RU2620342C1 - Cosmetic means on the basis of secrection products of mesenchemical stem/stormal cells of fatty fabric of human and the method of its production - Google Patents

Abstract

FIELD: cosmetology.

SUBSTANCE: group of inventions is a cosmetic for stimulating the regeneration of skin tissues, comprising as the main component a culture medium conditioned by mesenchymal stromal cells of human adipose tissue containing secretion products comprising VEGF at a concentration of at least 4 ng/million cells, FGF basic in concentration At least 5.8 pg/million cells, PEDF at a concentration of at least 10 ng/million cells, PAI-1 at a concentration of at least 35 ng/million cells, and a complex of at least 20 proteins as shown in table 1. The concentration of these components is presented in 10 ml of medium.

EFFECT: invention provides stimulation of skin regeneration, increase of skin elasticity and improvement of its aesthetic characteristics.

29 cl, 7 dwg, 2 tbl, 13 ex

Description

FIELD OF THE INVENTION

The group of inventions relates to the field of cosmetology and cosmeceuticals and can be used to stimulate regeneration processes in various layers of the skin, thereby exerting a cosmetic effect. The group of inventions also relates to a method of inhibiting skin aging, preventing and / or correcting cosmetic defects. The use of this group of inventions allows to stimulate the renewal of skin cells due to the regenerative effect of a balanced complex of bioactive factors secreted by human mesenchymal stem / stromal cells (MSCs).

State of the art

The prior art composition for stimulating the growth and regeneration of cells, as well as methods for its preparation (RU 2341270 C1). The composition contains disrupted peripheral blood leukocyte cells and stem and / or progenitor cells in an air-conditioned culture medium. A method of producing this composition for stimulating cell growth and regeneration involves obtaining a conditioned culture medium obtained by culturing peripheral blood leukocytes and stem and / or progenitor cells, destroying them in an conditioned medium, and combining the composition with a pharmaceutically acceptable carrier. Thus, the conditioned medium does not undergo a cleaning procedure and contains a large amount of ballast substances, which can have a negative effect on tissues. It should also be noted that the composition prepared by this method contains destroyed cells, which can be potentially dangerous and are not recommended for inclusion in cosmetics.

A composition is known in the art that enhances hair growth, the main component of which is a medium obtained by conditioning stem cells of human amniotic fluid (WO 2010107287 A2). It is stated that this composition is highly effective, however, the composition of the amniotic fluid varies significantly depending on the pathological condition, in addition, the procedure for obtaining the right amount of amniotic fluid is associated with certain risks, both for the mother (infectious complications, sensitization, amniotic fluid embolism, etc. ), and for the fetus (injury, up to death in rare cases).

In addition, a composition intended for regeneration of the skin and mucous membranes containing a culture medium including biologically active compounds based on low molecular weight peptides and cytokines, used for in vitro cultivation of eukaryotic cells and containing vital products and growth factors of eukaryotic cells is also known in the art. released by them into the nutrient medium in the process of cultivation (RU 2455354 C1). The composition of the composition includes human mesenchymal stromal cells in an amount of at least 105 cells / ml. Nutrient medium - an integral part of the composition contains bovine serum - xenogenic component.

Fetal bovine serum also contains a composition for preventing or treating skin defects, and a method comprising applying to a skin defect a composition containing bone marrow MSCs or conditioned medium obtained from MSCs, or a combination thereof (WO 2008155659 A2). The composition can be used to treat skin defects such as wrinkles, scar, age spot, cut or ulcer. The disadvantage of these compositions is that not defined a set of substances that directly provide the effect of the composition. The inclusion of human cells in the composition complicates the processes of its production, standardization, quality control and reduces the stability of the composition due to the limited viability of the cells.

An air-conditioned culture medium extract is also known in the art for external use to enhance hair growth and skin rejuvenation (WO 2010038232 A1). In this case, MSCs cultivated in a medium containing serum (bovine embryonic or obtained from human blood) are used, and growth factors are added to it to obtain the extract. However, the addition of growth factors from outside can disrupt the balanced composition of the composition and increase the potential risk to the health and life of the consumer, and also increases the cost of the composition.

Closest to the claimed invention is a tool for changing the growth rate or reproduction of cells and to slow down the aging of the skin obtained by culturing eukaryotic cells in three dimensions, and a method for its production (RU 2280459 C1). Cell cultures can be various types of cells, including neuronal, embryonic and mesenchymal stem cells, as well as fibroblasts, a distinctive feature is the cultivation in three dimensions. Removal of the cellular component is provided by the method for preparing the composition. The qualitative composition is described in sufficient detail, the emphasis is on the secretion of collagen as a structural protein of the skin, however, quantitative data are presented mainly for individual cytokines (IL-8, G-SCF) and some growth factors (VEGF, KGF, TGFb). In addition, the description does not indicate the quantitative content of other factors, which does not allow us to evaluate the reliability of the presented data on effectiveness. In addition, methods for purification of the product were not disclosed, the degree of preservation of activity after lyophilization and subsequent recovery was not evaluated.

Disclosure of invention

The objective of the invention is the creation of a cosmetic product for application to intact skin, capable of multifunctionally acting on skin with signs of aging - to enhance regeneration processes, increase elasticity and improve appearance.

The technical result to which the claimed invention is directed is to obtain a cosmetic product that effectively stimulates the renewal and regeneration of skin tissues due to the complex balanced action of growth factors and other biologically active molecules secreted by human mesenchymal stem / stromal cells (MSCs) in the culture medium, not containing xenogenic components. The effectiveness of the product is ensured by the selected content of key biologically active molecules that make up the main active component and ensure the implementation of the action of the cosmetic product. The qualitative and quantitative composition of the claimed agent is determined by testing obtained environmental samples of human MSCs that contain cell secretion products by their ability to stimulate the migration of human dermal fibroblasts on an in vitro model of a cell wound and by the presence of a complex of bioactive factors involved in skin regeneration processes. The active complex of the product includes structural skin proteins, enzymes and metabolic regulators of structural proteins, as well as factors that stimulate trophism and skin regeneration processes. The synergistic effect of this complex provides the versatility of the proposed tool. Conditioned media that do not meet stated requirements based on testing results are rejected.

The problem is solved in that the cosmetic product for stimulating the regeneration of skin tissue includes, as a main component, a culture medium conditioned by human mesenchymal stromal cells (MSCs) containing secretion products, including VEGF in a concentration of at least 4 ng / million cells, FGF basic in a concentration at least 5.8 pg / million cells, PEDF at a concentration of at least 10 ng / million cells, PAI-1 at a concentration of at least 35 ng / million cells, as well as a complex of at least 20 proteins shown in table 1, while the concentration I said components represented by 10 ml of medium.

The relative quantitative content of proteins in Table 1 is presented taking into account the allowable error.

In addition, the best result is achieved by using an agent that includes, as the main component, a culture medium conditioned by human MSC containing secretion products, additionally comprising a complex of at least one of the proteins listed in Table 2.

