RU2536992C1 - Method of increasing proliferative properties of diploid cells of human fibroblasts - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises scaling of diploid cells of line M-20 from cryobank of Institute of Poliomyelitis and Viral Encephalitis n.a. MP Chumakov of RAMS from the ampoule of the seed cell bank of 7 passage with obtaining the working cell bank of 16 passage. At that the cells of 20-33 passages, suitable for use in therapeutic and/or diagnostic purposes are produced by culturing in a nutrient medium containing 10% of human fibrinolytic active plasma (FAP), comprising platelet-derived growth factor PDGF in a concentration of from 155 to 342 pg/ml.

EFFECT: invention enables to increase proliferative activity of human fibroblast diploid cells.

2 cl, 2 tbl

Description

The invention relates to biotechnology, immunology, medicine, in particular to a method for increasing the proliferative properties of diploid cells of human fibroblasts for the use of such cells for therapeutic and diagnostic purposes, including for determining the antiviral activity of human interferons, for cell replacement therapy.

Lines of human diploid cells (LDPC) have undeniable advantages over all known types of cell cultures due to their ability to maintain stable biological and genetic characteristics in passages. Certification of LACCs for vaccine production is carried out in accordance with the uniform requirements developed by the World Health Organization [Requirements for Poliomyelitis Vaccine Oral: WHO Expert Committee on biological Standardization: 33 Report WHO. - Geneva, 1983]. These recommendations are taken as the basis of the national criteria for the certification of vaccine LDPC developed by GNIISiK MIBP them. L.A. Tarasevich and the Ministry of Health of the USSR [Methodical recommendations "Certification of transplantable cell lines - substrates for the production and control of medical immunobiological preparations" RD-42-28-10-89. Ministry of Health of the USSR. M., 1989. - S. 16]. The certified human diploid cell line has a limited lifespan and has stable biological, cultural and genetic characteristics, it is free from contaminants (bacteria, fungi, mycoplasmas, viruses) and does not cause the formation of tumors in immunosuppressed animals. The diploid cell line should have a certified seed cell bank at early levels of passages (up to 10 passages), consisting of at least 200 cryovials. When seed cells are passaged from one or several cryovials to passage level 16, a working cell bank is obtained from which necessary producer cultures for production or for research work can be obtained. In Russia and abroad there are only a few lines of human diploid cells (Wi-38, MRC-5, M-22, etc.), certified according to the listed requirements. Certified LHD are used in the manufacture of vaccines against poliomyelitis, measles, rubella, rabies, respiratory and cytomegalovirus infections, as well as interferon [T.K. Borisova, L.L. Mironova, O.I. Konyushko, V.D. Popova, V.P. Grachev, N.R. Shukhmina, V.V. Zverev. Domestic strains of human diploid cells are a substrate for the production of vaccines. Medical virology. Materials of the scientific-practical conference “Actual problems of medical virology dedicated to the 100th anniversary of M.P. Chumakova. " M. 2009. Volume XXVI. S. 305-307; L.L. Mironova, V.D. Popova, O.I. Konyushko. The experience of creating a bank of copyright transplantable cell lines and their application in virological practice. Biotechnology. 2000, p. 41-47]. LHD are widely used in vitro for the diagnosis of viral infections, toxicity analysis of various drugs and products, for replacement therapy [RF Patent No. 2373944, 06.23.2008. A method of treating a burn wound. A.S. Ermolov, S.V. Smirnov, V.B. Khvatov, L.L. Mironov; S.V. Smirnov, V.B. Grabs. Innovative technologies for local treatment of burns in the Research Institute of Emergency Medicine. N.V. Sklifosovsky. In the book: The New Economy. Innovative portrait of Russia. M., Center for Strategic Partnership, 2009. S. 388-390].

In IPVE them. M.P. Chumakova RAMS in the 80s of the 20th century, several lines of diploid cells from the skin and muscles of 8-10 week old human embryos were found. The present work is devoted to the modification of the production of human diploid cells for diagnostic purposes and cell replacement therapy, namely, the production of diploid cells of human fibroblasts with increased proliferative properties.

Prototype. RF patent №1440029 dated 03/22/93 [Mironova L.L., Preobrazhenskaya N.K., Solovyova M.N., Orlova T.G. Stobetsky V.I., Kryuchkova G.P., Karmysheva V.Ya., Kudinova S.I., Popova V.D., Alpatova G.A. IPVE and NIIEiM them. N.F. Gamalei. The strain of diploid cells of the skin and muscles of the human embryo, used as a test system for determining the antiviral activity of human interferons and the multiplication of viruses].

