Fibroblasts. Functions of fibroblasts

The main activity of modern aesthetic medicine- prevention of aging using high technology. As a result scientific research A pattern has been identified that cells have the ability to regenerate. Fibroblasts also have these properties, the regeneration of which leads to rejuvenation of the skin and the elimination of visible defects on them.

Functions and nature of fibroblasts

The term "fibroblasts" consists of two Latin words translated literally as "sprout" and "fiber". By their nature they are cells connective tissue, synthesizing the extracellular matrix (tissue structure that ensures the transfer chemical substances and mechanical support of skin cells). Fibroblasts produce substances that are precursors of collagen and elastin fibers, hyaluronic acid, fibrin.

They come from mesenchyme - germinal tissue that is found in the cells of the body of humans and animals. In the active state, the structure of fibroplasts implies the presence of a nucleus and processes; they have an increased size and contain a large number of ribosomes; in a resting state, they decrease in size and acquire a spindle-shaped shape.

Skin fibroblasts have wide range functions. Thanks to their presence in the body, the following processes occur:

• Activation of collagen and elastin synthesis processes.
• Formation of blood vessels.
• Directing immune system cells towards bacteria and foreign particles.
• Acceleration of tissue growth.
• Increased cell growth.
• Healing of damaged skin areas.
• Production of a number of proteins (proteoglycan, laminin and others).

Causes of age-related changes

The youth of the skin is determined by the cyclical process of production of collagen and elastin, which are subsequently broken down into their component parts, which are used by fibroblasts to re-produce them. Over time, the latter reduce their activity, ceasing to produce collagen and elastin fibers, which ultimately provokes skin aging.

Age-related changes begin to appear from 28 to 30 years of age. They are expressed in loss of elasticity and development of ptosis, changes in skin color, increased dryness, and the formation of wrinkles. And all this due to the fact that every decade fibroblasts decrease by 10% original number.

Replenishment of fibroblasts

So, in order to slow down aging and restore youth, it is necessary to restore fibroblasts. Most modern cosmetic techniques lead only to a temporary acceleration of the synthesis of collagen fibers, but do not increase the cells themselves. For a long time it was generally accepted that this was simply impossible.

Nowadays, science has made great strides forward, and the restoration of fibroblasts is no longer a fantasy. This procedure called SPRS therapy and is widely practiced in the States, European countries and, more recently, in Russia.

SPRS therapy: features and principle of implementation

Restoring fibroblasts is not easy; it requires going through a complex injection procedure. The results of its implementation are thickening of the skin and an increase in its elasticity, prevention and reduction of ptosis. Wrinkles are also reduced, pigmentation disappears and scars are smoothed out.

Therapy begins with the collection of the patient's cells from the skin located behind the auricle. The resulting sample is used for diagnosis and study, called biomaterial. It is used to develop a treatment regimen and artificially recreate fibroblasts, which will subsequently be injected back into the skin using injections.

Cells grown from patient biomaterials are not rejected by the body. After transplantation, they remain active for one and a half years, during which the skin condition improves.

Fibroblasts are not recommended to be administered by injection during exacerbation of chronic diseases, colds, viral infections accompanied by elevated temperature bodies. Contraindications include immunodeficiency, malignant formations, infections and chronic diseases V acute stage. Before carrying out the procedure, a preliminary consultation with a specialist is required to identify individual contraindications.

The procedure takes no more than an hour and is carried out in a course of 2 sessions with a break of 5 to 7 weeks. Before injections, local anesthesia is required.

Introducing fibroblasts is an expensive pleasure. A full range of services, including collection, storage, research and administration of biomaterials, is estimated at approximately 400,000 rubles.

Video: conducting SPRS therapy

Fibroblasts are connective tissue cells that ensure the production of collagen and elastin, therefore maintaining the youth of our skin. Over time, their number in the body steadily decreases, due to which external signs of age-related changes appear. The restoration of the number of fibroblasts is carried out using an injection technique based on artificially grown cells.

