RU2740152C1 - Method for activation of proliferative response of fibroblast-like cells of stromal-vascular fraction of adipose tissue - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to a method for activation of a proliferative response of fibroblast-like cells of stromal-vascular fraction (SVF) of adipose tissue. Method involves adipose tissue sampling, separation of SVF-cells and counting of recovered cells. Activity of glucose-6-phosphate dehydrogenase (G6PDH) enzymes, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) are then determined using a bioluminescent method. If fibroblast-like SVF cells reduce activity of one, two or three enzymes, then to increase efficiency of proliferative response in culture fibroblast-like cells of SVF added required amount of corresponding substrates for those enzymes, which activity is reduced: glucose-6-phosphate in amount of 2.12 mg/ml with G6PDH activity below 84.3 mcU/104, glyceraldehyde-3-phosphate in amount of 0.45 mg/ml with LDH activity below 250.42 mcU/104, glutamic acid in amount of 1.6 mg/ml with MDH activity below 212.34 mcU/104.EFFECT: invention allows for 30–40 minutes from the moment of biological material sampling to assess the fibroblast-like cell metabolism state, accelerate division and differentiation of fibroblast-like SVF cells by 35 % and more, as well as prolonged proliferative response.1 cl, 3 ex, 2 tbl

Description

The invention relates to medicine, namely to regenerative medicine, and can be used to increase the effectiveness of anti-burn therapy, as well as in the treatment of diabetic ulcers.

Despite the fact that a lot of time has passed since the discovery of the stromal-vascular fraction (SVF) of cells, SVF is most often considered as a source of mesenchymal stem cells (MSC) of adipose tissue [1]. It is known that MSCs have the ability to differentiate into fibroblast cells, but this process requires long-term (25-40 days) cultivation in a sterile box. This manipulation carries risks associated with unwanted MSC differentiation into non-target populations, the possibility of contamination and death of the culture during cultivation, and the need for constant monitoring of MSC stimulation to obtain a pool of target cells. At the same time, the use of SVF itself is devoid of these drawbacks and makes it possible to provide a treatment service almost immediately after hospitalization (after 4-5 hours). In some diseases, it is very important to shorten the time from the moment of sampling to the introduction of the cell preparation [2]. In this regard, it becomes necessary to develop a method that makes it possible to increase the efficiency of the proliferative response of fibroblast-like SVF cells, when used directly in SVF therapy.

A known method of activating the proliferative response of fibroblasts cultured on conductive nanotube substrates under the influence of a local electric field [4]. The cells are cultured on coverslips coated with a layer of nanotubes in Eagle's medium with the addition of 10% fetal calf serum in a CO ₂ incubator at 37 ° C for 72 hours. For electrical stimulation, a system of local supply of electric potential to cells through nanoscale electrodes is used. Electrical stimulation continues for another 48 hours. The specified method is able to stimulate the proliferation of fibroblasts, however, due to the need for a constant long-term effect on cells, its use on damaged tissues and in a short time is impossible. The method itself is very laborious and has a high cost due to the need to constantly apply a layer of nanotubes for each sample.

A known method of activating the proliferative response of fibroblasts under the influence of cytokines produced by mononuclear phagocytes [5, 6]. These include heparin-associated epidermal growth factor (HB-ECF), produced by macrophages and stimulating fibroblast proliferation, fibroblast growth factor (FGF) and its analogues [7, 8], as well as basic fibroblast growth factor (bFGF), which is part of the molecule natural fibroblast growth factor [9]. These agents are able to stimulate the proliferation of fibroblasts. However, their use in short-term cultivation is very difficult due to the complexity and high cost of industrial synthesis, as well as the possibility of the subsequent development of allergic reactions in a patient due to the high molecular weight of the drug.

A known method of activating the proliferative response of fibroblasts using periodontal peptides [10]. Periodontal peptides dose-dependently and selectively stimulate fibroblast proliferation. This effect is manifested in an increase in the number of cells and the proliferation index. Periodontal peptides are active in the concentration range from 0.5 to 50 μg / ml. Moreover, a dose-effect is traced - with an increase in the concentration of periodontal peptides in the culture medium, their specific activity increases. However, periodontal peptides have low biological activity. Thus, the maximum activity of a substance in a culture of fibroblasts is manifested at a concentration of 50 μg / ml. This requires the use of large doses of the drug and entails the possible occurrence of allergic and other unwanted side effects.

