RU2303631C1 - Method for production of mesenchymal stem cells and biotransplant using the same - Google Patents

Abstract

FIELD: biotechnology, in particular cell biology, medicine.

SUBSTANCE: medium for cryoconserving of human and animal cells contains blood serum autological to donor marrow in the next component ratio (vol.%): blood serum 45-65; dimethylsulfoxide 5-15; sulfanilamide and antibiotics 0.1-1.5 and balance: DMEM medium up to 100. Mesenchymal stem cells are cryoconserved in such medium to produce biotransplant.

EFFECT: cell material of prolonged storage time.

2 cl, 9 dwg, 2 tbl, 3 ex

Description

The invention relates to the field of cell biology, namely to the creation of a cell bank by cryopreservation of human and animal cells. It concerns the preservation medium and can be used in medicine, in particular, in the cryoconservation of mesenchymal stem cells (MSCs) to obtain a biograft.

Freezing cell cultures for their storage is widespread ("Culture of animal cells. Methods", under the editorship of R. Freshni, M .: Mir, 1989, pp. 108-120). Canned cells are used in medicine.

Famous review article Repin B.C. "Embryonic stem cell (from fundamental biology to medicine)." It is published in the journal Pathological Physiology. Experimental therapy ”, 2001, April-June (2), pp. 3-8. The article presents data on the history and possibilities of using the stem cell line, their phenotyping and other characteristics necessary for the medical use of the material.

The problems that need to be addressed in each individual case are the environment in which cryopreservation, the choice of freezing and thawing regimes are carried out so that the bulk of the cells remain alive and retain their sterility.

During the preservation process, cell damage and death can occur due to osmotic effects and the intracellular formation of ice crystals that break the cell membranes. In media, dimethyl sulfoxide (DMSO) is often used as a cryoprotectant, which has a toxic effect on cells, so conditions must be selected to minimize the negative effect of DMSO. Along with DMSO, glycerol is used as a cryoprotectant for freezing in nitrogen, but it has limited use. Polyvinylpyrrolidone has also received limited use (RF Patent No. 2161198 of 12/27/2000).

Known patent of the Russian Federation No. 2237712 from 10.10.2004, in which the medium for cryopreservation of human and animal cells is declared. It contains DMSO (5-15%), RPMI-1640 medium with the addition of 10-20% fetal calf serum, antibiotics and sulfonamides to protect cell cultures from infection, acetamide (10-30%), D-glucuronic acid, lecithin, cholesterol linoleic acid in a given ratio of components. Due to the presence of amino acids, vitamins and serum, this medium helps protect and adapt cells to cryopreservation conditions. The toxic effect on DMSO cells in it is reduced with the help of acetamide, D-glucuronic acid, lecithin, cholesterol, linoleic acid. These ingredients are stabilizers of cell membranes and colloidal structures, they increase the resistance of cells to freezing. In the composition of the medium for cryopreservation of cellular material, a nutrient medium, RPMI-1640, was used, characterized by a specific set of its constituent components in selected ratios. RPMI-1640 medium provides the viability of specific conserved cells. It contains a number of additional components.

The disadvantage of this environment is its multicomponent nature and the presence of serum of animal origin in human cell culture. Due to the fact that fetal calf serum is foreign in composition with respect to conserved, stored cells, cellular material cannot be used immediately after defrosting for medical purposes.

Autologous serum was used for cryopreservation of cells in the work of K. Abdulkadyrov and others. "The procurement and storage of cord blood. Comparison of three options for obtaining placental blood ”Actual issues of hematology and transfusiology: Materials of a scientific and practical conference, St. Petersburg, 2000. In each case, the combination plays a role: culture medium - serum. In this work, we used the RPMI medium with its own set of its constituent components and their ratio in the medium. Cryopreservative - DMSO, 20%. The repetition of proportions, the nutrient medium is serum, in a cryopreservation medium when replacing its components, for example, RPMI medium with DMEM, it will not necessarily lead to known results, which is associated with a difference in the component composition (qualitative and quantitative) of the nutrient medium and the mutual influence of the components on cells in the process of cryopreservation.