This strain of LDPC is designated M-21, however, the M-21 fibroblast culture had insufficient proliferative activity, which reduced the formation time of the monolayer and increased the consumption of cells and materials, and this ultimately led to a complete depletion of its reserves. As a result, a need arose for a new cell line suitable for determining the antiviral activity of human interferons and other biomedical purposes, more economically viable, characterized by high proliferative activity, with cans of seed and working cells. This line is marked M-20. At passage level 7, a seed bank was made. In 2012, a bank of working cells at passage 16 was made from the ampoule of bank 7 of the passage. Banks of sowing and working cells at levels 7 and 16 of the passages are stored in IPVE them. M.P. Chumakova RAMS and allow to ensure both production processes and scientific research.

The difference of the present invention from the closest analogue (prototype) is to increase the proliferative activity of M-20 cells using 10% fibrinolytically active plasma (FAP).

Thus, an object of the invention is a method of increasing the proliferative properties of diploid cells of human fibroblasts for biomedical purposes by culturing cells from a cryobank IPVE them. M.P. Chumakova RAMS, in which diploid cells of the characterized M-20 line are used, which scale from an ampoule of a seed bank of passage 7 and obtain a bank of working cells of passage 16, with cells of 20-33 passages suitable for use for medical and / or diagnostic purposes, obtained by cultivation in a nutrient medium containing 10% fibrinolytically active plasma (FAP) of a person. When culturing the cells, preferably DMEM nutrient medium with 10% FAP is used.

Human diploid cells of the characterized M-20 line, obtained by the above method, have high proliferative activity and are suitable for use in medical and / or diagnostic purposes.

The implementation scheme of the method:

1. One cryovial is used from the seed bank of passage 7 of the passage of IPVE named after M.P. Chumakova RAMS

2. Preparation of a bank of working cells at the 16th passage of IPVE named after M.P. Chumakova RAMS

3. Recovery of fibroblasts of the M-20 line from the bank of working cells of passage 16 (IPVE named after MP Chumakov RAMS).

4. Obtaining a monolayer culture of fibroblasts of the line M-20, 17 passage.

5. The restoration of the biological properties of fibroblasts of the M-20 line by triple passaging (up to 20 passages inclusive) to repair possible DNA damage during cryopreservation.

6. Obtaining cell cultures for diagnostic purposes and cell replacement therapy by replication of fibroblasts of the M-20 line from passage 20 to 33 using a nutrient medium containing 10% fibrinolytically active plasma (with a PDGF content of 155 to 342 pg / ml).

The proposed method provides for the production of cells with high proliferative activity and suitable for use in diagnostic and / or therapeutic purposes.

This technical result is achieved by culturing human fibroblasts of the M-20 line in a nutrient medium with the addition of 10% fibrinolytically active plasma (FAP), which has a growth-stimulating effect and enhances the proliferative activity of cell culture.

FAP is a clinically used transfusion medium, which is obtained from the blood of suddenly died from myocardial infarction, acute heart failure, cerebral hemorrhage in the first 6 hours after death [order of the Ministry of Health of the USSR No. 482 of 06/14/1972 "On improving the provision of medical and preventive institutions and clinics with cadaveric tissues, bone marrow and blood ”]. Post-mortem blood is a full-fledged transfusion medium with a number of biological properties - primarily increased fibrinolytic potential. In this regard, posthumous blood is also proposed to be called fibrinolysis. The main indications for post-mortem transfusion: acute hemorrhage, shock, anemia of various origins, burn injury, metabolic substitution in exogenous poisoning, filling of the AIK when using extracorporeal circulation in surgery [E.G. Tsurinova. Fibrinolysis blood transfusion. M., 1960, 159 s; S.V. Ryzhkov. Harvesting and the possibility of using fibrinolysis blood, depending on the time taken and the cause of death. Abstract. Doct. diss. L., 1968, 21 p .; G.A. Pafomov. Biological characteristics of blood of the suddenly deceased and its use in surgical practice. Diss. Doct. honey. Science. M., 1971, 355 p .; K.S. Simonyan, K.P. Gutiontova, E.G. Tsurinova. Post-mortem blood in the aspect of transfusiology. M., Medicine, 1975, 271 pp.]. The components of post-mortem blood are currently used: fibrinolytically active plasma, erythrocyte mass, leukocyte mass, platelet mass [G.Ya. Levin. Hemocoagulation properties and clinical use of cadaver blood plasma and platelets. Abstract. Doct. diss. M., 1978, 31 s; V.B. Grabs. Fibrinolytic and antiprotenase drugs from blood plasma of suddenly dead people. Diss. Doct. Medical Sciences, 1984, 417 p .; V.B. Khvatov Plasmakinase - a new thrombolytic preparation from postmortem plasma In: Thrombosis and Thrombolysis edd. E.I. Chazov, V.V. Smirnov). Consultants Bureau, N.Y., L, 1986, p. 283-310; V.B. Grabs. Medical and biological aspects of the use of post-mortem blood. Vestnik of the Academy of Medical Sciences of the USSR, 1991, 9. S. 18-24; V.B. Grabs. Cadaveric blood - the history and current status of the issue. Prob. hematol. and overflow. Blood, 1997, 1. S. 51-59]. Cadaveric blood components obtained from organ donors also received clinical use [dead individual with a beating heart according to the “Instructions for stating a person’s death based on a diagnosis of brain death” dated December 20, 2001 No. 460, registration of the Ministry of Justice No. 3170 dated January 17, 2002] . Transplantation of organs, tissues and cells is carried out in accordance with the Law of the Russian Federation "On transplantation of organs and (or) human tissues" - as amended. Federal Laws of June 20, 2000 No. 91-F3, dated October 16, 2006 No. 160-F3; V.B. Khvatov, S.V. Zhuravel, V.A. Gulyaev, E.N. Kobzeva, M.S. Makarov. Biological usefulness and functional activity of the cellular components of the blood of organ donors. Transplantology, 2011, 4, p. 13-19; Khubutia M.Sh., Khvatov V.B., Gulyaev V.A. and others. A method of compensating for the globular blood volume and immunomodulatory effects during transplantation. RF patent for the invention No. 2452519, publ. 06/10/2012, bull. No. 16].