Fibroblasts- leading cells of loose connective tissue, producing components of the intercellular substance. These are branched, spindle-shaped or spread-out cells measuring about 20 microns. The organelles of the internal metabolic environment are well developed in them. The fibroblast nucleus is oval in shape, contains evenly dispersed chromatin and 2-3 nucleoli. The cytoplasm is clearly divided into intensely stained endoplasm and weakly stained ectoplasm. The cytoplasm of fibroblasts (especially young ones) is basophilic. It reveals a well-developed endoplasmic reticulum with a large number of ribosomes attached to membranes in the form of chains of 10-30 granules. This ultrastructure of the granular endoplasmic reticulum is characteristic of cells that actively synthesize protein “for export.” There are also numerous free ribosomes and a well-developed Golgi complex. Mitochondria are large, their number is small. Cytochemical methods have shown the presence of glycolytic enzymes and hydrolytic enzymes of lysosomes (especially collagenase) in the cytoplasm of fibroblasts. Mitochondria oxidative enzymes are less active.

Musculoskeletal system of the cell ensures their mobility, change in shape, attachment to the substrate, mechanical tension of the film to which the cell is attached in culture. There are many microvilli and vesicular projections on the cell surface. Fibroblasts suspended in a liquid medium have a spherical shape. The fibroblast becomes spread out after adhering to a solid surface, along which it moves due to pseudopodia.

The main function of fibroblasts- synthesis and secretion of proteins and glycosaminoglycans used to form components of the intercellular substance of connective tissue, as well as the production and secretion of colony-stimulating factors (granulocytes, macrophages). Fibroblasts for a long time retain the ability to proliferate. Fibroblasts that have completed their development cycle are called fibrocytes. These are long-lived cells. The cell cytoplasm is depleted of organelles, the cell becomes flattened, and the proliferative potential decreases. However, the cell does not lose the ability to participate in regulation metabolic processes in fabric.

Intercellular substance. Consists of fibrillar and basic (amorphous) components. Using histoautoradiography methods with the introduction of labeled amino acids (3H-proline, 3H-glycine, etc.), it was established that protein molecules are synthesized in fibroblast polysomes. Fibroblasts can simultaneously synthesize several types of specific proteins and glycosaminoglycans. For the synthesis of collagen protein, the presence of vitamin C is essential, with a deficiency of which collagenogenesis is sharply inhibited. The synthesis of intercellular substances occurs more intensively under conditions of reduced oxygen concentration. Simultaneously with the synthesis of collagen, the fibroblast destroys approximately 2/3 of this protein using the enzyme collagenase, which prevents premature tissue sclerosis.

Synthesized procollagen molecules are brought to the surface of fibroblasts by exocytosis. In this case, the protein transitions from a soluble form to an insoluble one - tropocollagen. The combination of tropocollagen molecules into supramolecular structures - collagen fibrils - occurs in the immediate vicinity of the cell surface due to the action of special substances secreted by the cell. In particular, a protein was found on the surface of fibroblasts - fibronectin, which performs adhesive and other functions. Subsequent stages of fibrillogenesis occur by polymerization and aggregation of tropocollagen on previously formed fibrils. In this case, the maturation of collagen fibers can occur without direct connection with fibroblasts.
Glycosaminoglycans are regulators of collagen formation and are part of the main (amorphous) component of the intercellular substance.

Fibrillar component The intercellular substance of loose connective tissue includes three types of fibers - collagen, elastic and reticular. They have a similar mechanism of formation, but differ from each other in chemical composition, ultrastructure and physical properties. Collagen protein is identified by its amino acid composition and the sequence of amino acids in the collagen molecule. Depending on the variation of amino acids in the polypeptide chain, immune properties, molecular weight, etc., 14 or more varieties of collagen proteins are distinguished, which are part of the connective tissue of organs. They all make up 4 main types, or classes, of collagen.

