CN1678751A - Culture system, detection and analysis system and detection method for cancer cell colonies - Google Patents

Abstract

It is intended to provide a detection and analysis system for cancer cell colonies, whereby an environmental cell carcinogen causing cell carcinogenesis or a chemical or a food inhibiting cell carcinogenesis can be quickly and accurately detected, and a method therefor. Namely, a detection and analysis system for cancer cell colonies using a culture system (1) prepared by the agar overlaying method which comprises a bottom layer (1) being composed of a culture medium, soft agar and a carcinogenesis inducer and/or an anticancer agent and having a definite size and a top layer (12) being composed of a culture medium, soft agar and cells, and further provided with an optical microscope (21), an electronic data conversion unit (22) such as a digital camera and a computer system (23) for processing the data converted by the electronic data conversion unit (22). This computer system (23) has an image analysis software stored therein whereby the electronic data are grayed, calibrated and binarized by subtraction and single threshold and thus the presence or absence of colonies, the number of colonies, the size distribution of colonies, etc. are analyzed.

Description

Culture system, detection and analysis system and detection method of cancerous cell colony

technical field

The invention relates to a detection and analysis system for cancerous cell colonies, a method for detecting cancerous cell colonies, and a culture system for cancerous cell colonies used in the detection method for cancerous cell colonies. More specifically, the present invention relates to a culture system comprising soft agar and implemented by the soft agar multilayer method, and a system for semi-automating cell culture using the culture system and the detection of cancer cell colonies by cultured cells, and its method.

Background technique

Although medical technology has made remarkable progress, cancer has remained the leading cause of death in Japan since 1981. Therefore, the whole society began to pay attention to the prevention of cancer. Therefore, we must first understand the process of cancer on the basis of better prevention of cancer.

Usually, when carcinogenic substances remain in the body, most of the carcinogenic substances can be metabolized through the body's own defense capabilities and excreted from the body. A portion of carcinogens that remain in the body will eventually lead to cancer after going through three stages. First of all, these carcinogens will cause obstacles to cell genes in a short period of time (for example, a few days), resulting in the generation of mutated cells. The mutated cells undergo a incubation period of several years or even decades to become precancerous cells. Finally, precancerous cells proliferate at a rapid rate (eg, over a year) to cause cancer. We will not feel anything until we become such precancerous cells, and it is difficult to detect precancerous cells with current medical technology, so once cancer is diagnosed, there is not much time left.

On the other hand, through epidemiological investigations, it has been known that most of the causes of cancer are caused by the environment we live in, especially the living habits related to diet, and this view has been paid more and more attention; in order to achieve longevity and health society, has paid keen attention to the connection between "diet and cancer prevention".

However, as mentioned above, it takes a long time for a mutant cell to become a cancer cell, so in order to prevent cancer, it is necessary to continuously block this long process of carcinogenesis. At present, it is difficult to verify the cancer prevention effect achieved by improving diet, and only rely on epidemiological investigations to know the preventive effect. Therefore, it is urgent to develop an effective method for testing food materials to prevent cancer and to ascertain its mechanism of action.

As one of the methods to develop and test food materials to prevent cancer and to find out its mechanism of action, a test is described in Proceedings of the National Academy of Science of the United States of America. Volume 98, No. 13, pages 7510-7515 The method comprises calculating and measuring the number of colonies after forming colonies in the agar medium, so as to examine the canceration of normal cells and the development of cancer cells caused by carcinogens.

However, there are problems in the concentration and thickness of the agar medium in the prior art, and a stable number of cell colonies cannot be obtained. Moreover, the cultivation time is relatively long, requiring 20 to 30 days. Moreover, in the prior art, the detection of colonies can be observed with the naked eye under a microscope. Although the number of colonies can be calculated, the size and distribution of colonies cannot be analyzed. Moreover, counting the number of colonies requires a lot of time and physical strength. At the same time, the counting results will vary between individuals due to different experimenters, so there are problems in accuracy, which has become a problem to be solved by this test method. Moreover, in order to be able to use this test method to perform counting, the proficiency of the experimenter is also a problem that the test method needs to solve.

Contents of the invention

Therefore, the object of the present invention is to provide a detection and analysis system and detection and analysis method for cancerous cell colonies, which can quickly and accurately detect cell carcinogens that exist in the environment that will cause cell canceration and drugs that can inhibit cell canceration or food etc.

