JPH01265029A - Agent for suppressing proliferation of human malignant tumor cell - Google Patents

Abstract

PURPOSE:To provide the subject proliferation suppressing agent composed of a medium prepared by culturing a malignant tumor cell in a serumfree medium until the death of said cell, exhibiting no lethal effect on normal cell and free from the danger of acting as an antigen in the living body. CONSTITUTION:A malignant tumor cell is cultured and proliferated under the culture condition of normal higher animal cell and washed once with a culture liquid free from serum to remove the serum component and obtain a culture liquid free from serum and having a glucose concentration of 3-5g/l (culture liquid). The malignant tumor cell is cultured in the culture liquid prepared by the above process at 34-36 deg.C and 100% relative humidity in an atmosphere having a carbon dioxide gas concentration of 5% without exchanging the medium until the death of the malignant tumor cell. The objective stock liquid of the agent for suppressing the proliferation of malignant tumor cell can be produced by separating the cells from the cultured liquid by centrifugal separation.

Description

[Detailed description of the invention] Industrial applications The present invention relates to a human malignant tumor cell proliferation inhibitor.

Conventional engineer's malignancy II! A human malignant tumor cell proliferation inhibitor, which is obtained by culturing tumor cells in a medium supplemented with glucose at 30°C to 37°C and removing malignant tumor cells from the medium, is known as JP-A-59-162889. It is being

Problems to be Solved by the Invention An object of the present invention is to provide a human malignant tumor cell growth inhibitor that has a much greater growth-inhibiting effect on malignant tumor cells than those shown in the prior art. be.

Means for Solving the Problems This invention involves culturing and proliferating human malignant tumor cells under normal culture conditions for higher animal cells, washing them once with a serum-free culture medium, removing the serum valve, and increasing the serum-free glucose concentration. 3-5g
#! (Culture solution) using a culture solution prepared at 34° to 36°C.
°C (preferably 35 ± 1 °C), relative humidity 100%, 5%
malignant 1i without medium exchange in carbonate atmosphere.
After culturing (about 1 week) until m cells die, 10,00
A stock solution of malignant tumor growth inhibitor is obtained by centrifugation at 0 RPM for 10 minutes to remove malignant tumor II cells.

This invention discovered that in order to have the ability to suppress the growth of large malignant H tumor II cells, it is necessary to culture tumor cells until they die without changing the medium, and this cannot be achieved without the death of tumor cells. This was then used as a malignant tumor cell growth inhibitor.

Even if malignant tumor cells are cultured while changing the medium, they hardly produce anything that has the ability to suppress the growth of malignant tumor cells, and even if they can be sensitized with Sendai virus and produce physiologically active substances, they will still be sensitized. It is impossible to generate it without it. In addition, the stock solution of the malignant tumor growth inhibitor according to the present invention contains only amino acids, vitamins, glucose, minerals, which are nutrients used in culture, fetal bovine serum components with a slight R, and substances released from malignant tumor cells. , does not contain any substances that may have a negative effect on normal cells.

The glucose concentration in the culture medium should be at least 2 (1#!) to obtain stable maximum antitumor activity;
In another experiment (Experiment 2), the amount of glucose consumed was determined to be 2.5 (
1#! Therefore, the glucose concentration is adjusted to 4 to 5 g/l.

Furthermore, culturing at a temperature of 35°C shows the highest activity.

This malignant tumor cell proliferation inhibitor differs from high molecular weight substances such as Cytolyne, which is composed of glycoproteins, and has a high molecular weight of 300.
It is an extracted material after the death of malignant tumor cells with a concentration of 0 or less.

The human malignant tumor cell proliferation inhibitor of the present invention kills malignant tumor cells in vitro, but has no lethal effect on normal cells, only suppressing their proliferation, and has a small molecular barrier, so it can be used as an antigen in vivo. There is no fear that it will work, and it seems to have almost no side effects like conventional anti-tumor drugs.

There is no need to specifically limit the malignant tumor cells used in the present invention.

Example 1 Malignant tumor cells, such as human Be
After culturing ta Cells (uterine cancer cell line) until the container is saturated, the container is washed once with a serum-free medium to remove serum.

Next, a serum-free medium containing glucose added to the commercially available synthetic medium Eagle MEM at a concentration of 5Q/fl (medium) was added, and the cells were incubated at 35°C, 100% humidity, and 5% CO2 in the air without medium exchange. The cells are cultured for approximately one week until they die.

