RU2391990C1 - Composition for stimulation of cells' growth and regeneration (versions) and method of composition's manufacture (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of invention belongs to versions of pharmacologic composition designed for stimulation of cells' growth and regeneration and to methods of composition's manufacture. Composition contains lysate of peripheral blood leukocytes, stem and progenitor cells obtained from animal which preliminary undergone biopsy of those inner organs or skin, in which growth and regeneration must be stimulated. Another version of composition contains lysate of peripheral blood leucocites and/or stem and/or progenitor cells obtained from animal which preliminary undergone biopsy of those inner organs or skin, in which growth and regeneration must be stimulated. Peripheral blood leukocytes and/or stem and/or progenitor cells was preliminary cultivated with obtained by biopsy destroyed cells of inner organs or skin in which growth and regeneration must be stimulated. Versions of manufacture method include cells' acquisition followed by destruction.

EFFECT: invention provides improvement of regeneration process in affected organ.

14 cl, 2 tbl, 6 dwg, 7 ex

Description

The invention relates to medicine and veterinary medicine and can be used to treat diseases requiring stimulation of the regeneration of cells of organs and tissues in chronic inflammatory and degenerative processes of internal organs or skin integuments.

The prerequisites for the creation of this invention was the following.

Despite the continuous improvement of medical technologies, the treatment of patients with chronic diseases of the internal organs to this day remains an urgent task. Chronic inflammatory and / or degenerative process of internal organs, despite the efforts of doctors, as a rule, ends with decompensation and death of the patient. One of the factors significantly affecting the course of chronic diseases is the rate of regeneration in damaged organs. Chronic inflammation and hypoxia, impaired trophism of the damaged organ lead to inhibition of its function and the development of chronic stress, accompanied by the formation of functional insufficiency of immunoregulatory cells of the body.

The basis for this assumption was the work of G. Selye on the involution of the organs of the immune system under conditions of chronic stress, which is accompanied by a decrease in the functional activity of phagocytic blood cells [Hasper D., 1998; Levine T.V. et al., 2002] and a decrease in the morphogenetic activity of lymphoid cells, which are “carriers of regenerative information” in the body [Babaeva A.G. et al., 1982]. The severity of the involutional process in the bone marrow as the central organ of immunogenesis for chronic stress is determined by the total biological dose of the stress-damaging factor (severity and duration of the disease), as well as genetic (congenital) and phenotypic (acquired) reserves of bone marrow cell resistance (BMC) (age, physical training, etc.)

So, according to the literature, up to 70% of patients with chronic heart failure have signs of secondary immunodeficiency of varying severity, which indicates a pre-existing violation of proliferative, migration and information activity of both hematopoietic bone marrow cells and blood cells.

As a chronic disease develops, dysregulation of the immune system progresses, especially among those parts of it that are responsible for the regeneration of damaged organs. As a result, treatment of chronic processes becomes an insoluble task, because The body itself, creating an immune imbalance, seeks to weaken the ongoing activation therapy in order to slow down the process of wear and death of hyperfunctioning structures of vitally damaged organs.

One of the methods to increase the regeneration of a damaged organ is transplantation of bone marrow cells containing lymphocytes, stem and progenitor cells.

So, there is a known method for the treatment of chronic heart failure (Yu.L. Shevchenko. “Cell technology in cardiology,” Vestnik RAMS, 2003, No. 11, pp. 6-10), based on transplantation of fetal cells obtained from abortive material. The disadvantages of this method are:

1. The ban on the use of fetal material for transplantation to humans.

2. The risk of infection of the recipient with infections that may be contained in the graft.

3. The effectiveness of allogeneic material transplantation is reduced due to immune rejection.

The effectiveness of autologous bone marrow transplantation largely depends on the state of the immune system, which largely determines the morphogenetic and regenerative abilities of the body.

At the same time, another approach is possible using compositions designed to increase the regeneration of cells of organs and tissues.

Numerous studies have proved the participation of peripheral blood lymphocytes and bone marrow cells in the development of regenerative processes after acute damage or resection of organs. At least 3 basic patterns have been identified. Firstly, regeneration is accompanied by a change in the functional activity of lymphocytes and bone marrow cells. Secondly, in the process of development of the regeneration process, the population composition of T-lymphocytes and CMCs changes, which ensures the beginning of regeneration, development and completion of the process of restoration of the damaged organ. Thirdly, the functional activity of the cells of the immune system depends on which organ is damaged and how much damage [A.G. Babayeva, 1999], and in the process of acute damage, lymphocyte clones are formed in the body that carry “regenerative information” about that organ, which was damaged [Babaeva A.G., 1984; Babaeva A.G., Shubnikova, 1979; Babaeva A.G. et al., 1982; Shilova L.Ya. et al., 1982].

Moreover, the regeneration process in the body is accompanied by close cooperation of the nervous, endocrine and immune systems. The humoral growth and regulatory factors that stand out during the regeneration process provide a sequence of regeneration and proliferation phases [V.P. Shakhov, 1996; Goldberg E.D. et al., 1997; Digay A.M. et al., 1992; Digay A.M. et al., 1989; Digay A.M. et al., 1998].

An analogue of this approach is a method of treating acute and chronic dysfunction of internal organs with peptides derived from animal (pig) splenocytes [A.A. Makarov et al., 1993].

The disadvantages of this method are:

1. Only spleen cells are used to prepare splenoperfusate. However, it has been shown in the literature that with the development of acute stress damage, the functional, and most importantly, secretory activity of lymphoid cells in different organs differs [M.S. Blyakher et al., 1996; Yu.I. Zimin, 1974].

2. The cell is taken from the animal in a state of functional rest. As shown, acute stress causes a systemic restructuring of the functional activity of all regulatory (nervous, endocrine, immune) systems of the body. So, the introduction of serum obtained from patients with acute heart damage in experimental animals causes an increase in the proliferative activity of blood cells, bone marrow [Ya. G. Uzhansky et al., 1974]. In one of the works on the treatment of experimental diabetes mellitus, it was shown that 3 days after a partial pancreatic resection, an extract can be obtained from the remaining part of the organ, the introduction of which increases the regeneration of pancreatic tissue when an animal suffering from diabetes is introduced into the body [Anandwardhan A Hardikar, Ramesh R. Bhonde, 1999].