The cosmetic product can be presented in the form of a lyophilisate-conditioned medium, which may additionally contain transdermal penetration enhancers, while the lyophilisate is reduced in an aqueous solution of enhancers. It is proposed to use dimethyl isosorbide in the amount of not less than 1.5% with respect to the weight of the finished cosmetic product and / or hyaluronic acid or its derivatives in the amount of not less than 1% with respect to the weight of the finished cosmetic product and / or liposomes of various compositions as enhancers. . Sodium hyaluronate, various natural and chemical modifications of hyaluronic acid can be used as derivatives of hyaluronic acid. As liposomes of various compositions, liposomes based on soy phospholipids or any other suitable phospholipids are used. The secretion products of human MSCs contained in an air-conditioned environment can be partially or completely included in the composition of liposomes, while the agent is presented in the form of a solution or lyophilisate. Liposomes are prepared by forcing or removing a detergent. The incorporation of medium components into liposomes can be accomplished by lyophilization.

Upon receipt of the product in the form of a lyophilizate, it can additionally contain a pharmaceutically compatible with the lyophilizate and water-soluble polymer substance (for example, methyl cellulose), which forms a gel-like substance in a ratio by weight of not more than 1: 100 (per 1) by dissolving in an aqueous solution of transdermal penetration enhancers part of the lyophilisate is taken by not more than 100 parts of the polymer substance), with the formation of a stable gel-like substance, while the combination of the lyophilisate with the polymer and an aqueous solution of enhan Erov mixed together immediately before application to the skin. The enhancers listed above can also be used as enhancers in this product. Sodium hyaluronate and dimethyl isosorbide can be used as a 1% aqueous solution. In addition, the lyophilisate may also contain soy phospholipids based liposomes.

The problem is also solved by the fact that a method of obtaining a cosmetic product based on an air-conditioned medium containing products of secretion of MSCs of a person, including culturing MSCs of human VT up to 4-5 passage in a medium that supports the growth of undifferentiated human mesenchymal cells, washing the cells with a buffer solution, conditioning MSCs in serum-free and animal-free growth medium that supports cell viability of at least 70% and suitable for therapeutic use, selection a culture medium containing human MSC secretion products, removing cell debris from it, preliminary testing of the conditioned medium and sampling to obtain a cosmetic product, purification of the selected samples from low molecular weight components to obtain an agent containing human MSC secretion products including VEGF in a concentration of at least 4 ng / mln cells (not less than 200 pg / ml), FGF basic in a concentration of not less than 5.8 pg / ml cells (not less than 0.3 pg / ml), PEDF in a concentration of not less than 10 ng / ml (not less than 500 PCG / ml) PAI-1 at a concentration of at least 35 ng / million cells (at least 2 ng / ml), determined by enzyme-linked immunosorbent assay when restoring the lyophilisate in 10 ml of sterile physiological osmolarity buffer, as well as a complex of 20 proteins shown in table 1, determined proteomic analysis method.

In one of the embodiments of the method for obtaining funds selected purified samples are lyophilized.

In the best embodiment of the invention, the medium supporting the growth of undifferentiated human mesenchymal cells is a solution containing the base medium - AdvanceSTEM Cell Culture Media, and the additive - AdvanceSTEM Stem Cell Growth Supplement (HyClone, USA), while the additive is taken in a volume of 9-11 % to 100% of the volume of the base environment. Air conditioning is carried out in culture bottles or Petri dishes (Corning or similar), while human MSCs of human VT are placed on a flat substrate with a density of 5-15 * 10 ³ / cm ² in a conditioning medium in a volume of 0.1-0.2 ml / cm ² , then the culture containers with cells are placed for 6-8 days in a CO ₂ incubator at 37 ± 1 ° C, 5% CO ₂ and relative humidity ≥95%. A Hanks solution (PanEco or similar) is used as a buffer solution for washing cells from the components of the growth medium, while washing the cells is carried out by three to five-fold placement of cells in the mentioned solution at the rate of 0.1-0.2 ml of solution per cm 5 cells -10 minutes. As a conditioning medium, low glucose DMEM (HyClone, USA) or another growth medium supporting human MSC viability throughout the conditioning period is used. Removal of cell debris from the culture medium is carried out by centrifugation. Preliminary testing of samples of the conditioned medium is carried out according to their ability to stimulate the migration of human skin fibroblasts not less than 50% higher in comparison with the negative control, which is the basic growth medium used to condition human MSCs. To assess the migration of human skin fibroblasts, a cell wound model is used. The cultivation medium is purified by ultrafiltration with removal of low molecular weight compounds weighing less than 5 kDa. After purification of the culture medium from low molecular weight compounds, microfiltration of the obtained medium is additionally carried out through filters with a pore diameter of 0.22 μm. Lyophilization is carried out in 8-14 stages at a pressure of 0.013-0.027 kPa, temperature from -40 ° C to -5 ° C, lyophilization time is not more than 36 hours at maximum load of freeze drying. Centrifugation is carried out at 300 ± 5 g, temperature 6 ± 2 ° C for 10 minutes to remove cell debris. Purification of low molecular weight components is carried out using a Minim II tangential flow filtration system (Life Sciences, USA) or the like.

Brief Description of the Drawings

The invention is illustrated by drawings, where the figures show:

Figure 1. The migration rate of human skin fibroblasts. The graph illustrates the migration rates of human skin fibroblasts in μm / h, in vitro during cell stimulation with various solutions: DMEM base medium with low glucose (DMEM neg, negative control; n = 11), DMEM base medium with low glucose, conditioned MSCs (claimed composition) (DMEM 7d, test sample; n = 18), a low glucose DMEM base medium supplemented with 10 vol. % fetal bovine serum (DMEM pos, positive control; n = 12). Fibroblast migration was observed under a microscope with a life support system for 24 hours, the position of the cells was recorded using an automatic camera every 15 minutes. Low-glucose-conditioned DMEM basal medium, conditioned by MSCs (test samples), significantly (p <0.01) stimulates the migration rate of human skin fibroblasts by at least 50% compared to the negative control solution. The migration rate of human skin fibroblasts stimulated by a positive control solution also varies significantly compared with a negative control solution (p <0.01); no comparison of the groups of the test sample and the positive control was performed. Statistical processing of the results was carried out using the Kruskal-Wallis test, using the Dunn test in the variation of the comparison of values with the control group. A negative control solution was used as a control group.

Figure 2. The migration rate of human skin fibroblasts. The graph illustrates the rate of migration of human skin fibroblasts in μm / h, in vitro during cell stimulation with various solutions: DMEM base medium with low glucose content (DMEM neg, negative control), DMEM base medium with low glucose content conditioned by MSC (DMEM 7d , test sample), low glucose DMEM stock supplemented with 10 vol. % fetal bovine serum (DMEM pos, positive control). Fibroblast migration was observed under a microscope with a life support system for 24 hours, the position of the cells was recorded using an automatic camera every 15 minutes. The test sample stimulates the rate of migration of human skin fibroblasts, however, it is less than 50% compared with the negative control solution, therefore it is rejected according to the results of preliminary testing.

Figure 3. The migration rate of human skin fibroblasts. The graph illustrates the rate of migration of human skin fibroblasts in μm / h, in vitro during cell stimulation with various solutions: DMEM base medium with low glucose content (DMEM neg, negative control), DMEM base medium with low glucose content, conditioned by MSC, purified, lyophilized and then reconstituted in sterile saline (DMEM 7d, test sample), low glucose DMEM stock supplemented with 10 vol. % fetal bovine serum (DMEM pos, positive control). Fibroblast migration was observed under a microscope with a life support system for 24 hours, the position of the cells was recorded using an automatic camera every 15 minutes. The test sample stimulates the rate of migration of human skin fibroblasts by more than 50% compared with the negative control solution.

