CN105934513A - Method for obtaining cancer cells - Google Patents

Abstract

The object of the present invention is to sufficiently obtain cells that can be appropriately cultured in the culture process using the method for evaluating the anticancer effect of cancer cells in a short period of time regardless of the type of cancer. The present invention provides a method for obtaining cancer cells used in a method for evaluating the anticancer effect of drugs on cancer cells, and the method for obtaining cancer cells is characterized in that the method includes: (a) chemically slicing a sample from cancer tissue after finely cutting A step of crushing, (b) a step of physically crushing the sample obtained in the step (a), and (c) a step of chemically crushing the sample obtained in the step (b).

Description

How to get cancer cells

technical field

The present invention relates to a method for obtaining cancer cells. More specifically, the present invention relates to methods of obtaining cancer cells used in methods of evaluating the anticancer effects of drugs on cancer cells.

Background technique

Although anticancer agents sometimes act on normal cells and show strong side effects, it is known that the effectiveness of anticancer agents is less than 50% except in some cases, and the effectiveness of anticancer agents varies greatly from patient to patient. Therefore, it is required to avoid unnecessary administration of ineffective anticancer agents, reduce the physical and economic burden on patients, and prevent missed treatment opportunities. Therefore, before a cancer patient is treated with an anticancer drug, an anticancer drug sensitivity test for evaluating whether the anticancer drug to be administered is effective for the cancer patient is very important.

Anticancer drug sensitivity test typically involves in vitro growth of cancer tissue taken out by surgery or the like, administration of various anticancer drugs, and evaluation of life and death of cancer cells.

Examples of the anticancer agent sensitivity test include the SDI method, the HDRA method, the CD-DST method, and the ATP method. For example, the CD-DST method is described in Patent Documents 1 to 11, etc., and it shows a relatively high technology, but it is still an unstable technology for operation at a practical level. In order to perform accurate evaluation in these anticancer agent sensitivity tests, it is necessary to allow cancer cells to proliferate in a good state. For operations to be performed at a practical level as clinical examinations, it is required to reliably complete cell culture, ensure the amount of cells required for anticancer drug sensitivity tests even in biopsies with a small amount of tumor, and allow simultaneous evaluation of multiple samples.

prior art literature

patent documents

Patent Document 1: Japanese Patent No. 2879978

Patent Document 2: Japanese Patent Application Laid-Open No. 3-285696

Patent Document 3: Japanese Patent Application Laid-Open No. 7-31470

Patent Document 4: Japanese Patent Application Laid-Open No. 7-241190

Patent Document 5: Japanese Patent Application Laid-Open No. 10-115612

Patent Document 6: Japanese Unexamined Patent Publication No. 2003-9853

Patent Document 7: Japanese Patent Laid-Open No. 2008-11797

Patent Document 8: Japanese Patent Laid-Open No. 2005-95058

Patent Document 9: Japanese Unexamined Patent Publication No. 2010-227088

Patent Document 10: Japanese Patent Laid-Open No. 2011-115106

Patent Document 11: International Publication No. 2011/090068.

Contents of the invention

The problem to be solved by the invention

As a step before the anticancer drug sensitivity test, a tissue dispersion step in which cancer cells are extracted from the cancer tissue is required. Here, cancer tissue is usually composed of a parenchyma and a stroma. The parenchyma is composed of proliferating cancer cells, and the interstitium is composed of extracellular matrix such as collagen fibers, fibroblasts that produce these extracellular matrices, glycoproteins, mucopolysaccharides, etc. . The hardness of cancer tissue varies. If the stroma is rich in collagen fibers, it will be hard, and if it is rich in parenchymal cells, it will be soft.

In the conventional method, for example, the tissue is minced with a razor or the like until it becomes a paste, and then the extracellular matrix is digested by enzyme treatment, followed by tissue dispersion. However, this mincing process causes direct damage to cells regardless of the amount of stroma, and also causes damage such as desiccation caused by medium depletion. In the case of a sample with a lot of stroma, it takes time to become mushy, so the damage becomes severe. In both cases, the cells cannot be properly cultured in the subsequent culture process.

If the mincing treatment is completed in a short time in order to reduce damage to the cells, the separation of the interstitium and the cells may not be sufficient, and the cells may not be properly cultured in the subsequent culture process.

An object of the present invention is to provide a method for obtaining cancer cells used in anticancer agent sensitivity tests in a less damaged state than conventional methods, regardless of the type of cancer.

solutions to problems

In the conventional method, the cancer tissue is physically minced into a paste with a razor, and the interstitium is cut to expose the cells, and the tissue pieces are cut into small pieces to improve the efficiency of the digestion reaction by enzymes in the next step. efficiency. However, sometimes tumor cells cannot be properly cultured in subsequent steps. As a result of intensive research by the inventors of the present invention, it was found that in the conventional method, the damage to the cancer cells themselves due to the mincing treatment, the damage to the cells due to the depletion of the medium, and the leakage from the damaged cells Adverse effects on the subsequent cultivation process are the reason why proper cultivation cannot be performed.

It was also found that, in the case of a sample with a lot of stroma, it takes more time than usual to mince the cancer tissue with a razor, so that the cells are greatly damaged. Furthermore, even when the tissue is minced with a razor or the like, the cut of the tissue is not sufficient, and the yield of cells suitable for culture may be low.

