JPS6337623B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a serum-free or low serum concentration medium for culturing cells derived from mammalian lymphocytes.

Cells derived from mammalian lymphocytes are known to produce useful substances such as interferon, antibodies, enzymes, and lymphokine, and these useful substances can be obtained by culturing these cells. has been or is being considered.

Examples of culture of lymphoid cells in a serum-free medium include inducing interferon from human leukocytes or human lymphoblastoid cells with a virus, or culturing human or mouse lymphocytes with lectins or lipopolysaccharide. It is known that there are cases in which the cells become blastoids. However, the culture time in all of these methods is short and hardly accompanies cell proliferation. Conventionally, in order to culture and proliferate animal cells, it has been considered essential to add serum to the culture medium. Fetal bovine serum or calf serum is generally used as serum, and the amount added is usually about 10%. However, as a result of using a large amount of serum in the culture medium, serum accounts for most of the cost when culturing cells in large quantities.As a result, for example, when culturing human cells to produce interferon, There was a problem in that the product would be expensive when producing this type of virus. On top of that,
Serum may be contaminated with mycoplasma or viruses, and there may be unexplained differences between lots, so it is complicated to test the serum beforehand. Another problem was that it was not easy to separate and purify products such as interferon from the culture.

The present inventors have developed a medium for mammalian lymphocyte-derived cells that produce the above-mentioned useful substances, with a low serum concentration and even a serum-free medium that allows cell proliferation and subculture. As a result of extensive research, we found that adding mammalian serum albumin to a specific nutrient medium can lower the serum concentration, and that cells derived from mammalian lymphocytes can be cultured, proliferated, and passaged without the addition of serum. The present invention has been completed based on the discovery that it can be cultured and that the above-mentioned problems can be solved thereby.

That is, the present invention relates to a culture medium for mammalian lymphocyte-derived cells characterized by containing mammalian serum albumin separated from other serum components.

Mammalian serum albumin is, for example, human serum albumin, fetal bovine serum albumin, calf serum albumin, equine serum albumin.
These separation methods include ethanol fractionation,
This may be carried out by any known method for separating serum albumin, such as ammonium sulfate fractionation or gel filtration. Commercially available products such as crystalline albumin and Cohn's Fraction V can be used as they are. The amount added is 0.1 to 1% by weight in the case of serum-free medium.
The degree is appropriate. In the case of a medium containing serum, the serum already contains serum albumin.
It goes without saying that it may be better to have less than this.

Other media components include cell growth factors such as insulin and human transferrin, and nucleic acid precursors such as hypoxanthine, thymidine, deoxyadenosine, and deoxycytidine;
Sugar sources such as glucose, amino acids, vitamins,
Contains inorganic salts and other nutrient sources included in normal cell growth media.

Appropriate concentrations of both cell growth factors and nucleic acid precursors are approximately 1 to 5 mg/. Both cell growth factors and nucleic acid precursors are used singly or in an appropriate combination of two or more, depending on the type of cells to be cultured.

In addition to the above-mentioned serum albumin, cell growth factors, and nucleic acid precursors, the culture medium of the present invention contains nutrients contained in ordinary cell growth media, namely,
It is necessary to include sugar sources, amino acids, vitamins, and inorganic salts.

Glucose is usually used as the sugar source, and its concentration is preferably about 0.5 to 10 g/g/. others,
Add pyruvic acid etc. as appropriate. Amino acids are protein constituent amino acids, such as alanine,
Such as arginine, glutamine, styrene, threonine, lysine, valine, and phenylalanine. In ordinary culture media, essential amino acids are mainly added, but in the culture medium of the present invention, it is preferable to add not only essential amino acids but also a wide range of non-essential amino acids. The concentration of total amino acids is preferably about 0.5 to 5 g/. As for the amino acid composition, it is best to refer to a normal cell growth medium and supplement it with new amino acids. Vitamins include ascorbic acid, riboflavin, thiamine hydrochloride, calcium pantothenate, nicotinamide, pyridoxal hydrochloride, i-inositol, folic acid, VB ₁₂ , biotin, and inorganic salts include sodium chloride, potassium chloride, and calcium chloride. , magnesium sulfate, sodium dihydrogen phosphate, ferrous sulfate, zinc sulfate, sodium selenite, and the like. These vitamins and inorganic salts are preferably added to a normal cell growth medium as appropriate. In addition, metabolic intermediates such as choline bitartrate, glutathione, and putrescine dihydrochloride, and buffers such as baking soda, disodium β-glycerophosphate, and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid are added as appropriate. .

Although mammalian serum may not be added to the culture medium of the present invention, it is preferable to add serum when culturing at a high cell concentration, for example. In that case, it is not necessary to add a large amount of serum, and 5% by volume or less of the medium, usually 2% by volume or less, is sufficient.