Type 1 collagen found in connective and bone tissue, as well as in the sclera and cornea of ​​the eye; Type II - in cartilaginous tissues; Type III - in the wall of blood vessels, in the connective tissue of the fetal skin; IV-ro type - in basement membranes.

Fibroblasts(fibroblastocytes) (from Latin fibra - fiber, Greek blastos - sprout, germ) - cells that synthesize components of the intercellular substance: proteins (for example, collagen, elastin), proteoglycans, glycoproteins.

In the embryonic period, a number of mesenchymal cells of the embryo give rise to fibroblast differentiation, which includes:

· stem cells,

semi-stem progenitor cells,

· unspecialized fibroblasts,

differentiated fibroblasts (mature, actively functioning),

fibrocytes (definitive cell shapes),

myofibroblasts and fibroclasts.

WITH main function fibroblasts are associated with the formation of the main substance and fibers (which is clearly manifested, for example, during wound healing, the development of scar tissue, the formation of a connective tissue capsule around a foreign body).

Low-specialized fibroblasts are few-processed cells with a round or oval nucleus and a small nucleolus, basophilic cytoplasm, rich in RNA. The cell size does not exceed 20-25 microns. A large number of free ribosomes are found in the cytoplasm of these cells. The endoplasmic reticulum and mitochondria are poorly developed. The Golgi apparatus is represented by clusters of short tubes and vesicles.
At this stage of cytogenesis, fibroblasts have very low levels of protein synthesis and secretion. These fibroblasts are capable of mitotic reproduction.

Differentiated mature fibroblasts are larger in size. These are actively functioning cells.

In mature fibroblasts, intensive biosynthesis of collagen, elastin proteins, proteoglycans, which are necessary for the formation of the main substance and fibers, is carried out. These processes are enhanced under conditions of low oxygen concentration. Stimulating factors for collagen biosynthesis are also ions of iron, copper, chromium, ascorbic acid. One of the hydrolytic enzymes is collagenase- breaks down immature collagen inside cells, which regulates the intensity of collagen secretion at the cellular level.

Fibroblasts are motile cells. In their cytoplasm, especially in the peripheral layer, there are microfilaments containing proteins such as actin and myosin. The movement of fibroblasts becomes possible only after they are bound to supporting fibrillar structures using fibronectin- a glycoprotein synthesized by fibroblasts and other cells, ensuring the adhesion of cells and non-cellular structures. During movement, the fibroblast becomes flattened, and its surface can increase 10 times.

The plasmalemma of fibroblasts is an important receptor zone that mediates the effects of various regulatory factors. Activation of fibroblasts is usually accompanied by the accumulation of glycogen and increased activity of hydrolytic enzymes. The energy generated by glycogen metabolism is used to synthesize polypeptides and other components secreted by the cell.

Based on their ability to synthesize fibrillar proteins, the fibroblast family includes reticular cells of the reticular connective tissue of the hematopoietic organs, as well as chondroblasts and osteoblasts of the skeletal variety of connective tissue.

Fibrocytes- definitive (final) forms of fibroblast development. These cells are spindle-shaped with pterygoid processes. [They contain a small number of organelles, vacuoles, lipids and glycogen.] The synthesis of collagen and other substances in fibrocytes is sharply reduced.

Myofibroblasts- cells similar to fibroblasts, combining the ability to synthesize not only collagen, but also contractile proteins in significant quantities. Fibroblasts can transform into myofibroblasts, which are functionally similar to smooth muscle cells, but unlike the latter they have a well-developed endoplasmic reticulum. Such cells are observed in the granulation tissue of healing wounds and in the uterus during pregnancy.

Fibroclasts- cells with high phagocytic and hydrolytic activity, take part in the “resorption” of intercellular substance during the period of organ involution (for example, in the uterus after pregnancy). They combine the structural features of fibril-forming cells (developed granular endoplasmic reticulum, Golgi apparatus, relatively large but few mitochondria), as well as lysosomes with their characteristic hydrolytic enzymes. The complex of enzymes they secrete outside the cell breaks down the cementing substance of collagen fibers, after which phagocytosis and intracellular digestion of collagen occurs.