Based on these findings, the present inventors conducted further studies and found that, unlike the conventional methods, by performing chemical fragmentation, followed by physical tissue fragmentation, and then chemical fragmentation again, it was possible to obtain cancer with less damage regardless of the type of cancer. cell. In more detail, the method of the present invention carries out crushing through the process including the following treatment: firstly, chemical treatment such as enzyme treatment is carried out to decompose the interstitium to a certain extent, and after the interstitium is relaxed, physical crushing treatment such as pipetting is used again to disintegrate the interstitium. The tissue is crushed, and chemical treatment such as enzyme treatment is performed again. The method of the present invention does not require mincing, and the cell aggregates can be taken out while maintaining the state. In addition, it is possible to carry out all the treatments after the fine cutting treatment in the medium, and to prevent damage caused by depletion of the medium. Accordingly, it is considered that cancer cells can be efficiently extracted while reducing damage.

That is, the first aspect of the present invention provides a method for obtaining cancer cells used in a method for evaluating the anticancer effect of a drug on cancer cells, wherein the method for obtaining comprises:

(a) a step of chemically disrupting the finely sliced cancer tissue-derived sample,

(b) a step of physically crushing the sample obtained in step (a), and

(c) A step of chemically crushing the sample obtained in the step (b).

In addition, a second aspect of the present invention provides the method according to the first aspect, further comprising a step (d) of physically crushing the sample obtained in the step (c).

In addition, a third aspect of the present invention provides the method of the first aspect or the second aspect, wherein the cancer tissue-derived sample chemically disrupted in the step (a) is a sample that has not been minced.

In addition, a fourth aspect of the present invention provides the method according to any one of the first to third aspects, wherein the chemical fragmentation in steps (a) and (c) is a treatment with an enzyme-containing composition.

In addition, a fifth aspect of the present invention provides the method according to any one of the first to fourth aspects, wherein the chemical crushing in the step (a) is performed for 10 minutes to 60 minutes.

In addition, a sixth aspect of the present invention provides the method according to any one of the first to fifth aspects, wherein the physical crushing in the step (b) is performed smoothly for 10 seconds to 3 minutes.

In addition, a seventh aspect of the present invention provides the method according to any one of the first to sixth aspects, wherein the chemical crushing in the step (c) is performed for 10 minutes to 60 minutes.

In addition, the eighth aspect of the present invention provides the method described in any one of the first to seventh aspects, further comprising culturing the sample obtained in step (c) or (d) on a support to obtain The process of attaching cells to a support.

In addition, a ninth aspect of the present invention provides a method for evaluating the anticancer effect of a drug on cancer cells, the method comprising culturing the cancer cells obtained by the method described in any one of the first to eighth aspects, and allowing the drug to The process of acting on the cultured cancer cells.

In addition, the tenth aspect of the present invention provides a method for evaluating the anticancer effect of a drug on cancer cells, the method comprising embedding the cancer cells obtained by the method described in any one of the first to eighth aspects in a drip block The process of culturing in a drop-shaped (droplet-like) gel and allowing the drug to act on the cultured cancer cells.

The effect of the invention

According to the method of the present invention, suitable culturable cells can be obtained from a cancer tissue-derived sample regardless of the type of cancer in the step of culturing cells used in an anticancer drug sensitivity test. In addition, even when few cancer tissues are available from cancer patients, cells that can be used in anticancer agent sensitivity tests can be provided with a high probability. Furthermore, the time required for the tissue dispersion process can also be shortened to be shorter than that of the conventional technique, so the effect of shortening the time when a large number of experiments are performed can also be expected.

Description of drawings

FIG. 1 is a photograph showing pre-cultured cells in Comparative Example 1 and Examples 1 and 2. FIG. The sample number on the upper part of the photograph corresponds to the sample number described in Table 5.

detailed description

The present invention provides a method for obtaining cancer cells. The obtained cancer cells are used for drug sensitivity tests on cancer cells. The type of sensitivity test using the cells obtained in the present invention is not particularly limited, but a test using three-dimensional culture is preferred. Experiments for performing three-dimensional culture include, for example, evaluation of the results of culture of cancer cells in droplet bulk gels in which cancer cells are embedded. More preferably, the susceptibility test is the CD-DST method.

The type of cancer tissue to be used in the method of the present invention is not particularly limited. Examples include solid cancers such as digestive system cancer, head and neck cancer, breast cancer, lung cancer, cancerous chest/peritonitis, cervical cancer, uterine body cancer, and ovarian cancer. The methods of the invention are particularly suitable for cirrhous cancers. Examples of cancer tissue-derived samples include all or part of surgical material, all or part of a biopsy sample, and the like. As the surgical material, for example, tissue removed during surgical resection for therapeutic purposes can be used. In addition, for the purposes of pathological diagnosis, disease treatment, and prognosis determination, it is possible to use tissues collected by a minimally invasive collection method by a test resection method or a test puncture method. Examples of tissues collected by the minimally invasive collection method include various biopsy samples, thoracoscopic or laparoscopic materials, samples obtained from ascites, pleural effusion, and the like. The sample may be collected from the body and subjected to mechanical separation such as fine cutting with scissors, tweezers, or a razor. In addition, the sample may be a sample washed with a washing liquid containing medium components and antibiotics.

The method for obtaining cancer cells of the present invention includes, as step (a), a step of chemically disrupting a cancer tissue-derived sample.

The chemically crushed sample in step (a) is preferably a finely cut sample. By using the finely chopped sample in the step (a), the recovery amount of the obtained cells can be increased. Particularly in cirrhous cancer, the recovery rate of cells tends to be significantly improved in the case of a finely sliced sample. Finely sliced samples from cancer tissue can be obtained after collection from cancer patients using cutting instruments such as scissors, tweezers, and razors. For the sample from cancer tissue after finely cutting, it is preferably 1 mm to 10 mm square, more preferably 2 mm to 8 mm square, and further preferably 3 mm to 5 mm square. The content in the sample is preferably 80% by weight or more, more preferably 90% by weight or more, and even more preferably 95% by weight or more.