In view of the above-mentioned problems, the present inventors actively explored, and found that the culture conditions of cancerous cells were standardized, and the cancerous cells cultured according to the standards were stored in the form of digital data in a calculation mechanism such as a computer, and then the stored digital data was processed. By doing special treatment, the above-mentioned problems can be solved and the present invention has been completed.

The first invention is a culture system of cancerous cell colonies, which is characterized in that: it is prepared based on the agar multi-layer culture method, and includes a bottom layer and an upper layer; the upper layer has a thickness of 2.4mm, and contains medium, 0.5%- Soft agar at a concentration of 0.6%, and at least one substance selected from the group consisting of carcinogenic mutagenic substances and anticancer agents; the upper layer has a thickness of 1.6mm and contains medium, soft agar at a concentration of 0.3% to 0.4% and cells.

In the cancer cell colony culture system according to the first invention, it is important that the bottom layer and the top layer each have a predetermined composition. That is, by setting the culture system under constant conditions, reproducible detection and analysis results can be obtained.

In addition, in the first invention, it is preferable that the cells contained in the upper layer of the culture system are neonatal mouse skin cell strain JB6.

JB6 cells are a cell line established from the first generation of cultured cells from neonatal mouse skin. The cells are non-neoplastic and have the ability to form soft agar colonies only by treatment with cancer-promoting agents (TPA, TNF-α, EGF). Moreover, the cells can obtain colony energy after being treated with TPA for a period of time, and then they will be in a pre-cancerous state after being promoted. Therefore, it can be considered that the cells are excellent materials for cancer-promoting detection lines and analysis of their mechanism of action. Moreover, most compounds exhibiting cancer suppressing activity in the detection system of the present invention have demonstrated the same carcinogenic suppressing effect in various stages of animal experiments. Therefore, this detection system is currently being valued as a method capable of rapidly detecting the effect of promoting carcinogenesis suppression.

The second invention is a detection and analysis system for cancerous cell colonies, which is characterized in that: a culture system prepared based on the agar multilayer culture method is used, and the culture system includes a bottom layer with a specified size and an upper layer with a specified size; the bottom layer Contains medium, soft agar, and at least one substance selected from the group consisting of carcinogenic mutagenic substances and anticancer agents; the upper layer contains medium, soft agar and cells; and the detection and analysis system includes an optical microscope, which It is used to observe the cancerous colony cultivated by the culture system; it includes an electronic data conversion mechanism, which is used to convert the colony image developed in the optical microscope into electronic data; and includes a computer system, which is used to process the electronic data The electronic data converted by the conversion mechanism; an image analysis software is used in the computer system, which is used to gray out the electronic data, perform calibration, and perform binarization by subtraction and a single threshold to analyze the presence or absence of colonies selected from At least one condition in the group consisting of , colony number and colony size distribution.

With such a configuration, it is possible to rapidly and accurately detect cellular carcinogens existing in the environment that cause cancerous cells, and to quickly and accurately detect drugs or foods that inhibit cellular cancerous cells.

In the second invention, the computer system obtains analysis results based on standard data obtained from known carcinogenic mutagenic substances, preferably the computer system compares the analysis results of the above known carcinogenic mutagenic substances with The analysis results of at least one substance in the group consisting of substances and anticancer agents are compared.

Using known components as standard substances can easily investigate the cancerous trend of unknown components. In addition, the term "carcinogenesis tendency" used in the present invention means that a substance has either a cancer suppressing action or a mutagenic action.

Furthermore, in the second invention, it is preferable that the above-mentioned known carcinogenic mutagen is selected from the group consisting of TPA, TNF-α and active oxygen source.

The carcinogenicity of these substances is well known, and by using them as standard substances and comparing them with these substances, it is possible to easily grasp the carcinogenicity or cancer suppression of unknown substances.

The third invention is a method for detection and analysis of cancerous cell colonies, which is characterized in that: the detection and analysis system of the present invention is used; The stage of the culture system; (B) the stage of cultivating cancerous cells within the specified time in the above-mentioned culture system according to the specified conditions; (C) transmitting the above-mentioned cultured cancerous cells in the form of electronic image data through a microscope and an electronic data conversion mechanism to the stage of the computer system; and (D) grayscale the transmitted electronic image data, perform calibration, and perform binarization according to subtraction and a single threshold, and then analyze the presence or absence of colonies, the number of colonies and the distribution of colony size The stage of at least one condition in the formed group.

With such a configuration, it is possible to quickly and accurately detect cell carcinogens that cause cell canceration, or drugs or foods that can inhibit cell canceration that exist in the environment.