The closest analogue of the claimed invention is a composition comprising a means for changing the growth rate and reproduction of cells, containing conditioned cell culture medium obtained by culturing eukaryotic cells in three dimensions, as well as a method for its preparation (RF Patent 2280459 from 06/27/2006). However, this composition does not take into account the mechanisms of development of regeneration in response to the action of damaging factors.

At the same time, the claimed inventions are aimed at eliminating the above disadvantages and achieving a technical result, which consists in increasing the regenerative potential of cells of organs and tissues in chronic inflammatory and degenerative processes of internal organs and skin, which in turn provides an increase in the effectiveness of treatment of a wide range of chronic diseases .

The applicants of the present invention described the composition options: in a composition containing a peripheral blood leukocyte lysate, and / or stem and / or progenitor cells obtained from an animal which, before receiving cellular material, carries out tissue sampling of internal organs or skin integuments, the growth and regeneration of which is necessary stimulate, as well as the composition obtained after leukocytes of peripheral blood, and / or stem, and / or progenitor cells obtained from the animal, which before receiving cell material carried fence internal organs or tissues of the skin, growth and regeneration are necessary to stimulate the added lysate cells previously retrieved internal organs or tissues of the skin, co-cultivated and destroyed. Methods for preparing these compositions are also described.

The use of these compositions opens up new possibilities in obtaining products for use for various purposes, including tissue regeneration, for example, in the treatment of wounds and other tissue defects, as well as in the treatment of chronic inflammatory and degenerative processes of internal organs and skin integuments.

A cell lysate obtained from peripheral blood leukocytes, and / or stem, and / or progenitor cells obtained from an animal that undergoes tissue sampling of internal organs or skin integument, which is, for example, a biopsy study, obtained from cells, which are destroyed using physical (ultrasound, electric discharge, freezing-thawing), chemical (enzymes, chemicals), mechanical (grinding) and other factors, as well as their combinations , resulting in a violation of the integrity of cell structures. In the process of destruction of cultured cells, it is possible, but not necessary, to use physical (temperature reduction, use of inert gases), chemical (inhibitors of proteolytic enzymes) and other factors, as well as their combination to reduce the rate of destruction of cellular metabolites by proteolytic enzymes.

The inventive variants of the compositions can be used in any condition. Physical options for the compositions are, but are not limited to, liquid or solid media, frozen media, lyophilized media, or media dried to a powder state. In addition, this composition can be prepared in combination with a pharmaceutically acceptable excipient, used as a carrier for internal administration, for administration directly to food or food product, to obtain ointment or grinding for local. In addition, the composition may be further processed to increase or decrease the concentration of one or more factors or components contained in the composition. So, for example, the composition can be enriched with growth factors using immunoaffinity chromatography.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new compositions, as well as methods for their preparation, containing a peripheral blood leukocyte lysate and / or stem and / or progenitor cells obtained from an animal that is sampled from internal organs or skin, and the regeneration of which must be stimulated, as well as the composition obtained after leukocytes of peripheral blood, and / or stem, and / or progenitor cells obtained from the animal, which before receiving By the use of cellular material, tissues of internal organs or skin integuments are taken, the growth and regeneration of which must be stimulated, to which a lysate of cells, previously harvested tissues of internal organs or skin integuments are added, and they are jointly cultivated and destroyed.

Pharmacological compositions will contain various naturally secreted proteins, as well as intracellular metabolites synthesized by the cell and contained mainly in secretory granules.

The main steps for the first version of the composition:

Stage 1: behavior of tissue sampling of internal organs or skin using the example of a biopsy study

An animal under general anesthesia, under ultrasound control, undergoes a biopsy of internal organs percutaneously (we performed this manipulation on the liver, heart, pancreas, kidney), during which tissue from 10 to 100 μl is taken (depending on the size of the animal and organ punctured). A biopsy is not accompanied by severe pain. The level of physical and moral suffering in an animal may be comparable to the collection of peripheral blood during the preparation of immunobiological preparations.

A puncture biopsy is performed to collect the skin.

Stage 2: obtaining stem and / or progenitor cells and / or peripheral blood leukocytes

Bone marrow, peripheral blood, subcutaneous fat, etc. can be a source of stem and progenitor cells.

a) Obtaining cells from the bone marrow

Bone marrow was taken from an animal under general ether anesthesia from the femur, puncturing the bone and washing it with Hanks solution. Red blood cells were removed by adding to the resulting suspension a lysing solution in a ratio of 1:10. After adding the lysis solution, the cells were centrifuged and the resulting pellet was resuspended in Hanks solution.

Red blood cells are removed from the bone marrow suspension aspirate. To remove red blood cells, you can use gradient centrifugation, lysis of red blood cells with a lysis solution, or a combination of these methods with others, resulting in the separation of red blood cells and nucleated cells. These nucleated cells contain both stem cells and progenitor cells.

b) Obtaining progenitor cells from peripheral blood

To increase the number of stem and progenitor cells in peripheral blood, an animal was injected with G-CSF (granulocyte colony stimulating factor) for 2 days before taking the cells. Administration of the drug allows to increase the level of hematopoietic stem cells from 0.5% (control) to 3.5% of the total number of mononuclear cells in peripheral blood.

c) Obtaining stem cells from subcutaneous fat

After isolation, subcutaneous fat is treated with a collagenase solution that breaks down collagen to produce a cell suspension. After collagenase treatment, the cells are centrifuged and stem cells are isolated on a density gradient, which is confirmed by the cultivation method.

g) Obtaining peripheral blood leukocytes

Peripheral blood sampling is carried out by any known method, after which a leukocyte suspension is obtained: for this, 2 ml of heparinized blood is mixed with 1 ml of a 3% gelatin solution heated to 37 ° C and incubated in an incubator at 37 ° C for 30 minutes. After erythrocyte sedimentation, the plasma layer enriched in leukocytes is taken into plastic tubes and washed with a 10-fold volume of phosphate-saline buffer (pH 7.4). Then, the leukocytes by centrifugation for 10 min at 1000 rpm are resuspended in a Hanks solution without Ca ²⁺ and Mg ²⁺ . After counting the number of leukocytes, the cell concentration was adjusted to 10 ⁶ cells.

