JPS6320515B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for breeding a serum-free medium-grown strain derived from human leukemia cells that can grow in a serum-free medium. In other words, we can breed cells that proliferate in a serum-free medium, can induce differentiation into cells with macrophage-like functions in a serum-free medium, and produce monokines derived from macrophages in a serum-free medium, and develop an industrially inexpensive culture medium. The present invention relates to a cell mass culture technology for producing monokine, which is an in vivo physiologically active substance and can be applied as a pharmaceutical product.

(Prior Art) Few human-derived osteogenic leukemia cell lines have been established, and most cell lines are grown and cultured using fetal bovine serum. That is, human leukemia cell line THP-1 [International Journal of Cancer, Vol. 26, 171]
-176, 1980] was grown and cultured in a medium containing 20% fetal bovine serum, and HL-60 [Blood,
54, pp. 713-733, 1979], 10% fetal bovine serum medium, PL-21 [Gann, 74, 319-322]
Page, 1983] is 15% fetal bovine serum and 15% human umbilical cord serum.
% medium, KG-1 [Science vol. 200,
1153-1154, 1978] was grown and cultured in a medium containing 20% fetal bovine serum. and human leukemia cells
HL-60 is grown and cultured in a serum-free medium containing insulin and transferrin [Experimental Cell Research, Vol. 126, 497-
498, 1980], but most cell lines require serum for growth culture. Fetal bovine serum is expensive due to its limited production volume, and the quality of the serum varies widely due to individual differences between cows.Using serum in industrial cell culture is a cause of soaring process and equipment costs. It was hot. Furthermore, since serum contains many unknown proteins, in the production of monokine by cell culture, the process of purifying monokine from the culture solution is complicated and it is difficult to remove impurities.

(Means for Solving the Problems) In the present invention, research has been carried out on a method for industrially and inexpensively mass-preparing cells having functions equivalent to such human-derived macrophages. As a result, they succeeded in finding a method for reproducibly breeding a serum-free cultured cell line that proliferates vigorously in a serum-free medium from THP-1 cells, and completed the present invention.

Human leukemia cells THP-1 are subcultured in RPMI-1640 medium containing 10% fetal bovine serum. Therefore, RPMI-1640 was used as the basic medium, and insulin 5 mg/, transferrin 5 mg/,
A serum-free medium (named "ITESB medium") with ethanolamine 10 mg/, sodium selenite 0.05 mg/, bovine serum albumin 1 g/, penicillin G 50000 units/, streptomycin sulfate 50 mg/, and PH7.40 was designed and THP- When one cell was cultured, the proliferation rate was only 20 to 40% that of fetal bovine serum medium. Therefore, the present inventors modified the serum-free medium by adding commercially available growth factors, adhesion factors, and hormones to the serum-free medium ITESB. There was no promoting effect. That is, growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (LCGF), endothelial cell growth factor (ECGS), platelet-derived growth factor (PDGF), and T cells. Growth factors (TCGF) etc. were used. Further, as adhesion factors, fibronectin, polylysine, collagen, etc. were used. Further, as hormones, dexamethasone, glucagon, somatostatin, hydrocortisone, progesterone, triiodothyronine, etc. were used.

However, the present inventors found that serum-free medium ITESB
We discovered that the growth of human leukemia cells THP-1 was significantly promoted by adding a small amount (0.01 to 0.5%) of horse serum to a serum-free culture using a medium supplemented with a small amount of horse serum. They were able to complete a method for breeding cell lines. That is, cells cultured in a medium containing fetal bovine serum were first subcultured in ITESB medium containing 0.5% horse serum, and then
Subculturing is repeated while gradually decreasing the amount of horse serum added, and finally, subculture is performed in a complete serum-free medium ITESB, resulting in stable growth and physiologically homogeneous cells. I got the stock. The properties of the AH-01 strain are shown as an example of the cell line thus bred.