Fibrinolytically active plasma is obtained from the blood of suddenly dead people, prepared on the preservative Glyugitsir (blood: preservative ratio 4: 1) to maintain its fibrinolytically active properties. The plasma is separated from the cellular elements of the blood in a sterile box in compliance with all the rules of aseptic and antiseptic and similar to the receipt of donated plasma from canned donated blood. The clinical use of FAP in surgery and traumatology revealed the effect of stimulation of wound healing [I.Yu. Klyukvin, M.V. Zvezdina, V.B. Khvatov, F.A. Burdyga. A method of treating bitten wounds. Patent for the invention of the Russian Federation No. 2372927, publ., 20.11.2009, bull. No. 32]. We associated this effect with the presence of growth-stimulating factors in FAP secreted by activated platelets. Subsequently, in the FAP, we identified platelet-derived growth factor (PDGF). The growth-promoting effect of FAP in human cell culture has been shown in special studies. In a cell suspension of human fibroblasts of the M-20 line containing a known number of cells, the studied FAP samples were added at 10% concentration and 10 ml of the resulting mixture was placed in culture bottles with a growth surface area of 25 cm ² . Cells were grown for 3-4 days at 5% CO ₂ in the atmosphere and at 37 ° C. After 3-fold passaging, the grown cells were counted in a Fuchs-Rosenthal chamber and the ratio of the number of grown cells to the number of planted cells was determined — proliferation index (in table 1).

From the experiments it follows that the growth properties of FAP provide high proliferative activity and do not differ from that of embryonic cattle serum. Moreover, the FAP contains growth factors of human platelets, i.e. allogeneic type, in contrast to the fetal serum of cattle - xenogenic type. This fact is crucial for cell transplantation during replacement therapy. Note that the growth-promoting effect on the culture of M-20 cell lines is due, in particular, to the presence of PDGF in the FAP at a concentration of 155 to 342 pg / ml. These data were obtained using the Qantikine, Human PDGF-BB Immunoassay reagent kit from R & D Systems and the Multiskan ascent system from Thermo. The concentration of PDGF-BB in FAP is similar to its serum content. So, in the blood serum of blood donors and examined patients, the PDGF content ranged from 110 to 880 pg / l, on average 244 pg / ml, while in plasma the PDGF content ranged from 0-2 pg / ml.

For a better understanding of the proposed technical solution "the production of human diploid cells of the M-20 line for biomedical purposes" we give the following example.