The chemically crushed sample in step (a) is preferably a sample that has not been subjected to shredding treatment. By using a sample that has not been minced, it is possible to obtain cancer cells that are less damaged and proliferate appropriately in the subsequent culture process. According to the step (a) of the present invention, the interstitial tissue of the cancer tissue is decomposed, the binding force of the whole tissue is loosened, and a small physical force can be used for subsequent physical fragmentation. In this case, a sufficient amount of cancer cells can be obtained regardless of the type of cancer tissue.

Step (a) may be performed without or in the presence of a medium. Carrying out step (a) in the presence of a culture medium is preferable because damage to cancer cells due to depletion of the culture medium can be prevented. The state and type of the medium are not particularly limited as long as it is used for culturing, proliferating, and storing cancer cells. Preferably the medium is a liquid medium. Examples of a preferable medium include DF culture medium (DF: a mixed culture medium of 1 volume (volume) of Dulbecco's modified Eagle (DME) culture medium and 1 volume (volume) of Ham's F12 culture medium) and the like.

Since damage to cancer cells can be prevented, the time of step (a) is not particularly limited. It can take longer than previous methods, or it can be shorter. As an example of the time of preferable process (a), 10 minutes - 120 minutes, 10 minutes - 60 minutes, 10 minutes - 30 minutes etc. are mentioned. More preferably, the time of step (a) is 10 minutes to 60 minutes.

The chemical disruption in the step (a) refers to a process of chemically decomposing the intercellular substance to expose the cancer cells. Preferably, the chemical disruption in step (a) is performed by treatment with an enzyme-containing composition.

Enzymes contained in the composition include proteases such as collagenase, dispase, elastase, thermolysin, and trypsin, nucleolytic enzymes (nucleolytic enzymes) such as deoxyribonuclease, and hyaluronidase. These enzymes may be used alone or in combination. Preferably the composition contains collagenase. As the collagenase, any collagenase derived from Clostridium, actinomycetes and the like can be used. In addition, the purity of collagenase may be arbitrary, but it is preferable to contain crudely purified collagenase. Enzyme activity can be appropriately adjusted by those skilled in the art in consideration of the type of cells, time required for treatment, and the like.

The treatment time by the enzyme-containing composition is not particularly limited. Examples of preferable treatment time include 10 minutes to 120 minutes, 10 minutes to 60 minutes, 10 minutes to 30 minutes, 15 minutes to 30 minutes, and the like. The temperature of the treatment with the enzyme-containing composition can be appropriately adjusted by those skilled in the art according to the enzyme used. Examples of preferable processing temperature include 30°C to 40°C and 32°C to 38°C. The treatment with the enzyme-containing composition can loosen interstitial bonds, and then effectively expose cancer cells from a cancer tissue sample without becoming a paste. Therefore, even when the target cancer is a sclerosing cancer having a hard stromal tissue, strong physical treatment is not required to make a paste, and cancer cells with little damage can be obtained regardless of the type of cancer tissue.

The method of the present invention includes, as the step (b), a step of physically crushing the sample obtained in the step (a). Physical fragmentation can be performed with or without a knife such as a razor. For example, it can be performed by pipetting or mixing using a device. The physical crushing by pipetting can be performed using a disposable straw or the like. More specifically, the physical crushing by pipetting can be performed by placing the sample in a container such as a centrifuge tube, and performing multiple times, for example, 10 to 20 times of pipetting. In addition, physical crushing by mixing can be performed using an automatic dispersing device. The physical crushing by mixing, more specifically, can be performed by changing the tempo using the program of the device for 10 seconds to 3 minutes, preferably 30 seconds to 2 minutes.

In the method of the present invention, the interstitial tissue of the sample is decomposed by the chemical fragmentation in the step (a), and the bonding force of the whole tissue is loosened, so the physical force used in the step (b) can be relatively weak. By weakening the physical force, the damage to cancer cells can be reduced, and the efficiency of obtaining properly proliferating cancer cells in the subsequent culture process can be improved. In addition, in the present invention, the physical force can be weakened without performing the mincing process, so the tissue dispersion process of the sample can be completed in a short time. Furthermore, since the shredding process must be performed in the conventional process, it must be performed manually and cannot be automated. Step (b) of the present invention can be carried out manually, but the desired result can be obtained without manual operation. Therefore, the step (b) of the present invention can be performed as an automatic processing for a large number of samples, and further shortening of the processing time can be expected.

The physical disruption in step (b) can be performed in the presence of the enzyme-containing composition, culture medium, etc. used in the chemical disruption in step (a). In this case, the treatment of the step (a) and the treatment of the step (b) can be performed in the same reaction vessel, and the method of the present invention can be performed more simply and in a short time.

The method of the present invention includes, as the step (c), a step of chemically crushing the sample obtained in the step (b). The chemical crushing in the step (c) may be the same treatment as that in the above-mentioned step (a). The specific conditions may be the same as the above step (a), or may be changed.

