JPH04316483A - Serumless synthetic medium for suspended cell - Google Patents

Abstract

PURPOSE:To obtain the title medium for incubating suspended cells such as hybridoma for e.g. producing useful biologically active substances by adding specific ingredients to a low-serum medium prepared by incorporating an alpha MEM medium with choline tartrate and inositol. CONSTITUTION:(A) A low-serum medium prepared by incorporating an alpha MEM medium with at least choline tartrate and inositol is formulated with (B) at least one kind of saccharide selected from glucose, fructose, galactose, sucrose and lactose, (C) citric acid, (D) carnitine, (E) polyvinyl alcohol, (F) sodium selenite, (G) ethanolamine, (H) insulin, and (I) transferrin, followed by addition of distilled water; carbon dioxide is then introduced into the resulting product to adjust its pH value to 7.2-7.4 followed by sterilization through filtration, thus obtaining the objective medium for incubating suspended cells such as hybridoma or human lymphatic cells.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a serum-free synthetic medium used for culturing floating cells such as lymphocytes and hybridomas useful for producing biologically active substances.

0002

BACKGROUND OF THE INVENTION Advances in genetic engineering, cell engineering, and cell culture technology have made it possible to industrially produce physiologically active proteinaceous polymers, which was extremely difficult to achieve through conventional chemical synthesis. For example, production of monoclonal antibodies by mouse hybridomas, genetically modified CHO cells, genetically modified Nama
Production of interferon and erythropoietin by lwa cells has been developed and industrialized.

[0003] Generally, in tissue culture, it is necessary to culture cells using a medium containing amino acids, vitamins, inorganic salts, etc., to which serum, tissue extracts, etc. are added. However, these biologically derived additives contain unknown components, have large lot differences, are very expensive, and are difficult to manage in industrial production. Furthermore, when purifying useful substances from cultured cells, the presence of proteins contained in serum makes purification difficult. For this reason, serum-free media that do not contain biological components such as serum have been developed, and serum-free media for suspension cells such as hybridomas are particularly useful, and various media have been developed and commercialized. There is.

[0004] In these serum-free media, protein components such as insulin, transferrin, fatty acid-bound BSA, lipoproteins, proteinase inhibitors, catalase, and other cell growth factors are added instead of serum; Protein concentrations are often very high, for example as much as 1000 μg/ml. Although this high protein concentration is considerably lower than that of serum-supplemented media, it still poses a problem for purification. In addition, serum-free media with low protein concentrations are applicable to only a few types of cells, require long-term acclimatization, and are more likely to cause cells to lose their ability to produce substances during culture. It has drawbacks. Moreover, although these serum-free media can support growth of cells from high densities (approximately 1 x 10 5 /ml or higher), they cannot support growth from low cell densities (approximately 1 x 10 4 /ml or less). Conventional serum-free media have the above-mentioned problems, so when actually used, they must be acclimatized to the cells to be applied over a long period of time or have a high concentration of 1 x 105/ml or more. Care must be taken, such as culturing based on density,
Even if such operations are performed, it is often difficult to culture some cell lines.

[0005]

[Problems to be Solved by the Invention] Conventional serum-free media for suspended cells have high protein content, which makes it difficult to purify useful substances from cells, and low protein content, which limits the types of cells that can be used. , required a long period of acclimatization when applied. Also, the low density of cells (approximately 1 x 104/ml
It has the disadvantage that it cannot support proliferation from cells (below), and when it is applied, it must be acclimatized to the cells to be applied over a long period of time or cultured at a high density of 1 x 105 /ml or higher. It is necessary to take precautions such as not to Furthermore, depending on the cell line, it is often difficult to culture even if such operations are performed.

[0006]

[Means for Solving the Problems] The present inventor has conducted intensive research to overcome the problems of these serum-free media and to prepare a serum-free medium that can be used relatively easily. The present invention was completed by successfully obtaining a serum-free synthetic medium that met the above objectives by incorporating specific components into a low-serum medium, which was developed as a medium for the purpose of the present invention and will be described later.

That is, the present invention combines sugars, citric acid, carnitine, polyvinyl alcohol, sodium selenite, ethanolamine, insulin, and transferrin into a low serum medium prepared by adding at least choline tartrate and inositol to Alpha MEM medium. The present invention provides a serum-free synthetic medium for suspended cells characterized by the following.

[0008] The low serum medium used as the basal medium in the present invention has been developed by the applicant using genetically recombinant CH
Alpha ME was developed as a medium that has good proliferation and substance production with a low serum level of 1% for O cells.
This is M medium to which at least choline tartrate and inositol are added. As such a medium, UC202 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) having the composition shown in Table 1 is used.
, 5, 504-507, 1990].

