RU2803918C1 - Method of producing purified cultured blood serum depleted in growth factors - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the production of components of nutrient media for eukaryotic cell cultures.

EFFECT: obtaining a method of producing purified cultured blood serum depleted in microvesicles and containing a reduced amount of growth factors and other biologically active molecules.

1 cl, 2 dwg, 2 ex, 1 tbl

Description

The invention relates to the field of biotechnology, namely the production of components of nutrient media for eukaryotic cell cultures. A method has been proposed for obtaining purified cultured blood sera depleted in microvesicles and containing a reduced amount of growth factors and other biologically active molecules. The proposed method involves adding a chitosan solution, preferably with a molecular weight of at least 200 kDa, in a 1% (w/v) acetic acid solution to serum samples, incubating the mixture for 1 to 3 hours at 4°C, neutralizing, centrifuging and collecting supernatant, which is serum depleted of microvesicles and the growth factors they contain.

The technical result is the optimization of the process of obtaining serums depleted in vesicular growth factors, allowing to reduce the time spent and operating hours of expensive equipment.

BACKGROUND ART

Microvesicles are a heterogeneous collection of biological structures bound by membranes. Such structures have a bilipid layer, and they can be located inside the cell or in the extracellular space. The size of microvesicles ranges from approximately 20 to 1000 nm. Being fragments of the cell membrane, microvesicles retain a characteristic negative charge due to the difference in the concentration of ions inside and outside the vesicle.

Exosomes are microscopic extracellular vesicles with a diameter of 30 to 100 nm with a bilipid layer, secreted into the intercellular space by cells of various tissues and organs. Exosomes differ from other vesicles in that they are formed in the intracellular space and then secreted by the cell. The protein composition of exosomes largely reflects their origin from endosomes and differs somewhat depending on the type of cells in which they are formed (RU 2748234 C2).

Microvesicles and exosomes can be released by most types of cells and are found in many biological fluids, including blood plasma. Significant biological activity associated with the paracrine regulation factors included in their composition determines the interest of researchers in this group of subcellular structures. In this regard, a significant part of the work that makes up the current state of the art is associated with the collection of microvesicles or exosomes for their further use. In this case, the source of microvesicles is often conditioned culture media, i.e. cell culture media in which cells secreting microvesicles have been previously cultured. Collection of microvesicles is typically carried out using one of the four main methods described in Table 1, or a combination of them, with some methods being preparatory to the use of others.

At the same time, there is an opposite need - to obtain biological substances depleted in exosomes. These include, for example, horse blood serum, used as a growth factor-poor substitute for bovine fetal blood serum. Such serums can be used, for example, in myosatellite cell differentiation protocols, where a depleted environment is purposefully created to induce a cellular response. At present, a sufficient amount of data has been accumulated to suggest that the concentration of microvesicles in the serum determines its effect on the behavior and phenotype of cultured cells (DOP. 10.3402/jev.v3.24783).

The current situation makes the search for new ways to obtain depleted whey a promising area of research. The use of polymers for their deposition has a number of advantages, the main of which is the least dependence on the instrumentation of the institution in which the corresponding protocol is implemented. Thus, when using ultracentrifugation, it is necessary to carry it out on special expensive brands of centrifuges that provide acceleration of over 100,000 g for at least 1 hour, often with constant cooling, and immunoaffinity capture requires organization of work with antibodies in the institution.

There is a known method for the precipitation of microvesicles with polyethylene glycol or its mixture with protamine (DOT. 10.3892/ijmm.2016.2759). In this work, the authors collected microvesicles from the culture medium and biological fluids in which stem cells were grown. Precipitation was carried out by adding a solution of polyethylene glycol or a mixture of polyethylene glycol with protamine to the culture medium in a ratio of 1:4 and incubating overnight at 4°C, followed by centrifugation at 1,500 g for 30 minutes. In a similar manner, it is theoretically possible to remove microvesicles not only from culture media, but also from sera, as well as other solutions containing microvesicles that need to be purified from them.

The disadvantage of the method is its significant duration, which can lead to disintegration or damage of microvesicles and lead to the release of their components into the liquid phase of the treated solution, which prevents its depletion in growth factors. Another potential disadvantage is the use of polyethylene glycol, which cannot be biodegraded under cell culture conditions and will therefore be present in samples later and require additional purification steps to be included in the protocol.

There is a known method for the accelerated collection of microvesicles using polyethylene glycol (patent RU 2750928 C1) by preliminary centrifugation. In the invention, samples of conditioned cell medium were first centrifuged at 5,000 g for 30 minutes at 4°C, then the supernatant was collected and centrifuged at 17,000 g for 1.5 hours at 4°C. The sediment after the second centrifugation was resuspended in phosphate-buffered saline and incubated for an hour with a polyethylene glycol solution in a 1:1 ratio. After incubation, polyethylene glycol with microvesicles was pelleted by centrifugation at 1500 g for 30 minutes at 4°C.

