Method for extracting, purifying and concentrating exosomes from mesenchymal stromal cells

Abstract

The invention relates to methods for extracting, purifying and concentrating mesenchymal stromal cell exosomes enriched with micro-RNA, proteins and lipids, and can be used in cosmetology and pharmacology for rehabilitation and regeneration. The technical result of the invention is a more efficient process for producing exosomes. This technical result is achieved in that the present method for extracting, purifying and concentrating exosomes from mesenchymal stromal cells includes cultivating cells, collecting the conditioned culture medium, depleting high-molecular-weight proteins, extracting, purifying and concentrating exosomes, and freezing and lyophilizing the exosome concentrate; in order to enrich the culture medium with exosomes, multipotent mesenchymal stem cells (MSCs) are grown in a medium having a lowered glucose concentration and containing platelet lysate and 5 mM of L-alanyl-L-glutamine, wherein, in the case of static cell cultivation, the growth medium is placed in a culture vessel and once a level of not less than 80% monolayer confluence is reached, the culture medium is harvested and replaced once every 24 hours, and in the case of flow-through cell cultivation, cells are placed on cell carriers in a bioreactor and 72-96 hours after the start of cultivation, the culture medium is harvested and replaced once every 24 hours, the exosome-enriched culture medium is subjected to concentration, which includes successive stages of centrifugation, tangential flow filtration and isopycnic centrifugation: in the first stage, the culture medium is centrifuged for 10 mins at 2000 g to remove large cell particles and other inclusions; in the tangential flow filtration stage, the supernatant is concentrated using hollow fibre filters having a molecular weight cutoff of 500 kDa, after which the concentrate is subjected to fivefold diafiltration with an equal volume of DPBS phosphate buffer solution using the same hollow fibre cartridge; and in the isopycnic centrifugation stage, the concentrated supernatant is mixed with a saccharose solution and deuterium oxide and subjected to isopycnic centrifugation at 100000 g and 4˚С for 75 mins, after which the concentrate is subjected to further fivefold diafiltration with a buffer solution, then the concentrate is filtered through filters with a pore diameter of 0.22 µm and 0.1 µm, the exosome concentrate is subjected to initial freezing to -75˚C in a freezing chamber, and drying is carried out in a lyophilization chamber, wherein in a first drying stage, the concentrate is cooled to -40˚С and subsequently held for 100 mins at an absolute pressure in the chamber of not more than 10 Pa, then cooled to -30˚С and subsequently held for 610 mins at an absolute pressure in the chamber of not more than 10 Pa, then cooled to -10˚С and subsequently held for 315 mins at an absolute pressure in the chamber of not more than 10 Pa, after which it is cooled to 0˚С and subsequently held for 130 mins at an absolute pressure in the chamber of not more than 10 Pa, then heated to +10˚С and subsequently held for 130 mins at an absolute pressure in the chamber of not more than 10 Pa; in a second drying stage, drying is carried out at +22˚С for not less than 130 minutes at an absolute pressure in the chamber of not more than 10 Pa.

Description

[0001] METHOD FOR EXTRACTION, PURIFICATION AND CONCENTRATION OF EXOSOMES FROM MESENCHYMAL STROMAL CELLS

[0002] DESCRIPTION

[0003] The invention relates to methods for extracting, purifying and concentrating exosomes from mesenchymal stromal cells enriched with microRNA, proteins and lipids, and can be used in cosmetology and pharmacology for rehabilitation and regeneration [C12N 5 / 077].

