RU2662172C2 - Method for producing regenerative veterinary preparation based on extract of mesenchimal stem cells and conditioned medium - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention relates to veterinary medicine and can be used to produce a regenerative veterinary drug. Method for producing a regenerative veterinary drug comprises: culturing isolated de novo or deconserved mesenchymal stem cells derived from adipose tissue or bone marrow of an animal in a liquid nutrient medium supplemented with 10 % fetal bovine serum. Obtaining a conditioned medium with the content of interleukins: IL-1β, IL-2, IL-6 and IL-10 in concentrations of at least 2; 2; 10 and 2 pg/ml. Combining with the extract of MSC obtained by freezing-thawing the cell suspension. Glycine is added to a final concentration of 20 mg/ml of the solution for lyophilization, sterilization filtration and lyophilization are carried out.

EFFECT: use of this method of obtaining a veterinary drug using glycine at a final concentration of 20 mg/ml allows stabilizing the pH and the regenerative activity of the preparation.

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Description

Technical field

The invention relates to veterinary medicine and can be used to obtain and use for therapeutic purposes a regenerative preparation containing a set of biologically active substances isolated from an extract of mesenchymal stem cells (MSCs) of bone marrow (CM) or adipose tissue (VT) of animals and conditioned media obtained as a result of culturing these cells. The proposed method allows to obtain a drug that provides effective stimulation of the own recovery processes of the animal organism.

BACKGROUND OF THE INVENTION

Currently, intensive studies are being conducted on the possibility of obtaining and differentiating cells from various types of stem cells used to replace lost or damaged cells of vital organs and tissues by transplantation. In the course of growth and upon induction of differentiation of the initial stem cells into specialized ones, a conditioned medium is formed, which also has various biological activity, including a therapeutic effect.

The creation of a pharmaceutical preparation containing biologically active substances, in particular, growth factors, based on the extract of mesenchymal stem cells and the conditioned environment on which these cells are cultivated, involves the use of biotechnological approaches, rather than cellular technologies, which does not contradict the requirements of bioethics and law . Due to the replacement of cellular technologies with biotechnological approaches, obstacles are eliminated at all stages of the industrial introduction of the drug from preclinical studies to commercialization, followed by obtaining permission to sell.

It should be noted that degenerative diseases are caused by a violation of the ability of individual body tissues to recover due to the realization of their own regenerative potential and the development of dystrophic processes against the background of proliferation of sclerotic tissue. The pharmacological effect of the drugs used to treat such conditions is aimed at stimulating or replacing the functions of the preserved cellular elements. However, the concept of compensating for target organ dysfunctions through adaptation processes is often unsound. The facts presented indicate the need to develop a strategy of regenerative medicine, which consists in replacing dead or malfunctioning cellular elements with new ones.

State of the art

A number of conditioned media and pharmaceutical compositions are known that are obtained from culture media by culturing a mesenchymal stem cell in a stable culture; these media may also contain differentiation factors. Such analogs provide stimulation of regeneration processes, however, they have too narrowly targeted action. So, there are known environments aimed at restoring the musculoskeletal system - cartilaginous [application for the invention of the Russian Federation 2001116126 from 08.11.1999, priority Italy] and bone tissue [Mbalaviele G, Jaiswal N et al., Endocrinology, 1999]. A known composition containing bone marrow MSCs in humans in an amount of at least 10 ⁵ cells / ml and a culture medium that is used only for the regeneration of the skin [patent RU 2455354 C1]. A conditioned medium Majumdar MK, Thiede MA, et al., Stem Cell Res., 2000] was developed, which supports the differentiation of MSCs into mechanocytes or osteoblasts that secrete cytokines, which can activate the long-term growth of hematopoietic stem cells.