One of the disadvantages of this method is the use of resuspended sediment obtained after fractional centrifugation as a substrate for isolating microvesicles. Since centrifugation is carried out at relatively low accelerations, there is a risk of incomplete sedimentation of small microvesicles from solution. The authors qualitatively confirmed the presence of microvesicles smaller than 100 nm after isolation, but did not quantify their content. However, the need to centrifuge samples at 17,000 g imposes restrictions on institutions that do not have sufficiently powerful equipment. Also considered a disadvantage is the significant amount of polyethylene glycol in the final sample associated with the use of polyethylene glycol in a 1:1 ratio at concentrations up to 32%.

There is a known method RU 2 748 234 C2, which consists in preliminary purification of the conditioned medium by centrifugation at 500 g for 5 minutes and filtering through a filter with a pore diameter of 0.22 μm. The prepared medium was mixed with polymers immobilized on magnetic particles, including chitosan, cellulose sulfate, and polyethylene glycol, incubated for 5 hours at 4°C, the magnetic particles were separated and eluted with saline buffer overnight at 4°C.

The disadvantage of this method is the need to provide a substrate for the immobilization of exosome-binding ligands, which makes preparation for isolation, as well as the process of collecting microvesicles, extremely laborious. In addition, the use of magnetic particles as a substrate makes it necessary to have a magnetic separator in the laboratory. Expanding the list of equipment required to set up the technique is in many cases undesirable. Despite the apparent disadvantage associated with the duration of the method, it should be noted that the main part of the process occurs after the separation of microvesicles and medium, which makes it theoretically suitable for obtaining depleted sera.

The closest to the proposed solution, in terms of the underlying principle, is a method involving the binding of microvesicles to a chitosan solution and subsequent centrifugation (DOI: 10.1002/jev2.12138). Chitosan with a molecular weight of 60-120 kDa and a degree of deacetylation of 75% was used as a solution in 1% acetic acid with or without neutralization with sodium hydroxide. In this work, the isolation of microvesicles is achieved as follows. The biological fluid or conditioned culture medium was purified by sequential centrifugation at 4°C at 3,000 g and 17,000 g for 5 and 15 minutes, respectively. Chitosan was added to the medium thus cleared of large debris, and then incubated at room temperature for an hour. Then the mixture was centrifuged at 12,000 g for 15 minutes at 4°C, and the chitosan precipitate with microvesicles was washed with phosphate-buffered saline.

Despite the significant simplification and accessibility of the technique, this method has a number of disadvantages, which include the use of chitosan of medium molecular weight, which increases the requirements for acceleration during centrifugation. When using chitosan of a different molecular weight, different conditions may be required for sediment formation.

The purpose of the present invention is to optimize the process of removing microvesicles from culture serum to sustainably obtain a product depleted of vesicular growth factors, allowing to reduce the time and operating hours of expensive equipment.

To achieve this goal, a solution of chitosan, preferably with a molecular weight of at least 200 kDa, in a 1% (w/v) acetic acid solution is added to the serum samples. The mixture is thoroughly mixed and incubated for 1 to 3 hours at 4°C, after which it is neutralized until opalescence appears, preferably adding dropwise a 5% solution of potassium or sodium hydroxide. The opalescent mixture is incubated for an additional 30-60 minutes, after which it is centrifuged at 12,000 g for 20 minutes and the supernatant, which is a serum depleted of microvesicles and the growth factors they contain, is collected.

The goal is achieved as follows:

Prepare a solution of chitosan, preferably with a molecular weight of at least 200 kDa, at a concentration of 400 to 600 μg/ml. To achieve the best result, the calculated amount of chitosan is placed in half the volume of distilled water (800-1200 µg/ml), mixed thoroughly and this suspension is left at a temperature of 4°C to swell for a period of 2 to 16 hours. Separately, prepare a 2% (wt/vol) solution of acetic acid, namely, dissolve glacial acetic acid in distilled water at the rate of 0.32 to 0.36 mol (19.2-21.6 g) acid per 1 liter of water. The chitosan suspension is adjusted with a 2% acetic acid solution to a final concentration of 400 to 600 μg/ml, which corresponds to a twofold dilution. It is possible to prepare a chitosan solution directly by mixing the dry substance with a 1% solution of acetic acid (9.6-10.8 g/liter), however, with this approach the mixture contains a large number of bubbles, which, in turn, can make dosing difficult. Separately, prepare a 5% solution of potassium or sodium hydroxide by mass-volume method from dry alkali and distilled water. The chitosan solution can be prepared in advance and remains usable for at least 4 days.

Depleted serum samples do not need to be pre-cleaned by filtration or centrifugation because High molecular weight chitosan actively adsorbs large contaminants, such as blood cell debris.

The resulting chitosan solution with a molecular weight of at least 200 kDa, at a concentration of 400 to 600 μg/ml in a chitosan:serum ratio of 1:6 to 1:8, is added to the serum samples under aseptic conditions. Using less chitosan makes it difficult to observe precipitate formation, while using more significantly dilutes the serum, which may or may not be desirable depending on its further experimental application.

