RU2699754C1 - Fluorescent glioma cell line and method for production thereof - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and represents a fluorescent cell line C6-TagRFP-TurboFP635, which expresses red fluorescent proteins and is used to study brain glioma in vitro and in vivo, and comprises vectors pTagRFP-C and pTurboFP635-C. Invention also relates to a method for preparing said cell line.EFFECT: invention enables higher tumour cell survival during transfection and provides high survival rate of cells in the brain of animals after their implantation with broader technological capabilities of studying the dynamics of brain tumour.5 cl, 3 dwg, 4 ex

Description

The group of inventions relates to biotechnology, in particular to genetic engineering and can be used to obtain genetically transformed mammalian cells in cultures producing fluorescent proteins, used in scientific research to visualize cells in tissues and to develop new non-invasive methods for the early diagnosis of brain tumors, as well as effective methods for treating gliomas by targeted delivery of antitumor drugs across the blood-brain barrier.

Fluorescent cell lines are known that are obtained by transfection of a line through lentiviruses.

In particular, a fluorescent human leukocyte cell line MDS (myelodysplasticsyndrome) and a method for its production are known, which consists in transfecting the human leukocyte line MDS broadcast SKM-1 through lentiviruses carrying GFP genes (greenfluorescentprotein) (see patent application CN1056709967, IPC A 027, publ. 06/15/2016).

Also known are fluorescent glioblastoma cell lines (GBM) expressing red fluorescent proteins mKate2 and mCherry and a method for its preparation, which consists in transfecting the human Ulius glioblastoma line U251MG through lentiviruses carrying mKate2 and mCherry genes. Lines were evaluated as a tool for non-invasive imaging of orthotopic tumors. (Verreault M, Strutt D, Masin D, Fink D, Gill R, Bally MB. Development of glioblastoma cell lines expressing red fluorescence for non-invasive live imaging of intracranial tumors. Anticancer Res. 2011 Jun; 31 (6): 2161-71.)

Also known is the fluorescent cell line of human glioma SU3-RFP expressing red fluorescent protein (RFP) and the method for its production, which consists in transfecting the glioma line of human SU3 with lentiviruses carrying the RFP gene, the line was evaluated as a tool for non-invasive imaging of orthotopic tumors (Yuntian S, Quanbin Z , Ginshi Z, Zhaohui L, Aidong Wang, Xifeng Fei, Xingliang Dai, Jinding Wu, Zhimin Wang, Yaodong Z, Ye T, Jun D, Qing L and qiang H. Advantages of a dual-color fluorescence-tracing glioma orthotopic implantation model : detecting tumor location, angiogenesis, cellular fusion and the tumor microenvironment. Eexperimental and therapeutic medicine 10: 2047-2054, 2015).

However, the use of lentiviruses increases the risk of insertional mutagenesis. In addition, the disadvantages of these methods include the limited amount of insertion of encoded sequences (up to 8 thousand base pairs), which can be transmitted by lentivirus. The disadvantages of the transfection process include its complexity, since it is necessary to determine the sensitivity of the cell line to lentiviral transduction; select the optimal promoter for the expression of fluorescent protein; construct lentiviral vectors encoding fluorescent proteins and conduct transduction.

Closest to the claimed group of inventions is a fluorescent cell line melKor-TurboRFP and the method of its use for in vitro studies in experimental oncology (see RF patent No. 2458123, IPC C12N5 / 09, publ. 10.08.2012). A method of obtaining a cell line is to grow a cell line, add a mixture of plasmid DNA pTurboRFP-C (Evrogen, Russia), encoding fluorescent proteins, with Lipofectamine 2000 liposomes (Invitrogen, USA), incubate, add an antibiotic - geneticin (G418 antibiotic, analog neomycin), cultivation and selection of fluorescent colonies.

However, to obtain this cell line, we are talking about the introduction of only one vector of fluorescent protein, which may lead to insufficient expression of the fluorescent protein. In addition, the method is expensive due to the high cost of the commercial Lipofectamine 2000 reagent.

The technical problem of the group of inventions is the development of a glioma cell line for experimental medicine, which provides the development of new approaches to early diagnosis of a brain tumor, as well as effective methods of treating gliomas by targeted delivery of antitumor drugs through the blood-brain barrier.

The technical result is to increase the survival of tumor cells during transfection and to ensure high survival of cells in the brain of animals after implantation while expanding the technological capabilities of studying the dynamics of the development of brain tumors.

This problem and technical result is solved by the fact that a new fluorescent cell line C6-TagRFP-TurboFP635 was obtained by introducing genes encoding the expression of the red fluorescent proteins TagRFP and TurboFP635 and used to study brain gliomas in vitro and in vivo.

In a method for producing a fluorescent cell line, which consists in growing a cell line in a growth medium to a monolayer, removing the medium and adding a mixture of plasmid DNA encoding fluorescent proteins, liposomes and incomplete nutrient medium to the monolayer, incubating, adding an antibiotic, culturing and selecting fluorescent according to the invention, at least two vectors pTagRFP-C and pTurboFP635-C are used as plasmid DNA, and liposomes synthesized using phosphorus are used as liposomes tidilholina and the ganglioside.