Through the chemical fragmentation in step (c), the interstitium covering the cells can be decomposed, the binding force between tissues of the sample can be weakened, and the cancer cells can be effectively exposed. In the present invention, through the chemical crushing of the step (a) and the physical crushing of the subsequent step (b), the crushed sample in the step (c) is moderately relaxed and becomes a sample that easily exposes cancer cells. Therefore, the step (c) The chemical crushing can be finished in a short time. The time of step (c) is not particularly limited. As an example of the time of preferable process (c), 10 minutes - 120 minutes, 10 minutes - 60 minutes, 10 minutes - 30 minutes, 15 minutes - 30 minutes etc. are mentioned. More preferably, the time of the step (c) is 10 minutes to 60 minutes. When performing the treatment with the enzyme-containing composition, the treatment temperature can be appropriately adjusted by those skilled in the art according to the enzyme used. Examples of preferable processing temperature include 30°C to 40°C and 32°C to 38°C.

The chemical disruption of the step (c) can be directly performed in the reaction vessel used for the physical disruption of the step (b) in the presence of the enzyme-containing composition, medium, and the like used for the chemical disruption of the step (a). In this case, the time for re-supplementing the enzyme-containing composition or culture medium and the time for changing the reaction vessel can be shortened, and the method of the present invention can be carried out more simply. In addition, the time required for the overall process of the method of the present invention can be shortened, the total amount of damage to cancer cells obtained from the sample can be reduced, and the efficiency of obtaining properly proliferating cells in the subsequent culture process can be improved.

The method of the present invention preferably includes a step of physically crushing the sample obtained in the step (c) as the step (d). The physical crushing in the step (d) is the same treatment as that in the above-mentioned step (b). The specific conditions may be the same as the above step (b), or may be changed. In the method of the present invention, the physical fragmentation in the step (d) cuts the interstitial tissue whose binding force has been weakened by the chemical fragmentation in the step (c), and exposes the cancer cells. Since the cancer cells are exposed by the physical disruption in the step (d), even when the chemical disruption in the step (c) is performed for a short period of time, a large number of cancer cells can be obtained from the sample. By completing the chemical disruption in step (c) in a relatively short period of time, damage to cancer cells can be reduced, and the efficiency of obtaining appropriately proliferating cells in the subsequent culture step can be improved. Thus, the step (d) can shorten the chemical disruption time of the step (c) and improve the efficiency of obtaining desired cells.

The physical disruption in step (d) can be performed in the presence of the enzyme-containing composition, culture medium, etc. used in the chemical disruption in step (c). In this case, the treatment of the step (d) and the treatment of the step (c) can be performed in the same reaction vessel, and the method of the present invention can be performed more simply and in a short time.

The above steps (a)~(c) and (d) can be carried out continuously if necessary, or the next step can be carried out after a few minutes to several days after the end of a certain step. Therefore, the time it takes to disperse cancer cells by the method of the present invention is not particularly limited. In a certain embodiment, according to the method of the present invention, the time for distributing cancer cells can be shortened compared with conventional methods. Examples of the time for dispersion treatment in the present invention include 20 minutes to 6 hours, 20 minutes to 3 hours, 20 minutes to 2 hours, 20 minutes to 1 hour and 30 minutes, and 20 minutes to 1 hour.

In the present invention, the time from finely dissecting the cancer cells to adding them to the culture medium is not particularly limited. In a certain embodiment, according to the method of the present invention, the time from finely dissecting the cancer cells to putting them into the culture medium can be shortened compared with the conventional method. In the present invention, the time from finely dissecting the cancer cells to putting them into the culture medium is not particularly limited, and examples thereof include less than 6 hours, less than 3 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, Less than 5 minutes, less than 3 minutes, less than 1 minute, etc.

By the method of the present invention in which the above steps (a) to (c) and, if necessary, step (d) are performed, cancer cells with less damage than conventional methods can be obtained. The cancer cells can be cultured to obtain cancer cells for anticancer agent sensitivity tests.

In a specific embodiment, by culturing the cells obtained by the method of the present invention on a support to obtain cells attached to the support, cells that can proliferate appropriately in subsequent culture steps can be obtained. By obtaining cells attached to the support when culturing on the support, mesenchymal cells, dead cells, damaged and poorly proliferative cells contained in the sample can be removed, and in the subsequent culture, it can Proliferation is performed in a state suitable for drug susceptibility testing, and a composition containing cancer cells that can be successfully evaluated for anticancer effects is obtained with a high probability. The support for culturing cancer cells may be layer-coated with cell adhesion factors. Examples of cell adhesion factors preferably include extracellular matrices such as various types of collagen, fibronectin, laminin, vitronectin, cadherin, gelatin, peptides, and integrins. These cell adhesion factors One type may be used alone, or two or more types may be used in combination, but from the viewpoint of further improving cell adhesion and cell extensibility, it is more preferable to use various collagens, and among various collagens, it is particularly preferable to use type I collagen or IV collagen. type collagen. The cell adhesion factor coated on the surface of the support may be the same as the gelling agent in the drop block gel.

Cells adhering to the support can be obtained by, for example, removing the culture medium containing blood cells and unnecessary cell components, and then adding a cell detaching agent to detach the cells adhering to the support. As a cell detaching agent, EDTA-trypsin etc. are mentioned, for example. In the case of a coating on the support, detachment of cells adhering to the support can be performed by adding a detachment agent for the coating. When the coated material of the support is collagen, the peeling agent of the coated material is, for example, collagenase. When adding collagenase for cell detachment, before acting on the living cells, the collagen-gel layer to which the living cells are attached will be enzymatically hydrolyzed, thereby causing less damage to the living cells.

