KR101832133B1 - Method for the Isolation and Proliferation of Primary Lung Cancer Cells - Google Patents

Abstract

The present invention relates to culture conditions for the isolation and proliferation of lung cancer cells effectively from lung cancer tissue of a patient, and a method for screening a patient-specific cancer therapeutic agent using lung cancer cells cultured by the above method. More specifically, 2) culturing the incised tissue of step 1), 3) culturing the lung cancer cells in a size of 40-100 μm in diameter, 3) culturing the lung cancer cells Separating and culturing the lung cancer cells of step 3) in a serum-free N2 medium in a culture dish coated with a coating material to separate and culture only lung cancer cells, and 5) culturing the lung cancer cells of step 3) A method for isolating and propagating lung cancer cells from lung cancer tissue comprising culturing and proliferating lung cancer cells in a medium supplemented with serum and growth factors, And a method for screening a patient-customized cancer therapeutic using lung cancer cells.

Description

TECHNICAL FIELD The present invention relates to a method for isolating and proliferating lung cancer cells,

The present invention relates to culture conditions for the isolation and proliferation of lung cancer cells effectively from lung cancer tissue of a patient, and a method for screening a patient-specific cancer therapeutic agent using lung cancer cells cultured by the above method. More specifically, 2) culturing the incised tissue of step 1), 3) culturing the lung cancer cells in a size of 40-100 μm in diameter, 3) culturing the lung cancer cells Separating and culturing the lung cancer cells of step 3) in a serum-free N2 medium in a culture dish coated with a coating material to separate and culture only lung cancer cells, and 5) culturing the lung cancer cells of step 3) A method for isolating and propagating lung cancer cells from lung cancer tissue comprising culturing and proliferating lung cancer cells in a medium supplemented with serum and growth factors, And a method for screening a patient-customized cancer therapeutic using lung cancer cells.

Lung cancer is a term referring to malignant tumors of the lungs. Cancer cells can be divided into primary lung cancer, which first occurred in the bronchioles or alveoli, and metastatic lung cancer, which occurs in other organs and is caused by proliferation of blood vessels or lymph vessels. In general, primary lung cancer, called lung cancer, is classified microscopically into non-small cell lung cancer and small cell lung cancer depending on the type of cancer cells. The differentiation between non-small cell lung cancer and small cell lung cancer is due to the large difference in clinical course and treatment method. Although small cell lung cancer can be expected to be cured only by surgical treatment if it is diagnosed early in early stage of the disease, most cases of non-small cell lung cancer are found to be progressing to such a degree that surgical resection is difficult at the time of diagnosis, Rather than chemotherapy or radiation therapy. Generally, lung cancer refers to non-small cell lung cancer which accounts for about 80 to 85% of lung cancer. Such non-small cell lung cancer is subdivided into squamous cell carcinoma, adenocarcinoma, large cell carcinoma and other lung cancer.

In particular, lung cancer was a rare disease until the 19th century, but smoking began to increase rapidly in the 20th century. In the western world, it became the most common malignancy in both males and females. In Korea, It is the second most common cancer disease. The increase in incidence is caused not only by smoking but also by increased air pollution and industrial pollution. In addition, lung cancer is harder to treat than other cancers, and the incidence is not the highest, but deaths are the most common among cancer patients. The 5-year survival rate, which is the cure standard, is only 15%.

Treatment of these cancers varies according to stage, and basically there are three methods: surgical removal, radiation therapy, and chemotherapy. Surgical removal is the only method that can be expected to be the cure for lung cancer, and adjuvant therapies such as radiotherapy and chemotherapy are combined to reduce postoperative recurrence or metastasis. There are different treatments available for different types of lung cancer. Early detection of non-small cell lung cancer is important because it is likely to be cured by surgery if detected early. If not, surgical treatment, chemotherapy, and radiation therapy are performed in parallel. On the other hand, in the case of small cell lung cancer, surgical treatment is not performed in most cases because it grows relatively quickly and distant metastasis is easy. However, because of the high sensitivity of chemotherapy and radiation therapy, chemotherapy and chemotherapy will be used for limited stage, and for chemotherapy for scalable stage.