The following cells of fibrous connective tissue no longer belong to the fibroblast differentiation.

Skin fibroblasts form the basis of connective tissue. They are producers of hyaluronic acid, collagen fibers, and elastin. Age-related changes slow down the functioning of fibroblasts, causing the skin to become thin and flabby. Thanks to cellular injection technology the body independently launches the function of rejuvenating the structure of the dermis.

The essence of fibroblasts

Skin fibroblasts- these are cells of the connective tissue layer of the dermis, the predecessors of which were stem cells. They come in two forms:

1. Active - large cells, equipped with a flat oval-shaped nucleus, a large number of ribosomes and processes. They are characterized by intensive division, production of collagen and other matrix components.
2. Inactive (fibrocytes) - the cells are slightly smaller and have a spindle-shaped shape. They are formed from fibroblasts and cannot divide. Participate in fiber synthesis and wound regeneration.

As the body ages, the number of fibroblasts decreases and their activity decreases. This leads to a deterioration in the synthesis of intercellular substances. This process is reflected on the skin in the form of thinning, dryness, and sagging. It stretches and wrinkles form.

Functions

One of the main functions of fibroblasts is the production and regeneration of intercellular substance. By forming growth factors, components of the extracellular matrix, enzymes, they contribute to the destruction and new synthesis of collagen and hyaluronic acid. Thanks to the non-stop process, the intercellular substance is renewed. In addition, they produce cell growth factors:

• The main one is that the growth of all dermal cells is stimulated, fibronectin is produced for protective reactions;
• Transforming - collagen and elastin fibers are synthesized, blood vessels are formed, cells of the immune system are directed to foreign agents, bacteria;
• Epidermal - tissue proliferation, cell growth, and keratinocyte transport are activated;
• The growth of keratinocytes is epithelization, damage is regenerated.

Fibroblast growth factors are represented by multifunctional proteins that are mitogens and also perform endocrine, regulatory, structural function. Thanks to fibroblasts, proteins important for the skin are produced: proteoglycans, tinascin, nidogen and laminin.

The essence of the technique

SPRS therapy is a technique of injection skin rejuvenation using fibroblasts, eliminating the very cause of skin aging. The patent for the technology of intradermal transplantation of autofibroblasts belongs to FibrocellScience, an American company. Through cell technology, it has become possible to grow fibroblasts from a particle of human skin (biopsy). Own biomaterial eliminates the problem of tissue compatibility and the risk of infection. Autologous cells are positively perceived immune system and are able to function fully.

The sample can be taken at any age, but it is preferable to do it when young. Recommended age is from 20 to 30 years. You can save a piece of skin during any operation and place the cells isolated from it in cryogenic storage for many years. A temperature of -196 degrees allows you to store them throughout your life, using them as needed. This will allow you to carry out effective cosmetic procedures at any time.

Own fibroblasts, along with stem cells, have the property of maintaining potential during aging. Under local anesthesia A small sample of skin is taken from the patient behind the ear, navel, or forearm area. These areas are least exposed to ultraviolet radiation. Its size is about 4 mm. Fibroblasts isolated from it are placed in special vials.

When they are cultivated in a medium with fetal serum, the ability to proliferate is stimulated in young cells, and the old ones are washed away. There is a “rejuvenation” of culture. After a month, the number of cells increases several thousand times. After reactivation, the cell culture is transplanted into the patient and actively fills the dermis. After a month and a half, the multiplied fibroblasts are injected into the patient’s facial skin, including around the eyes, as well as into the neck, décolleté, and arms.

Procedure

The course consists of 3-5 sessions, intervals between which range from 3 to 6 weeks. Stages of the procedure:

• examination of the patient to identify existing contraindications;
• taking material;
• fibroblast cultivation;
• injection of cellular material into the skin using two methods: tunnel - into deep skin folds, papular mesotherapy;
• protecting the skin from ultraviolet radiation by applying cream.

