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.
FIELD: biotechnology, in particular isolation of mesenchymal stem cells from human tissues for treatment of various diseases.
SUBSTANCE: human mesenchymal stem cells are isolated from fat tissue, decidual placenta membrane, placenta caul, or placenta chorial stroma. Human tissue is crushed and treated with collagenase solution in DMEM (Dulbecco's Modified Eagle Medium) wherein fat tissue, decidual placenta membrane or placenta caul is treated with type I collagenase, and placenta chorial stroma is treated with type IV collagenase. Obtained suspension is purified from erythrocytes with lysing reagent followed by filtration through filters with pore size of 100 mum and 10 mum.
EFFECT: increased cell suspension homogeneity, target product yield, and cell viability.
2 cl, 4 tbl, 4 ex
FIELD: biotechnology, medicine.
SUBSTANCE: peritoneal macrophages are placed in medium 1999 and subjected for effect of helium plasma obtained at current strength 30 A, voltage 20 V and gas consumption 2 l/min/ Cells are irradiated from distance 20 cm to plasmatron nozzle for 30 s. Method provides reducing time of physical factor effect on cells and allows carrying out the local effect on macrophage functions both in cultured cells and within the whole body. Invention can be used in clinical practice for stimulation of biological processes in cells and tissues.
EFFECT: improved method for stimulation.
1 tbl, 2 ex
FIELD: medicine and biopharmacology.
SUBSTANCE: claimed biotransplant contains mesenchyme stem cells (MSC) obtained from fetal, donor or autologous material. Tissue is desagregated, obtained cell suspension is resuspended and cultivated on growth medium containing transferrin, insulin, fibroblast growth factor and heparin to accumulate mature stroma in cells. Method for treatment of lung hypertension includes intravenous drip-feeding of MSC in amount of 50-500 millions cells in 50-100 ml of physiological solution.
EFFECT: method for treatment of improved effectiveness having no undesired side effects.
FIELD: medicine and biopharmacology.
SUBSTANCE: claimed biotransplant contains culture of genetically non-modified mesenchyme stem cells (MSC), fibroblasts including epithelial cells. Biotransplant is used in intradermal administration. Biotransplant is administered topically in physiological solution with concentration from 1 to 10 millions per 1 ml. Total transplant amount is 1-10 millions depending on defect area.
EFFECT: treatment method with high reproducibility.
FIELD: medicine and biopharmacology.
SUBSTANCE: claimed biotransplant contains culture of genetically non-modified mesenchyme stem cells (MSC), including fibroblasts of hair follicule dermal papillae, epithelial cells including ones obtained from hair follicle stem niche. Biotransplant is used in intradermal administration. Method for alopecia treatment includes intradermal administration of biotransplant containing from 10 to 100 millions cells to patient, in procedure room by micropapulic method. Biotransplant is administered in physiological solution with concentration from 1 to 10 millions per 1 ml. Total transplant amount is 1-10 millions depending on lesion area and type of alopecia.
EFFECT: method for alopecia treatment with high reproducibility.
FIELD: medicine, hepatology, chemical-pharmaceutical industry, biotechnology.
SUBSTANCE: invention relates to a biotransplant used in treatment of chronic hepatitis and liver cirrhosis and comprising mesenchymal stem cells obtained from fetal or donor material. The parent tissue is subjected for disaggregation followed by culturing as fixed colonies in the growth medium containing fetal calve serum and glutamine and passage at low density value with change of medium composition and cultivation is carried out without accumulation cells with mature stroma in culture. Also, invention relates to a method for treatment of chronic hepatitis and liver cirrhosis that involves administration of indicated biotransplants by using venous catheter or by puncture in the portal system veins, or by using arterial catheter in splenic artery, or by puncture in spleen parenchyma, or by intraperitoneal route, or by puncture in gastrocolic omentum. Invention provides enhancing effectiveness in complex effect on damaged liver.
EFFECT: improved preparing method, improved treatment method.
FIELD: biotechnology, medicine.
SUBSTANCE: invention proposes a serum-free medium for growth and proliferation of cultured cells used for reconstruction of osseous and cartilage segments of the definite composite (variants). Invention provides preparing cells and their using for transplantation.
EFFECT: valuable properties of medium.
6 cl, 1 dwg, 2 tbl, 5 ex
SUBSTANCE: one should place bioplastic material in culture liquid with a culture of dermal fibroblasts to cultivate fibroblasts together with bioplastic material for 4 d, then it is necessary to sample culture liquid and detect the concentration of protein-bound hydroxyproline. One concludes upon the degree of collagenogenesis stimulation with tested bioplastic material by greater or lesser degree of increased concentration of protein-bound hydroxyproline in sampled culture liquid against the control. As a control sample it is useful to apply culture liquid in which one could observe the tested bioplastic material for 4 d but no fibroblasts. The innovation is simple and enables to quantitatively evaluate the stimulation of collagenogenesis with bioplastic materials without applying any laboratory animals.
EFFECT: higher accuracy of evaluation.
FIELD: biology, embryology.
SUBSTANCE: invention relates to a method for controlling oocytes quality and a composition for addition in medium for culturing oocytes. Method for controlling the oocytes quality involves placing at least one oocyte in cultural medium and carrying out culturing. The cultural medium comprises at least one anti-sense oligonucleotide of length 17-30 nucleotides wherein each of them is complementary to mRNA of at least one of the following genes: gene-inductors of apoptosis, such as HRK, FAS, FASL, BAX, Caspase-3, genes of growth factors, such as IGF1, gene-receptors of growth factors, such as IGF1R, genes regulating the dividing embryos rate, such as HLA-E, HLA-F, HLA-G, genes of cellular stress, such as HSF1 and HSF2. The composition added in cultural medium comprises one or more an anti-sense oligonucleotide describes above and acceptable solvent. The advantage of invention involves enhancing the quality and viability of oocytes and enhancing the therapeutic effectiveness in treatment of sterility.
EFFECT: improved controlling method, improved properties of composition.
15 cl, 2 dwg, 10 ex
FIELD: medicine, veterinary science, biology, cosmetology.
SUBSTANCE: invention proposes two strains of human diploid cells from embryo lung tissue and cutaneous-muscle tissue. Strains possess the high therapeutic effect in carrying out the substitution therapy in treatment of wounds of different etiology.
EFFECT: valuable medicinal properties of strain.
2 cl, 3 dwg, 4 ex