JPS58320B2 - Method for manufacturing mouse interferon - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing mouse interferon.

Mouse interferon is 5science Vol11
77 (1972), pages 797-799, it is known that although a relatively high activity is induced in normal mouse cells, this activity is extremely low in tumor cells.

However, normal mouse cells proliferate significantly slower than tumor cells, making them unsuitable for large-scale production of interferon.

The present inventor focused on mouse tumor cells, which proliferate at a high rate, and conducted research on a method for producing large amounts of interferon from mouse tumor cells.

As a result, cultured mouse tumor cells can be grown by inoculating them into a nutrient medium that is not supplied with body fluids, or transplanted into the mouse body while receiving the body fluids that serve as a nutritional source. Instead of propagating, it is implanted into the body of a warm-blooded animal other than mice and humans, or the body fluids of that warm-blooded animal are nourished in a diffusion chamber attached to or inside the body of a warm-blooded animal other than mice. Mouse-derived cells are grown while supplied as a source, and interferon is produced with high activity by acting on the obtained mouse-derived cells with an interferon inducer, and the produced interferon is purified and separated and collected to produce mouse interferon. They discovered that it could be easily produced in large quantities.

Furthermore, when using the method of the present invention, unlike when cells are grown using a nutrient medium that is not supplied with body fluids from a living body, a nutrient medium containing expensive serum etc. is not necessary or can be significantly saved. In addition to being extremely easy to maintain and manage during cell proliferation, it also has the characteristic that the activity of the induced interferon produced is significantly higher than when the cells are grown in the mouse body.

Furthermore, according to the present invention, cultured mouse-derived tumor cells are transplanted into the body of a warm-blooded animal other than mice and humans to receive body fluids from that animal, or By housing the warm-blooded animal in a diffusion chamber or the like that can receive the animal's body fluids without transplanting it into the body, and raising the animal normally, the body fluids supplied by the warm-blooded animal can be used as a nutritional source. and the cells can be easily proliferated.

Furthermore, compared to the case where cells are grown in a nutrient medium that does not receive body fluid supply from a living body, when using the method of growing cells using the body fluid of a warm-blooded animal of the present invention,
It has the characteristics of stable cell proliferation, high proliferation rate, and high interferon yield per cell.

The cultured mouse-derived tumor cells used in the present invention may be any cell that can be transplanted into the body of a warm-blooded animal other than mice and humans and easily proliferated. , 20 No. 6 (1975), pp. 616-643, L5178Y cells,
5M36 cells, L1210 cells, FAC-C cells, T3
Cells such as M1 cells, OUMS-2 cells, JTC-11 cells, ELD cells, and Sarcoma 180 cells are freely available.

Among them, L5178Y cells, L1210 cells, OUMS-
Lymphoid cells such as 2 cells and JTC-11 cells are preferred because of their high proliferation rate and high interferon activity.

The warm-blooded animal used in the present invention may be any animal in which cultured mouse tumor cells can proliferate, such as birds such as chickens and pigeons, dogs, cats, monkeys, goats, pigs, cows, horses, Mammals such as rabbits, guinea pigs, rats, nude rats, and hamsters can be used.

Transplanting mouse-derived tumor cells into these animals may cause an unfavorable immune reaction, so in order to suppress the reaction as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, fetuses, or Those in the newborn and early childhood stages are preferable.

Alternatively, these animals may be subjected to pretreatment such as irradiation with 200 to 600 rem of X-rays or gamma rays, or injection of antiserum or immunosuppressants to weaken the immune response before transplantation. good.

In addition, a porous filtration membrane capable of blocking the passage of animal cells without directly transplanting mouse-derived tumor cells into the animal body, such as membrane filter-1 ultrafiltration having a pore size of about 10-7 to 10 m-5, can be used. Diffusion chambers of various known shapes and sizes provided with membranes or hollow fibers are buried in the animal's body, for example in the abdominal cavity, and the diffusion chamber is supplied with body fluids containing nutrients from the animal body. It is also possible to proliferate any of the mouse-derived tumor cells that have been established in culture as described above.

In addition, if necessary, a diffusion chamber that connects and perfuses the body fluid containing nutrients in the diffusion chamber with the body fluid in the animal body is attached to the surface of the animal body, and tumor cells derived from mice are grown in the diffusion chamber. It is also possible to multiply
In addition, by making only a portion of this diffusion chamber removable and replaceable, cells can be multiplied to the fullest lifespan without slaughtering the animal.
It is also possible to further increase the amount of cells produced per animal.

Methods using these diffusion chambers do not allow mouse-derived tumor cells to come into direct contact with animal cells, making it easy to collect only mouse-derived tumor cells. There is no need for pretreatment to suppress the reaction, and various warm-blooded animals can be used freely.

Any method can be used to induce and produce interferon from mouse-derived living cells grown in this manner.

An interferon inducer can be applied to these cells while they are still in the animal body, or the proliferating cells derived from mice can be removed from the animal body and interferon inducers can be applied to them outside the body to induce interferon production. .

Furthermore, when mouse-derived tumor cells are grown in a diffusion chamber, the grown cells are left in the diffusion chamber or removed from the diffusion chamber and treated with an interferon-inducing agent to induce interferon production. It can also happen.

In addition, the interferon inducer used in the present invention may be a known agent such as a virus, bacterium, protozoa, rickettsiae, nucleic acid,
Endotoxin, polysaccharides, etc. are used liberally.

Interferon thus induced and produced can be easily purified and separated and collected by known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, concentration, and lyophilization.

