, etc. The base or base derivative may be supplied to the medium by measuring and operating the pump via a sensor and / or computer. Accordingly, the present invention, in a further aspect, comprises a container comprising a feed liquid comprising one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives, culture vessels, and An animal cell culture system including a pump is provided.

  In a further aspect, the present invention provides, in a further aspect, a feed solution added to a liquid medium of animal cells, comprising one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives. provide.

  In yet another aspect, the present invention provides an animal cell characterized in that one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives are added. A method for producing a culture medium is provided. Usually, a medium is produced by mixing a base or a base derivative and a medium component by a known means / method. The base or base derivative is as described above. The medium obtained by the method of the present invention may be a powder or a liquid. When the medium is a powder, it is diluted with a medium such as water. The amount of the base or base derivative added during the production of the medium is such that the growth of animal cells, enhanced activity, or increased yield of the target substance can be observed in the prepared liquid medium. Such an addition amount can be easily determined by a person skilled in the art by conducting a preliminary test. The amount of the base or base derivative added depends on the type of cell, the type of medium, other culture conditions, and the like. For example, the concentration in the medium may not be less than about 1000 mg / L. 1 mg / l to about 1000 mg / L, preferably about 1 mg / L to about 100 mg / L, such as about 2 mg / L to about 50 mg / L, about 5 mg / L to about 20 mg / L may be used. In the present specification, the concentration of the base or base derivative in the medium is the sum of the concentrations of the base or base derivative to be added.

  In yet another aspect, the present invention provides, in another embodiment, one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives in a medium before and / or during culture. A method for producing a useful substance is provided, which comprises culturing animal cells by addition. The useful substance refers to a substance useful for humans, and may be a high molecular compound such as a polypeptide or a medium to low molecular compound. Examples of useful substances include, but are not limited to, biopharmaceutical components. Specific examples of useful substances include, but are not limited to, interferon, interleukin, chemokine, erythropoietin, hematopoietic factor, growth factor, hormone, and antibody.

  As mentioned above, although this invention was demonstrated, an Example is shown below and this invention is demonstrated in more detail and concretely, An Example does not limit this invention. The terms in the present specification are understood to have the meanings usually understood in the field of cell culture and pharmaceuticals unless otherwise specified.

Human adipose tissue-derived multilineage progenitor cells are cultured for 5 days in a medium prepared by adding fetal calf serum to a medium having the composition shown in Table 1 (ITS composition is shown in Table 2), and the nucleosides and amino acids in the medium are cultured. The time course of the concentration of organic acids and sugars was examined. The results are shown in FIG. Although all the substances examined decreased with the culture time, it was found that uridine, cytidine, deoxycytidine, and hypoxanthine almost disappeared or disappeared completely on the 3rd to 5th day of culture.

Since the substances almost disappeared or completely disappeared in the above test were nucleosides, a solution (solution A) (1000-fold concentration) containing the nucleosides shown in Table 3 was prepared and added to the medium. Culturing was performed at the final concentration shown in Fig. 1, and the viability and the number of cells in the medium were examined.

The 5th passage human adipose tissue-derived multilineage progenitor cells (ADMPC) were seeded in a fibronectin-coated 10 cm culture dish at 2 × 10 ⁵ cells / dish, the medium was changed on the 3rd and 5th days of seeding, and on the 7th day. Cells were collected and counted. The test was conducted with two samples of the solution A added system and the non-added system. The basal medium was SteMedia containing 15% FBS and ITS. The results are shown in Table 4. The solution A addition system (sample 2) has a higher cell growth rate and speed than the solution A non-addition system (sample 1). By adding the solution A to the medium and using it for culture, efficient cell growth Was found to be obtained.

The 5th passage of ADMPC was seeded in a fibronectin-coated 10 cm culture dish at 2 × 10 ⁵ cells / dish. The medium was changed on the third and fifth days after seeding, and the cells were collected and counted on the seventh day. The basal medium was SteMedia containing 15% FBS and ITS. The results are shown in Table 5. In the case where the medium was not added for Solution A (Samples 1 and 2), when the medium was not changed for 7 days (Sample 2), compared with the case where the medium was changed on Days 3 and 5 (Sample 1) As a result, the cell growth rate and speed were decreased. As described above, cell growth is deteriorated unless the medium is exchanged. When the medium was exchanged using the medium in which the solution A was added to the basal medium in advance (Sample 3), the cell growth / speed improvement comparable to the medium without the Solution A (Sample 2) was shown. It was. It was found that by using a medium in which the solution A was added to the basal medium in advance, it was possible to extend the interval between medium exchanges and reduce the number of medium exchanges.

The fifth passage of ADMPC was seeded at 2 × 10 ⁵ fibronectin-coated 10 cm culture dishes / dish. The medium was changed on the third and fifth days after seeding, and the cells were collected and counted on the seventh day. The basal medium was SteMedia containing 15% FBS and ITS. The results are shown in Table 6. Compared to the case of culturing in the medium without addition of Solution A and changing the medium within 3 days (3rd day and 5th day of culture) (Sample 1), the solution was found on the 3rd and 5th day of culture. Even when A was additionally added (sample 2), the cell growth rate and speed were improved. It was found that the addition of solution A during the culture can extend the interval between medium exchanges and reduce the number of medium exchanges.

  The present invention can be used in the production of useful substances from animal cells. The present invention can be used in the field of biopharmaceuticals.

Claims (12)

  An animal cell characterized in that one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives are added to a medium before and / or during culture. Culture method.   The method of claim 1, wherein the base or base derivative is one or more selected from purine nucleosides, purine nucleoside derivatives, pyrimidine nucleosides, and pyrimidine nucleoside derivatives.   2. The base or base derivative is one or more selected from adenosine, deoxyadenosine, cytidine, deoxycytidine, guanosine, deoxyguanosine, thymidine, deoxythymidine, uridine, deoxyuridine, and hypoxanthine. The method described.   The method according to any one of claims 1 to 3, wherein the base or the base derivative is added so as to be 1 mg / L to 100 mg / L in the medium.   The method according to any one of claims 1 to 4, which is performed by fed-batch culture.   6. A system for animal cell culture for performing the method of claim 5, comprising one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives. A system comprising a container containing a feed solution, a culture container, and a pump.   A feed solution added to a liquid medium of animal cells containing one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives.   A method for producing an animal cell culture medium, comprising adding one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives.   9. The method of claim 8, wherein the base or base derivative is one or more selected from purine nucleosides, purine nucleoside derivatives, pyrimidine nucleosides, and pyrimidine nucleoside derivatives.   9. The base or base derivative is one or more selected from adenosine, deoxyadenosine, cytidine, deoxycytidine, guanosine, deoxyguanosine, thymidine, deoxythymidine, uridine, deoxyuridine, and hypoxanthine. The method described.   The method of any one of Claims 8-10 which adds a base or a base derivative so that it may become 1 mg / L-100 mg / L in a culture medium.   Animal cells are cultured by adding one or more bases or base derivatives selected from purine bases, purine base derivatives, pyrimidine bases, and pyrimidine base derivatives to a medium before and / or during culture. And a method for producing a useful substance.