The desire to create an environment for cryopreservation of cells, simple and reliable, cheap and acceptable for medical use immediately after defrosting the culture, i.e. without additional processing of the biograft, it requires the selection of medium components that are standard, applicable in medicine, readily available, characterized and included in optimal proportions in the composition of the medium. This is especially important for those who have found widespread use in medicine of MSCs.

Closest to the medium claimed according to the invention is a medium with a DMEM nutrient medium in which dimethyl sulfoxide (DMSO) acts as a cryoprotectant and in which serum is present.

So known publication, WO 9739104, op. 10/23/1997. The work is devoted to cryopreservation of human MSCs. For this procedure, the authors used a modified DMEM medium: a medium with a minimized amount of glucose, -12-DMEM-low, i.e. DMEM-lg. In the environment for cryopreservation use different amounts of serum of different origin. So the figure is 0%, but with such amounts of serum, the cells die. The use of fetal bovine serum (90%) as a cryopreservation medium can subsequently provoke an autoimmune reaction when MSC is administered to a patient.

The use of autologous serum (as a cryopreservation medium, without other nutrient media) in the amount of 90%, 95% requires for its preparation a large blood sampling from the patient, at least 200 ml, which in some cases is unacceptable for the patient, is difficult to tolerate by the donor. The composition was also tested: DMEM-lg and 5% autologous serum with cryoprotector DMSO (10%). Here, low glucose DMEM is modified. The amount of autologous serum (5%) in the composition is small, such a small amount of it in cryopreservation media negatively affects cell survival.

As a result of testing various compositions of the media for cryopreservation of MSCs, where they checked the cryoprotectant, preservatives, nutrient media for cells, their compatibility, the authors found the optimal medium for the cells, a description of the composition of which was not found in the literature.

Biopharmacology and medicine often use biotransplants based on cellular material in the treatment. Known RF patent No. 22589836 of March 19, 2004, which describes a biograft for the treatment of parkinsonism, the active component of which is a culture of genetically unmodified neuronal stem cells (NSCs). Cells after propagation under culturing conditions are administered endolumbally in physiological saline in an amount of 25-250 million NSC in 1 ml of solution. The introduced cells were not stored, the material was not subjected to cryopreservation, i.e. there was no effect on the cells with cold.

Known RF patent No. 2265443 dated 05/14/2004, protecting a biotransplant based on MSCs for the treatment of diabetes mellitus I and II degree. Genetically unmodified MSCs are administered by intravenous drip in physiological saline, the cells being freshly prepared, not stored. Having grown MSCs, it is necessary on their basis to have a ready-made graft with a long shelf life that completely preserves its homogeneity and cell viability - MSCs that do not require additional treatments before administration to patients, identical to a freshly prepared cell culture.

Known RF patent No. 2271819, op. 03/20/06, Biotransplant. The method of its production (options) and the method of treatment of alopecia (CJSC "REMETEX"). MSCs are used as a biograft. Sources of MSCs are the most diverse, including taking autologous material to create a biograft. The cultivation of cells on a growth medium is carried out without separating them from fibroblasts, which are useful in the treatment of alopecia. The treatment is carried out with a freshly prepared cell culture. Details of cryopreservation (the number of DMSO, modes, environment) are not given by the authors, as well as the characteristics of MSCs with markers and the percentage of homogeneity of the cell culture.

The essence of the invention is the use in the environment for cryopreservation as a stabilizer of cell membranes and colloidal structures of human serum, which was a bone marrow donor, i.e. autologous serum from peripheral blood. Due to the fact that the medium does not contain serum of cow embryos, autologous cellular material can be used immediately after thawing. Additional procedures for preparing cells for transplantation are not required.

The problem that this invention solves is the long (almost unlimited) shelf life of cells without changing the characteristics of the cell culture. This is achieved due to the composition of the medium, consisting of autologous human blood serum with the addition of DMEM with sulfonamides and antibiotics. The ingredients are balanced in the medium, DMSO is used as a cryoprotectant. After defrosting the cryopreserved cell culture, a ready MSC biotransplant is obtained.

