JPH0348767A - Method for testing drug sensitivity - Google Patents

Abstract

PURPOSE:To test the drug sensitivity of cancer cells by bringing the cells contg. the cancer cells into reaction with antihuman fibroblast antibodies and complement and subjecting the same to cell culture together with a drug. CONSTITUTION:The drug sensitivity of the cancer cells is tested without being affected by the fibroblast or macrophage by bringing the cells contg. the cancer cells into reaction with the antihuman fibroblast antibodies or antihuman macrophage antibodies and complement. The bactericide having an effect is determined by culturing the desired bacteria in media contg. various bactericides and investigating the presence or absence of colony formation. As a result, the bactericide contained in the culture liquid in which colonies are not formed is used as the sensitive drug for the bacteria. A liquid monolayer culture method with which the culture is relatively easy is preferably adopted at this time.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for testing drug sensitivity of cancer cells.

[Conventional technology]

As a method of drug sensitivity testing of cancer cells known so far, cells obtained from surgical sections or biopsies are made into a cell suspension by a cell culture medium reflux method or an enzyme treatment method.
There is a method of inoculating the cancer cells together with a culture solution containing an anticancer drug into a culture plate, culturing for several days to more than 10 days, and then performing acclimation or staining such as Giemsa to determine whether or not the cancer cells are sensitive to the anticancer drug used. It had been taken.

[Problem to be solved by the invention]

Anticancer drugs are administered quantitatively for the treatment of cancer patients. It is well known that it is not easy to select an anticancer drug suitable for each individual patient, and that incorrect selection can lead to adverse effects due to side effects.

Therefore, a drug sensitivity testing method for cancer cells has been developed as a method for selecting the most suitable anticancer drug for each cancer patient.

However, in conventional drug sensitivity testing methods, the highly proliferative normal fibroblasts contained in the sample proliferate more than the cancer cells, suppressing the proliferation of the target cancer cells, or surrounding the cancer cells. As a result, the determination of susceptibility was unclear and accurate results could not be obtained. For this reason, drug susceptibility testing methods are currently not widely used, even though they are said to be able to find anticancer drugs that can be expected to have the highest therapeutic effect on individual patients.

[Means to solve the problem]

The present inventors have solved these conventional problems (results of research have shown that by reacting cells, including cancer cells, with anti-human fibroblast antibodies or anti-human macrophage antibodies and complement, the fibroblasts or macrophages in question The inventors discovered that a method for testing the drug sensitivity of cancer cells can be carried out without being affected by the effects of cancer, and completed the invention.

Generally speaking, drug susceptibility testing methods are used to examine the resistance of pathogenic bacteria to drugs.

For example, penicillin-resistant strains of Staphylococcus are difficult to sterilize with penicillin, and it is necessary to find an effective sterilizing agent other than penicillin and use that to sterilize them. At this time, in order to determine which disinfectant is effective, the target bacteria are cultured in various media containing the disinfectant, and the presence or absence of colony formation is examined. As a result, the bactericidal agent contained in the culture solution in which no colonies are formed becomes a drug to which the bacterium is susceptible.

The basic principle of the present invention is the same as the above-mentioned method for testing the susceptibility of fungicides.

In carrying out the present invention, cells including cancer cells are cultured in a medium containing various anticancer agents. Various culture methods can be used, but it is desirable to use the liquid mole layer culture method, which is relatively easy to culture.

That is, cells including cancer cells are suspended, and the suspension is diluted with approximately 10%
Eagle Alpha containing tallow serum
a) Dilute with MEM culture solution and culture. Cells, including cancer cells, are cut into small pieces in a culture medium, and each cell is suspended individually in an enzyme solution containing trypsin or the like. An anti-fibroblast antibody or an anti-macrophage antibody is added to this suspension and allowed to react. The antibody concentration at this time is 0.01 μg to 10μg per 111ji′ of the cell suspension, preferably 1 μg to 1μg.
■It is. The reaction temperature at this time is a temperature at which cells function normally, and is usually room temperature to 45°C.

The antibody used here may be a polyclonal antibody obtained by immunizing goats or rabbits, or a monoclonal antibody obtained by cell fusion. Monoclonal antibodies can be easily produced, for example, according to the method described in "Monoclonal Antibodies and Cancer" (■Science Forum). When using antibodies, cross-reactivity can be avoided by using antibodies purified by affinity chromatography.

Incidentally, in the antibody reaction, an anti-human fibroblast antibody and an anti-macrophage antibody can also be used simultaneously.

Complement is added at the same time as the antibody or 5 to 6 minutes after the antibody reaction.
It is added after 0 minutes and allowed to react. The complement used is
Each component may be used separately or fresh serum may be used as it is. When using complement in serum, in order to remove foreign antibodies contained therein, it is necessary to react the sample cell suspension with the complement serum at a low temperature (0 to 4° C.) to absorb the foreign antibodies. Generally, commercially available complements are sufficient, and may be diluted and used as necessary. The amount of complement used is 1 to 1 per 11 of cell suspensions.
00 units (1 ttrlc guinea pig serum), preferably 3 to 8 units. Preparation of fresh complement is performed using the Immunology Experimental Procedures (
It can also be obtained by the method described in the Japanese Society of Immunology, etc. Add complement and then heat to 20°C to 4°C while stirring.
After reacting for 10 to 60 minutes or more at 0°C, preferably 35°C to 39°C, the culture solution used in the reaction is replaced with a culture solution containing an anticancer agent, and the culture is continued for another ten or more days. Cultivation is generally performed at room temperature to 45°C. As a result, it is determined whether or not there is drug sensitivity by examining the formation of colonies. The cells may be stained in advance with an appropriate staining solution to facilitate determination.

