WO1998000521A1

WO1998000521A1 - Serum-free media, method for culturing animal cells, and process for producing physiologically active substances

Info

Publication number: WO1998000521A1
Application number: PCT/JP1997/002167
Authority: WO
Inventors: Yasuo Amatsuji; Hiroshi Tamashima; Yahiro Uemura
Original assignee: The Green Cross Corporation
Priority date: 1996-06-28
Filing date: 1997-06-23
Publication date: 1998-01-08

Abstract

Serum-free media characterized by containing insulin and peptone but being substantially free from albumin and transferrin; a method for culturing animal cells characterized by culturing the cells in the above-mentioned media; and a process for producing physiologically active substances characterized by culturing animal cells capable of producing the substances in the above-mentioned media and recovering the substances thus produced from the cultures. These serum-free media allow sufficient proliferation of cells, facilitate the recovery of the physiologically active substances from the cultures, and are scarcely contaminated, thus enabling the provision of the above-mentioned culture method and production process.

Description

Specification

Serum-free medium, animal cell culture method and method for producing bioactive substance

Technical field

The present invention relates to a serum-free medium, a method for culturing animal cells using the same, and a method for producing a physiologically active substance.

Background art

In recent years, with the progress of DNA recombination technology, methods for producing bioactive substances such as proteins as cell products have been studied. Culture of animal cells becomes more important for the production of this cell product, but a large amount of medium is required to perform such cell culture on an industrial scale.

Conventionally, as a medium for growing (proliferating) animal cells, a very wide variety of media, for example, Dulbecco's modified Eagle's medium (DMEM) CMorton. HJJ (1970) In vitro 6, 89], F12 medium [Hara Natl. Acad. Sci. USA 53.288] and RPMI 1640 medium CGoding, JW (1980) J. Immunol. Methods 39.285; JAMA 199 (1957) 519]. Etc. have been used. However, in order to grow (proliferate) animal cells using these media, serum must be added to the media. For this reason, generally, serum of a fetus, a fetus, a human, or the like must be used at a concentration of about 1 to 15%.

However, there were the following problems when using a serum-containing medium.

① Serum itself is expensive, resulting in high costs.

② Serum has lot differences, which is disadvantageous for cultures that require reproducibility.

③ It is difficult to purify cell products from the culture supernatant.

がある May be a source of contamination such as viruses, mycoplasmas, or prions.

In view of such circumstances, methods for reducing the serum concentration in the medium have been studied. However, the decrease in serum concentration causes the cells to have a significant decrease in proliferation or death, and the yield of a desired cell product (for example, a physiologically active substance such as a protein) to be significantly reduced. It was difficult to reduce serum concentration. For these reasons, there has been a great deal of interest in serum-free media that do not contain serum and that allow cells to be cultured without loss of growth.

However, the conventional serum-free medium requires further addition of a number of protein components such as albumin, transfusin, and insulin as components instead of serum in order to maintain cell proliferation. However, even with the use of such a method, a complicated process is required to separate and purify only a physiologically active substance such as a target protein from a culture, and the problem still remains. In addition, these protein components are often used after being separated and purified from animals such as pigs and humans. Therefore, there is concern about contamination (eg, virus or prion) due to the origin.

Disclosure of the invention

The present invention has been made in view of the above-described problems, and provides serum-free, low-contamination, low-contamination, and easy-to-purify physiologically active substances from cultures, as well as sufficient cell proliferation. A primary purpose is to provide a medium.

A second object of the present invention is to provide a method for efficiently culturing animal cells. A third object of the present invention is to provide a method for producing a physiologically active substance in a large amount from animal cells capable of expressing and producing the physiologically active substance.

The present inventors have conducted intensive studies to achieve the above object, and as a result, by using a serum-free medium containing insulin and peptone and containing substantially no albumin and transpurin, the plant When a sex peptone is used, it is preferable to include an iron salt to obtain sufficient cell proliferation, and to reduce the addition of proteins such as albumin and the like, particularly proteins derived from animals, which have been conventionally used. That facilitates the separation and purification of biologically active substances from cultures, and reduces the possibility of contamination. They found that they could be produced well and completed the present invention.

