WO2024177532A1

WO2024177532A1 - Assay for evaluating the secretory activity of model cells

Info

Publication number: WO2024177532A1
Application number: PCT/RU2023/050293
Authority: WO
Inventors: Всеволод Арсеньевич ТКАЧУК; Жанна Алексеевна АКОПЯН; Наталия Андреевна БАСАЛОВА; Вадим Юрьевич Балабаньян; Анастасия Юрьевна ЕФИМЕНКО; Армаис Альбертович КАМАЛОВ; Анна Олеговна МОНАКОВА; Дмитрий Александрович ОХОБОТОВ; Владимир Сергеевич ПОПОВ; Георгий Дмитриевич САГАРАДЗЕ; Елена Владимировна ТАРАСОВА
Priority date: 2023-02-20
Filing date: 2023-12-15
Publication date: 2024-08-29

Abstract

The invention relates to the field of medicine and pharmaceutics and can be used for evaluating, in vitro, the efficacy of therapeutic agents intended for restoring a stem cell niche, inter alia to treat fertility disorders resulting from dysfunction of the spermatogenic tissue of the testes. Claimed is an efficient method for evaluating the specific activity of drugs for stimulating tissue regeneration which are intended to restore damaged stem cell niches by controlling the secretory activity of the cells that participate in niche formation. The technical result of the claimed invention is the development of a cellular model (assay) that makes it possible to efficiently evaluate the effect of a test substance on the restoration of the secretory activity of the cells supporting stem cell niches. Efficacy is determined using model cells which are contained in the assay and are specific for the damaged stem cell niches, and by means of calibrating the assay by taking two measurements of a specific secretion factor reflecting the secretory activity of the model cells, said measurements being made at different times during the culture of the cells in order to obtain the "scale" needed to evaluate the specific activity of the therapeutic agent.

Description

TEST SYSTEM FOR ASSESSING SECRETORY ACTIVITY OF MODEL CELLS

Field of technology to which the invention relates

The invention relates to the field of medicine and pharmacy and can be used to evaluate the effectiveness in vitro of therapeutic agents aimed at restoring the stem cell niche, including for the treatment of fertility disorders caused by dysfunction of the spermatogenic tissue of the testes.

State of the art

To test the biological activity of drugs, test systems are required that would allow assessing their effect on the function of a specific cell type (specific activity), especially under conditions of damage to these cells. However, such test systems are currently lacking, and the main source of information on the effect of drugs on cell behavior is the use of appropriate animal models.

For example, to study the effect of drugs on the damaged niche of the spermatogonial stem cell, including Leydig cells, Sertoli cells, endothelial cells, macrophages, etc., they use modeling of cryptorchidism (https://doi.org/10.3109/00365599609181297, https://doi.Org/10.1016/j.biopha.2018.10.174), animals knocked out for certain genes (10.1038/nrg911), modeling of drug-induced damage (https://doi.org/10.29188/2222-8543-2021-14-4-95-101, https://doi.Org/10.1002/j.1939-4640.2005.tb02883.x) and other approaches. However, the use of animals to study drugs requires significant labor costs and financial resources. It is important to emphasize that the use of laboratory animals for primary screening of drug activity already leads to the use of a significant number of individuals, which often contradicts the rule of "three Rs" (Replacement, Reduction and Refinement) introduced by Russell and Birch in 1959 (Directive 2010/63/EU). At the same time, the use of cell test systems allows for a reduction in the number of animals used, as well as a reduction in labor costs and the amount of funding required. From the state of the art

(https://www. sciencedirect.com/science/article/abs/Dii/0041008X91901204, https://sci- hub.ruZhttps://doi.org/10.1016/0890-6238(93)90066-G, 10.1159/000460822) a method for assessing the toxic effect of drugs is known, which involves the use of Leydig cells, mainly isolated from the testicles of 3-4 week old pigs. After isolation, the cells are cultured in a specific growth medium (DMEM: HAM F12 medium (I: I) supplemented with transfertin (5 pg/ml), vitamin E (10 pg/ml), insulin (5 &/ml), and fetal calf serum (0.1%)). To conduct tests on the third day after isolation, the synthetic activity of Leydig cells is stimulated by adding human chorionic gonadotropin (hCG) to the culture medium. Simultaneously with the addition of gonadotropin, agents such as ketoconazole (0.02 to 100 /IM), aminoghttethimide (0.4 to 400 PM), spironolactone (0.25 to 250 PM), cycloheximide (0.35 to 350 PM), and chlorpromazine (0.3 to 300 pM), MATE inhibitor cimetidine (1 mmol/L) or pyrimethamine (10 pmol/L) and others are added to the medium for 4 hours, after which the medium is replaced and the secretory activity of Leydig cells is assessed by assessing the content of testosterone or other components (progesterone, cAMP) in the culture medium after 4 hours, 24, 48, 72 hours using enzyme immunoassay or radioimmunoassay methods. The inhibitory activity of the drug is assessed in comparison with the control group, to the cells of which toxic agents were not added. The disadvantages of such models include, first of all, the possibility of assessing only the inhibitory effect of the drug on secretory activity. In addition, to conduct the test, it is necessary to stimulate the secretory activity of Leydig cells by introducing an expensive drug, human chorionic gonadotropin, into the culture. This method also does not reproduce the physiological damage to the cells of the spermatogenic niche. Also, these works do not discuss the possibility of using frozen cell samples for the experiment, which can potentially increase the number of animals required for the experiments.