Stage 3: obtaining the composition

After receiving the cells, together with the culture medium, they are transferred to test tubes in which the cells are destroyed using physical (ultrasound, freeze-thaw, electric pulse, etc.), and / or chemical (enzymes, chemical reagents, etc.), and / or mechanical (grinding, etc.) methods or their combinations, leading to disruption of cellular structures. The resulting composition, consisting of culture medium and destroyed cultured cells, can be used to prepare injection forms, tablet dosage forms, used as food additives, or dried, lyophilized, frozen, etc.

Ointments are prepared using medical vaseline as a base. The pharmacological composition is placed in a porcelain mortar and ground with a porcelain pestle to a finely divided state. Then, medical vaseline is added to the obtained powder and carefully triturated to obtain a homogeneous (homogeneous) substance. Ointment was applied to the surface of the skin.

Injection forms were prepared as follows. The sterile pharmacological composition was dissolved in distilled water. The solution was administered intraperitoneally, intramuscularly, intravenously or subcutaneously.

Liquid preparations for oral administration can be made, for example, in the form of solutions, syrups or suspensions. Such liquid preparations can be prepared by standard methods using pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia gum); anhydrous carriers (e.g., almond oil, fatty esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Additionally, the composition can be obtained with the addition of an antibiotic, anti-inflammatory agent, antiviral agent, antifungal agent, hormone, antitumor agent, analgesic, anesthetic, or any combination thereof. In this case, the addition of one or another agent is made taking into account the necessary therapeutic effect determined by a specialist.

The main stages for the second stage of composition

Steps 1 and 2 are carried out similarly to the above.

Stage 3: Obtaining a cell lysate (tissue-specific antigen) of pre-harvested tissues of internal organs or skin, the growth and regeneration of which must be stimulated.

To obtain a lysate, the obtained biopsy material was used.

To obtain a lysate, tissue cells are destroyed using physical (ultrasound, freezing-thawing, electrical impulse, etc.) and / or chemical (enzymes, chemicals, etc.), and / or mechanical (grinding, etc.) methods or their combinations leading to disruption of cellular structures. As an example: the tissue is mechanically homogenized into fragments of not more than 0.5 mm ³ . The resulting homogenate is poured with Hanks' solution 1: 1 and placed in the freezer for 1 hour, then the fabric is thawed, this cycle is repeated 3 times.

It was noted that the basis for the method of obtaining tissue-specific antigen (TA) is the destruction of cell structures, and it can be carried out:

1. Mechanically - rubbing the tissue of the parenchymal organs (for example, in a porcelain mortar using a porcelain pestle or in a glass homogenizer);

2. Using physical methods (for example, conducting freeze-thaw cycles or sonicating a cell suspension);

3. Using chemical methods (for example, treating a cell suspension with detergents that destroy the lipid bilayer of cell and intracellular membranes);

4. A combination of the above methods (mechanical grinding of tissue followed by freezing and thawing cycles).

In our work, we studied different methods of preparing TA and settled on the combination of mechanical abrasion followed by freeze-thaw cycles. We chose this method as the simplest and fastest executable. However, our studies have shown that others (the above methods) can achieve the same result (the manipulation time varies, a number of methods require additional equipment). In our work, we determined the level of activity of intralysosomal enzymes (in particular, acid phosphatase) after tissue destruction by various methods. We have not recorded significant differences when using different methods of tissue destruction. Thus, this "tissue-specific antigen", in our opinion, can be obtained by any known method.

Stage 4: cell culture

A cell culture medium for culturing cells can be any cell culture medium that adequately meets the nutritional needs of cultured cells. Examples of such media include, but are not limited to, Dulbecco’s Eagle’s modified medium (DMEM), Ham’s F12 medium, RPMI 1640, Iscov’s medium, McCoy’s medium, and other media compositions known to those skilled in the art, including those described in Methods For Preparation of Media, Supplements and Substrate For Serum-Free Animal Cell Culture, Alan R. Liss, New York (1984), and Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons Ltd., Chichester, England, 1996. This medium may be supplemented with any components necessary to maintain the desired cell or tissue culture. If necessary, fetal calf serum can be added, which is a complex solution of albumin, globulin, growth stimulants and hormones. Such serum should not contain pathogens and should be carefully checked for contamination with mycoplasma, bacteria, fungi and viruses.

The resulting cells, namely peripheral blood leukocytes, and / or stem and / or progenitor cells, are resuspended in a culture growth medium. A tissue-specific antigen obtained from the tissue of the organ whose cell regeneration is to be stimulated is also added to the cell culture medium. Cells and tissue-specific antigen are cultured together at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 1-60 days. A tissue-specific antigen homogenate was added to the culture medium in an average ratio of 1:30 (the range from 1:10 to 1:80 was checked).

Stage 5: obtaining a composition is carried out similarly to the stage set forth for the first version of the composition.

Examples confirming the suitability of these compositions

Example 1. The effectiveness of the pharmacological composition was studied on a model of myocardial damage in rats by cryodestruction (CD).

On the 30th day after causing damage to the left ventricle (LV) of the heart, the formation of rounded scar tissue with a diameter of 6-7 mm from the side of the epicardium and 3 mm from the side of the endocardium occurred; LV wall thickness in the center of the scar did not exceed 1 mm. As can be seen from table 1, the decrease in the relative mass of the left ventricle (per unit mass) after cryodestruction was approximately 13%, and for the free wall of the left ventricle this indicator was 17%.

The area of necrosis after cryodestruction with respect to the total area of the left LV wall was 25.1 ± 3.6% on the epicardial side and 23.0 ± 4.2% on the endocardial side. The area of transmural necrosis after cryodestruction with respect to the total area of the free wall of the LV was 11.4 ± 0.7% from the side of the epicardium, 6.4 ± 2.5% from the side of the endocardium.

Thus, the morphometry of macro preparations of the damaged heart made it possible to establish a significant decrease in LV muscle mass, which would inevitably lead to a decrease in its contractile activity.

Preparation of the pharmacological composition consisted of the following steps:

1) obtaining myocardial tissue in intact animals by biopsy under the control of ultrasound

In animals under ether anesthesia under the control of ultrasound and using biopsy needles, myocardial tissue was collected. The mass of biopsy tissue ranged from 1 to 10 mg. The collection of biopsy material did not affect the general condition of the animal and was not accompanied by their death.