That is, the present invention is as follows. (1) A serum-free medium containing insulin and peptone, and substantially free of albumin and transfusin.

(2) A method for culturing animal cells by culturing the animal cells in the above serum-free medium.

(3) A method for producing a physiologically active substance by culturing animal cells capable of producing the physiologically active substance in the serum-free medium to produce the physiologically active substance, and collecting the physiologically active substance from the culture.

Embodiment of the Invention

The serum-free medium of the present invention is substantially free of serum and contains insulin and peptone. The medium of the present invention can maintain sufficient growth of cells (especially animal cells) without substantially containing albumin and transfulin, and can be used in cultures because it does not contain albumin and transfulin. This makes it extremely easy to separate and purify biologically active substances such as proteins from proteins.

The serum-free medium of the present invention is prepared by adding insulin and peptone to a conventional basal medium. Examples of the basal medium include commonly used basal media. In other words, those containing a carbon source that can be assimilated by animal cells, a digestible nitrogen source, and inorganic salts are used, and trace active substances such as micronutrient promoting substances and precursors are added as necessary. May be. As the basal medium, any known medium for culturing cells can be used. For example, the above-mentioned DMEM medium, F12 medium and RPM116 medium are exemplified, and RPM164 medium is particularly preferable.

The insulin used in the present invention is not particularly limited in its origin, and preferably used is insulin derived from insulin or recombinant human insulin. The content of insulin is about 0.1 to 10 mg / L, more preferably about 0.1 to 2 mg ZL.

Peptone is the hydrolyzed form of various proteins and contains polypeptides, peptides and amino acids. For the hydrolysis, enzymes such as trypsin, pepsin, and papain or acids are used. The peptone used in the present invention is The origin and production method are not particularly limited, ie, conditions that are soluble in water, do not heat coagulate, do not precipitate in a saturated solution of ammonium sulfate, and that the pellet reaction is positive are satisfied. Preferably, peptone obtained by hydrolyzing a vegetable protein is used to reduce the possibility of contamination. Specifically, peptone derived from wheat or soybean is exemplified. Animal peptone can also be used. Specific examples include beef-derived peptone. The content of peptone varies depending on the quality and composition based on differences in the reaction conditions, such as the origin, the type of enzyme used in the production, and the reaction time, etc. is there. More preferably, it is about 1 to 10 g / L.

In the present invention, the serum-free medium containing insulin and peptone described above may further contain an iron salt. Particularly when a plant-derived peptone is used as the peptone, further addition of an iron salt is preferred. Examples of the iron salt used in the present invention include an inorganic salt, an organic acid salt, and a complex (complex salt) of iron. Examples of the inorganic salt include sulfate, chloride, nitrate and the like. Examples of the organic acid salt include an acetate, a citrate and the like. The iron complex is not particularly limited as long as it forms a complex with iron and can transport iron into cells, but it is easily soluble in water and stable at the pH of the medium (near neutral). That is, a synthetic non-proteinous substance which does not cause precipitation as iron hydroxide is selected. Specific examples include sodium ethylenediaminetetraacetate-iron complex (hereinafter abbreviated as iron EDTA), iron nitrosotriacetate complex, iron complex ethylenediaminedi (o-hydroxyphenylacetate), and the like. The amount of iron salt added is about 0.01 to 100 mg ZL. More preferably about 0.1 to 50 mg ZL o

The serum-free medium of the present invention may further contain, if necessary, 0.1 to 100 mg / L of hypoxanthine, preferably 10 to 1 S mgZL thymidine 0.01 to 10 OmgZl ^, especially 2 to 5 mg / L, selenium 0 1 to 100 / z gZL, especially 2 to 5〃 gZL, a-tocoprole (vitamin E) 0.01 to 10 mgZL, especially 0.1 to 0. 5 mg ZL can be added. When a transformed animal cell containing a vector having a resistance gene is to be cultured, the medium further contains the resistance vector contained in the vector to maintain the stability of the plasmid transformation. A selection agent corresponding to the gene may be added. Examples of the selection agent include those known to those skilled in the art, for example, neomycin, hygromycin, mycophenolic acid, hypoxanthine, xanthine, aminoptilin or methotrexate (MTX) and derivatives thereof.