[0009]

[Table 1]

Examples of sugars added to the serum-free synthetic medium of the present invention include monosaccharides such as glucose, galactose, mannose, and fructose, and disaccharides such as sucrose and lactose. The amount of sugar added is 0.
1 to 5 g/l, preferably 0.1 to 5 g/l. Each of these sugars may be added individually, but
They may be added in combination within the above concentration range.

[0011] Polyvinyl alcohol is 0.01 to 5 g/
1, preferably 0.05 to 1 g/l, and citric acid is 0.1 g/l.
1-2mM, preferably 0.25-1mM, carnitine 10-200μM, preferably 25-100μM
It is blended in an amount of .

[0012] In addition to these, it is necessary to add active ingredients required in conventional serum-free synthetic media, and sodium selenite, ethanol, amines, insulin, and transferrin are added in usual effective amounts. Furthermore, it is preferable to add less toxic dihydroxyethylglycine as a buffering agent, and the amount thereof is 2.5%.
~20mM, especially 5-15mM is preferred.

[0013]

[Examples] Next, the present invention will be explained in more detail with reference to Examples and Test Examples.

Example 1 With the composition shown in Table 2, other components were added to the UC202 medium, the total volume was made up to 1000 ml with distilled water, and the pH was adjusted to 7.2 to 7.4 by passing carbon dioxide gas therethrough. A serum-free synthetic medium was prepared by filter sterilization.

[0015]

[Table 2]

Test Example 1 Using the medium prepared in Example 1, cell proliferation and antibody production of mouse hybridoma (clone name: IV3-5) were investigated.

For cell proliferation, 1 ml of medium was added per well to a 24-well microculture plate, and 200 μl of a cell suspension of about 5×10 4 cells/ml was added thereto to give a concentration of about 1×10 4 cells/ml. CO2
The cells were cultured in an incubator, and the number of cells was counted using a hemocytometer at a predetermined time. The culture supernatant was used to measure the amount of antibody produced. The cells used in the test had been cultured in RPMI1640 medium supplemented with 10% fetal bovine serum until just before the test.

[0018] Antibody production ability was measured by enzyme immunoassay using a 96-well microplate on which the antigen was immobilized. That is, human IgG was immobilized in advance and 0.5% BSA was added.
The culture supernatant was diluted 10 times with PBS containing 0.5% BSA to a 96-well microplate that had been blocked with PBS, and 100 μl was added to each well, followed by reaction at room temperature for 1 hour. After the reaction and washing three times with PBS, add 100 μl of alkaline phosphatase-labeled anti-mouse IgG per well.
1 of the mixture was added, and the mixture was further reacted at room temperature for 1 hour. After washing in the same manner, the plate was colored using the Kind-King method, the absorbance at 490 nm was measured using a microplate reader, and the value was taken as the antibody production ability.

RPMI supplemented with 10% fetal bovine serum as a control
1640 medium was used for comparison with commercially available serum-free medium SFM1.
01 medium (Nissui Pharmaceutical; Yabe, N. et al.
, IN VITRO Cellular & Dev
Elopmental Biology, 22, 3
63-368, 1986) was used.

The results are shown in FIG. 1, and the medium of the present invention was superior to commercially available serum-free media in both cell proliferation and antibody production.

Test Example 2 Using the medium prepared in Example 1, the proliferation of human lymphoid cells Namalwa and Molt-4 was investigated. The results are shown in FIG. 2, and the serum-free medium of the present invention showed good growth in both cells, whereas the commercially available serum-free medium showed poor growth in Namalwa.

[0022]

Effects of the Invention The serum-free medium for floating cells of the present invention uses low serum medium UC202 as a basic medium, and by adding sugars, citric acid, carnitine, polyvinyl alcohol, a buffer, etc. It is possible to grow suspension cells such as cell lines at low densities, which is difficult with conventional serum-free media, and it is also capable of supporting the growth of a wider range of cells than conventional serum-free media. Since it is a medium, it can be used, for example, for culturing purposes such as producing useful biologically active substances.

[0023]

[Brief explanation of drawings]

FIG. 1 is a graph showing cell proliferation and antibody production when mouse hybridomas were cultured using the medium of the present invention.

FIG. 2 is a graph showing cell proliferation when Namalwa (a) and Molt-4 (b) were cultured using the medium of the present invention.

Claims (2)

[Claims] Claim 1: A low serum medium prepared by adding at least choline tartrate and inositol to Alpha MEM medium, which contains saccharides, citric acid, carnitine, polyvinyl alcohol, sodium selenite, ethanolamine, insulin, and transferrin. Serum-free synthetic medium for suspension cells. [Claim 2] The saccharide is one or two of glucose, fructose, galactose, saccharose, or lactose.
2. The serum-free synthetic medium for floating cells according to claim 1, wherein the serum-free synthetic medium is of at least one species.