The closest analogue of the claimed invention is a method for activating the proliferative response of fibroblasts under the action of the peptide Lys-Glu-Glu-Leu-Asn-Glu [11]. Fibroblasts are isolated according to a standard technique and introduced into medium 199 containing lactalbumin hydrolyzate and 20% fetal calf serum in a 1: 1 ratio. The peptide Lys-Glu-Glu-Leu-Asn-Glu is introduced into the culture of fibroblasts at concentrations from 0.005 to 0.5 μg / ml. The cells are cultured for 72 hours at 37 ° C. On microscopic assessment, under the influence of the Lys-Glu-Glu-Leu-Asn-Glu peptide, the number of fibroblasts increases, and the dose-effect takes place. Peptide Lys-GIu-Glu-Leu-Asn-Glu stimulates fibroblast proliferation in the concentration range from 0.01 to 0.5 μg / ml. This method is able to stimulate the proliferation of fibroblasts, however, due to the need for prolonged incubation in vitro, its use directly on the day of sampling is not possible. Also, the method does not take into account the initial metabolic state of SVF cells.

The objective of the invention is to provide a method for a personalized approach to activating the proliferative response of fibroblast-like cells obtained from SVF adipose tissue.

The task is carried out due to the fact that adipose tissue is taken from patients, from which SVF cells are isolated, after which the isolated cells are counted. Then, the bioluminescent method determines the activity of enzymes: glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). If the activity of one, two or three enzymes is reduced in the fibroblast-like cells of SVF, then to increase the efficiency of the proliferative response, the required amount of the corresponding substrates for those enzymes whose activities are reduced is added to the culture of fibroblast-like cells of SVF: glucose-6-phosphate in an amount of 2.12 mg / ml with G6PDH activity below 84.3 μE / 10 ⁴ . glyceraldehyde-3-phosphate in an amount of 0.45 mg / ml with an LDH activity below 250.42 μE / 10 ⁴ , glutamic acid in an amount of 1.6 mg / ml with an MDH activity below 212.34 μE / 10 ⁴ .

The values 84.3 μE / 10 ⁴ for G6PDH, 250.42 μE / 10 ⁴ for LDH and 212.34 μE / 10 ⁴ for MDH were obtained empirically, based on the bioluminescent analysis of the activity of these enzymes in proliferating fibroblast-like cells of SVF. At these values of enzyme activity, the proliferative activity of fibroblast-like cells in SVF was 65% and higher.

The concentration levels of glucose-6-phosphate, glyceraldehyde-3-phosphate and glutamic acid were also selected empirically on the basis that when the indicated concentrations of these metabolites were reached in the culture of proliferating fibroblast-like SVF cells (in the case of low enzyme activity), the proliferative activity of fibroblast-like SVF cells reached 65% or more. With an increase in the concentration levels of these metabolites in the culture of proliferating fibroblast-like SVF cells (in the case of low enzyme activity), the proliferative activity of fibroblast-like SVF cells did not increase.

In the treatment of skin lesions, the most promising candidates are multipotent MSCs isolated from the SVF of adipose tissue, which have a pronounced anti-inflammatory and regenerative potential and are capable of transformation into fibroblast differention cells. [13]. The number of MSCs. which can be isolated from adipose tissue is 100-1000 times higher than their amount contained in an equivalent volume of bone marrow [14]. In addition, MSCs isolated from adipose tissue are genetically more stable over a long term of cultivation, are characterized by a lower aging coefficient and high proliferative activity. SVF cells have anti-inflammatory and immunomodulatory effects. The functional activity, proliferation and differentiation of SVF cells are largely determined by the level of activity of their metabolic processes. Thus, G6PDH is an initializing and key enzyme of the pentose phosphate pathway, which supplies ribose and deoxyribose for macromolecular synthesis reactions, including for the synthesis of DNA and RNA [15]. Glucose-6-phosphate is a key metabolite for the stimulation of LDH and the final step in anaerobic glycolysis. Glyceraldehyde-3-phosphate is a key metabolite for stimulating the redox stage of anaerobic glycolysis [16]. Malate dehydrogenase is involved in catalysis at the final stage of the tricarboxylic acid cycle, which, in turn, is key for aerobic respiration. Glutamic acid, realizing the relationship between the tricarboxylic acid cycle and amino acid metabolism reactions, stimulates both metabolic processes with the activation of cell proliferation and differentiation.