From the state of the art (DOI: 10.23868/202104006 IN VITRO TEST SYSTEM FOR SCREENING DRUGS WITH IL-17A INHIBITORY ACTIVITY N.K. Osina et al., 2021) a cellular An in vitro test system for screening drugs with IL-17A inhibitory activity based on the use of juvenile fibroblasts isolated from postoperative material. For this purpose, a culture of human juvenile dermal fibroblasts was isolated from skin fragments obtained from male donors under 10 years of age during circumcision surgery. The cells were cultured up to passage 20-24, after which they were treated with IL-17A (series 1) or a mixture of IL-17A with the IL-17A inhibitor netakimab (series 2). IL-17A-dependent secretion of inflammatory mediators IL-6, IL-8, MCP-1 in the conditioned medium was assessed using enzyme immunoassay 18 hours after adding the agents. The obtained results demonstrated the ability of juvenile human fibroblasts cultured for >20 passages to respond to IL-17A stimulation with increased production of inflammatory cytokines — IL-6, IL-8, MCP-1. Neutralization of IL-17A with the inhibitor netakimab resulted in a dose-dependent decrease in the secretion of inflammatory cytokines into the culture medium. However, fibroblasts are non-specific cells that are part of many tissues, which does not reflect the mechanism of action specifically in this niche. In addition, fibroblasts are not the main producers of proinflammatory cytokines. Thus, despite the fact that the work showed the results that IL-17A leads to an increase in the secretion of inflammatory mediators, this model does not reflect the damage and mechanism of action in vivo, therefore, it is not considered as possible for assessing the specific activity of drugs. The developed model is suitable only for assessing drugs with inhibitory activity. Additional disadvantages of the model include the use of fibroblasts at late stages of cultivation and the lack of results on fibroblasts of early passages, which significantly lengthens the time of the experiment.

To assess the immunomodulatory activity of the secretome of mesenchymal stromal/stem cells (MSCs), a test based on changes in the secretion of interleukin 10 (IL-10) by peripheral blood mononuclear cells has been proposed (Jiao, J., Milwid, J. M., Yarmush, M. L., & Parekkadan, B. (2010). A Mesenchymal Stem Cell Potency Assay. Suppression and Regulation of Immune Responses, 221-231. doi:10.1007/978-l-60761-869-0_16 ). To perform the test, freshly isolated mononuclear cells are added to the wells of a 96-well plate at a rate of 100,000 cells/well, to which, after an unspecified period of time, samples of the secretome in a volume of 50 μl of MSC are added for 17-18 hours. After this, lipopolysaccharides (LPS, 30 mg / mL, 50 μl / well) are added to the wells of the plate for 5 hours, after which the cell supernatant is collected, in which the amount of IL-10 is assessed using the ELISA method. If the IL-10 level is high, then this is assessed as “high potency of treatment”. The disadvantages of this method include the use of this test only for one potential drug - the secretome of MSCs. It should also be noted that there are inaccuracies in the description of the protocol, which can interfere with the correct execution of the test, as well as the lack of an accurate assessment of the degree of effectiveness of the drug.

The closest to the claimed model can be considered to be the Insulin Secretion Assay (https://doi.org/10.3390/phl4080809, 10.1021/acssensors.6b00433) for testing drugs that affect insulin secretion. Primary or linear (e.g. MIN6) pancreatic P cells are used to perform this test. The cells are seeded in a 96-well plate, then deprived for 1 hour and incubated for 1 hour with the test substance. The amount of secreted insulin is measured by enzyme immunoassay, luciferase test, or similar approaches. The activity of the drug is assessed by changing the secretion of insulin by cells. The disadvantages of this model include the lack of damage modeling, as well as the lack of an accurate “scale” for assessing the biological activity of the drug. In addition, there are no direct data describing the possibility of using frozen cell samples. Also, differentiated pancreatic cells cannot be classified as cells that support the function of the stem cell niche.

Thus, the technical problem solved by the claimed invention consists in the need to overcome the shortcomings inherent in analogues and the prototype by creating an effective test system and a method for assessing the specific activity of drugs that stimulate tissue regeneration and are aimed at restoring damaged niches of stem cells through the control of the secretory activity of cells involved in the formation of niches. Disclosure of invention

The technical result of the claimed invention is the development of a cell model (test system) that allows for the effective evaluation of the effect of the test substance on the restoration of the secretory activity of cells that support stem cell niches. The effectiveness is determined by the use of model cells included in the test system and specific for damaged stem cell niches, calibration of the test system by double measurement of the specific secretory factor reflecting the secretory activity of the model cells at different stages of their cultivation in order to obtain a "scale" that is necessary for evaluating the specific activity of the therapeutic agent.

The technical result is achieved by a test system for determining the specific activity of a therapeutic agent stimulating tissue regeneration by assessing the secretory activity of supporting cells of the stem cell niche, including model cells preliminarily isolated and planted on the surface of a culture container, or frozen immediately after isolation from the tissue, a culture medium supporting their growth and secretory activity, a medium for conditioning the model cells, a solution for washing the cells, a kit for measuring the specific secretory factor by the enzyme immunoassay method in an amount of at least 12 experimental wells and the number of control wells specified in the kit for conducting one analysis. In this case, the number of model cells is at least 120 thousand per ^cm2 of the culture container in the growth medium, taken in an amount of at least 100 μl per 25-50 thousand cells, preferably in an amount of 120-200 thousand per ^cm2 of the culture container.

The claimed test system is intended to evaluate the specific activity of therapeutic agents or to clarify the cellular mechanisms of action of therapeutic agents through the influence on the secretory activity of model cells.