To avoid the damaging effect on DMSO cells, the amount of serum is balanced in the medium. Autologous serum in the composition of the cryopreservation medium is 45-65 vol.%. To exclude microbial infection of cells, antibiotics and sulfonamides are included in the medium. As a nutrient medium, the needle medium in the modification of Dyulbekko (DMEM) was used without any changes. In such ratios, RPMI media for cryopreservation were commonly used. The composition of each nutrient medium is a certain composition of substances, their effect on different cells is individual each time, it is necessary to check and select the environment for each specific case.

To reduce the damaging effect of DMSO, temperature conditions for working with cells are selected. For example, DMSO is added last to the medium, before the cryopreservation operation itself, and when the cells are thawed, which takes about two minutes, the ampoules with the cells are immediately stored at t = + 4 ° C, thus eliminating the damaging effect of DMSO before any actions with cells.

The composition of the medium, vol.%, For cryopreservation:

Human blood serum - 45-65

DMSO-5-15

Sulfanilamides and antibiotics - 0.1-1.5

Wednesday DMEM - the rest, up to 100.

Antibiotics and sulfonamides were used in the composition of the medium in generally accepted dosages: penicillin sodium salt - 100 mU / ml, streptomycin sulfate - 100 μg / ml, which in total was 0.1-1.5% in vol.%.

The technology for preparing the medium includes the following operations:

- mixing the DMEM medium with human serum, penicillin and streptomycin until the substances are completely dissolved;

- adding (immediately before use) to the medium of dimethyl sulfoxide and cooling it to room temperature;

- the concentrations of the components in the proposed environment for cryopreservation of autologous human cells are selected based on the interpretation of experimental data.

The cell population that was frozen in this medium, and then thawed and tested for viability and uniformity, is characterized by high homogeneity, according to positive markers (CD 90+, CD 105+, CD 106+, CD 44+) - markers of MSCs. The homogeneity of the cells is up to 99.9%. The percentage of viable cells changes slightly, remains practically unchanged, there is no infection. The cell cultures were checked every 4 months, the last test - after 24 months.

Table 1.
The average viability and quality of MSCs after cryopreservation. Before cryopreservation 4 months 8 months 12 months 16 months 20 months 24 months CD90 + 99.2 ± 0.4% 99 ± 0.6% 99.3 ± 0.4% 98.9 ± 0.7% 99.2 ± 0.4% 99.6 ± 0.2% 99.5 ± 0.4% CD44 + 99.1 ± 0.7% 99.1 ± 0.7% 99 ± 0.9% 99.3 ± 0.7% 99.2 ± 0.5% 99.3 ± 0.5% 99.3 ± 0.7% CD105 + 99.0 ± 0.9% 99.2 ± 0.7% 99.1 ± 0.6% 99.2 ± 0.5% 99.3 ± 0.6% 99.1 ± 0.9% 99.5 ± 0.3% CD106 + 99.4 ± 0.5% 99.2 ± 0.5% 99.1 ± 0.7% 99.5 ± 0.2% 99.2 ± 0.5% 99.3 ± 0.7% 99.1 ± 0.8% CD34 + 0% 0% 0% 0% 0% 0% 0% CD45 + 0% 0% 0% 0% 0% 0% 0% viability 98 ± 1% 88 ± 6% 87 ± 6% 85 ± 10% 88 ± 7% 86 ± 10% 88 ± 5%

Changing environmental parameters in the direction of increasing and / or decreasing the concentration of ingredients leads to a decrease in the homogeneity of the target product or the yield of cells.

Long-term storage of MSC cell culture is provided in the inventive medium during freezing in a program freezing apparatus according to a four-stage program, which includes the following steps:

Stage 1 - lowering the temperature from + 4 ° С to -20 ° С with a speed of 1 ° С / min;

Stage 2 - reducing the temperature from -20 ° C to -40 ° C at a speed of 2 ° C / min;

Stage 3 - reducing the temperature from -40 ° C to -80 ° C at a speed of 4 ° C / min;

Stage 4 - reducing the temperature from -80 ° C to -140 ° C at a speed of 20 ° C / min, followed by immersion of the tubes with cell suspension in liquid nitrogen (t = -196 ° C), where MSCs are stored for a long period.