[0004] A METHOD FOR EXTRACTING SPECIFIC EXOSOMES OF MESENCHYMAL STEM CELLS is known from the prior art [CN109097328, published 12 / 28 / 2018], which includes collecting supernatants of conditioned serum-free media of mesenchymal stem cells by culturing mesenchymal stem cells to a confluence of 60-70%, washing with PBS and replacing the medium with a serum-free one.This is followed by cell culturing, collection of conditioned medium, centrifugation and removal of cellular debris to obtain the original supernatant; centrifugation of the original supernatant at 2000 g for another 10 min to remove dead cells and large fragments, transfer of the supernatant after centrifugation to a centrifuge tube, centrifugation at 10,000 g for 30 minutes and collection of the supernatant containing microvesicles, centrifugation of the supernatant at 70,000 g for 60 minutes to obtain a pellet containing exosomes, washing, further centrifugation at 70,000 g for 70 minutes to obtain an exosome pellet, resuspension of the said pellet in PBS and filtration to obtain specific exosomes of mesenchymal stem cells.

[0005] Also known is a METHOD FOR PREPARATION TO INCREASE THE PRODUCTION OF EXOSOMES FROM MESENCHYMAL STEM CELLS [CN110195038A, published 09 / 03 / 2019], which uses the supernatant of the conditioned medium of umbilical cord mesenchymal stem cells, containing 100 ml of DMEM / F12 medium + 2 ml of nonessential amino acids + 2 ml of L-glutamine + 2 μg of VCAM1 reagent. After defrosting the cells from the bank, the cell suspension is transferred to a centrifuge tube containing 10 ml of cell culture medium; centrifuged at 1500 rpm for 5 minutes and the supernatant after centrifugation is discarded; the cells are resuspended by adding an appropriate amount of fresh medium, and the cells are counted and viability is determined; Depending on the number and viability of cells, the cell seeding density is adjusted to 1.0x10 5 viable cells / cm 2with the medium, add the cell suspension to the culture vessel and gently mix the cells to distribute them evenly throughout the vessel; observe cell growth, change the medium every other day; when the cells reach 80-90% confluency, they can be subcultured; exosomes are separated at 4°C: after 48 hours, the cell supernatant is extracted and centrifuged at 1500 rpm for 5 minutes, at 2000 g for 10 minutes and at 10,000 g for 20 minutes; exosomes are separated by ultracentrifugation at 100,000 g for 120 minutes using a Beckman Coulter Ultracentrifuge Type 70Ti rotor; the precipitated exosomes are collected and resuspended in sterile PBS and subjected to further purification by sucrose gradient centrifugation, stored at -80°C.

[0006] The disadvantage of the presented analogs is the absence of tangential filtration phases through hollow fiber filters and multiple diafiltration of the concentrate, which can lead to a decrease in the efficiency of the exosome production process.

[0007] The closest in technical essence is the METHOD OF OBTAINING AND CONCENTRATION OF MICRORNA-CONTAINING EXOSOMES

[0008] MULTIPOTENT MESENCHYMAL-STROMAL CELLS FOR USE IN COSMETICS AND MEDICINES TO STIMULATE REGENERATIVE PROCESSES AND SLOW DOWN THE AGING PROCESS [RU2710368, published 26.12.2019], containing a description of the process of isolating mesenchymal-stromal cells from the umbilical cord, adipose tissue and dental pulp, subsequent cultivation and collection of culture fluid enriched with exosomes, concentration of exosomes and depletion of high-molecular proteins in order to reduce the sensitizing ability of the drug, freezing or drying the resulting concentrate for long-term storage. To produce exosomes, MMSCs are cultured in an alpha-MEM or DMEM medium with a low glucose content, 5 mM L-alanyl-glutamine and umbilical cord blood platelet-activated plasma (UCPRP), cell cultivation in the above-described medium is carried out up to 80-100% confluence of the culture monolayer,Moreover, the cultivation can be static or flow-through: in static cultivation, the cells are placed in a culture vessel such as a flask or Petri dish, and when the monolayer reaches 80-100% confluency, the culture fluid is changed every 24 hours, the collected culture medium is saved for the isolation of exosomes, with 5-7 removals of the enriched medium from one culture, and in flow-through cultivation, the cells are placed in a flow-through incubator on cell carriers, which are microparticles, hollow fiber structures, when the cells reach a monolayer, the culture medium is completely changed to a new one, after which standard cultivation is carried out for 7 days, when a medium enriched with exosomes is obtained, it is concentrated with the removal of the protein fraction over 100 kDa,by using a tangential filtration system to remove large particles and concentrate: first, the culture medium is centrifuged for 30 minutes at 2000g or filtered with a 1-10 μm filter to remove large cell debris, then the supernatant is filtered with a tangential filter with pores of 100 kDa or 500 kDa, as a result of filtration, the volume of the liquid is reduced, and the exosomes are concentrated in the diafiltrate, the latter is then filtered by ultrafiltration to remove microvesicles and other large particles using filters with sizes of 0.22 μm and 0.1 μm.