Animal cells that can be cultured in the serum-free medium of the present invention may be those that are capable of producing a physiologically active substance themselves, or those that are transformed by genetic engineering to produce heterologous biologically active substances. Cells that produce bioactive substances themselves, such as hybridomas that produce monoclonal antibodies, interferon (IFN) — leukocytes that produce spleen, Namaluba cells, BALL-1 cells, and IFN—S Developing fibroblasts, lymphocytes producing IFN F, human kidney cells producing properokinase or perokinase (pro-UK or UK), melanoma cells producing tissue plasminogen activator (TPA) (For example, Bowes cells). The established host cells transformed by genetic engineering include Vero cells, He1a cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, HKG cells, human kidney cell lines and the like can be mentioned. Specifically, CHO—K, (Chinese hamster ovary cell ATCC CCL 61), BHK (hamster kidney cell: ATC C CCL 10), COS-7 (CV-1 Origin, SV—40 cell: ATCC CRL) 165 1), Vero cells (African green monkey kidney cells: ATCCCCL81), etc., and further, mouse myeloma cells (X63-Ag8-653; P3U1), human lymphoblast cells (IM-9: ATCC CCL 159), parent cells for producing human human hybridomas, and dhfr-deficient strains, HGPRT-deficient strains, and バ abain-resistant strains.

The method of culturing animal cells using the serum-free medium of the present invention is generally performed as follows. That is, for culturing animal cells using the serum-free medium of the present invention, A well-known culture vessel such as a dish, a flask, a roller bottle, a spinner flask, a microcarrier, a microcapsule, or a culture apparatus using hollow fibers can be used, but is not limited thereto. The culture method may be the subculture usually performed in the above-mentioned culture vessel or the like, or the old culture solution containing the cells may be continuously or intermittently contained in the culture tank or separated from the cells. A continuous culture method in which cells are cultured while extracting and extracting a corresponding amount of new culture medium and controlling the culture conditions for a long period of time. In addition, culture of animal cells using the serum-free medium of the present invention can be performed in the form of so-called high-density culture. Specifically performed in cell number 1 0 ⁷ Zml more dense. Other culture conditions include a culture temperature of 30 to 37 ° C. and a culture time of 1 to 10 days. If the medium is appropriately replaced, continuous culture for a longer period of time is possible.

Production of a physiologically active substance such as a protein by a cell may be performed by a means known per se. Examples of such means include the methods described in JP-A-61-177879, JP-A-63-147879, and the like.

According to the method for producing a physiologically active substance of the present invention, for example, antithrombin-II, pro-UK, TPA, hepatitis B surface antigen, pre-S-B hepatitis surface antigen, IFN, colony-forming stimulating factor, monoclonal A physiologically active substance such as an antibody can be produced.

Hereinafter, examples will be given in order to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1

Using 10.4 g of RPMI 1640 medium (J. Immunol. Methods 39, 285, (198 0), JAMA 199 (1957)) as the basic medium, 1 mg of insulin, 5 parts of beef-derived peptone 5 g, 13 mg of hypoxanthine, 4 mg of thymidine, 0.1 mg of tocopherol and 4 g of selenium to prepare 1 L of serum-free medium.

Example 2

Human kidney cells adhered to microphone mouth carrier beads were prepared in Example 1. As the number of cells in serum-free medium was added to a 1 0 ⁷ Zm 1. 3 7 ° were cultured under conditions of C 5% C0 _2, the production of 0. 6 2. 5 U / m 1 equivalent Purourokinaze was confirmed in culture medium at 2 days. Thereafter, the culture medium was changed every 23 days, and continuous cultivation for a long period of time (at least 3 months or more) was possible. Example 3

Using 10.4 g of RPMI 1640 medium as the base medium, adding 1 g of insulin lmg beef peptone derived from beef, 2 g of glucose and 2.5 g of sodium hydrogen carbonate to 1 L of serum-free medium Prepared.