The temperature difference in the chamber of the apparatus and test tubes with cell suspension for the period of crystallization of the liquid phase is set to Δ15 ° C.

A diagram of the process of cryopreservation of cell suspension is presented in figure 1.

The amount of DMSO in the experiment is 10%.

- The ordinate axis shows the time during which the cell suspension was cooled

- The temperature scale is shown on the x-axis

+ 4 ° С - the starting point for cooling the cell suspension

-9.4 ° С - crystallization onset temperature

-5.0 ° С - latent heat emission point

-9.4 ° С - transient phase

-20 ° С - transition to a cooling rate of 2 ° С / min

-40 ° С - transition to a cooling rate of 4 ° С / min

-80 ° С - transition to a cooling rate of 20 ° С / min

-140 ° С - the final point of cooling of the cell suspension and its transfer to containers with liquid nitrogen (-196 ° С / min)

After freezing the cell culture, the ampoules with MSCs are placed in a quarantine cryogenic storage.

If necessary, use MSCs to defrost them. Thawing of the cell suspension is carried out in a water bath at a temperature of + 40 ° C for two minutes or until the ice pieces in the ampoule are completely thawed, which are easily determined visually. After thawing, immediately, ampoules with cell culture are placed in an ice bath to maintain a temperature of + 4 ° C in order to minimize the toxic effect of DMSO on cells.

The cell suspension is thoroughly mixed, an aliquot of 0.3 ml of the suspension is taken and the safety of the cells is determined.

To assess viability, trypan blue dye solution is added to the cell suspension for 2-3 minutes. Dead cells are determined by blue-stained nuclei by microscopy in transmitted light. At the same time, cells are counted.

Infection of cell culture is checked by placing a cell suspension containing 2-5 million cells in an EDTA KE-filled tube to detect virus nucleic acids (HIV 1,2; HBV; HCV; CMV; HSV1; HSV2; HSV4; Epstein-Bar virus ; HTLV) in cellular material.

The data obtained are compared with data from studies of primary material.

Table 2 shows the tests performed with blood, bone marrow and MSCs.

Table number 2
Tests performed with blood, bone marrow and MSCs Research methods Before collection (in peripheral blood) After the fence After separation After defrosting Bone marrow volume + The number of OLS + + + CD 34+ ± + CD Count 71+ + + CD 44+ + CD 90+ + + Number of CD105 + four- Number of CD106 + + + SK vitality + + + Sterility backseeding + + Viral Hepatitis B (HBs Ag) + + (PCR) + (PCR) Viral hepatitis C + + (PCR) + (PCR) Syphilis + HIV infection + + (PCR) + (PCR) CMV + + (PCR) + (PCR) Toxoplasmosis + + (PCR) + (PCR) Herpes virus + + (PCR) + (PCR) Epstein-Bar virus + (PCR) + (PCR) HTLV-I and -II + + (PCR) + (PCR) ±

Cryopreserved MSCs are a ready-made biograft. Figure 2 presents the viability indicators of human MSCs (hMSCs) thawed at different storage periods. Assessment of the viability of cell material using methylene blue dye showed that during defrosting, cell losses are not large and amount to 3-13%, and the percentage of death at different storage periods is not significantly different. In the drawing, the abscissa axis shows months of storage, the ordinate axis represents% of viable cells. After cryopreservation, thawed MSCs retain all their characteristics.

Typical growth curves for human MSCs are shown in FIG. 3. The abscissa axis is the number of days, the ordinate axis is the number of viable cells.

The growth curves of human MSCs isolated from bone marrow of various donors Nos. 1, 2, 3 are graphs 1, 2, and 3. Cells were seeded at an initial density of 1270 cells per cm ² and counted every 2 days. The growth curves of MSC cultures have a long lag period (up to 8-10 days after seeding), after which they are characterized by high growth rates and reach a density of 50 thousand cells per square centimeter within 12-14 days, then the curves go to a plateau.

