CZ277691B6 - Micro-carriers with fibronectin suitable for cultivation of animal cells, process for preparing and use thereof - Google Patents

Abstract

The solution involves biologically active spheric gels from aldimine derivatives of gelatine on the surface of which fibronectin is adsorbed biospecifically. These microcarriers are suitable as a cultivation bed for cultivating animal cells. Microcarriers are formed by 50 to 350 μm spherical particles from the aqueous gel of aldimine derivatives of gelatine, which are reaction products of gelatine and a-amino acids with dysfunctional aldehydes, on the surface of which adhesive protein - fibronectin is adsorbed biospecifically. Microcarriers are prepared by putting spherical micro particles from a water gel of aldimine derivatives of gelatine into contact with a water solution containing fibronectin at pH 5 to 8 for 1 minute to 24 hours. Ballast components of the fibronectin solution are then washed away with a water buffer with the same pH and the product is preserved by freezing or lyophilization or by transferring it to a 20% to 99% solution of a suitable water-mixing organic solvent. After defrosting or swelling in water or after washing away the solvent and transferring to an aqueous physiological buffer with a pH 7 of 7.6 the product is used as a cultivation bed for cultivating animal cells under standard cultivation conditions.

Description

The present invention relates to biologically active spherical gels from aldimine gelatin derivatives to which fibronectin is adsorbed biospecifically. These microcarriers are suitable as a culture support for the cultivation of animal cells.

In laboratory work and especially in industrial production, it is often necessary to grow large amounts of cellular material. This is the case in the production of viral vaccines and various products of cellular metabolism, such as interferon, hormones), enzymes, nucleic acids and other valuable biological substances.

Various animal cell culture techniques have been developed for this purpose. The most effective technique is suspension cell culture. However, many cells need a suitable substrate to grow and divide, to which they attach and form a cell monolayer. They are mostly primary cells obtained from organs, tissues or embryos of various animals, for example primary cells from chicken, rat or mouse embryos. Further, these are diploid cell strains, for example, the LEP strain from the lungs of a human embryo.

The cell attachment, growth and multiplication mat must meet a number of requirements. It must have an optimum particle size, typically 70 to 320 microns, which is a compromise between the curvature of the particle surface and the overall particle size. Furthermore, it must have an optimum matrix density, typically 1.0 to 1.1 g / ml, so that the particles can be kept as fluid as possible by minimal agitation. It must not be cytotoxic but fully biocompatible.

Cell cultures are grown on microcarriers in culture media consisting of inorganic salts, amino acids, vitamins and coenzymes such as basal or minimal Eagle's medium and others. The medium is usually supplemented with animal sera, for example inactivated calf serum, fetal bovine serum, or chicken serum, at a concentration of 1-20% by volume. In some cases, the medium is supplemented with protein hydrolyzate, soy peptone, yeast hydrolyzate, and other nutrients. The culture media have an optimum pH in the range of 7.0 to 7.6 and an osmolality of 260 to 350 mOsm.

The microcarriers of cell culture pads are currently produced mainly from amino derivatives of dextran, agarose, cellulose, polyacrylamide, chemically crosslinked gelatin, but also of glass, silica gel and plastics, for example based on crosslinked polystyrene or copolymer polyacrylonitriles. Composite microcarriers are also produced in which the polymer or glass core is coated with a layer of peptides or proteins. In this case, the peptides or proteins are chemically bound to the core surface, or are chemically crosslinked to the insoluble coating on the surface. The most commonly used peptides and proteins are gelatin and collagen, which are more biocompatible and more suitable for cell attachment and growth than the core material.