Figure 4. The migration rate of human skin fibroblasts. The graph illustrates the rate of migration of human skin fibroblasts in μm / h, in vitro during cell stimulation with various solutions: DMEM base medium with low glucose content (DMEM neg, negative control), DMEM base medium with low glucose content, conditioned by MSC, purified, lyophilized, reconstituted in a 1% solution of sodium hyaluronate with dimethyl isosorbide (DMEM 7d, test sample), DMEM base medium with a low glucose content with the addition of 10 vol. % fetal bovine serum (DMEM pos, positive control). Fibroblast migration was observed under a microscope with a life support system for 24 hours, the position of the cells was recorded using an automatic camera every 15 minutes. The test sample stimulates the rate of migration of human skin fibroblasts by more than 50% compared with the negative control solution.

Figure 5. The results of determining the concentration of VEGF 165 by enzyme-linked immunosorbent assay in conditioned medium (72 h), collected from cultures of MSCs of human adipose tissue after genetic modification using a baculovirus vector. Data are given for control (untreated) MSCs of human adipose tissue after conditioning for 72 hours, as well as results after treatment with a virus with a titer of 75 or 100 particles / cell. The concentration of sodium butyrate to stimulate the expression of VEGF 165 was 5 mM for 15 h of cell incubation after removal of the baculovirus solution.

Figure 6. Hypoxia-induced changes in protein expression in human MSCs. MSCs of VT (n = 10) were cultured at 1% oxygen content (hypoxia) or under standard conditions (normoxia) for 48 hours. We analyzed the level of mRNA of proteins present in all cell samples by real-time PCR. A - The content of mRNA of proteins encoding some proteins secreted mainly during hypoxia. B - The content of mRNA of proteins encoding some proteins secreted mainly during normoxia.

Figure 7. Changes in the expression of G-CSF MSC in human VT during cell cultivation under hypoxic conditions. MSCs of VT (n = 10) were cultured at 1% oxygen content (hypoxia) or under standard conditions (normoxia) for 48 hours. Changes in the mRNA content estimated by real-time PCR and secreted protein in an air-conditioned environment (ELISA method) are presented.

The implementation of the invention

The preparation of a cosmetic product for application to the skin involves culturing human mesenchymal stromal cells of adipose tissue (MSC JT) of 4-5 passages in a serum-free culturing medium devoid of animal products, maintaining cell viability of at least 70% and suitable for therapeutic use, followed by sampling of the culture medium containing products of secretion of MSC in human VT, and the removal of cell debris from it. In order to determine the suitability of the air-conditioned environment for the production of cosmetics, the obtained samples are tested on the in vitro model of human skin fibroblast migration. The selected media samples are then purified from low molecular weight components by ultrafiltration followed by lyophilization to obtain the main active component of a cosmetic product containing human MSC secretion products, including key growth factors: VEGF at a concentration of at least 4 ng / million cells, FGF basic at a concentration of at least 5.8 PCG / million cells, PEDF at a concentration of not less than 10 ng / million cells, PAI-1 at a concentration of not less than 35 ng / million cells, determined by enzyme-linked immunosorbent assay when restoring the lyophilisate to sterile physiological osmolarity buffer, as well as a complex of 20 proteins (see Table 1), determined by proteomic analysis, which provide the effect of a cosmetic product.

At the same time, adrenal MSCs are grown in a commercial environment that supports the growth of undifferentiated human mesenchymal cells (AdvanceSTEM Cell Culture Media), containing 9-11% of a supplement to the growth medium (AdvanceSTEM Stem Cell Growth Supplement, both reagents - HyClone, USA), but before conditioning cell media are thoroughly washed from the medium components with Hanks solution (PanEco, Russia) and placed in a serum-free and animal-free growth medium, the latter using low glucose DMEM (HyClone, USA) or other growth medium decreasing viability of human MSCs (not less than 70%) during the entire period of conditioning and suitable for therapeutic use. The medium should not contain substances prohibited for use in cosmetics.

The cultivation can be carried out in culture bottles or Petri dishes (for example, Corning or the like), while the cells are grown on a flat substrate with a density of 5-15 * 10 ³ / cm ² in a growth medium in a volume of 0.1-0.2 ml / cm ² in a CO ₂ incubator at 37 ± 1 ° C, with 5% CO ₂ and relative humidity ≥95% for 7 ± 1 days. Removal of cell debris (cell debris) from the culture medium can be carried out by centrifugation, for example, at 300 ± 5 g, temperature 6 ± 2 ° C for 10 minutes. The cultivation medium is purified by ultrafiltration with the removal of low molecular weight compounds weighing less than 5 kDa, while microfiltration of the resulting solution is additionally carried out through filters with a pore diameter of 0.22 μm to obtain a sterile product. Cleaning can be carried out using a Minim II tangential flow filtration system (Life Sciences, USA) or similar. Lyophilization can be carried out in 8-14 stages at a pressure of 0.013 kPa, temperature from -40 ° C to -5 ° C, lyophilization time of not more than 36 hours at maximum load of freeze drying. For lyophilization use glass bottles with non-adhesive coating Adelphi Heakthcare, USA, Cat. VA 100-20 C or equivalent.

Lyophilization of the conditioned medium can be carried out in conjunction with other components of the product according to a protocol that ensures the pharmaceutical stability of the product.

In one of the embodiments of the invention, the cosmetic product is a solution - a culture medium, conditioned by MSC of VT, in another - a conditioned medium in the form of a lyophilisate obtained by the method described above, which contains key biologically active factors: VEGF in a concentration of at least 4 ng / million cells (at least 200 pg / ml), FGF basic at a concentration of at least 5.8 pg / million cells (at least 0.3 pg / ml), PEDF at a concentration of at least 10 ng / million cells (at least 500 pg / ml ), PAI-1 at a concentration of not less than 35 ng / million cells (not less than 2 ng / ml), determined by enzyme-linked immunosorbent assay during the restoration of 0.1 ± 0.005 g of lyophilisate in 10 ml of sterile physiological osmolarity buffer, as well as a number of key proteins shown in table 1.

The components of the cosmetic can be included in liposomes at a concentration of not less than 1% wt. (to the mass of the finished cosmetic product). Liposomes are a popular way of delivering pharmacologically active substances to the site of their therapeutic effects, providing better preservation of the content and biological activity of the delivered substances. In addition, liposomes can themselves be enhancers of transdermal penetration, helping to improve the penetration of the active components of the drug into the inner layers of the skin. Liposomes can be obtained by a variety of methods, including punching, detergent removal, and others well known in the art (Brunner J. Single bilayer vesicles prepared without sonication: physico chemical properties / J. Brunner, P. Skrabal, H. Hauser // Biochimica et Biophysica Acta. - 1976. - V. 455, No. 2. - P. 322-331; Dua JS, Rana AC, Bhandari AK Liposome: methods of preparation and applications // Int J Pharm Stud Res. - 2012. - T. 3 . - C. 14-20; Akbarzadeh A. et al. Liposome: classification, preparation, and applications // Nanoscale Res Lett. - 2013. - T. 8. - No. 1. - C. 102; Wagner A., Vorauer-Uhl K. Liposome technology for industrial purposes // Journal of drug delivery. - 2010 .-- T. 2011; Uhumwangho MU, Okor RS Current trends in the production and biomedical applications of liposomes: a review. - 2005).