The method is as follows.

In patients with burn injuries, diabetic ulcers and other injuries of the skin, adipose tissue is taken in a volume of at least 50 ml. Dilute the sample with an equal volume of buffered saline and centrifuge at 430g for 10 minutes. Then select the target cells. An equal volume of type II collagenase solution (with an activity of at least 125 U / mg) is added to the washed material and the mixture is incubated at 37 ° C for 30-60 minutes on a preheated orbital shaker with a rotation speed of 250 rpm. Upon completion of the incubation, an equal amount of buffered saline is added and the sample is poured into 50 ml tubes with a conical bottom, after which it is centrifuged for 10 minutes at 600g and a temperature of 4 ° C [12]. The resulting cell suspension is filtered through a filter with a hole diameter of less than 100 µm and the number of cells is counted. The result is a ready-made sterile suspension of SVF cells in saline. 1 million isolated SVF cells are used to determine the activity of G6PDG, LDH and MDH by one of the known methods, for example, bioluminescent [3]. For this, a suspension of isolated SVF cells (1.0 million / ml) is destroyed by osmotic lysis with the addition of distilled water (1: 5 by volume) and 1.0-2.0 mM dithiothreitol. Then directly determine the activity of G6PDG, LDH and MDH. For this, 50 μl of a suspension of destroyed cells is added to 150 μl of an incubation mixture containing an appropriate substrate and cofactor. Specific values of the concentrations of substrates and coenzymes, as well as the pH of the medium for the determined enzymes are presented in Table 1.

After incubation of the test samples at 37 ° C (for 30 minutes for G6PDG and MDG; 5 minutes for LDH), 50 μl of flavin mononucleotide (FMN) at a concentration of 1.5 × 10 ^-5 M, 50 μl 0, is added to 200 μl of the incubation mixture. 0005% myristic aldehyde and 10 μl of the enzymatic system NADH: FMNoxidoreductase-luciferase (all reagents of the bioluminescent system are diluted in 0.1 M K ⁺ , Na ⁺ -phosphate buffer with pH 7.0). After mixing the bioluminescent reagents and the incubation sample, the luminescence is measured using a biochemiluminometer, for example, the BLM-3607 brand.

Taking into account that the cells have a certain amount of substrates for the course of various metabolic reactions, including those catalyzed by the studied enzymes, the parameters conventionally called "the substrate background of enzymes" are determined. Determined under the same conditions as for the above dehydrogenases, but the buffer is added to the incubation mixture instead of the corresponding substrate. As a result of measuring the luminescence on a biochemiluminometer, the relative values of the activity of the studied enzymes are obtained. To obtain the absolute values of activity, graphs of the dependence of the intensity of bioluminescence on the concentration of NAD (P) H are plotted (calibration graph). To do this, 200 μl of a standard solution of NAD (P) H in the range of 10 ^-9 -10 ^-4 M is introduced into the cuvettes of the biochemiluminometer containing FMN, myristic aldehyde and NAD (P) H: FMNoxidoreductase-luciferase in the concentrations indicated above, after which they produce measuring the intensity of bioluminescence. Due to the wide range of pH buffers used to determine the dehydrogenase activity, as well as the pH dependence of the bioluminescence of the enzyme system from luminous bacteria, calibration curves are plotted for each pH buffer. The enzyme activity is calculated by the formula:

A = Δ [C] × V / T

Where:

A-activity of the enzyme, E per 1 × 10 ⁴ neutrophils (1E = 1 μmol / min [16]);

Δ [C] is the difference in the concentrations of NADH or NADPH in the "enzyme" and "enzyme background" samples, μmol;

V is the sample volume, ml;

T - incubation time, min.