The technical result is achieved by the method of using the test system according to paragraph 1, which consists in the fact that the model cells are preliminarily isolated from the tissue or frozen cells are used immediately after isolation, followed by defrosting, the cells are cultured until the number reaches at least 120 thousand per ^cm2 of the culture capacity in the growth medium taken in an amount of at least 100 μl per 25-50 thousand cells, the cells are washed, conditioning is carried out to accumulate the secretory factor, and then the level of secretion of a specific secretory factor by the cells is measured twice, while in parallel with the start of conditioning, a therapeutic agent is added to the test system to carry out the second measurement, followed by determining the change in the level of secretion of a specific secretory factor 48±24 hours after its addition. In this case, the model cells are planted in culture containers that promote attachment and spreading of the cells and maintain their viability in the nutrient growth medium. It is preferable to use 48-well and 96-well plates as culture containers, while 350±50 μl of the cell medium or the therapeutic agent being tested are applied to a 48-well plate and 150±50 μl to a 96-well plate. Leydig cells, Leydig cells or Sertoli cells, including immortalized cells, are used as model cells, and testosterone is measured in the Leydig cell medium. Testosterone is measured by enzyme immunoassay using a commercial kit that includes standard testosterone solutions, a wash buffer, a HRP conjugate concentrate solution, a TMB substrate, a solution for stopping the reaction, a 96-well plate with antibodies to testosterone, and the testosterone level 96-120 hours after the isolation of Leydig cells should be lower than the testosterone level 48-72 hours after the isolation of Leydig cells by at least 30%.

The therapeutic agent is diluted in a basal serum-free medium before use, and a therapeutic agent affecting the secretory activity of model cells is added to the cells in an amount of 100 μl per 25-50 thousand cells. Both drugs with a single active substance and multicomponent drugs can be used as therapeutic agents. The claimed test system can also be used to identify the key active component in the composition of a multicomponent therapeutic agent; inhibitor analysis is used for this purpose. In this case, changes in cell secretion are measured after adding an isolated component included in the therapeutic agent in different concentrations. Model cells are isolated from the testes of animals, mice or rats can be used as animals, with the assessment for rat cells being carried out 48 hours (control 1) and 96 hours (control 2) after isolation of model cells, and for mouse cells being carried out 72 hours (control 1) and 120 hours (control 2) after isolation of model cells. Model cells are used in culture immediately after isolation or after freezing and subsequent thawing, with freezing being carried out immediately after isolation. A culture medium supporting their growth and secretory activity is used for culturing model cells. It is preferable to use a solution containing the basic medium - DMEM-F12 with 1x antibiotic, 1x glutamate, 1x pyruvate, 2% fetal bovine serum and 1% ITS as a medium supporting the growth of Leydig cells and their secretory activity. A growth medium that maintains cell viability throughout the conditioning period and does not contain measurable factors in detectable quantities is used as a basal medium for conditioning model cells for the purpose of assessing their secretory activity. It is preferable to use DMEM with a glucose content of no more than 1 g/l, without phenol red, or another growth medium that maintains cell viability throughout the conditioning period and does not contain measurable factors in detectable quantities as a basal medium for Leydig cells for assessing secretory activity. Hanks' solution is used as a buffer solution for washing model cells from components of the growth medium, with the cells being washed by placing the cells in the said solution three to five times at a rate of 0.1-0.2 ml of solution per cm2 of cells for 5-10 minutes.

The specific activity of the therapeutic agent is determined by measuring the concentration of factors in the conditioned medium of model cells. To obtain the conditioned medium of model cells, it is necessary to collect the medium in the wells of KI, K2 and in the wells with the therapeutic agent, get rid of the debris by centrifugation at 300g for 5-10 minutes and collect the resulting supernatant for further analysis of the specific secretory factor. The concentration of factors secreted by target cells is measured by enzyme immunoassay, chromatographic or spectroscopic analysis, or by Western blotting methods. The conclusion about the specific activity of the therapeutic agent is made based on the comparison of the secretion level relative to the controls. In this case, if the therapeutic agent does not change the secretion level relative to control 2, it indicates the lack of effectiveness. In the case where the therapeutic agent reduces the secretion level relative to control 2, a conclusion is made about its inhibitory effect on secretion. If the therapeutic agent increases the secretion level relative to control 2, but does not reach the values of control 1, a conclusion is made about partial restoration of the secretory function. If the therapeutic agent reaches the values of control 1, a conclusion is made about complete restoration of the secretory function. In the case where the therapeutic agent leads to exceeding the values of control 1, a conclusion is made about hyperstimulating activity.

Brief description of the drawings

The invention is illustrated by the following drawings.

Figure 1 is a photomicrograph showing the morphology of rat testicular Leydig cells 24 and 96 hours after isolation. Leydig A cells 24 hours after isolation. Leydig B cells 96 hours after isolation.

Fig. 2 shows a graph illustrating the decrease in testosterone concentration during cultivation and the ability of the secretome of mesenchymal stromal cells to restore testosterone secretion relative to the negative control K K-1 (control 1): testosterone secretion level 48 hours after isolation; K-2 (control 2): secretion level 96 hours after isolation; secretome of MSCs - addition of the secretome of MSCs from human fat to Leydig cells at a concentration of 10x. N=5.

Fig. 3 shows a graph illustrating the relative concentration of testosterone in the medium of defrosted rat Leydig cells. K-1 (control 1): testosterone secretion level 48 hours after isolation; K-2 (control 2): secretion level 96 hours after isolation; MSC secretome - addition of MSC secretome from human fat to Leydig cells at a concentration of 10x. N=2.

Fig. 4 is a graph illustrating the relative concentration of testosterone in the mouse Leydig cell medium. K-1 (control 1): testosterone secretion level 48 hours after isolation; K-2 (control 2): secretion level 96 hours after isolation; MSC secretome - addition of the secretome of the stromal fraction of mouse testes without Leydig cells to mouse Leydig cells at a concentration of 10x. N=2.