Known pharmaceutical composition containing a conditioned nutrient medium that supports the growth of eukaryotic cells and a pharmaceutical carrier [RU 2001133453/13. Application of 05/12/2000, priority US: 09 / 313,538 of 05/14/1999]. The composition may have a different dosage form. A pharmaceutical carrier may include one or more extracellular products (components of a conditioned medium) secreted by protein separation methods. This composition is used to stimulate the healing of wounds and burns. The disadvantages of the pharmaceutical composition under consideration include the fact that the therapeutic effect is provided not by the use of the most conditioned medium, but by a complex of extracellular products and other elements of the composition. At the same time, the role of the medium is to maintain the vital functions of the cells and medium used to preserve the pharmaceutical carrier.

In regenerative medicine, the use of particles secreted by MSCs is possible. Thus, vesicles or exosomes can be used to treat skin diseases and burns [WO 2009105044 A1]. A method for producing particles is described, including obtaining a medium conditioned by MSCs, concentrating it, processing by gel filtration, selecting a fraction with dynamic light scattering, by UV detection at 220 nm (collecting fractions eluting with a retention time of 11-13 minutes). The resulting particle can be used as part of a pharmaceutical composition used to treat diseases. Despite the fact that there is evidence indicating the important role of exosomes in the realization of the paracrine effects of MSCs, there is no evidence that the fraction described in the invention is the key to the regenerative action of these cells. Also, the described technology is rather difficult to apply in the production process and does not provide proper standardization of the final product.

An analogue is the growth medium obtained by growing cardiomyocytes from bone marrow MSCs [K. Fukuda, Artificial Organs, 2001]. The basis of the medium under consideration is IMDM, to which antibiotics, antimycotics, L-glutamine and 20% fetal bovine serum are added, as well as a differentiation inducer 5-azacytidine (demethylating agent). During the growth and proliferation of MSCs and their differentiation into cardiomyocytes, the products of vital activity accumulate in the medium, which are also components of this medium. The disadvantages of this environment is that the properties of vital products are not identified and their effect has not been studied. In addition, the remains of the drug 5-azacytidine, which have a mutagenic effect, are preserved in the medium.

As a prototype of the invention, a conditioned medium is considered that has a therapeutic effect in thermal burns [patent RU No. 2292212, 01/27/07]. This medium contains mineral salts and amino acids, penicillin, amphotericin, L-glutamine and fetal calf serum, sufficient to maintain the viability and growth of human bone marrow MSCs; the medium is obtained at the stage of stationary growth of a stable cell line of mesenchymal stem cells located in the G ₀ period cell cycle. The described prototype has several drawbacks - the limited choice of raw materials (MSCs obtained only from bone marrow does not imply the use of previously accumulated and cryopreserved cultures), a narrow spectrum of regenerative action, and biologically active substances that enter the cells of MSCs in a significant amount are not included in the composition of the drug . Determining the time for collecting the conditioned medium indirectly (by the phase of the cell cycle) does not allow to obtain a product with a maximum content of cytokines.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION

A method of obtaining a regenerative preparation based on an extract of MSCs and a conditioned medium formed during cell growth includes the following steps: deconservation or isolation of de novo cellular material from the brain or adipose tissue of animals, culturing MSCs in a medium based on DMEM (Dulbecco's Modified Eagle's Medium) with a 10% content of fetal bovine serum (FBS) before the formation of a conditioned medium with a certain content of key interleukins - IL-β, IL-2, IL-6, IL-10, collection of conditioned medium, obtaining an MSC extract by freezing i-thawing the cell suspension, combining the extract with the conditioned MSC medium, stabilizing the components of the obtained preparation with glycine at a final concentration of 20 mg / ml, sterilizing filtration, filling into bottles and freeze drying.

DETAILED DESCRIPTION OF THE INVENTION

When creating the present invention, the task was set to apply the concept of regenerative medicine in veterinary medicine to restore the function of vital animal organs by replacing defective cellular elements with new ones by activating the progenitor cells of the affected tissue.

The technical result is achieved by developing a method for producing a drug for veterinary medicine, which has a complex effect on regenerative processes in the body.

The proposed method provides a preparation having high efficiency, which is due to the action of a balanced complex of cytokines contained in extracts and secreted by mesenchymal stem cells into the culture medium. To realize the regenerative effect of MSCs, the content of key interleukins was selected, namely IL-1β, IL-2, IL-6, IL-10, which are the main stimulants of the mechanisms of nonspecific defense of the body and a specific immune response, as well as activators of reparative processes in damaged tissues.