Then determine the substrate that must be added to the incubation medium to activate and maintain the proliferative response of fibroblast-like SVF cells. The culture of proliferating fibroblast-like cells SVF added glucose 6-phosphate in an amount of 2.12 mg / ml G6PDH activity at below 84.3 MKE / 10 ⁴ glyceraldehyde 3-phosphate in an amount of 0.45 mg / ml LDH activity at below 250 , 42 μE / 10 ⁴ , glutamic acid in an amount of 1.6 mg / ml with an MDH activity below 212.34 μE / 10 ⁴ .

With a decrease in the activity of two or three enzymes in the fibroblast-like cells of the SVF of the patient, several substrates, if necessary, are added to the culture of proliferating cells to increase the efficiency of the proliferative response.

Control of proliferative activity of fibroblast-like cells of SVF is carried out as follows.

The isolated fibroblast-like cells of the SVF are stained with the fluorescent dye carboxyfluorescein-diacetate-succinimidyl ether (CFSE) at a concentration of 3 μl / 10 ⁷ cells and placed in a thermostat for 20 minutes at 37 ° C, after which they are washed three times with a solution of complete nutrient medium (medium RPMI 1640, 10 % (of the total volume) of autologous serum, an antibiotic (for example, gentamicin at a concentration of 4-6 μl / ml)) and divide the cells into two samples. An experimental sample of cells is placed in an incubation mattress, 5 ml of complete nutrient medium is added, as well as glucose-6-phosphate, glyceraldehyde-3-phosphate, glutamic acid in accordance with the above method and placed in a CO ₂ incubator at 37 ° C for 120 hours ...

A control sample of cells is placed in an incubation mattress, 5 ml of complete culture medium is added and placed in a CO ₂ incubator at 37 ° C for 120 hours. After 120 hours of incubation, both cell samples are collected and washed twice from the nutrient medium with Hanks solution. In a cuvette, take 100 μl of a suspension of proliferating fibroblast-like SVF cells. Then add ready-made monoclonal antibodies CD34, CD45, CD73, CD90 carrying a fluorescent label FITC (fluoresceinisothiocyanate), PE (phycoerythrin), PC5 (phycoerythrin-cyanin 5) PC7 (phycoerythrin-cyanin 7), in an amount corresponding to the manufacturer's recommendations.

The mixture is thoroughly mixed and incubated at room temperature for 20 minutes in a place protected from direct sunlight. Evaluation of the proliferative response of fibroblast-like SVF cells is carried out on the basis of a decrease in CFSE fluorescence on a flow cytometer (for example, Navios (BeckmanCoulter, USA)) To improve the accuracy of the analysis, it is necessary to analyze at least 50,000 fibroblast-like SVF cells.

This method has been tested on 25 patients with burn injuries of varying severity table 2.

According to the results of examination of burn patients according to the claimed method, it was found that against the background of a decrease in the activity of G6PDG. MDH and LDH, when glucose-6-phosphate, glyceraldehyde-3-phosphate, glutamic acid are added to the SVF cell culture, the number (in percent) of activated SVF fibroblast-like cells involved in the proliferative response relative to control values increases in all 25 patients. According to the results of further clinical observation, the described dependence was found to coincide in 100% of cases.

Clinical example 1.

Patient D., 43 years old, was admitted to the burn department of the Krasnoyarsk Regional Hospital No. 1 on 05.22.2019. He received an injury due to contact with hot bitumen during repair work on the garage roof. After surgical treatment of the burn surface, a contact burn with hot bitumen of the left foot of IIIB degree with an area of up to 2% was diagnosed.

The study was carried out using the claimed method. Material was collected in a volume of 50 ml of adipose tissue. Produced the isolation of SVF cells by the method of sequential fermentation, in the amount of 8 × 10 ⁷ cells / ml. A bioluminescent study of enzyme activity was carried out. A decrease in LDH activity was found (below 250.42 μE / 10 ⁴ ). The culture was supplemented with the substrate glyceraldehyde-3-phosphate in the amount of 0.45 mg / ml. As a result, 12 × 10 ⁷ cells / ml were obtained. According to the results of phenotyping, an increase in the proliferative response of SVF fibroblast-like cells was found by 35%.