Serum-free cultured cell line AH-01 a Origin: Isolated from human osteogenic leukemia cell THP-1 b Morphology: 11-14 microns in diameter, almost spherical or monocytic cell-like c Number of chromosomes: Modal number of 88 chromosomes Chromosome number distribution mode characterized by exhibiting d Subculture: Infinite subculture e Functional characteristics: Differentiation is induced into non-proliferative cells with macrophage-like functions, producing macrophage-derived monokines.

f Cell proliferation: Proliferates well in suspension in serum-free medium. Generation doubling time is 31±6 hours.

g Storage conditions: Store frozen at -80°C to -196°C.

h Serum requirement: Does not require serum for growth.

In the present invention, in addition to RPMI-1640, basic media include Eagle Basal Medium (BME) medium, Minimum Essential Medium (MEM) medium, Iscove's medium, Ham's medium,
William's medium, Dulbecco's medium, Weymouth's medium and the like can be used alone or in appropriate mixtures. The compositions of these basic media are described in Animal Cell Culture Methods (Kyoritsu Shuppan, 1982) and the like. In addition, in breeding the serum-free cultured cell line of the present invention, in addition to the human leukemia cell THP-1, horse serum was used.
It can be applied to human leukemia cells that can grow in ITESB medium supplemented with 0.5%. For example, human leukemia cells
HL-60, KG-1, K-562 [Blood,
Vol. 45, p. 321, 1975] etc. contain horse serum medium.
Since it grows well on ITESB, the method of the present invention is applied.

Monokines produced by macrophages are physiologically active substances that act on the immune system and hematopoietic system, such as leukemia cell differentiation inducing factor (D-factor), macrophage activating factor (MAF), bone marrow cell colony formation promoting factor (CSF), Cytotoxic factor (Lymphotoxin), Cell growth factor (Growth)
factor) is known. The serum-free cultured cell line bred by the method of the present invention is characterized by having the ability to produce these monokines.

Next, as an example of these monokines, an assay method for leukemia cell differentiation-inducing factor (D-factor) will be described. An phagocytosis test of latex bead particles using mouse osteogenic leukemia cell line M-1 was conducted.
Hayashi's method (Toxicology Forum, Volume 7, 50)
~59 pages, 1984) according to the following procedure.

Eagle osteogenic leukemia cell M-1
Add 10% fetal bovine serum to a medium containing dissolved MEM (Nissui Pharmaceutical Co., Ltd.) and amino acid vitamin medium powder (Nissui Pharmaceutical Co., Ltd.), and inoculate the cells to a density of 1 x 10 ⁵ /ml.
It was cultured for 3 days. The cells were suspended in a new medium to a density of 2×10 ⁵ /ml, the test sample solution was added as appropriate, and the cells were cultured for 2 days. Cells were harvested by centrifugation, suspended in fresh serum-free medium, and
Polystyrene latex particles (1.099 micron diameter, manufactured by Dow Chemical) at a concentration of 2 μ/ml medium.
was added and cultured for an additional 4 hours. After thoroughly washing the cells with phosphate buffer to remove polystyrene latex bead particles in the medium, the cells were observed under a microscope, and cells that phagocytosed latex particles and non-phagocytosed cells were counted. The proportion of phagocytes was determined. The titer when giving 50% of the phagocytic cells is 50 units/
ml.

(Effects of the Invention) The present invention makes it possible to breed human leukemia cell lines that proliferate vigorously in a serum-free medium. Capable of mass culturing cell lines.
By appropriately producing and culturing the cells obtained in large quantities, it has become possible to culture and produce monokine in a serum-free medium. These monokines are useful substances for diseases of the immune system and hematopoietic system, and can be used as therapeutic or diagnostic agents.

(Examples) The present invention will be explained below with reference to Examples, but these are illustrative and do not limit the present invention in any way.