The proposed method involves the following operations: isolation of mesenchymal stem cells from the brain or adipose tissue of animals (de novo) or de-preservation of the finished cell culture, cultivation of MSCs in a DMEM-based medium with 10% PBS to form a conditioned medium (in which the content of key interleukins is determined : IL-1β - not less than 2 pg / ml, IL-2 - not less than 2 pg / ml, IL-6 - not less than 10 pg / ml, IL-10 - not less than 2 pg / ml, determined by enzyme immunoassay (ELISA)), collection of conditioned medium, obtaining extract M K, by freezing-thawing of cell suspensions, the combined extracts were conditioned with MSC medium, stabilization components resulting formulation (addition of glycine to a final concentration of 20 mg / ml), filling into vials of 1 ml and freeze drying.

In this invention, MSCs obtained independently by any known method can be used and in the form of commercial cultures of MSCs that have a quality certificate and are intended, including for clinical use (for example, such as ATCC).

Brief Description of the Figures

The invention is illustrated by photos, graphs and tables:

Photo 1. Mesenchymal stem cells from rat CM, null passage (light microscopy, 10 × eyepiece enlargement and 10 × objective lens).

Photo 2. Mesenchymal stem cells from rat CT, third passage (light microscopy, 10 × eyepiece and 40 × objective lenses).

Photo 3. Colony of MSCs from a rat VT, null passage (light microscopy, magnification of the eyepiece 10 × and lens 40 ×).

Chart 1. The effect of the drug on the basis of the extract and the conditioned medium of bone marrow mesenchymal stem cells (CS MSC KM) on the proliferative index of cell cultures of HPF and MSC.

Chart 2. The effect of the drug based on the extract and the conditioned medium of adipose tissue mesenchymal stem cells (SC MSCs VT) on the proliferative index of cell cultures of HPF and MSCs.

Chart 3. The effect of the drug based on the extract and the conditioned medium of deconserved mesenchymal stem cells (CS MSC CM decons.) On the proliferative index of cell cultures of PEC and MSC.

Figure 4. The effect of the drug on the basis of the extract and the conditioned environment of bone marrow mesenchymal stem cells (KS + MSC KM), adipose tissue (KS + MSC in VT) and deconserved bone marrow MSC (SC + MSC KM decons) on the activity of ALT in rat serum when modeling acute liver pathology (AKI).

Figure 5. The effect of the drug based on the extract and the conditioned medium of bone marrow mesenchymal stem cells (KS + MSC KM), adipose tissue (KS + MSC MF) and deconserved MSC of bone marrow (SC + MSC KM decons.) On the activity of AcT in serum rats in modeling acute liver pathology (AKI).

Figure 6. The effect of the drug on the basis of the extract and the conditioned environment of bone marrow mesenchymal stem cells (KS + MSC KM), adipose tissue (KS + MSC MF) and deconserved bone marrow MSC (SC + MSC KM decons.) On the protein content in blood serum rats in modeling acute liver pathology (AKI).

Table 1. The IP value of cell cultures of FEP and MSCs with the addition of different concentrations of the regenerative drug.

Table 2. Biochemical parameters of liver damage in the studied groups of animals.

The possibility of carrying out the invention and the effectiveness of its application are confirmed by the following examples:

Example 1. Bone marrow (CM) cells were isolated from rat thigh bones (in an amount providing an inoculum concentration of at least 1 × 10 ⁶ cells / ml) immediately after decapitation, placed in a cooled DMEM / F12 medium (Biowest, USA) containing gentamicin (80 μg / ml, Biolot, Russia) and amphotericin B (0.25 μg / ml, Biowest, USA) were washed twice from the blood by the indicated medium by centrifugation at 1200 rpm for 5 minutes.