The attachment of cells to the surface of microcarrier particles is not direct. It takes place via adhesive proteins, for example fibronectin. Fibronectin is a glycoprotein consisting of two

Polypeptide chains that are linked by disulfide bridges. Its molecular weight is about 440,000 daltons. Fibronectin in soluble form is present in blood plasma, at a concentration of about 300 μg / ml, and in serum, at approximately half the concentration. In insoluble form, bound in multimeric aggregates, it is present on the cell surface. The fibronectin cell receptors are gangliosides. Fibronectin is very sensitive to the action of various proteinases, including plasmin. In the isolated form, it is unstable (even in the lyophilized state) and thus gradually loses its biological activity. Also, its cost is considerably high and this does not allow its economical use in manufacturing cell culture. The natural representation of fibronectin in the cell wall is not sufficient, moreover, the proteolytic enzymes used to dissociate cells further reduce its amount. Therefore, fibronectin is added to the nutrient medium, usually as part of the serum. However, the use of serum is economically expensive.

These drawbacks eliminate microcarriers for animal cell cultivation according to the invention, characterized in that they are formed by spherical particles of 50 to 350 μη size from an aqueous gel of aldimine gelatine derivatives, on whose surface the adhesive protein - fibronectin is adsorbed biospecifically. Gelatin aldimine derivatives are the reaction products of gelatin and alpha-amino acids with difunctional aldehydes, for example glyoxal or glutaraldehyde. The microcarriers of the invention are prepared by contacting spherical microparticles of an aqueous gel of aldimine gelatin derivatives with an aqueous solution containing fibronectin, for example blood plasma or serum, at a pH of 5.0 to 8.0, for one minute to 24 hours whereupon the ballast components of the fibronectin solution are washed with an aqueous buffer of the same pH and the product is preserved by freezing or lyophilization or by transfer to 20% to 99% of an aqueous organic water-mixing solvent, for example methanol, ethanol, isopropanol or acetone. The microcarriers according to the invention, after thawing or swelling in water or after washing off the organic solvent and transferring to an aqueous physiological buffer at pH 7.0 to 7.6, are used as a culture support in the cultivation of animal cells under otherwise usual cultivation conditions, preferably in serum-free or on serum poor nutrient media.

Starting spherical aldimine gelatin derivatives suitable for the biospecific adsorption of fibronectin can be prepared according to AO CS 248.861. The method of contacting this gel material with a fibronectin solution can be accomplished in any manner. For example, statically (batchwise) or dynamically - by flowing a fibronectin solution through a bed of gel microparticles.

Since the surface of the microcarriers of the invention is fully saturated with fibronectin, and this biospecifically adsorbed, chemically intact, native fibronectin is also fully biologically active, thereby accelerating the phase of attachment of animal cells to the surface of the microcarriers.

Due to the use of a cheap source of blood plasma, prepared for example from waste bovine blood, the cost of preparing these microcarriers is very low. The starting blood plasma (or serum) is used either fresh or preserved (even in the long term), for example by freezing to -18 to -20 ° C. Appropriate inhibitors, such as ethylenediaminotetraacetic acid disodium, 6-aminocaproic acid, benzamide, and phenylmethanesulfonyl fluoride, may be added to plasma (sera) to prevent unwanted enzymatic cleavage of fibronectin by non-specific proteinases. The biospecific (sorption) interaction of fibronectin with aldimine derivatives of gelatin results in the formation of a solid complex in which otherwise unstable fibronectin is well stabilized against decomposition, so that microcarriers can be stored for long periods without loss of adhesion and other biological properties of fibronectin with respect to animal cells.

The microcarriers of the invention save their users expensive blood serum, which is added to the nutrient media. Serum levels can be reduced by up to one order, or, optionally, serum-free media can also be used.

The microcarriers of the invention can be prepared in a simple manner for use. According to the method of preservation: thawing, swelling in sterile physiological phosphate buffer, or washing the organic solvents with this buffer.

The examples below illustrate the preparation and use of microcarriers of the invention.

Example 1

The starting aldimine gelatin derivatives in the form of spherical gel microparticles having a particle size of 100 to 150 μιη were prepared according to Example 4, AO CS 248.861. The gel microparticle suspension in physiological phosphate buffer pH 7.2 was vacuum filtered. To a 300 g filter cake was added 85 ml of citrate blood plasma prepared from bovine waste. The suspension was stirred for 15 minutes, suctioned and filtered. The blood plasma ballasts were removed by washing with the same buffer as described above until the filtrate gave a positive reaction to the proteins with trichloacetic acid. The vacuum-aspirated microcarrier filter cake was dispersed in 275 ml of undenatured ethanol by vigorous stirring.