[0009] The main technical problem of the prototype is the absence of primary centrifugation processes after concentration of the conditioned culture medium, multiple diafiltration of the concentrate, and isopycnic centrifugation of the diafiltered supernatant during the implementation of the method, which can lead to a decrease in the efficiency of the exosome production process.

[0010] The objective of the invention is to eliminate the shortcomings of the prototype.

[0011] The technical result of the invention is to increase the efficiency of the process of obtaining exosomes.

[0012] The said technical result is achieved due to the fact that the method for extracting, purifying and concentrating exosomes from mesenchymal stromal cells includes culturing cells, collecting conditioned culture medium, depleting high-molecular proteins, isolating, purifying and concentrating exosomes, freezing and lyophilizing the exosome concentrate, to enrich the culture medium with exosomes, multipotent mesenchymal stem cells (MMSCs) are grown in a medium with a reduced concentration of glucose, platelet lysate and 5 mM L-alanyl-L-glutamine, while, during static cell cultivation, the growth medium is placed in a culture dish, upon reaching a level of at least 80% confluency of the culture monolayer, the culture medium is collected and replaced once every 24 hours, during flow cultivation, the cells are placed in a bioreactor on cell carriers, 72-96 hours after the start of cultivation, collection and replacement are carried out once every 24 hours of culture medium,the exosome-enriched culture medium is subjected to concentration, which includes successive stages of centrifugation, tangential filtration and isopycnic centrifugation, in the first stage the culture medium is centrifuged for 10 min at 2000 g to remove large cell particles and other inclusions, in the tangential filtration stage the supernatant is concentrated through hollow fiber filters with a molecular weight cutoff value of 500 kDa, after which the concentrate is subjected to fivefold diafiltration with an equal volume of DPBS phosphate buffer using the same hollow fiber cartridge, in the isopycnic centrifugation stage the concentrated supernatant is mixed with a sucrose solution and deuterium oxide and subjected to isopycnic centrifugation at 100,000 g and 4 ° C for 75 min, then the concentrate is subjected to an additional fivefold diafiltration with a buffer,After which the concentrate is filtered through filters with pore diameters of 0.22 µm and 0.1 µm. Specifically, MMSC is contained in an alpha-MEM-based medium.

[0013] In particular, MMSCs are maintained in a DMEM / F12-based medium.

[0014] In particular, MMSCs are contained in a medium with human platelet lysate.

[0015] In particular, MMSCs are contained in a medium with mammalian platelet lysate.

[0016] In particular, MMSCs are kept in the mentioned environment for at least 24 hours.

[0017] In particular, cell cultivation is carried out statically, with enriched culture medium being collected from the initial culture 4 to 5 times.

[0018] In particular, cell cultivation is carried out using a flow method, with the enriched culture medium being collected from the initial culture 8 to 10 times.

[0019] In particular, at the tangential filtration stage, the supernatant is concentrated using tangential filtration.

[0020] In particular, at the tangential filtration stage, the supernatant is concentrated using ultrafiltration. Implementation of the invention.

[0021] The present method involves the purification, concentration and storage of exosomes from multipotent mesenchymal stem cells (MMSCs) of the umbilical cord, Wharton's jelly, placenta, adipose tissue and other tissues of mesodermal origin of humans and mammals.