Cells were seeded with a concentration of 1 × 10 ⁶ cells / ml (for which preliminary cell counting was performed in the Goryaev’s chamber, taking into account viability by subvital staining with trypan blue), they were cultured in plastic bottles (SPL, Korea) with a growth surface area of 175 cm ² at a temperature of 37 ° C in an atmosphere with a 5% CO ₂ content. The growth medium included DMEM high glucose (Biowest, USA), L-glutamine, 10% PBS (Sigma Aldrich, USA), gentamicin (80 μg / ml), amphotericin B (25 μg / ml, Biowest, USA) . Isolation of the cell fraction of MSCs is based on the adhesion of these cells to the culture plastic, as a substrate.

A day after the start of cultivation, rinsing with an antibiotic medium was performed to remove the floating fraction of cells and the growth medium was replaced. Subsequently, the medium was replaced every 3-4 days. To achieve the confluence of the monolayer (visually checked with an inverted microscope) at least 85%, it was disaggregated with a dispersing solution (0.05-0.1% trypsin (Biowest, USA) in Versen's solution (PanEco, Russia) and inoculated in new culture bottles with a reseeding ratio of 1: 3.

To obtain a conditioned medium, the culture was carried out for 3-4 passages, then inoculation was carried out in culture bottles at the rate of 5-15 × 10 ³ cells per 1 cm ^{2 of} the surface area in 90 ± 5 ml of growth medium and cultivated under the conditions described above until the necessary concentration of interleukins in the environment.

Upon reaching the required concentration of biologically active substances, a conditioned medium containing all MSC secretion products was collected in sterile centrifuge tubes, purified by centrifugation at 3000 rpm and a temperature of 5 ± 1 ° C for 10 minutes from cell debris.

The resulting conditioned medium can be used directly for its intended purpose or stored for a long time under certain conditions (up to seven days at a temperature of 3 ± 1 ° C, longer storage requires a temperature of no higher than minus 20 ° C).

After collecting the conditioned medium, an extract of MSC KM was obtained: cell monolayer, with a confluency of at least 90%, was disaggregated with the above dispersing solution, 0.9% sodium chloride solution (5-10 ml per 1 culture bottle) was added, it was frozen temperature minus 20 ± 2 ° C, after which thawing was performed at room temperature. The obtained MSC KM extract was collected in sterile centrifuge tubes, purified from cell debris by centrifugation at 3000 rpm and a temperature of 5 ± 3 ° C for 10 minutes.

To obtain a regenerative preparation, the centrifugation pellet, which is an extract of MSC KM, was combined with a conditioned medium, the contents of IL-1, IL-2, IL-6, IL-10 were measured, glycine was added (to a final concentration of 20 mg / ml), Poured into vials of 1 ml and freeze-dried.

So, by the end of the fourth week from the beginning of the cultivation of MSC of KM rats, up to 3000 ml (at a seeding concentration at the stage of isolation of at least 1 × 10 ⁶ cells / ml) of a lyophilization solution based on the extract and conditioned medium of MSC KM can be obtained, which can be used for the preparation of a drug that stimulates the regenerative processes of the animal’s body.

Example 2. Cellular material was obtained from subcutaneous adipose tissue (VT) of a rat (in an amount providing an inoculum concentration of at least 1 × 10 ⁶ cells / ml) under sterile conditions after decapitation of the animal. Adipose tissue was placed in Dulbecco's Phosphate Buffered Saline (PBS) buffer solution (Biowest, USA) containing gentamicin (80 μg / ml, Biolot, Russia), fragmented to a homogeneous mass using sterile instruments, and then subjected to stepwise enzymatic treatment. The enzymatic treatment was carried out as follows: DMEM high glucose nutrient medium (Biowest, USA) was added to the crushed adipose tissue with the addition of an antibiotic and antimycotics, as well as trypsin enzyme to a concentration of 0.1-0.15%, kept for 30 ± 5 minutes at a temperature of 37 ± 1 ° С with constant stirring on a magnetic stirrer, then the medium with cells was filtered through 100 μm (BD) nylon membranes and 0.2-0.5% PBS (Sigma-Aldrich, USA) was added to inactivate trypsin. The described operation was repeated twice with a decrease in incubation time to 15 ± 5 minutes.