2) Obtaining peripheral blood cells and bone marrow cells

Bone marrow cells and peripheral blood cells were collected in animals in which a myocardial biopsy was performed. Cell collection is carried out on average after 5 days (from 1 to 25 days).

a) Obtaining stem and progenitor cells

Bone marrow was taken from an animal under general ether anesthesia from the femur, puncturing the bone and washing it with Hanks solution. Red blood cells were removed by adding to the resulting suspension a lysing solution in a ratio of 1:10. After adding the lysis solution, the cells were centrifuged and the resulting cell pellet was resuspended in Hanks solution. The isolated cells contained stem and progenitor cells, which was confirmed by cultivation and subsequent cytochemical staining.

b) peripheral blood leukocytes were obtained as follows. 2 ml of heparinized blood is mixed with 1 ml of a 3% gelatin solution heated to 37 ° C and incubated in an incubator at 37 ° C for 30 minutes. After erythrocyte sedimentation, the plasma layer enriched in leukocytes is taken into plastic tubes and washed with a 10-fold volume of phosphate-saline buffer (pH 7.4). Then, the leukocytes after centrifugation for 10 min at 1000 rpm are resuspended in a Hanks solution without Ca ²⁺ and Mg ²⁺ . After counting the number of leukocytes, the cell concentration was adjusted to 10 ⁶ cells. The selected cells were white blood cells and consisted of lymphocytes, monocytes and granulocytes.

3) Cell cultivation

The obtained peripheral blood cells and bone marrow cells were cultured in the culture medium for an average of 5 days at 37 ° C at 5% CO ₂ content.

4) Cell destruction

After cultivation, the cells were destroyed - 2 cycles of freezing and thawing and sonication.

5) Injection preparations were prepared from the obtained composition, which were administered intraperitoneally to the animal for 7 days (experimental group).

As the 2nd control, a composition consisting of cells obtained from an intact animal was used. The dose of the drug was taken the amount of protein obtained by the destruction of 10 ⁶ cells in 1 ml of distilled water.

When the control (2nd control group) composition, which was obtained from the cells of intact animals, was introduced to animals with cryodestruction (CD), we noted an increase in myocardial mass and an improvement in the contractility of the heart, however, in none of the experiments performed, a complete cardiac recovery was obtained. the muscles. It should be noted that the mass of the left ventricle in rats (experimental group) with CD, which was administered a composition that was obtained from a lysate of cells obtained from an animal in which myocardial tissue biopsy was taken before cell sampling, did not significantly differ from the myocardial mass of intact animals ( table 1) and was significantly higher compared to the 2nd control. The level of mitosis in the experimental group was significantly higher compared with the 2nd group.

Our experiments on animals demonstrated that the composition obtained from blood cells and / or bone marrow taken from an animal from which myocardial tissue was biopsy-harvested prior to sampling cellular material accelerates cell regeneration in the damaged organ.

Example 2. The effect of the pharmacological composition on the process of regeneration of the skin

The effectiveness of the use of the pharmacological composition was shown on a burn regeneration model.

In this model, animals (mini-pigs) under ether anesthesia were burned with an area of 50 cm ² .

The stages of obtaining a pharmacological composition

1) Obtaining skin tissue in intact animals

In animals under ether anesthesia, a patch of skin on the back was shaved. Then, from 50 to 150 μg of skin tissue were taken punctured from several skin punctures. The sampling site is treated with aseptic fluid. This procedure is harmless and safe and, subject to animal conditions, is not accompanied by the development of any complications (skin damage, suppuration at the site of the biopsy, etc.).

2) Cell production

a) Stem cells were obtained from subcutaneous fat from animals in which a skin biopsy was performed before cell sampling (an average of 5 days). After isolation using collagenase, the cells were resuspended in culture medium. Isolated cells are predominantly stem mesinchymal cells, which is confirmed by the method of cultivation and subsequent staining for specific intracellular markers, as well as surface proteins.

b) Obtaining white blood cells from peripheral blood

2 ml of heparinized blood was mixed with 1 ml of a 3% gelatin solution heated to 37 ° C and incubated in an incubator at 37 ° C for 30 minutes. After erythrocyte sedimentation, the plasma layer enriched in leukocytes was taken into plastic tubes and washed with 10-fold volume of phosphate-saline buffer (pH 7.4). Then, the leukocytes after centrifugation for 10 min at 1000 rpm were resuspended in a Hanks solution without Ca ²⁺ and Mg ²⁺ . After counting the number of leukocytes, the cell concentration was adjusted to 10 ⁶ cells.

3) Obtaining tissue-specific antigen

To obtain tissue-specific antigen, biopsy material obtained from the skin was used. The fabric is mechanically homogenized. The resulting homogenate is poured with Hanks' solution 1: 1 and placed in the freezer for 1 hour, then the fabric is thawed, this cycle is repeated 3 times. The homogenate is then centrifuged at 2000 rpm for 10 minutes. The supernatant is collected and filtered through a filter with a pore diameter of 0.22 μm to ensure sterility of the solution. The protein concentration in the solution is determined by the Lowry method and should be in the range from 0.5 to 1 mg / ml.

4) Cultivation of isolated cells with tissue-specific antigen

The resulting cells, namely peripheral blood leukocytes and stem cells, are resuspended in a culture growth medium. A tissue-specific antigen obtained from biopsy skin tissue is also added to the cell culture medium. Cells and tissue-specific antigen are cultured together at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 7 days. The tissue-specific antigen supernatant was added to the culture medium in an average ratio of 1:30 (the range from 1:10 to 1:80 was tested).

5) Cell destruction

After cultivation, the cells were destroyed - 2 cycles of freezing and thawing and sonication. The resulting cell lysate was combined with the medium in which the cells were cultured.

6) Injection preparations were prepared from the obtained composition, which were administered intraperitoneally to the animal for 7 days (experimental group), as well as ointment, which was applied to the wound surface. The composition of the ointment, along with an inert filler and pharmacological composition, included antibiotics (gentamicin) and an antiviral drug (acyclovir) to prevent infection.

As the 2nd control, a composition consisting of disrupted cells that were cultured without tissue-specific antigen obtained from skin tissue was used. The dose of the drug was taken the amount of protein obtained by the destruction of 10 ⁶ cells in 1 ml of distilled water.

