RU2790701C1 - Method for obtaining a solution of growth factors from platelets - Google Patents

Abstract

FIELD: regenerative medicine.

SUBSTANCE: claimed method for obtaining a solution of growth factors from platelet consists in taking the patient's venous blood, assessing the platelet count at the rate of at least 0.75x10⁹ platelets/ml, two-stage centrifugation of the blood sample, first at 100-1500 g for 3-15 minutes at a temperature of 12-26°C to obtain a plasma fraction with platelets uncontaminated with leukocytes or erythrocytes, then at 300-1500 g for 3-19 minutes at a temperature of 12-26°C for platelet precipitation, removal of the supernatant, followed by resuspension of the precipitated platelets in phosphate-buffered saline solution with the pH of 7.3 or isotonic 0.9% saline, activation of platelets, followed by release of factors by mixing the resulting volume of suspension with two volumes of glass granules 0.5-4.5 mm in diameter, followed by placement in a 10 ml syringe and incubation at a temperature of 18-37°C for 5-25 h, followed by separation of the liquid fraction from the glass granules by squeezing from a syringe and subsequent filtration through a filter with a pore diameter of 0.22 mcm with additional washing of the glass granules with phosphate-buffered saline, followed by isolation by filtration of a solution containing the growth factors, using a centrifuge filter with a pore diameter of 100-500 kDa by 10-60 minute centrifugation at the acceleration speed of 3200-5000 g at a temperature of 4-25° with the subsequent selection of the filtered fraction.

EFFECT: above method provides an increase in the content of growth factors from platelets in solution, which is able to increase the rate of regeneration of damaged tissues.

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Description

The present invention relates to the field of regenerative medicine and concerns a method for obtaining a solution of growth factors from platelets, which can be used to obtain a drug with a high content of autologous growth factors for the regeneration of damaged tissues.

Today, one of the most urgent problems of regenerative medicine remains the safety of biomedical cell products. The use of such drugs should not only promote the activation and maintenance of regenerative processes that contribute to the restoration of the structure and function of tissues, but should not lead to undesirable side effects associated with the impact of biologically active molecules.

Platelet-rich plasma (PRP), a fraction of plasma containing an increased concentration of platelets, has been widely studied and used in regenerative medicine in recent years. The clinical usefulness of PRP is confirmed by the fact that it contains high concentrations of growth factors contained in platelets and normal concentrations of fibrinogen, which synergistically promote the regenerative process [Lubkowska, Dolegowska, Banfi, 2012]. Growth factors (GF) released from activated platelets initiate and modulate healing in both soft and hard tissues [Kawase, 2015]. Platelet alpha granules contain numerous proteins: platelet growth factor (PDGF), transforming growth factor (TGF), interleukins, platelet angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor IGF and fibronectin. All these molecules are involved in such processes as directed transport, proliferation and differentiation of stem cells, having a complex effect on pro/anti-inflammatory and anabolic/catabolic processes in tissues. Based on the above, PRP is a simple, inexpensive, and minimally invasive procedure for delivering high concentrations of autologous RFs and cytokines to injured tissues in physiological proportions. This blood-derived treatment for damaged tissue has been widely used in various fields of medicine and has been shown to be effective.

PRP differs in factor content depending on the preparation protocol, and the main factors affecting the final composition of the product are usually: the initial platelet count, the nature of the anticoagulants used, the presence of leukocytes in the suspension and the activators used, leading to different biological results [Marques et al., 2015]. The term "activation" of platelets refers to two key processes that are initiated during PRP preparation: platelet degranulation to release growth factors from α-granules, and fibrinogen cleavage to initiate the formation of a matrix that allows the formation of a platelet gel and therefore restricts the secretion of molecules to a selected site. [Wasterlain, Braun, Dragoo, 2012]. An activation step prior to PRP administration is included in many protocols in use, usually by the addition of thrombin and/or calcium chloride (CaCl ₂ ), but it is sometimes preferred to administer PRP in an unactivated form, relying on spontaneous platelet activation occurring after exposure to native collagen present in the junctional tissues [Matteo Di et al., 2015]. One of the main disadvantages of using PRP is that in order to achieve the target level of biologically active platelet factors, not only the concentration of platelets is important, but also the form of the drug. The use of drugs with non-activated non-degranulated platelets can lead to unsynchronized and delayed secretion of growth stimulants and anti-inflammatory cytokines.