As mentioned above, the medium composition of the present invention is a normal cell growth medium as a basal medium, serum albumin, cell growth factors, nucleic acid precursors, and if necessary, sugar sources, amino acids, vitamins, inorganic salts, etc. It is best to decide by adding other items as appropriate. As such a basal medium, Dalbeco-modified
MEM medium is preferred, but any other known cell growth medium such as RPMI-1640 medium, Eagle MEM medium, Ham F-12 medium, etc. may be used.

To prepare a medium, for example, all components except serum albumin are dissolved in water to a predetermined concentration, and then serum albumin is added and dissolved while adjusting the pH to 7.2 to 7.2 using 1N NaOH. 7.4
The resulting solution may be sterilized by passing it through a membrane filter under pressure.

As an example of mammalian lymphocyte-derived cells cultured in the medium of the present invention, Epstein-Barr Virus (EBV)
human lymphoblastoid cells transformed with
Examples include UNCL, C5180Y, Namalva cells derived from human Burkitt's lymphoma, and hybridomas derived from mouse lymphocytes such as SPI cells. These cells produce human interferon and human or mouse antibodies in culture.

The method for culturing lymphocyte-derived cells using this medium can generally be carried out in the same manner as when culturing using a medium supplemented with serum. 10×15 ⁵
Cells/ml may be added and cultured at 35 to 37°C while aerating air containing 4 to 5% by volume of _CO2 . After culturing, the proliferated cells may be separated by a conventional method.

It goes without saying that the medium of the present invention can be used not only for cell proliferation but also for culturing cells to produce target substances such as interferon and antibodies.

Using the medium of the present invention, it is possible to obtain cell proliferation almost comparable to that of a conventional medium supplemented with a large amount of serum. In addition, lymphocyte-derived cells can be subcultured over a long period of time in the same way as conventional serum media, regardless of the presence or absence of serum, making it possible to obtain proliferating cells and the target products produced by them at an extremely low cost compared to conventional media. be able to. Furthermore, since serum is not added at all or in a small amount, it is easy to separate and purify the target substance. In particular, when human lymphocytes are cultured in a medium containing human serum albumin, all protein components in the culture medium are of human origin, so the target substance produced in the culture medium does not need to be completely purified. It has the advantage that it can be administered to

UNCL cell production example 1 Approximately 20 ml of blood was aseptically collected from the umbilical cord of a newborn baby with heparin, and the lymphocytes were immediately fractionated by specific gravity centrifugation using Ficoll Isopac. This lymphocyte compartment receives 3 times the amount of
After repeating washing three times by adding Eagle MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and centrifuging, and discarding the supernatant, RPMI containing 10 v/v% fetal bovine serum - 1640 medium (Nissui Pharmaceutical)
Washed lymphocytes (manufactured by Co., Ltd.) were added to a nucleated cell density of 3 x 10 ⁶ cells/ml and suspended. EBV grown in B-95-8 cells was added to this lymphocyte solution at a concentration of 5×10 ⁵ TD ₅₀ /ml.
After culturing at 37° C. for 2 hours, the cells were collected by centrifugation, and washed three times by adding EagIe MEM medium, centrifuging, and discarding the supernatant. Washed cells were placed in RPMI containing 10v/v% fetal bovine serum.
Inoculate into 1640 medium at 3 x ¹⁰ cells/ml,
Culture was performed at 36-37°C and 5v/v% _CO2 .
Cultures lasted for 1.5 months, during which time v/v
Either an equal volume of RPMI-1640 medium containing % fetal bovine serum was added, or a half volume of the medium was exchanged with this medium.

Three cases of umbilical cord blood were treated in this manner.
The results of measuring the amount of spontaneous production of IF after each cell was transformed are shown in the table below. This production amount was determined by implanting each cell into RPMI-1640 medium containing 10v/v% fetal bovine serum at a concentration of 5 x ¹⁰⁵ cells/ml under conditions of 5v/v% CO2 _and 37°C. So 5
This was obtained by measuring the activity of the culture supernatant after culturing for one day. IF activity x 10 ³ U/ml UNCL-1 2.5 Umbilical cord blood 〃 -2 6.1 Lymphocytes 〃 -3 4.5 Example 1 Sodium chloride 6400.0mg/ Potassium chloride 400.0 〃 Calcium chloride (anhydrous) 200.0 〃 Magnesium sulfate (anhydrous) 97.7 〃 Sodium dihydrogen phosphate (dihydrate)
125.0 〃 Ferric nitrate (nonahydrate) 0.1 〃 Glucose 1000.0 〃 Sodium pyruvate 110.0 〃 L-Arginine hydrochloride 84.0 〃 L-Cystine dihydrochloride 62.6 〃 Glycine 30.0 〃 L-Glutamine 584.0 〃 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 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 Amine hydrochloride 4.0 i-inositol 7.2 〃 Phenol Red 5.0 〃 The following components were added and dissolved in Dalbeco's modified Eagle medium (Dalbeco's modified MEM) having the composition as described above.