[0022] The source of exosomes for the concentrate are characterized multipotent mesenchymal stem cells from young and tested healthy donors with high proliferative activity of cells (from the 1st to the 8th passage), maintaining karyotype stability over the 8th passage.

[0023] The source of their production is dental pulp, bone marrow, adipose tissue, placenta, and other tissues of mesodermal origin in humans and mammals.

[0024] The proposed method includes culturing the appropriate cell type, collecting the conditioned culture medium rich in exosomes, depleting high-molecular proteins from the culture medium, isolating, purifying and concentrating the exosomes, and freezing or lyophilizing the exosome concentrate produced in this manner for long-term storage and transportation.

[0025] To enrich the culture medium with exosomes, MMSCs are kept for at least 24 hours in a medium based on alpha-MEM or DMEM / F12 with a reduced glucose concentration, human platelet lysate (hPL) or mammalian platelets and 5 mM L-alanyl-E-glutamine, while cell cultivation is carried out to 80-100% confluence of the culture monolayer.

[0026] Cultivation is carried out statically or by flow method.

[0027] In static culture, cells are grown in specialized containers (e.g., Petri dishes, culture plates, culture flasks, or cell factories). Once the monolayer reaches 80-100% confluency, the culture medium is collected and replaced every 24 hours. The enriched culture medium is collected from the original culture 4 to 5 times. The resulting culture medium is stored for exosome isolation.

[0028] In flow cultivation, cells are placed in a bioreactor on cell carriers (in particular, hollow fiber carriers, macro- or microparticles); 72-96 hours after the start of cultivation, the culture medium is collected and replaced once every 24 hours, while the enriched culture medium is collected from the original culture 8 to 10 times.

[0029] The resulting exosome-enriched culture medium is subjected to concentration, during which particles larger than 100 nm and protein fractions smaller than 100 kilodaltons (kDa) are removed, while the concentration includes successive stages of centrifugation, tangential filtration and isopycnic centrifugation.

[0030] At the first stage, the culture medium is centrifuged for 10 minutes at 2000 g to remove large cell particles and other inclusions.

[0031] In the second step, the supernatant is concentrated by tangential filtration or ultrafiltration through hollow fiber filters with a pore size of 500 kDa. Filtration reduces the volume of the medium by 10-15 times, after which the concentrate is diafiltered five times with an equal volume of DPBS phosphate buffer using the same hollow fiber cartridge. The concentrated (diafiltered) supernatant is mixed with a sucrose solution and deuterium oxide and subjected to isopycnic centrifugation at 100,000 g and 4°C for 75 minutes. The resulting concentrate is then diafiltered five times with buffer.

[0032] In the final step, filters with pore sizes of 0.22 µm and 0.1 µm are used to remove microvesicles and some other remaining particles. The filtrate obtained during the stepwise concentration contains concentrated and purified exosomes, the quality of which is tested, measured, and assessed using flow cytometry, quantitative PCR, or alternative methods for analyzing microRNA and other components.

[0033] Lyophilization is performed using a freeze-drying system. The exosome concentrate is first frozen to -75°C in a freezer in a special drying container.

[0034] Next, drying is carried out in a lyophilization chamber, wherein at the primary drying stage the concentrate is cooled to -40°C with subsequent holding for 100 minutes at an absolute pressure in the chamber not exceeding 10 Pa, then cooled to -30°C with subsequent holding for 610 minutes at an absolute pressure in the chamber not exceeding 10 Pa, then cooled to -10°C with subsequent holding for 315 minutes at an absolute pressure in the chamber not exceeding 10 Pa, after which it is cooled to 0°C with subsequent holding for 130 minutes at an absolute pressure in the chamber not exceeding 10 Pa and heated to +10°C with subsequent holding for 130 minutes at an absolute pressure in the chamber not exceeding 10 Pa, and at the second stage drying is carried out at +22°C for at least 130 minutes at an absolute pressure in the chamber not exceeding 10 Pa.

