JPS63202377A - Novel cell strain - Google Patents

Abstract

PURPOSE:To obtain a human lymphoblast cell which can be proliferated in a serumfree medium free from glutamine, by acclimatizing a specific human lymphoblast cell in a medium containing glutamic acid or glutamic acid and acetyglutamine. CONSTITUTION:A human lymphoblast cell which can be proliferated in a medium free from glutamine and containing glutamic acid and/or acetylglutamine is prepared from a human lymphoblast cell which cannot be proliferated in a glutamine-free medium by acclimatizing the human lymphoblast cell in a medium containing glutamic acid or glutamic acid and acetylglutamine. Preferable example of a parent strain for the growth of glutamine-nonrequiring strain is Burkitt lymphoma, Namalwa cell strain ATCC, etc., which are kinds of human lymphoblast cell. The acclimatization can be carried out e.g. by subculturing the cells at a cell density of about 0.5X10<5>-8X10<5> cells/ml in a medium free from glutamine and containing glutamic acid and/or acetylglutamine for 3 weeks - 1.5 month or longer.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for acclimating human lymphoblastoid cells, which do not proliferate in a glutamine-free medium, to a serum-free medium that does not contain glutamine and contains glutamic acid and/or acetylglutamine instead of glutamine. This invention relates to a novel human lymphoblastoid cell that can be grown in a medium and a method for preparing the same.

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel human lymphoblastoid cell and a method for its preparation. The cells of the present invention can be grown in a serum-free medium that does not contain glutamine, making this cell line advantageous for producing lymphokines such as interferon, cell growth factors, and cell growth inhibitory factors. Therefore, it can be applied to medicines, chemicals, foods, etc. that use these substances.

Conventional technology Conventionally, when culturing animal cells, cow or horse serum was added at a concentration of 0.5 to 2
A medium with 0% added is used, and in addition, glutamine,
Antibiotics have been added. Recently, attempts have been made to consider serum-free media due to loft differences in serum and cost issues. In addition, some attempts have been made to cultivate cells grown in serum-free medium (Japanese Patent Application Laid-Open No. 60-6190).

Problems to be Solved by the Invention Animal cells are generally prepared using 0.5 to 20% bovine or horse serum.
It can be cultured in vitro using the containing medium. Established cells such as cancer cells can now be cultured in large quantities on an industrial scale. Cell culture produces useful substances such as interferon, interleukin, and plasminogen activator.

Glutamine is usually added to the medium used for cell culture. Glutamine is unstable to heat and becomes 9 when sterilized by steaming.
Since 5% or more of it decomposes into 5-oxoproline, it is usually used after filtration sterilization. Glutamine is metabolized with cell growth and releases ammonia into the medium. This ammonia is known to cause inhibition of cell growth, including CHO-Kl in Chinese hamsters and B-B in cows.
It has been reported that 50% growth inhibition occurs in HK cells even at a concentration of about 4mM. When using the perfusion culture method for Namalva cells, 3 m
It is known that the ammonia concentration is M. Therefore, in order to proliferate and maintain cells at even higher density, it is necessary to reduce the amount of glutamine added, which is a source of ammonia.

Means to Solve the Problem The present inventors have already shown that it is possible to acclimate human lymphoblastoid cells to a serum-free medium by a conditioning method and to grow cells grown in a serum-free medium. (Japanese Patent Application Laid-Open No. 1986-1999--The present inventors conducted research to obtain a glutamine-independent strain of Burkitt's lymphoma Namalva cells, which are human lymphoblastoid cell lines that can grow in a serum-free medium by an acclimation method.

First, when we tried to use acetylglutamine, which can be sterilized by steaming, instead of glutamine, we found that acetylglutamine was not assimilated by human lymphoblastoid cells, and they gradually died, decreasing the survival rate, and eventually the cell number decreased to 30%. It decreased to below.

If you continue to passage to fresh medium, you will not see cell proliferation, but you will see an increase in survival rate, and if you continue to passage the cells for an even longer period of time, you will start to see cell proliferation.
The starting cell line is close to growth. In this way, we were able to obtain a strain that does not require glutamine.