In medicine, in addition to PRP itself, factors isolated from platelets during activation, as well as recombinant factors, can be used to activate regeneration processes. Platelets contain various biologically active molecules with a natural balance of anabolic and catabolic functions, which helps to optimize the tissue environment and promote the healing process [Qian et al., 2017; Tschon et al., 2011]. Growth factors derived from PRP can promote tissue regeneration by promoting cell migration, proliferation, differentiation, and extracellular matrix synthesis [Gulotta et al., 2011; Isaac et al., 2012]. The main method for obtaining factors from platelets is the preparation of a platelet lysate.

It is known that the ability to adhere to platelets is one of the key functions responsible for the implementation of their biological functions, incl. thrombus formation begins with platelet attachment to the wall of the damaged vessel, followed by the involvement of other platelets and immune cells in the process of thrombus formation and subsequent regeneration. In this case, adhesion leads to the activation of platelets, followed by the release of the contents of their granules [Wu, 2015]. A known way to activate platelets through their adhesion (and subsequent aggregation) is the method of passing blood or plasma through a column containing glass beads [Dobrovolsky, Titaeva, 2015]. This principle was used to ensure the activation of platelets in the composition of PRP, which ensured the release of growth factors into the medium by cells.

Currently, a sufficient number of different methods are known for obtaining and maintaining growth factors in the proper quality, followed by their use in various preparations that can increase the rate of regeneration of damaged tissues. However, almost all known and used methods have a number of disadvantages: insufficiently fast regeneration process after application, the use of substances that can have a negative effect on the body.

Therefore, the search for a more efficient and safer way is the most urgent task.

Known organ-specific regenerant GI, containing the stage of centrifugation of the platelet concentrate, followed by filtration. (RU 2462255 C1, class A61K 35/16, A61L 27/22, A61L 27/44, published on September 27, 2012).

This regenerant includes a fibrin matrix with thrombin, a cryoprecipitate obtained from quarantined fresh frozen donor plasma, which has a destructive effect on the human body.

A known method of obtaining a growth supplement based on a platelet lysate from a platelet mass of donors to a medium for increasing the cell mass of stem, progenitor, differentiated and tumor cells, including normalizing a platelet mass sample to a given platelet concentration by centrifuging the platelet mass and resuspending the sediment in the supernatant of a given volume to the specified platelet concentration. (RU 2648162 C2, class C12N5/071, publ. 22.03.2018).

This method does not allow obtaining the required amount of autologous growth factors due to the destruction of growth factors by freezing and further thawing.

A known method of preparing a platelet lysate with a high content of growth factors, including the following steps: sampling the patient's venous blood, assessing the content of platelets with granules, centrifuging the blood and the resulting plasma, and removing the supernatant. (RU 2739515 C1, class A61K 35/19, G01N 33/49, B01D 21/26, A61P 43/00, published on 12/25/2020).

This method does not allow obtaining the required amount of autologous growth factors due to the lack of stages of mixing the suspension with subsequent separation of the liquid fraction.

The objective of the invention is to develop a method for obtaining a solution of growth factors from platelets in a plasma-free environment.

The technical result of using the invention is to increase the content of growth factors from platelets in solution, which can increase the rate of regeneration of damaged tissues.

The task is achieved in that the method for obtaining a solution of growth factors from platelets includes taking the patient's venous blood, assessing the content of platelets in the blood at a rate of at least 0.75×10 ⁹ platelets/ml, two-stage centrifugation of the blood sample, first at 100-1500 g in for 3-15 minutes at a temperature of 12-26°C to obtain a plasma fraction with platelets without admixture of leukocytes and erythrocytes, then at 300-1500 g for 3-19 minutes at a temperature of 12-26°C to precipitate platelets, remove the supernatant from subsequent resuspension of the precipitated platelets in a phosphate-buffered saline solution with a pH of 7.3 or an isotonic 0.9% sodium chloride solution, platelet activation followed by the release of factors by mixing the resulting suspension volume with two volumes of glass granules with a diameter of 0.5-4, 5 mm, followed by placement in a 10 ml syringe and incubation at a temperature of 18-37°C for 5-25 hours, followed by separation of the liquid fraction from glass granules by squeezing out from a syringe and subsequent filtration through a filter with a pore diameter of 0.22 μm with additional washing of glass granules with phosphate-buffered saline, followed by isolation by filtration of a solution containing growth factors using a centrifuge filter with a pore diameter of 100-500 kDa by centrifugation for 10-60 minutes at an acceleration speed of 3200-5000 g at a temperature of 4-25°C, followed by the selection of the filtered fraction.