The cosmetic may contain dimethyl isosorbide in a concentration of not less than 1.5% wt. (to the mass of the finished cosmetic product), hyaluronic acid and its modifications in a concentration of not less than 1% wt. (to the mass of the finished cosmetic product) and other transdermal enhancers that enhance the penetration of active components into the skin, as well as in some cases have their own positive cosmetic effect on the skin condition.

Thus, the technical result is achieved due to the fact that the main active component of the cosmetic product for skin application is obtained by culturing human MSC in a serum-free medium lacking xenogenic components, i.e. with a composition that is safe for use by the consumer and which maintains cell viability during the selected conditioning period, by collecting the conditioned medium containing biologically active components secreted by the cells in the culture medium, preliminary testing of the medium, and selecting samples that meet the specified requirements, and cleaning the selected medium samples using ultrafiltration and stabilization by lyophilization.

The effectiveness of the developed product suggests the presence of a whole range of effects aimed at stimulating the proliferation and migration of specialized cells involved in the repair and regeneration processes (endothelial cells, fibroblasts, etc.). The use of a cultivation medium, conditioned with human MSC in human VT, will provide a comprehensive solution to problems, both medical and aesthetic, that arise with aging of the skin. It is assumed that hydration and nutrition of the outer layers of the skin will be carried out due to the action of additional active ingredients (vitamins and nutrients), which can be chosen by specialists depending on the application, and rejuvenation and stimulation of skin renewal, including by enhancing the synthesis and renewal of collagen, keratin and other structural proteins of the skin can be carried out using serum-free medium containing a combination of growth factors and other biologically active components, secretion human MSCs that promote skin regeneration. This solution allows us to obtain a combination of protein factors secreted by human MSC in human gastrointestinal tract and, accordingly, having undergone the necessary modifications and dissolved in a medium that does not contain animal proteins and bacterial fragments.

To obtain a conditioned medium, it is proposed to use multipotent mesenchymal stem / stromal cells (MSCs) of a person responsible for tissue repair and regeneration. One of the most accessible sources of MSCs in humans is subcutaneous adipose tissue; MSCs contained in it can be easily isolated as a result of enzymatic processing of adipose tissue samples obtained as a result of cosmetic liposuction or during the surgical removal of fat deposits. The cultivation of the isolated MSC in adipose tissue results in a relatively homogeneous population of multipotent stromal fibroblast-like cells.

In the claimed invention, MSCs obtained independently by any method known from the prior art can be used, and in the form of commercial MSC preparations having a quality certificate and intended, including for clinical use, for example, such as the ATCC product (ATCC® PCS -500-011), which is human MSCs isolated from lipoaspirate, cultured up to 2 passages and subjected to cryopreservation, or similar.

Previously, on models of limb ischemia and myocardial infarction and subcutaneously implanted matrigel in animals, the authors of the invention showed that local and systemic administration of VT MSCs increases the number of vessels in tissues with impaired blood supply, leads to an improvement in blood flow in these tissues and a decrease or disappearance of symptoms of ischemia. In addition, it was found that the introduction of MSC in the VT promotes the germination of nerve endings in the subcutaneously implanted matrigel and improves the recovery of peripheral nerves after traumatic injury. According to the studies of the authors of the present invention and the work of other scientific teams, the main mechanism underlying the regenerative effect of MSC in adipose tissue is the production by these cells of a wide range of biologically active cytokines and growth factors that accelerate the healing and restoration of tissues after damage. The qualitative and quantitative composition of secreted MSC components varies significantly between donors, and various factors, such as age, sex of the donor, and the presence of chronic diseases, can significantly affect it. Proposed in this invention a method of obtaining funds based on secreted MSC products allows you to achieve a certain qualitative and quantitative composition of components that provide a cosmetic effect. The resulting composition can be adjusted in case of discrepancy with the claimed parameters, as well as to increase the effectiveness of the product using methods known from the prior art. So, individual components can be added directly to the conditioned medium, the cells can be pretreated before and / or during conditioning (the methods used include, but are not limited to, culturing cells under hypoxic conditions, in the presence of biologically separate substances, as part of various matrices and on three-dimensional carriers, genetic modification of cells and other methods). In addition, the conditioned medium, in addition to the cleaning method of the invention, can also be treated to remove unwanted or excess components. Used methods for purifying an air-conditioned environment that allow maintaining optimal activity well known to those skilled in the art include, but are not limited to, gel chromatography (using matrices such as Sephadex), ion exchange chromatography, metal chelate affinity chromatography with an insoluble matrix, and HPLC purification . Depending on the purpose of the application of the conditioned medium and / or the products obtained from it, if necessary, the sterility of the final product should be maintained.

Most protocols for isolation and cultivation of MSC in adipose tissue use a medium supplemented with fetal calf serum (ETS) or fetal bovine serum (FBS). These serums contain the necessary cocktail of growth factors, hormones and vitamins that stimulate cell adhesion and proliferation in vitro. At the same time, more and more information is accumulating that cells grown using animal sera are potentially unsafe, in particular, there is a risk of infection of the cell culture by prions or viruses during cultivation using animal sera, as well as the possibility of an immune reaction from the recipient’s body . In addition, it was shown that long-term cultivation in the presence of PBS significantly changes the properties of cells of various origins. Thus, bone marrow mesenchymal cells cultured in the presence of PBS are characterized by transcriptome instability, including a change in the expression of genes responsible for the regulation of the cell cycle, apoptosis and cell adhesion.

A distinctive feature of this invention is the use of a specialized medium for the cultivation of MSC in adipose tissue that supports the growth and functional properties of undifferentiated human MSCs at the stages preceding conditioning of MSCs: AdvanceSTEM Cell Culture Media (HyClone, USA) with the addition of Penicillin-Streptomycin antibiotic solution (HyClone, USA) and additives to the growth medium AdvanceSTEM Stem Cell Growth Supplement (HyClone, USA), which are taken in the following ratio of components, vol. %:

- AdvanceSTEM Cell Culture Media - 89-90;

- Penicillin-Streptomycin antibiotic solution - 0.95-1.05;

- Additives to the growth medium AdvanceSTEM Stem Cell Growth Supplement - 9.0-10.0.

MSCs are isolated from human subcutaneous adipose tissue and cultured in growth medium prepared according to the above scheme up to 4-5 passage. Then the cells are thoroughly washed from the components of the growth medium with Hanks solution (PanEco company or similar) and placed in a serum-free and animal-free growth medium; DMEM with a low (1000 mg / L) glucose content (HyClone, USA) or another is used as the latter growth medium that supports the viability of human MSCs (at least 70%) throughout the entire conditioning period and suitable for therapeutic use. The specified medium for conditioning cells is added in a volume of 0.1-0.2 ml / cm ² and cultured in a CO ₂ incubator at 37 + 1 ° C, 5% CO ₂ and relative humidity ≥95% for 7 ± 1 days. The conditioned medium containing all the secretion products of human MSCs is collected in sterile centrifugation containers, the remaining cells are removed by centrifugation, tested on a model for stimulating the migration of human skin fibroblasts, and if the test results are positive, the resulting supernatant is purified using the ultrafiltration procedure, removing all low molecular weight compounds weighing less than 5 kDa. At the ultrafiltration stage, using appropriate equipment and observing aseptic conditions, a sterile product can be obtained. If it is not possible to ensure sterility of the product during the ultrafiltration procedure, then the microfiltration step is added. To do this, the concentrated medium is filtered through disposable sterile filters with a pore diameter of 0.22 μm, sufficient to purify microorganisms potentially contained in the product, and then subjected to a lyophilization procedure to obtain the final product (lyophilisate of the conditioned medium containing human MSC secretion products) containing including key biologically active factors that standardize the final product. So, when restoring 0.1 ± 0.005 g of lyophilisate in 10 ml of a sterile solution with physiological osmolarity, the method of enzyme-linked immunosorbent assay in solution should determine VEGF at a concentration of at least 4 ng / million cells (at least 200 pkg / ml), FGF basic at a concentration of less than 5.8 pg / million cells (at least 0.3 pg / ml), PEDF at a concentration of at least 10 ng / million cells (at least 500 pg / ml), PAI-1 at a concentration of at least 35 ng / million cells (no less than 2 ng / ml), as well as a complex of at least 20 proteins, presented in table 1, determined by proteomic analysis.

