JPS6338190B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a medium for culturing animal cells. [Prior Art] Research aimed at culturing animal, especially human cells, and obtaining useful physiologically active substances from the culture supernatant or cells has been active, and interferon (IFN) and other useful substances have been intensified. Remarkable progress has been made in the development of monoclonal antibodies, as they have the potential to prevent and treat cancer. However, there are major technical problems in obtaining a large amount of physiologically active substances from semen through cell culture, purifying them, and utilizing them. One of the problems is that the culture medium must be sterilized by filtration using a filter such as a membrane filter. This sterile filtration method usually
A filter with a pore size of 0.2 to 0.5 mm is used, but this filtration method is difficult to remove microorganisms smaller than bacteria, such as mycoplasma and viruses, which are known to cause problems in animal cell culture. . Also,
Filtration sterilization must be performed under pressure or reduced pressure, and depending on the material of the filter, trace amounts of active ingredients in the culture medium may be adsorbed and lost, or the pressure applied to the membrane or the non-uniformity of the material may for,
It is rare for trace amounts of bacteria to leak out, so
Media prepared by filter sterilization must be subjected to various assays. The above problems are especially serious in large-scale cell culture, and the running costs of membranes and equipment also increase. Heat sterilization can solve these problems. As a method for preparing a medium that can be heat sterilized, a method for preparing a medium that has a pH of 4.0 to 4.5 and can be sterilized by autoclaving is known (Proc.
Soc.EXp.Biol.Med., vol. 127, p. 335, 1968). However, in these media, L-glutamine and sodium bicarbonate cannot be sterilized by heat, and L-glutamine still needs to be sterilized by filtration. L-glutamine derivatives such as L-alanyl-L-glutamine are known to be stable against heat (J. Appl. Biochem. vol. 4, p. 280, 1982). It was unclear whether it would be used. [Problems to be Solved by the Invention] Therefore, an object of the present invention is to find an L-glutamine derivative that is stable against heat and can be used as L-glutamine in animal cells, and to thereby achieve heat sterilization. The objective is to find a culture medium for animal cells that can [Means for solving the problem] Under the circumstances described above, the inventors
It has been found that a medium prepared by autoclaving an aqueous solution of a dipeptide represented by -L-glutamine (X is an L-amino acid) is suitable for culturing animal cells. The medium of the present invention is a medium that is slightly acidic and can be sterilized by heat, such as Eagle MEM medium, Dulbecco's MEN medium.
Remove sodium bicarbonate and L-glutamine from culture medium, RPMI1640 medium, mixed culture thereof, etc.
In addition, XL-glumitan was added. These media also include serum-supplemented media or serum-free media containing serum or various growth factors as serum substitutes. X of the dipeptide represented by XL-glutamine of the present invention is glycine, L or DL-alanine, L-aspartic acid, L-glutamic acid, L-
-valine, L-leucine, L-serine, L-lysine, L-phenylalanine, etc., but glycine, L- or DL-alanine is preferred from the viewpoint of raw material cost. The animal cells used in the present invention include:
For example, WI-38, HeLa, L, UMCL, BALL-
1, X-5563, Molt-4, peripheral blood lymphocytes, various hybridomas, etc. XL-glutamine is originally the L-glutamine that should be added.
Dissolve to the same molar concentration as glutamine (1 to 4mM) and sterilize with normal heat (120℃, 20 minutes)
It is better to do this. In addition, only XL-glutamine is made into an aqueous solution with a concentration 100 to 500 times its added concentration,
It can also be prepared by adding 1/100 to 1/500 of the amount to the culture solution after heat sterilization. All of the low molecular weight medium components can be prepared by heat sterilization by adding a 10% aqueous sodium bicarbonate solution to the medium prepared in this manner so as to have a predetermined amount in the medium after heat sterilization. 〔Effect of the invention. Effect] The animal cell culture medium of the present invention is superior in that all of the low molecular weight medium components can be heat sterilized. Another advantage is that the medium can be stored for a long period of time. Moreover, X of XL-glutamine according to the present invention
can select amino acids that match the needs of the cell. Since these dipeptides have increased solubility, necessary amino acids can be supplied in cell culture by adding high concentrations of amino acids with low solubility, such as tyrosine, or amino acids that are largely consumed by cells. In addition, by supplying these amino acids as dipeptides, they are absorbed into cells in accordance with cell proliferation or peptidase activity, so they are utilized more effectively than when they are used as amino acids alone. Example 1 10.2 g of heat-sterilizable RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) shown in Table 1 and glycyl-L-glutamine,
L-glutamine or N-acetyl-L-glutamine was dissolved in distilled water 1 to the concentration shown in Table 2, and heat sterilized at 120° C. for 15 minutes in an autoclave. Placed in a separately sealed culture medium bottle, 20 ml of 10% sodium bicarbonate solution heat-sterilized at 120°C for 15 minutes was added, and then 50 ml of fetal bovine serum (FBS) was added.
UMCL cells and BALL using 100ml of medium each
-1 cells were seeded in a 100 ml spinner flask manufactured by Belco at an initial cell concentration of 5 x 10 ⁵ cells/ml, and the cells were heated at 5% CO ₂ at 37°C and at a rotational speed of 80 rpm.
UMCL cells were cultured for 4 days, and BALL-1 cells were cultured for 5 days. After culturing, each cell density was measured using an eosin Y staining method and a hemocytometer.

【table】

[Table] The results are 10% prepared by normal filtration sterilization.
Table 2 shows a comparison with the results obtained using FBS-supplemented RPMI1640 medium as a control medium.

[Table] Example 2 0.5% bovine serum albumin (BSA) was added to the RITC57-1 medium (without L-glutamine) shown in Table 3.
was added, and the pH was adjusted to 7.4 with 1N NaOH solution, and a medium was prepared by filter sterilization. In this medium, an aqueous solution 100 times the concentration of the L-glutamine derivative shown in Table 4 was prepared, one of which was sterilized by filtration using a 0.22 μm membrane filter manufactured by Millipore, and the other was
After heat sterilization in an autoclave at ℃ for 20 minutes, 1/100 amount of each was added to the above medium.
Put 20 ml of each medium into a 3024 flask made by Falcon, and add 20 ml of each medium to UMCL cells, BALL-1 cells and X5563 cells.
4 x ¹⁰ cells/ml, 3 x ¹⁰ cells/ml and 1 cell, respectively.
x10 to give an initial concentration of ⁵ cells/ml, and 5%
The cells were cultured for 4 days, 5 days, and 4 days under CO ₂ and 37°C, respectively. The viable cell density after culturing was measured in the same manner as in Example 1. The results are shown in Table 4.

【table】

[Table] Umu
HEPES 1200

【table】

【table】

Claims (1)

[Claims] 1 A medium for culturing animal cells containing a dipeptide represented by XL-glutamine (X is an L-amino acid).