[0035] The implementation of the proposed method allows the use of the produced concentrated exosomes for two weeks in an unchanged form for clinical or cosmetic use.

[0036] Exosomes frozen at -20°C, -40°C, -60°C, and -80°C have a shelf life of up to six months. Lyophilization for long-term storage allows exosome concentrate to be stored for up to a year at room temperature up to +25°C, and for over two years at temperatures below +4°C.

[0037] The exosome concentrate obtained by the proposed method is used parenterally (in particular, by subcutaneous, intravenous, intraperitoneal, intratumoral, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intrafocal and intracranial injection or infusion), intraventricularly, orally, by inhalation, externally, rectally, nasally, buccally, vaginally or by means of an implantable reservoir for the purpose of regenerating superficial and deep damage and stimulating the processes of restoration of lost functions.

[0038] Exosome concentrate is also used as an additive for cosmetic products. Adding the concentrate helps slow down the skin aging process, stimulates the synthesis of the extracellular matrix, and improves the overall condition of the skin. It is also used for the treatment of alopecia and burns, including deep burns.

[0039] The stated technical result - increasing the efficiency of the exosome production process - is achieved due to the following factors:

[0040] - concentration of the culture medium enriched with exosomes includes centrifugation for 10 minutes at 2000 g, tangential filtration or ultrafiltration through hollow fiber filters with a pore size of 500 kDa with fivefold diafiltration of the concentrate and isopicric centrifugation of the diafiltered supernatant at 100,000 g and 4°C for 75 min, which allows for the removal of large cell particles and other inclusions, in particular particles larger than 100 nm and protein fractions less than 100 kDa, and thus increases the efficiency of the exosome production process;

[0041] - after the concentration stage, an additional five-fold diafiltration of the concentrate with a buffer is performed, which also helps to increase the efficiency of the exosome production process;

[0042] - the concentrate is dried at the mentioned temperatures from -40°C to +22°C, followed by holding for 100 to 610 minutes at an absolute pressure in the chamber no higher than 10 Pa, which, thanks to the sequential multi-stage process, also contributes to the final increase in the efficiency of the exosome production process.

[0043] The choice of parameter values ​​used to carry out the processes of purification of exosome preparations is determined by both the physicochemical properties of exosomes and the characteristics of stem cell biology:

[0044] - the frequency of changing the culture medium is selected in such a way that, on the one hand, the maximum concentration of exosomes in the culture fluid is achieved (no more than 5 times for static and no more than 10 times for flow methods, otherwise the costs of nutrient media and additives increase and, thus, the efficiency of obtaining exosomes decreases), and on the other hand, an influx of new nutrients is provided to maintain the metabolism of MSCs (no less than 4 times for static and no less than 8 times for flow methods, otherwise, metabolic disturbances occur), which ultimately increases the efficiency of the process of obtaining exosomes;

[0045] - the characteristics of the membranes used for tangential filtration of the exosome preparation were selected in such a way as to retain exosomes in the concentrate, but allow soluble proteins to pass into the filtrate, which helps to increase the efficiency of the exosome production process;

[0046] - the temperature values ​​(stepwise change of -40°C, -30°C, -10°C, 0°C, +10°C, +22°C), holding time intervals (from 100 to 610 minutes at the corresponding stages) and pressure (not exceeding 10 Pa) during lyophilization were selected in such a way as to achieve a minimum moisture concentration in the exosome preparation and at the same time avoid prolonged exposure of the lyophilisate to unfavorable conditions, thereby ultimately increasing the efficiency of the exosome production process; at temperatures above the specified temperature and / or with a reduction in the specified time interval (for each drying stage), the risk of retaining residual unwanted moisture in the exosome preparation increases, and at temperatures below the specified temperature and / or with an increase in the specified time interval (for each drying stage), the role of the factor of exposure of the lyophilisate to unfavorable conditions increases, which can reduce the efficiency of the exosome production process;