In the stage (A) of the third invention, it is preferable that the substance having a carcinogenic effect or an anticancer effect is a food or a food-derived substance.

The anti-cancer effect and carcinogenicity of food are the most important issues now. The present invention can detect cancerous tendencies of these foods or substances derived from foods.

Furthermore, in the stage (B) of the third invention, the culture conditions of the culture system are preferably about 37°C and 5% carbon dioxide gas for 15 days to 30 days.

By standardizing the culture conditions, detection can be performed with high precision and high reproducibility under the same conditions.

Furthermore, in the stage (D) of the third invention, it is preferable to carry out the analysis using image analysis software running in the computer system, and perform transparency homogenization of the agar gel, deal with scattered light from the microscope, and then analyze the selected free colonies. At least one situation in the group consisting of shape, size and quantity calculation and measurement and size distribution; and, preferably through image differential processing to identify useless objects and the shape of the colony, and the value of the long axis/short axis is less than or equal to 1.6 Cell colonies Identified as colonies of cancerous cells.

By using this image analysis software, detection can be performed with high precision and high reproducibility. Furthermore, by using image analysis software, inspection can be performed without skilled skills.

Description of drawings

Fig. 1 is a schematic diagram showing the outline of the cancer cell colony detection and analysis system described in this example.

Fig. 2 is a graph showing the results obtained when the concentrated potato juice solution is added to the bottom layer of the culture system using the cancer cell colony detection and analysis system described in this embodiment.

Fig. 3 is a graph showing the results obtained when the blueberry anthocyanin is added to the bottom layer of the culture system using the cancerous cell colony detection and analysis system described in this embodiment.

Fig. 4 is a graph showing the results obtained by using the cancerous cell colony detection and analysis system described in this embodiment to measure the bottom layer of the culture system with rhein derived from the traditional Chinese medicine rhubarb.

Fig. 5 shows that paeoniflorin, mallow pigment, geranium pigment, anthocyanin (cyanidin) and delphinium contained in anthocyanins are added to the bottom layer of the culture system using the cancerous cell colony detection and analysis system described in this embodiment. The curve chart of the results obtained by measuring the situation of flower element.

Detailed ways

Embodiments of the present invention will be described below.

First, Fig. 1 schematically shows the cancer cell colony detection and analysis system of the present invention. It can be seen from Figure 1 that the cancerous cell colony detection and analysis system of the present invention mainly includes a culture system 1 and a detection system 2; the culture system 1 is prepared based on a prescribed agar multilayer culture method; For detection and analysis of cancerous cells cultured through the above culture system.

(training system)

The culture system 1 used in the present invention is prepared based on the agar multilayer culture method, and includes a bottom layer 11 and an upper layer 12; At least one substance in the group consisting of mutagenic substances and anticancer agents; the upper layer 12 has a specified size and contains culture medium, soft agar and cells.

The medium used in the bottom layer 11 can be appropriately selected from known mediums, for example, EMEM (Eagle's minimal basal medium) plus 5% fetal bovine serum can be used. In addition, as the soft agar medium used for the bottom layer 11, a conventionally known medium can also be used, but in consideration of reproducibility, it is particularly preferable to use a medium produced by the same manufacturer. In the present invention, the concentration of the soft agar forming the bottom layer 11 is, for example, 0.5% to 0.6%, preferably a fixed amount of 0.5%. Furthermore, the thickness is also specified as a fixed thickness of, for example, 2.4 mm. To the bottom layer 11 constituted in this way, a predetermined amount of carcinogenic derivative or anticancer agent is added to adjust the bottom layer 11 in the culture system 1 of the present invention.

In addition, for the agar gel concentration and thickness of the upper layer 12 and the bottom layer 11, in order to facilitate microscope shooting, the agar gel needs to have a certain transparency, so it is required to study the agar gel concentration and thickness, and the selection will not affect the cell culture and The concentration and thickness of the agar gel can also be easily photographed by the microscope.

As for the carcinogenic mutagen used at this time, known carcinogenic mutagens whose carcinogenicity has been sufficiently established, such as TPA, TNF-α, active oxygen sources, and carcinogenic substances of unknown carcinogenicity may be added. As described later, add a known carcinogenic mutagen, use the culture system 1 of the present invention prepared under specified conditions to cultivate, use the data that has been tested and analyzed as the standard data, and add an equal amount of unknown components that require detection and analysis After comparing with the standard data, the carcinogenicity of unknown ingredients can be analyzed.