Insulin 10mg/ Human transferrin 5 Hypoxanthine 4 Thymidine 0.7 Deoxycytidine 0.03 Deoxyadenosine 1.0 6,8-dihydroxypurine 0.3 Glyose 1000 L-Alanine 20 L-Asparagine (monohydrate) 56 〃 L-Aspartic acid 20 L-Cysteine hydrochloride (monohydrate) 40 L-Glutamic acid 20 L-Proline 20 Ascorbic acid 10 Biotin 0.2 Folinic acid 0.01 VB ₁₂ 0.1 FeSO ₄・7H ₂ O 0.8 〃 ZnSO ₄・7H ₂ O 0.02 〃 Na ₂ SeO ₃ 0.004 〃 CaCl ₂ 100 〃 Glutathione 1.0 〃 Putrescine dihydrochloride 0.1 〃 Disodium β-glycerophosphate 1500 〃 Bicarbonate of soda 1300 〃 RITC thus obtained 55 Bovine serum albumin (BSA) in the amount shown in Figure 1 was added to the -9 medium, the pH was adjusted to 7.3 with 1N NaOH, and the resulting solution was filtered through a membrane filter. Various
UNCL-3 cells in a BSA-concentrated medium were added at 5×10 ⁵ cells/ml and cultured for 5 days. The results of measuring the cell concentration of the culture solution and the amount of interferon produced are shown in FIG.

Furthermore, the activity of interferon was determined in human amnion-derived FL cells and VSV (Vesicular Stomatitis Virus).
It was measured by the cell degeneration inhibition method using (Masahiko Iizuka et al., Kinka Igaku, Vol. 29, p. 660 (1974)).

Meanwhile, fetal bovine serum (FBS) was added to RPMI-1640 medium.
The right side of Figure 1 shows the results of culturing UNCL-3 cells in the same manner by adding them to a medium supplemented with 10% by volume of .

in RITC 55-9 medium containing 5 mg/ml BSA.
UNCL-3 cells were added at 5×10 ⁵ cells/ml and cultured for 3 months by passage every 5 days, but no significant decrease was observed in their proliferation ability and interferon production ability.

Example 2 A medium prepared by adding 0.5% by weight of human serum albumin to RITC 55-9 medium in the same manner as in Example 1.
UNCL-3 cells were added and cultured in the same manner as in Example 1 to RPMI-1640 medium supplemented with 10% FBS by volume, and the cell concentration and interferon production amount of the culture solution were measured over time. The results are shown in Figure 2.

Example 3 RITC 55-9 medium was prepared in the same manner as in Example 1.
Example 1
Namalva cells derived from human Burkitt's lymphoma were added and cultured in the same manner as above, and the changes in cell concentration of the culture solution over time were measured. The results are shown in FIG.

In addition, these cells were cultured for 2 months by passage every 4 days at 3 × 10 ⁵ cells/ml using RITC 55-9 medium supplemented with BSA, but the amount of interferon produced by Sendai virus induction was It was 80% of that when serum-supplemented medium was used.

Example 4 Human lymphocytes transformed with EBV
C5180Y cells (anti-sheep red blood cell antibody producing strain)
RITC 55-9 medium and FBS supplemented with 0.5% BSA by weight
The cells were cultured in the same manner as in Example 1 using RPMI-1640 medium supplemented with 10% by volume, and the cell concentration of the culture solution was measured over time. The results are shown in FIG.

[Brief explanation of the drawing]

FIG. 1 shows the results of measuring the influence of BSA concentration on the cell concentration and interferon activity of the culture solution for UNCL cells. Second
Figures 4 to 4 show the results of measuring the cell concentration (also interferon activity in Figure 2) of the culture medium for the medium of the present invention and a conventional serum medium. In each figure, the white circles represent the cell concentration of the culture medium of the present invention, the white squares represent the interferon activity, the black circles represent the cell concentration of the conventional serum medium, and the black squares represent the interferon activity.

Claims (1)

[Scope of Claims] 1. A culture medium for mammalian lymphocyte-derived cells, characterized by containing mammalian serum albumin separated from serum. 2. The medium according to claim 1, wherein the mammalian serum concentration is less than 5%. 3. The culture medium according to claim 2, wherein the mammal is a human, a cow, or a horse. 4. Claim 1 or 3, wherein the mammalian lymphocyte-derived cells are interferon-producing cells.
Medium as described in section. 5. The medium according to claim 4, which is substantially free of mammalian serum.