Patients note that the procedure is painful, so Emla anesthetic cream is used. The amount of the drug used is up to 3 ml per session. The recovery period lasts for 2-3 days. After the procedure, it is prohibited to apply cosmetics. For two weeks you need to refrain from visiting the sauna or bathhouse. The skin must be protected from the sun by lubricating it with a cream with a high degree of protection. It is recommended to repeat the procedure once annually. The result is an improvement in the condition of the facial skin over several months.

Efficiency and advantages of the method

Rejuvenation with fibroblasts gives the first results after 1.5 or 2 months. Full effect The effect of the procedure appears after six months and lasts for 2-3 years. Enhanced production of growth factors and extracellular matrix begins. Fibroblasts go through natural phases of the cycle: they are activated, synthesize elastin, collagen and other substances, then the degradation phase begins, replacing them with new fibroblasts.

Their use is widespread in medicine - against burns, for tissue regeneration during trophic ulcers, wounds Their importance in cosmetology is great. The youth of the skin is formed by the number of fibroblasts. Placed in the dermis, grown fibroblasts are embedded in tissues, beginning the production of collagen and elastin. As a result, the skin becomes elastic, acquires an even color, and fine wrinkles disappear.

But you should not expect a tightening effect from the procedure. This technique is aimed at improving the quality characteristics of the skin. The main advantages of SPRS therapy:

• the drug works with genes, which eliminates disruption of the primary structure of the skin;
• natural rejuvenation processes are activated;
• safety, no risk of rejection, allergic reaction;
• long-term preservation of the result.

Within 6 months, wrinkles around the eyes are smoothed out by 90%. The décolleté and neck look younger by 95%, cheeks by 87%. Folds around the mouth are reduced by 55%.

Contraindications

Despite complete safety, the procedure has some contraindications:

• pregnancy, lactation period;
• impaired blood clotting;
• malignant neoplasms;
• autoimmune diseases;
• predisposition to scar formation;
• ARVI;
• inflammation on the skin.

During the day after the session, redness on the skin and microhematomas may be observed. The next day the symptoms disappear.

Autofibroblast transplantation technology has official permission from Roszdravnadzor. Its safety is confirmed by laboratory monitoring of cell viability.

Owners of patent RU 2536992:

The invention relates to the field of biotechnology, specifically to cellular technologies, and can be used in medicine. The method involves scaling diploid cells of the M-20 line from the cryobank of the IPVE named after. M.P. Chumakov Russian Academy of Medical Sciences from an ampoule of a seed cell bank of passage 7 to obtain a bank of working cells of passage 16. In this case, cells of 20-33 passages, suitable for use for therapeutic 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 in a concentration of 155 to 342 pg/ ml. The invention allows to increase the proliferative activity of diploid human fibroblast cells. 1 salary files, 2 tables.

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

Human diploid cell lines (HDCL) have undeniable advantages over all known types of cell cultures in their ability to maintain stable biological and genetic characteristics during passages. Certification of LDCC intended for the production of vaccines is carried out in accordance with uniform requirements developed by the World Health Organization. These recommendations are taken as the basis for the national criteria for certification of vaccine LDKCH, developed by the State Research Institute of Clinical Infectious Diseases named after. L.A. Tarasevich and the USSR Ministry of Health [ Guidelines“Certification of continuous cell lines - substrates for the production and control of medical immunobiological preparations» RD-42-28-10-89. USSR Ministry of Health. M., 1989. - P. 16]. The certified line of human diploid cells 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 tumor formation in immunosuppressed animals. The diploid cell line must have a certified seed cell bank at early passage levels (up to passage 10), consisting of at least 200 cryovials. By passaging seed cells from one or several cryovials to the 16th passage level, a working bank of cells is obtained, from which the necessary producer cultures can be obtained for production or for research work. 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 LDCVs are used in the production of vaccines against polio, 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 scientific-practical conference « Actual problems medical virology, dedicated to the 100th anniversary of M.P. Chumakov." M. 2009. Volume XXVI. pp. 305-307; L.L. Mironova, V.D. Popova, O.I. Konyushko. Experience in creating a bank of original lines of transplantable cells and their use in virological practice. Biotechnology. 2000, p. 41-47]. LDCN are widely used in vitro for the diagnosis of viral infections and toxicity analysis various drugs and products for replacement therapy [RF Patent No. 2373944, 06/23/2008. Method of treatment burn wound. A.S. Ermolov, S.V. Smirnov, V.B. Khvatov, L.L. Mironov; S.V. Smirnov, V.B. Hvatov. Innovative technologies local treatment of burns at the Research Institute of Emergency Medicine named after. N.V. Sklifosovsky. In the book: New Economics. An innovative portrait of Russia. M., Center for Strategic Partnership, 2009. pp. 388-390].