Cancer chemotherapy is one of the effective cancer treatment methods, but continuous administration of chemotherapy is pointed out as a cause of failure of chemotherapy. This is because the cells acquire resistance to the anticancer drug, and when they acquire resistance to an anticancer drug, they have cross resistance that the drug is resistant to other anticancer drugs. Thus, multidrug resistance (MDR) is known as the ability of cancer cells to acquire nonspecific resistance regardless of the anticancer drug structure [Gottesman et al ., Nat . Rev. Cancer , 2: 48-58, 2002; Ambudkar et al ., Oncogene , 22: 7468-85, 2003). MDR acquisition of these cancer cells is well known as one of the major causes of failure of chemotherapy. The anticancer drug resistance can be categorized into two major categories. One is the inability of the drug to enter the cell, and the other is that the cancer cell itself may be less susceptible to the anticancer drug due to genetic or epigenetic changes. Anticancer drugs also have unique side effects. The most important is the reduction of leukocytes and platelets due to myelosuppression, which is a fatal side effect to patients who may be linked to the immune function and lead to infection. In addition, side effects such as causing abnormalities in the digestive or reproductive system or causing alopecia are limiting the application of anticancer drugs.

The most important reason for the difficulty in treating lung cancer is diversity. In addition to having various genetic characteristics for each tumor, cancer cells having various genetic characteristics in the same tumor of the same patient are mixed with various normal cells, so that one drug kills specific cancer cells, The effect of shrinking does not kill all the cancer cells with different characteristics. Therefore, when a small number of cells are divided again and the tumor grows, the result is changed to a cell which is resistant to the anticancer drug after the chemotherapy. Currently, there are about 200 kinds of anticancer drugs that block the growth mechanism of these various cancer cells. However, there are no test methods for determining which drugs should be used for which tumors, and only about 6 drugs per patient can be used . Therefore, it is very important to treat anticancer drugs suitable for lung cancer patients, and it is necessary to secure the cells of patients in order to screen patient-customized anticancer drugs in advance. However, effective methods for the isolation and proliferation of lung cancer cells from lung cancer patients have yet to be reported.

Therefore, the present inventors have conducted intensive researches to prevent side effects caused by indiscriminate use of anticancer drugs that have not been tested for their beneficial effects on patients, and as a result, they have found that optimal culture conditions for isolating, culturing, and proliferating lung cancer cells from lung cancer tissues And a method for screening a patient-customized cancer therapeutic agent using the cells cultured by the above-mentioned method, and finally completed the present invention.

It is an object of the present invention to provide a method for treating lung cancer, comprising the steps of 1) incising lung cancer tissue isolated from a patient to a size of 40-100 탆 in diameter, 2) culturing the incised tissue of step 1), 3) Separating the lung cancer cells from the cultured cells using a desipase, 4) culturing the lung cancer cells in step 3) in a serum-free N2 medium in a culture dish coated with the coating material, and isolating and culturing only lung cancer cells And 5) culturing the separately cultured lung cancer cells of step 4) in a medium supplemented with serum and a growth factor, thereby proliferating the lung cancer cells.

Another object of the present invention is to provide a method for screening a patient-customized cancer therapeutic agent, which comprises culturing lung cancer cells prepared by the above method and treating the cancerous cancer cells with cancer candidate substances.

According to one aspect of the present invention, there is provided a method of treating cancer, comprising the steps of: 1) dissecting a lung cancer tissue isolated from a patient to a size of 40-100 m in diameter; 2) culturing the incised tissue of step 1); 3) isolating lung cancer cells using the dyspazyme in the cultured cells of step 2); 4) culturing lung cancer cells in step 3) in a serum-free N2 medium in a culture dish coated with a coating material to separate and culture only lung cancer cells; And 5) culturing the isolated and cultured lung cancer cells of step 4) in a culture medium supplemented with serum and a growth factor, and proliferating the lung cancer cells from the lung cancer tissue.

The step 1) is a step of dissecting the tissue to effectively proliferate the lung cancer cells in the tissue isolated from the patient. The size of the tissue incision to obtain the most optimal proliferation rate is not limited but is preferably 40 to 70 μm , From 40 to 100 μm, from 70 to 100 μm, and most preferably from 40 to 100 μm. The tissue can be cut so that the incised tissue can be attached to the culture dish at a high rate, and the optimal size, that is, the size of 40 to 100 mu m, can be obtained using the porous membrane. According to one embodiment of the present invention, it was confirmed that the tissue coagulation ratio was the highest at 40 to 100 μm (FIG. 1).

