JPS6233875B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for culturing lymphoblastoid cell lines at high density. BACKGROUND OF THE INVENTION Research has been conducted on culturing lymphoblastoid cell lines and obtaining useful physiologically active substances from the culture supernatant. However, there are major technical problems in obtaining a large amount of physiologically active substances from cell culture supernatants, purifying them, and using them. One of these is that cells produce extremely small amounts of physiologically active substances. The primary reason for this is thought to be that the saturation density of cells, which allows cells to proliferate and produce physiologically active substances, is low. For example, UMCL cells, which are a cell line that spontaneously produces interferon, are one of the cell lines with good cell proliferation, but their saturation density is only about 2×10 ⁶ /ml. For this reason, the conventional method has been to increase the amount of culture solution, but handling a large amount of culture solution requires the development of culture engineering technology, and requires large capital investment and maintenance costs. There is a problem. Therefore, methods have been considered to increase the amount of physiologically active substances produced by cells by increasing the saturated cell density instead of increasing the amount of culture solution. For example, a method of culturing cells at a high density of 5 to 10 x 10 ⁶ /ml by patching or continuously exchanging the culture medium (Japanese Patent Application Laid-Open No. 169261/1982), A method is known in which the saturated cell density is increased and the production of interferon is increased by adding nutritional components consumed during the culture. (Japanese Patent Application Laid-open No. 58-162035). The second major problem is that it is necessary to add approximately 10% serum such as fetal bovine serum (FBS) to the cell culture medium. These sera are very expensive and have unexplained differences between lots, making it difficult to culture large quantities of cells. Furthermore, since these serums contain many types of foreign proteins, it is difficult to separate and purify useful active substances. To date, methods have been developed to culture cells using culture media containing various growth factors and proteins with clear characteristics as an alternative to serum (D.
Barnes, GHSato, Cell, vol. 122, p. 649, 1980
In addition, as a substitute for serum albumin, a serum-free medium containing cyclodextrin and unsaturated fatty acids (Japanese Patent Application Laid-Open No. 1981-81600) is known. On the other hand, a serum-free medium to which L-glutamic acid is added at a concentration of 20 to 150 mg has been known (Satoko Takaoka, Tissue Culture Vol. 9, p. 196,
1983, Japanese Patent Publication No. 56-81600). However, all serum-free media reported so far support cells at low density (1 to 5 x 10 ⁵ /
This culture method is not suitable for high-density cell culture (more than 1 x 10 ⁷ /ml) because it can only proliferate cells at a cell density of 1 to 2 x 10 ⁶ /ml. Measures to Solve the Problems Based on the above problems, we have conducted intensive studies and found that (1) 1 to 4 g/α-cyclodextrin (α-CD) and (2) 2 to 4 g/ Using a medium containing galactose or 0.3 to 1 g/L-glutamic acid and substantially free of serum,
We have discovered that by culturing a B lymphoblastoid cell line, these cells can be cultured at high density. As the medium used in the present invention, any conventionally known serum-free medium can be used. For example, RITC57-1 medium, RITC57-8 medium, Iscove's medium, Ham's F-12 and Dulbecco's MEM
Examples include a serum-free medium in which various growth factors are added to a mixed medium, but a serum-free medium rich in amino acids, vitamins, etc. is generally preferred. The serum-free culture solution of the present invention contains 1 to 4 g/α-
These components containing CD and further containing 0.3 to 1 g/L-glutamic acid and/or 2 to 4 g/galactose are recognized to be toxic at higher concentrations. L-glutamic acid may be in a free form or may be added as a metal salt such as a sodium salt and a salt such as a hydrochloride. The α-CD of the present invention is a compound in which six glucose residues are arranged in a ring. Its concentration range is 1
~4g/. Below this range, no effect is observed, and above this range, toxicity is observed and at the same time solubility deteriorates. The B lymphoblastoid cell line in the present invention is
Human B lymphoblastoid cell lines mutated by Epstein-Barr virus, such as UMCL cells, C5 cells,