The mixture is thoroughly mixed and incubated for 1 to 3 hours at 4°C, after which a 5% solution of potassium or sodium hydroxide is added dropwise under aseptic conditions until opalescence appears. Opaseescence most often takes the form of thin whitish fibers at the surface of the solution. When it appears, stop adding alkali and leave the neutralized mixture for additional incubation for 30-60 minutes. After this, the mixture is centrifuged at 12,000 g for 20 minutes and the supernatant, which is serum depleted of microvesicles and the growth factors they contain, is collected. Cooling the mixture during centrifugation is not necessary, but may be desirable if the depleted whey is not intended to be used immediately after receipt.

The resulting serum is depleted in microvesicles and the growth factors they contain, but contains an increased amount of sodium or potassium acetate, so if it is added to the culture medium in an amount of more than 10%, preliminary purification from salts may be required by dialysis through a membrane with a throughput of at least 3. 5 kDa.

Chitosan is a natural polyaminosaccharide with a wide range of biological properties. Chitosan is a water-insoluble deacetylated form of chitin. Acetylation of chitosan ensures its solubility in polar solvents. The presence of amino groups creates areas on the surface of the chitosan molecule with a partial positive charge, which are capable of electrostatically attracting microvesicles with a negative charge. This allows microvesicles to be adsorbed on dissolved chitosan molecules during incubation of the serum with a chitosan solution. After neutralizing the solution with a 0.2 M alkali solution, chitosan ceases to dissolve and precipitates in the form of fibers, along with adsorbed microvesicles.

The high molecular weight of chitosan allows it to be precipitated from solution in centrifuges that create accelerations of up to 12,000 g, which is much less than in ultracentrifugation units (over 100,000 g) and makes it possible to deplete whey using the claimed method without the use of ultracentrifuges.

Thus, a reduction in the number of manipulations with serum and requirements for laboratory hardware is achieved. In addition, since the neutralization of chitosan and its conversion to an insoluble form is carried out in situ, immediately after incubation of chitosan with serum, the technique can be adapted to chitosan with different solubilities, for example, obtained from non-traditional sources.

Application example 1

A 5% (v/v) emulsion of petroleum jelly in water was prepared with the addition of 0.5% (v/v) TWIN-20. An artificially produced emulsion was used to ensure that the amount of solids was well above the detection limit. A chitosan solution at a concentration of 600 μg/ml was added to the emulsion in a ratio of 1:6 and incubated for 3 hours.

After centrifugation, the precipitate was washed with phosphate-buffered saline, hexane was added, and incubated for 15 minutes to dissolve the petroleum jelly adsorbed on the chitosan fibers. A drop of hexane was mixed on a glass slide with an equal volume of an alcohol solution of iodine and left until completely dry. Since Vaseline oil is not volatile, after the mixture dries, microscopic drops remain on the glass, colored dark with dissolved iodine. In fig. Figure 1 shows crystals formed after drying a solution of iodine mixed with hexane. In this case, hexane was used, obtained after incubation with the precipitate formed after the adsorption of vaseline oil from the emulsion. In fig. Figure 2 shows crystals formed after drying a solution of iodine mixed with hexane. In this case, hexane was used, obtained after incubation with the precipitate formed after experimental procedures with a control sample of distilled water.

The supernatant was collected separately and the dry residue was assessed on an A&D MS70 moisture analyzer at 60°C. Since petrolatum oil cannot evaporate at a given temperature, the method makes it possible to quantitatively measure its content in the emulsion before and after the adsorption of petrolatum oil droplets with chitosan. After drying, the weight of the native emulsion aliquot decreased from 3.140 to 0.170 g (5.41% solids), while the weight of the chitosan-treated emulsion decreased from 3.070 to 0.122 g (3.97% solids).

The data obtained qualitatively and quantitatively indicate that the proposed method allows the adsorption of fat-soluble fractions of heterophase mixtures. Incomplete adsorption is associated with the lack of electrostatic interaction between the molecules of chitosan and compounds in the composition of petroleum jelly.

Application example 2

Isolation of microvesicles from serum was performed with FBS (fetal bovine serum) samples. A chitosan solution at a concentration of 500 μg/ml was added to the serum in a ratio of 1:8 and incubated for 1.5 hours. After centrifugation, the supernatant was discarded, the chitosan precipitate was washed with phosphate-buffered saline and hydrolyzed with an excess of 1 M hydrochloric acid solution at 80°C for 1 hour. The hydrolyzate was cooled to room temperature and the formation of free fatty acid spots on the surface of the fatty acids was observed, which is qualitative evidence that the sediment contained adsorbed microvesicles, which included membrane phospholipids, hydrolyzed to fatty acids, glycerol and phosphate ions.

Claims (1)

A method for obtaining culture sera depleted of microvesicles and vesicular growth factors, characterized in that depletion is carried out by binding microvesicles with a solution of chitosan with a molecular weight of at least 200 kDa, in a 1% (wt./vol.) solution of acetic acid, followed by neutralization, centrifugation at no more than 12,000 g and collection of the supernatant.