The step 2) is a step of adhering and culturing the tissue having a certain range of sizes cut and filtered in step 1) to a culture dish. In this stage of the culture, the growth of lung cancer cells begins from the outgrowth clump attached to the culture dish. A coated culture dish can be used for better adhesion. Preferably, the coating material is not limited to any material that can be used for attachment of lung cancer cells, but examples thereof include collagen, gelatin or matrigel, more preferably matrigel.

The term " outgrowth clump "of the present invention means a lump in which a cell grows and spreads out from a tissue lump that has been cut into an appropriate size and applied to a culture dish. All tissue fragments applied to the culture dish adhere and do not propagate the cells. Therefore, the present invention is applied to find the optimal tissue incision size by calculating the ratio of proliferation to the number of applied tissue fragments.

The term " matrigel "of the present invention is a gelatinous extract secreted in Engelbreth-Holm-Swarm mouse sarcoma cells, which contains extracellular matrices (ECMs) composed of a specific protein. In the present invention, lung cancer cells can be propagated after attaching matrigel as a scaffold.

Step 3) is a step of isolating lung cancer cells extending from the proliferative coagulation in step 2). In order to remove the mixed fibroblasts and purify pure lung cancer cells, it can be separated by enzyme treatment with distearase have.

The term "Dispase" of the present invention is a proteolytic enzyme that cleaves fibronectin, collagen IV and a small amount of collagen I. Dyspazes are found in several bacteria and can be isolated from the culture filtrate from Bacillus Polymik. It can also be extracted and purified for research purposes. It is suitable for light separation of various tissues and recovery of cultured cells while minimizing damage to cells. According to the embodiment of the present invention, the cancer colony is separated purely by using a disagget, and in the case of the collagen degrading enzyme, not only the treatment time is prolonged, but also cancer cells are not effectively separated, and trypsin- And the feeder cell, which is a cell, was also dropped, confirming that the conventional cell separation material was inadequate.

The step 4) is a step for starting the cultivation of the purified lung cancer cells in the step 3), wherein only the lung cancer cells are purified and cultured. In particular, it is a step of obtaining pure lung cancer cells by inhibiting the proliferation of the remaining fibroblasts and killing them by using serum-free N2 medium for about 5 to 7 days at the beginning of culture. This step can be carried out using a culture dish coated with a coating material. At this time, the coating material to be used may be selected from the group consisting of collagen, gelatin or matrigel, and may preferably be matrigel. According to the examples of the present invention, the adhesion and propagation power of the collagen, matrigel and gelatin-coated culture dishes were compared and it was confirmed that the adhesion (FIG. 3A) and the growth rate (FIG. 3B) of the matrigel-coated culture dish were high.

The term "serum-free N2 medium" of the present invention refers to a medium supplemented with N2 supplement to a basic culture medium from which serum is excluded. The "N2 supplement" is a chemically synthesized serum-free supplement based on the N-1 formulation of Buttenstein. Human hormone transferrin and recombinant insulin, and progesterone, putrescine and selenite as active ingredients. It is generally added to Neurobasal medium or DMEM together with growth factors to induce differentiation into neural cells. The primary culture medium can be used without limitation in the culture medium for lung cancer cell growth, for example, RPMI1640, DMEM, DMEM / F12. However, in the present invention, in the initial culture of lung cancer cells, in order to purify lung cancer cells from fibroblasts, preferably, DMEM (Dulbecco's Modified Eagle Medium) medium containing L-glutamine, penicillin-streptomycin, non- essential amino acid and mercaptoethanol Can be used as a basic culture medium. According to the embodiment of the present invention, the culture conditions of the purified lung cancer cells are as follows: (1) N2, (2) FBS, (3) FBS and growth factor, Lt; RTI ID = 0.0 > (FIG. 4A). ≪ / RTI >

Step 5) is a step of propagating lung cancer cells isolated and cultured in step 4). In this step, a medium containing serum and growth factors can be used for effective proliferation of lung cancer cells. Preferably, serum may be fetal serum but is not limited thereto. At this time, the concentration of serum may be 10%.