Analysis of the distribution of hMSCs over the phases of the cell cycle by flow cytometry after the third passage also showed that they actively proliferate in culture and enter the G2 block only when a dense monolayer is reached (data not shown).

To confirm that thawed cells are MSCs, the cell composition of populations of cells isolated from human bone marrow was studied. For this, we used the method of surface staining of cell suspensions with antibodies against hematopoietic cell markers CD45 and CD34 and against MSC markers CD90, CD44, CD105, CD106. The number of CD45 ⁺ cells (hematopoietic cells) ranged from donor to donor in the range of 0-0.5% of the total number of cells. The number of CD34 ⁺ cells (hematopoietic cells) ranged from donor to donor within 0-0.1% of the total number of cells, the number of mesenchymal stem cells (CD90 ⁺ , CD44 ⁺ , CD105 ⁺ , CD106 ⁺ ) ranged from 99 to 99 , 8% of the total number of cells (figure 4). The picture of the cellular composition did not change significantly from reseeding to reseeding during the first three passages after isolation.

To further characterize hMSC cultures, their ability to differentiate in orthodox directions (osteogenic, chondrogenic and adipocytic) (three lineage assay) was tested. Induction of MSC differentiation in orthodox directions led to significant changes in the morphology of both individual cellular elements and the entire population. With osteogenic differentiation by the seventh day, the formation of mineral conglomerates over individual cellular elements was observed. By the fourteenth day in the culture, mineralization fields were formed, covering the entire population of cells. During chondrogenic differentiation on the 7-10th day, a dense cell conglomerate was formed from the cell monolayer, similar in structure to the forming cartilage. With adipogenic differentiation, the preservation of the cell monolayer and the appearance of cells morphologically similar to human adipocytes were observed.

As a result of cryopreservation, biologically active material was obtained in a medium with the patient's blood serum, for which MSCs are subsequently intended. The cell concentration is 4-5 · 10 ⁶ in ml biotransplant.

Autologous blood serum is important both in the process of cryopreservation and when working with a biograft: for example, the patient’s blood group is no longer relevant for treatment with MSCs after their defrosting.

Similar procedures with a positive result were performed on MSCs of animals. Cryopreservation was subjected to MSC pigs. After thawing of cryopreserved cellular material (storage for 12 months), the cells retain viability, are homogeneous in phenotype CD 90+, CD 105+, CD 106+, CD 44+, CD 45-, CD 34-. The number of cells is 91% of the stored.

Cryopreservation does not affect the phenotypic characteristics of MSCs and their proliferative and differentiation potentials. The proposed composition of the ingredients of the medium for cryopreservation of human and animal cells allows you to save cellular material in an autologous environment and maintain cell survival during cryopreservation.

Examples of the invention

Example 1. Cryopreservation of human MSCs.

Mesenchymal stem cells (MSCs) were isolated from the patient’s bone marrow. At the same time as bone marrow sampling for MSCs, 100 ml of peripheral blood was taken from the patient, from which serum was obtained by standard methods under sterile conditions. For this, venous blood is collected in a sterile plastic tube. Before the formation of a clot, the blood is left for 30 minutes - 1 hour at room temperature or placed in a thermostat at 37 ° C. The clot formed is separated from the walls with a glass rod, sometimes shaking the tubes is enough. After that, the blood is centrifuged in glass tubes for 10-15 minutes at 1000-1500 rpm or in plastic tubes centrifuged at 2000-3000 rpm for less time. The resulting serum is transferred to another clean tube. Blood serum is autologous with respect to MSCs and was used for cryopreservation and as a filler in the delivery of cellular material.

