The test system for studying cell tumors (neoplasia)

 

(57) Abstract:

The invention relates to biotechnology, experimental Oncology and cell biology and can be used for comparative studies of tumor cells and metastasis of one type of cancer. The test system for studying various cell neoplasm is a line of transplantable cells isolated from tissue tumors and consists of two lines of transplantable cells isolated from the tumor tissue and metastasis of Lewis lung carcinoma. The invention allows the study of anticancer activity and to screen drugs. table 2.

The invention relates to the field of biotechnology, experimental Oncology, cell biology and can be used for a comparative study of the properties of tumor cells and metastasis of one type of cancer, and also to study the anticancer activity of new drugs,

Solid tumors, especially tumors of the lung, contain distinct populations of cells, which complicates diagnosis and treatment of cancer. Currently available small number of models limits the study in vitro.

The problem of creating a TEC system for a comparative study is also to explore the biological activity of new drugs is important.

A known test system (Herrmann D. B. et. al. Antitumor activity of ilmofosine in the Lewis-Lung carcinoma model, Lipids, v. 26, 12, 1991). To obtain cells of Lewis lung carcinoma conducted a series of perepevok intramuscularly tumors. Then the tumor was dissected. Mechanical and enzymatic method received a suspension of cells suitable for transplantation.

The test system cannot be used to study cell metastasis. This system is not sustainable in transplantable cell line.

Known closer to the stated test system. Tumor lung carcinoma excised, crushed into small pieces. Minced tissue was placed in a solution of enzymes: collagenase, Gnkazy and hyaluronidase. The cells are then washed and placed in culture medium using the substrate of Matrygel. Cell line TL-1 was 100 passages and were used to study the expression of cell adhesion molecules, tumorogenic activity and metastatic potential in SD mice. (Sakakibara , T., et al. Doxorubicin encapsulated in sterically stabilized liposomes is superior to free drug or drug-containing conventional liposomes at suppressing growth and metastases of human lung tumor Xenografts. Cancer Researsh 56, 15, 3743-3746, 1996). The test system also cannot be used for comparative studies of tumor cells and metastasis.

to tumor growth and metastasis of Lewis lung carcinoma.

The problem is solved due to the fact that the test system to study the differences in cell neoplasia (or tumors), which is a transplantable cell line isolated from a tissue tumors, consists of two lines of transplantable cells isolated from the tumor tissue and metastasis of Lewis lung carcinoma.

The proposed test system differs in that it is a line of tumor cells and metastasis of Lewis lung carcinoma obtained without the aid of enzymes and cultured on conventional substrates. Both lines were about two hundred passages and keep tumorogenesis. Both cell line deposited at the Russian cell culture collection 12.01.1999, numbered SCC(P) N 652-D and SCC(P) N 653-D, respectively.

Tumor Lewis lung carcinoma arose spontaneously and may be registered in the United States 1951.

The test system are as follows. Excised tumors and multiple metastases. Washed in saline solution, crushed and placed in a solution of Versene (R. I. Freshney. Culture of animal cells. A manual of basic technique. New York. 1983). The cells are then washed and placed in a nutrient medium.

Morphological and cultural properties. Cells were cultured in medium RPMI-1610 from dobavlaut to form spheroids, have healthy tumorogenicity.

The contamination. Contaminants were not detected.

Cryopreservation. Cells are removed from the substrate with a solution of Versene. Precipitated at 800 rpm Preparing a suspension in a nutrient medium 106cells/0.5 ml, pipeinput and transferred to vials for cryopreservation. Make a 0.5 ml solution of 20% DMSO in serum and placed at -70oC. 10-14 days transferred to -160oC.

Conditions of defrost. Standard.

The invention is illustrated by examples.

Example 1. Washed from the environment of the cultivation of cells suspended in phosphate buffer. A drop of suspension (106cells/ml) is applied onto a glass slide. The product is dried in air, fixed in pairs 10% formaldehyde for 3 minutes. Washed with distilled water. Dried in the air. Fixed cells cause the solutions of carbohydrate vectors-fluorescence glycoconjugates on polyacrylamide matrix (50-60 ál, Sug-PAA-Fl, 0.3 mg/ml) and incubated 1 hour at 37oC. Washed with distilled water and dried in air. Linking cells with vector evaluated using a fluorescent microscope (495 nm, 530 nm). The results obtained for the determination of ligand-receptor binding 25 preceptors both types of cells (PL. 1)

Example 2. Cells were washed off the environment of the cultivation, suspended in phosphate buffer, 1 ml cell suspension was added 100 μl fluorescence carbohydrate vectors (Sug-PAA-Fl), the ligand-receptor binding is determined quantitatively by using flow cytometry. The results showed that the binding defined by the average value of the fluorescence of a single cell (mean), probe SiaLexwith cell tumor of 3.45, and cell metastasis was 7.45, linking vector Lexwith cell tumor - 14.4V, cell metastasis - 6,89.

Example 3. Drug delivery to the cell neoplasms are available liposomes include carbohydrate vector, providing specific binding of liposomes with cell lectins. Cells are removed from the substrate with a solution of Versene, washed from the culture medium. 1 ml of cell suspension in physiological solution (106cells/ml) add 100 ál of the drug fluorescenceenhanced liposomes (drug N 1 and of the drug such as liposomes carbohydrate vector Lex(drug # 2). Using flow cytometry measure the dynamics of binding within 15 minutes of the tumor cells and metastasis. In table. 2 shows the average values floorsa a line of transplantable cells, obtained from tissue neoplasms, characterized in that the test system consists of two lines of cells : tumor SCC (P) 652-D and metastasis SCC (P) 653-D Lewis lung carcinoma.

 

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