After filtration, the cell suspension was centrifuged for 10 min at 1200 rpm, the supernatant was completely removed. The resulting pellet was resuspended in DMEM high glucose growth medium (Biowest, USA) with 10% PBS (Sigma-Aldrich, USA), after which the cells were seeded in culture vials (70 ± 5 ml of cell suspension / vial) with a growth surface area of 175 cm ² (SPL, Korea) with a concentration of 1 × 10 ⁶ cells / ml and cultured at 37 ° C with a 5% concentration of CO ₂ .

The selection of the cell fraction of MSCs, as in the previous example, is based on the adhesion of these cells to the culture plastic, as a substrate. The accumulation of cell mass, the formation of a conditioned medium and the preparation of a regenerative preparation were carried out in the same manner as in Example 1. The production time of the preparation was also 4 weeks, the amount of the product was up to 3000 ml (at the inoculation concentration at the extraction stage of at least 1 × 10 ⁶ cells / ml) of the solution lyophilization based on extract and conditioned medium

Example 3. Cellular material was obtained by deconservation of cell cultures of MSCs (KM or VT rats). A cryovial with cells (a cell concentration of at least 1 × 10 ⁶ cells / ml, a cell suspension volume of 1 ml) was placed in a water bath with a water temperature of 37 ± 1 ° C, shaking periodically, until completely thawed. Cells were washed from the cryoprotectant using DMEM high glucose growth medium (Biowest, USA). For this, the cell suspension was transferred into a sterile culture vial with a growth surface area of S = 25 cm ² , small doses were added to exclude osmotic damage, DMEM high glucose medium (10-15 ml), the resulting suspension was uniformly distributed into two centrifuge tubes and centrifuged at 1200 rpm for 10 minutes. The supernatant was removed, the precipitate was resuspended in a growth medium containing DMEM high glucose (Biowest, USA), L-glutamine, 10% PBS (Sigma-Aldrich, USA), gentamicin (40 μg / ml, Biolot, Russia), amphotericin B (25 μg / ml, Biowest, USA). Cells were sown (70 ± 5 ml of cell suspension / vial) at a concentration of 8-10 × 10 ⁴ cells / ml in culture bottles with a growth surface area of 175 cm ² (SPL, Korea) and cultivated at 37 ° C with 5% CO ₂ . The growth medium was changed every 3-4 days. The accumulation of cellular biomass, obtaining a conditioned environment and the drug was carried out similarly as in Example 1.

Example 4. The properties of the drug based on the extract and the conditioned environment of MSCs of bone marrow and adipose tissue were evaluated by the effect on the proliferation of cell culture of human embryo fibroblasts (PEC) and mesenchymal stem cells isolated from rat bone marrow (in vitro).

To do this, model cell cultures were taken at the third passage, seeded in 12-well culture plates (SPL, Germany) with the test regenerative preparation added to the growth medium at various concentrations (1%, 5%, 10%), cultured for three days under standard conditions (incubated in a humid chamber in a CO ₂ incubator at 37 ° C and 5% CO ₂ ) with the addition of 10% PBS (Sigma-Aldrich, USA) to medium 199 (MPE Chumakov Institute of Applied Mathematics and Epidemiology) , Russia) for PMF and DMEM / F12 (1: 1) (Biowest, USA) - for MSCs. The inoculum concentration of FEC cells is 8-10 × 10 ⁴ cells / ml, MSC - 7-9 × 10 ⁴ cells / ml medium. As a control, cells grown in growth medium without the addition of a regenerative preparation were used.

At the end of the incubation, the grown cultures were removed from the plastic surface and the proliferative index was determined. The proliferation index (IP) was determined as the ratio of the number of cells in 1 ml of the suspension obtained by removing the cells from the substrate to achieve confluence of the monolayer to the inoculum cell concentration (the number of cells in ml of the solution when plated on culture plates).

The data obtained for cultures of two morphological types indicate (Table 1, charts 1-3) that the level of proliferative properties of cells of both cultures is increased when the proposed regenerative preparation (obtained by all three process modifications described in the previous examples) is added to growth media) by 10-85% for PEC and 2–25% for MSC as compared with the growth medium, while a linear relationship was observed between the concentration of the drug and the value of the proliferation index.

