JPS61289039A - Serum-free culture cell strain - Google Patents

Abstract

NEW MATERIAL:Cell strain AH-01 derived from human bone marrow leukemic cell, capable of being multiplicated in a serum-free medium. Derivation: separated from human bone marrow leukemic cell THP-1. Shape: having 11-14mu diameter, almost spherical or monocyte cell-like form. Number of chromosomes: distribution mode of numbers of chromosomes showing model number of 88 chromosome number. Subculture: infinite. Functional characteristics: being differentiated and induced to a nonvegetative cell having macrophage-like functions, producing monokine derived from macrophage. Cell multiplication: multiplying well in a serum-free medium in a suspension state, doubling time of generation is 31+ or -6hr. Preservation condition: in cold storage at -80-196 deg.C. USE:Mass production of monokines which are physiologically active substances in organism and applicable as drugs. PREPARATION:Human bone marrow leukemic cell THP-1 is subjected to subculture in a medium comprising PRMI-1640 as a base medium, low serum and a very small amount of equine serum and subculture is repeated in a serum-free medium containing no equine serum with increase in the subculture.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to human myeloid cells that proliferate in a serum-free medium, can be induced to differentiate into non-proliferative cells with macrophage-like functions, and produce macrophage-derived monokines. Leukemia cell line A
This is related to H-Of, and is a useful technical field for industrially mass-producing monokine, which is an in vivo physiologically active substance and can be applied as a pharmaceutical, using an industrially inexpensive culture medium. be.

(Prior Art) Monokine is produced by macrophages in the living body and is an important physiologically active substance in the living body related to biological defense mechanisms such as the immune system and the hematopoietic system. For example, myeloid colony forming factor (CSF), macrophage activating factor (MA
F) Leukemia cell differentiation-inducing factor 7. These are physiologically active substances that act on the immune system and are useful as pharmaceuticals for the treatment and prevention of bacterial infections and carcinomas, and for the treatment of hematopoietic disorders.

(Problems to be Solved by the Invention) However, in order to collect human macrophages that produce monokine, lung lavage surgery was required, and it was humanely impossible to prepare large quantities. Furthermore, large amounts of fresh human blood were required to prepare large amounts of monocytes, which are thought to be precursor cells of macrophages found in peripheral blood. Therefore, it is difficult to prepare large quantities of cells with macrophage functions and to produce large quantities of monokine from these cells, so industrial production of monokine has not yet been put to practical use. .

In order to maintain their metabolic activity, these macrophages or monocytes obtained from the human body are cultured in a serum medium containing tallow serum, etc., and it has been difficult to obtain monokine samples with high purity. .

(Means for Solving the Problems) In the present invention, in order to solve these drawbacks, human proliferative cells were cultured in large quantities in a serum-free medium, and then cell lines were successfully obtained. This serum-free culture strain was named AH-01 and cryopreserved. Since AH-01 maintained stable properties such as cell morphology, cell proliferation rate, generation doubling time, and ability to induce differentiation into cells with macrophage functions, it was It was confirmed that it was a serum cultured cell line.

That is, the present invention was completed based on the above research results, and relates to serum-free cultured cell line AH-01 having the following properties.

a) Origin: Isolated from human myeloid leukemia cell THP-1 b) Morphology: 11-14 microns in diameter, almost spherical or monocytic cell-like C) Chromosome number: Showing a modal number of 88 chromosomes Distribution mode of chromosome number characterized by d) Subculture: Infinite subculture e) Functional characteristics: Differentiation is induced into non-proliferative cells with macrophage-like functions, and macrophage-derived monokine is produced.

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

g) Storage conditions: Cryopreservation at -196°C h) Serum requirement:
Does not require serum for growth.

The method for establishing the serum-free cultured cell line AH-01 obtained by the present invention will be described. Human myeloid leukemia cell THP-1
(International Journal of Cancer)
Cancer), vol. 26, pp. 171-176, 1980
) was used as the original cell. These cells are normally maintained in RPMI-1640 medium containing 20% tallow serum.