The presence of glutamic acid in the medium is considered to be the reason why this strain that does not require glutamine was obtained. The basal medium used was RPMI-1640 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) containing Q, 2mM glutamic acid. If glutamic acid, ammonia and ATP are present, glutamine is biosynthesized by the action of cellular glutamine synthetase. This biosynthetic reaction is thought to be in an equilibrium relationship with glutaminase, and excess glutamic acid is thought to shift the equilibrium in glutamine synthesis.

For the above reasons, addition of excess glutamic acid is thought to be effective for cell conditioning, so we attempted conditioning using a medium supplemented with 4mM glutamic acid instead of acetylglutamine, and found that this method also resulted in cell conditioning. Similarly, a glutamine non-auxotrophic strain was obtained.

The content of the invention will be explained in detail below.

The present invention provides novel human lymphoblastoid cells that are free of glutamine and can be grown in serum-free media containing glutamic acid and/or acetylglutamine. The present invention also includes:
Human lymphoblast cells that do not proliferate in a medium that does not contain glutamic acid, and human lymphoblasts that do not contain glutamine and can proliferate in a serum-free medium that contains glutamic acid and/or acetylglutamine by acclimatizing them to a medium that contains glutamic acid or glutamic acid and acetylglutamine. A method of preparing cells is provided.

As a parent strain for cultivating a glutamine-nonrequiring strain, any human lymphoblastoid cell that requires glutamine can be used. Suitable examples include Burkitt's lymphoma, which is a human lymphoblastoid cell, and the Namalva cell line ATCCCRL-1432. Furthermore, the KJM-1 strain (Japanese Patent Application Laid-open No. 60-61
If the strain is adapted to a serum-free medium, such as the homologous synonym of the KT-1 strain disclosed in 1990, various analyzes are easier.

As a method of acclimatization, 0.5X10' to 8X105 pieces/
A method is used in which cells of approximately ml are subcultured in a medium that does not contain glutamine but contains glutamic acid and/or acetylglutamine for 3 weeks to 1.5 months or more. It is necessary to replace the medium and subculture so that the pH of the medium does not fall below 6.5.

Since cell density may decrease significantly at the early stage of passage,
Concentrate by centrifugation or removing the supernatant and adjust the cell density to the above range. Namalva cell strain KJM adapted to serum-free medium
The acclimatization method will be specifically explained using the -1 strain as an example.

Using KJM-1 strain as seed cells, remove glutamine from serum-free medium and add 0.4 to 40mM acetylglutamine 7 (AG
) or inoculated at a cell density of 10' to 1 x 10 cells/ml in a medium further supplemented with 0.4 to 40 mM glutamic acid (GA). After culturing this at 36-37°C or lower for 1-5 days, the temperature was 1.50 Orpm.

Centrifuge for 5 minutes and replace with fresh medium. These operations are repeated for 1.5 months or more until growth is stable and cell proliferation approaches that of the parent strain.

Cells do not necessarily grow, so 4-
It is necessary to repeat the procedure 5 times in a row, or if the cells die, it is necessary to repeat the same procedure from the beginning. As the basal medium of serum-free medium, RPMI-1640 (manufactured by Tamizu Pharmaceutical Co., Ltd.), MIE
M (manufactured by Tadami Pharmaceutical), Dulbetsco MEM (manufactured by Tadami Pharmaceutical), Ham F10, Ham F12 (manufactured by Flow Lab), D
M160. DM170 (manufactured by Kyokuto Pharmaceutical Co., Ltd.) or the like is used.

As an additive in serum-free medium, penicillin 5-100
u/ml, x Treptomycin 5-100 ■/ml
, Hepes buffer 10-300mM. Transferrin 0.1-100 g/ml as serum replacement
Preferably 1-1 Ox/ml, insulin 0.3-1
00 g/ml preferably 1-10 u/ml, sodium selenite 0.1-100 X 10-'M preferably 1-10 X 10-'M, sodium pyruvate 0.1-
100mM, galactose 0.01-100mg/ml
Preferably 0.1 to 10 mg/ml, Pepol B-1
88 (manufactured by Toho Chiba Chemical Co., Ltd.) 0.001 to 10%, preferably 0.01 to 1%, dimethyl-α- or β-cyclodextrin (manufactured by Toshin Chemical Co., Ltd.) 1 kg to 1 Q mg/
m+, low molecular weight gelatin (MW, about 10,000) manufactured by Tsuta Gelatin Co., Ltd.) 0.01 to 100 mg/ml, preferably 0.5 to 30 mg/ml, etc. are used.

