RU2742244C1 - Human bladder cancer cell line 587 blcan tvv - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to a human bladder cancer cell line 587 BlCan TVV. Said line is used for preparing biomedical cell products, creating experimental models and testing anticancer preparations, expresses tumour-associated antigens NY-ESO-1, MAGEA1, BAGE, GAGE, HER-2new and stored in Specialized collection of vertebrate cell cultures of Russian collection of cell cultures under registration number RCCC (P) 792D.

EFFECT: invention allows extending the range of human tumour cell lines.

1 cl, 3 ex

Description

The invention relates to the field of biotechnology. The resulting new bladder cancer cell line 587 BlCan TVV has stable morphological and cultural characteristics. This cell line can be used in active specific immunotherapy of solid tumors to create biomedical cell products, suitable for laboratory studies in order to develop diagnostic kits and test systems for determining the activity of pharmaceuticals. The cell line was deposited in the Specialized collection of vertebrate cell cultures of the Russian collection of cell cultures under the registration number RKKK (P) 792D.

Despite the tremendous success achieved in recent decades in the field of surgical treatment and drug therapy of malignant neoplasms, the problem of high mortality among patients with metastatic forms of cancer remains. The use of fundamentally new methods of treatment based on cell technologies expands the arsenal of methods for influencing the tumor process. One of these approaches is antitumor vaccine therapy, including based on the use of autologous and / or allogeneic tumor cells [Seledtsov V.I., Goncharov A.G., Seledtsova G.V. Clinically feasible approaches to potentiating cancer cell-based immunotherapies // Hum Vaccin Immunother. - 2015. - Vol. 11 (4). - P. 851-869. doi: 10.1080 / 21645515.2015.1009814].

The objects of biomedical research are living organisms, but working with them is extremely difficult, and the study of molecular interactions is practically impossible, since it is very difficult to distinguish specific processes from millions of other events simultaneously occurring in the body. In addition, the results of all studies conducted on animal model systems cannot be directly extrapolated to humans, and human experimentation is prohibited. Therefore, obtaining and cultivating cells isolated from the human body is currently a very promising approach that allows you to obtain an unlimited amount of cellular material for any research and conduct them on an identical material, which is important for developing standard approaches to solving a number of problems in biology and medicine [ Menzorov A.G., Serov O.L. Why do we need collections of cell lines // Vavilov Journal of Genetics and Breeding. - 2016. - T.20 (6). - S. 945-948. doi10.18699 / VJ16.215].

Banking cell lines intended for clinical use is an important strategic task, without the solution of which it is impossible to date the further development of medical technologies [Rachinskaya O.A., Chaplenko A.A., Melnikov E.V., Semenova I.S. ., Olefir Yu.V. World practice of storing cell lines intended for clinical use // BIOpreparations. Prevention, diagnosis, treatment. - 2018; 18 (4): 216-224].

In the structure of oncological morbidity, bladder tumors account for 2 to 5% of all neoplasms, ranking second among urological cancers in Russia and third in mortality from them [Grigoriev E.G., Frolova I.G., Usynin E.A., Velichko S.A., Okunev V.V. Bladder cancer: the possibilities of radiation diagnostic methods (literature review) // Siberian Journal of Oncology. - 2013. - No. 3 (57). - S. 75-81].

Bladder cancer can be classified as one of the most aggressive forms of malignant neoplasms. Surgical methods, chemotherapy and radiation therapy are the standard treatment for this type of malignant tumors, however, their high ability to metastasize and the formation of drug resistance create the prerequisites for the search and creation of new therapeutic approaches, especially since the immunogenicity of bladder cancer is beyond doubt, since its characteristic feature is a high level of mutational load [Zabolotneva A.A., Zhavoronkov A., Garazha AV, Roumiantsev SA, Buzdin AA Characteristic patterns of microRNA expression in human bladder cancer // Front. Genet. 2013; 3:31. doi: 10.3389 / fgene.2012.00310].

The development of new treatments for this type of malignant neoplasms requires the involvement of cellular technologies based on the production and use of well-characterized bladder cancer cell lines.

The technical result of the invention is to expand the arsenal of tumor cell lines, which can be used for experimental and clinical purposes, namely, to create antitumor biomedical cell products, the use of which makes it possible to increase the effectiveness of treatment and increase life expectancy in the treatment of malignant neoplasms. In addition, the resulting new cell line can be used to test the activity of various pharmaceuticals, create diagnostic kits, test systems and experimental models for the development of new therapeutic approaches and drugs.

The specified technical result is achieved by the fact that a new cell line of human urinary bladder cancer 587 BlCan TVV was obtained, expressing specific tumor-associated antigens, used for the preparation of biomedical cell products, creating experimental models and testing antitumor drugs, is stored in the Specialized collection of vertebrate cell cultures of the Russian collection of cell cultures under the registration number RKKK (P) 792D.

The resulting cell line 587 BlCan TVV has stable cultural and morphological characteristics.

The genealogy of the 587 BlCan TVV cell line is presented as follows.