The cell culture of MSC obtained by cultivation was removed from the mattresses using trypsin solution with DMEM. Cells were tested for viability and phenotype compliance by generally accepted methods using light microscopy (methylene blue staining) and flow cytometry. Cells have homogeneity in markers CD 90+, CD 105+, CD 106+, CD 44+, CD 45-, CD 34- and are viable (no color when treated with propidium iodide 98 ± 1% of cells). They counted the cells in Goryaev’s cell. The number of cells was 10 million. After this, the cell suspension was centrifuged for 5 minutes at a speed of 1500 rpm. The precipitate was suspended in 5 ml of DMEM medium, then the nutrient medium DMEM = 5 ml was added to a cell concentration of 10-12 · 10 ⁶ in 1 ml, after which a suspension of cells of 3.75 ml of autologous plasma was diluted to a concentration of 8-10 · 10 ⁶ 1 ml .

For cryopreservation, we calculated a medium containing 10% by volume of dimethyl sulfoxide (DMSO), autologous plasma - 20%, antibiotics and sulfonamides (100 IU / ml of penicillin sodium salt, 100 μg / ml streptomycin sulfate), DMEM nutrient medium - 40%. All components are designed for a final concentration in a volume equivalent to the volume of the suspension with the cells to be cryopreserved. DMEM medium was mixed with human blood serum, penicillin, streptomycin until the substances were completely dissolved. In this example, it was 13.5 ml.

Directly before the cryopreservation procedure, DMSO was added to the medium and the mixture was cooled to room temperature (+ 18 ° C - + 22 ° C). Wednesday was added to the MSC.

The cell suspension prepared for cryopreservation was separated by 2 ml into sterile cryovials, then each tube was closed with a lid and transferred to a program freezing apparatus, the cell suspension was cooled to + 4 ° С - + 8 ° С. The same cryopreservation medium and the same concentration of cell suspension obtained from the blood of an adult donor were used as a biological simulator of cell suspension.

They made a freeze in the apparatus according to a four-stage program:

initially cooled to -140 ° C.

Then the material was transferred to liquid nitrogen. They kept MSCs, checking their viability every 4 months, for two years. After thawing, the quality of MSCs and the number of surviving cells were evaluated as described above. After 24 months, the cells were viable (90 ± 2% remained), their uniformity was high:

CD90 + = 99.5 ± 0.4%, CD44 + = 99.3 ± 0.7%, CD105 + = 99.5 ± 0.3%, CD106 + = 99.1 ± 0.8%, CD34 + = 0%, CD45 + = 0%.

Example 2. Cryopreservation of MSCs of animals.

Bone marrow was taken from the head of the femur (a possible fence from the bones of the lower leg: tibia or tibia) pigs. To obtain autologous serum, venous blood of the same animal was used. Pig MSC cells are phenotyped at 6 markers.

MSCs were isolated from pig bone marrow, as in Example 1, by centrifugation at the media boundary: a bone marrow suspension in DMEM - ficoll. 200 ml of blood were drawn from a pig’s vein, from which autologous serum was obtained according to the standard method of Example 1. The cell culture of pig MSCs was accumulated by passaging on mattresses with a nutrient medium. The result was 9.7 × 10 ⁶ cells. Cell suspension was centrifuged for 5 minutes at a speed of 1500 rpm. The precipitate was suspended in 5 ml of DMEM medium, then DMEM = 5 ml was added to a cell concentration of 10-11 · 10 ⁶ in 1 ml, after which a suspension of cells of 3.75 ml of autologous plasma was diluted to a concentration of 8-10 · 10 ⁶ 1 ml .

For cryopreservation, the medium was calculated and prepared according to the method of Example 1.

Cells were placed in a medium for cryopreservation, the suspension was distributed in 2 ml aliquots according to Example 1.

Cells were frozen in the apparatus according to a 4-step program, as for human MSCs.

After defrosting the cellular material, which underwent cryopreservation for 12 months, the cells retain viability, are homogeneous in phenotype CD 90+, CD 105+, CD 106+, CD 44+, CD 45-, CD 34-. The number of cells is 91% of the stored.

Example 3. Cryopreservation of human skin fibroblast cells.