Fig. 4 shows a graph illustrating the dependence of the intensity of testosterone secretion by rat Leydig cells on the concentration of the secretome of mesenchymal stromal cells.

Fig. 5 shows a graph illustrating the dependence of the intensity of testosterone secretion by Leydig cells on the concentration of VEGF in the secretome of mesenchymal stromal cells.

Fig. 6 shows a diagram illustrating the contribution of the MSC secretome and VEGF in its composition to the restoration of spermatogenesis.

Fig. 7. Doxorubicin administration results in damage to seminiferous tubules. A single administration of MSC secretome does not restore seminiferous tubule morphology (A), but there is a trend toward an increase in restored tubules 150 days after doxorubicin injection (B). Data are presented as median, 25th and 75th percentiles, minimum and maximum values are shown for each group. Total (C) and motile (D) sperm fractions 150 days after completion of doxorubicin injections. The number of animals with total counts exceeding the threshold is shown in yellow, and the number of animals with total counts below the threshold is shown in gray, p > 0.05 between the doxo + MSC secretome group and the doxo + MSC secretome + VEGF AT group. Fisher's exact test was used. N = 8 for the doxo + MSC secretome group, n = 5 for the doxo group, N = 2 for the doxo + MSC secretome + VEGF AT group, and N = 3 doxo + MSC secretome + isotypic VEGF AT. (E) Micrographs of mouse testis sections, hematoxylin and eosin staining. 1 - doxo, 2 - doxo + MSC secretome, 3 - doxo + MSC secretome + VEGF AT.

Implementation of the invention

The proposed test system is based on the use of specialized model cells, previously isolated from tissues whose effect on regeneration by a therapeutic agent must be assessed, or frozen immediately after isolation with subsequent defrosting and cultivation in a medium that supports their growth and secretory activity, until achieving a quantity sufficient for detection of a specific secretory factor.

Unlike the analogue, the efficiency and physiological correspondence of the model to damage in vivo is ensured by using cells whose secretory activity (secretion of a marker specific factor involved in maintaining the functioning of the niche) decreases during cultivation. A decrease in secretory activity during cultivation allows this test system to be considered relevant for displaying “physiological damage” to niche cells, which also results in a decrease in the secretory activity of niche cells.

Unlike the analogue, in which terminally differentiated organ cells whose function is aimed at maintaining the entire organism are used as a model object, this invention proposes to use cells whose paracrine activity is aimed primarily at maintaining the normal functioning of the stem cell niche. As such cells, it is permissible to use tissue-specific niche cells described in the literature, such as Leydig cells, Sertoli cells, including immortalized cells, or others that meet the specified properties. An individual culture medium is selected for the isolated cells. Thus, in the closest analogue for culturing pancreatic b-cells, a DMEM medium with 15% fetal bovine serum, 1% penicillin/streptomycin antibiotic, 0.1% b-mercaptoethanol and 0.5% G418 is used. For Leydig cells, DMEM-F12 medium with 1x antibiotic, 1x glutamate, 1x pyruvate, 2% fetal bovine serum and 1% ITS was used.

Unlike the analogue, the accuracy of the drug efficiency assessment is achieved due to the presence of two control groups in the model, unlike the prototype, which used one control group in which the baseline level of secretion of a specific secretory factor was measured. In the proposed test system, the first control group is model cells, the secretory activity of which is assessed immediately after the start of testing during the first 48-72 hours of cultivation, which allows us to assess the physiological behavior of cells close to the norm (control group 1, “norm”). The test substance (therapeutic agent) is added to the cells at the time of cultivation when their secretory activity is reduced, which allows us to evaluate the restoration of the functional activity of the cells. The second control group consists of cells to which the test substance is not added, which makes it possible to evaluate the secretory activity of the cell in a situation of niche damage in vitro (control group 2, “damage”). The therapeutic agent diluted in a basal serum-free medium is applied to the remaining experimental wells. In this case, the method for obtaining the secretome of model cells includes washing the cells with a buffer solution, conditioning in a serum-free medium with or without the addition of a therapeutic agent, collecting the culture medium containing the secretion products of the model cells, and cleaning from cell residues. Specific secretory factors in the secretome of model cells are measured by enzyme-linked immunosorbent assay (10.1038/jid.2013.287), chromatographic analysis, spectroscopic analysis (10.1016/j .abb.2012.11.007) or Western blot (10.1155/2014/361590).

Thus, unlike the prototype, which does not provide a method for accurately assessing the effectiveness of the drug, but only a comparative assessment of the secreted marker substance was carried out (more than in the control - less than in the control), using the proposed test system, it is possible to more accurately assess a drug that does not have cytotoxicity by the level of effectiveness: suppression of secretory activity (statistically below the level of the "damage" group) no effect (statistically no different from the level of the "damage" group) increase in secretory activity of cells (statistically higher than the "damage" group, but less than the "norm" group) restoration of secretory activity of cells (statistically no different from the "norm" group) stimulation of hypersecretion of cells (statistically higher than "norm") The technical advantage of the test system is the proven ability to work on frozen cell samples. Unlike the prototype, which does not specify the possibility of working on frozen and thawed cell samples, the possibility of working on frozen cell samples was shown, which does not lead to a decrease in the possibility of obtaining reliable results on models, which potentially reduces the amount of donor tissue or the number of animals required for the work.

Another advantage of the model is the ability to evaluate the mandatory quality indicator of biological products - specific activity. The model can be used to study in vitro the mechanisms of action of therapeutic agents. As therapeutic agents, the proposed test system can be used to study drugs, including high-tech drugs, herbal preparations, small molecules, dietary supplements, drugs based on somatic cells and other agents whose supposed mechanism of action is stimulation of tissue regeneration by influencing the secretory activity of cells in the stem cell niches. In addition, for therapeutic agents that are a multicomponent mixture of active substances, this test system can be used to study the contribution of each component. Determination of key factors will allow studying the mechanisms of action, as well as selecting a surrogate factor for indirect determination of specific activity.