Example 2

The same spherical gel microparticles from aldimine gelatin derivatives as in Example 1 were used to adsorb fibronectin, except that their size was 200 to 250 μιη. A suspension of these particles in 0.05 M TRIS-HCl buffer pH 7.5 containing sodium chloride in one molar concentration was poured into a filter bottom column to form a bed of gel microparticles, a 4 cm diameter cylinder and a 20 cm length. 150 ml of serum prepared from bovine waste blood was fed to the top of the column at a rate of about 5 ml / min. The column bed was then eluted at the same rate with the above buffer until a negative reaction to the proteins in the filtrate. Finally, the microcarrier bed was washed with two column volumes of physiological phosphate buffer at pH 7.2 and vacuum aspirated. Half of the carrier thus prepared was frozen by freezing to -20 ° C and the other half of the microcarrier was lyophilized.

Example 3

Primary chicken embryonic cells differentiate in vitro into muscle fibers. In this case, a nutrient medium with 10% by volume of fetal calf serum is normally used. Using serum-free media, the cell attachment phase was very long (8-10 hours).

Using gelatin microcarriers with adsorbed fibronectin, the time required to attach the cells was significantly reduced (2 to 3 hours). This also increased the percentage of cells that survived the explanation period, as well as the total number of cells attached and the number of myotubes formed there was significantly higher.

Example 4

For large-scale production of viral antigens on chicken embryonic cells, culture media with the addition of 5% to 10% bovine serum are used. A low serum medium may be used to cultivate said cells on fibronectin sorbed microcarriers. 5,000 ml of Hanks' saline solution with 0.3% lactalbumin hydrolyzate and 2% bovine serum, 300 ml microcarrier sediment and 5 x 10 ⁹ chicken embryonic cells are placed in a 20 liter fermenter. The phase of attachment of the cells to the microcarriers takes 8 to 12 hours, with intermittent stirring - 1 minute stirring and 30 minutes at rest. Then another 5,000 ml of the same culture medium is added to the fermenter and the suspension is stirred without interruption. Complete cell monolayers are formed on microcarriers on the third to fifth day of culture. The total number of cells propagated on microcarriers was determined and the cells were subsequently infected with Aujeszky's disease virus per cell at 0.1 units (PFU) - the number of plaques formed in the lysed cells. The virus is allowed to adsorb to the cells for 4 hours. After virus adsorption, the suspension is further agitated, 48 hours after infection of the cells, an overall cytopathic effect is produced. From the fermenter was then aspirated fluid and infectious Tube method determines the infectious titer, this amounts to typically 1 ml 10 ^{7 '0-10 8} * ^5.

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PATENT CLAIMS

Claims (4)

PATENT CLAIMS Microcarriers for animal cell culture, characterized in that they are formed by spherical particles of 50 to 350 microns in size from an aqueous gel of aldimine derivatives of gelatin, on whose surface the adhesive protein - fibronectin is adsorbed biospecifically. Microcarriers according to claim 1, characterized in that the aldimine derivatives of gelatin are the reaction products of gelatin and alpha-amino acids with difunctional aldehydes, for example glyoxal or glutaraldehyde. Process for preparing microcarriers according to claims 1 and 2, characterized in that the spherical microparticles of the aqueous gel of aldimine gelatin derivatives are contacted with an aqueous solution containing fibronectin, for example blood plasma or serum, at a pH of 5.0 to 8.0, for one minute to 24 hours, after which the ballast components of the fibronectin solution are washed with an aqueous buffer of the same pH and the product is preserved by freezing or lyophilization or by transferring to 20% to 99% aqueous organic water-mixing solvent, for example methanol, ethanol, isopropanol or acetone. Microcarriers according to claims 1 to 3, used after thawing or swelling in water or washing of the organic solvent and transferred to an aqueous physiological buffer of pH 7.0 to 7.6 as a culture support in the cultivation of animal cells under otherwise conventional culture conditions, preferably in serum-free or serum-poor nutrient media.