Clinical example 2.

Patient V., 35 years old, was hospitalized in the burn department of the Krasnoyarsk Regional Hospital No. 1 on 08/04/2019. He was injured as a result of overturning a pot of hot water. Surgical treatment of the burn surface was performed under anesthesia, and a thermal burn of both thighs and legs of I-II-IIIA degree with a total area of 12% was diagnosed.

The study was carried out using the claimed method. Material was collected in a volume of 50 ml of adipose tissue. Produced the isolation of SVF cells by the method of sequential fermentation in the amount of 4.7 × 10 ⁷ cells / ml. A bioluminescent study of enzyme activity was carried out and a decrease in the activity of enzymes was established: G6PDH (below 84.3 μE / 10 ⁴ ) and MDH (below 212.34 μE / 10 ⁴ ). Substrates glucose-6-phosphate in the amount of 2.12 mg / ml and glutamic acid in the amount of 1.6 mg / ml were added to the culture of fibroblast-like cells of SVF. As a result, 7.6 × 10 ⁷ cells / ml were obtained. According to the results of phenotyping, an increase in the proliferative response of SVF fibroblast-like cells was established by 38%.

Clinical example 3.

Patient S., 28 years old, was hospitalized in the burn department of the Krasnoyarsk Regional Hospital No. 1 on March 12, 2019, the injury was received from falling into a fire and burning clothes. Surgical treatment of the burn surface under anesthesia was performed; a thermal burn with a flame of the lumbar region, the anterior abdominal wall IIIA-IIIB degree with a total area of 15% was diagnosed.

The study was carried out using the claimed method. Material was collected in a volume of 49 ml of adipose tissue. Produced the isolation of SVF cells by the method of sequential fermentation in the amount of 5.3 × 10 ⁷ cells / ml. A bioluminescent study of enzyme activity was carried out and a decrease in the activity of enzymes was established: G6PDH (below 84.3 μE / 10 ⁴ ), MDH (below 212.34 μE / 10 ⁴ ) and LDH (below 250.42 μE / 10 ⁴ ). The corresponding substrates were added to the culture of fibroblast-like SVF cells: glucose-6-phosphate in the amount of 2.12 mg / ml, glyceraldehyde-3-phosphate in the amount of 0.45 mg / ml, and glutamic acid in the amount of 1.6 mg / ml. As a result, 9.2 × 10 ⁷ cells / ml were obtained. According to the results of phenotyping, an increase in the proliferative response of SVF fibroblast-like cells by 41% was found.

The technical result of the proposed method:

- The ability to assess the state of metabolism of fibroblast-like cells within 30-40 minutes from the moment of taking biological material;

- Individual metabolic activation of the proliferative response of fibroblast-like cells, accelerating their division and differentiation by 35% or more;

- Possibility of long-term maintenance of the proliferative response due to periodic introduction of substrates and constant monitoring of the state of cells;

- Obtaining active fibroblast-like cells capable of exerting a pronounced therapeutic effect.

Thus, the method is effective, meets modern requirements for methods of regenerative medicine, and allows to obtain activated fibroblast-like cells in a short time.

Information sources

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Claims (1)

A method of activating the proliferative response of fibroblast-like cells of the stromal-vascular fraction (SVF) of adipose tissue, including the collection of adipose tissue, isolation of SVF cells, counting the isolated cells, characterized in that the bioluminescent method determines the activity of enzymes: glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH), and if the activity of one, two, or three enzymes is reduced in the fibroblast-like cells of SVF, then to increase the efficiency of the proliferative response, the required amount of the corresponding substrates for those enzymes whose activities are reduced: glucose 6-phosphate in an amount of 2.12 mg / ml with G6PDH activity below 84.3 μE / 10 ⁴ , glyceraldehyde-3-phosphate in an amount of 0.45 mg / ml with LDH activity below 250.42 μE / 10 ⁴ , glutamic acid in the amount of 1.6 mg / ml with an MDH activity below 212.34 μE / 10 ⁴ .