Example 1 Human monocytic leukemia cell line THP-1 was subjected to RPMI-
10 ml of medium containing 1640 with 20% fetal bovine serum in diameter
Place in a 10cm plastic culture dish until the number of cells is 1.
The cells were inoculated at × ¹⁰⁵ cells/ml. 5% of this
When cultured for 3 days in a cell culture incubator with CO ₂ , 95% air, and 100% humidity, 6.5×10 ⁵
The number of cells had reached 2 cells/ml. Cells were collected by centrifugation, washed thoroughly with phosphate buffer, and cells were collected again by centrifugation. prepared separately
10 ml of ITESB medium containing RPMI-1640 as the basic medium and 1.0% horse serum was added to a plastic culture dish with a diameter of 10 cm, and the collected cells were cultured 2x.
The cells were transplanted at a concentration of ¹⁰⁵ cells/ml. The cells were cultured in a cell culture incubator for 4 days, and the cells were collected as described above and transplanted again into ITESB medium containing 1.0% horse serum, and the culture was repeated. After repeating subculture 5 times, the medium was changed to ITESB medium containing 0.5% horse serum, and subculture was repeated 5 times. As cell growth began to stabilize, the horse serum was diluted to 0.1%.
Further subculture was performed five times. At this time, the cell concentration on the fourth day of culture was 10 to 12 x ¹⁰⁵ cells/ml. As the cells gradually became accustomed to the low-serum medium, we switched to the complete serum-free medium ITESB and subcultured them.
As a result of 80 days (20 subcultures), it was possible to obtain cells that stably proliferated to a cell concentration of 12 to 14 x ¹⁰⁵ cells/ml in the serum-free medium ITESB. From microscopic observation, morphology by cell staining, and karyotype observation,
Since it had become a uniform cell line, it was transferred to AH-
It was designated as 01 strain and stored frozen. Note that the number of cells was counted using a Coulter Counter cell counting machine (manufactured by Coulter Electronics).

Example 2 RPMI-
10 ml of medium containing 1640 with 10% fetal bovine serum in diameter
Place in a 10cm plastic culture dish until the number of cells is 1.
The cells were inoculated at × ¹⁰⁵ cells/ml. 5% of this
When cultured for 3 days in a cell culture incubator with CO ₂ , 95% air, and 100% humidity, 8.9×10 ⁵
The number of cells had reached 2 cells/ml. Using these cells, in the same manner as in Example 1, 1.0% horse serum was first added.
The cells were subcultured 5 times in ITESB medium, further subcultured 5 times each in 0.5% horse serum medium and 0.1 horse serum medium, and then subcultured in complete serum-free medium ITESB for 40 days (10 subcultures). I went. As a result, a cell line that grows in serum-free medium was obtained. This familiar strain
16×10 ⁵ cells/ml after 4 days of culture in ITESB medium
It grew to.

Example 3 50 ml of serum-free ITESB medium was dispensed into two 250 ml plastic flasks. in advance
AH-01 cells cultured in ITESB for 4 days were transplanted to an initial cell concentration of 2.0 x ¹⁰ cells/ml.
When cultured for 4 days in a cell culture incubator at 37°C under 5% CO ₂ , 95% air, and 100% humidity, the total number of cells was 137×10 ⁵ . The cells were collected by centrifugation, suspended in 15 ml of ITESB medium in each of 10 culture dishes with a diameter of 10 cm, and 0.1 μg of phorbol ester (12-O-tetradecanoyl phorbol-13-acetate) was added to the plates. /ml in a cell culture incubator.
Culture was performed for 24 hours. Collect the cells with a pipette, wash twice with phosphate buffer, and then remove the cells.
The cells were suspended in 150 ml of ITESB medium (3 plastic culture dishes with a diameter of 15 cm) and cultured for 4 days in a cell culture incubator. The cell culture supernatant was collected by centrifugation, and the activity was measured by the leukemia cell differentiation-inducing factor assay method described above.
450 units/ml of leukemia cell differentiation inducing factor was produced.

Claims (1)

[Claims] 1. A method for breeding a cell line that proliferates in a serum-free medium by culturing a human osteogenic leukemia cell line in a low-serum medium containing horse serum and then in a serum-free medium.