The term "growth factor" of the present invention is a protein involved in the differentiation and growth of cells, and is an essential element in the normal cell cycle, and is a crucial factor in animal life maintenance. Furthermore, it regulates fetal development, plays a crucial role in maintaining and repairing the tissue, and stimulates the production of blood cells. It is also involved in cancer progression. In the present invention, it is added to the culture and proliferation of purified lung cancer cells so that the proliferated lung cancer cells are transplanted into the body or still maintain the characteristics of lung cancer cells in an in vitro environment. The growth factor may be, but is not limited to, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or insulin-like growth factor (IGF). (1) N2, (2) FBS, (3) FBS and growth factors (bFBF, EGF and IGF) and (4) ACL4 were added to the basic medium as the culture conditions of the purified lung cancer cells according to the embodiment of the present invention It was confirmed that the purified lung cancer cells proliferated most effectively in culture medium supplemented with FBS and growth factors (bFBF, EGF and IGF) in one medium (Fig. 4B).

The cultures of steps 4) and 5) may co-culture lung cancer cells alone with a monolayer adherent culture or feeder cells in a culture dish. At this time, mouse embryo fibroblast (MEF) may be used as a supplier cell, but it is not limited thereto. Preferably a single-layer adherent culture.

The term "adhesion culture" of the present invention means that cells are cultured on any surface, and most cell lines except for hematopoietic cells of vertebrates tend to prefer adhesion culture. For in vitro culture, a pre-coated culture dish may be used as a material capable of improving adhesion in order to enhance the adhesion ability. As the coating material for improving the adhesion, materials such as collagen, gelatin, and matrigel can be used.

The term "supplier cell" of the present invention is a cell used for assisting the growth of cells requiring various tricky cultures including stem cells, and is produced from spleen cells, macrophages, thymocytes or fibroblasts, It helps to grow the cells to be cultured. The supplier cells are inactivated before use through gamma irradiation or enzyme treatment in advance. When cultured, the donor cell acts as the basal layer of the cells to be cultured, and thus provides important metabolites without growth or division. Preferably, mouse embryonic fibroblasts can be used as the supplier cells.

The term " co-cultivation "of the present invention means that the cells to be cultured are mixed with other cells in one culture dish and cultured. Generally, the cells are cultured together with the supplier cells. The gamma-ray or the enzyme treatment can provide the growth factors or the metabolites without the growth or disruption of the supplier cells. Thus, Can be improved. However, there is a disadvantage that target cells must be separated from these donor cells after culturing.

According to one embodiment of the present invention, it was confirmed that culturing and proliferation of cells were possible when cultured using single layer adhesion culture (2D) and co-culture technique (FIG. 5). However, in the case of co-culture, the target cell must be separated from the supplier cell. However, since the growth of cells can not be observed when cultured using the floating culture (3D) technique, a single layer adhesion culture technique or a co-cultivation technique can be preferably used as a culture method for growing lung cancer cells of the present invention However, more preferably, a single-layer adhesion culture technique can be used.

In the present invention, the lung cancer may be non-small cell carcinoma, and non-small cell carcinoma may be squamous cell carcinoma, large cell carcinoma or adenocarcinoma.

In another aspect, the present invention provides a method for producing a cancer cell, comprising the steps of: culturing lung cancer cells prepared by isolating and propagating lung cancer cells from the lung cancer tissue; And treating the lung cancer cell with a cancer treatment candidate substance.

According to an embodiment of the present invention, the lung cancer cell prepared according to the cell isolation and culture conditions of the present invention is the same cell as the lung cancer tissue of the patient. Specifically, according to another embodiment of the present invention, lung cancer cells produced by the method of the present invention showed cancer cell-specific cell division and abnormal chromosome number increase (Fig. 6), and in the mouse transplantation experiment, And survived in the mouse body and existed as a tumor-like tissue (Fig. 7). In addition, abnormal tumor cell proliferation and cell size, shape, and nucleus diversity were observed after tumor transplantation, indicating that tumors formed from transplanted lung cancer cells have typical tumor characteristics. That is, the lung cancer cells prepared by the method of the present invention show the same characteristics as the lung cancer cells in the living body, and thus they can be used for the screening of the patient-customized cancer therapeutic agents.

Accordingly, a cancer treating cell can be obtained by treating a patient's lung cancer cells obtained in this way with a series of candidate cancer treating agents, thereby confirming the patient-specific anticancer activity, thereby finding a cancer treating agent suitable for the patient for effective treatment. That is, when the cancer cell is treated by treating the lung cancer cell of the patient prepared using the isolation and culture conditions of the present invention, the cancer therapeutic material is a customized anti-cancer drug for the patient who has isolated the lung cancer cell and the cancer cell is not killed The cancer treatment material can be judged to be an inadequate anticancer drug for this patient.