Moreover, anticancer agents, such as various anticancer agents for medicine or food, can also be added to the bottom layer 11 in the same manner. Furthermore, for example, by adding a carcinogenic mutagen and an anticancer agent together to the bottom layer 11, the anticancer effect of the anticancer agent can also be analyzed.

The upper layer 12 in the culture system 1 of the present invention has a predetermined size and contains soft agar at a predetermined concentration and cells for growing cancer cells.

The soft agar used in the upper layer 12 needs to be set to predetermined conditions such as a concentration of 0.3% to 0.4% and a thickness of 1.6 mm.

There is no particular limitation on the cells used in the present invention, preferably JB6 cells.

JB6 cells are a well-known cell line established from the first cultured cells of neonatal mouse skin. (N. H. Colburn et al., Nature, 281, 589 (1979)). The cells are non-neoplastic and have the ability to form soft agar colonies only by treatment with cancer-promoting agents (TPA, TNF-α, EGF). Moreover, the cells can obtain colony energy after being treated with TPA for a period of time, and then they will be in a precancerous state after being promoted. Therefore, it can be considered that the cells are excellent materials for detecting carcinogenesis promotion or analyzing the mechanism of carcinogenesis promotion (refer to Z.Dong et al. ., Proc.Natl.Acad.Sci.USA, 91,609(1994); Z.Dong et al., MolCarcinog., 19,204(1997); J.J.Li et al., Cancer Res., 57,3569( 1997); C. Huang et al., Proc. Natl. Acad. Sci. USA, 95, 156 (1998); and J. Y. Chung et al., Cancer Res., 59, 4610 (1999)). Moreover, in the same detection line as the present invention as described later, most compounds exhibiting cancer inhibitory activity have demonstrated the same tumorigenic inhibitory effect in various animal experiment stages (refer to M.R.Young et al., Proc.Natl. Acad. Sci. USA, 96, 9827 (1999); and Y. Zhao et al., Cancer Res., 61, 6082 (2001)). Therefore, the detection method using this JB6 cell is now being regarded as a method capable of rapidly detecting the effect of promoting and suppressing carcinogenesis. By using the cells in the present invention, measurement errors due to differences in experimenters can be minimized without much time and physical effort.

In the present invention, the soft agar thus constituted is placed on a petri dish and dissolved at a predetermined temperature, for example, 42° C., thereby forming the culture system 1 .

The culture system 1 thus prepared is cultured under predetermined conditions. By specifying the culture conditions of the culture system 1, tissue culture of cancer cells can be performed stably in a short period of time, and reproducible detection and analysis results can be obtained.

For example, in the present invention, the culture condition is about 37° C. in 5% carbon dioxide gas for 15 to 30 days, preferably in an incubator for 15 days, so that reproducible detection and analysis can be implemented.

(Detection Department)

The detection system 2 in the cancerous cell colony detection and analysis system of the present invention mainly includes a microscope 21 , an electronic data conversion mechanism connected to the microscope 21 , namely a digital camera 22 and a computer system 23 .

The microscope 21 used in the present invention is an optical microscope with a known magnification, and is not particularly limited as long as it can be connected to an electronic data conversion mechanism such as a digital camera 22 .

The digital camera 22 is an electronic data conversion mechanism that converts an analog image magnified by the microscope 21 into digital image data as electronic data, such as digital image data in JPEG, TIFF, PCX, and GIF formats. Through the digital camera 22, the digital image data will be stored on the recording medium built in the digital camera 22, and can be stored in the computer system 23 via the recording medium, preferably directly transmitted to In the computer system 23.

The computer system 23 used in the detection system 2 of the present invention mainly includes a memory, an arithmetic processing device (CPU, central processing unit) for running programs stored in the memory, RAM (random access memory), and a digital camera 22 for accessing The interface for the captured digital image data is, for example, a mechanism for accessing a recording medium built in the digital camera 22 such as a USB interface or a memory card reader/writer. The memory of the computer system 23 stores programs such as an operating system and image analysis software, digital image data, and the like.