Example 5. The regenerative effect of a preparation based on an extract and a conditioned medium of a culture of mesenchymal stem cells from bone marrow or adipose tissue was studied in an acute in vivo model of liver damage (MOPP) in rats. The model of acute liver damage was performed by a single injection of rats into the stomach through a probe of paracetamol (N- (4-hydroxyphenyl) acetamide) in the form of a suspension at a dose of 700 mg per 1 kg of body weight. The treatment procedure after MOPP consisted in daily intramuscular administration of 0.5 ml of the studied drug to the animals of the experimental group for 5 days. As a control, rats with MOPP that were not subjected to therapeutic effects were used.

A day after the end of the course of treatment, the rats were removed from the experiment by decapitation, having previously provided anesthesia due to the intraperitoneal administration of chloral hydrate at a dose of 400 mg per 1 kg of animal body weight. In the blood serum, the activity of the aspartate- (AcT) and alanine aminotransferase (ALT) enzymes was determined using a standard Vital kit and the total protein content was determined using the biuret method. The studied biochemical parameters reflect the functional state of the liver. The enzymes AcT and Alt are present in significant quantities in the liver and kidneys, therefore, normally, the concentrations of AcT and Alt in the blood are low. When hepatocytes are damaged, the enzymes AcT and Alt are lost, and the concentration of these biochemical markers in the blood increases. A change in the level of total protein (a sign of gross pathology of the liver) was not observed.

The results of the study (Table 2 and graphs 4-6) show that the use for 5 days of the proposed regenerative preparation based on the extract and conditioned medium using mesenchymal stem cells, both de novo obtained from bone marrow and adipose tissue, and deconserved, led to a significant improvement in the condition of animals with AKI, which is confirmed by the concentration of the studied markers of liver damage (indicators of ACT, ALT levels). Thus, the concentrations of these enzymes in the group of animals treated with the regenerative preparation did not significantly differ from those in the group of intact rats (not exposed to MOPP).

Thus, the proposed regenerative drug has a therapeutic effect in acute liver damage of animals. Significant differences in the effectiveness of the drug obtained from various sources of mesenchymal stem cell cultures were also not detected.

Information sources:

1. Adams R. Methods of cell culture for biochemists - M .: Mir, 1983. - 264 p.

2. Digay A.M., Zyuzkov G.N. Cell therapy: new approaches // Science in Russia. - Moscow: Publishing House "Science", 2009. - Volume. 169. - No. 1. S. 4-8.

3. Goldberg ED, Dygay AM, Zhdanov VV, Zyuzkov GN, Miroshnichenko LA, Simanina EV, Stavrova LA, Udut EV, Fonima T. N., Vetoshkina T.V., Khrichkova T.Yu., Ermakova N.N., Plotnikov M.B., Aliev O.I., Chernysheva G.A. Pharmacological aspects of regenerative medicine // Bull. an experiment. biol. and medicine. - 2008. - Appendix 2. - S. 14-21.

4. Pathophysiology: Textbook for medical universities. / Ed. V.V. Novitsky, E.D. Goldberg. - Tomsk: Publishing house of Tomsk University, 2001. - 681-687.

5. Digay A.M., Zyuzkov TN, Zhdanov VV, Madonov PG, Artamonov AV, Bekarev AA, Udut VV Immobilized granulocyte colony stimulating factor. Pharmacological properties and prospects of use. - Tomsk: Publishing House: LLC Printing Manufactory, 2011. - 149 p.