The burn area was evaluated in the work, histological studies were performed. The results are presented in figures 1 and 2.

In animals that were administered a pharmacological composition obtained from cells that were cultured without tissue-specific antigen, a pronounced regeneration of damaged skin was observed for 3-4 days. This is accompanied by a decrease in edema, a decrease in inflammation, an increase in granulation and the development of microvessels. However, in animals that were administered a composition obtained from animal cells that were cultured together with a tissue-specific antigen, starting from day 7, a significantly faster wound closure and a decrease in epithelization time were observed compared with the 1st and 2nd control. The wound closure time in such animals is fully correlated with the number of microvessels, which was determined on histological sections of tissues that were taken during biopsies (figure 2)

Thus, the pharmacological composition obtained from animal cells, which were cultured with tissue-specific antigen, helps to accelerate the regeneration of damaged organs and tissues.

Example 3

The effectiveness of the use of the composition obtained from lymphoid and progenitor cells obtained from the peripheral blood of animals for the treatment of toxic hepatitis

Guinea pigs with a model of toxic hepatitis were used in the study (administration of subcutaneous 0.1 ml of a 40% CCL ₄ oil solution for 14 days).

1) Obtaining cells

A pharmacological composition was prepared from cultured lymphoid and progenitor cells obtained from peripheral blood. To increase the number of progenitor cells in peripheral blood, the animal was injected with G-CSF (granulocyte colony stimulating factor) for 2 days before taking the cells. Administration of the drug allowed to increase the level of hematopoietic stem cells from 0.5% (control) to 3.5% of the total number of mononuclear cells in peripheral blood. 7 days before cell collection, the animal under the control of ultrasound in an anesthetized state conducted a biopsy sampling of liver tissue.

The isolated cells consisted of peripheral blood leukocytes and progenitor cells, which was confirmed by flow cytometry.

2) Obtaining tissue-specific antigen

To obtain tissue-specific antigen, biopsy material obtained from liver tissue was used. The fabric is mechanically homogenized. The resulting homogenate is poured with Hanks' solution 1: 1 and placed in the freezer for 1 hour, then the fabric is thawed, this cycle is repeated 3 times. The homogenate is then centrifuged at 2000 rpm for 10 minutes. The supernatant is collected and filtered through a filter with a pore diameter of 0.22 μm to ensure sterility of the solution. The protein concentration in the solution is determined by the Lowry method and should be in the range from 0.5 to 1 mg / ml.

3) Cultivation of isolated cells with tissue-specific antigen

The obtained cells, namely peripheral blood leukocytes and progenitor cells, are resuspended in a culture growth medium. A tissue-specific antigen obtained from liver tissue by biopsy is also added to the cell culture medium. Cells and tissue-specific antigen are cultured together at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 5-7 days (checked from 1 to 15 days). The tissue-specific antigen supernatant was added to the culture medium in an average ratio of 1:30 (the range from 1:10 to 1:80 was tested).

5) Cell destruction

After cultivation, the cells were destroyed - 2 cycles of freezing and thawing and sonication. The resulting cell lysate was combined with the medium in which the cells were cultured.

6) The resulting pharmacological composition was administered to animals subcutaneously daily for 7 days.

The effectiveness of the treatment was evaluated according to blood biochemistry (figure 3).

As can be seen from the data presented in Fig. 3, 1 week after the start of CCL ₄ administration, a sharp (2.5-fold) increase in the level of transaminases (AcT) is observed in animals, which indicates the cytolytic effect of the toxin. The management of the composition obtained from the destroyed cultured cells and the culture medium in which these cells were cultured leads to a pronounced and reliable therapeutic effect, which is manifested by the normalization of transaminases. (The enzyme level in the blood of intact animals is taken as 100%.)

Example 4

The effectiveness of the use of a substrate obtained from cultured bone marrow cells was studied in experiments with modeling dyslipidemic liver damage in rabbits.

In rabbits with chronic dyslipidemia, fatty degeneration of the liver develops within 4 months and the morphometric characteristics of hepatocytes are sharply impaired (Table 1).

1) Obtaining cells

Cells were obtained from bone marrow by the method described above. The isolated cells contained stem and progenitor cells, which was confirmed by cultivation and flow cytometry.

7 days before the bone marrow cells were taken from an animal under anesthesia, a liver biopsy was performed under ultrasound control with a sampling of liver tissue (from 50 to 200 μg).

2) Tissue-specific antigen was prepared according to the method described above. To obtain tissue-specific antigen, biopsy material obtained from liver tissue was used. The fabric is mechanically homogenized. The resulting homogenate is poured with Hanks' solution 1: 1 and placed in the freezer for 1 hour, then the tissue is thawed, this cycle is repeated 3 times. The homogenate is then centrifuged at 2000 rpm for 10 minutes. The supernatant is collected and filtered through a filter with a pore diameter of 0.22 μm to ensure sterility of the solution. The protein concentration in the solution is determined by the Lowry method and should be in the range from 0.5 to 1 mg / ml.

3) Cell cultivation

The resulting cells, namely stem and progenitor cells, are resuspended in culture growth medium. A tissue-specific antigen obtained from liver tissue by biopsy is also added to the cell culture medium. Cells and tissue-specific antigen are cultured together at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 5-7 days (checked from 1 to 15 days). The tissue-specific antigen supernatant was added to the culture medium in an average ratio of 1:30 (the range from 1:10 to 1:80 was tested).

4) Cell destruction

After cultivation, the cells were destroyed - 2 cycles of freezing and thawing and sonication. The resulting cell lysate was combined with the medium in which the cells were cultured.

5) The resulting pharmacological composition was administered to animals subcutaneously daily for 7 days.

As control No. 1, we used a substrate isolated from cultured bone marrow cells obtained from an animal in which a liver biopsy was not performed before cell collection. As control No. 2, we used a substrate isolated from cultured cells obtained from an animal in which a liver biopsy was performed before collection, but whose cells were cultured without tissue-specific antigen.

The protein content in the control and experimental substrate did not differ.

As can be seen from the data presented (table 2), the introduction of the composition (control 1) increased the regenerative ability of hepatocytes with fatty liver. However, this effect was not sufficiently pronounced. The introduction of a composition obtained from animal cells to which a liver biopsy was performed before cell sampling (control 2) leads to a significant increase in hepatocyte regeneration processes in the liver of experimental animals.