In the present invention, the culture result read from the digital camera 22, ie digital image data, can be analyzed by image analysis software. The image analysis software can, for example, grayscale the digital image data, perform calibration, and perform binarization according to subtraction and a single threshold, and then analyze the presence or absence of colonies, the number of colonies and/or the distribution of colony sizes. At this time, it is preferable to make the transparency of the agar gel uniform according to the purpose of the microscope photography, and to process the light scattered by the microscope, and it is more preferable to perform image difference processing by a method well known in the technical field, thereby distinguishing between useless substances and cancerous cell colonies. shape. At this time, the cancerous cell colony is usually circular, so if the value of the long axis/short axis in the cancerous cell colony judgment condition is 1.6 or less, the analysis can be carried out after removing elongated useless objects and the like.

In addition, when photographing with a microscope, the amount of light in the central part must be large, and the field of vision around the central part is dark. Therefore, image difference processing is performed in order to correct the influence of the light quantity distribution. As for the method of image differential processing, the agar gel culture dish without adding cells is photographed first, and then the digital image data is used as a control image. If the control image is removed from the sample image to be detected and analyzed, a sample image with uniform brightness can be obtained.

With such a configuration, cells cultured in the culture system 1 under predetermined conditions can be detected and analyzed efficiently and with high reproducibility.

Next, the differences between the detection and analysis of the cancerous cell colony detection and analysis system of the present invention and the existing system are listed.

Differences between the existing system and the system of the present invention

         Prepare samples, culture cells

              ↓

Induce cell carcinogenesis (37°C, 5% CO ₂ , 14 days)

    ↓                                                                                                

  Colony microscope shooting device   Counting with the naked eye under a microscope

↓ ↓ ↓

Result Result Result

(number, shape, size, distribution) (number of colonies only)

Evaluation results: Accuracy improvement, time reduction Calculation of individual differences of measurers

Analysis Items Increased Analysis Items Restricted

Ease of mass analysis Difficulty of mass analysis

                                              

[Example]

Next, using the cancerous cell colony detection and analysis system of the present invention, add food-derived substances with anticancer effects to the bottom layer 11 of the culture system 1 and then culture it. the result of.

(Example 1)

In Example 1, the culture system 1 was prepared by adding potato juice concentrate and TPA to the agar in the bottom layer 11 . The culture system 1 was cultured under predetermined conditions, and the results of detection and analysis using the cancer cell colony detection and analysis system of the present invention are shown in FIG. 2 . In the graph shown in FIG. 2 , the vertical axis represents the canceration inhibition rate, and the horizontal axis represents the concentration of potato juice, and the canceration inhibition rate of each concentration of potato juice is plotted.

According to the graph shown in Fig. 2, it can be known that if the concentration of potato juice increases, the rate of inhibition of carcinogenesis is improved.

(Example 2)

In Example 2, the culture system 1 was prepared by adding blueberry anthocyanins contained in blueberries to the agar of the bottom layer 11 . The culture system 1 was cultured under the prescribed conditions, and the detection and analysis results using the cancer cell colony detection and analysis system of the present invention are shown in FIG. 3 . The graph shown in Fig. 3 is the same as the graph shown in Fig. 2, wherein the vertical axis represents the canceration inhibition rate, and the horizontal axis represents the concentration of blueberry anthocyanin, and the canceration inhibition rate of blueberry anthocyanin at each concentration is plotted.

According to this graph, it can be known that if the concentration of blueberry anthocyanin increases, the rate of inhibition of carcinogenesis is improved.

(Example 3)

In Example 3, the culture system 1 was prepared by adding rhein derived from rhubarb in traditional Chinese medicine to the agar in the bottom layer 11 . This culture system 1 was cultured under predetermined conditions, and the detection and analysis results using the cancer cell colony detection and analysis system of the present invention are shown in FIG. 4 . The graph shown in FIG. 4 is the same as the graph shown in FIG. 2 , wherein the vertical axis represents the canceration inhibition rate, and the horizontal axis represents the concentration of rhein, and the canceration inhibition rate of each concentration of rhein is plotted.

From this graph, it can be seen that when the concentration of rhein derived from rhubarb increases, the carcinogenesis inhibition rate increases.

(Example 4)

In Example 4, culture system 1 was prepared by adding paeoniflorin, mallow pigment, geranium pigment, anthocyanin, and delphinidin contained in anthocyanins to the agar of the bottom layer 11, respectively. The culture system 1 was separately cultured under the prescribed conditions, and the detection and analysis results using the cancer cell colony detection and analysis system of the present invention are shown in FIG. 5 . The graph shown in Figure 5 is the same as the graph shown in Figure 2, wherein the vertical axis represents the canceration inhibition rate, and the horizontal axis represents the concentration of each component derived from anthocyanidin, and the canceration inhibition of each component at each concentration is plotted Rate.