A more detailed description of the claimed invention is provided below. The present invention may be subject to various changes and modifications that will be clear to a specialist based on reading this description. Such changes do not limit the scope of the claims.

All reagents used were commercially available and all procedures, unless otherwise stated, were performed at room or ambient temperature, i.e., in the range of 18 to 25°C.

Example 1. Obtaining human adipose tissue MSCs.

MSCs are isolated from the subcutaneous fat deposits of healthy donors of both sexes. The cells are isolated from material obtained during minor surgery under local anesthesia or during planned surgeries. The surgical material (15 g of adipose tissue taken from the umbilical region or other area of subcutaneous fat localization) is placed in a disposable tube with HBSS buffer (HyClone) containing a 5-fold concentration of antibiotic 500 units/ml (HyClone). Under sterile conditions of a culture box, the biopsy is fragmented to a homogeneous mass using sterile instruments, after which it is subjected to enzymatic treatment in a solution containing AdvanceSTEM™ medium (HyClone) /500 units/ml antibiotic (HyClone), 200 U/ml collagenase type I and dispase (40 U/ml) (Worthington Biochemical) (the ratio of enzymes to fat should be 1:1:1) at 37°C for 30-45 minutes with constant stirring. After incubation, an equal volume of medium with serum 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 200g, after which the supernatant is completely removed. The cell pellet is treated with erythrocyte lysis buffer (BD) until the solution turns red, after which it is centrifuged for 5 min at 200g. The supernatant is completely removed, and the pelleted cells are resuspended in the MSC growth medium. For culturing mesenchymal cells, the AdvanceSTEM™ support expansion and maintenance of undifferentiated human MSCs (HyClone) medium is used in combination with the addition of a 10% solution of AdvanceSTEM™ Stem Cell Growth Supplement (HyClone) and 100 U/ml antibiotic (HyClone). This medium was developed by the manufacturer (HyClone) for culturing 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 cultivated in a growth medium in an incubator at 37°C and 5% CO2 concentration. When the AT MSC monolayer reaches 80%, the cells are passaged. For this purpose, the cells are washed three times with a Versen solution before being removed from the surface of the culture plastic. Then 1 ml of the HyQTase cell dissociation reagent (HyClone) is added to each dish. When the cells acquire a round shape and detach from the substrate, 9 ml of the growth medium is added to each culture container and the cell suspension is intensively pipetted. Then the cells are seeded in a ratio of 1:4.

Example 2. Obtaining the secretome of human adipose tissue MSCs.

To obtain the secretome of MSCs, 4-5 passage adipose tissue that had reached 80% confluency were washed three times with Hanks' solution with calcium and magnesium salts (PanEco, Russia). DMEM containing low glucose, GlutaMAX™ Supplement, pyruvate (Gibco, USA), hereinafter referred to as DMEM, or a medium developed to support the growth of MSCs, NutriStem XF Medium (Biological Industries, Israel) (hereinafter referred to as NutriStem), containing 100 U/ml penicillin/streptomycin were added to the dishes. The cells were cultured for 3-14 days, after which the conditioned medium, containing components of the MSC secretome, were collected and purified from cellular debris by centrifugation for 10 min at 300 g.

Example 3. Concentration of MSC secretome

For concentration, centrifuge filters "Apicon" (#Z740186, Merck, Germany) with a pore size designed to cut off molecules smaller than 10 kDa were used. New filters were cleaned and "clogged" with protein by concentrating 5 ml of sterile 1% fetal bovine serum (Gibco, USA) in a phosphate-buffered saline solution (PanEco, Russia) to a minimum residual volume. Then the filter was washed 3 times with 70% ethanol by passing the full volume of alcohol through the filters, and 3 times with a mixture of 2% penicillin-streptomycin solution in phosphate-buffered saline. Then the MSC secretome was placed in the filters and spun on the filters at a speed of 3000 g until the concentrate volume was reduced by 25 times compared to the initial one. The resulting concentrate was collected for further use. After work, the filters were washed with a solution of phosphate buffered saline containing 100 U/ml penicillin/streptomycin. The filters were stored in the same solution at a temperature of +4C until the next use.

Example 4. Method for assessing the concentration of VEGF, which indirectly stimulates spermatogenesis, in the secretome of mesenchymal stromal cells of human adipose tissue.

To determine the concentration of VEGF using the ELISA method, a commercial kit from R&D Systems (Human VEGF Quantikine ELISA Kit, cat#DVE00) or similar is used.

Using a standardized VEGF solution, prepare calibration samples of known concentration so that sample #1 contains 500 pg/ml VEGF, each subsequent sample contains 2 times less VEGF. Sample #0 should contain only the Sample Diluent solution. The test samples in the amount of 100 μl, as well as standardized samples of the VEGF 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 mouse monoclonal antibodies against human VEGF are added to each well. After 3 hours of incubation, the plates are washed as described above. 100 μl of the streptavidin-horseradish peroxidase complex solution are applied to each well. After 1 hour, the plate is thoroughly washed and 100 ml of the solution is added to each well. µl of the TMB/E substrate solution supplied with the kit. The plate is incubated at room temperature for 15 minutes, then 100 µl of stop buffer is added to each well, and the reaction is assessed 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. The device is operated in accordance with the instructions.

Example 6. Method for stimulating restoration of spermatogenesis by introducing human MSC secretome into a model of chemotherapeutic damage by doxorubicin.