The introduction of a composition (experiment) isolated from cultured cells obtained from an animal in which a liver biopsy was performed before collection into cells that were cultured with a tissue-specific antigen exerted the maximum regenerative effect.

Example 5

The effectiveness of the use of compositions obtained from progenitor cells obtained from peripheral blood of animals for the treatment of chronic toxic hepatitis

We used rats with a model of chronic toxic hepatitis (administration of subcutaneous 0.1 ml of a 40% CCL ₄ oil solution for 60 days).

1) Obtaining cells

A pharmacological composition was prepared from cultured progenitor cells obtained from peripheral blood. To increase the number of progenitor cells in peripheral blood, the animal was injected with G-CSF (granulocyte colony stimulating factor) for 2 days before taking the cells. Administration of the drug allowed to increase the level of hematopoietic stem cells from 0.5% (control) to 3.5% of the total number of mononuclear cells in peripheral blood. To isolate the population of progenitor cells, the CliniMAX device was used, which allows using a magnetic bead coated with antibodies to CD34 receptors to isolate a pure fraction of the progenitor cells. 7 days before cell collection, the animal under the control of ultrasound in an anesthetized state conducted a biopsy sampling of liver tissue.

The isolated cells consisted of progenitor cells, which was confirmed by flow cytometry.

2) Obtaining tissue-specific antigen

To obtain tissue-specific antigen, biopsy material obtained from liver tissue was used. The fabric is mechanically homogenized. The resulting homogenate is poured with Hanks' solution 1: 1 and placed in the freezer for 1 hour, then the tissue is thawed, this cycle is repeated 3 times. The homogenate is then centrifuged at 2000 rpm for 10 minutes. The supernatant is collected and filtered through a filter with a pore diameter of 0.22 μm to ensure sterility of the solution. The protein concentration in the solution is determined by the Lowry method and should be in the range from 0.5 to 1 mg / ml.

3) Cultivation of isolated cells with tissue-specific antigen

The resulting cells, namely progenitor cells, are resuspended in a culture growth medium. A tissue-specific antigen obtained from liver tissue by biopsy is also added to the cell culture medium. Cells and tissue-specific antigen are cultured together at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 5-7 days (checked from 1 to 15 days). The tissue-specific antigen supernatant was added to the culture medium in an average ratio of 1:30 (the range from 1:10 to 1:80 was tested).

5) Cell destruction

After cultivation, the cells were destroyed - 2 cycles of freezing and thawing and sonication. The resulting cell lysate was combined with the medium in which the cells were cultured.

6) The resulting pharmacological composition was administered to animals subcutaneously daily for 7 days.

The effectiveness of the treatment was evaluated according to blood biochemistry (figure 4).

As can be seen from the data presented in figure 4, 1 week after the start of the introduction of CCL ₄ in the blood of animals there is a sharp (2.5 times) increase in the level of transaminases (ACT), which indicates the cytolytic effect of the toxin. The management of the composition obtained from the destroyed cultured cells and the culture medium in which these cells were cultured leads to a pronounced and reliable therapeutic effect, which is manifested by the normalization of transaminases. (The enzyme level in the blood of intact animals is taken as 100%.)

Example 6. The effect of the pharmacological composition on the process of regeneration of the skin

The efficacy of using the pharmacological composition has been shown in a burn regeneration model.

In this model, animals (rats) under ether anesthesia were burned with an area of 5 cm ² .

The stages of obtaining a pharmacological composition

1) Obtaining skin tissue in intact animals

In animals under ether anesthesia, a patch of skin on the back was shaved. Then, a skin scraping (from 10 to 50 μg of skin tissue) was performed with a scalpel from a small area. The scraping site is treated with aseptic fluid. This procedure is harmless and safe and, subject to animal conditions, is not accompanied by the development of any complications (skin damage, suppuration at the site of the biopsy, etc.).

2) Cell production

a) Stem cells were obtained from the bone marrow from animals in which skin scraping was performed before cell collection (average 5 days). After removal of red blood cells, bone marrow cells were scattered on Petri dishes. After 2 days, the culture medium was changed with the removal of non-adherent cells. Attached cells were cultured to obtain a monolayer. Attached cells are predominantly stem mesinchymal cells, as evidenced by the method of cultivation and subsequent staining for specific intracellular markers, as well as surface proteins.

3) Obtaining tissue-specific antigen

To obtain tissue-specific antigen used material obtained from the skin. The fabric is mechanically homogenized. The resulting homogenate is poured with Hanks' solution 1: 1 and placed in the freezer for 1 hour, then the tissue is thawed, this cycle is repeated 3 times. The homogenate is then centrifuged at 2000 rpm for 10 minutes. The supernatant is collected and filtered through a filter with a pore diameter of 0.22 μm to ensure sterility of the solution. The protein concentration in the solution is determined by the Lowry method and should be in the range from 0.5 to 1 mg / ml.

4) Cultivation of isolated cells with tissue-specific antigen

Tissue-specific antigen obtained from skin tissue is added to the obtained stem cells during cultivation. Cells and tissue-specific antigen are cultured together at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 7 days. The tissue-specific antigen supernatant was added to the culture medium in an average ratio of 1:30 (the range from 1:10 to 1:80 was tested).

5) Cell destruction

After cultivation, the cells were destroyed - 2 cycles of freezing and thawing and sonication. The resulting cell lysate was combined with the medium in which the cells were cultured.

6) Injection preparations were prepared from the obtained composition, which were administered intraperitoneally to the animal for 7 days (experimental group), as well as ointment, which was applied to the wound surface. The composition of the ointment, along with an inert filler and pharmacological composition, included antibiotics (gentamicin) and an antiviral drug (acyclovir) to prevent infection.

As the 2nd control, a composition consisting of disrupted cells that were cultured without tissue-specific antigen obtained from skin tissue was used. The dose of the drug was taken the amount of protein obtained by the destruction of 10 ⁶ cells in 1 ml of distilled water.

The burn area was estimated in the work. The results are presented in figure 5.