From this graph, it can be seen that when the concentration of each component is increased as a whole, the rate of inhibition of carcinogenesis is improved. Among them, delphinidin has been found to have a particularly high inhibitory effect on cell carcinogenesis.

As described above, as described in Examples 1 to 4, it was found that by using the culture system and system of the present invention, the cancer cell inhibition rate can be quantitatively measured.

[industrial availability]

According to the present invention, it is possible to provide a test method that can quickly and accurately detect cell carcinogens that will cause cell canceration and drugs or foods that can inhibit cell canceration that exist in the environment. Food materials are effective in preventing cancer.

Claims (10)

1. the culture system of a cancerous tumor cell colony is characterized in that: modulation forms based on agar multilayer culture method for it; And comprise

Bottom, it contains the soft agar of substratum, 0.5%～0.6% concentration and is selected from by carcinogenic mutagen and carcinostatic agent is formed at least a material in the group and is had 2.4mm thickness; And

The upper strata, it contains the soft agar of substratum, 0.3%～0.4% concentration and cell and has 1.6mm thickness.

2. the culture system of cancerous tumor cell colony according to claim 1 is characterized in that: the cell that is contained in the described upper strata is newborn mice skin cells JB6 strain.

3. the check and analysis system of a cancerous tumor cell colony is characterized in that: use based on the synthetic culture system of agar multilayer culture method, this culture system comprises

Bottom, it contains substratum, soft agar and is selected from by carcinogenic mutagen and carcinostatic agent is formed at least a material in the group and is had specified dimension, and

The upper strata, it contains substratum, soft agar and cell and has specified dimension;

And described check and analysis system comprises

Opticmicroscope, it is used to observe the canceration colony of cultivating by described culture system;

Electronic data throw-over gear, its colony video conversion that is used for being developed in described opticmicroscope is an electronic data; And

Computer system, it is used to handle described electronic data;

Adopted a kind of image analysis software in the described computer system, it is used for described electronic data gray processing, calibrate, and carry out binaryzation, and then analyze and to be selected from by having or not colony, colony quantity and colony distribution of sizes to be formed at least a situation in the group according to subtraction and single threshold value.

4. the check and analysis system of cancerous tumor cell colony according to claim 3 is characterized in that: described computer system is used for the analytical results and the analytical results that is selected from by at least a material of carcinogenic mutagen and group that carcinostatic agent is formed of more known carcinogenic mutagen.

5. the check and analysis system of cancerous tumor cell colony according to claim 4 is characterized in that: described known carcinogenic mutagen is selected from the group of being made up of TPA, TNF-α and active oxygen source.

6. the check and analysis method of a cancerous tumor cell colony is characterized in that: use according to the described check and analysis of arbitrary claim system in the claim 4 to 6; And comprise

(A) select the stage have the material of carcinogenesis or antitumous effect and modulate described culture system;

(B) condition stage of cultivating cancerous tumor cell at the appointed time according to the rules in described culture system;

(C) by microscope and electronic data throw-over gear, with the described stage that is sent to computer system through the cancerous tumor cell of cultivating with the form of electronic image data; And

The described electronic image data gray processing that (D) will transmit is calibrated, and is carried out binaryzation according to subtraction and single threshold value, and then analysis is selected from by having or not colony, colony quantity and colony size to be formed the stage of at least a situation in the group.

7. the check and analysis method of cancerous tumor cell colony according to claim 6 is characterized in that: in the described stage (A), described material with carcinogenesis or antitumous effect is food or the material that is derived from food.

8. the check and analysis method of cancerous tumor cell colony according to claim 6 is characterized in that: in the described stage (B), the culture condition of described culture system is to cultivate 15 days～30 days in 37 ℃, 5% carbon dioxide.

9. the check and analysis method of cancerous tumor cell colony according to claim 6, it is characterized in that: in the described stage (D), implement described analysis by the image analysis software of in described computer system, being moved, and evenly, after the scattered beam that adjusts the telescope to one's eyes handles, analyze to be selected from and measure and size distribution is formed at least a situation in the group by colony shape, size and quantity Calculation in the transparency that makes agar gel.

10. the check and analysis method of cancerous tumor cell colony according to claim 9, it is characterized in that: in the described stage (D), described analysis is handled the shape distinguish dud and cancerous tumor cell colony by image difference, and the numerical value of major diameter/minor axis is characterized as the cancerous tumor cell colony smaller or equal to 1.6 cell.

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