Skin fibroblasts were isolated from the patient’s skin flap. At the same time as the skin flap was taken, 100 ml of peripheral blood was taken from the patient, from which serum was obtained according to the standard method under sterile conditions. For this, venous blood is collected in a sterile plastic tube. Before the formation of a clot, the blood is left for 30 minutes - 1 hour at room temperature or placed in a thermostat at 37 ° C. The clot formed is separated from the walls with a glass rod, sometimes shaking the tubes is enough. After that, the blood is centrifuged in glass tubes for 10-15 minutes at 1000-1500 rpm or in plastic tubes centrifuged at 2000-3000 rpm for less time. The resulting serum is transferred to another clean tube. Blood serum is autologous with respect to skin fibroblasts and has been used for cryopreservation and as a filler in the delivery of cellular material.

The cell culture of skin fibroblasts obtained by cultivation was removed from the mattresses using trypsin solution with DMEM medium. Cells were tested for viability by conventional methods. Cells are homogeneous in morphometric flow cytometry and viable. They counted the cells in Goryaev’s cell. The number of cells was 10 million. After this, the cell suspension was centrifuged for 5 minutes at a speed of 1500 rpm. The pellet was suspended in 5 ml of DMEM medium, then DMEM = 5 ml was added to a cell concentration of 10-12 · 10 ⁶ in 1 ml, after which a suspension of 3.75 ml of autologous serum cells was diluted to a concentration of 8-10 · 10 ⁶ 1 ml .

For cryopreservation, we calculated a medium containing 10% by volume of dimethyl sulfoxide (DMSO), 20% autologous serum, antibiotics and sulfonamides (100 IU / ml of penicillin sodium salt, 100 μg / ml streptomycin sulfate), and DMEM nutrient medium - 40%. All components are designed for a final concentration in a volume equivalent to the volume of the suspension with the cells to be cryopreserved. DMEM medium was mixed with human blood serum, penicillin, streptomycin until the substances were completely dissolved. In this example, it was 13.5 ml.

Directly before the cryopreservation procedure, DMSO was added to the medium and the mixture was cooled to room temperature (+ 18 ° C - + 22 ° C). Wednesday was added to the MSC.

The cell suspension prepared for cryopreservation was separated by 2 ml into sterile cryovials, then each tube was closed with a lid and transferred to a program freezing apparatus, the cell suspension was cooled to + 4 ° С - + 8 ° С. The same cryopreservation medium and the same concentration of cell suspension obtained from the blood of an adult donor were used as a biological simulator of cell suspension.

They made a freeze in the apparatus according to a four-stage program:

initially cooled to -140 ° C.

After defrosting the skin fibroblast cells, the cells were tested for viability and phenotype compliance by generally accepted methods using light microscopy (methylene blue staining) and flow cytometry. Cells are homogeneous in morphometric flow cytometry and viable (no color when treated with propidium iodide 97 ± 1.5% of cells).

Example 3. Cryopreservation of human stem cells.

Cord / placental blood stem cells are a heterogeneous cell population similar in composition to the red bone marrow. Usually they are characterized by a single marker CD 34+.

A cord / placental blood sample was separated by centrifugation into blood plasma and cell suspension. A stem cell concentrate was isolated from a cell suspension of a patient's umbilical / placental blood sample. At the same time, serum was obtained from umbilical cord blood plasma using a standard technique using a coagulant under sterile conditions. The resulting serum is transferred to another clean tube. Blood serum is autologous with respect to stem cell concentrate, and it was used for cryopreservation and as a filler in the delivery of cellular material.

Cord / placental blood stem cell concentrate was obtained by primary processing of cell material (standard procedure) without using in vitro culture techniques. Cells were tested for viability and phenotype compliance by generally accepted methods using light microscopy (methylene blue staining) and flow cytometry. The cell population has standard characteristics for morphometric indicators of flow cytometry (CD34 + - 1.2 ± 0.2%) and viable (no color when treated with propidium iodide 95 ± 1% of cells). They counted the cells in Goryaev’s cell. The number of cells was 10 million. After this, the cell suspension was centrifuged for 5 minutes at a speed of 1500 rpm. The pellet was suspended in 5 ml of DMEM medium, then DMEM = 5 ml was added to a cell concentration of 10-12 · 10 ⁶ in 1 ml, after which a suspension of 3.75 ml of autologous serum cells was diluted to a concentration of 8-10 · 10 ⁶ 1 ml .