The term "cancer treatment candidate substance" of the present invention means a substance which is applied to a cancer patient to improve or change the symptom of a patient by cancer, and the substance showing the above characteristics such as an anticancer agent, antitumor antibiotic, .

Preferably, the cancer candidate candidate may be a cancer therapeutic agent selected for the treatment of all primary and metastatic cancers that develop in the lung, such as metastatic lung cancer, metastatic lung cancer, lung cancer, breast cancer, lymph node metastasis, and sarcoma besides primary non-small cell and small cell cancer . It is currently used in the treatment of lung cancer, including Carboplatin (paraplatin), Cisplatin, Cyclophosphamide, Ifosfamide, Nidran, Nitrogen Mustard (Methylethanolamine hydrochloride), Nitrogen mustar (Mechlorethamine HCL), Bleomycin, Doxorubicin, Mitomycin C, Cytarabine, Flurouracil, Gemcitabine ), Trimetrexate, Methotrexate, Etoposide, Vinblastine, vinorelbine, Alimta, Altretamine, Such as Procarbazine, Taxol, Taxotere, Topotecan, Irinotecan, Bevacizumab, Gefitinib, Erlotinib, Picibanil Vial, Aclarubicin, Tegafur, Epi, May be a cancer treatment selected from other carcinomas, as well as epirubicin, belotecan, anthracycline, idarubicin, or dactinomycin.

The method of the present invention can effectively isolate and proliferate only lung cancer cells from lung cancer patients and can be effectively used for establishing a patient-specific lung cancer cell line. The lung cancer cells isolated by the method of the present invention do not lose the characteristic characteristic of the cancer. Thus, such a lung cancer cell line may be useful for the selection of a patient-customized therapeutic agent. Therefore, it will be effective in the patient-customized treatment that can provide the optimal anticancer agent for the patient having the anticancer drug resistance.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphical representation of the shape of the outgrowth clump and the percentage of protruding clots according to the incision size of the tissue.
Fig. 2 shows the effective separation of the cell group when using dispase. Fig.
FIG. 3 is a diagram showing cell adhesion rate and cell proliferation according to the culture dish coating material. FIG.
FIG. 4 is a diagram showing a cell culture form according to medium conditions. FIG.
FIG. 5 is a diagram showing the morphology of cells according to culture dish attachment culture (2D) and co-culture with MEF. FIG.
6 is a diagram showing the results of chromosomal analysis of cultured lung cancer cells.
FIG. 7 is a graph showing tumor formation ability and tumor formation in vivo after transplantation into a mouse model of lung cancer cells cultured by the method of the present invention. FIG.
8 is a diagram showing the histological analysis results of the tissue formed by the cells 6 weeks after transplantation into a mouse.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: Comparison of separation results according to tissue incision size

The following experiments were performed to isolate the tissue from the patient under optimal conditions.

The lung cancer tissues of the patient were cut using a surgical scissor at a size of 0.5 to 1 cm for about 5 to 10 minutes, and then the incised tissues were treated with a porous membrane (Strainer, BD Bioscience) having various pore sizes of 40, 70, Were divided into five groups in which the size of the incised tissue was less than 40 占 퐉, 40 to 70 占 퐉, 40 to 100 占 퐉, 70 to 100 占 퐉, and more than 100 占 퐉.

The tissue from which the cells spread out from the incised tissue at the time of culture was classified as an outgrowth clump (Fig. 1A). As a result of dividing the ratio of the tissue agglutination according to the tissue incision size, the proportion of proliferation agglutination was highest in the group separated from 40 to 100 μm among the five experimental groups classified according to the size of the incisional tissue (FIG. 1B). Therefore, it was found that cutting lung cancer tissue to 40 to 100 mu m is most effective.

Example  2: Optimal Enzyme Screening for the Purification of Lung Cancer Cells

The following experiment was conducted to find the most suitable enzyme for isolating lung cancer cells from lung cancer tissues.