A feature of this invention is the preliminary testing of environmental samples containing cell secretion products obtained by conditioning MSCs of adipose tissue on the ability to stimulate the migration of human dermal fibroblasts on an in vitro model of a cell wound in order to determine suitability for the production of cosmetics.

Another feature of this invention is the ability to clean and, if necessary, simultaneously concentrate the conditioned medium using a Minim II tangential flow filtration system (Life Sciences, USA) or similar, which can significantly speed up the cleaning and / or concentration process and contributes to better preservation of biologically active substances in an air-conditioned environment.

As a result of the implementation of this invention receive a cosmetic product based on the secretion products of human MSCs, which can be used to stimulate skin regeneration, increase its elasticity and improve aesthetic characteristics.

The sterile solution of the conditioned medium enriched with biologically active factors is a biologically engineered cosmetic product that can be easily obtained in large volumes and can be used both separately and as an additive to various skin care products, cosmetic and dermatological products, in order to replenish the levels of growth factors and matrix proteins in the skin, hair and nails of a person. The inventive tool may contain a liquid lubricating agent, for example glycerin, glycogen, maltose, etc. Organic polymeric base materials such as polyethylene glycol and hyaluronic acid, as well as various forms of collagen, can also be used as lubricants.

The tool of the present invention can be used in the form of solutions or lyophilisate in various forms of cosmetics, such as creams, gels, masks, hair products, including for microinjections in the form of a solution, as part of hydrogels, etc., and administered to a patient in various ways using standard procedures well known in the art. Compositions of conditioned media can be prepared as a topical preparation using transdermal penetration enhancers to facilitate the penetration of this compound into the skin, and applied as topical application. In addition, the claimed tool can be made taking into account the controlled prolonged release of the active components.

Below is a detailed description of the invention by examples of specific performance, which demonstrate the practical feasibility of the invention, but do not limit the possibility of carrying out the invention using other means and methods.

Example 1. Obtaining MSCs of human adipose tissue.

MSCs are isolated from subcutaneous fat deposits of healthy donors of both sexes. Cells are isolated from material obtained during minor surgery under local anesthesia or during routine surgical operations. Surgical material (10-15 g of adipose tissue taken from the umbilical region or other area of localization of subcutaneous fat) is placed in a disposable tube with HBSS buffer (HyClone) containing a 5-fold antibiotic concentration of 500 units / ml (HyClone).

Under sterile conditions of the culture box, the biopsy is fragmented to a homogeneous mass using sterile instruments, and then subjected to enzymatic treatment in a solution containing AdvanceSTEM ™ medium (HyClone) / 500 u / ml antibiotic (HyClone), 200 u / ml type I collagenase and dispase (40 units / ml) (Worthington Biochemical) (the ratio of the volume of enzymes and fat should be 1: 1: 1), at 37 ° C for 30-45 minutes with constant stirring. At the end of the incubation, an equal volume of serum medium is added to the sample and filtered through nylon membranes with a pore size of 100 μm (BD). The filtered suspension is centrifuged for 5 min at 200 g, after which the supernatant is completely removed. The cell pellet is subjected to treatment with erythrocyte lysis buffer (BD) until the solution becomes red, then centrifuged for 5 min at 200 g. The supernatant is completely removed, and the precipitated cells are resuspended in MSC growth medium. AdvanceSTEM ™ support expansion and maintenance of undifferentiated human MSCs (HyClone) combined with 10% AdvanceSTEM ™ Stem Cell Growth Supplement (HyClone) and 100 u / ml antibiotic (HyClone) are used to cultivate mesenchymal cells. This medium was developed by the manufacturer (HyClone) for the cultivation of undifferentiated MSCs, in particular cells derived from adipose tissue, without the addition of serum (http://www.thermo.com).

The isolated MSCs are seeded at a concentration of 200 thousand per ml in Petri dishes and cultured in a growth medium in an incubator at 37 ° C and at a 5% concentration of CO ₂ . Upon reaching 80% of the monolayer of MSC ZhT (0 passage), they are frozen in liquid nitrogen and stored in a master bank for further use in order to accumulate the medium.

To produce the medium, MSCs of VT are scaled. Before removing from the surface of the culture plastic, the cells are washed three times with Versen's solution. Then, 1 ml of HyQTase (HyClone) cell dissociation reagent is applied to each dish. When the cells acquire a rounded shape and detach them from the substrate, 9 ml of growth medium is introduced into each culture container and the cell suspension is intensively pipetted. Then the cells are seeded in a ratio of 1: 4.

Example 2. Obtaining a conditioned medium containing the secretion products of MSCs in human adipose tissue.

To obtain a conditioned environment, MSCs of VT are increased to 2-5 passage. MSCs of human gastrointestinal tract are added in an amount of 5-15 * 10 ³ / cm ² in culture containers for growing eukaryotic cells, after attachment, the culture vessels with cells are washed with Hanks saline solution (Paneko, Russia) (in an amount of 0.1-0.2 ml / cm ² three times for 10 minutes) and fill them with fresh growth medium for conditioning in the amount of 0.1-0.2 ml / cm ² . As the latter, a non-specific universal growth medium for different types of DMEM with Low Glucose cells (HyClone, USA) or a specific growth medium for supporting the growth of human MSC MSC NutriStem® XF Basal Medium (Biological Industries, Israel) with the addition of Penicillin-Streptomycin antibiotic solution is used (HyClone, USA) (1.00 ± 0.05 ml per 100 ± 5 ml of medium). Then the cells were cultured in a CO ₂ incubator at 37 + 1 ° C, 5% CO ₂ and relative humidity ≥95% for 7 days.

As culture containers for growing eukaryotic cells, culture bottles or Petri dishes (Corning or the like) are used, and the cells are grown on a flat substrate with a density of 5-15 * 10 ³ / cm ² .

Example 3. Preliminary testing of the conditioned environment of MSCs of human adipose tissue in an in vitro model in order to determine the suitability for the production of cosmetic products.

To assess the ability of the developed product to effectively stimulate skin regeneration, primary human dermal fibroblasts of passage 4-5 are used, obtained from healthy donors. The effect of the obtained samples of the conditioned medium containing the secretion products of MSCs from adipose tissue of various donors on the migration rate of skin fibroblasts in a cell wound model is investigated.

Human skin fibroblasts are cultured in low glucose DMEM supplemented with 10% fetal calf serum. To model the conditions for wound overgrowth, a cell wound model (scratch assay) is used. For this, fibroblasts are planted on 12-well plates at a concentration of 10 thousand cells / ml and cultured under standard conditions until a subconfluent monolayer is reached. Then the cells are deprived 24 hours.