[0047] - the values ​​of temperature, time and centrifugal acceleration during centrifugation were optimized in order to pre-remove cellular debris that can hinder the tangential filtration process and reduce the productivity and efficiency of the system (at least 10 minutes and 2000 g during pre-centrifugation and at least 4°C, 75 minutes and 100,000 g during isopicric centrifugation), and at the same time to avoid excessive acceleration (no more than 10 minutes and 2000 g during pre-centrifugation and no more than 4°C, 75 minutes and 100,000 g during isopicric centrifugation), which can lead to undesirable sedimentation of exosomes and reduce the efficiency of the process of their production.

[0048] The given parameter values ​​were obtained empirically and are optimal in terms of increasing the efficiency of the processes of extraction, purification and concentration of exosomes from MMSCs.

[0049] The advantage of the proposed method is the high speed of all processes, which allows exosomes to be preserved intact (without damage). Furthermore, the elimination of a high-speed centrifugation step minimizes the formation of exosome aggregates.

[0050] An example of achieving a technical result.

[0051] MMSCs from donor adipose tissue with high proliferative activity of cells up to the 8th passage were selected as a source for obtaining exosomes.

[0052] To enrich the culture medium with exosomes, the indicated MMSCs were maintained for 48 hours in a medium based on DMEM / F12 with a reduced glucose concentration, human platelet lysate (hPL) and 5 mM L-alanyl-L-glutamine, while cell cultivation was carried out to 90% confluence of the culture monolayer.

[0053] Cultivation was performed statically. The culture medium was placed in cell factories. Once the monolayer reached 90% confluency, the culture medium was collected and replaced every 24 hours. The enriched culture medium was collected from the original culture five times. The resulting culture medium was stored for exosome isolation.

[0054] The resulting exosome-enriched culture medium was centrifuged for 10 min at 2000 g to remove large cell particles and other debris. The supernatant was then concentrated by ultrafiltration through 500 kDa hollow fiber filters. The concentrate was then diafiltered five times with an equal volume of DPBS phosphate buffer using the same hollow fiber cartridge. The concentrated supernatant was mixed with a sucrose solution and deuterium oxide and subjected to isopic centrifugation at 100,000 g and 4°C for 75 min. The resulting concentrate was then diafiltered five times with buffer.

[0055] To remove microvesicles and other remaining particles, a filter with pore sizes of 0.22 μm and 0.1 μm was used, after which the quality of the obtained exosomes was monitored using flow cytometry methods.

[0056] The exosome concentrate was preliminarily frozen to -75 °C in a freezer, after which it was dried in a lyophilization chamber: at the primary drying stage, the concentrate was cooled to -40 °C with subsequent holding for 100 minutes at absolute pressure in the 1Pa chamber, then cooled to -30 °C with subsequent holding for 610 minutes at absolute pressure in the 1Pa chamber, then cooled to -10 °C with subsequent holding for 315 minutes at absolute pressure in the 1OPa chamber, after which it was cooled to 0 °C with subsequent holding for 130 minutes at absolute pressure in the 1Pa chamber and heated to +10 °C with subsequent holding for 130 minutes at absolute pressure in the 1OPa chamber, and at the second stage, drying was performed at +22 °C for 150 minutes at absolute pressure in the 1OPa chamber.