In FIG. 1 is a diagram illustrating example 1, showing that the proliferative activity of fibroblasts after exposure to growth factors from platelets increases, where: OD is the optical density of the solution; 1 - non-activated PRP; 2 - PRP activated with 20 mm CaCl ₂ ; 3 - solution containing platelet growth factors obtained after incubation of PRP with glass beads.

In FIG. 2. shows a diagram and photomicrographs illustrating example 2, showing the indicators of fibroblast migration in vitro after adding growth factors from platelets to their culture, where: A - comparison of the number of fibroblasts that migrated to the scraping site; B - image of scrapings after fibroblast migration, obtained using an inverted light microscope; 1 - phosphate buffered saline (pH 7.3); 2 - non-activated PRP; 3 - activated with 20 mm CaCl ₂ PRP; 4 - solution containing platelet growth factors obtained after incubation of PRP with glass beads.

In FIG. 3 is a graph illustrating example 3 showing the concentrations of platelet epidermal growth factor in a solution obtained using the method of obtaining growth factors from platelets compared to PRP and activated CaCl ₂ PRP, determined using enzyme-linked immunosorbent assay, where: 1 - non-activated PRP; 2 - activated with 20 mm CaCl ₂ PRP; 3 - solution containing platelet growth factors obtained after incubation of PRP with glass beads.

The way to obtain a solution of growth factors from platelets is as follows.

Venous blood is taken from the patient into a standard tube with an anticoagulant, which interrupts the clotting process.

Tubes with collected venous blood are placed in a centrifuge and centrifuged for 3-15 minutes at an acceleration speed of 100-1500 g and a temperature of 12-26°C to obtain platelet-free plasma without leukocytes and erythrocytes. Then the content of platelets is assessed at a rate of at least 0.75×10 ⁹ platelets/ml. The platelet count is assessed by cell count by optical microscopy. Table 1 shows the data on the concentration of platelets in plasma after applying different modes of the first centrifugation.

Table 1
Plasma platelet concentrations depending on the first centrifugation mode (acceleration, time, temperature) Acceleration value for the first centrifugation, g Time, min Temperature, °C Concentration
platelets, cells/ml 100 3 12 0.80⋅10 ⁹ 100 6 12 0.83⋅10 ⁹ 100 15 12 0.90⋅10 ⁹ 100 3 19 0.87⋅10 ⁹ 100 6 19 0.83⋅10 ⁹ 100 15 19 0.92⋅10 ⁹ 100 3 26 0.78⋅10 ⁹ 100 6 26 0.86⋅10 ⁹ 100 15 26 0.95⋅10 ⁹ 700 3 12 0.90⋅10 ⁹ 700 6 12 0.84⋅10 ⁹ 700 15 12 0.94⋅10 ⁹ 700 3 19 0.76⋅10 ⁹ 700 6 19 0.78⋅10 ⁹ 700 15 19 0.80⋅10 ⁹ 700 3 26 0.98⋅10 ⁹ 700 6 26 0.93⋅10 ⁹ 700 15 26 0.95⋅10 ⁹ 1500 3 12 1.40⋅10 ⁹ 1500 6 12 1.32⋅10 ⁹ 1500 15 12 1.23⋅10 ⁹ 1500 3 19 1.75⋅10 ⁹ 1500 6 19 1.52⋅10 ⁹ 1500 15 19 1.57⋅10 ⁹ 1500 3 26 1.48⋅10 ⁹ 1500 6 26 1.42⋅10 ⁹ 1500 15 26 1.53⋅10 ⁹

Tubes with collected plasma are centrifuged for 3-19 minutes at an acceleration speed of 300-1500 g and a temperature of 12-26°C to sediment platelets at the bottom of the tube. Then the plasma part is partially removed - 2/3 of the volume of the tube containing platelets, followed by resuspension of platelets in the remaining volume of plasma. The concentration of platelets in the suspension is calculated by optical microscopy. Table 2 presents data on the concentration of platelets in plasma after applying different modes of the second centrifugation.