The cell culture of bladder cancer 587 BlCan TVV was prepared from a sample of the primary tumor - muscular non-invasive bladder cancer from patient T.V.V., 63, obtained as a result of cystectomy carried out in a specialized oncological medical institution.

Obtaining cell line 587 BlCan TVV was carried out as follows.

The tumor tissue was obtained by surgery. Tumor samples crushed with a sterile scalpel (1-3 mm ² ) were subjected to mechanical disaggregation in an automatic mode, placing them on sterile knives for 30 s - 1 min. The resulting cell suspension was passed sequentially through sterile nylon filters 100 µm, 70 µm, 50 µm, suspended in complete medium and transferred to a culture dish, in which the cultivation was carried out for a long time. For better attachment of cells to the substrate, a 2% gelatin solution was used, which was preliminarily covered with culture dishes. A stable growing cell line was obtained at passage 40.

The morphological features of the 587 BlCan TVV cell line are characterized as follows.

The culture is represented by cells of predominantly polygonal shape with rounded light nuclei containing 1-2 nucleoli. The cytoplasm has a pronounced granularity, concentrated around the nucleus. The cells easily form symplasts with a characteristic “cobblestone” pattern.

The markers of the 587 BlCan TVV cell line are as follows.

By flow cytometry, the surface localization of the testicular cancer NY-ESO-1 antigen and antigens of the MAGE, BAGE, GAGE families was revealed.

By the method of immunocytochemistry, hyperexpression of the HER-2new antigen was revealed.

The PCR method revealed hyperexpression of the testicular cancer gene MAGEA1.

The cultural properties of the 587 BlCan TVV cell line are presented as follows.

The cell line is cultivated in a nutrient medium DMEM / F12 (80%) with the addition of 20% fetal calf serum, antibiotics (penicillin with streptomycin at a concentration of 100 U / ml and 100 μg / ml, respectively); 37 ° C, 5% CO ₂ , 100% humidity. For better attachment of cells to the substrate, a 2% gelatin solution is used, which is preliminarily covered with culture dishes. The culture has an adhesive monolayer type of growth. In vials with a volume of 25 cm ² in 5 ml of medium, 2 × 10 ⁶ cells are inoculated. Subculture 1 time per week in a 1: 1 ratio using equal volumes of 0.25% trypsin solution and 0.02% versene solution.

The conditions for cryopreservation are as follows.

For long-term storage, cells are preserved by freezing in liquid nitrogen. Cryoenvironment: DMEM / F12 50%, fetal calf serum 40%, DMSO 10%; 3 x 10 ⁶ cells / ml per ampoule. Cells of the cell line are resuspended in freezing medium. Freezing mode: liquid nitrogen, temperature decrease by 1 ° С per minute to -25 ° С, then quick freezing to -70 ° С. Storage in liquid nitrogen at -196 ° C. Fast defrosting at 37 ° C. The cells are diluted in 10 ml of serum-free medium and precipitated by centrifugation, resuspended in 5 ml of the same medium containing 20% fetal calf serum, and transferred to a 25 cm ² culture flask. Cell viability is assessed by the inclusion of trepan blue. Cell viability after cryopreservation is 85%.

Contamination was investigated as follows.

During long-term observation, bacteria and fungi were not found in the culture. The PCR method was used to test for the presence of Mycoplasma pneumonia, genitalium, hominis - the test for mycoplasma is negative.

The karyological characteristics of the 587 BlCan TVV cell line are as follows.

46, XY, -9.21p (del (9) (p21) - 67.5% cells.

42, XY, -9, -15, -16, -17, -19, + mar -29.5% cells.

The use of the 587 BlCan TVV cell line is illustrated by the following examples.

Example 1. Creation of cell 3D-models based on the 587 BlCan TVV cell line.

1. Cell line 587 BlCan TVV was cultured as described above. Upon reaching a confluent monolayer, the cells were removed from the substrate using an equilibrium mixture of 0.25% trypsin solution and 0.02% versene solution.

2. The cells were washed twice in complete culture medium, and the cells were counted and their viability assessed.

3. Cells 587 BlCan TVV in the amount of 500, 1000, 3000 were placed in a drop of complete nutrient medium (DMEM / F12, 20% FCS, glutamine) with a volume of 25 μl, localized on the lid of a 35 mm Petri dish. A 0.9% NaCl solution was added to the Petri dish itself.

4. Petri dishes were placed in a CO ₂ incubator (37 ° C, 100% humidity) for 7 days. In the process of cultivation on the 3rd day, the nutrient medium in drops was changed.

5. At the end of the cultivation, the spheroids removed from the drops were washed in a single PBS solution, then planted into the wells of a 48-well plate, pre-coated with polymerized Matrigel (100 μl per well), and 500 μl of culture medium was added.

6. Plates were placed in an automated Cell-IQ system for monitoring living cells (Chip-Man Technologies Ltd, Finland) and the efficiency of spheroid formation was determined by calculating the diameter of the spheroid and its area.

7. Made the following measurements: spheroids obtained from 500 cells 587 BlCan TW, had an average diameter of 302.5 ± 1.63 μm, an average area of 71734.4 ± 115.66 μm ² ; 1000 cells - average diameter 623.4 ± 4.43 μm, average area 266120.0 ± 52.32 μm ² ; 3000 cells - average diameter 795.8 ± 12.71 μm, average area 497138.6 ± 55.92 μm ² .