With a sterile tip of 200 μl, a standard-width scratch is applied to the cell monolayer and the cells are poured with a conditioned medium containing the secretion products of human MJ VT. As a negative control, a low glucose DMEM base medium is used. As a positive control, low glucose DMEM stock supplemented with 10% fetal calf serum was used.

A fibroblast plate is placed in a Nikon Ti CO ₂ camera for intravital observation, using the phase contrast shooting mode with a 5x lens for 24 hours with a shooting step of 15 minutes. The resulting series of images are analyzed using ImajeJ software, counting the migration speed of 50 random cells in each field of view in microns / hour. The test is considered positive if the activity level of the samples of the conditioned medium exceeds the corresponding value for the negative control by at least 50% (Figure 1). Samples that failed the test are discarded (Figure 2), and the cells with which they were produced are not further used. Selected samples of conditioned medium should demonstrate significant specific activity (Figure 1) and can be used for further stages of the process.

Example 4. The purification of the conditioned medium containing the secretion products of MSCs of human adipose tissue, ultrafiltration.

Samples of the conditioned medium containing all the secretion products of human MJ VT were collected in sterile containers for centrifugation, centrifuged at (5000 ± 10) rpm, temperature (6 ± 2) ° C, for 10 minutes to clean the cell debris removal . The resulting supernatant is taken into new sterile containers with a volume of 250-500 ml.

Additional purification of the medium from low molecular weight components is carried out using a Minim II tangential flow filtration system (Life Sciences), using ultrafiltration cartridges (Minimate TFF capsule, PALL, 5 kDa) or similar. For this, the feed tank is filled with 100 ml of conditioned medium. The pump performance is set at 90-120 ml / min, the pressure is not more than 2 atmospheres, the filtrate flow rate is not more than 20-30 ml / min. 5-6 rounds of purification are carried out, replenishing the volume by adding Hanks solution to the feed tank. After that, turn off the pump, transfer the return hose to the vessel to collect the purified solution, turn on the pump and wait for the drainage of the supply tank. To displace the residues of the purified medium, Hanks solution is added to the feed tank, the pump is turned on and the purified medium is collected under visual control until the entire solution leaves the system. If necessary, these methods are used to concentrate samples.

If it is not possible to maintain sterility during ultrafiltration, the purified medium is subjected to microfiltration. Filtration of the purified medium is carried out through disposable sterile filters with a pore diameter of 0.22 μm, ensuring sterilization of the product. The medium is fed to the filters by a peristaltic pump. The filtrate is collected in a sterile polypropylene container.

Example 5. Lyophilization of purified conditioned medium containing the secretion products of MSCs in human adipose tissue.

The purified filtered medium is subjected to a lyophilization procedure to obtain the final product (lyophilisate of an air-conditioned medium containing human MSC secretion products). To do this, pour purified filtered medium of (10.0 ± 0.1) ml into sterile glass bottles with a capacity of 100 ml. Vials are sealed with butyl rubber stoppers for lyophilization. Then the bottles are placed in freeze drying. It is cooled to -45 ° C with a temperature control of the product, then it is kept at the indicated temperature for 1 hour. After that, turn on the condenser, cool it to -45 ° C, turn on the vacuum pump and bring the pressure to 0.027 kPa. Perform the following steps:

1) Step cooling to 10 ° C in 20 minutes at a pressure of 66.6 kPa;

2) Smooth cooling to -15 ° C in 25 minutes at a pressure of 66.6 kPa;

3) Holding at -15 ° C for 120 minutes at a pressure of 66.6 kPa;

4) Smooth cooling to -40 ° C in 25 minutes at a pressure of 66.6 kPa;

5) Holding at -40 ° C for 150 minutes at a pressure of 0.013 kPa;

6) Smooth heating to -30 ° C in 150 minutes at a pressure of 0.013 kPa;

7) Smooth heating to -20 ° C in 150 minutes at a pressure of 0.013 kPa;

8) Smooth heating to -10 ° C in 150 minutes at a pressure of 0.013 kPa;

9) Smooth heating to -5 ° C in 150 minutes at a pressure of 0.013 kPa;

10) Holding at -5 ° C (within 20 hours before shutting down by the operator) at a pressure of 0.066 kPa;

11) Devacuation up to 66.6 kPa;

12) Full automatic corking with rubber stoppers;

13) Vacuum discharge;

14) Unloading bottles.

The total duration of the lyophilization procedure does not exceed 36 hours with a full load of freeze drying.

Example 7. A method for assessing the concentration of key factors mediating the cosmetic effects of an environment conditioned by MSCs of human adipose tissue.

Evaluation of the content of VEGF, FGF basic (FGFb), PEDF and PAI-1 in reconstituted in 10 ml of sterile physiological solution lyophilisate conditioned medium containing the secretion products of MSC in human VT is carried out by enzyme-linked immunosorbent assay (ELISA). VEGF at a concentration of at least 4 ng / million cells (at least 200 pkg / ml), FGF basic at a concentration of at least 5.8 pkg / million cells (at least 0.3 pkg / ml), and PEDF at a concentration of at least 10 ng / million cells (at least 500 pg / ml), PAI-1 at a concentration of at least 35 ng / million cells (at least 2 ng / ml).

Evaluation of FGFb content using the R&D Systems Human FGF basic Quantikine ELISA Kit.

To determine the concentration of FGFb by ELISA, a commercial kit from R&D Systems (Human FGF basic Quantikine ELISA Kit, cat # DFB50) or a similar one is used.

Using a standardized FGFb solution, calibration samples of known concentration are prepared so that sample No. 1 contains 500 pg / ml FGFb, each subsequent one contains 2 times less FGFb. Sample No. 0 should contain only Sample Diluent. The test samples in an amount of 100 μl, as well as standardized samples of the FGFb solution, are applied three times to the wells of a 96-well plate and incubated on a shaker for 12 hours at 4 ° C. The plate is washed with Wash Buffer, and 100 μl of biotinylated murine monoclonal anti-human FGFb antibodies are added to each well. After 3 hours of incubation, the plate is washed as described above. In each well, 100 μl of horseradish streptavidin-peroxidase complex solution is applied. After 1 hour, the plate was washed thoroughly and 100 μl of TMB / E substrate solution supplied with the kit was added to each well. The plate was incubated at room temperature for 15 minutes, then 100 μl of a stop buffer was added to each well, and the reaction was evaluated on an ELISA READER MICROPLATE READER Spectro spectrophotometer at a wavelength of 450 nm. The optical density level at a wavelength of 570 nm is used as a reference. Work on the device is carried out in accordance with the instructions.

Evaluation of PEDF content using a Cusabio Human PEDF ELISA Kit.

To determine the concentration of PEDF by ELISA, a commercial kit from Cusabio (Human PEDF ELISA Kit) or the like is used.