The cancer cell-containing composition obtained by obtaining the cells attached to the support can be directly embedded in the drip block gel and cultured without subsequent further isolation or purification, and the sensitivity test of the anticancer effect can be performed. Thus, if a composition obtained by obtaining cells attached to a support is used, sensitivity tests of anticancer effects can be performed with fewer steps, and cancer cells can be obtained from samples derived from cancer tissues. Therefore, the damage to the cells can be further reduced, and the probability of success in the sensitivity test of the anticancer effect can be increased, and a large number of samples can be evaluated.

Another aspect of the present invention provides a method of evaluating the anticancer effect of a drug on cancer cells using the obtained cancer cells. The method includes culturing the obtained cancer cells, and further includes allowing target drugs to act on the cultured cancer cells. It is preferred to allow the drug to act in vitro . In a preferred embodiment, the method includes the step of embedding the obtained cells in a bulk gel and culturing them.

In a specific embodiment, the method of evaluating the anticancer effect of a drug on cancer cells using the cancer cells obtained by the obtaining method of the present invention can be carried out by a method comprising the following steps:

(A) A step of embedding the obtained cancer cells in a bulk gel,

(B) a step of contacting the droplet block gel obtained in the step (A) with a drug-containing solution,

(C) a step of culturing cancer cells in the bulk gel after step (B), and

(D) A step of evaluating the culture result of the step (C).

Examples of the droplet block gel in the step (A) include gels that exhibit a convex surface shape on a flat substrate. One example of the volume of the drop block gel is 3~300 μL, 3~150 μL, 5~100 μL, 15~50 μL, etc. The height of the dripping block gel is, for example, 2 mm or less. Examples of the droplet block gel include, for example, droplet block gels that exhibit a transparency of 1 to 95% with respect to light of 400 nm. The viscosity of the drop block gel is, for example, 50 to 2000 centipoise, 100 to 1000 centipoise, etc. from the viewpoint of both ease of handling and maintaining the shape of the drop block gel. The block gels may contain a gelling agent. Examples of the gelling agent include type I collagen, extracellular matrices such as Matrigel, soft agar, and the like. From the viewpoint of maintaining the shape of the bulk gel, the content of collagen in the bulk gel is, for example, 0.1 to 2.0% by weight. Furthermore, the bulk gel may contain polymer materials such as polysaccharides and other extracellular matrices, medium components such as serum medium, and the like. The drop block gel can be set to, for example, pH 6.2-7.6, pH 6.8-7.4, etc., by using, for example, a buffer solution. The salt strength (salt strength) or ionic strength of the bulk gel is, for example, 100 to 180 mmol, 140 to 160 mmol, or the like.

The embedding of cancer cells in the above step (A) can be carried out, for example, in the following manner: mix the solution containing the gelling agent with components such as cancer cells, ice-cool the resulting mixture, drop it on the substrate with a pipette, Let stand at ~45°C for 30 minutes to 2 hours. The substrate is a substrate having a surface on which a droplet bulk gel can be immobilized. Examples of substrates include Petri dishes such as petri dishes and multi-dish; glass bottles and other commonly used culture containers; glass or plastic sheet-like coverslips or Culture plates such as cell disks, etc. The base material is preferably an optically transparent base material from the viewpoint of easiness in evaluating the results of the culture of cancer cells. The concentration of cancer cells in the bulk gel embedding cancer cells is, for example, 1 to 10 ⁷ cells/mL, preferably 10 ² to 10 ⁶ cells/mL.

The drug in the above step (B) is a drug to be evaluated for its anticancer effect on cancer cells. As a treatment, prevention, or improvement agent for cancer, in addition to anticancer agents that directly act on cancer cells, there are also anticancer agents that do not directly attack cancer cells, but exert their effects by cooperating with immune cells or other drugs in the body. Drugs that inhibit the proliferation of cancer cells, slow down the activity of cancer cells, or kill the function of cancer cells. Examples of anticancer agents include metabolic antagonists such as 5-FU; irinotecan-based anticancer agents such as SN-38; microtubule depolymerization inhibitors such as docetaxel; and platinum agents such as cisplatin and oxaliplatin. preparations, etc. In addition, selective modification of growth factors and their receptors related to cell proliferation, molecules or enzymes related to cell proliferation, cell cycle, apoptosis and signal transduction, etc., to obtain anticancer effects molecularly targeted drugs. For example, there are drugs that act on growth factor receptors such as trastuzumab, cetuximab, and gefitinib; drugs that act on signal transduction of fusion genes such as imatinib and crizotinib; Drugs that inhibit angiogenesis in cancer tissue, such as valizumab, etc. Other drugs include prodrugs of anticancer agents, drugs that regulate the activity of intracellular metabolic enzymes involved in the metabolism of anticancer agents or their prodrugs, immunotherapeutic agents, and the like.

In the above step (B), the contact with the drug-containing solution is preferably carried out in the following manner: for example, on the drip block gel on the substrate, the drug-containing solution is overlapped in multiple layers, and the entire drop block is covered with the drug-containing solution. Gel so that drops of lumpy gel do not dry into a flat dry mass. The drug-containing solution to be contacted may contain a medium such as a serum medium in addition to the drug. The concentration of the drug in the solution is preferably set arbitrarily according to the purpose based on the concentration of the drug near the cells when the drug is administered to the organism from which the cells originate.

The culture of the cancer cells in the above step (C) is preferably carried out by bringing the droplet bulk gel embedding the cancer cells into contact with a liquid medium. The liquid medium to be contacted is preferably a serum-free medium from the viewpoint of suppressing the proliferation of fibroblasts mixed therein. The contact with the liquid medium is preferably carried out by covering the entire droplet bulk gel with liquid medium so that the droplet blockgel does not dry out. The time for culturing is, for example, 1 to 10 days, preferably 3 to 8 days. The droplet bulk gel contacting the liquid medium can be washed away with the drug after contacting with the drug-containing solution.