Example 2. Use of the 587 BlCan TVV cell line in an experimental study of migration and invasion of solid tumor cells.

1. Cell line 587 BlCan TVV was cultured as described above. Upon reaching a confluent monolayer, the cells were removed from the substrate using an equilibrium mixture of 0.25% trypsin solution and 0.02% versene solution.

2. The cells were washed twice in complete culture medium, and the cells were counted and their viability assessed.

3. Cells of line 587 BlCan TVV were seeded into inserts for a 24-well plate with a pore diameter of 8 μm with / without Matrigel (BD Bioscience, USA) in an amount of 5 × 10 ⁴ cells per ml, 500 μl of serum-free medium per insert. Complete nutrient medium DMEM / F12 with 20% fetal calf serum was added to the wells of a 24-well plate in an amount of 750 μl. Serum components acted as chemoattractants for cultured tumor cells.

4. The plate was incubated overnight under standard cultivation conditions in a Heracel CO ₂ incubator (Termo Electron LTD GmbH, Germany).

5. After incubation, the nutrient medium with Matrigel and non-invasive cells was removed from the inserts using a cotton swab soaked in the medium. The procedure was repeated twice.

6. The cells located on the underside of the membrane were fixed with buffered 3.5% formalin, permobilized with 100% methanol and stained with Giemsa's dye.

7. The invasive cells were counted in three fields of view using x40 and x100 microscope objectives. The percentage of cells with invasive properties was calculated as the ratio of the number of cells migrating through the matrigel to the number of cells migrating uncoated.

8. The invasive potential of 587 BlCan TVV cells was 10.5%.

Example 3. Use of cell line 587 BlCan TVV for the preparation of antitumor vaccines based on activated dendritic cells.

1. Cell line 587 BlCan TVV was cultured in plastic flasks in complete nutrient medium DMEM / F12 supplemented with 20% fetal calf serum, penicillin (100 U / ml), streptomycin (100 μg / ml). The cultivation was carried out at 37 ° C, 5% CO ₂ , 100% humidity in a CO ₂ incubator "Heracel" (Termo Electron LTD GmbH, Germany).

2. When the confluent monolayer was reached, the cells were subcultured and the culture was further passaged with a 1: 1 seeding.

3. The culture medium was removed from the vial, the surface of the vial was washed with a small amount of an enzyme solution (0.25% trypsin and 0.02% versene solution in equal proportions) and then 1-3 ml of this solution was added to the cells. The vials were placed in a CO ₂ incubator and visual control of the state of the cells was carried out.

4. The cells were harvested with a serological pipette, washed by double centrifugation in 10 ml of 0.9% saline solution at 1000 rpm for 10 minutes.

5. The cells were counted and their viability was assessed.

6. To prepare a tumor lysate of 587 BlCan TVV cells, 6 consecutive cycles of instant freezing to -196 ° C and thawing to room temperature in phosphate-buffered saline without cryoprotectant were carried out; the quality of lysis was monitored using 0.1% trypan blue and a light microscope.

7. Precipitation of cell detritus was carried out by centrifugation (10 min, 3000 rpm), filtration of the supernatant fraction through a 0.2 µm filter and packaging of the tumor lysate into cryovials for storage at -20 ° C until use.

8. Lysate with 587 BlCan TVV cell line was introduced into the cultures of immature dendritic cells of patients with bladder cancer on the 5th day of cultivation in a ratio of 3 lysed tumor cells: 1 DC, growth factors were added: granulocyte-macrophage growth factor (72 ng / ml ), interleukin 4 (20 ng / ml) and tumor necrosis factor alpha (20 ng / ml). It was incubated under the conditions of 37 ° C, 5% CO ₂ , 100% humidity in a CO ₂ incubator "Heracel" (Termo Electron LTD GmbH, Germany) for 48 hours.

9. As a result, we obtained activated DCs with an immunophenotype.

CD14 ^- / CD1a ^- / 7CD83 ⁺ / CD80 ⁺ / CD86 ⁺ / HLADR ⁺ / CD209 ⁺ / CCR7 ⁺ .

Cell line of human urinary bladder cancer 587 BlCan TVV expands the arsenal of tumor cell lines that can be used for experimental and clinical purposes, namely for the creation of antitumor biomedical cell products, the use of which makes it possible to increase the effectiveness of treatment and increase life expectancy in the treatment of malignant neoplasms. In addition, the resulting new cell line can be used to test the activity of various pharmaceuticals, create diagnostic kits, test systems and experimental models for the development of new therapeutic approaches and drugs.

Claims (1)

Cell line of human urinary bladder cancer 587 BlCan TVV, expressing tumor-associated antigens NY-ESO-1, MAGEA1, BAGE, GAGE, HER-2new, used for the preparation of biomedical cell products, creation of experimental models and testing of anticancer drugs, is stored in the Specialized collection of cell cultures vertebrates of the Russian collection of cell cultures under registration number RKKK (P) 792D.