When cell growth has recovered to the same level as the parent strain, the concentration of ammonia metabolized in the medium is measured and compared with the parent strain. As a measurement method, the production of glutamic acid from ammonia and α-ketogeltaric acid by glutamate dehydrogenase (GLDH) is determined by the difference in absorbance of NADPH2.

As a reagent, an ammonia quantitative reagent Determiner NHs (manufactured by Kyowa Medex Co., Ltd.) is used. Furthermore, after collecting the cells and subjecting them to sonication, the activity of intracellular glutamine synthetase is measured. The activity measurement uses a system that measures the production of γ-glutamyl hydroxamic acid from glutamine and hydroxyamine by glutamine synthetase.

The medium used to culture conditioned cells does not contain glutamine. If glutamine is contained in the culture solution after culturing, glutamine synthesis is proven.

Glutamine analysis of the culture solution is performed using an automatic amino acid analyzer JLC-200A manufactured by JEOL Ltd.

Examples of the present invention will be shown below.

Example 1 Glutamine-nonrequiring cells were grown using the KJM-1 strain, which is a serum-free medium-accumulated strain of human lymphoblastoid Namalva cells.

RPMI-1640 was used as the basal medium, and 3 g/
n+1 insulin, 5g/ml) Lanceferrin, 5m
M Sodium Pyruvate, 1.25 X I O-''M Sodium Selenite, 1 mg/ml Galactose, 4mM Glutamine, 10mM Hepes Buffer, 25u/ml Penicillin G, 25g/ml Streptomycin and 0°1%
The glutamine-requiring cell KJM-1 strain was subcultured using a serum-free medium supplemented with baking soda (this medium is referred to as ITPSG medium). The cells were inoculated into a medium containing 4mM acetylglutamine and cultured. The culture is
After 2-4 days at 37°C in 25 cd culture flasks, the cultures were centrifuged at 1,50 Orpm for 5 min.
The resulting cells were passaged by resuspending them in fresh medium. As cells are passaged, their survival rate deteriorates and the number of cells decreases. Seven days after the initial culture, the cell number decreased to 27% and was able to maintain this cell number thereafter.

After 1.5 months, the cell number recovered to more than 90% of the time of inoculation,
The number of cells could now be increased to I x 10 cells/ml. Since then, the cells have continued to be passaged and have shown stable proliferation for over 24 months. 0.9 X I O'1III/m
After 55 days, the cells inoculated with 100 ml of cells proliferated to 4.4×10 5 cells/ml, and the proliferation rate was almost the same as the 3.5×10 5 cells/ml of the parent strain KJM-1 cultured in glutamine-supplemented medium. At that time, the amount of ammonia metabolized in the medium was reduced to 41% of that of the parent strain.

Example 2 Human) Glutamine-nonrequiring cells were grown using the KJM-1 strain, which is a serum-free medium-accumulated strain of Namalva cells, which are IJ lymphoblast cells. Using ITPSG medium as a medium, cells subcultured with glutamine-requiring cotton 111KJM-1 strain were transferred to the above IT to a concentration of 5 x 10' cells/ml.
The cells were inoculated into a PSG medium to which 4mM glutamic acid was added instead of glutamine, and cultured. Culture was carried out in a 25 cd culture flask at 37°C for 2 to 4 days, and then the culture was centrifuged at 150 rpm for 5 minutes, and the resulting cells were resuspended in fresh medium for subculture. Yes.

In the medium supplemented with glutamate, there was not much of a decrease in cell number, but no increase in cell number was observed either. After 3 weeks, the cells started to proliferate, and after 3 months, they approached the growth of the parent strain. then 1
It has shown stable growth for 4 months. cells 0.9X
The number of cells after 5 days of inoculation at 10' cells/m1 was compared with that of the parent strain. Both the adapted strain and the parent strain showed 3.5×10' cells/ml. The ammonia concentration at that time was 75% of the parent strain.

Example 3 The activity of glutamine synthetase and the production of glutamine were investigated. The parent strain, the glutamine-incompatible acetylglutamine and/or glutamic acid supplemented strain were cultured, and 2 ml of each cell suspension (5 x 10'N/...1) was taken and centrifuged to collect the cells and supernatant.