The ratio of vectors to liposomes is chosen 1:10.

In a preferred embodiment of the method, phosphatidylcholine is used from egg yolk, and ganglioside is used from the brain of a bovine. Geneticin is used as an antibiotic.

The method is illustrated by illustrations, which show:

in FIG. 1 - micrographs of tumor cells of fluorescent C6-TagRFP-TurboFP635 glioma after in vitro cultivation (A) and rat brain sections after infection of tumor cells of fluorescent glioma (B);

in FIG. 2 - the result of magnetic resonance imaging of the development of glioma in rats; the arrows indicate the accumulation of contrast medium - gadolinium in the tumor tissue, indicating its development; nearby - control animals (without tumor);

in FIG. 3 - data of confocal and fluorescence microscopy, as well as histological analysis of the development of fluorescent glioma in rats:

A - fluorescent glima (red) with blood vessels (green);

B - histological preparation of rat glyme; arrows indicate the migration of tumor cells from daughter tissues to healthy areas of the brain; at a large magnification, pliomorphism of glioma is shown as a morphological sign of its development;

C - confocal microscopy of glioma growth in the vessels of the brain;

D is the same at high magnification;

E - data of fluorescence microscopy on the development of glioma (red) and the opening of the blood-brain barrier (green means passing through the blood vessels of the brain in the area of glioma green dye) as a functional sign of a severe degree of tumor development;

F - brain vessels (green) next to the tumor (red); the border of the tumor is shown near healthy tissues, where the vessels are not yet damaged;

G - fluorescence microscopy of the migration of the tumor (yellow by merging the two colors of green and red) through the vessels of the brain (green).

The method is as follows.

The transfected cell line was obtained from rat C6 glioma cell line (RKKK (P)) as follows: cells were transfected with pTagRFP-C and pTurboFP635-C plasmid DNA using liposome transfection followed by selection with geneticin (antibiotic G418, neomycin analogue).

The obtained cell line C6-TagRFP-TurboFP635 has stable cultural and morphological characteristics.

Morphological signs of the cell line.

The resulting cell line is characterized by the presence of multiple dense growth centers of spindle-shaped tumor cells that form contacts with other cells in the culture. There are giant multinucleated cells, figures of mitosis.

Cultural properties of the cell line.

It is cultivated in a DMEM growth medium containing 10% fetal calf serum and antibiotics (penicillin and streptomycin at a concentration of 100 units / ml and 100 μg / ml, respectively). In culture vials with a volume of 25 cm ² in 5 ml of medium sown 1x10 ⁶ cells. The cultivation temperature is 37 ° C. A monolayer of cells forms in 2-5 days.

Cells are removed using a standard solution of Trypsin-EDTA (Paneko, Russia).

Cryopreservation conditions.

For long-term storage, cells are preserved by freezing in liquid nitrogen. Cells are resuspended in growth medium for freezing (BioRad, Russia). Freezing mode: liquid nitrogen, temperature decrease by 1 ° С per minute to minus 25 ° С, then quick freezing to minus 70 ° С. Storage in liquid nitrogen at a temperature of minus 196 ° C.

Defrosting is fast at 37 ° C. Cells are diluted in 10 ml of serum-free medium and precipitated by centrifugation, resuspended in 5 ml of the same medium containing 10% fetal calf serum, and transferred to a 25 cm ² culture vial. Cell viability is assessed by the inclusion of trypan blue. Cell viability after thawing is 85-90%.

Contamination.

With prolonged observation, bacteria and fungi were not found in the culture.

The stability of the expression of fluorescent protein in the absence of a selection medium is observed for more than 10 cycles of cultivation.

Cells of the obtained cell line express fluorescent proteins, which makes it possible to effectively investigate the effect of antitumor agents on the monolayers of these cells in vitro without using laborious cell counting using the Goryaev chamber or functional cell viability tests that require additional manipulations with the appropriate reagents.

The method is illustrated by examples.

Example 1. Obtaining a complex of liposomes with the vectors pTagRFP-C and pTurboFP635-C.

Liposomes based on a matrix of egg yolk phosphatidylcholine (ePC) and GM1 ganglioside from bovine brain (GM1 liposomes) were constructed.

Liposomes were obtained by the standard extrusion method after hydration of the lipid film according to the method of Kuznetsova N., Kandyba A., Vostrov I., Kadykov V., Gaenko G., Molotkovsky J., Vodovozova E.,. Liposomes loaded with lipophilic prodrugs of methotrexate and melphalan as convenient drug delivery vehicles. J. Drug Delivery Science Technol. 19 (2009) 51-59. The bilayer sample contained 1 mole% phosphatidylcholine.

In equal proportions, the vectors pTagRFP-C and pTurboFP635-C were added at the time of liposome synthesis in a mass fraction of 1:10, at a concentration of 400 ng / μl.

After extrusion through membrane filters with a pore diameter of 100 nm, liposomal dispersions in physiological saline (phosphate buffer, pH 7.1, total lipid concentration of 25 mM) were obtained in a Lipex setup (NorthernLipids, Canada). According to dynamic light scattering (Malvern Zetasizer Nano ZS, Malvern, UK), the average diameters and polydispersity indices for liposomes were 104 nm and 0.076.