In IPVE im. M.P. Chumakov RAMS in the 80s of the 20th century, several lines of diploid cells were established from the skin and muscles of 8-10 week old human embryos. This work is devoted to modifying the production of human diploid cells for diagnostic purposes and cell replacement therapy, namely the production of diploid human fibroblast cells with increased proliferative properties.

Prototype. RF Patent No. 1440029 dated March 22, 1993 [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 im. N.F. Gamaleya. A strain of diploid human embryonic skin and muscle cells used as a test system for determining the antiviral activity of human interferons and virus propagation].

This LDCC strain is designated M-21, however, the fibroblast culture M-21 had insufficient proliferative activity, which reduced the time of monolayer formation and increased the consumption of cells and materials, and this ultimately led to the 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 medical and biological purposes, more cost-effective, characterized by high proliferative activity, and having banks of seed and working cells. This line is designated M-20. At passage level 7, a seed cell bank was prepared. In 2012, a bank of working cells at the 16th passage level was made from an ampoule of the bank of passage 7. Banks of seed and working cells at levels 7 and 16 passages are stored at the Institute of Vessels of Experimental Physics named after. M.P. Chumakov RAMS and allow us to provide both production processes, and scientific research.

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

Thus, the object of the invention is a method for increasing the proliferative properties of diploid human fibroblast cells for medical and biological purposes by cultivating cells from the cryobank of the IPVE named after. M.P. Chumakov Russian Academy of Medical Sciences, in which diploid cells of the characterized M-20 line are used, which are scaled from the ampoule of a bank of seed cells of passage 7 and a bank of working cells of passage 16 is obtained, with cells of passages 20-33 suitable for use for therapeutic and/or diagnostic purposes, obtained by cultivation in a nutrient medium containing 10% fibrinolytically active plasma (FAP) of a person. When culturing cells, preferably the nutrient medium DMEM 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 for therapeutic and/or diagnostic purposes.

Scheme of implementation of the method:

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

2. Preparation of a bank of working cells at the level of passage 16 of the 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 M.P. Chumakov, Russian Academy of Medical Sciences).

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

5. Restoration of the biological properties of fibroblasts of the M-20 line by three-fold passaging (up to the 20th passage inclusive) to repair possible DNA damage during the cryopreservation process.

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

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

This technical result is achieved by cultivating 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 the cell culture.