table 2
Plasma platelet concentrations depending on the second centrifugation mode (acceleration, time, temperature) Acceleration value for the first centrifugation, g Time, min Temperature, °C Platelet concentration, cells/ml 300 3 12 1.30⋅10 ⁹ 300 eleven 12 1.67⋅10 ⁹ 300 19 12 1.80⋅10 ⁹ 300 3 19 1.36⋅10 ⁹ 300 eleven 19 1.43⋅10 ⁹ 300 19 19 1.45⋅10 ⁹ 300 3 26 1.73⋅10 ⁹ 300 eleven 26 1.84⋅10 ⁹ 300 19 26 1.85⋅10 ⁹ 900 3 12 ^1.91⋅109 900 eleven 12 1.87⋅10 ⁹ 900 19 12 ^1.94⋅109 900 3 19 1.72⋅10 ⁹ 900 eleven 19 1.78⋅10 ⁹ 900 19 19 1.82⋅10 ⁹ 900 3 26 1.93⋅10 ⁹ 900 eleven 26 1.95⋅10 ⁹ 900 19 26 1.92⋅10 ⁹ 1500 3 12 2.42⋅10 ⁹ 1500 eleven 12 2.29⋅10 ⁹ 1500 19 12 2.23⋅10 ⁹ 1500 3 19 2.27⋅10 ⁹ 1500 eleven 19 2.32⋅10 ⁹ 1500 19 19 2.72⋅10 ⁹ 1500 3 26 2.41⋅10 ⁹ 1500 eleven 26 2.56⋅10 ⁹ 1500 19 26 2.35⋅10 ⁹

The resulting volume of platelet suspension is mixed with two volumes of glass granules (as an example, it is necessary to indicate what materials the granules are used from) with a diameter of 0.5-4.5 mm, previously washed with a phosphate-saline solution with a pH of 7.3 or isotonic 0.9 % sodium chloride solution, followed by placement in a 10 ml syringe and incubation at a temperature of 18-37°C for 5-25 hours. The concentration of EGF is determined using a commercial ELISA kit for determining the concentration of EGF in biological fluids SEA560Hu (CCC, USA). Table 3 shows the concentration of EGF depending on the diameter of the glass beads, the time and temperature of plasma incubation (PRP) with beads.

Table 3
Plasma EGF concentration (PRP) as a function of glass bead diameter and incubation time Diameter of glass granules, mm Time, h Temperature, °C EGF concentration, pg/ml 0.5 5 37 115±15 0.5 15 28 216±22 0.5 25 18 230±30 2.5 5 37 217±37 2.5 15 28 225±15 2.5 25 18 240±59 4.5 5 37 261±71 4.5 15 28 280±35 4.5 25 18 293±53

The liquid fraction from the glass beads is separated by squeezing from a syringe and then passed through a filter with a pore diameter of 0.22 μm, followed by washing the glass beads with a phosphate-brine solution with a pH of 7.3.

The selection of the fraction containing growth factors is carried out using a centrifuge filter with a cut-off threshold for molecular weight of 10-500 kDa by centrifugation for 10-60 minutes at an acceleration rate of 3200-5000 g at a temperature of 4-25°C, followed by selection of the passed through the filter factions. Table 4 shows the concentrations of EGF in solution after passing the obtained liquid fraction through the filter, depending on the speed of centrifugation, cut-off threshold by molecular weight, duration of centrifugation and temperature.

Table 4
EGF concentration in solution as a function of the molecular weight cut-off of the filter used and centrifugation time The value of acceleration during centrifugation, g Cut-off threshold (limit) by mol. mass, kDa Time, min Temperature, °C EGF concentration, pg/ml 5000 10 10 4 40±10 4000 10 35 15 35±9 3200 10 60 25 57±35 5000 300 10 4 150±15 4000 300 35 15 171±45 3200 300 60 25 183±35 5000 500 10 4 160±32 4000 500 35 15 210±10 3200 500 60 25 315±23

An increase in the content of growth factors from platelets is provided by contact activation of platelets, followed by secretion of the contents of the granules into the solution when interacting with the glass granulate. The method for obtaining growth factors includes the stage of mixing the volume of the required platelet suspension with two volumes of glass granules, previously washed with phosphate-buffered saline or isotonic sodium chloride solution, which subsequently does not have a destructive effect on the human body, followed by incubation at a temperature of 18-37°C for 5-25 hours with further separation from glass beads and filtration of the resulting solution.

Below are examples of a specific implementation of the invention, proving the achievement of the specified technical result.

Example 1

Activation of fibroblast proliferation by growth factors derived from platelets.