Example 2. Cultivation of the cell line C6.

2 days before transfection, the cells were seated in the wells of a 24-well plate in 0.5 ml of growth medium (DMEM) containing 10% fetal calf serum containing no antibiotics, so that during transfection the cells would be 70-90% - monolayer.

100 μl of the DNA liposome complex was resuspended in 1 ml of serum-free DMEM, incubated for 5 minutes at room temperature. Then, 200 μl of the DNA liposome complex was added to each well of the plate containing cells and 0.5 ml of serum-free DMEM medium without antibiotics. Gently rocking the plate, the added complex was distributed over the entire area of the wells. Cells were incubated at 37 ° C in a CO ₂ incubator for 6 hours, then the old nutrient medium was removed and 0.5 ml of DMEM medium containing 10% fetal calf serum without antibiotics was added.

After transfection, the mixture of transfected and non-transfected cells was selected using geneticin at a concentration in the growth medium of 800 μg / ml. The selection medium with antibiotic was changed every 4th day, selection was made within 2 weeks.

The number of viable cells was determined by the standard method in the Goryaev chamber using a 0.5% solution of trypan blue in PBS. 1 × 10 ⁶ cells were seeded in culture vials of 25 cm ² volume in 5 ml of medium. The cultivation temperature is 37 ° C. Cells were cultured in DMEM containing 10% fetal calf serum, penicillin (100 units / ml), streptomycin (100 μg / ml) in culture bottles (Costar).

Example 3. In vitro fluorescence of the C6-TagRFP-TurboFP635 cell line was confirmed by fluorescence microscopy (Fig. 1A).

Figure 1 B shows micrographs of slices of the brain of a rat after the engraftment of tumor cells of fluorescent glioma C6-TagRFP-TurboFP635.

Example 4. Studies were conducted on 170 sexually mature outbred rats. The experiments were performed on sexually mature female rats in accordance with the "Rules for the Work Using Experimental Animals" (Appendix to the Order of the Ministry of Higher and Secondary Education of the USSR of November 13, 1984, No. 742). In vivo glioma modeling was performed using intracerebral stereotactic implantation of C6 fluorescent cells. The rat was pre-sedated with seduxen (50 μg / ml), then deeply anesthetized with intraperitoneal administration of zoletil (100 μg / kg). They were immobilized on a stereotactic table by fixing the head according to the technique, the hair was removed at the site of the planned operation, an incision was made in the area of the planned introduction at the selected coordinates with a length of 2 mm. Prepared glioma cells (5x10 ⁵ cells per rat) were implanted into the caudoputamen region using a Narishige stereotactic apparatus (USA) at the following coordinates Ar -1; L 3.0; V 4.5, TBS -2.4 mm. Cells were injected using a Hamiltonian microsyringe connected to an infusate at a rate of 3 μl / min in a volume of 1015 cells / ml. After the introduction of the cells, the wound was sutured in layers, treated with a 2% solution of brilliant green. The animal was placed in a clean cage. The duration of implantation did not exceed 10-15 minutes. The animal woke up on its own 30 minutes after anesthesia. No painful or other unpleasant manifestations were noted. The results of stereotactic implantation were evaluated according to an MRI scan of the brain using a Brivo MR 355 1.5 T scanner in T2 mode (GE Healthcare, UK).

In FIG. Figure 2 shows photographs of an MRI scan of glioma development in rats. In FIG. Figure 3 presents confocal microscopy data confirming the presence and development of glioma (A, C, D, F, and G) and the opening of the blood-brain barrier in severe stages of tumor development (E), as well as the result of histological analysis of the development of fluorescent glioma in rats (B).

Thus, a new glioma cell line has been developed for experimental medicine, which provides the development of new approaches to early diagnosis of brain tumors, as well as effective methods of treating gliomas by targeted delivery of antitumor drugs through the blood-brain barrier.

Claims (5)

1. Fluorescent cell line C6-TagRFP-TurboFP635, expressing red fluorescent proteins and used to study brain gliomas in vitro and in vivo, containing the vectors pTagRFP-C and pTurboFP635-C. 2. A method of obtaining a fluorescent cell line according to claim 1, which consists in growing the cell line of rat C6 glioma in a growth medium to a monolayer, removing the medium and adding to the monolayer a mixture of plasmid DNA encoding fluorescent proteins, liposomes and serum-free nutrient medium, incubation, addition antibiotic cultivation and selection of fluorescent colonies, characterized in that at least two vectors pTagRFP-C and pTurboFP635-C are used as plasmid DNA, and liposomes synthesized as liposomes are used used with phosphatidylcholine and ganglioside, with a ratio of vectors to liposomes of 1:10. 3. The method according to p. 2, characterized in that the use of phosphatidylcholine from egg yolk. 4. The method according to p. 2, characterized in that they use ganglioside from the brain of a bull. 5. The method according to p. 2, characterized in that geneticin is used as an antibiotic (G418).

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