The damaging agent doxorubicin was administered to young sexually mature male Black mice intraperitoneally in the form of a solution prepared from lyophilisate to create a research model. Animal studies were conducted in accordance with EU Directive 201/63/EU and in agreement with the Bioethics Committee of Lomonosov Moscow State University (application number 90-zh). Animals were taken from the nursery of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences.

The experiments were performed on 45 sexually mature young male Black mice of standard weight characteristics. All experiments were performed in accordance with the requirements of the Helsinki Accords on humane treatment of animals. These animals were injected intraperitoneally with doxorubicin as a solution prepared from the lyophilisate. Doxorubicin was injected intraperitoneally once every 2 days at a dose of 1 mg/kg to achieve a total dose of 10 mg/kg. After the completion of the doxorubicin course, the animals were injected with MSC secretion under the protein coat of the testicle. Before the administration, MSC secretion in a 50-fold concentration was mixed with 2% porcine collagen gel (McMedi, Moscow, Russia) in a volume ratio of 1:4 to achieve a 10-fold concentration for injections.

The severity of damage developed in the testicles after a course of doxorubicin and the effectiveness of MSC secretome therapy were assessed after 1 and 5 months. For this purpose, the testicles with appendages were removed and testicular tissue samples were taken for histological examination, which was carried out using a standard technique with staining of histological sections with hematoxylin and eosin. The severity of spermatogenesis impairment was assessed by determining the proportion of normal, recovering and damaged seminiferous tubules. The appendages of the testicle were homogenized in a 5% glucose solution in a tissue/solution ratio of 1:10 s subsequent determination of the number of actively motile and immobile spermatozoa in the supernatant fluid in the Goryaev chamber using the standard spermogram analysis method.

The number of tubules in the experimental group does not change significantly compared to the group of untreated animals.

The results of the study of the number of spermatozoa and their motility showed that in the group receiving the injection of the MSC secretome, 5 months after the administration, the total number of spermatozoa was 6.2512*10 ^L 6, and the number of motile spermatozoa was 0.12316875*10 ^L 6, which significantly exceeds these indicators in animals with damage without therapy: 0.412* 10 ^L 6 and 0.000687* 10 ^L 6, respectively.

Example 7. Method for studying the contribution of VEGF to spermatogenesis restoration in a model of chemotherapeutic damage by doxorubicin.

To study the contribution of VEGF to the restoration of spermatogenesis, we modeled spermatogenesis damage by doxorubicin using the method described in Example 6. Groups of intact mice, mice with spermatogenesis damage, mice with MSC secretome therapy, mice that received an injection of MSC secretome with VEGF antibodies at a concentration of 0.1 μg/ml (ab36424, Abeam, USA), and mice that received an injection of MSC secretome with isotype control antibodies at a volume concentration of 1:10,000 (910801, Biolegend, USA) were formed. Before administration, MSC secretome or the MSC secretome product with antibodies at a 50-fold concentration was mixed with 2% porcine collagen gel (McMedi, Moscow, Russia) at a volume ratio of 1:4 to achieve a 10-fold concentration for injections.

The severity of the effect of VEGF blocking by antibodies was assessed after 1 and 5 months. For this purpose, the testicles with appendages were removed and testicular tissue samples were taken for histological examination, which was carried out using a standard technique with staining of histological sections with hematoxylin and eosin. The severity of spermatogenesis impairment was assessed by determining the proportion of normal, recovering and damaged seminiferous tubules. The epididymis were homogenized in a 5% glucose solution in a tissue/solution ratio of 1:10, followed by determination of the amount of active motile and immobile spermatozoa in the Goryaev chamber using the standard spermogram analysis method.

The number of tubules in the experimental group does not change significantly compared to the group of untreated animals and animals with MSC secretome therapy.

The results of the study of the number of spermatozoa and their motility showed that in the group receiving the injection of the MSC secretome, 5 months after the injection, the total number of spermatozoa is 6.2512*10 ^L 6, and the number of motile spermatozoa is 0.12316875*10 ^L 6, while in the group receiving the injection of the MSC secretome with antibodies to VEGF, 5 months after the injection, the total number of spermatozoa is 0.25*10 ^L 6, and the number of motile spermatozoa is 0, which indicates a significant decrease in the effect of spermatogenesis restoration when blocking VEGF in the MSC secretome.

Example 8. Method for obtaining Leydig cells from rat testicles.

Eight-month-old male Wistar rats were anesthetized in a desiccator using CO2 and a flow rate that displaced at least 20% of the chamber volume per minute. After breathing ceased and there was no response to footpad compression, the rats were euthanized by cervical dislocation. Rat testes were isolated in Hanks' solution (PanEco, Russia) with 5% penicillin/streptomycin antibiotic (Gibco, Thermo Fisher Scientific, USA) or similar, transferred to 100-mm culture dishes, the tunica albuginea removed, and then washed with Hanks' solution 3 times with 5 ml each. The testes were then transferred to 7-10 ml of DMEM medium (Gibco, Thermo Fisher Scientific, USA) with 2.5 mg/ml trypsin and 10 μg/ml DNase for 4-10 min at 34-35°C, shaking periodically. As a result, the interstitial cells are separated from the seminiferous tubules and are in suspension in DMEM medium. To inactivate the enzymes, 15-20 ml of DMEM + 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) were added, gently mixed by inverting the tube, and then left for 5-20 min until the tubules settled. The liquid fraction of the supernatant with interstitial cells was transferred to a new tube. The procedure of mixing the tubules and removing the supernatant was repeated 5 times. The resulting cell suspension was centrifuged at 300*g and resuspended in DMEM:F12 with 1x penicillin/streptomycin, lx Glutamax-I (Gibco, Thermo Fisher Scientific, USA), lx pyruvate (Gibco, Thermo Fisher Scientific, USA), 2% fetal bovine serum and 1x insulin-transferrin-selenium supplement (ITS, PanEco, Russia). The Leydig cell suspension was then passed through a 100 μm filter. Leydig cells were seeded in 48-well plates and placed in a CO2 incubator at 35°C.