For cryopreservation, we calculated a medium containing 10% by volume of dimethyl sulfoxide (DMSO), 20% autologous serum, antibiotics and sulfonamides (100 IU / ml of penicillin sodium salt, 100 μg / ml streptomycin sulfate), and DMEM nutrient medium - 40%. All components are designed for a final concentration in a volume equivalent to the volume of the suspension with the cells to be cryopreserved. DMEM medium was mixed with human blood serum, penicillin, streptomycin until the substances were completely dissolved. In this example, it was 13.5 ml.

Directly before the cryopreservation procedure, DMSO was added to the medium and the mixture was cooled to room temperature (+ 18 ° C - + 22 ° C). Wednesday was added to the MSC.

The cell suspension prepared for cryopreservation was separated by 2 ml into sterile cryovials, then each tube was closed with a lid and transferred to a program freezing apparatus, the cell suspension was cooled to + 4 ° С - + 8 ° С. The same cryopreservation medium and the same concentration of cell suspension obtained from the blood of an adult donor were used as a biological simulator of cell suspension.

They made a freeze in the apparatus according to a four-stage program:

initially cooled to -140 ° C.

After thawing the cell culture, their number decreased by 0.1%.

MSC cells undergone cryopreservation were tested with a positive result in the treatment of cancer, coronary heart disease, idiopathic dilated cardiomyopathy, and allergic diseases. An important role in the treatment is played by the fact that the cells are autologous, and the blood serum for their storage is also autologous. The mechanism of action in each case is different, but everywhere MSCs improve the patient’s quality of life, prevent complications, and provide long-term remission. Typically, MSCs are administered intravenously, 1 million cells per 1 kg of patient weight.

An example of the use of cryopreserved autologous MSCs as a medical biotransplant is the treatment of cancer, in particular invasive bladder cancer. Bone marrow for puncture of MSCs was punctured prior to treatment with chemotherapy, radiation therapy. Cell culture was accumulated by passaging and stored in a cryogenic storage. Cells were homogeneous in 6 markers of MSCs, cells are viable.

Autologous MSCs in a cryopreservation medium (with autologous serum) were introduced to reduce the toxic effect of therapeutic therapy during the course of treatment and at the end of it. The treatment effect was noticeable - patients did not develop complications on the main course of treatment, which usually take place (hematological, cardiological, etc.), prolonging the life of patients significantly exceeds the known terms.

The introduction of a biograft - cryopreserved autologous MSCs in autologous serum - gives a lasting therapeutic effect in the treatment of all diseases where a biograft was used, the effect was especially unexpected in the treatment of allergies.

The result of the invention is the possibility of widespread use of MSCs for medical, cosmetic purposes due to the preservation of cells unchanged. After thawing, the cells are suitable for biotransplantation to the patient, because are his own and are in autologous serum.

Analyzing the results of numerous experiments, it is clear that in the proposed medium, MSCs are preserved during cryopreservation by 85-95%, without changing their cellular characteristics.

Claims (2)

1. The environment for the cryopreservation of human and animal cells, containing a nutrient medium, a cryoprotectant dimethyl sulfoxide, autologous blood serum to conserved cells, characterized in that it additionally contains sulfonamides and antibiotics, and as the nutrient medium, the needle medium in the Dulbecco modification (DMEM) is used in the following the ratio of components, vol.%: Blood serum  45-65 Dimethyl sulfoxide  5-15 Sulfonamides and antibiotics 0.1-1.5 Wednesday DMEM  The rest is up to 100 2. Biotransplant for medical use, characterized in that it is a suspension of phenotypic homogeneous CD 90+, CD 105+, CD 106+, CD 44+, CD 45-, CD 34- and viable mesenchymal stem cells cryopreserved in the medium according to claim 1, wherein the concentration of 6 cells is 4-5 · 10 ⁶ in 1 ml of biotransplant.

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