The cells derived from the proliferative coagulation of Example 1 are mixed with a lung cancer cell group and fibroblasts (Fig. 2, left panel). Therefore, in order to effectively isolate only the lung cancer cell group from the cells derived from the initially cultured tissue, a chemical method using various enzymes and a previously reported physical separation method [SK Oh et al ., Stem Cells , 2005; 23: 605-609]. The enzymes used are Gibco, Gibco, collagenase type IV (Gibco) and hyaluronidase (Sigma). Each commercially available enzyme was diluted to 1% with distaspase and type 4 collagenase, and trypsin-EDTA and hyaluronidase were used at concentrations of 0.25% and 100 units / ml, respectively.

As a result, it was confirmed that the lung cancer cell group was effectively separated only in the test group treated with the distearate (Fig. 2, right panel). On the other hand, collagenase has a long treatment time, no effective cancer cell mass has been isolated, and trypsin-EDTA has been observed to fall off with neighboring supplier cells, indicating that it is not suitable for cell purification.

Example  3: Optimal Petri dish  Selection of coating materials

Cells were cultured using matrigel, gelatin or collagen coated culture dishes, respectively, for effective attachment of the initially incised tissue and isolated lung cancer cell lines. The concentrations of the coating materials used are 1% Matrigel (BD Bioscience), 0.1% Gelatin (Sigma) and 1% collagen (Bioland). Each coating material was applied to a culture dish and then coated at room temperature for about 10 to 15 minutes. The matrigel was used for 24 hours, the gelatin was used for 30 minutes to 1 hour, and the collagen was also dried for 30 minutes to 1 hour.

As a result, when the matrigel-coated culture dish was used, the excellent adhesion rate of proliferation (FIG. 3A) and the growth rate of the same number of cells under the same condition for 7 days (FIG. 3B and C) were also high.

Example  4: Optimal culture selection

Purification of lung cancer cells from cultures of early incised tissues and experiments were carried out using the following four kinds of culture media in order to find an appropriate culture medium for culture and proliferation of lung cancer cells. (Gibco-BRL), 1% penicillin-streptomycin (Gibco-BRL), 1% non-essential amino acid (Gibco-BRL) in DMEM (Dulbecco's Modified Eagle Medium, Gibco-BRL) , And 0.1 mM mercaptoethanol (Gibco-BRL) were used. 10% FBS, 10% FBS, 10 ng / ml bFGF (R & D system), and 10% fetal bovine serum (FBS) 10 ng / ml EGF (R & D) and 10 ng / ml IGF (R & D)), and ACL4 [N. Masuda et al ., CHEST , 1991; 100: 429-438].

As shown in FIG. 4, the culture medium supplemented with 10% FBS + BEI containing serum and growth factors induces excessive proliferation of fibroblasts during the early stage of culture, that is, in the purification of lung cancer cells, (Fig. 4A upper panel). On the other hand, it was confirmed that the growth of unnecessary fibroblasts was suppressed under the condition of N2 addition medium without serum, so that only pure lung cancer cells were purified (Fig. 4A lower panel). However, in experiments for the proliferation of purified lung cancer cells under conditions of serum-free N2 addition medium, effective proliferation was observed in the medium supplemented with 10% FBS + BEI containing serum and growth factors (Fig. 4B top panel) In the medium, death of lung cancer cells was observed over time (Fig. 4B lower panel). Therefore, in order to purify lung cancer cells, the cells are cultured for 5 to 7 days in a serum-free N2 supplemented medium to remove fibroblasts and to allow only proliferation of pure lung cancer cells. After that, for the proliferation of purified lung cancer cells The culture with 10% FBS + BEI supplemented with serum and growth factors was confirmed to be suitable.

Example  5: Selection of optimal culture technique

In order to confirm the effect of the culture technique on the culture and proliferation of the early refined lung cancer cells, simultaneous culture with the single cell adhesion culture method (2D), suspension flocculation culture method (3D) and mouse embryo fibroblast (MEF) Culture method.

As a result, the growth of cells was not observed when cultured by suspension agglomerate culture (3D), but when cells were grown using single layer adhesion culture method (left panel in FIG. 5) and co-culture method with MEF (right panel in FIG. 5) Culturing and proliferation were possible. However, in the case of co-cultivation, there is a disadvantage that it is troublesome to remove feeder cells.

Example  6: Chromosomal analysis of cultured lung cancer cells

The lung cancer cells obtained by culturing incised tissues of 40-100 mu m size in a culture dish separated and proliferated under optimal culture conditions established in Examples 1 to 5, that is, a matrigel-coated culture dish, Purified from the mixed fibroblasts, the lung cancer cells thus purified were initially cultured in serum-free N2 supplemented medium for a certain period of time to remove excess fibroblasts, followed by the addition of 10% FBS + BEI supplemented medium containing FBS and growth factors In order to confirm whether the proliferated lung cancer cell lines still have cancer characteristics, chromosomal fragmentation was examined by immunohistochemistry.