In animals that were administered a pharmacological composition obtained from cells that were cultured without tissue-specific antigen, a pronounced regeneration of damaged skin was observed for 3-4 days. This is accompanied by a decrease in edema, a decrease in inflammation, an increase in granulation and the development of microvessels. However, in animals that were administered a composition obtained from animal cells that were cultured together with a tissue-specific antigen, starting from day 7, a significantly faster wound closure and a decrease in epithelization time were observed compared with the 1st and 2nd control.

Thus, the pharmacological composition obtained from animal cells, which were cultured with tissue-specific antigen, helps to accelerate the regeneration of damaged organs and tissues.

Example 7

The effectiveness of the use of a substrate obtained from cultured peripheral blood cells was studied in experiments with modeling hyperglycemia in rats.

In rats, after administration of streptozotocin, persistent hyperglycemia develops (Fig. 6).

1) Obtaining cells

A pharmacological composition was prepared from cultured lymphoid cells obtained from peripheral blood. 7 days prior to cell collection, the animal under the control of ultrasound in anesthesia conducted a biopsy sampling of pancreatic tissue. The presence of insulin producing cells was confirmed histochemically using dithizone.

The isolated cells consisted of peripheral blood leukocytes, which was confirmed by flow cytometry.

2) Obtaining tissue-specific antigen

To obtain tissue-specific antigen, biopsy material obtained from pancreatic tissue was used. The fabric is mechanically homogenized. The resulting homogenate is poured with Hanks' solution 1: 1 and placed in the freezer for 1 hour, then the tissue is thawed, this cycle is repeated 3 times. The homogenate is then centrifuged at 2000 rpm for 10 minutes. The supernatant is collected and filtered through a filter with a pore diameter of 0.22 μm to ensure sterility of the solution. The protein concentration in the solution is determined by the Lowry method and should be in the range from 0.5 to 1 mg / ml.

3) Cultivation of isolated cells with tissue-specific antigen

The obtained cells, namely peripheral blood leukocytes, are resuspended in the culture growth medium. A tissue-specific antigen obtained from pancreatic tissue by biopsy is also added to the cell culture medium. Cells and tissue-specific antigen are cultured together at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 5-7 days (checked from 1 to 15 days). The tissue-specific antigen supernatant was added to the culture medium in an average ratio of 1:30 (the range from 1:10 to 1:80 was tested).

5) Cell destruction

After cultivation, the cells were destroyed - 2 cycles of freezing and thawing and sonication. The resulting cell lysate was combined with the medium in which the cells were cultured.

6) The resulting pharmacological composition was administered to animals subcutaneously daily for 7 days.

The effectiveness of the treatment was evaluated according to blood biochemistry (Fig.6).

As can be seen from the data presented in Fig.6, in rats after the administration of streptozotocin there is a sharp increase in glucose level. The introduction of a composition obtained from destroyed cultured cells and the culture medium in which these cells were cultured leads to a pronounced and reliable therapeutic effect, which is manifested by a decrease in the level of glucose in peripheral blood.

Table 1 Comparison of the mass of intact hearts and hearts after CD in animals that were injected intraperitoneally with a composition obtained from:
1) cells of peripheral blood and bone marrow isolated from an intact animal (2nd control);
2) cells of peripheral blood and bone marrow isolated from animals in which myocardial biopsy was performed before the collection of cells (experimental group) The mass of the animal, g The mass of the free wall of the left ventricle, (mg) / weight of the animal (g) LV mass, (mg) / animal mass (g) Intact animals 356.7 ± 5.8 1.701 ± 0.031 2.298 ± 0.062 Animals after cryodestruction (1st control) 352.1 ± 27.8 1.25 ± 0.09 * 1.98 ± 0.01 * Animals after cryodestruction and administration of a composition obtained from cells of an intact animal (2nd control) 363.1 ± 10.1 1.50 ± 0.02 * # 2.05 ± 0.02 * # Animals after cryodestruction and administration of a composition obtained from the cells of an animal to which myocardial biopsy was performed before collecting the cells (experimental group) 355.1 ± 13.1 1.61 ± 0.04 + 2.15 ± 0.08 + - * - significant in relation to the group of intact animals, p <0.05
- # - significant in relation to the 2nd control, p <0.05
- + - significantly in relation to the 1st and 2nd control, p <0.05

table 2 The effect of the introduction of the pharmacological composition on the regeneration of hepatocytes of the damaged liver Group of animals Number of norms. hepatocytes (NG) The number of degenerative hepatotics (DG)% NG / DG Volume fraction of cores% Binuclear hepatocytes% Kernels with two or more nucleoli% Intact (n = 5) 88 12 8 19 2 5 Chr. dyslipidemia (n = 11) 55 32 1.5 ± 0.1 10 2.1 ± 0.4 13 Control No. 1 (n = 12) 65 25 2.9 ± 0.5 13 5.0 ± 0.3 fifteen Control No. 2 (n = 12) 71 eighteen 5.1 ± 0.1 eighteen 9.1 ± 0.3 eighteen Experience (n = 12) 88 fourteen 6.9 ± 0.4 22 11.4 ± 0.4 24

Literature

1. Babaeva A.G. The cellular basis of regeneration in mammals. M .: Medicine, 1984, 212 p.

2. Babaeva A.G. The unity and opposite of the cytogenetic activity of lymphocytes and their antibodies forming function during restoration processes in organs. Bull. experimental biology and medicine, 1999, vol. 128, No. 11, p. 484-490.

3. Babaeva A.G., Shubnikova E.A. The structure, functions and adaptive growth of the salivary glands. M .: Publishing house of Moscow State University, 1979, 191 p.

4. Babaeva A.G., Nesterenko V.G., Yudina N.V. Strengthening the ability of mice with repeated liver resections to transfer "regenerative information". Bull. experimental biology and medicine, 1982, No. 6, pp. 98-99.

5. Blyacher M.S., Gutorova N.M., Fedorova I.M. et al. The number of lymphocyte subpopulations in the spleen and the level of hematopoietic tissue proliferation in mice during surgical interventions. Bull. experimental biology and medicine, 1996, No. 3, pp. 301-303.

6. Goldberg E.D., Dygay A.M., Khlusov I.A. The role of the autonomic nervous system in the regulation of hematopoiesis. - Tomsk, 1997, 218 p.

7. Digay A.M., Klimenko N.A. Inflammation and hematopoiesis. - Tomsk, 1992, 276 p.

8. Digay A.M., Shakhov I.P. The role of intercellular interactions in the regulation of hematopoiesis. Tomk, 1989, 224 p.