Prepare serial dilutions of the PEDF standard, according to the instructions attached to the kit. A solution of biotinylated antibodies to human PEDF and a 1x solution of horseradish peroxidase conjugated with avidin are prepared. Apply 100 μl of a standard solution or sample to each well of the microplate. Cover with adhesive film and leave at 37 ° C for 2 hours. After two hours of incubation, the adhesive film is carefully peeled off and the liquid removed from all wells without rinsing. 100 μl of 1x biotinylated antibody solution is added to each well. Cover with a sticky film and leave the microplate for 1 hour at 37 ° C. After re-incubation, remove all contents from the wells. Wash the wells 4 times by adding 200 μl of washing buffer to each of them. Leave the wash buffer in the well for two minutes and carefully select the liquid after each washing step. At the end of washing, turn the microplate upside down and blot with a sheet of filter paper. Add to the wells 100 μl of 1x solution of horseradish peroxidase conjugated with avidin. Cover the microplate with adhesive tape and allow to incubate for 1 hour at 37 ° C. After incubation, the wells are washed six times according to the previously described procedure. Add 90 μl of TMB Substrate to each well. Incubate the wells at 37 ° C for 15-30 minutes. Add 50 μl of Stop Solution) to the wells), mix the contents of each well. Not later than 5 minutes after the addition of Stop Solution), the optical density of the solution in each well is determined at a wavelength of 450 nm. As a reference, the level of optical density at a wavelength of 540 nm is used.

The remaining key proteins are evaluated in the same manner as described in the instructions for ELISA assay kits.

Example 8. Evaluation of the content of the complex of active proteins in the condensed medium of MSCs of human adipose tissue by proteomic analysis.

350 microm*0.5 mm (Eksigent, Dublin, CA, USA) при скорости потока 3 мкл/мин и элюируют сквозь разделяющую колонку со скоростью 300 нл/мин. Градиент буфера Б в буфере А варьируют от 5 до 40% в течение 120 минут.Proteomic analysis was performed on a TripleTOF 5600+ mass spectrometer with an NanoSpray III ion source (ABSciex, Concord, Ontario, Canada) combined with a NanoLC Ultra 2D + nano-HPLC system (Eksigent, Concord, Ontario, Canada) or the like. To load the samples, buffer A (a mixture of 98.9% water, 1% methanol, 0.1% formic acid (v / v)) and buffer B (99.9% acetonitrile, 0.1% formic acid (v / v)) are used. The trap column is conditioned with the same solutions (A and B) for 25 minutes at a flow rate of 300 nl / min. Samples are loaded onto a Chrom XP C18 3 microm 120 column

350 microm * 0.5 mm (Eksigent, Dublin, CA, USA) at a flow rate of 3 μl / min and elute through the separation column at a speed of 300 nl / min. The gradient of buffer B in buffer A varies from 5 to 40% for 120 minutes.

The mass spectrum is obtained in the mode of positive ions, the spread of m / z can be 300-1250, the signal accumulation time is 250 ms. For identification of proteins, file type. wiff is analyzed in ProteinPilot 4.5 revision 1656 (ABSciex) using the search algorithm Paragon 4.5.0.0 revision 1654 (ABSciex) with a set of standard settings for searching the database or a similar analysis method.

The following parameters are used: cysteine-iodoacetamide alkylation, trypsin melting, TripleTOF 5600 equipment settings, additional FDR analysis. Peptides are identified using standard settings using the ProteinPilot software package or similar. This approach allows you to identify and evaluate the content of all proteins presented in table 1. Samples of the medium are selected for the presence of these proteins to obtain a cosmetic product.

Example 9. Evaluation of the content of additional active proteins in the conditioned environment of MSCs of human adipose tissue by proteomic analysis.

Selected samples of the conditioned medium are additionally checked for the presence of biologically active factors secreted by human MSCs by the proteomic analysis method as described in Example 8. As a result, an additional complex of 47 proteins in the cosmetic composition is determined (Table 2).

Example 10. An example of obtaining a cosmetic product based on the secretion products of MSCs in human adipose tissue.

Selected samples of a conditioned medium containing a complex of products of secretion of MSC in human VT are used as a cosmetic product in the form of a solution or for inclusion in other forms of cosmetic products (creams, gels, masks, etc.). To assess the ability of the obtained product to effectively stimulate skin regeneration, the effect of the samples on the rate of migration of human skin fibroblasts on a cell wound model (Figure 3) or other relevant in vitro models is investigated. As a negative control, a low glucose DMEM base medium is used. As a positive control, low glucose DMEM stock supplemented with 10% fetal calf serum was used.

To stimulate the penetration of the product into the skin and enhance the cosmetic effect, a 1% solution of sodium hyaluronate with dimethyl isosorbide is added to the product in the form of a lyophilisate. The specific activity of the drug is evaluated on a model of human skin fibroblast migration (Figure 4) or other relevant in vitro models.

Example 11. Obtaining a cosmetic product based on the secretion products of MSCs of human adipose tissue, modified genetically to increase the production of vascular endothelial growth factor (VEGF165)

To obtain a conditioned medium with an increased content of one of the key active ingredients, a human MSC culture of human adipose tissue is used, which is treated with a viral vector before the conditioning procedure, with the gene for vascular endothelial growth factor (VEGF 165), i.e. subjected to transient genetic modification.

For this, adipose tissue MSCs in 60-75% confluency are used, which are washed from the growth medium with phosphate-buffered saline, after which a Hanks solution containing recombinant human baculovirus VEGF 165 in a titer of 75-150 viral particles per cell is applied to the culture. Then the cup is covered with foil to protect the photosensitive baculovirus and left for 6 hours at room temperature with slow (50 rpm) stirring on an orbital shaker. At the end of the incubation, the virus solution is selected, the cells are washed twice with phosphate-buffered saline, and a growth medium is applied to the culture, to which 5 mM sodium butyrate is added for 15 hours to increase the production of VEGF 165. At the end of the incubation, the medium containing sodium butyrate is removed, the cells are removed washed twice with phosphate-buffered saline, and then incubated for 24-48 hours in a growth medium. Thus obtained culture is used to obtain a conditioned medium according to the methods described in the application. When using genetically modified MSCs in human adipose tissue, there is a sharp increase in the content of VEGF 165 (Figure 5), which is one of the key protein active components of the conditioned medium (data for conditioning for 72 hours are given).

Example 12. Obtaining a cosmetic product based on the secretion products of MSCs of human adipose tissue, cultured under conditions of low oxygen content

As one of the methods for correcting the composition of the secretory components of human MSCs, the method of culturing cells under hypoxia can be used. MSCs of VT (n = 10) of passage 4-5 were cultured at 1% oxygen content (hypoxia) or under standard conditions (normoxia) for 48 hours, then the conditioned medium was collected. This method allows you to increase the production of some proteins and reduce the production of others (Figures 6, 7) and can be applied depending on the purpose of the cosmetic product.

Example 13. Obtaining a combined cosmetic product based on the secretion products of MSCs of human adipose tissue, ready for use.

At the first stage, a lyophilisate of liposomes of an conditioned medium is obtained; for this, liposomes based on soy phospholipids (average size of at least 500 nm) are mixed in a ratio of 1:25 with a purified conditioned medium that underwent preliminary testing, and then lyophilization is carried out as described in example 5. To obtain a cosmetic product, the lyophilisate of the conditioned medium or the lyophilisate of the liposomes of the conditioned medium is mixed with granules of carboxymethyl cellulose (particle size - not more than 0.1 mm) in a ratio of 1: 2 by weight in b an Araban mixer until a homogeneous powdery substance is obtained - hereinafter - the solid-phase component. At the same time, a 1% solution of sodium hyaluronate with dimethyl isosorbide is mixed in a ratio by weight of 50: 1 - then the liquid-phase component. The finished product is obtained by mixing solid-phase and liquid-phase components in a ratio by weight of 1:20 with vigorous stirring and, if necessary, heating.