The evaluation of the culture result in the above-mentioned step (D) is performed, for example, by comparing the number of surviving cancer cells before and after culture, or comparing the number of surviving cells when cultured with drug addition and the number of surviving cells when cultured without adding drug. Compare. The number of surviving cancer cells can be measured by visual observation with a microscope. In addition, the number of surviving cancer cells can also be measured by using a staining method for selectively staining living cells, and measuring the color development caused by the staining. Staining methods for selectively staining living cells include staining methods utilizing phagocytosis of cells, such as neutral red staining; and staining utilizing intracellular enzyme activity, such as latex particle staining and fluorescein diacetate staining. method; staining method using other fluorescent reagents, etc. The stained cancer cells can be fixed by formalin fixation or the like. Thereby, the elution of the staining agent can be temporarily prevented, and highly sensitive staining can be performed. The stained block gel can be dried. Thereby, deterioration and deterioration can be prevented. Drying of the bulk gel can be performed, for example, by air drying, forced drying by heating at about 10 to 50° C., or the like. The measurement of color development by staining can be evaluated by photographing the stained bulk gel and digitizing the photographed image. The numerical evaluation may include numerical correction based on the shape of the stained cell image. Cancer cells tend to be massive and form dark images, and fibroblasts tend to be thin and fibrous and form light images. By correcting the numerical value of the blocky stained image, the survival can be measured more accurately and easily. of cancer cells.

Example

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the scope of these examples.

Collagenase activity assay method

The collagenase activity in this specification is measured according to the following FALGPA decomposition activity measurement method.

FALGPA decomposition activity assay method:

The following method is published on the website of Sigma-Aldrich Company (http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-collagenase-using-n-3-2furylacryloyl-leu-gly-pro -ala.html).

(1) Abbreviation:

[Table 1]

.

(2) Principle:

When FALGPA was decomposed into FAL and Gly-Pro-Ala by the action of collagenase, the decrease variable of 345 nm absorbance (A345nm) derived from FALGPA was measured, and the activity was calculated.

(3) Method:

a. Reagents:

(a) Reagent B 50mM Tris (Tricine), 10mM CaCl ₂ , 400mM NaCl, pH7.5 (25°C) buffer:

Dissolve 0.896g of trismethylolglycine (Sigma-Aldrich, T0377), 2.34g of NaCl (Sigma-Aldrich, S9888), 0.147g of CaCl ₂ 2H ₂ O (Sigma-Aldrich, C3881) in 80mL of distilled water, and wash with 1M NaOH Solution (Sigma-Aldrich, S2567), or 1M HCl solution (Sigma-Aldrich, H3162) adjusted to pH 7.5 (25°C), add distilled water to 100mL;

(b) Reagent C 1.0 mM N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala(FALGPA):

Add 9.6 mg of FALGPA (Sigma-Aldrich, F5135) into 20 mL of the solution of A, and stir for more than 30 minutes to completely dissolve it;

(c) Reagent D distilled water:

(d) Reagent E enzyme solution:

Dissolve the enzyme in distilled water to achieve a concentration of 5-10 times that used.

b. Conditions:

Reaction solution pH=7.5, reaction temperature=25°C, absorbance=A345nm, optical path=1cm

c. Reagent composition and operation of the reaction solution:

[Table 2]

.

Put 2.9 mL of reagent B into a detection cell with a light path of 1 cm, and heat to 25°C. After A345nm is stable, add 0.1mL reagent C (blank test) or reagent D (this test), mix immediately and record the decrease of A345nm at 25°C for 5 minutes.

(4) Definition and calculation method of activity unit

Under the above conditions, pH 7.5, 25° C., and the presence of calcium ions, the amount of enzyme that hydrolyzes 1.0 μmole of FALGPA within 1 minute was defined as 1 FALGPA unit (unit). The FALGPA unit is obtained by the following formula,

FALGPA unit/mL={(E1-E2)×3/(F×0.1)}/0.53

The symbols or numerical values in the above formula are as follows.

[table 3]

.

Reference example 1:

In the conventional method, washing of the specimen, finely dissecting the tissue, mincing the tissue, dispersing the tissue, recovering it, pre-cultivating it, and recovering the cells are performed in the following steps 1 to 7.

1. Sample washing:

Specimens are taken from solid cancer tissues such as gastric cancer, colon cancer, pancreatic cancer, and other digestive system cancers, breast cancer, lung cancer, head and neck cancer, cancerous chest/peritonitis, cervical cancer, uterine body cancer, and ovarian cancer in cancer patients. As shown below, remove bacteria adhering to the surface of the specimen. First, three 10 cm Petri dishes were prepared, and 20 mL of antibiotic-containing medium solution (specimen washing solution) was added to each Petri dish. Put the specimen into the specimen washing solution in the first Petri dish to fully wash the specimen surface. Transfer the specimens to the second and third Petri dishes for washing. The sample washing solution used was: piperacillin (manufactured by Toyama Chemical Co., Ltd. , PENTCILLIN for injection) so that its final concentration relative to the base medium (base medium) was 1 mg/mL, and kanamycin (manufactured by Nacalai Tesque Company, Kanamycin Sulfate Reagent) was added to make it relative to the base medium concentration of 0.5 mg/mL, and amphotericin B (manufactured by Wako Pure Chemical Industries, Ltd.) was added to make the final concentration 2.5 μg/mL.