The medium is collected and centrifuged at 10.00 ORPM for 10 minutes to separate and remove malignant tumor cells to obtain a stock solution of the malignant tumor cell proliferation inhibitor.

Example 2 10% in a synthetic medium such as Eagle's MEM or in a medium to which glucose has been added to a concentration of 5 g/l.
After culturing malignant tumor cells in a medium supplemented with tallow serum until the container is saturated, the cells are washed once with a serum-free medium to remove serum.

Next, glucose is 5111#! (Medium) Add serum-free medium added to the commercially available synthetic medium Eagle MEM so that the temperature is 11 degrees, 35 degrees Celsius, 100% humidity, 5% CO211.
The cells are cultured for approximately 1 week until the cells die, without changing the medium, in the air at a temperature of 30°C.

Collect the medium and centrifuge at 10.00 ORPM for 10 minutes.
Malignant tumor cells are separated and removed to obtain a stock solution of a malignant tumor cell growth inhibitor.

Experiment 1 Differences in the ability to suppress malignant tumor cell proliferation depending on the presence or absence of medium exchange during serum-free culture.

A. Comparative Inhibitor ■ Inhibitor cultured by the method of Examples 1 and 2 without replacing the medium until m cells died.

(2) In Examples 1 and 2, the inhibitor was cultured for 10 days without culturing the cells until they died, with medium replacement every other day.

B. Commercially available synthetic medium prepared by adding 10% tallow serum in a plastic petri dish with a diameter of 15111111, such as Eagle's MEM111j! and He1la Ce1ls1×104
After culturing at 35°C for 24 hours, the medium was replaced with a medium containing each of the inhibitors ① and ②.From then on, the medium was replaced with the same medium every other day, and the growth status of m. ,1lell
The number of viable cells was counted.

In addition, as a control, a similar operation was performed using a medium with the same composition that did not contain an inhibitor.

Table 1 Table 1 shows the results, and the suppression rate is It is indicated by.

Experiment 2 Optimum glucose concentration A during malignant tumor cell culture, method for producing inhibitors Culture-proliferated 1llella Ce1s were washed once with serum-free medium to remove serum, and 2x107 or more 11ella Ce1s were placed in a culture bottle with a bottom area of 150cn+2.
ls and serum-free medium to which glucose was added at a concentration of 1, 2, and 5 g per 11 of the medium, respectively.
In addition, the cells were cultured at 37° C., 100% humidity, and 5% CO2 air for about one week without changing the medium until the cells died.

Each medium was centrifuged at 10.00 ORPM for 10 minutes to remove tumor H cells, and the glucose concentration was 1°2 and 5Q1.
0, and cultured to obtain a sample stock solution.

B. Assay method for inhibitors: A commercially available synthetic medium containing 10% tallow serum in a 15 mm diameter plastic petri dish, such as Eagle's MEMI.
111j! and He1la Ce1ls1 Measured on day 1.

In addition, as a control, a similar operation was performed using a medium with the same composition that did not contain an inhibitor.

C4 Results Table 2 shows the results, and shows the inhibition rate in the case where no inhibitor was added as a control. Based on the glucose concentration of a commercially available synthetic medium of 1g/71 hours, the number of viable cells is 2g/j! 1/3 hour.
1/4 at 5g71 hours. The suppression rate will be three times and four times higher, respectively.

In addition, since it has been confirmed in another experiment that the required amount of glucose is 2.5 g/l of the medium, 4 g/l is set as the optimum level.

Table 2 Experiment 3 Malignant II! Optimal Temperature A for Tumor Cell Culture: Production of Tragic Tumor Suppressor at 25°C, 35°C, and 42°C Culture grown 1llella Cells were washed once with serum-free medium to remove serum, and cultured with a basal area of 150c12. Eagle's ME as a commercially available serum-free synthetic medium in bottles
M50m 1 with 1lella COIIS2 x 1
Q: Insert 7 or more specimens, and the experimental groups are 42°C, 35°C, and 25°C.
3 groups at ℃, control at 37℃ and relative humidity 90~io
The cells were cultured for about 1 week in an atmosphere of 0% and 5% CO2 without replacing the medium until they died.

The culture medium was collected from each group, centrifuged at 10.0008 PH for 10 minutes to remove tumor cells, and incubated at 42°C, 35°C, and 25°C.
Sample stock solutions cultured at 37°C and 37°C were obtained.