Thus, the claimed composition is a secretion product of human MSCs, forming a biologically active complex with the following positive functions / characteristics:

- the absence of xenogenic components and stability - a broad and detailed composition, including growth factors, biologically active peptides, chemoattractants, proteolytic enzymes and molecules that regulate the activity of regenerative processes;

- the ability to cause regeneration of the skin;

- the ability to activate the function of fibroblasts, thus exerting a beneficial effect on the mechanical and functional properties of the skin

- the possibility of widespread use in various cosmeceutical formulations for various applications, as well as the creation of combined cosmetics (including enhancers of transdermal penetration, liposomes, etc.).

Claims (29)

1. A cosmetic product for stimulating the regeneration of skin tissue, which includes as the main component a culture medium conditioned by mesenchymal stromal cells of human adipose tissue, containing secretion products, including VEGF at a concentration of at least 4 ng / million cells, FGF basic at a concentration of at least 5.8 pcg / million cells, PEDF at a concentration of not less than 10 ng / million cells, PAI-1 at a concentration of not less than 35 ng / million cells, as well as a complex of at least 20 proteins shown in table 1, while the concentration of these components presented in 10 ml of medium. 2. The tool according to claim 1, characterized in that the cosmetic product is presented in the form of a lyophilized conditioned medium. 3. The tool according to claim 2, characterized in that it further comprises transdermal penetration enhancers, wherein the lyophilisate is reduced in an aqueous solution of enhancers. 4. The tool according to p. 3, characterized in that as enhancers used dimethyl isosorbide in an amount of at least 1.5% relative to the weight of the finished cosmetic products, and / or hyaluronic acid or its derivatives in an amount of at least 1% with respect to the mass of the finished cosmetic products, and / or liposomes of various compositions. 5. The tool according to claim 4, characterized in that sodium hyaluronate is used as derivatives of hyaluronic acid. 6. The tool according to p. 4, characterized in that as liposomes of various compositions use liposomes based on soybean phospholipids. 7. The tool according to claim 1, characterized in that the secretion products of mesenchymal stromal cells of human adipose tissue contained in an air-conditioned medium are partially or fully included in the composition of liposomes, while the tool is presented in the form of a solution or lyophilisate. 8. The tool according to p. 7, characterized in that the liposomes are obtained by pressing. 9. The tool according to p. 7, characterized in that the liposomes are obtained by the method of removal of detergent. 10. The tool according to claim 7, characterized in that the inclusion of the components of the medium in liposomes is carried out by lyophilization. 11. The tool according to claim 2, characterized in that it further comprises a polymer substance compatible with the lyophilisate and soluble in water, which forms a gel-like substance in a ratio by weight of not more than 1: 100 when dissolved in an aqueous solution of transdermal penetration enhancers, ensuring formation a stable gel-like substance, while the combination of the lyophilisate with the polymer and an aqueous solution of enhancers are mixed together immediately before application to the skin. 12. The tool according to p. 11, characterized in that the lyophilisate contains liposomes, which are used as liposomes based on soy phospholipids. 13. The tool according to claim 11, in which a compatible polymer substance, which forms a gel-like substance when dissolved in water, is methyl cellulose. 14. The tool according to p. 11, characterized in that as the enhancers of transdermal penetration using sodium hyaluronate in a concentration of not less than 1% wt. to the mass of the finished cosmetic and dimethyl isosorbide in a concentration of not less than 1.5% wt. to the mass of the finished cosmetic product. 15. The tool according to p. 14, characterized in that sodium hyaluronate and dimethyl isosorbide are used in the form of a 1% aqueous solution. 16. A method of obtaining a cosmetic product according to claim 1 on the basis of a conditioned medium containing secretion products of mesenchymal stromal cells of human adipose tissue, comprising cultivating mesenchymal stromal cells of human adipose tissue up to 4-5 passages in an environment that supports their growth, washing the cells with buffer solution, their conditioning in a serum-free and animal-free growth medium supporting cell viability of at least 70% and suitable for therapeutic use, selection a culture medium containing the secretion products of mesenchymal stromal cells of human adipose tissue, removing cell debris from it, preliminary testing of the conditioned medium and sampling to obtain a cosmetic product, purification of the selected samples from low molecular weight components to obtain an agent containing secretion products of mesenchymal stromal cells of adipose tissue human, including VEGF at a concentration of at least 4 ng / million cells, FGF basic at a concentration of at least 5.8 pg / million cells, PEDF in concentrations of at least 10 ng / million cells, PAI-1 at a concentration of not less than 35 ng / million cells, determined by enzyme-linked immunosorbent assay when restoring the lyophilisate in a sterile buffer with physiological osmolarity, as well as a complex of 20 proteins shown in table 1, determined by the method proteomic analysis. 17. The method according to p. 16, characterized in that to obtain funds according to p. 2, the selected purified samples are lyophilized. 18. The method according to p. 16, characterized in that the medium that supports the growth of human mesenchymal cells is a solution containing the base medium - AdvanceSTEM Cell Culture Media, and the additive - AdvanceSTEM Stem Cell Growth Supplement, while the additive is taken in a volume of 9- 11% to 100% of the volume of the base environment. 19. The method according to p. 16, characterized in that the conditioning is carried out in culture bottles or Petri dishes, while the mesenchymal stromal cells of human adipose tissue are placed on a flat substrate with a density of 5-15 * 10 ³ / cm ² in a medium for conditioning in a volume of 0 , 1-0.2 ml / cm ² , then the culture containers with cells are placed for 6-8 days in a CO ₂ incubator at 37 ± 1 ° C, 5% CO ₂ and relative humidity ≥95%. 20. The method according to p. 16, characterized in that as a buffer solution for washing cells from the components of the growth medium using a Hanks solution, while washing the cells is carried out by three to five-fold placement of cells in said solution at a rate of 0.1-0.2 ml solution per cm ² cells for 5-10 minutes. 21. The method according to p. 16, characterized in that the medium for conditioning use DMEM with a low glucose content or other growth medium that supports the viability of mesenchymal stromal cells of human adipose tissue throughout the entire period of conditioning. 22. The method according to p. 16, characterized in that the removal from the culture medium of the cell debris is carried out by centrifugation. 23. The method according to p. 16, characterized in that the preliminary testing of samples of the conditioned medium is carried out by the ability to stimulate the migration of human skin fibroblasts not less than 50% higher compared to the negative control, which is the basic growth medium used for conditioning mesenchymal stromal cells of human adipose tissue. 24. The method of claim 23, wherein a cell wound model is used to evaluate human skin fibroblast migration. 25. The method according to p. 16, characterized in that the purification of the cultivation medium is carried out by ultrafiltration with the removal of low molecular weight compounds weighing less than 5 kDa. 26. The method according to p. 16, characterized in that after purification of the culture medium from low molecular weight compounds, microfiltration of the obtained medium is additionally carried out through filters with a pore diameter of 0.22 μm. 27. The method according to p. 17, characterized in that the lyophilization is carried out in 8-14 stages at a pressure of 0.013-0.027 kPa, temperature from -40 ° C to -5 ° C, lyophilization time of not more than 36 hours at maximum load of freeze drying. 28. The method according to p. 22, characterized in that the centrifugation is carried out at 300 ± 5 g, a temperature of 6 ± 2 ° C for 10 minutes to remove cell debris. 29. The method according to p. 26, characterized in that the cleaning of low molecular weight components is carried out using a filter system in the tangential flow Minim II.

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