If a higher level of purification is required, it is possible to further combine known methods such as adsorption to ion exchanger, elution, gel filtration, affinity chromatography, isoelectric focusing, and false aerophoresis. It is often possible to obtain interferon of the highest purity.

In the present invention, the activity of interferon is determined by Wagner's
r R,R, Biological 5tndies
ofinterferon, Virology Vo
13 (1961), pp. 323-337, vesicular sunkake virus (V
esi-cular Stomatitis Viru
s) was measured by the plaque half-life method.

In addition, the hemagglutination titer is determined by Journal by J, E, 5alk.
lof Immunology Vol, 49 (1
944), p. 87.

Examples will be described below.

Example 1 Newborn hamsters were previously injected with an antiserum prepared from rabbits using a known method to weaken the hamster's immune response, and mouse-derived tumor cells L1 were subcutaneously cultured into the hamsters.
210 cells were transferred and then the hamsters were housed in the usual manner for 3 weeks.

As a result, the subcutaneous tumor was excised and cut into small pieces, and then suspended in trypsin-containing physiological saline (4°C) to disperse the cells and fractionated.

These cells were added to pH 7, 2, 4 containing 5% v/v of calf serum.
After washing with Eagle's minimal basic medium at 37°C, the cells were diluted in a medium with the same composition to a cell concentration of 107/ml, and partially purified mouse interferon was added at a rate of about 50 units/ml, and incubated at 37°C. It was kept for about 6 hours.

Newcastle disease virus was added to this at a rate of about 300 hemagglutination titer/ml and kept for 20 hours to induce interferon production.

This was centrifuged at about 4°C and about 1000g to remove precipitates such as cells, and the resulting supernatant was adjusted to pH 2.0°0.1M.
Dialyzed against potassium hydrochloride buffer at 4°C for 70 hours, then dialyzed against physiological saline containing pH 7.2, 0.01M phosphate buffer for 12 hours, microfiltered, and concentrated the filtrate to remove interferon. I got the liquid.

This interferon solution was used as a partially purified interferon solution.

As a control, tumor cells generated by transplanting L1210 cells into mice were used, and interferon was induced to be produced in the same manner as in hamsters, followed by partial purification.

The activity of these produced interferons was measured and shown in Table 1.

As is clear from the results in Table 1, very highly active interferon is induced and produced from mouse-derived tumor cells transplanted into hamsters, and the activity of partially purified interferon is also significantly high, compared to control mice. It reached more than 100 times.

Example 2 After injecting antiserum into a newborn hamster to weaken its immune response, mouse tumor cells JTC-11 were injected intraperitoneally into the newborn hamster.
The cells were transplanted, and then the hamsters were raised in the usual manner for 4 weeks.

As a result, cells were collected from approximately 7 ml of ascites fluid, and then treated in the same manner as in Example 1 to induce production of interferon, which was partially purified and concentrated.

The interferon solution obtained had an activity of approximately 1,700,000 units per hamster.

Example 3 After a grown hamster was irradiated with gamma rays of about 300 rem to weaken the immune response, mouse-derived tumor cells OUMS-2 cells were subcutaneously transplanted into the hamster and reared for 3 weeks.

Cells were collected from the resulting tumor in the same manner as in Example 1, and interferon was induced to be produced, which was partially purified and concentrated.

Next, this concentrate was freeze-dried to obtain a powder containing interferon.

The activity of the interferon obtained was approximately 9,400,000 units per hamster.

Example 4 After a grown rat was irradiated with approximately 400 rem of X-rays to weaken the immune response, cultured mouse-derived tumor cells L5178Y cells were implanted into the peritoneal cavity of the rat,
Thereafter, the rats were housed in the usual manner for 2 weeks.

In this intraperitoneal cavity, approximately 5,0
Newcastle disease virus with a hemagglutination titer of 0.00 was injected, and two days later the animals were sacrificed and ascites fluid was collected.

This was partially purified in the same manner as in Example 1, and an interferon solution having an activity of about 300,000 units per rat was collected.

Example 5 Mouse-derived tumor cells OUM were cultured in a plastic cylindrical diffusion chamber with an internal capacity of about 20 m1 equipped with a membrane filter with a pore size of about 0.55 microns.
Two S-2 cells were suspended in physiological saline and implanted into the abdominal cavity of a dog.

After the dog was housed in the usual manner for 4 weeks, the diffusion chamber was removed.

The mouse-derived cell concentration obtained in this way was approximately 4×1
09 ml, which is about 102 to 103 times that when grown in an in vitro nutrient medium in a carbon dioxide gas incubator.

These cells were treated in the same manner as in Example 1 to induce the production of interferon, purified and concentrated, and then lyophilized to obtain a powder having interferon activity.

The interferon activity obtained was approximately 50 per dog.
,000,000 units.

Example 6 A cultured mouse-derived tumor cell, JTC-11 cells, was transplanted into a fertilized chicken egg that was kept at 37°C for 5 days, and then kept at 37°C for 5 days.

Proliferating cells were collected from the eggs, treated in the same manner as in Example 1 to induce the production of interferon, and then purified and concentrated to obtain an interferon solution.

The activity of the interferon obtained was approximately 700,000 units per 10 fertilized eggs.

Claims (1)

[Claims] 1. Cultured mouse-derived tumor cells are transplanted into the body of a warm-blooded animal other than mice and humans, or in a diffusion chamber attached to or inside the body of a warm-blooded animal other than mice and humans. The method is characterized in that it is grown while being supplied with body fluids from a warm-blooded animal, the resulting mouse-derived cells are treated with an interferon inducer to induce interferon production, and the produced interferon is purified and separated and collected. Method for producing mouse interferon.