JPS6147183A - Serum-free medium for human-human hybridoma cultivation - Google Patents

Abstract

PURPOSE:A serum-free medium for human.human hybridoma cultivation, containing lipoprotein, extremely suitable for human.human hybridoma cultivation, capable of carrying out improved multiplication and a large amount of high-density cultivation, wherein an antibody is easily purified and recovered. CONSTITUTION:A serum-free medium for desired human.human hybridoma cultivation, containing lipoprotein. A human.human hybridoma obtained by fusing a human lymphoma cell with a normal antibody-forming cell is preferably used as the human.human hybridoma applied. Especially a human lymphoma cell, such as a cancerous cell, e.g., human lymphoma cell and a fused cell of a strain deficient in hypoxanthine-guanine-phosphoribosyltransferase and a human B cell is preferable as the human.human hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a serum-free medium for culturing human-human hybridomas. More specifically, the present invention relates to a serum-free medium that promotes the growth of human-human hybridomas and is suitable as a high-density medium.

Recently, attempts have been made to produce useful physiologically active substances such as antibodies, interferon, insulin, and various growth hormones using animal aerocytes, and some of these have reached the stage of practical use. Such animal cells often include, for example, lymphoblastoid cells, leukocytes, fibroblasts [l
cells, hybridomas, etc. are used.

Generally, when culturing animal cells, serum obtained from calves or bovine fetuses is used as a medium, but this serum is not only expensive, but also has uneven quality, instability, and Due to manual labor difficulties, it cannot be said to be suitable as a medium for culturing animal cells in Inuyama and obtaining a large amount of useful physiologically active substances.

On the other hand, so-called serum-free media, which are artificially synthesized without using these serums, are being actively developed. Serum-free media are synthetically synthesized by artificially combining the types and proportions of various inorganic salts, amino acids, vitamins, sugars, antibiotics, and sometimes growth-promoting substances.
It has excellent advantages such as availability, low price, and ease of purification and recovery of cultured products. However, there are many types of animal cells, and therefore it is necessary to develop serum-free media suitable for the growth of each type.

Recently, many monoclonal antibodies that act specifically on specific cells have been discovered for use as diagnostic and therapeutic agents, and the cells that produce them, hybridomas, have also been developed due to rapid advances in biotechnology. I started getting more.

Therefore, the present inventors used a human-human hybridoma that produces a useful antibody and tried using a serum-free medium. However, when human-human hybridomas were cultured using a serum-free medium in which normal human animal tiAl1211 mouse-mouse hybridomas or human-mouse hybridomas grow relatively well, sufficient retention was achieved. It wasn't the result. Therefore, in order to improve the growth of human-human hybridomas, we conducted further research and found that the addition of lipoproteins greatly improved the growth effect, and that this effect was noticeable in high-density culture. This discovery led to the present invention.

The present invention was achieved based on this knowledge, and is a serum-free medium for culturing human-human hybridomas containing lipoproteins.

By using a serum-free medium supplemented with lipoproteins, human
The effect of the addition of lipoproteins is specific in that the growth of human hybridomas is promoted, especially when they are cultured at high density.

The human-human hybridoma applied to the serum-free medium of the present invention may be any hybridoma obtained by fusion of various human-derived cells, but especially human lymphoma
Advantageously, the hybridoma is a human-human hybridoma obtained by fusing Il cells with human antibody-producing normal cells. Furthermore, hybrid-7, which is a fusion of human Burkitt's lymphoma-derived cells and human antibody-producing normal cells, is particularly desirable.

Especially human lymphoid ramocytes, e.g. human lymphoma cells,
A fusion cell of a cancer cell such as a human myeloma cell or a human melanoma that is deficient in HGPRT and a human B11Q cell is used as the human-human hybridoma of the present invention.

Human lymph 11111'IN includes, for example, NA
T-30, NAT-3o-8, L T CR-4ON-
Specific examples include HMy2゜G M4672, which are used as parent strains of hybridomas, and are fused with lymphocytes collected from cancer patients, such as lung cancer and gastric cancer, to produce human cells.・Human hybridomas can be obtained. 'The serum-free medium used in the present invention may be a serum-free medium that is usually developed and used for culturing animal cells and is prepared by adding lipoproteins. Lipoproteins are fractionated from living organisms as a type of complex protein, and are themselves known substances. The amount of lipoprotein, as a protein, is 5 to 10 μg per 1 d of serum-free medium, preferably 10 to 8 μg per 1 d of serum-free medium.
It is advantageous to add 0 μ3.

The basic medium for forming the serum-free medium may be one that is used for culturing animal cells, as mentioned above, but specifically, ``tissue culture'' edited by Junnosuke Nakai et al.
(Published by Asakura 3 Store, 1981), pages 7 to 24 of synthetic media can be used as the basal medium. In addition, ethanolamine (5 to 30%
μM), phospholipid (10-100μ9/rtti)
, insulin (2-10 μg/d), transferrin (10-35 μg/d).

Selenium (10-11~104M), Hiruhin M (
0.5-5mM), thymidine (1X10-7 to 1x10
``5M).

It is more desirable to add one or more types of hyboxanthin (1×10″G to 1×10→M).Specific media include TES, ITES, MITES [(1)
H, Murakaa+i et, at, P'roc
, Natl.

Acad, Sci, LJSA, Vol, γ9.
pp1158-[62°February 198
2. Ce1f 31oloay, (n)H, Mur
akami et, al, Aoric, Biol
,Chem,, 46(71183i-1837(19
82), GiO Murakami, Shimozai, ``Machine Culture'''903
), 515-519 (1983)]. Among these, those to which ethanolamine is added are more preferred.

The lipoprotein-containing serum-free medium of the present invention is extremely suitable for culturing human-human hybridomas, and the development of this medium has made it possible to achieve excellent proliferation and high-density culture of Ofuna. Furthermore, since it is a serum-free medium, it is easy to purify and recover antibodies from hybridomas, and the practical production of monoclonal antibodies as diagnostic and therapeutic agents can be expected.

The present invention will be described in detail below with reference to Examples.

Example 1 - Hybridoma producing monoclonal antibodies against lung cancer obtained by fusion of lymphoma cell NAT-30 with cells collected from a lung cancer patient (
The following culture experiment was conducted using HB4-G7).

In a petri dish, add 2 d of a medium containing 10 m9/Id of lipoprotein as a protein to each of the following media at a concentration of 10 μu/2, and add the above hybridoma (H84-
G7) at a concentration of 5 x 10" cells/d. After standing at 37°C for 3 days, the cell density was examined. The results are shown in Table 1 below, relative to the serum medium as 100. Shown as a value.

Table 1 Example 2 A hybridoma (H9) producing a monoclonal antibody against lung cancer was obtained by fusion with a IIl cell taken from a human lung cancer patient. Culture was carried out in the same manner as in Example 1. The results are shown as relative values when the result when serum is used as a medium is taken as 100.

<Margin below> Table 2

Claims (1)

[Claims] 1. A serum-free medium for human/human hybridoma culture containing lipoproteins. 2. The medium according to claim 1, wherein the human-human hybridoma is a hybridoma obtained by fusion of human lymphoma cells and human antibody-producing normal cells.