Example 9. Method for reproducing the model for assessing the stimulation of secretory activity of Leydig cells.

The fraction enriched with Leydig cells was seeded in a 48-well plate in an amount of 100 to 200 thousand per 1 cm2. Leydig cells were seeded in a 48-well plate in DMEM:F12 medium with the addition of 1x penicillin/streptomycin, I ^x Glutamax-I, I ^x pyruvate, 2% fetal bovine serum and I ^x insulin-transferrin-selenium (ITS). The volume of the medium was 350 μl per well. Then the plates with Leydig cells were placed in a CO2 incubator at 35 °C. The next day, all wells were washed three times with 500 μl of Hanks' solution (PanEco, Russia) and fresh culture medium was added. Day 2 control wells were supplemented with phenol red-free, low glucose DMEM:F12 containing I ^x penicillin/streptomycin, I ^x glutamax-I, and I ^x pyruvate. The next day, the wells were washed three times with 500 μl of Hanks' solution and either MSC secretome or phenol red-free, low glucose DMEM:F12 containing I ^x penicillin/streptomycin, I ^x glutamax-I, and I ^x pyruvate were added. Leydig cell secretome was collected from day 2 control wells and centrifuged at 300 ^x g for 10 min. The resulting supernatant was placed at -80°C. After two days, Leydig cell secretome was collected from the remaining wells and centrifuged at 300 ^x g for 10 min. The resulting supernatant was placed at -80°C.

Example 9. Method of using frozen Leydig cells.

Since it is not always possible to use Leydig cells immediately for the experiment (for example, time is needed to obtain the MSC secretome), a test system was tested on frozen Leydig cells to check their viability and the ability to respond to stimulation with the MSC secretome after freezing. In addition, the ability to freeze a large number of cells will allow using a smaller number of animals, which is appropriate from an ethical point of view.

The Leydig cell suspension obtained in Example 8 was centrifuged at 300g for 10 minutes, and the supernatant was removed. The resulting sediment was resuspended in the required amount of a mixture containing 10% DMSO and 90% fetal bovine serum until a concentration of 1 million cells in 1 ml of the mixture was achieved. The resulting suspension was then frozen in aliquots in 1 ml cryovials at - 80C. The aliquots were stored in liquid nitrogen.

If necessary, aliquots were defrosted, diluted with two volumes of DMEM medium, centrifuged at 300 g for 5 minutes, and then diluted with the required amount of culture medium. Leydig cells were seeded in 48-well plates and placed in a CO2 incubator at 35°C.

Leydig cells have been shown to survive freezing and retain the ability to respond to the signals studied.

Example 10. Method for obtaining Leydig cells from mouse testicles.

Three-month-old male Black mice were anesthetized in a desiccator using CO2 at a flow rate that displaced at least 20% of the chamber volume per minute. After breathing ceased and there was no response to footpad compression, the rats were euthanized by cervical dislocation. Rat testes were isolated in Hanks' solution (PanEco, Russia) with 5% penicillin/streptomycin antibiotic (Gibco, Thermo Fisher Scientific, USA) or similar, transferred to 100-mm culture dishes, the tunica albuginea removed, and then washed with Hanks' solution 3 times with 5 ml each. The testes were then transferred to 7-10 ml of DMEM medium (Gibco, Thermo Fisher Scientific, USA) with 2.5 mg/ml trypsin and 10 μg/ml DNase for 4-10 min at 34-35°C, shaking periodically. As a result, the interstitial cells are separated from the seminiferous tubules and are in suspension in the DMEM medium. To inactivate the enzymes, 15-20 ml of DMEM + 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) were added, gently mixed by inverting the tube, and then left for 5-20 min until the tubules settled. The liquid fraction of the supernatant with interstitial cells was transferred to a new tube. The procedure of mixing the tubules and removing the supernatant fluid was repeated 5 times. The resulting cell suspension was centrifuged at 300*g and resuspended in DMEM:F12 with 1x penicillin/streptomycin, lx Glutamax-I (Gibco, Thermo Fisher Scientific, USA), lx pyruvate (Gibco, Thermo Fisher Scientific, USA), 2% fetal bovine serum and 1x insulin-transferrin-selenium supplement (ITS, PanEco, Russia). Then the Leydig cell suspension was passed through a 100 μm filter and diluted in 1 ml of DMEM medium. The resulting volume was applied to a Percoll gradient and centrifuged at 800g for 30 minutes and 200g for 10 minutes. The Percoll fraction, containing Leydig cells were collected, diluted in a volume of DMEM medium, mixed and centrifuged at 300g for 10 minutes. The supernatant was collected, and the pellet with Leydig cells was resuspended in the required solution of the culture medium. Leydig cells were planted in 48-well plates and placed in an incubator with CO2 at 35°C.

Claims

CLAUSE OF THE INVENTION

1. A test system for determining the specific activity of a therapeutic agent that stimulates tissue regeneration by assessing the secretory activity of model cells, including model cells that have been previously isolated and planted on the surface of a culture container, or frozen immediately after isolation from the tissue, a culture medium that supports their growth and secretory activity, a medium for conditioning the model cells, a solution for washing the cells, a kit for measuring a specific secretory factor using an enzyme immunoassay in a quantity of at least 12 experimental wells.

2. The test system according to item 1, characterized in that it is used to evaluate the specific activity of therapeutic agents, or to clarify the cellular mechanisms of action of therapeutic agents through the influence on the secretory activity of model cells, or to identify the key active component in the composition of a multicomponent therapeutic agent.