[0057] The first portion of the concentrated exosomes produced in this manner was used unchanged for two weeks for cosmetic use. The second portion, frozen at -40°C, was stored for six months without loss of quality, which is required for successful cosmetic procedures, particularly for skin tightening on the face and neck. The third portion of the exosomes, lyophilized for long-term storage, was stored for 12 months at +25°C. In 2024,The applicant, in accordance with the present description, carried out laboratory tests: a culture medium enriched with exosomes obtained in various experiments from umbilical cord MMSCs, Wharton's jelly, placenta, adipose tissue, dental pulp and other tissues of human mesodermal origin was concentrated by centrifugation for 10 minutes at 2000 g, tangential filtration and ultrafiltration through hollow fiber filters with a pore size of 500 kDa with fivefold diafiltration of the concentrate, as well as isopicric centrifugation of the diafiltered supernatant at 100,000 g and 4 °C for 75 min, then an additional fivefold diafiltration of the concentrate with a buffer was carried out, and the concentrate was dried at temperatures from -40 °C to +22 °C, followed by holding for 100 to 610 minutes at an absolute pressure in the chamber not exceeding 10 Pa.

[0058] The efficiency of the process of obtaining, purifying and concentrating exosomes exceeded by an average of 12% the similar process used in the method described in the prototype and was assessed in terms of the following criteria:

[0059] - achieving the maximum concentration of exosomes in the culture fluid;

[0060] - achieving a minimum concentration of moisture in the exosome preparation;

[0061] - absence of cellular debris before centrifugation;

[0062] - preservation of exosomes in an intact form;

[0063] - minimization of the formation of exosome aggregates.

[0064] Thus, the test results confirmed the achievement of the declared technical result.

Claims

FORMULA 1. The method for extracting, purifying and concentrating exosomes from mesenchymal stromal cells includes culturing cells, collecting conditioned culture medium, depleting high-molecular proteins, isolating, purifying and concentrating exosomes. To enrich the culture medium with exosomes, multipotent mesenchymal stem cells (MMSCs) are grown in a medium with a reduced concentration of glucose, platelet lysate and 5 mM L-alanyl-L-glutamine. In this case, during static cell cultivation, the growth medium is placed in a culture dish. Upon reaching a level of at least 80% confluency of the culture monolayer, the culture medium is collected and replaced once every 24 hours. In flow cultivation, the cells are placed in a bioreactor on cell carriers. 72-96 hours after the start of cultivation, the culture medium is collected and replaced once every 24 hours. The exosome-enriched culture medium is concentrated.which includes successive stages of centrifugation, tangential filtration and isopycnic centrifugation, in the first stage the culture medium is centrifuged for 10 min at 2000 g to remove large particles of cells and other inclusions, in the tangential filtration stage the supernatant is concentrated through hollow fiber filters with a molecular weight cutoff value of 500 kDa, after which the concentrate is subjected to fivefold diafiltration with an equal volume of DPBS phosphate buffer using the same hollow fiber cartridge, in the isopycnic centrifugation stage the concentrated supernatant is mixed with a sucrose solution and deuterium oxide and subjected to isopycnic centrifugation at 100,000 g and 4 °C for 75 min, then the concentrate is subjected to an additional fivefold diafiltration with a buffer, after which the concentrate is filtered through filters with pore diameters of 0.22 μm and 0.1 μm.

2. The method according to item 1, characterized in that MMSC are contained in an alpha-MEM-based medium.

3. The method according to item 1, characterized in that the MMSCs are contained in a medium based on DMEM / F12.

4. The method according to claim 1, characterized in that the MMSCs are contained in a medium with human platelet lysate.

5. The method according to claim 1, characterized in that the MMSCs are contained in a medium with a mammalian platelet lysate.

6. The method according to paragraph 1, characterized in that the MMSC are kept in the said medium for at least 24 hours.

7. The method according to item 1, characterized in that the cell cultivation is carried out statically, while the enriched culture medium is collected from the initial culture 4 to 5 times.

8. The method according to item 1, characterized in that the cell cultivation is carried out using a flow method, while the enriched culture medium is collected from the initial culture 8 to 10 times.

9. The method according to item 1, characterized in that at the tangential filtration stage the supernatant is concentrated using tangential filtration.

10. The method according to item 1, characterized in that at the stage of tangential filtration the supernatant is concentrated using ultrafiltration.