6. Culture of animal cells. Methods / Ed. R. Francis - Moscow: Mir, 1989 .-- 333 p.

7. “A conditioned environment with a therapeutic effect”, patent number 2292212, publication date 01/27/07, authors: Konoplyannikov A.G., Kolesnikova A.I., Saenko A.S., Bardychev M.S., Pasov V. .V., Kurpesheva A.K., Krikunova L.I .;

8. "Biologically active drug" lymphokinin, with immunomodulating properties ", patent number 2048816, application number 93031657/14, application filing date 06/07/1993, publication date 11/27/1995, authors: Bykovskaya S.N., Shadrin O.V. ., Dvoryanchenko D.Yu., patent holder of Simera CJSC;

9. "The tool for the treatment of burns and wounds based on cytokines and growth factors secreted by human mesenchymal cells, a method of obtaining a means and method of treating burns and wounds", patent number 2574017, authors: Stambolsky DV, Tkachuk VA, Semina E.V., Tarasova E.V., Rubina K.A., Kochegura T.N., Sysoeva V.Yu., Efimenko A.Yu., Akopyan J.A .;

10. "Means for changing the growth rate or reproduction of cells, a method for its production, a method for stimulating wound healing or treatment of burns, a method for correcting a cosmetic defect, a method for inhibiting skin aging and a method for stimulating hair growth", Patent No. 2280459, Application No. 2001133453/13 publication date of the application 01/20/2005, publication date 07/27/2006, authors: Noton G.K. (Us), Horvits D.L. (Us), Applegate M.A. (Us), Zeltinger J. (Us), Mansbridge J.N. (Us), Kern A. (Us), Landin L.K. (Us), Ratcliffe E. (Us), Pinney R.E. (Us); patent holder: Skinmedics, Inc. (Us);

11. "Mesenchymal stem cell conditioned medium", patent number WO2008020815 A1, application number PCT / SG2007 / 000257, publication date 02.21.2008, authors: Sai Kiang Lim, Elias Lye, patent holder Agency For Science, Technology And Research;

12. A. van Koppen, J.A. Joles, B. W. M. van Balkom et al., "Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease," PLoS ONE, vol. 7, no. 6, Article ID e38746, 2012;

13. B.R. Zhou, Y. Xu, S.L. Guo et al., "The effect of conditioned media of adipose-derived stem cells on wound healing after ablative fractional carbon dioxide laser resurfacing," BioMed Research International, vol. 2013, Article ID 519126, 9 pages, 2013;

14. M.K. Jr. Patterson, Measurement of growth and viability of cells in culture. In: W.B. Jakoby, I.H. Pastin, eds., Methods in Enzymology, New York: Academic Press Inc., Vol. 58, 141-152, 1979;

15. Mbalaviele G, Jaiswal N, Meng A, Cheng L, Van Den Bos C, Thiede M. Human mesenchymal stem cells promote human osteoclast differentiation from CD34 + bone marrow hematopoietic progenitors // Endocrinology. 1999 Aug; 140 (8): 3736-3743;

16. S.H. Bhang, S. Lee, J.Y. Shin, T.J. Lee, H.K. Jang, and B.S. Kim, "Efficacious and clinically relevant conditioned medium of human adipose-derived stem cells for therapeutic angiogenesis," Molecular Therapy, vol. 22, no. 4, p. 862, 2014;

17. Leo Timmers, Sai Kiang Lim, Imo E. Hoefer "Human mesenchymal stem cell conditioned medium improves cardiac function following myocardial infarction", Stem Cell Research vol. 6, pp. 206-214, 2011;

18. Reza Moghadasali, Henricus A.M. Mutsaers, Mahnaz Azarnia "Mesenchymal stem cell-conditioned medium accelerates regeneration of human renal proximal tubule epithelial cells after gentamicin toxicity" - Experimental and Toxicologic Pathology - vol. 65, pp. 595-600, 2013.

Claims (1)

A method of obtaining a regenerative veterinary preparation includes: culturing isolated de novo or deconserved mesenchymal stem cells obtained from adipose tissue or bone marrow of an animal in a liquid nutrient medium with the addition of 10% fetal bovine serum, obtaining a conditioned medium containing interleukins: IL-1β, IL -2, IL-6 and IL-10 in concentrations of at least 2; 2; 10 and 2 pg / ml, the addition of MSC extract obtained by freezing and thawing of a cell suspension, the addition of glycine to a final concentration of 20 mg / ml lyophilization solution, sterilizing filtration and lyophilization.

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