3. A method for assessing secretory activity using the test system according to claim 1, characterized in that the model cells are preliminarily isolated from the tissue or frozen cells are used immediately after isolation with subsequent defrosting, the cells are cultured until they reach a quantity of at least 120 thousand per ^cm2 of the culture capacity in a growth medium taken in an amount of at least 100 μl per 25-50 thousand cells, the cells are washed, conditioning is carried out to accumulate the secretory factor, and then the level of secretion of a specific secretory factor by the cells is measured twice, while in parallel with the start of conditioning, a therapeutic agent is added to the test system according to claim 1 to carry out the second measurement, followed by determining the change in the level of secretion of a specific secretory factor 48±24 hours after its addition.

4. The method according to item 3, characterized in that the therapeutic agent is diluted in a basal serum-free medium.

5. The method according to paragraph 3, characterized in that a therapeutic agent affecting the secretory activity of model cells is added to the cells in an amount of 100 μl per 25-50 thousand cells.

6. The method according to item 3, characterized in that the model cells are planted in culture containers that facilitate the attachment and spreading of cells and maintain their viability in the growth nutrient medium.

7. The method according to I.6, characterized in that 48-well and 96-well plates are used as culture containers.

8. The method according to I.7, characterized in that 350±50 µl of the cell medium or test agent is applied to a 48-well plate and 15(H-50 µl to a 96-well plate.

9. The method according to paragraph 3, characterized in that Leydig cells or Sertoli cells, including immortalized cells, are used as model cells.

10. The method according to paragraph 3, characterized in that the model cells are isolated from the testes of animals.

11. The method according to claim 10, characterized in that mice or rats can be used as animals, wherein the first measurement of the level of secretion of a specific secretory factor by cells is carried out after 48 hours for rat cells and after 72 hours for mouse cells (control 1), and the second measurement is carried out after 96 hours for rat cells and after 120 hours for mouse cells (control 2) after the start of testing.

12. The method according to item 11, characterized in that the model cells are used in culture immediately after isolation or after freezing and subsequent thawing, with freezing being performed immediately after isolation.

13. The method according to item 3, characterized in that testosterone is measured in the Leydig cell medium.

14. The method according to item 13, characterized in that testosterone is measured by the enzyme immunoassay method using a commercial kit, which includes standard testosterone solutions, a washing buffer, a solution of HRP conjugate concentrate, a TMB substrate, a solution for stopping the reaction, and a 96-well plate with antibodies to testosterone.

15. The method according to item 13, characterized in that the testosterone level 96-120 hours after the isolation of Leydig cells should be lower than the testosterone level 48-72 hours after the isolation of Leydig cells by at least 30%.

16. The method according to item 3, characterized in that a culture medium that supports their growth and secretory activity is used to cultivate model cells.

17. The method according to claim 16, characterized in that the medium supporting the growth of Leydig cells and their secretory activity is a solution containing a basic medium - DMEM-F12 with 1x antibiotic, 1x glutamate, 1x pyruvate, 2% fetal bovine serum and 1% ITS.

18. The method according to item 3, characterized in that a growth medium that maintains the viability of the cells throughout the entire conditioning period and does not contain measurable factors in detectable quantities is used as a medium for conditioning the model cells for the purpose of assessing their secretory activity.

19. The method according to claim 18, characterized in that DMEM with a glucose content of no more than 1 g/l, without phenol red, or another growth medium that maintains the viability of the cells throughout the entire conditioning period and does not contain measurable factors in detectable quantities is used as a conditioning medium for Leydig cells for assessing secretory activity.

20. The method according to item 3, characterized in that the conditioning of model cells for the accumulation of the secreted factor is carried out for 24-48 hours.

21. The method according to item 3, characterized in that Hanks' solution is used as a buffer solution for washing model cells from components of the growth medium, wherein the cells are washed by placing the cells in the said solution three to five times at a rate of 0.1-0.2 ml of solution per cm2 of cells for 5-10 minutes.

22. The method according to paragraph 3, characterized in that the concentration of a specific secretory factor is measured in the conditioned medium of model cells.

23. The method according to i. 28, characterized in that in order to obtain the conditioned medium of the model cells, it is necessary to collect the medium in the wells KI, K2 and in the wells with the added therapeutic agent, remove the cellular debris by centrifugation at 200-300g for 5-10 minutes and collect the resulting supernatant for further analysis of the specific secretory factor.

24. The method according to item 22, characterized in that the measurement of the concentration of factors secreted by target cells is carried out by an enzyme-linked immunosorbent assay, or a chromatographic assay, or a spectroscopic assay, or a Western blotting method.

25. The method according to paragraph 3, characterized in that conclusions about the specific activity of the therapeutic agent are made on the basis of a comparison of the level of secretion relative to controls.

26. The method according to claim 25, characterized in that if the therapeutic agent does not change the level of secretion relative to control 2, a conclusion is made about the lack of effectiveness.

27. The method according to item 25, characterized in that if the therapeutic agent reduces the level of secretion relative to control 2, then a conclusion is made about its inhibitory effect on secretion.

28. The method according to claim 25, characterized in that if the therapeutic agent increases the level of secretion relative to control 2, but does not reach the values of control 1, then a conclusion is made about partial restoration of the secretory function.

29. The method according to item 25, characterized by the fact that if the value of the therapeutic agent reaches the control value 1, then a conclusion is made about the complete restoration of the secretory function.

30. The method according to item 25, characterized in that if the therapeutic agent leads to an excess of control values 1, then a conclusion is made about hyperstimulating activity.

31. The method according to paragraph 3, characterized in that both drugs with one active substance and multicomponent drugs can be used as therapeutic agents.