9. Dygay A.M., Skurikhin EG, Suslov N.I. and others. Byul. the ex. Biology and Medicine, 1998, No. 12, p. 628-631.

10. Zimin Yu.I. An increase in the number of hematopoietic parent cells in mice in the initial period of the stress reaction. Bull. experimental biology and medicine, 1974, No. 12, p.17-19.

11. Makarov A.A., Tsypin A.B., Vityazev G.A., Dolina O.A. et al. The use of xenosplenoperfusate for the treatment of purulent-septic diseases. Surgery, 1993, No. 11, pp. 10-15.

12. Uzhansky Y. G., Skuratov V. L., Yushkov B. G. et al. On the leukopoietic and leukoinhibitory properties of the blood serum of patients with myocardial infarction. Pat. Physiology, 1974, No. 3, pp. 55-59.

13. Shakhov V.P. A drifting cascade-like slowly developing adaptation mechanism under the influence of extreme factors on the body. Bull. experimental biology and medicine, 1996, No. 5, p.571-574.

14. Shilova L.Ya., Poltorakin B.C., Suslov A.G. The effect of donor spleen lymphocytes treated with carbon tetrachloride on the mitotic activity of the liver and the production of α-fetoprotein of usengi mice. Bull. experimental biology and medicine, 1982, No. 6, pp. 99-101.

15. Anandwardhan A. Hardikar, Ramesh R. Bhonde. Modulating experimental diabetes by treatment with cytosolic extract from the regenerating pancreas. Diabetes Research and Clinical Practice 46 (1999), 203-211.

16. Hasper D., Hummel M., Kleber F.X. Systemic inflammation in patients with heart failure. European Heart Journal (1998) 19, 761-765.

17. Levine T.V., Levine A.B., Bolenbaugh J., Green P.R. Reversal of heart failure remodeling with age. Am J Geriatr Cardiol. 2002, Sep-Oct; 11 (5): 299-304. PMID: 12214168 [PubMed - indexed for MEDLINE].

18. Selye H. Nature. 1936, vol. 138, N3479, p. 32.

Claims (14)

1. A composition for stimulating the growth and regeneration of cells of organs and tissues in chronic inflammatory and degenerative processes of internal organs or skin, containing a lysate of peripheral blood leukocytes, stem and progenitor cells obtained from an animal that undergoes a biopsy of tissues of internal organs before receiving cellular material or skin integument, the growth and regeneration of which must be stimulated. 2. The composition according to claim 1, characterized in that the cell lysate is in the form of a liquid, in solid form, in lyophilized form, in the form of a powder, gel or film. 3. The composition according to claim 1, characterized in that it further comprises an antibiotic, anti-inflammatory agent, antiviral agent, antifungal agent, hormone, antitumor agent, analgesic, anesthetic, or any combination thereof. 4. A method of obtaining a composition according to claim 1 for stimulating the growth and regeneration of cells of organs and tissues in chronic inflammatory and degenerative processes of internal organs or skin integuments, including
a) conducting an animal biopsy of tissues of internal organs or skin, the growth and regeneration of which must be stimulated;
b) obtaining peripheral blood leukocytes from the animal, stem and progenitor cells of the bone marrow, followed by their cultivation;
c) destruction of cells to obtain a cell lysate;
d) obtaining a composition consisting of a peripheral blood leukocyte lysate, bone marrow stem and progenitor cells;
e) combining said composition with a pharmaceutically acceptable carrier. 5. The method according to claim 4, characterized in that it further processes said composition to obtain it in the form of a liquid, solid product, lyophilized product, powder, gel or film. 6. The method according to claim 4, characterized in that they additionally add an antibiotic, anti-inflammatory agent, antiviral agent, antifungal agent, hormone, antitumor agent, analgesic, anesthetic, or any combination of these compositions. 7. The method according to claim 4, characterized in that the destruction is carried out using physical methods and / or chemical methods. 8. The method according to claim 7, characterized in that the destruction of the cells is carried out by conducting cycles of freezing - thawing or sonication of the cell suspension. 9. A composition for stimulating the growth and regeneration of cells of organs and tissues in chronic inflammatory and degenerative processes of internal organs or skin, containing a lysate of peripheral blood leukocytes and / or stem and / or progenitor cells obtained from an animal that is undergoing biopsy before sampling cellular material tissues of internal organs or skin integument, the growth and regeneration of which must be stimulated, while peripheral blood leukocytes and / or stem and / or progenitor cells redvaritelno with disrupted cultured cells previously retrieved biopsies of internal organs or tissues of the skin, which growth and regeneration is necessary to stimulate; 10. A method of obtaining a composition according to claim 9 for stimulating the growth and regeneration of cells of organs and tissues in chronic inflammatory and degenerative processes of internal organs or skin integuments, including
a) conducting an animal biopsy of tissues of internal organs or skin, the growth and regeneration of which must be stimulated;
b) obtaining from the animal peripheral blood leukocytes and / or stem and / or progenitor bone marrow cells;
c) destruction of cells previously collected by biopsy of tissues of internal organs or skin, the growth and regeneration of which must be stimulated to obtain a cell lysate;
d) adding a lysate of cells of tissues of internal organs or skin integuments obtained previously in step c) to previously obtained peripheral blood leukocytes and / or stem and / or progenitor bone marrow cells and their subsequent cultivation;
d) destruction of cells after cultivation to obtain a cell lysate;
f) preparing a composition by combining the cell lysate obtained in step e) with the medium in which the cells were co-cultured in step d);
g) combining said composition with a pharmaceutically acceptable carrier. 11. The method according to claim 10, characterized in that it further processes said composition to obtain it in the form of a liquid, solid product, lyophilized product, powder, gel or film. 12. The method of claim 10, wherein the antibiotic, anti-inflammatory agent, antiviral agent, antifungal agent, hormone, antitumor agent, analgesic, anesthetic, or any combination thereof are added to the composition. 13. The method according to claim 10, characterized in that the destruction is carried out using physical methods and / or chemical methods. 14. The method according to p. 13, characterized in that the destruction of the cells is carried out by conducting cycles of freezing - thawing or processing the cell suspension with ultrasound.

Images