B. Inhibitor Assay Method 151m diameter plastic shell I! -He1laCe in Les
After implanting llsl C4 and culturing it in MEM medium supplemented with 10% tallow serum for 24 hours, add amino acids, vitamins, glucose, etc. to the sample solution so that it has the same composition as the medium MEM (commercially available as powder). After that, the medium was changed every other day, and the humidity was kept at 100% for 3 days.
Cultured at 7°C in air with 5% CO2, survival 1 [11tl
The number of cells was counted, and the growth inhibition rate was calculated using .

C4 Results Table 3 shows each culture for the three inhibitor samples. Table 4 shows the growth inhibition rate at fJW degree.
It shows the growth inhibition rate at other temperatures compared to the growth inhibition rate at 35°C, and it can be seen that around 35°C is the optimal temperature for malignant tumor cells.

Table 3 Table 4 Experiment 4 Inhibitory effects of inhibitors on various cells Figures 1 to 4 are growth curves showing oral changes in cell proliferation in the culture medium of various cells. In both experiments, the experimental group used Amicon YM5 (M, W,
After ultrafiltration with 103), a medium supplemented with glucose, amino acids, vitamins, and 10% serum was used as a nutrient source, and for the control group, a fresh medium BME supplemented with the same nutrient source as in the experimental group was used. Initial concentration of cells is 1 x 104ce
In the experiment shown in Figure 1, a culture medium with a diameter of 35 mm was used. In the experiments shown in Figures 2 to 5, the diameter of
) An incubator was used.

The experiment shown in Figure 1 investigated the changes over time in the HRC #1 cell line derived from human renal cell carcinoma. Compared to the control group, the number of cells increased. The experimental group containing substances extracted from the medium after HRC culture clearly showed that growth was suppressed.

Figure 2 shows the results of examining the changes over time in normal human diploid skin cell lines NAS63. For the experimental group, a medium extracted from the culture medium of an established cell line MK derived from human gastric cancer was used. There is. These growth curves clearly show that the experimental group of human gastric cancer-derived established cell line MK has a growth inhibiting effect compared to normal cells.

Experiment 5 Inhibition effect by a mixture of inhibitor and fresh medium Figures 3 and 4 show the concentration response at various mixing ratios of fresh medium made from the culture medium of established cell line HRC derived from human renal cell carcinoma. FIG. 3 shows the concentration-response curve of HRC, an established strain derived from human renal 10-cell carcinoma, and FIG. 4 shows the concentration-response curve of normal human diploid skin line miscellaneous cell NAS63. Both are membranes made by Amicon that sieve molecules of ff1100 from the culture medium after cultivation! FJYM 2 filtered inhibitor was used, and the percentages indicate the proportion of inhibitor medium to the sum of inhibitor and fresh medium. These figures show that although the inhibitor exhibits a growth-inhibiting response to normal cells as well, a stronger growth-inhibiting effect is observed in the established cell line HRC derived from human renal cell carcinoma, which is an example of malignant seed water.

D. Acute toxicity test The cells used in Figures 2 and 4, namely [normal human diploid skin fibroblasts NAS63J, are normal cells collected from the anterior chest skin of a 63-year-old normal male; As is clear from the above, although growth is inhibited to some extent, there is no lethal effect.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the proliferation of established cell line HRC derived from human renal cell carcinoma with the inhibitor of the present invention and fresh medium, and FIG. 2 is a diagram showing the proliferation of normal human diploid cells with the inhibitor of the present invention and fresh medium. Figure 3 shows the proliferation of skin fibroblasts NAS63. Figure 3 shows the concentration response of human renal cell carcinoma-derived established cell line HRC at various mixing ratios of the inhibitor and fresh medium. It is a figure showing the concentration response of normal human diploid skin fibroblast cell NAS63 similar to the figure. Figure 1 Number of days □ Figure 2 Number of days □ Figure 3”/.

Claims (3)

[Claims] (1) A human malignant tumor cell proliferation inhibitor comprising a medium in which malignant tumor cells are cultured in a serum-free medium until the cells die. (2) The glucose concentration of the medium is 3 to 5 g per liter of medium.
The human malignant tumor cell proliferation inhibitor according to claim (1), characterized in that h. (3) The human malignant tumor cell proliferation inhibitor according to claim (1) or (2), wherein the culture temperature is 35±1°C.