2. Fine cutting of tissue:

Move the washed specimen into a new petri dish, and use scissors and tweezers to quickly and finely cut it on the petri dish, and cut the cancer tissue into 3-5mm squares.

3. Shredded tissue:

Mince the finely chopped specimen in a Petri dish with a razor blade clipped on a needle holder until it becomes a paste. It should be noted that when dealing with multiple specimens, the scissors, tweezers, and needle holders should be washed with 70% ethanol and baked with a burner for repeated use. Add 20 mL of DF culture medium to the minced cancer tissue, and recover the tissue together with the DF culture medium into a 50 mL centrifuge tube. Furthermore, 10 mL of DF culture solution was added to fully recover the tissues attached to the culture dish. Centrifuge at 400 xg for 3 minutes in a tabletop centrifuge.

4. Decentralized organization:

After centrifugation, the supernatant was removed by aspiration. Add 9 mL of DF culture medium to the centrifuged pellet, shake the centrifuge tube to mix and fully untie the tissue pieces. Add 1 mL of tissue dispase enzyme composition adjusted to 10 times the concentration of the prescription, and adjust the tissue dispersion solution of the prescription concentration. 10% FBS (FBS is fetal bovine serum) can be added according to the enzyme composition. Stirring and shaking were performed in a thermostat at 37° C. for about 2 hours using a desktop shaker. Typically, the composition of the added tissue dispase is adjusted such that the adjusted enzyme activity reaches a collagenase activity of 0.01-15 U/mL, preferably 0.03-10 U/mL.

5. Recycling:

Add 10 mL of DF culture medium to make the total amount to 20 mL, and centrifuge at 400×g for 3 minutes to remove supernatant. After removing the supernatant, gently shake the centrifuge tube to loosen the cells. After adding 10 mL of medium, perform a strong pipette to physically loosen the cell clumps. The cell-containing suspension was filtered using a nylon sieve with a pore size of 300 μm. Add 10mL DF culture medium, and wash the centrifuge tube and the nylon mesh thoroughly.

6. Pre-culture:

The cells obtained in the recovery treatment were recovered by centrifugation, and the supernatant was removed by suction. The cell pellet after centrifugation was suspended in 5 mL of the preculture medium (PCM-1) of the Primaster Kit (manufactured by Kurashiki Bosho Co., Ltd.). Inoculate the PCM-1 culture fluid with suspended cells in the collagen/gel/glass bottle. Culture overnight in a CO ₂ incubator. After culturing overnight, the culture medium containing blood cells and unnecessary cell components was removed by suction. Observe the engraftment of tumor cells on the collagen/gel/glass bottle.

7. Cell recovery:

Aspirate and remove the culture medium in the collagen/gel/glass bottle, add 5mL DF culture medium for washing, then add 2mL DF culture medium. Furthermore, 0.2 mL of an enzyme composition for tissue dispersion adjusted to a concentration 10 times the prescribed concentration was added to obtain an enzyme solution with an adjusted prescribed concentration. Shake at 37°C for 15-30 minutes to dissolve the collagen/gel in the vial. Recover the cells stripped from the collagen/gel/bottle into a 50 mL centrifuge tube. It should be noted that when the adhesion of the cells to the glass bottle was confirmed, 3 mL of EDTA-trypsin was added and shaken for 5 minutes. After confirming the detachment of the cells, add 5 mL of 10% serum medium, fully co-wash (co-wash) the inside of the glass bottle, and recover it into a 50 mL centrifuge tube. After adding 10 mL of DF medium to constant volume, the cells were recovered by centrifugation.

Example 1: Dispersion of tissue by manual manipulation

Specimen washing/fine cutting

Cancer tissues of non-small cell lung cancer, gastric cancer, and colorectal cancer were taken out as specimens, and the specimens were washed according to step 1 of the above reference example 1. According to step 2 of the above-mentioned reference example 1, the washed specimen was finely cut into 3~5mm squares.

Organization Distributed Processing

Immediately after finely cutting, 5 mL of reaction solution (a mixture of 4.5 mL of DF medium and 0.5 mL of enzyme solution) was added to 0.3 g of the tissue piece, put into a 15 mL capacity centrifuge tube, and enzyme digested at 37°C for 30 minutes. The enzyme solution was prepared to have a collagenase activity of 0.31U/mL.

After enzymatic digestion, stir the contents by pipetting 10-20 times with a 5 mL disposable pipette. After stirring, the enzyme digestion was carried out again at 37° C. for 30 minutes, and the stirring was repeated.

Add EDTA solution to stop the enzyme reaction. Then, the cell mass was sedimented by centrifugation, and the enzyme solution was removed.

Recovery/Preculture/Cell Recovery

According to steps 5-7 of the above-mentioned Reference Example 1, the cells were recovered.

Example 2: Tissue Dispersion Using Device

Specimen washing/fine cutting

Cancer tissues of non-small cell lung cancer, gastric cancer, and colorectal cancer were taken out as specimens, and the specimens were washed according to step 1 of the above reference example 1. According to step 2 of the above-mentioned reference example 1, the washed specimen was finely cut into 3~5mm squares.

Organization Distributed Processing

For each cancer tissue, cells were recovered in the same manner as in Example 1, except that the tissue dispersion treatment was performed using a gentleMACS Dissociator (manufactured by Miltenyi Biotec). The details of organization dispersion processing using "gentleMACS Dissociator" are as follows.