Example 4

Using 10.4 g of RPMI 1640 medium as the base medium, 5 mg of insulin, 1 mg of soybean-derived peptone, 50 mg of sodium sodium iron ethylenediaminetetraacetate. 2 g of glucose and bicarbonate Add 2.5 g of sodium to prepare 1 L of serum-free medium 7 ο

Example 5

Using 10.4 g of RPMI 1640 medium as the base medium, lmg insulin-derived soybean-derived peptone 5 g, sodium iron ethylenediaminetetraacetate 1 mg, ferrous sulfate 10 mg, glucose 2 g and 2.5 g of sodium bicarbonate was added to prepare 1 L of a serum-free medium.

Example 6

Using 10.4 g of RPMI 1640 medium as base medium, lmg of insulin lmg soybean-derived peptone 5 g of sodium iron ethylenediaminetetraacetate 1 mg, 10 mg of ferric chloride, and 2 g of glucose And 2.5 g of sodium bicarbonate were added to prepare 1 L of a serum-free medium.

Example 7

Using 10.4 g of RPMI 1640 medium as the basal medium, 1 g of insulin, 5 g of soybean-derived peptone, 10 mg of ferrous sulfate, 2 g of glucose, and 2.5 g of sodium hydrogen carbonate This was added to prepare 1 L of serum-free medium. Experimental example 1

The cell growth of the serum-free medium of the present invention (Examples 3 and 4) was examined.

Properokinase-producing CHO cell line pUH-7 cells were seeded in each medium at a cell density of 5 X 10 'cells / ml. After culturing for 4 days, the cells were treated with trypsin, separated from the vessel wall and suspended, and then the suspension was stained with 0.1% erythrocycin BZPBS. After staining, the number of live cells and the number of dead cells were each measured with a microscope on a hemocytometer, and the degree of cell number proliferation was examined.

As a result, whereas in the medium of Example 3 0. Showed 9 6 X 1 0 ⁶ or Zm 1 cell proliferation, in the medium of Example 4, 1. 1 4 X 1 0 6 or Zm I Met. Furthermore, even when the serum-free medium of the present invention contained EDTA iron at a concentration of 10 mg L, a cell growth effect comparable to that of the medium of Example 3 was observed.

Experimental example 2

The cell proliferation of the serum-free medium of the present invention (Examples 3, 5, 6, and 7) was examined. All were carried out in accordance with Experimental Example 1 except that the cell density was 15 × 10 ⁴ Zm 1 and the number of culture days was 3 days. As a result, the medium of Example 3 showed a cell growth of 1.25 X 10 ⁶ Zm1, whereas the medium of Example 5 showed 1.28 X 10 ⁶ cells / ml. in the medium of example 6, 1. 4 X 1 0 6 or Roh m 1, the medium of example 7 was 1. 4 4 X 1 0 ⁶ or Zm 1. According to the present invention, it is possible to provide a serum-free medium in which cells can be grown sufficiently and proteins produced by the cells can be easily separated and purified from the medium. Furthermore, in the serum-free medium of the present invention, cells do not need to be gradually acclimated, and can be grown as they are. Therefore, cell culture can be performed efficiently, and a large amount of protein can be efficiently obtained.

In addition, the serum-free medium of the present invention, in particular, contains plant-derived peptone and iron salt to maintain effects such as cell proliferation and bioactive substance production equivalent to or higher than those of conventional serum-free medium. In addition, additives that have been concerned so far It is possible to reduce the possibility of contamination of viruses, mycoplasmas, prions, etc., which are derived from marine products. This application is based on Japanese Patent Application No. 168759/1996 filed in Japan, the contents of which are incorporated in full herein.

Claims

The scope of the claims

1. A serum-free medium containing insulin and peptone, and substantially free of albumin and transfusin.

2. The serum-free medium according to claim 1, which is capable of exhibiting a growth ability in animal cells equal to or higher than that of a serum-free medium containing insulin and animal-derived peptone.

3. The serum-free medium according to claim 2, wherein the peptone is a plant-derived peptone.

4. The serum-free medium according to claim 3, further comprising an iron salt.

5. The serum-free medium according to claim 4, wherein the iron salt is an iron inorganic salt, an iron organic acid salt, or an iron complex.

6. A method for culturing animal cells, comprising culturing animal cells in the serum-free medium according to claim 1.

7. An animal cell capable of producing a physiologically active substance is cultured in the serum-free medium according to claim 1 to produce the physiologically active substance, and the physiologically active substance is collected from the culture. A method for producing a physiologically active substance.