L929 mouse fibroblast cells were thawed after cryopreservation and cultured in DMEM nutrient medium supplemented with 10% FBS, 4 mM L-glutamine after 1-2 passages. Cells were removed from the culture mat with a trypsin/EDTA solution (0.25%). The cell concentration was adjusted to 50,000 cells/ml and the suspension was transferred to a 96-well plate at 100 μl per well. The cell plate was incubated overnight at 37°C, 5% CO ₂ . The next day, non-activated PRP, PRP activated with 20 mM CaCl ₂ were added to the cells - for this, CaCl ₂ was added to the platelet suspension at a final concentration of 20 mM and incubated for 30 min, or a solution containing platelet growth factors obtained after incubation of PRP with glass granules. The solutions were normalized according to the content of EGF in them - the final concentration of EGF in the solution should be 50 ng/ml (the concentration is preliminarily estimated using enzyme immunoassay and a commercial kit for determining the concentration of EGF in plasma). After dilution, the solutions were further filtered through a 0.22 µm filter. Then, 20 μl of solutions were added to the wells containing cells. Incubated 24 hours at 37°C, 5% CO ₂ . The proliferative activity of fibroblasts was assessed using a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). The optical density of the solution was read at 570 nm using a spectrophotometer. The addition of a solution containing platelet growth factors, obtained after incubation of PRP with glass beads, led to the activation of the proliferative activity of fibroblasts (Fig. 1).

Example 2

Activation of fibroblast migration in vitro by growth factors derived from platelets.

L929 mouse fibroblast cells were thawed and cultured in DMEM nutrient medium supplemented with 10% FBS, 4 mM L-glutamine after 1–2 passages. The cell concentration was adjusted to 500,000 cells/ml and the suspension was transferred into 6-well plates (2 ml per well) and then incubated at 37°C, 5% CO_2.When 95% culture density was reached, each well was scraped in a straight line using a 200 µl pipette tip. The well was washed three times with sterile phosphate-buffered saline pH 7.3 and filled with solutions: phosphate-buffered saline (pH 7.3), non-activated PRP; PRP activated with 20 mM CaCl₂; a solution containing platelet growth factors obtained after incubation of PRP with glass beads. Cell culture plates were placed in an incubator for 24 h (37°C, 5% CO₂) and then examined with an inverted optical microscope. In each well, the number of migrated cells was counted. The addition of platelet-derived growth factors to the fibroblast culture resulted in the activation of fibroblast migration in vitro (FIG. 2).

Example 3

Preparation of a solution containing platelet growth factors: a solution containing growth factors of platelets obtained after incubation of PRP with glass beads contains a concentration of EGF greater than PRP activated by CaCl ₂ or non-activated PRP.

The concentration of epidermal growth factor was determined by immunoassay using a commercial kit for determining the concentration of human EGF (Cloud-Clone corp., USA) in the following solutions: non-activated PRP, PRP activated with 20 mM CaCl ₂ , and also in a solution containing growth factors platelets obtained after incubation of PRP with glass beads. The obtained results of measuring the concentration of EGF showed that the proposed method allows to obtain a solution with a concentration of EGF higher than for PRP activated with CaCl ₂ (Fig. 3).

Claims (1)

A method for obtaining a solution of growth factors from platelets, including taking the patient's venous blood, assessing the content of platelets in the blood at a rate of at least 0.75x10 ⁹ platelets/ml, two-stage centrifugation of the blood sample, first at 100-1500 g for 3-15 minutes at a temperature 12-26 °C to obtain a plasma fraction with platelets without admixture of leukocytes and erythrocytes, then at 300-1500 g for 3-19 minutes at a temperature of 12-26 °C to precipitate platelets, remove the supernatant, followed by resuspension of the precipitated platelets in phosphate- buffered saline solution with a pH of 7.3 or isotonic 0.9% sodium chloride solution, activation of platelets, followed by the release of factors by mixing the resulting volume of suspension with two volumes of glass beads with a diameter of 0.5-4.5 mm, followed by placement in syringe for 10 ml and incubation at a temperature of 18-37 ° C for 5-25 hours, followed by separation of the liquid fraction from the glass granules by issuing pouring from a syringe and subsequent filtration through a filter with a pore diameter of 0.22 μm with additional washing of the glass granules with phosphate-buffered saline, followed by isolation by filtration of a solution containing growth factors using a centrifuge filter with a pore diameter of 100-500 kDa by 10-60 minute centrifugation at an acceleration rate of 3200-5000 g at a temperature of 4-25 ° C, followed by the selection of the filtered fraction.

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