Immediately after finely cutting, 5 mL of the same reaction solution as in Example 1 was added to the 0.3 g tissue piece, recovered into the C tube of the special kit, and treated with enzyme at 37°C for 30 minutes. After processing, place the C tube on the gentleMACS Dissociator and mix for 30 seconds. Thereby, the tissue that has been digested to some extent is unraveled, exposing the undigested tissue. After mixing, it was further enzymatically treated at 37°C for 30 minutes. After processing, place the C tube on the gentleMACS Dissociator and mix for 30 seconds. Add EDTA solution to stop the enzyme reaction. Then, the cell mass was sedimented by centrifugation, and the enzyme solution was removed.

Recovery/Preculture/Cell Recovery

According to steps 5-7 of the above-mentioned Reference Example 1, the cells were recovered.

Comparative example 1:

Cancer tissues of non-small cell lung cancer, gastric cancer, and colorectal cancer were used as specimens, and cells were recovered according to steps 1 to 7 of Reference Example 1.

Comparison of time required

In Comparative Example 1 and Examples 1 and 2, the times required for washing the specimen, finely cutting the tissue, mincing the tissue, dispersing the tissue, and recovering the tissue are shown in Table 4 below.

[Table 4]

.

As shown in A of Table 4, in the example, it does not require time (no time) until it is put into the culture medium, so the damage to the cells is reduced. In addition, as shown in B of Table 4, in the examples, the enzyme treatment time was shortened by repeated effective dispersion treatment, and the damage to the cells by the enzyme was reduced.

Thus, according to the method of the present invention, cells with less damage can be obtained in a short time if necessary.

Comparison of cell recovery

The amount of recovered cells in Comparative Example 1 and Examples 1 and 2 using cancer tissues of non-small cell lung cancer, gastric cancer and colorectal cancer are shown in Table 5 below. The 3-5 mm square tissue slices after the fine-cutting process of the tissue by the method described in 1-2 of Reference Example 1 were divided into two groups, and the tissue dispersion process described in Comparative Example 1 and the tissue fragments described in Examples 1 and 2 were carried out. Distributed processing of the organization described above. After the dispersion treatment, the cancer cells were recovered according to the method described in 5 of Reference Example 1, and after pre-cultured overnight according to the method described in 6 of Reference Example 1, the cells were recovered according to the method described in 7 of Reference Example 1. The number of viable cells was determined by blue staining.

The tissue weights in the table represent the weight per group of cancer tissues used for cell recovery. The comparison of the number of recovered cells represents the value obtained by dividing the number of cells in Examples 1 and 2 of the same sample by the number of cells in Comparative Example 1. By measuring the number of cells immediately after pre-incubation, the number of uninjured cells can be more accurately determined, as opposed to the number of cells in recovered pasty tissue pieces.

It should be noted that for comparison, data immediately after pre-incubation were used for comparison. The reason why the recovered cells are not used for confirmation is that it is impossible to determine whether the recovered cells are damaged.

[table 5]

.

If the relative difference in the number of recovered cells is less than 25% as the same level, and more than 25% is regarded as one side being more than the other side, then as shown in Table 5, when the method of the embodiment is adopted, the same degree as that of the method of the comparative example is adopted. The same degree or more cells can be recovered from cancerous tissue than in the case of . It was shown that cells with less damage can be obtained according to the method of the present invention.

Observation of pre-cultured cells

Using cancer tissues of non-small cell lung cancer, gastric cancer and colorectal cancer, the pre-cultured cells in collagen/gel/glass bottles obtained in Comparative Example and Examples 1 and 2 were observed with an optical microscope. The results are shown in Figure 1. As shown in FIG. 1 , it was confirmed that the cells obtained in the examples proliferated significantly compared with the comparative examples. It was shown that cells with less damage can be obtained according to the method of the present invention.

Claims (10)

1. the preparation method of cancerous cell used in evaluation medicine is to the method for the antitumaous effect of cancerous cell, it is characterised in that The method includes:

(a) by the operation of the sample chemically fragmenting from cancerous tissue after frittering process,

(b) will by the broken operation of the sample physics of operation (a) gained and

C () will be by the operation of the sample chemically fragmenting of operation (b) gained.

Method the most according to claim 1, wherein, also includes the operation that will be crushed by the sample physics of operation (c) gained As operation (d).

Method the most according to claim 1 and 2, wherein, in operation (a), the sample from cancerous tissue of chemically fragmenting is not Through the sample that chopping processes.

The most according to the method in any one of claims 1 to 3, wherein, the chemically fragmenting of operation (a) and operation (c) be by The process carried out containing enzymatic compositions.

Method the most according to any one of claim 1 to 4, wherein, the chemically fragmenting of operation (a) carries out 10 minutes ~ 60 Minute.

Method the most according to any one of claim 1 to 5, wherein, the physics of operation (b) is broken carries out 10 seconds ~ 3 points Clock.

Method the most according to any one of claim 1 to 6, wherein, the chemically fragmenting of operation (c) carries out 10 minutes ~ 60 Minute.

Method the most according to any one of claim 1 to 7, wherein, also includes by operation (c) or the examination of (d) gained Sample is cultivated on the support, it is thus achieved that be attached to the operation of the cell of holder.

9. evaluating the medicine method to the antitumaous effect of cancerous cell, wherein, the method includes using in claim 1 ~ 8 arbitrary The cancerous cell that method described in Xiang obtains is cultivated, and makes medicine act on the operation of the cancerous cell after cultivation.

10. evaluating the medicine method to the antitumaous effect of cancerous cell, wherein, the method includes to appoint in employing claim 1 ~ 8 The cancerous cell that one described method obtains is embedded in drip in block gel and cultivates, and makes medicine act on the cancer after cultivation thin The operation of born of the same parents.