As a result, abnormal cell division (Fig. 6A) and abnormal chromosome number increase (Fig. 6B), which are characteristic of cancer cells, were observed, as shown in Fig. Thus, it was confirmed that the lung cancer cells separated and cultured by the method of the present invention have the same characteristics as the cells in the patient's living body.

Example  7: In vivo Tumor formation  Ability analysis

In order to confirm that the lung cancer cells cultured by the culture method for optimized separation, culture and proliferation of the present invention used in Example 6 had the characteristics of cancer cells, Cy5.5 fluorescent substance was labeled on the lung cancer cell line, And xenogens were used to confirm the viability of the cells and tumor formation.

As a result, the transplanted lung cancer cells were alive even after 6 weeks, and they were observed to be present as a tumor-like tissue (Fig. 7).

Example  8: Histological analysis of the formed tissue

The lung cancer cells cultured by the culture method for optimized isolation, culture, and proliferation of the present invention obtained in Example 7 were transplanted into a mouse and cryo-sectioned for 6 weeks to cryo-section the tumor tissue to obtain hematoxylin and Eosin and Masson's trichrome, and the results are shown in FIG.

As a result, abnormal cell proliferation and cell size, shape, and cell nucleus diversity indicate that tumor tissues formed from the transplanted lung cancer cells have typical tumor characteristics.

These results suggest that the lung cancer cells separated and cultured by the method of the present invention have the same characteristics as the cancer cells in the living body, suggesting that they can be suitably used for screening of patient-customized anticancer agents .

Claims (9)

A method for isolating and propagating lung cancer cells from lung cancer tissue comprising the steps of:
1) incising lung cancer tissue isolated from a patient to a size of 40-100 탆 in diameter;
2) culturing the incised tissue of step 1);
3) isolating lung cancer cells using the Dispase in the cultured tissue of step 2);
4) culturing lung cancer cells in step 3) in a serum-free N2 medium in a culture dish coated with a coating material to separate and culture only lung cancer cells; And
5) Culturing the isolated and cultured lung cancer cells of step 4) in a medium supplemented with serum and growth factors and propagating.
The method according to claim 1,
Wherein the culture is performed by co-culturing the lung cancer cells with a monolayer adherent culture or a feeder cell alone in a culture dish.
3. The method of claim 2,
Wherein the donor cell is a mouse embryonic fibroblast.
The method according to claim 1,
Wherein the lung cancer is non-small cell carcinoma.
5. The method of claim 4,
Wherein said non-small cell carcinoma is selected from the group consisting of squamous cell carcinoma, large cell carcinoma and adenocarcinoma.
The method according to claim 1,
Wherein the coating material is a matrigel.
The method according to claim 1,
Wherein the growth factor is a basic fibroblast growth factor (bFGF), an epidermal growth factor (EGF), or an insulin-like growth factor (IGF).
8. A method for treating cancer, comprising the steps of: isolating and proliferating lung cancer cells from lung cancer tissue by the method of any one of claims 1 to 7; And
And treating the lung cancer cells with a cancer treatment candidate substance.
9. The method of claim 8,
The cancer treatment candidate substance may be selected from the group consisting of carboplatin (paraplatin), cisplatin, cyclophosphamide, ifosfamide, Nidran, nitrogen mustard (Methylethanolamine hydrochloride), Nitrogen mustar (Mechlorethamine HCL), Bleomycin, Doxorubicin, Mitomycin C, Cytarabine, Flurouracil, Gemcitabine ), Trimetrexate, Methotrexate, Etoposide, Vinblastine, vinorelbine, Alimta, Altretamine, Such as Procarbazine, Taxol, Taxotere, Topotecan, Irinotecan, Bevacizumab, Gefitinib, Erlotinib, Picibanil Vial, Aclarubicin, Tegafur, Epilou, The method of sour (Epirubicin), Vero Tecan (Belotecan), anthranilic ssayikeulrin (anthracycline), idarubicin (idarubicin), and shut actinomycin that the cancer therapeutic agent is selected from the group consisting of (dactinomycin).

Images