Cells of line M-20 of passage 16 are restored from a working bank. For this, a cryovial with cells is removed from liquid nitrogen and placed in a water bath at a temperature of 38 ° C and after thawing, the contents are transferred to a culture vessel with DMEM nutrient medium containing 10% FAP (with PDGF content from 155 to 342 pg / ml), add gentamicin antibiotic at the rate of 1 ml of 4% solution per 1 liter of culture medium. To form a monolayer, cells are cultured for 4-5 days at 37 ° C and 5% CO ₂ in the atmosphere. After the formation of a monolayer of cells, 3 consecutive passages are necessary for DNA repair after cryopreservation. Then, cells are replicated from passage 20 to passage 33. The cells of these passages are intended for biomedical purposes. The resulting cell line is characterized in detail in accordance with the requirements of WHO and GNIISiK MIBP them. L.A. Tarasevich, including HLA typing of cells of the M-20 line, as well as the study of its cytokine spectrum. We give a comparative characteristic of the properties of the M-20 line and the M-22 line (table 2). The M 22 line (human diploid fibroblasts) is licensed as a vaccine substrate and approved for the production of any types of medical viral vaccines, and is also used to treat burn wounds of the II-IIIA degree [RF Patent for invention No. 2373944, 23.06.2008. A method of treating a burn wound. A.S. Ermolov, S.V. Smirnov, V.B. Khvatov, L.L. Mironova, O.I. Klnyushko, E.A. Zhirkova, BC Bocharova].

Line M-20 installed in IPVE them. M.P. Chumakova RAMS in 1986 from the skin and muscles of a 10-week human embryo obtained as a result of an abortion from a healthy woman. Cancer, sexually transmitted diseases, hepatitis, tuberculosis in the anamnesis are not found; no genetic or congenital diseases were observed in the family. DMEM cell culture medium supplemented with 10% FAP. The sieving coefficient is 1: 3-1: 4 twice a week with a seed dose of cells of 7 × 10 ⁴ cells / ml. The cell monolayer consists of oriented homogeneous spindle-shaped cells with oval nuclei containing 1-3 nucleoli and small blocks of chromatin. In the life cycle of the line, 3 phases of development can be distinguished: formation of 1-3 passages, active growth of 4-40 and aging of 41-52, then death occurred. The cells of the line have a human karyotype 2t = 46, XU. The line is characterized by high genetic stability: 93.3-96.9% of the cells have a diploid set of chromosomes, cells with a polyploid set of not more than 1.6%. Gaps and breaks, as well as ring chromosomes were not observed. The number of bands of G-6FDE and LDE isoenzymes and their electrophoretic mobility coincide with those for human red blood cells. G-6FDG slow type. When seeding on selective nutrient media, contamination with bacteria, fungi, mycoplasmas was not detected. In addition, mycoplasma contamination was not detected when staining with Hochst 33258 DNA fluorochromes and olivomycin, as well as by PCR. Virus contamination in experiments on suckers and adult white mice, guinea pigs, rabbits and chicken embryos, as well as on homologous and heterologous cell cultures, was not detected. Tumorigenicity control. When cells were introduced, immunosuppressed animals did not form tumors. Reverse transcriptase was not detected. HLA markers: Class I: A * (02.03) / B * (07.40) / CW * (03.07). Class II: DRB1 * (15.16) / DQB1 * (05.06). Cells of the M-20 line at passage level 20 produce mRNA of α-interferon (IFNα) and interleukins: IL1β, 2, 4, 6, 8, 10, 18.

Thus, the proposed line is diploid - has a limited lifespan, maintains the karyotype of normal human cells throughout life, is free from contaminants and does not have oncogenic potentials. It is characterized by safety in accordance with the recommendations of WHO and the requirements of GNIISiK MIBP them. L.A. Tarasevich. In IPVE them. M.P. Chumakova RAMS there are banks of sowing and working cells that can meet all the needs of production and scientific research. M-20 cells are susceptible to infection by various viruses. Additionally studied the cytokine spectrum of the M-20 line. Knowledge of the cytokine spectrum of cells allows us to more accurately assess the results in determining the interferon status of patients and give reasonable recommendations on the use of therapeutic and prophylactic drugs.

Human diploid cells — fibroblasts of strain M-20 with increased proliferative activity obtained by the proposed method can be used for diagnostic purposes, in particular for determining the activity of interferon (IFN) in human serum, as well as for therapeutic purposes, for example, for local treatment of pressure sores , bitten wounds, non-healing and burn wounds.

Claims (2)

1. A method of increasing the proliferative properties of diploid cells of human fibroblasts, characterized in that the diploid cells of the characterized line M-20 from the cryobank IPVE them. M.P. Chumakova RAMS scaled from the ampoule of the seed bank of passage 7 and receive a bank of working cells of passage 16, while cells of 20-33 passages suitable for use for medical and / or diagnostic purposes are obtained by culturing in a nutrient medium containing 10% fibrinolytically active plasma (FAP) of a person containing platelet-derived growth factor PDGF at a concentration of 155 to 342 pg / ml. 2. The method according to claim 1, in which when culturing cells using a nutrient medium DMEM with 10% FAP.