The present inventors conducted basic research on serum-free culture methods for human cells, using RPMI-1640 as the basic medium, and
Insulin (5-50mg/ff), transferrin (2-20mg/42), ethanolamine (1-2
0 mg/l), sodium selenite (0,01
~0.08 mg/l), bovine serum albumin (0,
A serum-free medium containing 5-2 g//) was developed. Although THP-1 did not grow well in this medium, a low-serum medium containing a trace amount of horse serum (0.01-0.45%) is normally used under conditions that allow THP-1 to grow. 5% CO2, 95% air, 100% humidity, temperature 37℃
The cells were cultured in a cell culture incubator. Centrifuge the proliferated cells to collect the cells from the culture medium and suspend them in a new medium at 2.0 x 10' cells/m1.
It was cultured for 4 days. As a result of such subculturing, THP-1, which initially did not grow well on serum-free medium,
A serum-free cultured cell line with vigorous growth ability in serum-free medium was obtained. These cells maintain the ability to induce differentiation into cells with macrophage functions, and have the functional characteristic of producing macrophage-derived monokines in a serum-free medium. The cultured cell line was named AH-01.

Original cell THP-1 and serum-free cultured cell line A according to the present invention
A comparison of H-Of is shown in Table 1.

Margin table below 1 Number of chromosomes 88 46 (Modal number) (Tetraploid after clotting) (Diploid) Cell proliferation Propagation in serum-free medium Propagation in beef tallow serum Generation doubling time 31± 6 hours 60-70 hours *Literature International Journal
of Cancer+ vol. 26, pp. 171-176, 1
980 Thus, AH-01 is a cell that has industrially advantageous conditions such as a high growth rate and the ability to grow in a serum-free medium.

The basic serum-free medium used in the present invention includes R
PMI-1640 medium, Eagle Basal Medium (
BME) medium, Minimum Esche (Japanese 56), etc.

An example of a typical serum-free medium is RPMI-1640.
as the basic medium, insulin 5mg/β, transferrin 5mg/j! , ethanolamine Omg/l,
Sodium selenite 0.05 mg/l, bovine serum albumin 1 g/l, penicillin G 50,000 units/l, streptomycin sulfate 50 mg/l, pH 7.40 are used.

In this serum-free medium, at a temperature of 37°C, 5% cot,
Subculturing is possible without limit in a cell culture incubator with 95% air and 100% humidity. Figure 1 shows the growth curve. From numerous culture experiments, the generation doubling time of AH-01 in serum-free medium was 31±6 hours.

The number of chromosomes was determined by analyzing the distribution mode using a standard method for cell experiments, and as shown in FIG. 2, it was found to be pseudotetraploid with a modal number of 88 chromosomes.

AH-01 could be induced to differentiate into cells with macrophage-like functions by a phorbol ester compound. The edibility test for the microbead particles thus obtained was conducted using Hayashi's method (Toxicology Forum, 7.
Vol. 50-59, 1984), the following procedure was performed.

Mouse myeloid leukemia cells M-1 were dissolved in Eagle MEM (Tamizu Pharmaceutical Co., Ltd.), and 10% fetal bovine serum was added to a medium containing amino acid/vitamin medium powder (Tamizu Pharmaceutical Co., Ltd.), and the number of cells was 1X10.
'/mj! The cells were inoculated and cultured for 3 days. The cells were suspended in a new medium at a density of 2×10'/mf, the test sample solution was added as appropriate, and the cells were cultured for 2 days. Cells were collected by centrifugation, suspended in fresh serum-free medium, and distributed at 2 μm/m j! Polystyrene latex particles (1,099 micron diameter, Dow
(manufactured by Chemica 1) was added thereto and cultured for an additional 4 hours. After thoroughly washing the cells with phosphate buffer to remove polystyrene latex particles in the medium, the cells were observed under a microscope to count cells that had actually phagocytosed the latex particles and non-phagocytic cells. The ratio of The titer when giving 50% of actual phagocytes was determined to be 50 units/ml.

The serum-free medium used for this purpose contains 20% tallow serum and 10% dimethyl sulfoxide, and the number of cells is 5-10) < 10.
By suspending cells/ml and storing them frozen at 180°C to 1196°C, cells that retain their proliferation ability can be reproducibly preserved.

(Effects of the Invention) The present invention provides a cell line AH-01 that proliferates vigorously in a serum-free medium, can be induced to differentiate into cells with macrophage-like functions, and also produces monokine. It has become possible to culture and produce monokines derived from human macrophages homogeneously and in large quantities in a serum-free medium, and to provide substances useful in the fields of therapeutic and diagnostic drugs for diseases of the immune system and hematopoietic system. I can do it.

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

Example 1 Human monocytic leukemia cell line THP-1 was transformed into RPMr1640
Add 20% beef tallow serum to Oml of medium to 10cm in diameter.
The cells were placed in a plastic culture dish of 500 mL, washed thoroughly with phosphate buffer so that the number of cells was 1 x 10 S cells/m It, and the cells were collected again by centrifugation.