Examples include TAPC301 cells, B-lymphocyte-derived leukemia cells such as BALL-1 cells, and X-5563 cells. The culture method of the present invention may use known culture vessels and devices, but devices that can increase oxygen supply and minimize mechanical cell damage are suitable. For example, a method of culturing in a flask or a sialet by gently shaking the container at a rate of 10 to 20 times per minute, a method of aerating oxygen gas into a culture tank developed for microcarriers,
Alternatively, an airlift culture method may be used. Effects While conventional culture methods could only maintain B lymphoblastoid cell lines at 1 x 10 ⁷ /ml for up to 12 hours, the method of the present invention can maintain up to 3. B lymphoblastoid cell lines can be cultured at a higher density. As a result, for example, spontaneous interferon production by UMCL cells could be significantly increased compared to conventional methods. Moreover, the frequency of medium replacement could be reduced to less than half. Example 1 The culture solution was 0.5% of the RITC57-1 medium shown in Table 1.
A medium supplemented with % bovine serum albumin (BSA) (RITC57-13 + 0.5% BSA) was used as the basal medium, and the components shown in Table 2 were further added, followed by filtration sterilization using a membrane filter with a pore size of 0.22 μm. Table 1 (mg/) Sodium chloride 6240.0 Potassium chloride 390.0 Calcium chloride (anhydrous) 200.0 Magnesium sulfate (anhydrous) 97.7 Sodium dihydrogen phosphate (dihydrate) 125.0 Ferric nitrate (nanahydrate) 0.1 Glucose 2000.0 Sodium pyruvate 110.0 L-arginine hydrochloride 84.0 L-cystine dihydrochloride 62.6 Glycine 30.0 L-histidine hydrochloride (monohydrate) 42.0 L-isoleucine 104.8 L-leucine 104.8 L-lysine hydrochloride 146.2 L-methionine 30.0 L-phenylalanine 66.0 L-serine 42.0 L-threonine 95.2 L-tryptophan 16.0 L-tyrosine disodium (anhydrous) 89.5 L-valine 93.6 Choline bitartrate 7.2 Folic acid 4.0 Nicotinamide 4.0 Calcium pantothenate 4.0 Pyridoxal hydrochloride 4.0 Riboflavin 0.4 Thiamine hydrochloride 4.0 i-inositol 7.2 phenol 5.0 L-alanine hydrate 20.0 L-asparagine 56.0 L-aspartic acid 20.0 L-cysteine hydrochloride hydrate 40.0 L-glutamic acid 20.0 L-proline 20.0 Bithion 0.2 Vitamin B ₁₂ 0.1 Mannose 500 .0 Galactose 500.0 Lecithin 2.5 Hypoxanthine 4.0 Thymidine 0.7 Deoxycytidine 0.03 Deoxyadenosine 1.0 6,8 dihydroxypurine 0.3 Zinc sulfate heptahydrate 0.02 Sodium selenite 0.004 L-Glutamine 584 Putrescine dihydrochloride 0.1 Fornic acid 0.01 Glutathione 10 Sulfuric acid 1st Iron heptahydrate 0.8 Disodium β-glycerophosphate 1500 HEPES 1200 Sodium bicarbonate 1300 Kanamycin sulfate 60 Crystalline insulin 10 Human transferrin 5 Culture UMCL cells in RITC57-1 + 0.5% BSA medium and replace the medium (total amount). The cells were grown twice every 4 days to reach a cell density of 4×10 ⁶ /ml, and then collected by low-speed centrifugation (1000 R.PM, 5 minutes). Cells were immediately incubated 5x in each medium listed in Table 2.
10 ⁷ /ml, and 5 ml of the suspension was suspended at a speed of 15 times/min using a Falcon 3003 Schare with a Belco locking plate installed in an incubator with a 5% carbon dioxide concentration. Culture was performed with rocking for 30 hours. After culturing, each cell density was measured using an eosin Y staining method and a hemocytometer. Also,
After culturing, a portion of the cell suspension was centrifuged to collect the culture supernatant, and the interferon titer was measured. Interferon was measured using WISH cells and Bezukyulastomateisis virus, and the cytopathic inhibition rate in a microtiter plate was measured using standard human interferon as a standard. The results are shown in Table 2.

【table】

[Table] Example 2 As the culture solution, RITC57-1+ shown in Table 1 was used.
A culture solution containing 0.5% BSA medium as the basal medium and the components shown in Table 3 was used. RITC57
BALL-1 cells were cultured in -1 + 0.5% BSA medium and grown to 6 x 10 ^{6 cells} , then collected by low speed centrifugation (1000 R.PM, 5 minutes). Immediately, the cells were suspended in each medium shown in Table 3 at a concentration of 5×10 ⁷ /ml, and 10 ml of the suspension was cultured in a Falcon 3024 flask for 48 hours in the same manner as in Example 1. After culturing, the cell density and the amount of IgM produced in the culture supernatant were measured in the same manner as in Example 1. The results are shown in Table 3. Immunoglobulin class M (IgM) produced by BALL-1 cells was measured by a fluorescence immunoassay using Bio-Rad ImmunoFlow beads.

【table】

Claims (1)

[Claims] 1 (1) 1-4 g/α-cyclodextrin and (2) 2-4 g/galactose or 0.3-4 g/
A method for high-density culture of a B-lymphoblastoid cell line, which comprises culturing the B-lymphoblastoid cell line using a medium containing 1 g/L-glutamic acid and substantially free of serum. .