FAP is a clinically used transfusion medium, which is obtained from the blood of people who suddenly died from myocardial infarction, acute heart failure, cerebral hemorrhage, in the first 6 hours after death [Order of the USSR Ministry of Health No. 482 of June 14, 1972 “On improving the provision of therapeutic and prophylactic institutions and clinics with cadaveric tissues, bone marrow and blood"]. Postmortem blood is a complete transfusion medium, which has a number of biological properties - primarily increased fibrinolytic potential. In this regard, it is also proposed to call postmortem blood fibrinolysis. The main indications for postmortem blood transfusion: acute blood loss, shock, anemia of various origins, burn injury, metabolic replacement during exogenous poisoning, filling the AIK when using extracorporeal circulation in surgery [E.G. Tsurinova. Transfusion of fibrinolysis blood. M., 1960, 159 p.; S.V. Ryzhkov. Preparation and possibilities of using fibrinolysis blood depending on the time of collection and the cause of death. Author's abstract. doc. diss. L., 1968, 21 pp.; G.A. Pafomov. Biological characteristics of the blood of suddenly deceased and its use in surgical practice. Diss. doc. honey. Sci. M., 1971, 355 pp.; K.S. Simonyan, K.P. Gutiontova, E.G. Tsurinova. Post-mortem blood in the aspect of transfusiology. M., Medicine, 1975, 271 pp.]. Currently, postmortem blood components are used: fibrinolytically active plasma, red blood cell mass, leukocyte mass, platelet mass [G.Ya. Levin. Hemocoagulation properties and clinical application plasma and platelets of cadaver blood. Author's abstract. doc. diss. M., 1978, 31 p.; V.B. Hvatov. Preparations of fibrinolytic and antiprotenease action from the blood plasma of suddenly deceased people. Diss. doc. Med Sciences, 1984, 417 pp.; 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. Hvatov. Medical and biological aspects of the use of posthumous blood. Bulletin of the USSR Academy of Medical Sciences, 1991, 9. pp. 18-24; V.B. Hvatov. Corpse blood - history and current state of the issue. Problem hematol. and overflow. blood, 1997, 1. S. 51-59]. Components of cadaveric blood obtained from organ donors have also received clinical use [a deceased individual with a beating heart according to the “Instructions for ascertaining the death of a person based on a diagnosis of brain death” dated December 20, 2001 No. 460, Ministry of Justice registration 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 Human Organs and (or) Tissues” - as amended. Federal laws dated 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 cellular components of the blood of organ donors. Transplantology, 2011, 4, p. 13-19; Khubutia M.Sh., Khvatov V.B., Gulyaev V.A. etc. A method for compensating globular blood volume and immunomodulatory effects during transplantation. RF patent for invention No. 2452519, publ. 06/10/2012, bulletin. No. 16].

Fibrinolytically active plasma is obtained from the blood of suddenly deceased people, prepared with the preservative Glyugitsir (blood: preservative ratio 4:1) to preserve its fibrinolytically active properties. The separation of plasma from the cellular elements of blood is carried out in a sterile box in compliance with all the rules of asepsis and antiseptics and is similar to obtaining donor plasma from canned donor blood. Clinical use of FAP in surgery and traumatology has revealed the effect of stimulating wound healing [I.Yu. Klyukvin, M.V. Zvezdina, V.B. Khvatov, F.A. Burdyga. A method for treating bite wounds. Patent for invention of the Russian Federation No. 2372927, publ., November 20, 2009, bulletin. No. 32]. We associated this effect with the presence of growth-stimulating factors in FAP, secreted by activated platelets. We subsequently identified platelet-derived growth factor (PDGF) in FAP. The growth-stimulating effect of FAP in human cell culture has been shown in special studies. The studied FAP samples at a 10% concentration were added to a cell suspension of human fibroblasts of the M-20 line, containing a known number of cells, and 10 ml of the resulting mixture was placed in culture flasks with a growth surface area of ​​25 cm 2 . Cells were grown for 3-4 days in an atmosphere of 5% CO 2 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 - the proliferation index (in Table 1).

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

For a better understanding of the proposed technical solution “production of human diploid cells of the M-20 line for medical and biological purposes,” we provide the following example.