Insulin 5mg to separately prepared RpMx-1s4o
/i! ) Lanceferrin 5mg/J, ethanolamine 10ml 1g/11 Sodium selenite 0.05n+
g/l, bovine serum albumin 1 g/l, penicillin G5
0.000 units/l, streptomycin sulfate 50II
Ig/2, pH 7,40 (named serum-free medium ITESB) with 1.0% horse serum added to the diameter IQ
Place the collected cells in a 2 cm plastic culture dish.
The cells were transplanted at x10' cells/ml. After culturing in a cell culture incubator for 4 days, cells were collected as described above, transplanted again into ITESB medium containing 1.0% horse serum, and culture was repeated. After repeating subculture five times,
The medium was changed to ITESB medium containing 0.5% horse serum, and subculture was repeated five times. As cell proliferation began to stabilize, the horse serum was diluted to 0.1% and subculture was carried out five more times. At this time, the cell concentration on the 4th day of culture was 1
It was 0-12X10' pieces/ml. Gradually 7ES
We were able to obtain cells that stably proliferated up to a cell concentration of 12-14 x lO' cells/ml in B.

From microscopic observation, morphology by cell staining method, and observation of cutting molds,
Since the cell line was homogeneous, it was designated as AH-01 strain and cryopreserved.

The number of cells was counted using a Coulter Counter cell counter (manufactured by Coulter Electronics).

Example 2 AH-01 grown and cultured for 4 days in serum-free medium ITESB
Cells were pre-cultured with 5 Qm of serum-free medium ITBSB.
1 was dispensed into two 250 ml plastic flasks, and the cells were transplanted to a culture vessel with a total number of 2 x 105 cells/m1, and incubated in a cell culture incubator for 4 hours.
Cultured for 1 day. The average number of cells in the two flasks is 14.
．． It was 5 1xlO/ml.

After collecting all the cells, add 14 mj of ITESB medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide.
2. After one month, remove one tube from the cell suspension and thaw it in warm water at 37°C.
Cells were washed twice with TESB medium to remove fetal bovine serum and dimethyl sulfoxide. When living cells were observed using dead cell staining using erythrosin B89
．． It was 2%.

Example 3 Instead of the serum-free medium ITESB's basal medium RPMI-1640, various basal media shown in Table 2 were used, and 50 ml of each was dispensed into 250 m, g capacity plastic flasks. A cultured in ITESB for 4 days in advance
H-Of cells were collected at an initial cell concentration of 2.0 x 10' cells/
The cells were transplanted to a volume of 1.0 ml and cultured statically in the same manner as in Example 2. The number of cells proliferated after 5 days of culture was determined by Coulter.
It was measured with a counter cell counter and the results shown in Table 2 were obtained.

Table 2 Basic medium Growth rate (cell number) Williams medium 9. QX105 cells/m j2 Example 4 AH-01 cells were prepared by culturing in the same manner as in Example 2. Since the total number of cells was 135 x 10' cells, these were suspended in 15 ml of ITESB medium in 10 culture dishes with a diameter of 10 cm, and then phorbol ester (12-0-tetradeca2-noyl phorbol- 13-
acetate) at 0.1゛1: μg/m It I,
After culturing for 18 hours in a cell culture incubator to which G was added to give Z, the cells were collected with a pipette and washed twice with phosphate buffer. 10 cells
After suspending in 0 ml of ITESB medium (2 plastic culture dishes with a diameter of 15 cm) and culturing in a cell culture incubator for 3 days, the culture supernatant was collected by centrifugation, and the above leukemia cell differentiation-inducing factor was suspended. When the activity was measured by an assay method, 500 units/m (l) of leukemia cell differentiation-inducing factor were produced.

[Brief explanation of drawings]

Claims (1)

[Scope of Claims] A cell line derived from human myeloid leukemia cells that can be grown in a serum-free medium and having the following characteristics Aa) Origin: isolated from human myeloid leukemia cells THP-1 b) Morphology: Diameter 11 ~14 microns, almost spherical or monocytic cell-like c) Chromosome number: Distribution mode of chromosome number characterized by showing a modal number of 88 chromosomes d) Subculture: Infinite subculture e) Functional characteristics: Induced to differentiate into non-proliferative cells with macrophage-like functions and produces macrophage-derived monokines. f) Cell proliferation: Proliferates well in suspension in serum-free medium. Generation doubling time is 31 ± 6 hours. g) Storage conditions: Cryopreserved at -80°C to -196°C h) Serum requirement: Does not require serum for growth.