Cells were suspended in 2 ml of PBS (phosphate buffer containing saline) and tlL-TRASONICDISRllPTORM
After destroying the cells with ultrasonic waves for 2 minutes at 140 W under water cooling using odel LIR-200P (manufactured by Tomy Seiko), 150
The supernatant was collected by centrifugation at 0 rpm for 10 minutes. 100 μs of this supernatant was added to 500 mM imidazole-HCJ for 4 m
M ADP.

200mM glutamine, 200mM hydroxylamine, 200mM sodium arsenate, and 3mM n CR2 were each added to a reaction solution containing 90IjIl and 360ρ of distilled water, and reacted at 37°C for 24 hours. After reaction, 2
mM iron chloride reagent (10%FeCR* 6H20,6N
HCI, 24% trichloroacetic acid (TCA) = 1:
1:1) was added, and the amount of γ-glutamylhydroxamic acid produced by the reaction was determined by the absorbance at 540 nm (
It was measured using a Shimadzu spectrophotometer UV-240).

The absorption value at 540 nm and the calibration curve of T-glutamylhydroxamic acid are shown in Table 1.

Table 1 The amount of T-glutamylhydroxamic acid is defined as the amount of glutamine synthetase, and Table 2 shows the concentration of glutamine synthetase produced by each cell line.

Table 2 Parent strain -4mM O, 2mM O,013-Also, the change in glutamine in the medium after 7 days of culture was
3 JLg/ml decreased to 109 g/ml, whereas the acetylglutamine-acclimated strain decreased from 0 to 8 JLg/ml.
and increased from 0 to 21 μ/m in glutamate-acclimated strains.
It was increasing to l.

Example 4 Metabolism of parent strain KJM-1 strain and glutamine non-requiring strain,
The biosynthetic pathway was compared by amino acid analysis of the culture supernatant after culturing.

As culture media, the medium of Example 1 was used for the parent strain, and the medium to which acetylglutamine and glutamic acid were added instead of glutamine was used for the conditioned strain, respectively.

As a result, in addition to glutamine and glutamic acid, KJ
In the M-1 strain, arginine is not consumed much and there is no production of ornithine or citrulline, but in the strain grown in a medium containing glutamine-free acetylglutamine and glutamic acid,
Both were endowed with the ability to consume arginine and synthesize ornithine and citrulline. Table 3 shows the amounts of arginine, ornithine, and citrulline produced.

Table 3 Parent strain 223 0 0 Glutamic acid 0 92 51
Acclimated Strain Acetylglu 21 75 65 Tamine-adapted Strain (Amino acids in medium after 7 days of culture) Effects of the Invention According to the present invention, novel human lymphoblastoid cells that can be expanded in a serum-free medium that does not contain glutamine are provided.

Patent applicant (114) Director of the Agency of Industrial Science and Technology Sachi Itadzuka

Claims (4)

[Claims] (1) Novel human lymphoblastoid cells that do not contain glutamine and can grow in a serum-free medium containing glutamic acid and/or acetylglutamine. (2) Serum-free medium is RPMI-1640, Eagle ME
M, Dulbecco MEM, Ham F10, Ham F12, D
A basal medium selected from M160 and DM170,
Insulin, transferrin, selenite, pyruvate, hepes buffer, penicillin, streptomycin,
Galactose, Pepol B-188, dimethyl-α- or β-cyclodextrin and low molecular weight gelatin (M
10. The cell according to claim 1, characterized in that the medium is supplemented with one or more types selected from the group consisting of: W, about 10,000). (3) By acclimatizing human lymphoblastoid cells, which do not proliferate in a medium containing glutamine, to a medium containing glutamic acid or glutamic acid and acetylglutamine, they can proliferate in a serum-free medium that does not contain glutamine but contains glutamic acid and/or acetylglutamine. Method of preparing human lymphoblastoid cells. (4) Serum-free medium is RPMI-1640, Eagle ME
M, Dulbecco MEM, Ham F10, Ham F12, D
A basal medium selected from M160 and DM170,
Insulin, transferrin, selenite, pyruvate, hepes buffer, penicillin, streptomycin,
Galactose, Pepol B-188, dimethyl-α- or β-cyclodextrin, and low-molecular-weight gelatin (
4. The method according to claim 3, wherein the medium is supplemented with one or more types selected from the following: MW of about 10,000).