Cells of the M-20 line, passage 16, are recovered from the working bank. To do this, the cryovial with cells is removed from liquid nitrogen and placed in 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), the antibiotic gentamicin is added at the rate of 1 ml of 4% solution per 1 liter of nutrient medium . To form a monolayer, cells are cultured for 4-5 days at 37°C and 5% CO 2 in an atmosphere. After the formation of a monolayer of cells, 3 successive passages are carried out, necessary for DNA repair after cryopreservation. Then cells are replicated from passage 20 to passage 33. Cells from these passages are intended for biomedical purposes. The resulting cell line was characterized in detail in accordance with the requirements of the WHO and the GNIISiK MIBP named after. L.A. Tarasevich, including HLA typing of the M-20 cell line, and also a study of its cytokine spectrum. We provide a comparative description of the properties of the M-20 line and the M-22 line (Table 2). Line M 22 (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 for the treatment of burn wounds of II-IIIA degrees [RF Patent for invention No. 2373944, 06/23/2008. Method for treating a burn wound. A.S. Ermolov, S.V. Smirnov, V.B. Khvatov, L.L. Mironova, O.I. Klnyushko, E.A. Zhirkova, B.C. Bocharova].

Line M-20 was installed at IPVE named after. M.P. Chumakov 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. There was no history of cancer, sexually transmitted diseases, hepatitis, or tuberculosis; genetic and congenital diseases was not observed in the family. Cell culture medium DMEM supplemented with 10% FAP. The seeding ratio is 1:3-1:4 twice a week with a seeding 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 clumps of chromatin. IN life cycle The line can be distinguished into 3 phases of development: formation 1-3 passages, active growth 4-40 and aging 41-52, then death occurs. The cells of the line have a human karyotype 2m=46, XY. The line is characterized by high genetic stability: 93.3-96.9% of cells have a diploid set of chromosomes, cells with a polyploid set are no more than 1.6%. No gaps, breaks or ring chromosomes were observed. The number of bands of isoenzymes G-6PDE and LDE and their electrophoretic mobility coincide with those for human erythrocytes. G-6FDG slow type. When sowing on selective nutrient media, no contamination with bacteria, fungi, or mycoplasmas was detected. In addition, no mycoplasma contamination was detected when staining with DNA fluorochromes Hochst 33258 and olivomycin, as well as PCR method. Contamination with viruses in experiments on sucklings and adult white mice, guinea pigs, rabbits and chicken embryos, as well as on homologous and heterologous cell cultures. Control of tumorigenicity. When the cells of the line were administered to immunosuppressed animals, tumors did not form. No reverse transcriptase was 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 the 20th passage level produce mRNA for α-interferon (IFNα) and interleukins: IL1β, 2, 4, 6, 8, 10, 18.

Thus, the proposed line is diploid - it has a limited lifespan, retains the karyotype of normal human cells throughout life, is free from contaminants and does not have oncogenic potential. It has been characterized for safety in accordance with WHO recommendations and the requirements of the GNIISiK MIBP named after. L.A. Tarasevich. In IPVE im. M.P. Chumakov RAMS there are banks of seed and working cells that can meet all the needs of production and scientific research. Cells of the M-20 line are sensitive to infection by various viruses. Additionally, the cytokine spectrum of the M-20 line was studied. Knowledge of the cytokine spectrum of cells makes it possible to more accurately evaluate the results when determining the interferon status of patients and give informed 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 to determine the activity of interferon (IFN) in human blood serum, as well as for medicinal purposes, for example, for local treatment of bedsores , bite wounds, long-term non-healing and burn wounds.

1. A method for increasing the proliferative properties of diploid human fibroblast cells, characterized in that diploid cells of the characterized M-20 line from the cryobank of the Institute of Vessels named after. M.P. Chumakov RAMS is scaled from an ampoule of a bank of seed cells of passage 7 and a bank of working cells of passage 16 is obtained, while cells of passages 20-33, suitable for use for therapeutic and/or diagnostic purposes, are obtained by cultivation in a nutrient medium containing 10% fibrinolytically active plasma (FAP) human containing platelet-derived growth factor PDGF in a concentration of 155 to 342 pg/ml.

2. The method according to claim 1, in which the nutrient medium DMEM with 10% FAP is used when culturing cells.

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