[For production]

The present invention is a method for testing drug sensitivity of cancer cells, in which cancer cells can be stably cultured and the cancer cells are not affected at all.

〔Example〕

The present invention will be explained below with reference to Examples.

Example 1 1 g of a solid tumor obtained by biopsy was kept aseptic and washed with medium (10 U heparin, Eagle Alpha MEM culture solution), then the tissue was cut to 1 mm or less with scissors, and treated with 01075% collagenase and 0005% D
F containing Nase and 10% tallow blood fi (Fe2)
-12 culture solution was stirred at 37°C for 16 hours. This was centrifuged at 11,000 rpm for 10 minutes), and alpha MEM culture medium (10% FCS, 100 J/ml peninrin strebtomycin, 5 μg/m4 insulin, 10 μl
g7mj2 transferin, 0.27H/
mi! estradiol, 10.5 μg/mj!
Hydrocortisone, 5μg/mj!
EGF (containing 0.6% methylcellulose). Next, 1 μ of a sterile anti-fibroblast mouse monoclonal antibody and 5 units of fresh guinea pig complement fluid that had previously absorbed foreign antibodies were added to each ml of this cell suspension at 37°C.
The mixture was heated while stirring for 1 hour. Centrifuge this again 11
000rp.

10 minutes), alpha MEM culture solution (10% FC5,1,
00U/ml penicillin-streptomycin, 5μg
/+++f insulin, 10 t1g/lIl tra
nsferin, 0.27 μg/m1estra
dioL 10.5ug/ml hydroort
isone (containing 5 μg/mAEGF, 0.6% methylcellulose). The cells were diluted to 2×10 3 cells and plated in a 24-well plate. After 24 hours, the same culture medium was replaced once, and the cells were cultured at 37°C in a 5% CO2 environment for 13 days. As a control, the cells were cultured in the same manner as above without adding antibody or complement.

After keeping 1 g of solid tumor obtained from the surgical materials of 41 patients in a sterile state and washing it with medium (IOU heparin, Eagle Alpha MEM culture solution), this tissue was cut into pieces of less than 1 piece with scissors, and the size was 0.075. % collagenase, 0.005% DNA
F-12 with se and 10% tallow serum (Fe2)
The culture solution was stirred at 37°C for 16 hours. Centrifugally 1
000rpn+, 10 minutes) Eagle Alfy M E
M culture solution (10% FCS, 100U/ml penicillin-streptomycin, 5 μg/1lIll insulin, 10 μg/ml transferin,
0.27 μg/ml estradiol, IQ,
5 μg/lsi, hydrocortisone,
5μg/mj! EGF (containing 0.6% methylcellulose). Next, 1 m of this cell suspension
1 volume of a sterile anti-fibroblast mouse monoclonal antibody and 5 units of Mormont fresh complement fluid that had previously absorbed foreign antibodies were added per A', and the mixture was heated at 37°C for 1 hour with stirring. Centrifuge this again at 11,000 rpm for 10 minutes), and
7MEM culture solution (10% FCS, 100U/rB
f Beninrin Strebutomain, 5μg/mj! Insulin, 10 μg/rnl transferin
, 027 pg/ml estradiol, 10
．． 5ug/mi! hydrocortjsone.

5 μg/m4 EGF, containing 0.6% methylcellulose). Place these cells in a 24-well plate.
The cells were diluted to become X103 cells and plated. After 24 hours, the same culture medium was replaced once, and each anticancer drug (adriamycin 0.001-0
．． 03μg/m4, 5-fluorouracil 0.05~
The cells were cultured in the above cell culture medium containing 0.7 μg/ml'). As a control, the cells were cultured in the same manner as above without adding antibody or complement.

Table 1 shows the results of the drug sensitivity testing method for cancer tissues obtained as various surgical materials, with and without treatment with antibodies and complement.

Table 1 shows no macrophages and can be said to be an extremely effective method for testing drug sensitivity of cancer cells.

[Brief explanation of drawings]

Figure 1 shows 13 cells after anti-fibroblast antibody and complement treatment.
This is a stained image after culturing for one day. FIG. 2 shows a melanoma cultured for 13 days without anti-fibroblast antibody and complement treatment.

Claims (8)

[Claims] (1) A method for testing the drug sensitivity of cancer cells, which involves reacting cells containing cancer cells with an anti-human fibroblast antibody and complement, and then culturing the cells together with a drug. (2) A method for testing drug sensitivity of cancer cells, which involves reacting cells including cancer cells with an anti-human macrophage antibody and complement, and then culturing the cells together with a drug. (3) The method according to claim 1, wherein the anti-human fibroblast antibody is a polyclonal antibody. (4) The method according to claim 1, wherein the anti-human fibroblast antibody is a monoclonal antibody. (5) The method according to claim 2, wherein the anti-human macrophage antibody is a polyclonal antibody. (6) The method according to claim 2, wherein the anti-human macrophage antibody is a monoclonal antibody. (7) The method according to claim 1 or 2, wherein the drug is an anticancer drug. (8) The method according to claim 1 or 2, in which an anti-human fibroblast antibody and an anti-human macrophage antibody are used simultaneously.